GOOD MORNING, EVERYBODY.
WE'RE GOING TO TRY TO START ON
TIME BECAUSE WE HAVE A FULL
SCHEDULE AND WE'RE VERY EXCITED.
SO ON BEHALF LF PARK MY
CO-CHAIR, WHICH HE BASICALLY HAS
DONE ALL THE WORK AND SAID OH
YOU SHOULD STAND UP HERE AND I'M
NAOMI TAYLOR, YOU SHOULD ALL
KNOW ME HAD U NETS A HUGE
PLEASURE TO HAVE YOU HERE.
I'LL SAY A COUPLE WORDS BUT I
WANTED TO TAKE THE OPPORTUNITY
TO INTRODUCE GLENN MERLINO AND
THANK GLENN MERLINO AND ALL THE
DIRECTION.
CENTER FOR CANCER RESEARCH FOR
THEIR INCREDIBLE SUPPORT AND
REALLY A HUGE THANKS FOR
STARTING THIS MEETING.
>> THANKS, NAOMI.
I AM REALLY EXCITED TO BE HERE.
THE IMMUNOLOGY MEETINGS HERE ARE
ALWAYS THE BEST, IN MY OPINION,
AND THIS I'M SURE LOOKING AT THE
SCHEDULE AND SEEING WHO IS IN
THE AUDIENCE, THIS WILL BE NO
DIFFERENT.
I WOULD LIKE TO WELCOME YOU TO
BETHESDA, FOR THOSE OF YOU
DHEENT LIVE HERE, AND TO THIS
WONDERFUL MEETING, AND I WOULD
ALSO LIKE TO DO SO ON BEHALF
THOMAS BELLY AND CCR RESEARCH.
I'M GOING TO SPEND A COUPLE
MINUTES SO YOU CAN GET RIGHT TO
T BUT THIS IS GOING TO BE A
LITTLE BIT OF A CCR-CENTRIC
INTRODUCTION.
FOR THOSE ON THE OUTSIDE, YOU
GET TO SEE A LITTLE BIT ABOUT
WHAT WE'RE DOING HERE AND WHAT
WE'RE ABOUT.
FOR THOSE OF YOU WHO LIVE AND
WORK HERE, MAYBE YOU'LL REMEMBER
HOW GREAT THIS PLACE REALLY IS
FROM THE INTRODUCTION.
AND THANK YOU NAOMI AND I'LL
COME BACK TO THAT FOR ALL YOU
ORGANIZERS AT THE END.
OKAY.
SO BECAUSE OF OUR SITUATION HERE
IN TERMS OF HOW WE DO BUSINESS
AND HOW WE GET FUNDED AND THE
RESOURCES THAT WE HAVE, IT GIVES
US REALLY SCIENTIFIC -- GIVES US
REALLY AMAZING SCIENTIFIC
FREEDOM, BY SOLVING THE MOST
IMPORTANT CHALLENGING AND
NEGLECTED PROBLEMS IN CANCER
RESEARCH, PREVENTION AND ALSO
PATIENT CARE, AND OUR VISION IS
SIMPLY TO CREATE THE MEDICINES
OF TOMORROW.
SO CCR IS A BIG PLACE.
NCI IS A BIGGER PLACE.
BUT WE ARE THE LARGEST OF THE
INTRAMURAL COMPONENTS, AND WE DO
FROM SOUP TO NUTS.
WE HAVE VERY STRONG BASIC
RESEARCH PROGRAM, WE DO
TRANSLATION, CLINICAL CARE, AND
WE HAVE, AS YOU'LL SEE, THE
LEARNLGST CLINICAL RESEARCH
CENTER IN THE -- LARGEST
CLINICAL VERCH IN THE WORLD.
TODAY COURAGE WE HAVE 233 PI'S
ORGANIZED INTO FOUR FOUR
BRANCHES, CENTERS AND PROGRAMS
MOSTLY DPEENLDING ON OUR MODE I
THINK MORE THAN ANYTHING ELSE,
WE HAVE TWO WIG BIG CAMPUSES,
BETHESDA, ABOUT 50 MINUTES NORTH
IN FREDERICK WHICH HAS ABOUT 25%
OF OUR PI'S, 3300 STAFF
ALTOGETHER AND 40% TRAINEES, SO
I WANT TO EMPHASIZE THAT WE DO
THE FULL SPECTRUM.
AND FOR THE RESEARCH THAT WE'LL
BE TALKING ABOUT TODAY, I WANT
TO EMPHASIZE THE
DISCOVERY-DRIVEN BASIC RESEARCH
COMPONENT BECAUSE TOM AND I AND
ALL OF THE LEADERSHIP BELIEVES
THAT THIS IS THE FOUNDATION OF
THANKS WE DO HERE AND LEADS TO
TRANSLATION, CLINICAL RESEARCH
AND OF COURSE EVENTUALLY PATIENT
CARE BUT THE FOUNDATION IS
REALLY THE BASIC RESEARCH.
SOY MENTION THIS HAD BEFORE BUT
REALLY WHAT'S UNIQUE ABOUT OUR
PROGRAM IS THE SCIENTIFIC
FREEDOM THAT ALLOWS US TO ALMOST
LITERALLY WAKE UP, GO INTO THE
SHOWER, HAVE AN IDEA AND IF YOU
CAN CONVINCE YOUR POOR GULLIBLE
POST-DOC THAT YOU SHOULD DO THIS
NEW PROJECT, YOU'RE ON YOUR WAY.
AND WE WANT YOU TO TAKE RISKS.
WE ACTUALLY CHALLENGE YOU TO
TAKE RISKS AND WEKS DO THINGS
HERE THAT TAKE -- AND WE CAN DO
THINGS THAT TAKE A LONG TIME.
FOR EXAMPLE, WAS RTDING TO BUILD
MEL LANOMA MOUSE MODEL SAYS, I'M
PRETTY SURE HAD THERE I COULD DO
THE FIRST BUT WOULDN'T GET THE
COMPETITIVE MODEL AFTER THREE OR
FOUR YEARS.
BUT YOU HAVE THE TIME AND IT
WORKS OUT REALLY WELL AND THE
HEART OF OUR PLACE HERE IN
BETHESDA IS NIH CLINICAL CENTER,
LARGEST CLINICAL CENTER IN THE
WORLD, WE HAVE A FULLY DEDICATED
STAFF, HOSPITAL, AND BASICALLY
ALL THE PHYSICIAN SCIENTIST HERE
HAVE ACTIVE RESEARCH
LABORATORIES AS WELL AS THEIR
CLINICAL TRIALS, AND WE TRY TO
HAVE SEAMLESS TRANSLATION SAYS
AND FIRST IN HUMAN TRIALS.
THESE ARE SOME OF THE NUMBERS.
LAST YEAR WE HAD ELEVEN HUNDRED
ARTICLES AND PEER REVIEWED
JOURNALS, CURRENTLY 346 OPEN
TRIALS, CLINICAL TRIALS COMING
ALL THE TIME, 27,000 PATIENTS IN
VAROUS STUDIES, THOUSANDS OF
COLLABORATIONS IN ALL THE STATES
AND MANY COUNTRIES.
OH, SINCE I BEGAN TO TALK RS WE
HAVE TWO MORE P -- WE HAVE TWO
MORE PI'S THAN WE HAD BEFORE,
YOU SEE HOW ACTIVE OUR PROGRAM
IS.
BOIRKS I'VE GOT TO TALK TO
SOMEBODY ABOUT THAT.
18 NEW FACULTY RECRUITS JUST
THIS YEAR AND THREE NEWLY
TENURED INVESTIGATORS, STAFF
SCIENCE IS ACTUAL BACKBONE OF
OUR PROGRAM AND FRMENTS OUTSIDE
AND REALLY THE HEARTBEAT OF WHAT
GOES ON HERE AND OF COURSE WE
HAVE HUNDREDS OF POSTDOCTORAL
FELLOWS, POSTBACK STUDENTS
REALLY INTO MENTORING AND
PROMOTING CAREERS OF YOUNG
INVESTIGATORS, LOTS OF PATENTS
WHAT, WE CALL CRADA'S AND ACTIVE
LICENSES.
AS ANY AT THAT THIS ROGERS AND
MIKE BETA, CLOSE TO THEM IN
THEIR LAB, LOWER RIGHT-HAND
CORNER, PIONEERING IMMUNOTHERAPY
FROM STEVE ROSENBERG ON WE'VE
HAD A LOT TO DO WITH -- YEARS
AGO AND IT TURNED OUT IT WAS
ABSOLUTELY ESSENTIAL IN THE
CLINICAL TRIALS.
AS I SAID THE PEOPLE IN HERE ARE
GOING TO SET THE STAGE FOR THAT.
TO JUST DRIVE THAT HOME, THESE
ARE SOME OF THE EXISTING
STRENGTHS, WE HAVE OF COURSE
DIFFERENT KINDS OF CANCER
MECHANISMS, WE HAVE A LOT OF
WORK ON RARE CANCERS IN
PARTICULAR, WE REALLY FOCUS ON
THOSE.
EPIGENETICS, SOME OF THE FIRST
MICROBIOME WORK WAS DONE HERE,
DRUG DISCOVERY ALL THE TIME, OF
COURSE PRECISION MEDICINE, OF
COURSE HIGHLIGHTING IN
IMMUNOLOGY AND HOW IMPORTANT THE
FUNDAMENTAL IMMUNOLOGY IS TO THE
ENTIRE PROGRAM AND LEADS TO
EVERYTHING ELSE THAT YOU SEE
HERE.
AND AS I SAID, WE'RE VERY PROUD
OF THE IMMUNOLOGY MEETINGS AND
SEMINARS AND SYMPOSIUMS WE'VE
HAD OVER THE YEARS.
SO THIS IS JUST FROM 2017, OUR
RECENT FDA DRUG APPROVALS, YOU
CAN SEE THERE'S QUITE A FEW OF
THEM IN ALL KINDS DIFFERENT
AREAS.
I'M VERY PLEASED IN PARTICULAR
ABOUT THE 2018 HAIRY CELL
LEUKEMIA BECAUSE I WORKED FOR
IRA PAST SOME TIME AGO AND HIS
IMMUNOTOXINS REALLY WERE THE KEY
TO THAT DRUG AND YOU CAN SEE
THEY CONTINUE ALL THE WAY INTO
2023.
IN TERM OF GLOBAL REACH, WE HAVE
THOUSANDS OF COLLABORATIONS
THERE.
WE WORK WITH MANY, MANY
INSTITUTIONS AND MANY, MANY
DIFFERENT TRAINING
OPPORTUNITIES.
WE TRY TO BE AS INFLUENTIAL AS
WE CAN THROUGHOUT THE WORLD.
JUST LET ME QUICKLY, I'M NOT GRG
TO GO THROUGH THE SCHEDULE, BUT
THE SCHEDULE IS REALLY QUITE
AMAZING.
HAD YOU HAVE LUMINARIES FROM
INTRAMURALS AND EXTRAMURALS,
SCIENTIFIC COUNCILMEMBERS, I
THINK THE ORGANIZERS DID AN
AMAZING JOB OF PUTTING THIS
PROGRAM TOGETHERS.
SO ON DAY ONE YOU'LL BE TALKING
ABOUT THE THYMUS MOSTLY AND
MOSTLY IMMUNITY FRRKS
CROSS-TALK, DICIALT KINDS OF
SIGNALLING, DAY TWO INNATE
IMMUNITY AND MORE ON CELL
SIGNALLING AND CELL FUNCTION AND
SECOND PART FINALLY OF THE
CANCER IMMUNOLOGY PROGRAM.
IT LOOKS LIKE NAOMI IS GOING TO
WRAP THINGS UP FOR US, WHICH
TAKES ME TRITED -- TAKES ME
RIGHT TO WHERE I WANT TO BE, IN
THANKING EVERYBODY FOR PUTTING
THIS WONDERFUL SYMPOSIUM
TOGETHER.
I WANT TO PARTICULARLY THAT I
NAOMI AND HYUN AND OTHERS ALSO
AND I WOULD LIKE TO SHOUT OUT
THE PEOPLE WHO REALLY MAKE THIS
STUFF HAPPEN, JULIA LAMB,
CRYSTAL --
[CHEERING]
[APPLAUSE]
HAD BECAUSE OTHERWISE WE WOULD
ALL BE SITTING HERE AND HAVING
COFFEE AND THAT WOULD BE IT.
OKAY.
SO THAT'S REALLY ALL I HAD TO
SAY.
I'M GOING TO HAVE NAOMI COME UP
AND REALLY KICK THINGS OFF RIGHT
NOW.
BUT THANK YOU AGAIN FOR THE
INVITATION TO TALK, AND PLEASE
HAVE A WONDERFUL SYMPOSIUM.
[APPLAUSE]
>> THANK YOU, GLENN, FOR
STARTING THAT OFF ON A WONDERFUL
FOOTING.
IT'S REALLY A HUGE PLEASURE TO
BE HERE.
AND I THINK WHAT GLENN TALKED
ABOUT IS THAT THIS IS A MEETING
SPORCH SOARED BY THE CENTER FOR
CANCER RESEARCH, BUT IT REALLY
IS SUPPOSED TO BE -- SPONSORED
BY THE CENTERED FOR CANCER
RESEARCH, REALM IS SUPPOSED TO
BE TO SHOWCASE IMMUNOLOGY.
I SPENT MOST OF MY CAREER IN
FRANCE AND HAD THE OPPORTUNITY
TO COME BACK TO U.S. A COUPLE
YEARS TO JOIN THE PEDIATRIC
ONCOLOGY BRANCH.
LAST WEEK WE HAD SOME
INDIVIDUALS COME FROM CENTER FOR
CHILDHOOD CANCER ALLIANCE COME
TO VISIT THE LAB, AND, YOU KNOW,
CLEARLY PEOPLE WHO ARE NOT
NECESSARILY INTERESTED IN BASIC
IMMUNOLOGY, THEY'RE THERE
BECAUSE THEY WANT A CURE -- THEY
WANT TO CURE CHILDREN WITH
CANCER.
BUT I THINK WHEN WE SHOW THEM
THE PICTURES OF WHAT WE WERE
DOING IN THE LAB AND WHAT WAS
IMPORTANT, YOU KNOW, ONE OF THE
THINGS THAT WAS ON THE SCREEN IS
PEOPLE HADN'T SPENT A HUGE
AMOUNT OF TIME REALIZING THAT
GREEN FLUORESCENT PROTEIN FROM A
JELLYFISH COULD BE USED TO
FIGURE OUT WHERE CELLS WERE
GOING.
WE COULDN'T BE ABLE TO LOOK AT
CANCERS IN MICE IF WE DIDN'T
KNOW HOW LUCIFERASE WORKED AND
TO BE ABLE TO STUDY FIREFLIES.
WHILE SOME OF THESE THINGS YOU
WOULD MAKE THE FRONT PAGE OF THE
WASHINGTON POST AND SAY WHY ARE
WE SPENDING MONEY ON THIS?
THE POINT IS WE WHERE THOSE
FRONTIERS COME, I THINK THAT'S
WHY WE'RE HERE.
IT'S A HUGE PLEASURE TOWF ALL
HERE IN PERSON BECAUSE I THINK
MAKING THOSE LEAPS AND
BOUNDARIES CAN'T HAPPEN IF
PEOPLE DON'T TALK TO EACH OTHER.
THE PLEASURE OF BEING HERE ON
SABBATICAL WOULD SAY THE MOST
IMPORTANT THINGS YOU LEARN YOU
LEARN FROM GOING AND HAVING
COFFEE AND TALKING AND I THINK
THAT WITH COVID AND WITH THE
FACT THAT PEOPLE WEREN'T HERE IN
PERSON, WE REALLY LOST SOME OF
THAT.
SO IT'S REALLY A HUGE PLEASURE
TO SEE SO MANY SMILING FACES AND
WE HAVE TRIED TO KEEP THE TALKS
DOWN TO A MINIMUM BECAUSE WE
WOULD LIKE TO MAKE SURE THAT WE
HAVE QUESTIONS.
SO PLEASE ASK QUESTIONS.
WE WILL GIVE AWARDS FOR TRAINEES
WHO ASK QUESTIONS BECAUSE THAT'S
WHERE WE WANT THE QUESTIONS TO
COME FROM.
SO IF YOU SEE SOMEBODY WHO LOOKS
OLDER AND MORE GRAY HAIRS IN
FRONT OF YOU, YOU CAN GO IN
FRONT OF THEM.
IF THERE'S SOMEONE BEHIND YOU
WHO HAS LESS GRAY HAIRS THAN
YOU, THEY GET TO GO FIRST.
THAT GOES FOR YOU TOO.
SERIOUSLY.
THE POINT IS REALLY TO HAVE IT
BE AN INTERACTIVE ENVIRONMENT.
WE REALLY WANT THIS TO BE A
PLACE WHERE WE CAN DISCUSS,
WHERE WE CAN INTERACT.
SO IT'S A HUGE PLEASURE TO BE
ABLE TO DO THIS, TO THINK ABOUT
WHERE WE'RE GOING TO GO AND TO
NOT HAVE ANY IDEA WHAT THAT
MEANS.
SO WE HAVE SPEAKERS WHO ARE
HERE, WHO WILL BE SHOWCASING THE
CCR, AND THAT IS TOGETHER WITH
PEOPLE FROM ALL OVER THE WORLD,
AND IT'S REALLY A HUGE PLEASURE
TO HAVE PEOPLE WHO COME HERE
FROM QUITE SOME NUMBER OF
DIFFERENT CONTINENTS TO REALLY
BE ABLE TO SEE WHERE THE
EX-QUESTIONS ARE.
AND IT'S -- WHERE THE NEXT
QUESTIONS ARE.
THE QUESTIONS ARE IMPORTANT
IMRRKS THEN WE CAN DEVISE THE
EXPERIMENTS TO ANSWER THEM ONCE
WN WHAT THE QUESTIONS ARE.
IT WOULD BE REALLY PATE TI NOT
TO BE DISCUSSING THOSE
QUESTIONS.
PLEASE DO ASK THAT.
SO I'M STANDING HERE BECAUSE
LOTS OF PEOPLE CAME BEFORE ME TO
PUT THIS IN PLACE.
THIS FRONTIERS IN BASIC
IMMUNOLOGY PROGRAM.
THIS IS P THE SEVENTH ONE.
I WASN'T EVEN BORN FOR THE FIRST
ONE.
I'M KIDDING.
BUT IT WAS STARTED BY AL SINGER
AND IT WAS STARTED BY ALTOGETHER
WITH LARRY SAMULSON, JOHN ASHWA
AND RAINY, A SHOUT OUT TO ALL OF
YOU TO COME BACK AND ORGANIZE
THIS.
SO WITHOUT FURTHER ADO, WELCOME,
AND THANK YOU AGAIN.
PLEASE ASK QUESTIONS.
[APPLAUSE]
>> GOOD MORNING TION EVERYONE,
WELCOME AGAIN.
I'M STANLEY ADORO TOGETHER WITH
CHRISTIAN MAYER WE'LL BE
STARTING THE MORNING QUESTION.
I WOULD LIKE TO WELCOME AL
SINGER TO GIVE THE FIRST TALK
TIED TO TRANSCRIPTIONAL
DETERMINATION OF T LINEAGE FATE.
WELCOME, AL.
>> THANK YOU.
SOPHIA.
I'VE ACTUALLY CHANGED THE TOPIC
OF MY TALK THIS MORNING.
AND I'LL DESCRIBE IT TO YOU IN A
MOMENT.
THANKS.
THANK YOU.
WELL, LISTEN, SO GOOD MORNING
EVERYONE AND WELCOME OF THE
WE'VE BEEN LOOKING FORWARD TO
HAVING YOU ALL HERE.
SO IT'S GREAT TO SEE YOU.
AS MECHANISM OF CD8 T CELL
TOLERANCE WHICH WE CAME UP WITH
REALLY BASED ON OUR
UNDERSTANDING OF HOW T LYNN
LINEAGE FATE IS DMERND THYMUS.
I WAS ORIGINALLY GOING TO TALK
TO YOU ABOUT HOW IT IS
DETERMINED IN THE THYMUS I
THOUGHT I WOULD TELL YOU ABOUT
THIS NEXT GENERATION OF STUDY
ABOUT WHAT WE'VE LEARNED AS A
CONSEQUENCE OF IT.
SO AGAIN, I'M AL SINGER FROM THE
EXPERIMENTAL IMMUNOLOGY BRANCH
HERE IN THE INTRAMURAL PROGRAM
OF THE CANCER INSTITUTE AND --
WAS DONE BY SHARIFF BODDER WHO
PUSHED THIS FROM THE BEGINNING
TO THE END AND YOU'LL (AWAY FROM
MIC) ABOUT DLSZ THESE INSIGHTS.
SHERIF BADR.
YOU'LL LEARN ABOUT A NEW
MECHANISM OF T CELL TOLERANCE
WHICH WE THINK IS THE ACTIVE
MECHANISM OF T CELL TOLERANCE
NAMED CLONAL EVICTION.
AS YOU KNOW IN THE THYMUS,
IMMATURE THYMUS SITES DEVELOP
FROM STEM CELL PRECURSORS AND
THESE GUYS THROUGH AN ORDERED
SERIES OF DEVELOPMENTAL EVENTS
IN THE THYMUS.
THOSE THAT BECOME ALPHA-BETA T
CELLS EXPRESS THE ALPHA-BETA T
CELL RECEPTOR AND THEY FIRST
EXPRESS THE ALPHA-BETA T CELL
RECEPTOR AS AN IMMATURE DOUBLE
POSITIVE THYMUS SITE AND IT'S
DOUBLE POSITIVE BECAUSE IT HAS
BOTH CD4 AND CD8 CODE RECEPTORS.
DEVELOPMENT STOPS AT THIS POINT
UNLESS THE DOUBLE POSITIVE
THYMUS SITES FIND A LIGAND FOR
THE T CELL RECEPTOR THAT SIGNALS
THEM TO GO FURTHER.
SO TO GET SIGNALED, THE CELLS
NEED TO GET, NEED TO SOMEHOW
ENCOUNTER LCK AND THE LCK IS
SEQUESTERED BY THE CD4 AND CD8
CO-VEEP TORS.
CD4 ENGAGES CLASS TWO AS YOU ALL
KNOW, SAILT ENGAGES CLASS ONE
THAT, BRINGS IN THE LCK
ASSOCIATED WITH THE TAIL, YOU
GET A SIGNAL.
SO THE FIRST STEP IN SELECTION
OF THE THYMUS IS THIS TCR
SIGNALLING STEP.
AS A CONSEQUENCE OF THAT T -- OF
THOSE TCR SIGNALS AND AS I'LL
TELL YOU IN A MOMENT, DEPENDING
ON THE QUALITY OF THOSE TCR
SIGNALS, THE DOUBLE POSITIVE
THYMUS CYTES ARE INDUCED TO
EXPRESS ONE OR THE OTHER OF THE
LINEAGE FACTORS THAT DETERMINE
THEIR T CELL LINEAGE FATE, SO
THERE'S A TCH CRZ FACTOR YOU'LL
HEAR ABOUT A LOT TODAY THAT
HELPS HELPER LINEAGE FATE ON THE
THERE CELLS OR RUNX3D, D WHICH
INDUCES THE CD8 CYTOTOXIC TELZ.
WE'VE BEEN STUDYING HOW THOSE
EVENTS HAPPEN FOR MANY YEARS NOW
AND A LONG TIME AGO, ACTUALLY IN
2000, WE THERE PUBLIC PUBLISHED
A MODEL CALMED THE KINETIC
SIGNALLING MODEL OF T LIJAL FATE
DETERMINATION.
THE FUNNY THING ABOUT THE MODEL
WAS THAT IT WAS BASED NOT ON THE
CO-RECEPTOR PROTEINS BUT ON THE
CO-REACCEPTOR GENES AND IT CAME
FROM THE OBSERVATION WHICH WAS A
SURPRISE TO US AND WITH THOUGHT
FROM THE MOMENT WE SAW IT THAT
THERE WAS SOMETHING IMPORTANT
HERE, BUT IT'S TAKEN A WHILE TO
FIGURE OUT WHAT THAT WAS, THE
TCR SIGNALLING, SO THIS
SIGNALLING EVENT IN THYMIC
SELECTION HAS A CONSEQUENCE ON
CD4 AND CD8 GENE EXPRESSION,
THAT IS, THAT TCR SIGNALLING
EVENTS SELECTIVELY TERM NAILTS
THE TRANSCRIPTION OF CD8 GENE.
SO YOU GET A TCR SIGNAL, THE CD8
GENE IS SHUT OFF.
SO TCR SIGNALLING THAT'S
DEPENDENT ON THE CD8 ENCODED
CO-RECEPTOR PROTEIN, THOSE
SIGNALS GRADUALLY WEAKEN,
STEADILY WEAKEN EARN UNTIL THE
SIGNAL IS FINALLY DISRUPTED
DURING SELECTION.
THAT DISRUPTION OF TCR ARE
SIGNALLING FLRKS CYTOKINE, WE'VE
IDENTIFIED SIX OR SEVEN OF THEM,
THAT WILL INDUCE SIGNAL OF THESE
CELLS TO EXPRESS LLZ -- ON THE
OTHER HAND IF THE TCR SIGNAL IS
DEPENDENT NOT ON THE CD8 ENCODED
CO-RECEPTOR BUT ON THE CD4,
NOTHING HAPPENS TO IT, IN FACT,
THAT SIGNAL INCREASES AND
PERSISTS THROUGH OUR POSITIVE
SELECTION AND THAT PERSISTS IN
TCR SIGNAL AND THIS IS THPOK TO
DEPENDENT THE HELPER CELL.
THIS IS HOW LYNN YAM FATE IS
DECIDED, DEPENDENT ON WHETHER
THE SIGNAL PERSIST OR CREASES
AND THE CD8 DEPENDENT TCR SIGNAL
CEASES.
SO IT WILL CD8 RECEPTOR SIGNALS
ARE DISRUPTED AND CEASE DURING
THE PROCESS OF POSITIVE
SELECTION.
SO WE'VE BEEN INTERESTED IN
UNDERSTANDING HOW THIS CAN I
KNELT I CAN SIGNALLING MODEL
IMABLGHTS ON T CELL TOLERANCE --
IMPACTS ON T CELL TOLERANCE.
THIS IS WHAT I'LL TELL YOU ABOUT
TODAY, BECAUSE IF YOU THINK
ABOUT WHAT THIS KINETIC
CERTAINTY MODEL PREDICTS FOR CD8
T CELL TOLERANCE THERE'S QUITE
AN INTERESTING PREDICTION H THAT
IS THAT THE CD8 DEPENDENT
SIGNALS IN THE THYMUS BECAUSE
THEY PROGRESSIVELY WEAKEN UNTIL
THEY'RE DISRUPTED, DEVELOPING
CD8 T CELLS MAY NOT BE SIGNALED
STRONGLY ENOUGH AND MAY NOT BE
SIGNALED LONG ENOUGH TO BE ABLE
TO INDUCE CLONAL DELETION DURING
THYMIC SELECTION, TONT SAY
THERE'S NO CLONAL DELETION, BUT
THERE CAN'T BE ENOUGH CLONAL
DELETION TO ACCOUNT FOR T CELL
TOLERANCE SO WE WONDERED, WHAT'S
THE BASIS FOR CD8 T CELL
TOLERANCE?
IS IT REALLY CLONAL DELETION THE
WAY WE ALWAYS THOUGHT ABOUT
ABOUT IT?
I MEAN, EVERYBODY THINKS THAT
CLONAL -- AND CAN TELL YOU THAT
NO, IT'S ACTUALLY NOT TRUE FOR
CD8 T CELLS.
THE STUDY I'LL TELL YOU ABOUT
THAT WAS PURSUED MADE USE OF
THIS MOUSE, TRANSGENIC MOUSE
THAT WAS GENERATED BY TROY
BALDWIN AND CHRIS CHRIS HEDCRIS,
MADE USE OF THE HY TRANSGENIC
GENE, THIS IS A CLASSIC T CELL
RECEPTOR SPECIFICITY.
WELL, WHAT CHRIS HOGQUIST AND
BALDWIN MODIFIED IN TERMS OF
THIS TRANSGENIC MOUSE WAS THEY
PREVENTED EXPRESSION OF THE TCR
TRANSGENE SO THAT IT WASN'T
EXPRESSED UNTIL THE DOUBLE
POSITIVE STAGE.
SO THIS REFLECTED EXACTLY WHAT
HAPPENED IN THE NORMAL MOUSE.
T CELLS DEVELOPED WHEN YOU HAVE
THE DOUBLE POSITIVE IT THYMUS
CYTE STATES AND FINALLY
EXPRESSED T CELL RECEPTOR.
SPECIFIC HY T CELLS AND YOU CAN
SEE IN THE FEMALE MOUSE THESE
CELLS UNDERGO POSITIVE SELECTION
AND GRURND CD8 POSITIVE.
IN THE MALE MOUSE THERE ARE NO
CD8 POSITIVE CELLS AND THE
PRESUMPTION AS TO WHY THERE WAS
NEVER ANY CD8 SIGNAL POSITIVE
CELLS SINCE THE VERY FIRST
TRANSGENIC MOUSE, VERY FIRST
TRANSGENIC MOWD MADE BY BOB
CLOMER THOUGHT DUE TO DELETION.
ARE THESE CELLS MISSING BECAUSE
THEY AND THE PRECURSORS HAVE
BEEN CLONALLY DELETED?
BUT IN FACT IF YOU LOOK IN THE
PERIPHERY OF THESE MALE MICE,
THE PERIPHERY HAVE LOTS OF STAT.
AND IN FACT, THE MALE MOUSE HAS
MANY MORE CD8 SIGNAL POSITIVE
CELLS IN THE PERIPHERY THAN THE
POSITIVELY SELECTING MALE --
FEMALE MOUSE.
WE THOUGHT THIS CANNOT BE CLONAL
DELETION.
SOS CLONAL DELETION IN FACT
EXPLAINS HOW IT IS THAT CD8
CELLS ARE MISSING F YET THOSE
SAME CELLS APPEAR IN THE PRIFER
RI.
HOW DOES THIS WORK?
SO WE LOOKED IN THE THYMUS AT
THESE HY CD4 MALE CELLS AND WE
FOUND OF COURSE AS MINK OF YOU
KNOW THAT IN THE THYMUS WHAT YOU
SEE IS A POPULATION OF CD4 CD8
DOUBLE DULL CELLS WHICH HAVE
BEEN STRONGLY SIGNALLED BECAUSE
THEY'VE UNDERGONE NEGATIVE
SELECTION AFTER ALL THEY'RE HY
SPECIFIC AND ENCOUNTER THE HY
MALE ANTIGEN OF THE THYMUS SO
THAT STRONG SIGNALLING
UPREGULATES EXPRESSION OF THE
PD1 WHICH IS A FACT OF TCR
SIGNALLING AND ATTENUATOR.
SO IT'S AN ATTEMPT BY THE CELL
TO RESPOND IT THAT STRONG TCR
SIGNAL AND UPREGULATES PD1 AND
THESE CELLS ARE ACTUALLY
IMMATURE SO THEY DON'T EXPRESS
CD24, THEY DON'T DOWN REGULATE,
THEY EXPRESS HIGH LEVELS OF CD
24-RBGS THEY DON'T DOWN REGULATE
CD4 AND THEY DON'T EXPRESS QA2,
THESE ARE IMMATURE PD1 POSITIVE
DOUBLE DULL CELLS SO WE WONDERED
WHAT HAPPENS TO THESE CELLS?
IS IT POSSIBLE THESE GUYS
ACTUALLY GET OUT OF THE THYMUS?
NORMALLY THE ONLY CELLS IN THE
THYMUS THAT LEAVE THE THYMUS ARE
MATURE CELLS.
THESE RNLT MATURE CELLS.
AS YOU SAW, THESE ARE IMMATURE
CELLS BUT WE WONDERED, MAYBE
THEY DO LEAVE THE THYMUS ANYWAY.
TO TEST THAT WE INJECTED THESE
MICE WITH A CONJUGATED
MONOCLONAL ANTIBODY AND LOOKED
FIVE MINUTES LATER.
THE IDEA BEING THAT IN FIVE
MINUTES THE ONLY CELLS THESE
ANTIBODIES WOULD ENCOUNTER WOULD
BE THOSE THAT WERE IN CONTACT
WITH THE BLOODSTREAM BECAUSE THE
ANTIBODY DIDN'T HAVE ENOUGH TIME
TO GET INTO TISSUES.
IN FACT, IF YOU LOOK AND SEE,
YOU SEE THAT THE CELLS THAT GOT
LABELED BY THE ANTIBODIES ARE
THESE DOUBLE DULL CELLS EVEN
THOUGH MOST OF THE CELLS IN THE
THYMUS ARE ACTUALLY PRESELECTION
DOUBLE POSITIVE THYMOSIGHTS SO
THEY DON'T GET LABELED.
SUGGEST THESE TELZ ARE IN
CONTACT WITH THE BLOODSTREAM
BECAUSE THEY'RE IN PROCESS OF
LEAFING THE THYMUS IT.
IF THEY LEAVE THE THYMUS WE
SHOULD BE ABLE TO FIND THEM IN
THE PERIPHERY, YOU SHOULD
REFLECT BOTH IN THE LYMPH NODE
AND SPLEEN AND YOU SEE IN THE
MALE YOU IN FACT FIND THESE
CELLS, YOU FIND THESE DOUBLE
DULL CELLS PRESENT IN THE MALE
PERIPHERY.
AND THESE ARE ACCIDENT OCCURLY
LIKE THE DOUBLE DULL CELLS IN
THE THIEMSZ, CD24 OHIO, THEY'RE
IMMATURE AND QA2 NEGATIVE.
THESE CELLS APPEAR IN THE
PERIPHERY AS IMMATURE CELLS SO
IS IT POSSIBLE THESE CELLS
ARE -- OUTSIDE OF THE THYMUS TO
BECOME IMMATURE CELLS?
TO TEST THAT WE TOOK THE CD4/8
MICE PARK THEM IN A RAG
KNOCK-OUT MICE HERE AND YOU SEE
HERE IN TWO OR TWO AND A HALF
DAY IN THE RAG KNOCK-OUT MOUSE
THESE DOUBLE DULL CELLS ALL BACK
CD8 POSITIVE.
YOU CAN ALSO DO THAT BY SIMPLY
TAKING THESE CELLS AND PUT THEM
INTO CULTURE WITH IL SEVEN IN
VITRO FOR A COUPLE DAYS, SAME
THING HAPPENS, THESE CELLS GO
FROM BEING DOUBLE DULL TO
BECOMING CD8 POSITIVE N THE
REACT IS PD1 POSITIVE NEGATIVELY
SELECTED CD4 CD8 THYMOCYTES,
PREMATURELY EVICTED AS CONTINUE
ARE DIFFERENTIATION IN THE
PERIPHERY, CALLED CLONAL
EVICTION.
HOW DO THESE CELLS GET EVICTED?
HOW IN THE WORLD IS THIS
ACTUALLY POSSIBLE THAT THESE
IMMATURE CELLS GET EE VICK
UNDERSTAND FROM THE THYMUS?
WE KNOW THAT MATURE CELLS
NORMALLY LEAVE THE THYMUS BY
EXPRESSION OF THESE PHOSPHATE
TYPE ONE RECEPTOR, KNOWN AS S1P1
THIS IS THE GENE THAT ENCODES
S1P1 AND YOU CAN SEE NORMALLY
MATURES DON'T NORMALLY EXPRESS
S1PR1, ONLY MATURE, BUT IN
NEGATIVELY SELECTED MALE MOUSE
WE SEE THE CD8 DULL IMMATURE
PRECURSOR CELLS THAT HAVE BEEN
NEGATIVELY SELECTED ALSO EXPRESS
S1P1.
IS THIS IMPORTANT FOR EXPRESSION
OF DEPARTURE LEAVING THE THYMUS?
TO TEST THAT, WE MADE THE HY CD4
MALE MOUSE S1P1 DEFICIENT BY
MAKING THEM S1P1 CONDITIONAL
KNOCK-OUT.
SO WHEREAS THE MALE MOUSE
NORMALLY DOESN'T HAVE ANY CD8
SIGNAL POSITIVE THYNOCYTES WE
FOUND SOMETHING REALLY DRAMATIC,
THAT IS, IF YOU PREVENT THE
CELLS FROM LEAVING THE THYMUS BY
REMOVING S1P1 EXPRESSION, NOW
YOU SUDDENLY SEE CD8 POSITIVE
CELLS APPEARING IN THE
NEGATIVELY SELECTING MALE MOUSE.
SO THIS IS NOT CLONAL DELETION.
THE CELLS HAVEN'T BEEN DELETED
HERE, BECAUSE IF YOU SIMPLE STOP
THEM FROM LEECHG THE THYMUS, THE
CELLS APPEAR.
AND THESE CELLS IN FACT NOW HAVE
BECOME MATURE, THEY'VE MATURED
IN THE THYMUS TO BECOME MATURE
QA2 POSITIVE CD8 SIGNAL POSITIVE
CELLS, THE WAY THAT HAPPENS IS
YOU JUST HAVE TO KEEP THEM FROM
LEAVING THE THYMUS.
SO I THINK THIS EXPERIMENT IN
ITSELF PROVIDES I THINK, I DON'T
KNOW IF IT'S PROOF, BUT IT'S
CERTAINLY COMPELLING EVIDENCE
THAT THE ABSENCE OF CD8 SIGNAL
POSITIVE CELLS IN THESE
NEGATIVELY SELECTING MICE,
EVICTION, THERE THEY ARE.
OKAY.
SO THAT'S OUR CONCLUSION FOR
THIS PART, THAT THE ABSENCE OF
CD8 POSITIVE THYMOCYTES IS DUE
TO CLONAL EVICTION, NOT
DELETION.
HOW IS IT THAT S1P1 WHICH IS
NORMALLY EXPRESSED ONLY ON
MATURE CELLS, HOW IS IT THAT
THESE GUYS, THAT IMMATURE
THYMOCYT HE S ARE INDUCED TO
EXPRESS S1P1?
THERE WAS REALLY A REMARKABLE
SET OF EXPERIMENTS WHAT SHERIF
FOUND, THESE ARE IMMATURE
THYMOCYTES STIMULATED IN VITRO
FLRKS FOR ANTIBODY.
WITHOUT STIMULATION, THYMOCYTES
DON'T EXPRESS S1P1.
SO IMMATURE TRRLS THYMOCYTES
ACTUALLY REQUIRE SIGNAL TO
UPREGULATE AND IF YOU SIGNAL
THEM YOU GET S1P1 EXPRESSION.
IN MATURE CELLS, THE EXACT
OPPOSITE HAPPENS.
BUT HOW IS THIS WORKING?
THE WAY IT WORKS IN FACT IS THAT
UPSTREAM OF S1P1 IS
TRANSCRIPTIONAL PRSES REPRESSOR
PRRKS EXPRESSED AT HIGH LEVEL
IMMATURE THYMOCYTES, RESPONSIBLE
FOR PREVENTING S1P1 BEING
EXPRESSED.
ARE.
SO HOW DO WE PROVE THAT?
REALLY N A REMARKABLE EXPERIMENT
THAT SHARIF CAN HE USED THIS
FLEX SWITCH MS SYSTEM TO TRY TO
INDUCE EXPRESSION OF GFI1 IN
THESE IMMATURE THYME SIGHTS.
SO -- THYMOCYTES, IT WE HAD A
LENTIVIRUS, WE LOOKED AND THE
CONTROL LENTIVIRUS DIDN'T HAVE
CD8 CELLS.
SO HAD YOU CAN SEE THESE CELLS
EXPRESSED HIGH LEVEL OF GFI1
FROM LENTIVIRUS, THESE CELLS
RETAINED IN THYMUS AND DEVELOPED
INTO MATURE CD8 T CELLS.
OKAY.
SO AT THIS POINT, WE WOULD SAY
THAT HERE IS OUR UNDERSTANDING
OF IT.
AND THAT IS THAT STRONG TCR
SIGNALLING DURING NEGATIVE
SELECTION DOWN REGULATES GFI ONE
IN THE NEGATIVELY SELECTED
IMMATURE THYMOCYTES WHICH ALLOWS
THESE CELLS TO EXPRESS S1T1 THAT
CAUSES THESE IMMATURE THYMOCYTES
TO LEAVE THE THYMUS AND GO INTO
THE PERIPHERY.
AND IN THE PERIPHERY, THESE
CELLS ENCOUNTER IL7, PERHAPS
OTHER FACTORS AS WELL, AND THAT
INDUCES THE DIFFERENTIATION FROM
THE DOUBLE DULL CELLS TO BECOME
MATURE CD8 T CELLS.
SO THE NEXT QUESTION WAS, ARE
THESE MATURE CD8 T CELLS THAT
NOW ARISE IN THE PERIPHERY FROM
THESE PRECURSOR CELLS, DO THESE
CELLS R THESE CELLS
SELF-TOLERANT?
AND THE ANSWER IS, YES.
AND THE WAY THIS IS SHOWN WAS
THAT WE TOOK EXPHAIL FEMALE HYC
CD4 MOUSE AND INJECTED THEM WITH
PEPTIDE AND FEMALES RESPONDED BY
UPREGULATING CD107A, YOU CAN SEE
THE FEMALES RESPONDED BUT MALE
CD8 CELLS DID NOT RESPOND.
SO TO SHOW THAT THEY'RE TOLERANT
AS WELL, THEY'RE NONRESPONSIVE.
OKAY.
SO WE THINK THE SOLUTION TO THE
PROBLEM OF HOW YOU GET CELLS
MATURE CD8 CELLS IN THE
PERIPHERY WHEN NONE OF THEM ARE
PRESENT IN THE THYMUS IS THAT
PRECURSORS GET PREMATURELY
CLONEALLY EVICTED BY THE STRONG
TCR SIGNALLING DOWN REGULATION
OF GF IN I1 AND EXPRESSION OF
S1P1 AND THEN COMPLETE THE
DIFFERENTIATION IN THE PERIPHERY
IN THE PRESENCE OF THEIR EXTRA
THYMIC LIGAND, AUTO REACTIVE
LIGAND TO BECOME SELF-TOLERANT
CD8 T CELLS.
IN THE LAST MINUTE OR SO I WANT
TO SHOW YOU THAT THIS IS NOT
SOMETHING UNIQUE TO THE CD8
TRANSGENE OR ANY TRANSGENE
BECAUSE WE LOOKED TO SEE IF THIS
WAS HAPPENING IN NORMAL
POLYCLONAL MICE.
THESE ARE ALL A SET OF QUITE
SURPRISING EXPERIMENTS.
TO LOOK AT POLYCLONAL SELECTED
THYMOCYTES, WE LOOKED IN
KNOCK-OUT MOUSE, HERE NORMAL
MODEL DOESN'T HAVE CD4 T CELLS,
BUT IT DOES HAVE CD8 T CELLS.
THERE'S A SMALL SUBSET OF CELLS
EXPRESS PD1 INDICATING THEY'VE
BEEN STRONGLY SIGNALLED DURING
NEGATIVE SELECTION.
WHAT'S INTERESTING IS THAT IF
YOU LOOK AT THE PD1 NEGATIVE
CELLS THE VAST MAJORITY OF THE
CELLS YOU SEE IT LOOKS JUST LIKE
NORMAL CD8 SIGNAL POSITIVE
SELECTION.
IT LOOKS JUST LIKE THE FEMALE
MOUSE, IF YOU WILL, IN THE
TRANS-- BUT IF YOU LOOK AT THE
PD1 POSITIVE CELLS, DRAMATIC
OBSERVATION, YOU SEE DULL CELLS
IN THE POLYCLONAL MOUSE AND IT
LOOKS JUST LIKE THE HYCD4
NEGATIVELY SELECTING MALE MOUSE
AND THOSE -- OH, SORRY.
AND THOSE DOUBLE DULL CELLS IN
FACT HAVE DOWN REGULATED GFI1
AND UPREGULATED LOGICAL S1P1.
YOU SEE THE SAME IN THE
PERIPHERY AS YOU SEE ON
TRANSGENIC AND THOSE DOUBLE DULL
CELLS IF YOU CULTURE THEM IN
FROM IL7 GO FROM BEING DOUBLE
DULL TO MATURING INTO CD8 T
CELLS JUST AS WE SAW IN THE
TRANSGENIC.
SO THESE CELLS DIFFERENTIATE
INTO THE MATURE CD8 CELLS.
MY LAST DATA SLIDE HERE IS THAT
IF WE LOOK AT A NORMAL B6 MOUSE,
LOOKING AT MALE AND FEMALE MICE
AND LOOK AT HY SPECIFIC CLASS 2
CELLS OR HY SPECIFIC CD8 CLASS
ONE CELLS, AND WHAT YOU CAN SEE
IS IF YOU ACTUALLY USE STAIN TO
GO IDENTIFY HY SPECIFIC T CELL,
YOU SEE IN THE MALE CELLS
THERE'S A LOSS BECAUSE OF CLONAL
DELETION OF THE HY SPECIFIC
CLASS 2 T CELLS.
BUT IF YOU LOOK AT THE CD8 CELLS
IN BOTH EXPHAIL FEMALE THERE'S
NO -- MALE AND FEMALE THERE'S NO
LOSS WHATSOEVER, THESE GUYS HAVE
NOT UNDERGONE CLONAL DLEEKS ARE
THEY'VE UNDERGONE CLONAL
EVICTION.
CONCLUSION WE THINK THATS
POLYCLONAL THYMOCYTES ARE
EVICTED DURING NEGATIVE
SELECTION, COMPLETED
DIFFERENTIATION OF THE TOLERANT
CELLS IN PERIPHERY AND I'VE
SHOWN YOU NOW STUDIES IN BOTH
TCR TRANSGENIC AND NORMAL
POLYCLONAL MOUSE REVEAL THAT
CLONAL EVICTION, NOT CLONAL
DELETION, IS THE BASIS OF CD8 T
CELL TOLERANCE.
OKAY.
SO HERE IS MY LAB.
HERE IS SHERIF, AND MIHO WHO DID
THE FAMOUS FLIP-FLOP MOUSE AND
LAID THE INTELLECTUAL GROUND FOR
THESE EXPERIMENTS AND THIS IS
JUST A PICTURE OF OUR LITTLE
EXPERIMENTAL IMMUNOLOGY BRANCH
AND OUR PICK KNACK LAST YEAR.
SO THANK YOU VERY MUCH -- LAST
JUNE.
SO THANK YOU VERY MUCH.
[APPLAUSE]
>> CAN YOU HEAR NECESSITY?
DO THESE CELLS EXPRESS, THOSE
EVICTED CELLS, THE ONES THAT YOU
FIND, DO THEY EXPRESS CD8 BAY
TASS, THAT'S THE FIRST PART OF
THE QUESTION?
DO THEY EXPRESS BETA?
>> YES, ALPHA ALPHA-BETA T
CELLS.
>> ALSO DO YOU FIND SOME THAT
ARE AM ALPHA IN THE GUT?
>> NO.
>> SO THAT'S REALLY SOMETHING
ENTIRELY DIFFERENT FROM
WHATLY -- AND OTHERS HAVE FOR
INSTANCE PROPOSED.
>> THANK YOU, AL.
DO YOU HEAR ME FROM THE
MICROPHONE?
IS IT ON?
>> YES.
>> FIRST, BOTH REMI AND I BOTH
HAVE TOO MANY GRAY HAIRS TO BE
THE ONLY ONES ASKING QUESTIONS.
TWO QUICK QUESTIONS, AL, FIRST,
IT'S A GREAT STORY, CONGRATS, SO
I DON'T UNDERSTAND HOW YOU SEE
SUCH A DOWN REGULATION OF CD4
WHEN THE HALF-LIFE OF CD4 IS
ACTUALLY QUITE LONG, THAT'S THE
FIRST PART.
THE SECOND PART IS THAT WHEN YOU
LOOKED AT THOSE CELLS THAT WERE
LOW IN THE CD5 ANTIBODY STAINING
THERE WERE A BUNCH OF CELLS THAT
WERE NEITHER CD8 NOR CD4 THAT
YOU MANAGED TO NOT TALK ABOUT AT
ALL.
WHAT WERE THOSE?
>> SO THE DOUBLE-NEGATIVE CELLS
THAT EXPRESS NEITHER CD4 OR CD8
REMAIN TO BE WORKED OUT STILL.
IT'S NOT CLEAR WHAT THOSE ARE
ENTIRELY.
>> AND WHY DO YOU LOSE CD4?
I LOOKED IT UP, THE HALF-LIFE IS
SIX TO TWELVE HOURS.
WHY AFTER AN OVERNIGHT CULTURE
WHY WOULD YOU SEE NO CD4?
>> IT'S A TWO DAY CULTURE
ACTUALLY.
>> OH, OKAY.
>> SO IT TAKES A COUPLE OF DAYS
TO LOSE ALL THE CD4.
>> IT'S ALL GOOD.
HELLO.
HOW DO YOU EXPLAIN THE
APPEARANCE, SINGLE POSITIVE CD4
T CELL SUBPOPULATION IN THE
FEMALE MICE?
ARE THEY BEING CLONEALLY EVICT
IN A SIMILAR FASHION?
>> NO.
SO WHEN YOU SAY -- SO THESE
ARE -- SO IN THESE MICE, YOU
HAVE THESE RAG POSITIVE MICE
BECAUSE FOR A NUMBER OF REALLY
JUST I THINK TECHNICAL REASONS,
THERE MAY BE SOMETHING MORE
BEHIND AT THAT.
SO THE CD4 CELLS THAT YOU SEE
ARE ACTUALLY PROBABLY EXPRESSING
ENDOGENOUS ALPHA THAT GIVES
THEM, THAT ALLOWS THEM TO SEE
CLASS TWO AND BECOME A CD4 T
CELL.
SO THOSE ARE NOT EVICTED.
ARE THE CELLS WE KNOW SO FAR
FROM THIS STUDY ARE EVICTED ARE
THE CELLS THAT GET A STRONG TCR
SIGNAL IN THE THYMUS WHICH WOULD
BE THE NEGATIVELY SELECTED
CELLS.
IF YOU THINK ABOUT IT FOR A
WHILE, YOU REALIZE THIS MAY
APPLY TO OTHER THYMIC SUBSETS AS
WELL.
THESE ARE THE ONLY ONES WE KNOW
ABOUT SO FAR.
IS THAT OKAY?
>> THANK YOU.
YES.
>> THANK YOU, AL.
[APPLAUSE]
>> JUST TO QUICKLY REMIND ALL
THE SPEAKERS TO ALLOW TIME FOR
QUESTIONS AND SOME DISCUSSION,
PLEASE KEEP YOUR TALK TO A MAX
15 MINUTES SO WE HAVE AT LEAST
FIVE MINUTES FOR QUESTIONS.
SO I'M GOING TO GO AHEAD AND
INTRODUCE OUR NEXT SPEAKER,
REMY BOSSELUT TO TELL US ABOUT
CD4 T CELL DIFFERENTIATION.
>> IT'S A TOUGH THING TO DO,
I'LL TRY MY BEST HAD -- I'LL TRY
MY BEST.
OKAY.
S.
>> OKAY.
YES, IP THE --
>> YES, I WANT THE POINTER,
PLEASE.
THANK YOU.
THANK YOU VERY MUCH FOR YOUR
TIME WAITING FOR ME THSM I WOULD
LIKE TO START BY THANKING THE
COMMITTEE FOR INVITING ME TODAY.
MOST OF THE WORK I AM GOING TO
SHOW WAS DONE BY HIGH --
STUDENT, NOW IN MICHIGAN IN
MEDICAL SCHOOL, SHE'S VERY
TALENTED ALTOGETHER.
THIS WAS REALLY, WITH HELP BE -D
THERE WERE PLENTIFUL PEOPLE IN
THE LAB WHO HELPED WITH THIS AND
ARE GOING ON SHOW A -- DRIVING
FORCE BEHIND THIS, WHEN WE
STARTED.
WE HAD FANTASTIC COLLABORATOR
JEFF CHU, MIKE KELLY, AND WE GOT
MICE FROM -- AND NONE OF THIS
WOULD HAVE BEEN POSSIBLE WITHOUT
THE ASSISTANCE OF THE CENTER FOR
CANCER RESEARCH FOR FUNDING NEW
TECHNOLOGY AND INSTRUMENTAL FOR
THIS.
SO THIS IS WHAT I AM GOING TO
ADDRESS, REALLY TWO SHORT
VIGNETTES ON SINGLE CELL
TRANSCRIPT TORE THE ROLE OF
THESE TWO FACTORS, IN THE THYMUS
LEADING TO THE TRANSITION.
SO AS MANY PEOPLE HERE ARE
INTERESTED IN THE FUNCTION OF T
CELL AND DIFFERENTIATION IN
PARTICULAR CONVENTIONAL T CELLS
THAT BIND WITH PEPTIDES AND
EXPRESS CD4, BIND CLASS ONE
EXPRESS CD8 SO EMERGE FROM
THYMUS I THINK I ALREADY
INTRODUCED THIS EXPRESS BOTH CD4
AND CD8 AND SO BECOME CD4
POSITIVE AND THIS IS A VERY
SIMPLIFIED VIEW OF WHAT WE THINK
HOW THIS IS HAPPENING.
THE CD4 THYMUS EXPRESS
TRANSCRIPTION FACTOR, EXPRESSION
OF THIS OTHER FACTOR EXPRESSED
IN CD8 CELLS, SO THAT THE CELL
EXPRESSED NORMALLY -- SIGNAL
BECOMES CD4 AND DON'T EXPRESSES
ARE CLASS ONE SIGNAL, BECOME
CD8.
NOW, IF NOT COMPLETE AT THIS,
WHAT HAPPENS THERE BEFORE THE
CELLS EXPRESS BECAUSE POSITIVE
EXPRESS NEITHER OF THEM, DOESN'T
MEAN MUCH LESS CLEAR IN PART
BECAUSE THE CELLS THAT MAKE THIS
DECISION ARE IN THIS -- MY MOUSE
ISN'T MAKING IT, BUT WHERE THEY
ARE CELLS THAT ARE POSITIVE AND
SELECTION ARE PRECURSOR AND OF
COURSE THEY ARE EXPRESSING THE
CELLS.
SO TRY TO DISENTANGLE THIS
COMPLEXITY SOME YEARS AGO, THIS
IDEA WHAT WAS AT THE TIME IN
THIS TECHNOLOGY, SINGLE CELL
SEQUENCING, AND STARTED FROM
CLASS ONE TO CLASS TWO KNOCK-OUT
MOUSE AND THE CD69 POSITIVE
THYMOCYTES, ALREADY A LOOSE
GATE.
SO THIS IS A KNOCK-OUT RECEPTOR
SIGNALLING, THE CELL EXPRESSED
IN 69 I'VE SEEN SOME SIGNALLING.
SHE STARTED THOSE CELLS,
CAPTURED THEM WITH THIS 10X
DROPLET SYSTEM AND DID A NUMBER
OF BIOINFORMATICS.
I WILL NOW DETAIL.
THIS WAS PUBLISHED A COUPLE
YEARS AGO.
IT AND THIS IS WHAT YOU SEE THE
WHOLE FAMILY, SHOWING CELLS, AND
THE CELLS ARE ARRANGED IN SUCH A
WAY THAT THEIR DISTANCE IN THE
BLOOD MORE OR LESS MATCHES THEIR
TRANSCRIPT TORE SIMILARITY.
NOW, PUTTING TOGETHER CELLS THAT
ARE CLOSE TO EACH OTHER, THESE
BROWN-RED CELLS ARE CLOSER TO
EACH OTHER THAN THE ORANGE
CELLS.
WE CAN IDENTIFY THOSE CRYSTALS
BY GENE EXPRESSION, -- THE ONES
CD4 AND CD8 AND SO ON AND SO
FORTH.
THIS AS I'VE BEEN TOLD IN THE
PAST IS A LITTLE BIT, HAS A
LITTLE BIT FEELING OF DEJA VU.
NOW, WE CAN DIETLE MORE THING,
WE CAN TRY TO USABLE CIS TO DO
TRAJECTORY MAPPING AND FIND A
WAY -- FIND WHERE THE LINEAGES
BRANCH OUT FROM EACH OTHER AND
ALSO INDEED WHAT HAPPENS VERY
EARLY, SO THOSE ARE THE DOUBLE
POSITIVE THYMOCYTES AND WE CAN
TRY TO LOOK AT THIS SH TO SEE
OUR TRY TO SEE HOW GENE
EXPRESSION WORKS AND IF WE DO
THIS, YOU CAN SEE WE REALLY
CATCH THE CELLS IN THE ACT OF
SIGNALLING, THESE ARE CD5 AND
CD69 TO SIGNALLING AND WE CAN
SEE THIS INCREASE AS THE CELL
MOVES FROM DP1 TO DP2 AND
CONVERSELY IF WE LOOK AT GENES
EXPRESSED BY TCR ORANGE CELLS,
WE SEE THEM GOING DOWN AS THE
CELLS STOP SIGNALLING.
AND WE CAN ALSO TRACK THE
EXPRESSION AT LEAST IN THIS
EARLY PHASE, MORE OR LESS THE
SAME WAY REGARDLESS OF IMAGE
SPECIFICITY.
WITH THIS, WE CAN TRY TO LOOK
INTO THOSE CELLS INTO THEIR
PROGENY AND SEE WHICH
TRANSCRIPTION FACTORS COULD BE
IMPORTANT FOR LINEAGE
DIFFERENTIATION AND WE FIND A
NUMBER OF PATTERNS I'M GOING ON
SHOW YOU EVERYTHING, OF COURSE,
THIS WOULD BE WAY TOO BIG.
BUT THIS PATTERN, THE GENE
EXPRESSION IN CLASS ONE SIGNAL
CELL, NO EXPRESSION IN CLASS ONE
SIGNAL CELL, AND EXPRESSION AS
THE CELLS BECOME IMMATURE MATURE
CD4.
THERE ARE OTHER FACTORS THAT ARE
EXPRESSED MUCH EARLIER THAT WE
CAN SEE IN THESE DP2 CELLS IN
WHICH THERE ARE STILL ARRANGING
CELLS AND WHAT I AM GOING TO
DISCUSS TODAY IS THIS ADDITIONAL
FACTOR, 281 THAT'S UNIQUE TO
SPECIFIC AND COMES EARLY IN THE
PROCESS.
SO THIS IS JUST TO SUMMARIZE
WHAT I JUST SAID.
AND THE QUESTION WE'RE ASKING IS
WHETHER, YOU KNOW, 281 IS
IMPORTANT FOR DIFFERENTIATION.
SO WE CONTACTED, WE DID SOME
RESEARCH AROUND TO SEE WHAT WAS
DONE ON 281 AND REALIZED THAT IN
CHINA THEY HAD IN FACT MADE A
KNOCK-OUT OF 281 SO WE CONTACTED
LI AND OF COURSE TO SOME EXTENT
WE USE A HOAX BECAUSE 281 WAS
INDEED IMPORTANT FOR T CELL
FUNCTION BUT DIDN'T FIND MUCH IN
APPROXIMATE T CELL
DIFFERENTIATION IN THE THYMUS.
SO IT TOOK MORE THAN THIS AND
REALIZE THAT IN FACT IN ADDITION
TO 281 THERE IS THIS OTHER
FACTOR 148, VERY SIMILAR TO 281
AND THIS IS HOW IN FACT, THERE
IS NO -- OUTSIDE ITSELF BETWEEN
THESE TWO FACTORS AND ANYTHING
ELSE. SO WE OBTAINED FROM
JUANITA WHO WAS AT MICHIGAN THE
COMMISSIONER MOUSE FOR 148 AND
WE DECIDED TO ALSO THIS IS JUST
SORRY THE EXPRESSION FACTOR OF
281 THAT'S EXPRESSED IN TWO
SIGNAL CELLS, 148 IS EXPRESSED
IN THYMOCYTES REGARDLESS OF I AM
MARGE SPECIFICITY.
SO 148 AND 281 USING CD4 THYMUS
AND FB DISPAISH WE DELETED THESE
TWO GENES WITH THE PDK. I'LL BE
BRIEF.
SO IN SHOWING THIS, OKAY, THE
281 DOESN'T HAVE MUCH OF -- THE
148 DOESN'T HAVE MUCH.
BOTH THOSE DHOINGTZ SEEM TO GO
VERY WELL, YOU HAVE THIS
POPULATION OF DOUBLE POSITIVE
CELLS AND THEY'RE ALL INDEED
DOUBLE POSITIVE THYMOCYTES, BUT
HERE WE SEE THAT THE EXPRESSION
GOES DOWN BY 50%, BOTH IN THYMUS
AND WHAT I'M SHOWING YOU ON
THYMUS IS ALSO TRUE IN THE -- SO
THAT'S SOMETHING BUT NOT
GIGANTIC.
WE LOOK AT SINGLE CELL AND THERE
IS MUCH BOLDER IN FACT THAT
STAINING CAN TELL US.
SO THIS IS SINGLE CELL
SEQUENCING ON WILD-TYPE CELLS
WITH THOSE SAME IN THE
ORGANIZATIONS.
AND I WANT YOU TO PAY ATTENTION
ON THESE ORANGE CELLS, IMMATURE
CD4 IN THE KNOCK-OUT.
THERE IS NO ORANGE CELL INSIDE
THE CD4 AREA, THERE ARE CRYSTAL
SEPARATING, SO THERE'S A MAJOR
TRANSCRIPTOR CHANGE BETWEEN THIS
AND THIS AS WE REMOVE THOSE TWO
FACTORS.
WE TRIED TO UNDERSTAND HOW THIS
WAS WORKING, SO ANALYSIS ON THE
THPOK GENE AND FOUND THAT
THPOK 281 BINDING TWO TWO --
BINDING TO TWO SITES, THOSE RED
BLOTS, YEARS AGO, THIS WAS
BINDING TO THE TRANSCRIPTION
FACTOR AS I DISCUSSED EARLIER.
SO WHAT THIS SUGGESTED TO US IS
THAT THERE MIGHT BE AN
INTERACTION BETWEEN WHAT THE BE
GATA3 AND 218 CHT IF YOU EXPRESS
BOTH IN CELL LINE YOU WILL FIND
281 WILL BIND TO GATA3 AND WE
FOUND TREATMENT OF THE MICE AND
DOESN'T SIMPLY AFFECT THE
BINDING DNA IN A MANNER THAT
DOESN'T INVOLVE PROTEIN-PROTEIN
INTERACTIONS.
SO THIS TO SUMMARIZE THIS, SO
THESE TWO FACTORS,
DIFFERENTIATION WITH THYMUS,
BIND THPOK AND EXPRESSION.
THE FACT THEY BIND TO 3, I WAS
THINKING ABOUT WHAT ELSE THEY
COULD DO TOGETHER, AND THIS IS
HOW WE GOT INTERESTED INTO THE
POTENTIAL FUNCTION OF TH2
DIFFERENTIATION.
SO WE DID A NUMBER OF
EXPERIMENTS, IN THE INTEREST OF
TIME I'M NOT GOING TO SHOW, BUT
WHAT WE DID WITH TH2 IS LOOK AT
THESE MICE ASK SEE HOW THEY
RESPOND WITH MEUNTD, PRODUCES AN
AIRWAYLY P -- IN A WILD-TYPE
MOUSE YOU CAN FIND IN THE LUNG
OR LYMPH NODE SOME CELLS THAT
DIFFERENTIATE INTO TH2 AND
PRODUCE INTO CYTOKINES.
AND THIS IS STRONGLY AFFECTED IN
THE PDKO MOUSE IN WHICH THE T
CELLS IS NOT EXPRESSED, MUCH
LESS OF THESE TWO CYTOKINES,
MUCH LESS AS FOUR AS WELL.
WHAT IS SOMEWHAT REMARKABLE, THE
FACTOR IS CRITICAL TO MS
DIFFERENTIATION, NOT MUCH
AFFECTED BY THE KNOCK-OUT.
WHAT WE SUSPECTED IS THAT THE
CELLS PERHAPS DIDN'T MAKE THE
CYTOKINES, THAT MICE ABLE TO
DIFFERENTIATE INTO SOMETHING
LIKE -- SO TO TRY TO UNDERSTAND
WHAT WAS GOING ON IN THOSE MICE,
AGAIN SIGNIFICANT HE WILL CELL
STUDIES, THIS TIME SHE DID
TECHNOLOGY THAT DOES TOGETHER ON
THE SAME SLIDE.
THIS WAS DONE.
AND THE SAME KIND AS SHE DID
BEFORE.
AGAIN, SO WE GET THIS MAP AND
CRYSTALS THAT WE CAN NUMBER AND
WE CAN TRY TO IDENTIFY THOSE
CRYSTALS AND YOU CAN SEE
EXPRESSED, IN4 IS EXPRESSED THIS
IN THIS BRANCH, AND WE FIND
EXPRESSION OF L21 OR ASCL2 ON
THE VERY BOTTOM OF THIS WING SO
WITH THIS SOME CRYSTAL AT LEASTW
YOU, THIS EXPRESS.
WHAT HAPPENS IF WE LOOK NOW,
WELL, NOT MUCH, AND THAT WAS
SOMETHING THAT WAS A SURPRISE,
BECAUSE IF YOU REMEMBER ALREADY
IN THE THYMUS THERE WAS A CLEAR
SHIFT OF THE KNOCK-OUT CONTROL
CELLS, HERE THERE IS NOT MUCH
THAT HAPPENS.
IN FACT, THERE IS INDEED A
CHANGE IN L4 EXPRESSION, THAT
EXPRESSION IS NOT AFFECTED IN
WHICH WE DO A BOLD SEARCH WE CAN
SHOW YOU, THERE ARE PROBABLY A
FEW GENES THAT DIFFERENTIATE
EXPRESSION BUT NOT MANY AND
THERE'S REALLY NOT MUCH THAT'S
GOING ON.
IF YOU DO ATTACK SIGNAL THINGS
ARE SLIGHTLY DIFFERENT.
WHAT YOU CAN SEE IS THAT WE CAN
PROJECT THE TRANSCRIPTOME ON
THIS MAP L WE CAN SEE A VERY
GOOD CAUSE BETWEEN THE
TRANSCRIPTOME TRK THE RAFN AND
THE WAY THE CELLS ARE AND YOU
CAN ALSO SEE THAT THE TH2 CELLS
ARE SOMEWHAT MOVED AND YOU FIND
THE CRYSTALLINE DIFFERENT, I
HOPE YOU CAN SEE THIS BLUISH.
THIS SUGGESTED TO US THAT IN
FACT THERE IS A DIFFERENCE, IT'S
NOT SO MUCH TRIPTD TORE
DIFFERENCE BUT THERE IS A
DIFFERENCE AT THE LEVEL OF
CONNECTING ACCESSIBILITY.
SO TO LOOK INTO THIS, TH2
EXPRESSION, WE DO THIS WITH
CRYSTALS IT, THIS IS WHERE
TISSUE CYTOKINE WILL BE ALL THE
WAY ON YOUR RIGHT ARE ENCODED.
THIS IS A CRYSTAL THEETS NOT A
TISSUE CRYSTAL, TH1 CELLS AND
YOU CAN SEE THAT THERE ARE A FEW
PEAKS THAT HAPPEN.
BUT THERE ARE OTHER AREAS IN
WHICH THERE IS A MUCH LESS
COMPARED TO THIS WILD-TYPE TH2
CRYSTAL -- CLUSTER, AND THIS IS
PARTICULARLY TRUE AT A SITE
THAT'S BEEN IDENTIFIED YEARS AGO
AS BEING IMPORTANT FOR TH2
CYTOKINE EXPRESSION, CGRE, CNS1
AND 4.
YOU CAN SEE THERE IS DEFINITELY
AN CRERKS HERE, HERE, HERE.
SO THE ACCESSIBILITY OF THE
CONNECTION IS NOT NORMAL AND WE
BELIEVE THAT THIS IS IN FACT IN
IN THE EXPRESSION OF THE
CYTOKINES.
SO IF WE TRY TO UNDERSTAND A
LITTLE BIT MORE WHAT'S
HAPPENING, THIS IS THE SAME BUT
SMALLER, WE NEED TO SEEK
ANALYSIS FOR THE FP281 --
ZFP281, THIS CAN BE DONE IN
VIVO.
WE CAN FIND BINDING TO MOST OF
THESE PEAKS IN FACT, THERE'S NO
BINDING, NO NEGATIVE CONTROL
BINDING.
THOSE PEAKS CORRESPOND TO AREAS
WHERE -- CAN BIND AS WELL AS
SHOWN HERE.
ONE THANK WE THOUGHT MIGHT BE
POSSIBLE IS THAT 281 WILL BRING
THAT BINDING, BUT THAT'S
ACTUALLY N THE CASE, AS YOU CAN
SEE, THE BINDING EXACTLY THE
SAME, WHETHER WE HAVE 281 OR
WHETHER WE DON'T HAVE IT.
SO OUR CURRENT NODLE EXPLAIN HOW
THIS WORKS IS THAT THOSE TWO --
CURRENT NODLE EXPLAIN HOW THIS
WORKS, THOSE TWO OR THREE
FACTORS HAVE TO BIND TOGETHER TO
PROMOTE NORMAL CONNECTING AND
TH2 CYTOKINE EXPRESSION.
IF ONLY HERE, THE CELLS CAN
STILL ADOPT A TH2 FATE SO THEY
ARE NOT BECOMING TH1 OR ANYTHING
ELSE BUT THEY WILL NOT PROPERLY
EXPRESS THE CYTOKINES THE TH2
CYTOKINES.
ONE POSSIBILITY WE CONSIDERED
WAS THAT THIS WAS IN FACT A
GENERIC MECHANISM AND THAT EVERY
GENE WOULD ALSO DEPEND ON 281,
BUT WE DON'T THINK THAT'S THE
CASE BECAUSE WHEN WE LOOK AT
BINDING ON THE GENOME BASIS,
CHIPSEQ WE CAN FIND PLENTY OF
ZFP281 PEAKS BUT RELATIVELY FEW.
AND AMONG THOSE -- CONTROLLED
BOTH BY GATA3 AND BY ZFP281.
WITH THIS I'LL STOP HERE AND TO
SUMMARIZE WHAT I SAID, WE THINK
ZFP281 -- DIFFERENTIATION IN THE
THYMUS, THAT THOSE TWO FACTORS
ARE IMPORTANT FOR P THE
EXPRESSION OF CYTOKINES BUT WE
DON'T THINK THEY'RE ESSENTIAL
FOR T CELLS IT TO ADOPT A TH2
TRANSCRIPTOME THERE.
MAKES US SUSPECT THAT THEY
FUNCTION AS GATA3 COFACTORS AT
SPECIFIC GENE LOCI IN DEVELOPING
AND MATURE CD4 T CELLS.
THANK YOU VERY MUCH.
[APPLAUSE]
>> HELLO.
HAD.
>> I WILL SPEAK LOUD.
HAVE YOU LOOKED AT THE EFFECT OF
ZFP DEFICIENCY IN BINDING OF
FIVE IN FM CYTOKINE ORGANISMS?
>> YOU'RE ASKING ME WHETHER IT
AFFECT THE BINDING OF STAT5?
>> AFFECT SND.
>> NO, WE HAVE NOT LOOKED.
>> FIVE PROBABLY WITH EACH OTHER
TO INDUCE TH2 CYTOKINES SPHWHR
IT'S ABSOLUTELY POSSIBLE, BUT WE
HAVE NOT LOOKED INTO THAT.
>> CAN YOU HEAR ME, REMY?
>> YES.
>> VERY NICE TALK.
SO IF YOU LOOK AT TRRMS
CHROMATIN YOU'LL SEE THAT MASTER
TRANSCRIPTION FACTORS LIKE
GATA3, IF YOU LOOK AT THOSE LOCI
THAT YOU SEE BOTH POSITIVE AND
REPRESSIVE MARKS IN TH1 AND TH2
WHEREAS THE CYTOKINES ONLY SHOW
OPEN CHROMATIN OR H3 WHATEVER,
THE POSITIVE MARKS, THE
APPROPRIATE LINEAGE.
SO WHAT DO YOU SEE, HAVE YOU
LOOKED AT ZPF281 AND 148 IN
TERMS OF THEIR CHROMATIN
MODIFICATION?
>> SO WE HAVEN'T LOOKED AT
WHETHER -- SO THE SITE I WAS
POINTING AT ARE MARKS BY -- AND
OTHERS, BY ITS 27, SO THIS
ACTIVITY MARKER, AND WE HAVEN'T
LOOKED IF THAT IS AFFECTED BY
THE 281 AND 148 KNOCK-OUT, IF
THAT WAS YOUR QUESTION.
>> THANKS.
>> HELLO.
SO --
>> I LIKE THAT --
>> I WOULD LIKE TO ASK A
QUESTION ABOUT TRANSCRIPTION
FACTOR AND THE -- CELLS, WHY
SPECIFICALLY UPREGULATED IN
CLUSTER SELECTED NOT CLASS ONE,
WHAT SIGNAL INDUCES THEIR
EXPRESSION OR BINDING TO GATA3?
>> IT'S A GREAT QUESTION.
YOU KNOW, I THINK TO BE FAIR, IF
YOU ASK -- WOULD BECOME A
DIFFERENT ANSWER.
BUT I THINK THE FAIR ANSWER IS
THAT SO FAR, IT'S NOT ENTIRELY
KNOWN.
ONE OF THE PROBLEMS HERE IS THAT
THE SIGNALS THAT WOULD
UPREGULATE THESE FACTORS ARE
MISSING TO -- ARE SIMILAR TO
THOSE THAT DRIVE POSITIVE
SELECTION IN THE THYMUS SO THAT
IT'S GENETICALLY VERY DIFFICULT
FOR ONE AND THE OTHER TO TRY TO
ADDRESS THIS QUESTION IN THE
2020 STUDY TWRM WE USED SINGLE
CELL ATTACK AND RNA SEQUENCING
TO TRY TO PREDICT FACTORS THAT
WOULD BIND THE ZFP281 AND 148
WAS ONE OF THEM.
AND OTHER FACTORS, IT LIKE --
WERE ALSO, SO IT IS POSSIBLE
THAT THESE FACTORS ARE INVOLVED,
BUT THE TRUTH OF THE MATTER IS
THAT WE DON'T KNOW.
>> OKAY.
THANK YOU.
>> WRONG ANSWER TO SAY NOTHING,
RIGHT?
>> THANKS, REMY.
DO YOU HAVE ANY NOAKSZ ABOUT
STABILITY -- DO YOU HAVE ANY
NOTIONS ABOUT STABILITY OF
GATA3?
YOU SHOWED THAT IT'S THERE,
BUT -- EVEN AT THE LOCUS, IF YOU
HAVE A STABLE EFFECT OF GATA3'S
FUNCTION.
SO IF YOU WOULD LOOK AT OTHER
KINDS THIS PERTUBATIONS WHERE
YOU MIGHT SEE PLASTICITY OR
THINGS LIKE THAT.
>> YOU MEAN THE STABLGHT OF
GATA3 TO THE LOCUS?
I DON'T KNOW HOW EASY THAT WOULD
BE.
WHAT I CAN SAY IS THAT IF WE DO
-- EXPERIMENTING WE DON'T
FIND A SHIFT TO TH1 OR GVMENT
THE CELLS REMAIN GATA3
EXPRESSING, THEY DON'T MAKE THE
CYTOKINES VERY WELL, BUT THEY
DON'T BECOME TH1.
TO SNREAS WILL NOT BECOME TH1.
THANK YOU VERY MUCH.
[APPLAUSE]
>> OUR NEXT SPEAK ARE IS YOUSUKE
TAKAHAMA, TITLE OF HIS TALK IS
THYMIC FORMATION OF CD8 T CELL
REPERTOIRE.
>> THANK YOU VERY MUCH.
MY NAME IS YOUSUKE TAKAHAMA HAD,
EXPERIMENTAL IMMUNOLOGY BRANCH.
TALKING ABOUT SELECTION OF CD8 T
CELLS BUT I DECIDE TO DO CHANGE
MY MIND.
BUT I DECIDED TO CHANGE MY
MIND.
DOES IT WORK?
THIS SORRY.
I WOULD LIKE TO FOCUS ON MY
RECENT WORK ON THE MAJORITY OF T
CELL 21 WHICH IS CHEMOKINE WHICH
IS IMPORTANT FOR T CELLS.
T CELL SELECTION, THYMIC
CONTEXT, THE CORTICAL
MICROENVIRONMENT, MAINLY VIA
CORTICAL THYMIC CELLS IN THE
REGION OF THYMOCYTES SELECTED
AND IN THIS ENVIRONMENT, SLECTDZ
THYMOCYTES MOVE TO THE
ENVIRONMENT TO ESTABLISH ARE THE
REPERTOIRE.
IN THE REGION WHAT IS -- IN THE
MEDULLA REGION WHAT IS IMPORTANT
IS THYMIC EPITHELIAL CELLS ARE
IMPORTANT FOR T CELLS TO
ESTABLISH SELF-TOLERANCE.
MTEC'S FOR INSTANCE IN
TRANSCRIPTION FACTOR CAUSE
SEVERE AUTISM UNITY, AND NOT
ONLY THAT, NUCLEAR PROTEIN
CALLED AIRE, AUTO IRREGULAR
EXPRESSED IN EPITHELIAL CELLS
PROMOTE GENOME-WIDE EXPRESSION,
CONTRIBUTING TO NEWLY GENERATED
T CELLS.
AND RECENTLY, THE IT IS
HIGHLIGHTED TO ENHANCE THE
DISPLAY.
ANYWAY, IT'S IMPORTANT TO
PROVIDE VAST MAJORITY OF
REMEMBER BE SELF-ANTIGENS IN THE
THYMUS FOR SELF-TOLL RANGS.
THE QUESTION THAT I WANT TO
ADDRESS HERE AND I HAVE BEEN
ADDRESSING IS HOW IT'S POSSIBLE
TO SET UP THE THYMOCYTES TO BE
GENERATED IN THE CORTEX MIGRATE
FROM THE CORTICAL ENVIRONMENT TO
THE MEDULLARY ENVIRONMENT.
THAT IS A QUESTION THAT I HAVE
BEEN ADDRESSING, AND WE HAVE
FOUND THAT THE ARE 7 CHEMICAL
RECEPTOR IS IMPORTANT TO THE
MIGRATION OF T CELLS AND THAT IS
IMPORTANT TO ESTABLISH
EVENTUALLY ESTABLISH CENTRAL
TOLERANCE, WITH SEVERAL
EXPERIMENTAL RESULTS, CCR7 IS
EXPRESSED BY MATURE MATURE
THYMOCYTES BUT NOT BY IMMATURE
THYMOCYTES AND IN IMMATURE
THYMOCYTES THIS IS INDUCED FOR
SELECTION, SIGNALS INDUCE OR
PROMOTE THE EXPRESSION OF CCR7.
MEDULLARY ACCUMULATION OF MATURE
THYMOCYTES IS DEFECTIVE IN CCR7
ANIMALS AND MORE IMPORTANTLY
THEY FAIL TO ESTABLISH T CELLS
WITHOUT T CELL 7 MEDULLARY
MIGRATION COULD AUTOIMMUNE
TISSUE LESIONS.
SO WHAT IS THE REASON FOR THIS?
THERE ARE THREE POTENTIAL
PROTEINS ENCODED, ONE IS CCR21 A
GENE, ENCODE TO CCL21 PROTEIN
AND MULTIPLE GENE ELM CODE IN
CCL21 PROTEIN, TO LOU SEEN.
-- TO LEUCINE.
THE GENE ENCODED, THE CCL19
PROTEIN, AND NOT ONLY THAT, BY
MAKING A SERIES OF KNOCK-OUT
MICE WE HAVE IDENTIFIED THAT
CCR21A ENCODED IN CCR21
PROTEIN -- CCL21 SPREENT MOST
IMPORTANT ONE IN THIS CONTEXT.
HOW CCL21A PROTEIN WORKS IN THIS
CONTEXT, IN CCL21 DEFICIENT
DEFICIENT MICE, IN THE MEDULLA.
MEDULLARY MIGRATION OF
THYMOCYTES DEFECTIVE IN CCL21A
DEFICIENT MICE, KNOCK-OUT
MYSELF, BUT NOT CCL19 KNOCK-OUT
MICE, DEFECTIVE IS THIS CCL21A
KNOCK-OUT MICE AND WHAT IS
INTERESTING HERE IS THAT CCL21A
EXPRESSED IN THE CELLS WITHIN
THE THYMUS ARE PRIMARILY
MEDULLARY THYMIC CELLS OR
MTEC'S.
WHAT ARE THOSE MTEC'S?
AS I MENTIONED THAT MTEC'S ARE
SO POPULAR IN TERMS DISPLAYING
CELL FUNCTIONS BUT NOW MTEC'S DO
EXPRESS CERTAIN PARTICULAR AND
WE FOUND THAT CCL21-EXPRESSING
MTEC'S WITHIN MTEC POPULATION,
CCL21 EXPRESSING IN THE CELLS
ARE LARGELY DISTINCT FROM AIRE
EXPRESSING CELLS WHICH IS NOT
JUST BY THE POPULATION
EXPERIMENT ANALYSIS, BUT
EXPRESSING THE EXPRESSION.
SO MTEC'S ARE NOT JUST
HOMOGENEOUS POPULATION TO
DISPLAY CELL FUNCTIONS.
MTEC'S INCLUDE FUNCTIONALLY
HETEROGENEOUS SUBSET, THAT IS
IMPORTANT FOR CENTRAL
SELF-TOLERANCE, ONE IS THE
FUNCTION AND THYME SIETSZ
ATTRACTING FMS PRODUCING CELLS.
I WOULD LIKE TO SHOW TODAY SOME
OF THE RESULTS OF HOW THOSE
DIFFERENT HETEROGENEOUS SUBSET
CONTRIBUTE TO ESTABLISH CENTRAL
SELF-TOLERANCE.
WE ACTUALLY MADE CCL21 DOUBLE
DEFICIENT MICE TO CHARACTERIZE
HERE.
INITIALLY WE FOUND THAT OF
COURSE IN DEFICIENT MICE AS HAS
BEEN VERY WELL KNOWN ABOUT THE
TISSUE LESIONS IN MULTIPLE
TISSUES, SO THE CCL21 MUCH MORE
SEVERE TISSUE LESIONS IN A
VARIETY OF TISSUES.
HERE ON THE RIGHT SIDE IS AN
EXAMPLE OF THE LIVER SHOWING
THAT AIRE DWICIALT SINGLE CELL
KNOCK-OUT, CCL21 KNOCK-OUT MICE
SHOWED MINOR TISSUE LESIONS BUT
DOUBLE KNOCK-OUT MICE SHOWED
QUITE SEVERE TISSUE ISSUES.
AND THOSE TISSUE LESIONS IS NOT
ONLY TISSUE PROBLEMS BUT ALSO
THE LIVER ENZYMES TO THE
BLOODSTREAM AND ALSO THE
OBSTRUCTION CAWDZED HERE
SPLENOMEGALY, NOT JUST LIMITED
TO THE LIVER BUT PRODUCING THE
ORGAN IS ALSO SEVERE IN HERE, IN
T CELL IT CCL21 DEFICIENT MICE.
HOWEVER, CCL21 EXPRESSED
SPECIFICALLY IN UNTIL MTEC'S BUT
THE EXPRESSION OF THOSE ARE NOT
FLIMENTD THYMUS, FOR EXAMPLE,
THE EXTRA THYMIC EXPRESSED IN
THE CELLS CONTRIBUTING TO THE
SELF-TOLERANCE.
SO DO THOSE MTEC'S COOPERATE TO
PREVENT THE TISSUE LESIONS?
TO ADDRESS IT, THE THYMUS IT
TRANSPLANTATION INTO AM ATHYMIC
MICE FRMS NUDE MICE TO
RECONSTITUTE T CELL DEVELOPMENT,
TO FIND THAT THE LOSS OF THOSE
TWO POPULATIONS CAWDZED MOST
SEVERE AUTOIMMUNE DISEASE TR,
EXPRESSING MTEC'S IT, WHICH IS
CLEAR AND ALSO THE SCORING OF
THE IN VIVO LUNG, SO ARE SHOWING
MUCH MORE SEVERE PROBLEMS IN THE
BODIES.
SO THIS RESULT SHOWS THAT LIKE
AIRE POSITIVE ARE MTEC'S
CONTRIBUTE TO SELF-TOLERANCE,
AND MORE IMPORTANTLY, THOSE TWO
DIFFERENT SUBSETS COOMENT TO --
COOPERATE TO ESTABLISH CENTRAL
TOLERANCE.
WHAT IS MOST INTERESTING WITH
THAT, THAT COOPERATION SEEMS TO
BE ATTRIBUTED ON THE LOSS OF
ANTI-MEDULLARY REGION WHICH IS
WHAT'S SHOWN HERE ON THE RIGHT
SIDE BY RELB KNOCK-OUT MICE,
APPROXIMATE SHOWING SIGNIFICANT
PROBLEMS WITH THEIR AIRE CCL21
THYMIC ENVIRONMENT.
SO THE COMBINATION RESULTS IN
DIFFERENT SUBSET, FUNCTIONALLY
EQUIVALENT TO MTEC TOLERANCE.
HOW DO THEY COOPERATE?
DO THEY CONTRIBUTE TO ELIMINATE
LY THE REGULATORY T
CELLS AND FOR THE ELIMINATION OF
A SENSE OF REACTIVITY AS WE
MEASURE THE NUMBER OF THE TWO
DIFFERENT IT SELF-REACTIVE T
CELLS TO REACTIVE TO THOSE CELL
FUNCTIONS IN THE ACTIVATION OF
SELF-REACTIVE T CELLS.
ALSO HAD IN THE DEVELOPMENT OF
REGULATORY T CELLS FROM THE
MATURE STATE -- IMMATURE STAGE
TO THE MATURE STAGE, DOUBLE
DEFICIENT ANIMALS SHOW MUCH MORE
SEVERE PROBLEMS THAN THE SINGLE
POSITIVE HAD SINGLE DEFICIENT
ANIMALS.
SO THE CONCLUSION HERE IS THAT
AIRE DEFICIENT MTEC'S AND CCL21
POSITIVE COOPERATE TO ESTABLISH
SELF-TOLERANCE BY OPTIMIZING
NEGATIVE SELECTION AND TREG
GENERATION.
SO THEY COOPERATE, HIGHLIGHTING
THE FUNCTIONAL IMPORTANCE OF
THOSE CHEMICAL IN CCL21
EXPRESSING MTEC'S IN CENTRAL
TOLERANCE.
IN THE LAST FEW MINUTES I WOULD
LIKE TO ADD THAT THE RECENT
RESULTS OF SHOWING HOW THOSE, TO
ANALYZE HOW CCL21 THYMOCYTES ARE
INTERACTING WITH MTEC'S CAN BE
INTEGRATED INTO THE DEVELOPMENT
OF WELL ESTABLISHED FOR THE
DEVELOPMENT OF SELF-ANTIGEN
DISPLAYING MT HE C'S, THEN TO
BECOME THE POST AIR HE
POPULATION.
HOW DO THOSE CCL21 POSITIVE
CELLS COULD BE INTEGRATED INTO
THIS AIRE MTEC PATHWAY?
TO ADDRESS IT, WE CREATED TWO
DIFFERENT IT MICE TO MEASURE THE
PRESENT EXPRESSION OF CCL21 GENE
AND PAST CCL21 GENE BY CROSSING
CCL21 MICE WITH LOXP ANIMALS, SO
IN THE MICE YOU CAN MEASURE THE
PRESENT EXPRESSION IN THE MICE,
PRESENT AND PAST EXPRESSION OF
CCL21 GENE.
TO FIND THAT, CCL21A ATDTOMATO
EXPRESSED BY 40% MTEC'S WHICH IS
DIFFERENT FROM AIRE POSITIVE
CELLS, OKAY, THIS IS THE
PROTOTYPIC CAN AIRE, THEY ARE
DIFFERENT CELLS, THE CELLS ARE
DIFFERENT, BUT CCL21 CRE SHOWS
THAT THE VAST MAJORITY OF MTEC'S
ARE LABELED BY EGFP INCLUDING
THE AIRE POSITIVE CELLS ANDLY --
POSITIVE CELLS.
SO DERIVED FROM CCL21
TRANSCRIBING CELLS.
THOSE ARE JUST GENE EXPRESSION
OF CCL21 LOCUS, THE PROTEIN
PRODUCED IN THYMOCYTES ARE
INTERACTING FUNCTIONAL INDEX
COULD RETAIN THAT CAPABILITY TO
GIVE RISE TO SELF-ANTIGEN
DISPLAYING MTEC'S?
TO ANALYZE THAT, WE
RECONSTITUTED CCR21 PROTEIN
POSITIVE ARE TDTOMATO RE:
AGGREGATED WITH DEFICIENT IT --
TO MAKE REAGGREGATED THYMUS TO
TRANSPLANT INTO THE KIDNEY
CAPSULE.
SOME FIVE WEEKS LATER WE FOUND
THAT THYMUS REAGGREGATED WITH
TDTOMATO.
SHOWING THE DEVELOPMENT OF IT
AIRE POSITIVE CELLS, THAT'S
POSITIVE, AND ALSO THE MAJORITY
REAGENT IS DEVELOPED.
SO TO FINALIZE MY TALK, I JUST
WOULD LIKE TO GIVE YOU A
CONCLUSION THAT T CELL IF --
THAT CCL21 POSITIVE SLS ATTAIN
DEVELOPMENT THE CAPABILITY TO
DIFFERENTIATE INTO AIRE POSITIVE
MTEC'S.
SO ONE OF THE BIGGEST
CONCLUSIONS HERE IS THAT CCL21
POSITIVE FUNCTIONAL INDEX
INCLUDE THE CAPABILITY TO
FUNCTIONALLY CONVERT FROM CCL21
POSITIVE CELLS TO AIRE POSITIVE
CELLS TO ESTABLISH CENTRAL
TOLERANCE AND THAT IS IMPORTANT,
WE BELIEVE, TO ASSEMBLE THE
FUNCTIONAL DIVERSITY IN THE
THYMUS MEDULLA.
LY THANK YOU VERY MUCH FOR YOUR
ATTENTION.
[APPLAUSE]
SORRY, FMS I SHOULD THANK MY
COLLABORATORS.
THIS STURDY WAS INITIATED IN
JAPAN, ANDLY BY MY TEAM.
>> I HAVE A QUESTION ON WHAT THE
FUNCTION OF THIS.
SO DO YOU THINK THAT THE --
>> SPEAK UP.
I CANNOT HEAR YOU MUCH.
>> IS THIS BETTER?
SO DO YOU THINK THAT THE 21
EXPRESSING MTECS ARE
FUNCTIONALLY THERE TO BRING
THYMOCYTES INTO A POCKET OF
OTHER PRESENTING CELLS THAT ARE
GOING TO ACTUALLY DO THE
TOLERANCE INDUCTION, OR DO YOU
THINK THAT THE ANTIGENS
PRESENTED ARE --
>> THAT'S A VERY GOOD QUESTION.
ONE THING THAT WE HAVE RECENTLY
NOTICED FROM P -- FROM THE
SINGLE TYPE OF EXPERIMENT IS
THAT THEY DO EXPRESS A TYPICAL
CELL FUNCTION CALLED C-REACTIVE
PROTEIN.
CCL21 POSITIVE CELLS DO EXPRESS
OR THE MAJOR SOURCE FOR THE
C-REACTIVE PROTEIN, WHICH IS
QUITE --
[LAUGHTER]
WHAT?
OH?
YES.
ANYWAY, SO THEY COULD PROVIDE
SOME SELF-SO-CALLED TISSUE
RESECTOR CELL FUNCTIONS INTO THE
MEDULLARY REGION.
BUT WE BELIEVE THAT THEIR MAJOR
JOB IS TO ATTRACT AND ACCUMULATE
AND PERHAPS HOLD THOSE
DEVELOPING THYMOCYTES FOR THEM
TO ESTABLISH OR FOR THEM TO
INTERACT WITH A VAST MAJORITY OF
AIRE POSITIVE OR THYMIC CELL
FUNCTIONS PROVIDED BY OTHER
POPULATIONS IN MT HE C'S.
>> THANK YOU.
>> REMY WAS FIRST BUT HE'S BEING
ULTRA CHIVALROUS.
I WANT TO ASK YOU ABOUT AGING.
WHAT HAPPENS TO WANT CCL21
POSITIVITY AS THE ANIMAL
EXPAIDGES BY EXTENSION PEOPLE
LIKE OURSELVES?
>> THAT'S, WELL, I WOULD BE
HAPPY TO EXAMINE CCL21
EXPRESSION, BUT, YOU KNOW,
THAT'S A GREAT QUESTION.
WE DON'T KNOW.
I MEAN, I DON'T KNOW MUCH.
DEPENDING ON -- SO WE HAVEN'T
EXAMINED ANYTHING MORE THAN DID
HOW YEAR OLD OF AGE IN ANIMALS,
SO AS WELL AS WE TALK ABOUT
MIDDLE-AGED ANIMALS, WE DON'T
SEE MUCH OF THE REDUCTION IN
AIRE POSITIVE CELLS OR CCL21
POSITIVE CELLS, BUT I UNDERSTAND
THAT THE AIRE, SHOWING THAT THE
EXPRESSION OF AIRE IS QUITE
IMPORTANT AT THE PERINATAL
STAGE -- LATER LIFE.
MAYBE FOR MATURE T CELLS FOR
THEIR DEVELOPMENT, PERINATAL
DEVELOPMENT, MAYBE THE KEY
PERIOD FOR THEM TO ESTABLISH
SELF-TOLERANCE.
IF THAT IS THE CASE, CCL21 IN
THE THYMUS MAY BE MOST IMPORTANT
IN EARLY STAGE OR OF THE THYMUS
DEVELOPMENT PERINATALLY.
>> I'M JUST THINKING ABOUT THE
DEFECTS IN TOLERANCE ESTABLISHED
IN ADULTS WHEN THEY HAVE
TRANSPLANTATION AND IF THE T
CELLS NEWLY DEVELOPED GET GET TF
MEDULLA BECAUSE OF T CELLS BE
COULDN'T DEVELOP.
>> THAT'S EXIENTING TOPIC.
>> I'LL ASK -- SO ASKED ALL THE
QUESTIONS I WANTED TO ASK BUT
ONE THEY DIDN'T HAVE, THAT IS IF
YOU YOU'VE ALSO LOOKED AT ANY --
SETTINGS AND YOU KNOW WHAT
HAPPENS DURING TIMES OF
REGENERATION WITH THESE
DIFFERENT --
>> THAT'S A GREAT QUESTION.
WE HAVE NO ANSWERS ACTUALLY TO
YOU, BUT -- I MIDDLE EASTERN, TO
ANYBODY, BUT WE'LL BE HAPPY TO
LEARN MORE ABOUT THE REGION AND
PROCESS IN TERMS OF THOSE
CHEMOKINE OR CHEMOKINE RECEPTOR
INTERACTION.
THANK YOU.
>> GREAT.
THANK YOU.
>> IS THERE ANY TRANSCRIPTION
SIMILARITY WITH FRC EXPRESSED --
>> YES, COMMON CHARACTER, BUT
THERE WHAT IS MOST IMPORTANT IS
THAT FRC'S IN LYMPH NODE THEY DO
PRODUCE A LOT OF CHEMOKINE
CALLED CCL19, WHICH FROM THE
COMPARISON OF KNOCK-OUT MICE IN
ANIMALS CCL19 SEEMS TO PLAY MUCH
MORE IMPORTANT ROLE THAN CCL21
IN MAJORITY OF LYMPH NODE.
SO DEPENDING ON THE ORGANS.
>> JUST A QUICK QUESTION.
SO TRANSCRIPTIONALLY ALL THE
CCL21-EXPRESSING CELLS, ALL THE
CELLS EXPRESS CCL21 AT SOME
POINT AND EVENTUALLY SOME OF
THESE WILL BECOME AIRE
EXPRESSING CELLS.
WHAT TRIGGERS THIS CONVERSION?
IS THERE ANYTHING SPECIFIC?
WHY WOULD THERE BECOME --
>> WHAT TRIGGERS THE CONVERSION?
YEAH, EXCELLENT QUESTION FROM
REVIEW OF NUMBER ONE.
[LAUGHTER]
WE DON'T KNOW YET.
WE WOULD LOVE TO LEARN HOW THIS
CONVERSION OR WHATEVER THE
DEVELOPMENT PROCESS IS OPERATED.
THANK YOU FOR YOUR INTEREST.
APPLAUSE PLU.
>>>
[APPLAUSE]
>> OUR NEXT SPEAKER IS JONATHAN
ASHWELL AND HE WILL TALK ABOUT
GLUCOCORTICOIDS IN THE THYMUS.
I'VE GOT IT.
OKAY.
THANK YOU.
OKAY H WELCOME.
THINGS ARE RUNNING A LITTLE
LATE.
A BREAK IS NEXT, SO THERE'S
RELIEF COMING.
I'LL BE TALKING ABOUT THE
ANATOMY OF THE -- SORRY.
THE ANATOMY, REGULATION AND
FUNCTION OF GLUCOCORTICOIDS
PRODUCED IN THE THYMUS, AND I'LL
START LIKE EVERYBODY ELSE BY
REVIEWING VERY QUICKLY THYMUS
SELECTION -- THYMOCYTES
SELECTION AND THIS IS OUR REVIEW
OF IT, MOST PEOPLE'S REVIEW OF
IT, DURING EVICTION -- IGNORING
EVICTION, THAT THIS SHOWS THE
RELATIONSHIP BETWEEN THE
AFFINITY OF T CELL RECEPTORS
THAT ARE BETA RECEPTORS
GENERATED IN THE THIEMTION AND
THE FATE OF THE CELLS.
THE NOTION IS SHOWN HERE
SCHEMATICALLY ON THE RIGHT THAT
THOSE CELLS THAT HAVE
SUBTHRESHOLD AFFINITY FOR
SELF-PEPTIDE MHC, DEATH BY
NEGLECT, THE THYMOCYTES
ALPHA-BETA RECEPTORS WITH VERY
HIGH AFFINITY FOR SELF AND ARE
POTENTIALLY DANGEROUS ARE
ELIMINATED VIA AN ACTIVE PATHWAY
THAT INDUCES APOPTOSIS,
SELECTION, CELLS SHOWN IN GREEN
THAT THREE OR FOUR PERCENT OF
CELLS IN THE THYMUS THAT HAVE --
AND THAT'S POSITIVE SELECTION.
THESE CELLS GO ON TO THE
PERIPHERY WHERE THEY FORM THE T
CELL REPERTOIRE, THAT IS THE
UNIVERSE ADVANTAGE TO WHICH THE
T CELLS CAN RESPOND.
NOW, OTHER STORY GOES BACK EVEN
FURTHER THAN AL'S STORY TO THE
1990S, WHERE WE HAD DISCOVERED
THAT ACTIVATION DUE TO T CELL
APOPTOSIS AND WE HAD THOUGHT AT
THE TIME THAT THIS MIGHT BE A
MODEL FOR NEGATIVE SELECTION OF
THYMUS BECAUSE AT THE TIME THAT
WAS THE ONLY SITUATION WE KNEW
IN WHICH TCR OCCUPANCY WOULD
RESULT IN DEATH OF T CELLS
RATHER THAN ACTIVATION.
WE ALSO KNEW THAT DOUBLE
POSITIVE THYMOCYTES JUST LIKE T
CELL -- WAS SENSITIVE TO
APOPTOSIS INDUCED BY
GLUCOCORTICOIDS AND POST-DOC IN
THE LAB WANTED TO KNOW WHETHER
THESE TWO DEATH STIMULI WERE
ADDITIVE OR SYNERGISTIC AND
SURPRISINGLY THEY WERE NEITHER.
THAT WHEN BOTH THE CELLS WERE
STIMULATED BOTH VIA TCR AND
GLUCOCORTICOIDS THE CELL
SURVIVED AND WE CALLED THAT MEW
TUT ANTAGONISM AND IMMEDIATELY
OF COURSE -- MUTE ACTUAL AND --
MUTUAL.
AGAIN THIS IS THE CONVENTIONAL
SLIDE SHOWN ON THE LEFT SHOWING
THE RELATIONSHIP BETWEEN
AFFINITY AND FATE, AND WHAT WE
POSTULATED WAS THAT
GLUCOCORTICOIDS ACTUALLY WERE
IMPORTANT FOR SETTING THAT RIGHT
THRESHOLD, THE GATE BETWEEN
POSITIVE AND NEGATIVE SELECTION,
THAT TCR SIGNALLED THYMOCYTES
WOULD UNDERGO DEATH NEGATIVE
SELECTION BUT AT THE LOWER RANGE
OF THAT HIGH AFFINITY, THAT
WOULD BE ANTAGONIZED BY
GLUCOCORTICOIDS AND THOSE CELLS
WOULD SURVIVE, THEY WOULD BE
RESCUED FROM NEGATIVE SELECTION.
OKAY?
THAT WOULD MAINTAIN A ROBUST AND
FUNCTIONAL TCR REPERTOIRE.
AND NECESSARY AND TESTABLE
PREDICTION OF THIS MODEL IS THAT
IF ONE WERE TO DECREASE
SENSITIVITY OF THYME SIETSZ TO
GLUCOCORTICOIDS THAT NOW THOSE
CELLS THAT HAVE BEEN RESCUED BY
GLUCOCORTICOIDS WOULD GO ON TO
GO NEGATIVELY SELECTED, NO MORE
AN TAG NISM AND THEREFORE THAT
GREEN POPULATION THAT POOL THAT
GOES TO THE PERIPHERY IS GOING
TO BE REDUCED IN NUMBER AND
COMPLEXITY.
SO YOU WOULD END UP BECAUSE
THOSE CLONES HAVE BEEN LOST IN
THE REPERTOIRE.
OKAY?
SO YOU END UP WITH BOTH A LESS
COMPLEX REPERTOIRE AND ALSO A
LESS -- A LOWER AFFINITY
REPERTOIRE BECAUSE YOU'RE
ELIMINATING THOSE CELLS THAT ARE
IN THE HIGHER END OF AFFINITY
FOR SELF-PEPTIDE PLUS MHC.
THERE WAS A PROBLEM WITH THIS
MODEL WE KNEW RIGHT AWAY, AND
THAT WAS THAT GLUCOCORTICOIDS
ARE MADE BY THE ADRENALS, RIGHT,
AND THEY'RE DISTRIBUTED IN THE
BLOOD AS A HORMONE.
BUT FETAL MICE OR FETAL MAMMALS
AND POSTNATAL MICE AND HUMANS
HAVE VERY LOW LEVELS OF SICK
LATING GLUCOCORTICOIDS.
IN MICE THEY DON'T ACHIEVE ADULT
LEVELS UNTIL ABOUT FOUR WEEKS OF
AGE AND MUMS I THINK IT TAKES
ABOUT A YEAR BEFORE THEY REACH
LEVELS SIMILAR TO OLDER
INDIVIDUALS, IN HUMANS.
IF THIS MODEL HAD ANY CHANCE OF
BEING CORRECT THE -- ITSELF
WOULD HAVE TO BE A SOURCE OF THE
GLUCOCORTICOIDS.
WE'LL TESTS THAT.
ON THE LEFT, ALL YOU SEE RIGHT
NOW IS THE SCHEME SHOWING THE
PATHWAY OF THE NOVA SYNTHESIS
GLUCOCORTICOIDS ALL THE WAY FROM
CHOLESTEROL TO CORTICOSTERONE
WHICH IS THE PROMINENT
GLUCOCORTICOID MOUSE, WHAT YOU
REMEMBER FROM THE SLIDE HOWEVER
IS THIS LAST STEP, AND THAT'S
THE CONVERSION OF THE OXY
CORTICOSTERONE WHICH IS INACTIVE
TO THE GLUCOCORTICOID STER OWN,
AND FROM THIS SLIDE WHAT I WANT
YOU TO REMEMBER IS SIP IS 11B1
BECAUSE THIS WILL PLAY A
DOMINANT ROLE IN THE REST OF
THIS TALK.
SO WE TESTED IF THE THYMUS
GLUCOCORTICOIDS, A POST-DOC IN
THE LAB DID A SIMPLE EXPERIMENT,
TOOK THYMUS, GROUND IT UP,
COLLAGENOUS TREATED, AND FOR THE
PRESENCE OF ACTIVE
CORTICOSTERONE AND WE DIDN'T SEE
ANYTHING.
BUT IF SHE THYMOCYTES DEPLETED
FIRST AND THEN GROUND UP THE
THYMUS ET CETERA AND NOW WE
ENRICHED FOR THE HE EPITHELIAL
COMPONENT IS PUT THE SAME NUMBER
OF CELLS ABOUT 5 MILLION CELLS
IN THE WELL NOW SHE COULD SEESLY
SEE THE PRODUCTION OF DEOCTY.
SO DOES PRODUCE GLUCOCO-COURT
STOID T -- GLUCOCORTICOID, NEXT
FEW SLIDES.
LET'S TEST OUR MODEL.
VERY FIRST PREDICTION F WE
REDUCE RESPONSE WE SHOULD ALTER
SELECTION AND REPERTOIRE.
IS THAT THE CASE?
MANY DIFFERENT STUDIES
ADDRESSING THIS, BUT I THINK THE
MOST CONVINCE AND GO WHAT I'LL
SHOW YOU A FEW EXAMPLES OF IS
THIS STUDY HERE THRAFS DONE BY
PAUL MIDDLE DEM STAT A STAFF --
MIDDLE DEM STAT A STAFF
SCIENTIST, KNOCKED OUT THE
GLUCOCOURTED COID RECEPTOR HAD
OR AND KNOCKED OUT AND MAKING
MICE AND CROSSING THEM TO
APPROXIMATE CRE MICE, NOW ONLY
THYMOCYTES ARE GOING TO BE
AFFECTED AND WE SHOWED THAT IN
FACT THEY DO THIS GR -- EXCUSE
ME.
AND THE ADRENAL IS PERFECTLY
NORMAL, CIRCULATING PERFECTLY
NORMAL.
THYMUS IN THESE MICE IS ABOUT A
THIRD HAD OR SMALLER THAN
WILD-TYPE.
DESPITE THAT THE PERIPHERAL T
CELL POPULATION IN THE POOL
LOOKS GROSSLY NORMAL, NORMAL
NUMBERS OF LYMPH NODE T CELLS,
CD4 CD8 DISTRIBUTIONS ARE VERY
SIMILAR.
OKAY?
SO AMONG THE MANY EXPERIMENTS
THESE ARE I'LL SHOW YOU JUST A
COUPLE.
THIS IS ONE WHERE PAUL IMMUNIZED
THESE MICE WITH PIGEON
CYTOCHROME C CHOSEN FOR A
REASON, THE RESPONSE IN B10A
MICE DEPICTED IN C IS QUITE
TYPICAL, CD4 RESPONSE I SHOULD
SAY, THEY'RE ALL RESTRICTED BY
IBFK, THEY ALL SEE THE FRAGMENTS
81 TO 104 AND TEFNED ON USE A T
CELL RECEPTOR CONTAINING D
ALPHA-1 1 BETA 3, WE IMMUNIZED
MICE, WAITED A WEEK, TEN DAYS,
TOOK OUT DRAINING LYMPH NODES
AND ASSAYS WITH INCREASING
AMOUNT OF PEPTIDE AND YOU SEE
THE WILD-TYPE T CELLS RESPONDED
PERFECTLY NORMALLY TO ANTIGEN
BUT THE MICE FROM THE GR
KNOCK-OUT MICE, GR KNOCK-OUT
THYMOCYTES MICE THE RESPONSE WAS
VERY POOR, AT LEAST 100 FOLD
SHIFT IN RESPONSE TO THE RIGHT.
NOT BECAUSE THE T CELLS WERE
SICK OR HAD SOME PROBLEM FROM
DIVIDING, ET CETERA, BECAUSE IF
WE CROSS LINK T CELL RECEPTOR,
TO BYPASS THE SPECIFICITY OF THE
T CELL RECEPTORS WE GET
PROLIFERATION AND EVEN BETTER
EXPERIMENT I THINK WAS THAT WHEN
WE CROSS THESE NIES THE AND
TRANSGENIC MOUSE, WHICH HAS A
RECEPTOR THAT SEES PIGEON
CYTOKINE C, ALL THE T CELLS
THERE'S NO POLYCLONAL REPERTOIRE
NOW, ALL THE T CELLS SEE
CYTOCHROME C AND NOW ACTIVATE
WITH ANTIGEN WE SEE THEY'RE
IDENTICAL TO THE WILD-TYPE T
CELLS SO THIS IS A REPERTOIRE
ISSUE, IT'S NOT AN ISSUE WITH
SPECIFICITY -- OF THE T CELLS
THEMSELVES, BUT THIS WOULD BE I
WOULD CONSIDER A CLASSIC KIND OF
HOLE IN THE REPERTOIRE AND WE
FOUND MANY OTHER EXAMPLES OF
THIS.
WE ALSO HAVE SOME EVIDENCE-BASED
ON THE REPERTOIRE CHANGE MORE A
PHYSICAL BASIS, THIS IS A WHILE
AGO IN THE EARLY DAYS OF HIGH
THROUGH PRESIDENT SEQUENCING,
BUT WE SEQUENCED THE CDR3 REGION
OF NAIVE T CELLS THAT EXPRESS V
ALPHA ELEVEN BETA 3, I WANT TO
EXPRESS WE SORTED FOR NAIVE T
CELLS AND WE EVENTUALLY LOOKED
AT THE POST THYMIC REPERTOIRE
PRIOR TO ANY PERTUBATIONS BY
EXPOSURE TO ANTIGEN, THIS IS THE
NAIVE REPERTOIRE.
AND ON THE FREQUENCY OF THE 100
OR 110 MOST USED CDR3'S IN THE
WILD-TYPE MICE AND THAT'S IN THE
CLOSED DOTLE IF YOU CAN SEE
THAT, AND ASKED WHETHER THE GR
KNOCK-OUT MICE HAD THE SAME
REPERTOIRE BASED ON CDR3 AND THE
ANSWER IS NO IT'S VERY
DIFFERENT, WE SAW IF WE DID THE
OPPOSITE EXPERIMENT WE SAW THE
SAME THING THAT, THE USE OF CDR3
IS QUITE DIFFERENT.
SO THIS ACTUALLY, THAT MODEL
ACTUALLY, THIS TEST OF THAT
MODEL IS PASSED, IF YOU KNOCK
OUT GLUCOCORTICOID
RESPONSIVENESS YOU GET MICE THAT
ARE IMMUNOLOGICALLY UNFIT, HAVE
AN ALTERED REPERTOIRE AND
INEFFECTIVE REPERTOIRE.
SO LET'S GOT BACK TO THE THYMUS
AND GLUCOCORTICOID PRODUCTION.
WE NOW KNOW THAT GLUCOCORTICOID
IS MADE IN THE THYMUS BUT WE
DON'T KNOW WHETHER THEY'RE
BIOLOGICALLY RELEVANT R THEY
MADE IN A HIGH ENOUGH QUANTITY,
IN THE RIGHT LOCATION, ET
CETERA, OR IS THE EFFECT OF
GLUCOCORTICOIDS MEDIATED VIA
CIRCULATED ADRENAL SUPPLY POOL.
TO ANSWER THIS QUESTION, PAUL
MADE ANOTHER KNOCK-OUT MOUSE,
THIS TIME KNOCKED OUT CIF11B1
THE ENZYME THAT SYNTHESIZES
GLUCOCORTICOIDS AND KNOCKED OUT
EPITHELIUM, CROTIONED TO FOX
N1CRE MOUSE SO ONLY THYMIC
EPITHELIUM WILL BE ABLE TO MAKE
THE -- CIRCULATING PERFECTLY
NORMAL, STRETCH RESPONSE IN
THESE MICE IS PERFECTLY NORMAL.
HIGHLY LOCALIZED KNOCK YOWLT OF
GLUCOCORTICOIDS.
THIS SHOWS YOU THAT THE MICE ARE
WHAT WE SAY THEY ARE, THIS IS
DONE BY CALIN DONAHUE A GRAD
STUDENT IN THE LAB WHO CULTURED
THYMUS AND NOW OUR SAAS SAYS --
NOW OUR ASSAYS ARE BETTER NOW
AND CAN MEASURE IF
GLUCOCORTICOID DIRECT FROM
THYMUS AND SEE IN WILD-TYPE
MOUSE THEY MAKE PLENTY OF, BUT
IN THE KNOCK YOWLT MICE YOU
DON'T SEE IT.
SO WE CAN NOW ELIMINATE THYME
SIETSZ PRODUCTION -- THYMUS
PRODUCTION OF GLUCOCORTICOIDS.
WHAT HAPPENS NOW?
DOES THIS HAVE ANY -- WHAT'S THE
EFFECT OF THIS?
DO WE SEE ANY DIFFERENCE DPIERTD
WILD-TYPE MICE?
SO -- COMPARED TO THE WILD-TYPE
MICE?
TO ADDRESS THIS, WE ASKED, WHAT
DO THYMOCYTES PERCEIVE?
SO WE LOOKED FROM AT STEROID
RESPONSIVE GENE, GLUCOCORTICOID
RESPONSIVE GENES OF THYMOCYTES
FROM THESE DIFFERENT MICE.
WE SHOW IN LAB ONE, PAUL DID OUR
PCR TO QUANTITATE THEM AND GIVE
WILD-TYPE MICE THYMOCYTES THE
LEVEL OF ONE AND FIRST LOOKED AT
THE GR1 KNOCK-OUT THYMOCYTES,
THYMOCYTES THAT CANNOT RESPOND
TO GLUCOCORTICOIDS AT ALL
REGARDLESS OF THEIR SOURCE AND
WE SEE A 40 TO 50 PEST REDUCTION
IN -- 40 TO 50% REDUCTION IN
EXPRESSION OF THESE GENES, BASAL
PRODUCTION.
WHAT WAS IMPORTANT HERE IS WHEN
WE LOOKED AT THE CIF -- AT THE
CIF -- WE SEE THE EXACT SAME
REDUCTION IN EXPRESSION OF THESE
GLUCOCORTICOID RESPONSIVE GENES
MEANING EFFECTIVELY THE ONLY
SOURCE OF STEROIDS THAT ARE
PERCEIVED BY THYMOCYTES AT LEAST
TO THE LEVEL OF UPREGULATING
THESE GENES AND OTHERS IS
PRODUCED LOCALLY, THAT SYSTEMIC
LEVELS DON'T MAKE A DIFFERENCE,
IT IS ONLY LOCAL PRODUCTION THAT
MATTERS.
AND COMFORTINGLY, THESE MICE
COPIED THE GENE KNOCK-OUT MICE,
T CELL RESPONSES WERE POOR,
SHOWING -- INTOWNTS THE T CELLS
ARE FINE BECAUSE IF YOU CROSS T
CELL RECEPTOR, THEY RESPOND
NORMALLY.
SO WHO IS MAKING THE
GLUCOCORTICOIDS?
CORTICAL EPITHELIUM OR THYMIC
EPITHELIUM, MEDULLARY
EPITHELIUM?
SORRY.
THANK YOU FOR GOING THROUGH ALL
THAT SO I CAN JUST IGNORE THE
DIFFERENCES BETWEEN THESE CELLS,
WITH THE EXCEPTION I'M UNDER
STRESS, THE POINT THAT AIRE IS
EXPRESSED BY MEDULLARY TEC AND
THAT AIRE DRIVES -- AS FAMOUSLY
PROCESSED TO ANTIGENS THAT ARE
THEN USED TO DELETE CAUSE
NEGATIVE SELECTION OF CELLS THAT
BEAR -- RECEPTORS THAT RECOGNIZE
THEM, IMPORTANT IN THE
ESTABLISHMENT OF CENTRAL
TOLERANCE.
SO WE COULDN'T USE ANTIBODIES TO
CIF112B1 TO IDENTIFY THE CELLS
OF INTEREST BECAUSE THE
ANTIBODIES CROSS-REACT, THESE
ENZYMES ARE VERY SIMILAR IN
THESE PATHWAYS, AND THE ANTIBODY
CROSS-REACTS.
SO MATT TAVES A POST-DOC IN THE
LAB DECIDES TO MAKE A REPORTER
MOUSE, SCARLET TO CIP11B1 SHOWN
HERE AND KNOCK THAT INTO THE
ENDOGENOUS LOCUS IN B6 MICE AND
YOU CAN SEE ON THE FAR RIGHT
THAT IT WORKED BECAUSE THAT'S A
CROSS-SECTION OF THE ADRENAL AND
YOU CAN SEE THE ZONAFOCICULADA
LIGHTS UP, SO TO ADDRESS THE
QUESTION WE'VE BEEN THINKING
ABOUT SINCE EARLY 190S AND THAT
IS WHICH CELL IS ACTUALLY MAKING
GLUCOCORTICOIDS?
IF YOU LOOK AT THE RIGHT, LOWER
RIGHT, THAT'S FOLK AM MICROSCOPY
THAT KAITLYN DONAHUE PERFORMED
IN WHICH SHE STAINED THE
CORTICAL IF TEC'S WITH L51
THAT'S BLUE NO, RED IN THAT
REGION, REGULAR TEXT IS STAINED
IN GREEN UEA1 AND MEDULLA
EXPRESSION I HOPE YOU CAN SEE T
CAPABILITY SEE IT FROM THIS
ANGLE, THAT QUIEPT A FEW RED
CELLS, YELLOW CELLS, ET CETERA.
SO IT'S THE MEDULLARY TEXT THAT
ARE MAKING FROM CYP11B1
CONFIRMED ON THE RIGHT BY FLOW
CYTOMETRY WHERE KAITLYN NUND IF
YOU LOOK AT THE BOTTOM -- FOUND
IF YOU LOOK AT THE BOTTOM, THE
REPORTER MICE, THAT THYMOCYTES
DON'T MAKE IT, APC, MACROPHAGES
DON'T MAKE TCTEC DON'T MAKE IT,
MTEC LOWS WHICH ARE THE MORE
MAIM TOUR MTEC'S START TO MAKE
CYP11B1 AND ABOUT TEN PERCENT OF
THE CELLS ARE POSITIVE.
AT THE TIME IN 2022 THE PAPER
FROM NICHOLSON AND FROM
MATHESON'S LAB, I'VE REFERRED
TO, WHERE THEY SORTED MHC LOW
CELLS AND LOOKED AT CELLLY SEQ,
WE PREFERRED CELL HIGH, WE WOULD
TAKE WHAT WE WOULD GERKTS PLEASE
TO DO SEE WHEN WE INTERROGATED
THEIR DATASET, WE COULD SEE A
POPULATION OF CYP11B1 THAT
CLUSTERED IN THIS ANALYSIS.
IMPORTANTLY FOR US, IT TURNED
OUT THAT THAT CLUSTER ALSO WAS
ENRICHED IN AIRE.
SO WE ASKED THE QUESTION, IS
AIRE RELATED IN FACT TO THE
EXPRESSION OF CYP11B1?
KAITLYN LOOKED AT CYP11B1
POSITIVE CELLS AND COUNT THE
NUMBER THAT ARE EITHER IF AIRE
POSITIVE OR NEGATIVE AND AS YOU
CAN SEE ABOUT 60% OF THOSE CELLS
THAT MAKE GLUCOCORTICOIDS ARE
AIRE POSITIVE.
AN EXAMPLE SHOWN THE CONFOCAL
EXAMPLE I SHOWED THERE SHOWS AN
AIRE POSITIVE CELL IS ALSO
CYP11B1 POSITIVE.
WHAT ABOUT THE 40% AIRE
NEGLECTITY?
DOES THAT MEAN IT'S NOT
NECESSARILY INVOLVED?
MTEC'S DON'T STOP
DIFFERENTIATING AFTER THEY
BECOME AIRE POSITIVE, THEY FORM
A STATE WHERE THEY LOSE AIRE
EXPRESSION AND GAIN OTHER
ANTIGENS LIKE OR MOLECULES LIKE
KERATIN 10.
THESE ARE CALLED POST AIRE
CELLS.
KAITLYN THEN ASKED WHAT
PERCENTAGE OF THESE 40%, WHAT
PERCENTAGE ALSO EXPRESS KERATIN
10 AND THE ANSWER WAS 80%,
THAT'S IN THE UPPER RIGHT, MY
POINTER IS NOT WORKING SO I
CAN'T POINT TO T BUT THE ANSWER
IS THAT BY THIS ANALYSIS, ALMOST
ALL THE CELLS THAT MAKE UP
CYP11B1 ARE EITHER POSITIVE AT
THAT TIME OR MORE RECENTLY AIR
HE POSITIVE, CONFIRMED WHEN
KAITLYN CROSSED THE AIRE
KNOCK-OUT MICE TO THE REPORTER
MICE AND YOU CAN SEE THAT IF YOU
LOOK AT THE BOTTOM LEFT YOU CAN
SEE THAT THE AIRE KNOCKOUTS LOSE
EXPRESSION OF CYP11B1.
NOW, CYP11B1 BY ITSELF DOESN'T
MAKE GLUCOCORTICOIDS, NEEDS ALL
THE OTHER ENZYME PS IN THE SUITE
AS SHOWN THERE, SO LOOKED AT THE
SEQ DATA IN THE THYMUS WHICH
POPULATION CAN MAKE ALL THE
ENZYMES AND THE ONLY POPULATION
THAT COULD MAKE ALL THE ENZYMES
IS THE TEC'S.
IF YOU LOOK TO THE RIGHT OF
THAT, HE ALSO ASKED NOW IF YOU
SUBDIVIDE THE TEC'S INTO
DIFFERENT POPULATIONS, WHAT
ENZYME DID THEY EXPRESS, AND
ONLY THE MTEC HIGHS MADE ALL THE
ENZYME SAYS REQUIRED FOR
GLUCOCORTICOID SYNTHESIS.
THAT DOESN'T MEAN THEY'RE ALL ON
THE SAME CELL, OF COURSE,
HOWEVER, THEY ARE ON THE SAME
CELL.
IF YOU LOOK TO THE FAR RIGHT AS
I'VE ALREADY SHOWN YOU WE CAN
DETECT THYMOCYTES PRODUCTION OR
GLUCOCORTICOID PRUK FROM THE
THYMUS AND AS YOU SEE HERE, WHEN
YOU KNOCK OUT AIR HE IT MOSTLY
GOES AWAISM THIS IS THE LAST
DATA SLIDE.
WE THEN ASKED WHAT OTHER
PATHWAYS MIGHT BE EXPRESSED IN
MTEC'S?
KAITLYN DID RPCR ON MTEC'S AND
INVOLVING BIOSUN THINK CIS, NOT
JUST GLUCOCORTICOIDS BUTS
PROGESTERONE AND SEX STEROIDS
AND YOU CAN SEE THEY ALL THESE
ENZYMES ARE PRESENT IN MTEC'S
AND THEY'RE ALL DEPENDENT ON AIR
HE.
WHEN YOU KNOCK OUT AIRE THEY ALL
GO DOWN, PROMPTING US TO
EXPERIMENT WE NEVER HAD THOUGHT
OF DOING BEFORE, AND THAT IS
TO -- PROGESTERONE, TESTOSTERONE
AND HE IS STROH DIAL AND --
ESTRADIOL AND THEY'RE ALL
MARKEDLY REDUCED IN THE ABSENCE
OF AIRE.
TAKE AWAY, CONCLUSION IS THAT
AIRE DRIVES EXPRESSION OF NOT
JUST TRA'S BUT ALSO ENTIRE
SYNTHETIC PATHWAYS, GENES THAT
CAN PRODUCE SECONDARY REAGENTS
THAT ARE INDIRECTLY REGULATED BY
AIRE BUT ARE ACTUALLY PRODUCED
BY SYNTHETIC PATHWAYS.
NOW, THERE'S A PARADOX HERE, AND
THAT IS WHY WOULD AIRE PROMOTE
BOTH TRA MEDIATED DELETION,
WHICH IS REQUIRED FOR TOLERANCE,
AS WELL AS GLUCOCORTICOID
MEDIATED POSITIVE SELECTION,
WHICH IS REQUIRED FOR EFFECTIVE
IMMUNITY?
I THINK THE ANSWER ACTUALLY IS
PRETTY CLEAR, AND THAT IS TO
EX-PABD THE T CELL RECEPTOR.
IF MT HE C'S WERE ONLY DELETING
AND EXPRESSING THOUSANDS AND
THOUSANDS OF -- GENERAL OUGHTING
MANY THOUSANDS OF SPECIFICITIES
THEN YOU WOULD LOSE ALL THOSE
CELLS FROM THE REPERTOIRE AND
YOU WOULD HAVE A RESTRICTED
REPERTOIRE.
BUT BECAUSE THE SAME CELL THAT'S
PRESENTING THE ANTIGEN THAT
WOULD OTHERWISE DLEELT AND
PRODUCES GLUCOCORTICOIDS THAT
ARE THERE THAT SYNAPSE OR IN
THAT IF YOU WILL IN THAT REGION,
NOTICES CELLS CAN BE SELECTED
AND GO ON -- THOSE CELLS CAN BE
SELECTED AND GO ON TO PROMOTE
POSITIVE IMMUNE RESPONSE AND
THIS IS EXACTLY WHAT WE SAW WHEN
WE KNOCKED OUT GLUCOCORTICOID
RECEPTOR IN THYME SIETSZ, WE SAW
ARE REPERTOIRE AND WEAK IMMUNE
SYSTEM.
I'VE ALREADY MENTIONED I THINK
PEOPLE INVOLVED IN THIS WORK.
I WANT FOINT OUT AGAIN THAT MATT
TASE AND KAITLYN WERE INVOLVED
FROM THE POINT FROM THE -- AND
DID ALL THE WORK FROM THE
REPORTER MOUSE ON, MATT IS AT
CORNELL WRITING GRANTS AND
KAITLYN IS IN THE LAB BUSY
WRITING HER THESIS.
SO THANK YOU VERY MUCH.
[APPLAUSE]
>> THIS TIME I THINK WE'LL ASK
EXACTLY THE SAME QUESTION.
YOU MIGHT KNOW THE OLD WORK BY
RICHARD BOYTES THAT HAS
DEMONSTRATED THAT SEX STEROIDS
DURING AGING MIGHT BE ONE OF THE
REASONS --
>> IGD HARDLY HEAR, I'M SORRY.
>> I'M SURE IF I CAN GET THIS
ANY LOUDER.
OKAY.
SO ACTUALLY DEMONSTRATING THAT
SEX STEROIDS BLOCK AIDS CAN LEAD
TO IMPROVED THYMIC FUNCTION H HE
ALSO STIPULATED THAT WITH AGE
THAT SEX STEROID LEVELS GO UP
AND THAT THAT MIGHT BE ONE OF
THE REASONS FOR THE EVOLUTION.
-- INVOLUTION.
MY QUESTION IS OF COURSE IF YOU
SEE ANY CHANGES IN THE
GENERATION OF THESE SEX STEROIDS
WITH AGE BY THESE TEC'S.
>> GOOD QUESTION.
IT'S IMPORTANT TO DO THE WORK.
>> WE CAN'T HEAR YOU ON THE
ZIESH OH, I CAN SPEAK LOUDER.
YES, THAT'S A VERY GOOD
QUESTION, AND YES, OF COURSE THE
WORK, IN FACT, MATT AND I WROTE
A REVIEW LAST YEAR ON THE ROLE
OF SEX STEROIDS AND THYMUS AND
IT TURNS OUT THEY MOSTLY
REGULATE THE EPITHELIUM, RATHER
THAN THE THYMUS ACTUALLY
THEMSELVES.
THIS INFORMATION ABOUT THE SEX
STEROIDS IS RATHER NEW.
WE HAVE NOT EXPLORED THAT,
ANYWHERE CLOSE TO BEING DONE
WITH GLUCOCORTICOIDS HAD.
WHAT I CAN TELL YOU IS THAT
GLUCOCORTICOID SYNTHESIS IS
HIGHEST IN FEEBLGHTS LIFE AND
NEONATAL LIFE, AND IT GOES DOWN
POST-BIRTH AND REACHES ADULT
LEVELS ALTHOUGH ABOUT 50% AT
ABOUT FOUR WEEKS OF AGE, WHICH
YOU MENTIONED THE ROLE OF AGING
AND GLUB CORTICOIDS.
I SUSPECT THAT'S GOING TO BE
TRUE FOR THE OTHER PATHWAYS
INVOLVED BECAUSE THEY ARE
DEPENDENT.
OKAY?
BUT WE HAVE NOT INVESTIGATED
THAT.
>> I ACTUALLY AGAIN WANTED TO
ASK A RELATED QUESTION, BUT THIS
IS REALLY ABOUT DIOXINS AND IT'S
VERY OLD LITERATURE ABOUT
BASICALLY THE ENDOCRINE
DISRUPTING EFFECT OF DIOXINS ON
THE THYMUS AND IT SEEMS TO ME
THAT WITH THE TOOLS THAT YOU
HAVE RIGHT NOW IT WOULD BE
REALLY INTERESTING TO REEXAMINE
THIS WHOLE ISSUE AND TO ASK
WHICH CELLS ROLES ARE REALLY
BEING EITHER MIMICKED OR SUB
VERLTED BY THE DIOXINS.
>> YOU SEE WITH AHR SIGNALLING?
>> YES, ALSO THERE WAS SOME
POSSIBILITY THAT IT HAD AN
ESTROGEN EFFECTED AND I WAS
CURIOUS IF THAT HAD COME UP IN
ANY OF THE CONNECTIONS OF THE
WORK THAT YOU'RE SEEING.
>> THAT'S POSSIBLE.
WE DO THINK, I WANT TO JUST
MENTION, WE SAW OTHER PALT WAYS
THAT WEEFNLT INVESTIGATED YET --
PATHWAYS THAT WE'VE NOT
INVESTIGATED YET, ENZYME
PATHWAYS IN FEALT ACID SIFN
THINK CIS AND PHOSPHATE
GENERATION SO WE THINK LOTS OF
THESE PALT WAYS THAT MAY BE
INVOLVED HERE.
>> JOHN, COULD YOU JUST SIMPLY
-- THE MTEC'S, THE
SUBSET?
>> YOU'RE TALKING ABOUT THE
TISSUE MTEC'S THAT MATHIS WAS
HERE TALKING ABOUT RECENTLY?
>> YES.
>> THOSE WOULD BE THE POST
AIRE MTEC'S.
WE MAPPED OUR CLUSTER WITH THE
CLUSTER SHE HAD DEFINED AND THE
CLUSTER THAT CONTAINS CYP11B1 IS
DIFFERENT, THE NEUROENDOCRINE
CLUSTERS OR THE DIFFERENT TISSUE
SHE FOUND SO IT SEEMS LIKE IT
DOESN'T CLUSTER WITH HERS, BUT
THEN WE'RE LOOKING AT, WE SEE
THIS IN MTEC HIGHS AND SHE'S
REALLY LOOKING AT MTEC LOWS, A
POPULATION THAT'S NOT AS
RELEVANT HERE FOR
GLUCOCORTICOIDS.
>> BUT DID YOU LOOK AT LIKE
TRANSCRIPTION FACTORS THAT ARE
SPECIFIC FOR -- THE WAY SHE
IDENTIFIES --
>> I'M SAYING WE GO DO ON THE
SINGLE CELL SEQ WE DID, YES, AND
THERE'S NO OVERLAP WITH THOSE
POPULATIONS, WITH HER GROUPS
THAT SHE HAD DEFINED.
IT'S A NEW, UNIQUE GROUP.
>> THANK YOU.
>> HAVE YOU LOOKED TO SEE IF
THERE'S ANY CHANGES IN HAD T REG
POPULATIONS DEPENDENT ON
GLUCOCORTICOID LEVELS?
>> THAT'S A GREAT QUESTION, AND
WE THOUGHT THRMED BE -- AND WE
THOUGHT THERE WOULD BE, IN FACT,
WE DIDN'T.
THE T REGS, IN NUMBER THEY
AREN'T CHANGED.
WE DID NOT -- WE DID ONE
EXPERIMENT WHERE WE TRIED TO SEE
IF THE FUNCTION NORMALLY, WITH
THE ADOPTED COLLIDUS MODEL, THEY
LOOKED PRETTY NORMAL SO WE HAVE
NOT PURSUED THAT.
>> YOU ONLY LOOKED AT IN FOX #
POSITIVE?
>> YES, WE ONLY LOOKED AT FOX,
YES.
>> IT SOUNDS LIKE YOUR MUTANT
MICE MIGHT HAVE A MORE SEVERE
PHENOTYPE EARLIER IN LIFE.
HAVE YOU LOOKED IN NEONATAL
MICE?
>> IN WHAT REGARD?
>> WELL, YOU SAID THE CIRCLE A
AND GLUCOCORTICOIDS DON'T GO
THERE EARLY IN LIFE, RIGHT?
>> NELLER LIFE.
>> YOU SAID THAT THE
GLUCOCORTICOIDS ARE HIGHER IN
FETAL THYMUS.
>> RIGHT.
>> AS OPPOSED TO ADULT THYMUS.
>> IN THE THYMUS, YEAH.
>> THAT MADE ME THINK THAT THE
PHENOTYPE MIGHT BE MORE SEVERE
EARLIER IN LIFE AS OPPOSED ZIESH
THE PHENOTYPE WE'RE LOOKING
AT -- YOU WOULD HAVE TO LOOK AT,
TAKE UP --
>> IF YOU LOOK UP THE
REPERTOIRE, FOR EXAMPLE.
>> WE HAVE LOOKED AT THYMUS AT
DIFFERENTIATION AND AS I SAID
QUALITATIVELY WE HAVE A SMALL
THYMUS, POPULATIONS FROM DOUBLE
POSITIVE ON DOWN ARE REDUCED,
OKAY, DOUBLE-NEGATIVES ARE
PRESERVED, WE SEE PERFECTLY
NORMAL DOUBLE-NEGATIVE
GENERATION.
SO WE THINK IT'S MOSTLY
AFFECTING ONLY TCR SIGNAL CELLS,
AND WE CAN, WE'VE ALSO STHEEN
THE MHC BACKGROUND MAKES A BIG
DIFFERENCE.
THE BACKGROUND HAPLOTYPE MAKES A
BIG DIFFERENCE.
MORE COMPLEX HAPLOTYPE THE
SMALLER THE THYMUS BECOMES,
BECAUSE WE THINK BECAUSE THERE'S
MORE CELL PEPTIDE AND MORE
DIVERSITY PEPTIDE AND MORE
RECEPTORS ARE GOING TO BE
OCCUPIED.
>> JOHN, IT'S A WONDERFUL STORY,
I'M GLAD IT'S BEEN RESURRECTED.
TWO QUICK QUESTIONS.
ONE IS, DO YOU KNOW WHEN YOU SEE
ALL THESE DIFFERENT GENES
EXPRESSED IN THE CORTICOSTEROID
PATHWAY, IS IT JUST THE GENES
EXPRESSED OR DO YOU KNOW IF ALL
THE PROTEINS ARE EXPRESSED AS
WELL ALONG THE WAY?
>> WELL, BY CYP11B1 REPORTER
IT'S A FUSION PROTEIN, RIGHT?
SO IF YOU SEE IN SCARLET YOU'RE
SEEING CYP1 <!0> B1 AND WE ALSO
KNOW THAT TRP THE CYP11B1 IS
FUNCTIONAL BECAUSE THE ADRENALS
ARE PERFECTLY FUNCTIONAL,
OTHERWISE THE MICE WOULD BE
HYPOADRENAL AND THEY'RE NOT.
CIRCULATING LEVELS ARE NORMAL,
STRESS RESPONSE IS NORMAL.
THAT'S IDENTIFYING THE PROTEIN.
OKAY?
LOOKING AT THE OTHER PROTEINS,
THERE ARE OTHER PEOPLE WHO HAVE
IDENTIFIED -- NOT MT HE C'S OF
COURSE BUT IN THE THYMUS SOME
PEOPLE YES HAVE BY ANTIBODY
MEDIATED MECHANISMS TECHNIQUES
HAVE SEEN SOME OF THE OTHERS
EXPRESSED.
>> THE OTHER QUESTION WAS, THE
ORIGINAL --
>> YOU REALIZE THEY'RE MAKING
GLUCOCORTICOIDS.
SO GENES DON'T MAKE
GLUCOCORTICOIDS, JUST TO
CLARIFY, RESURRECT THAT NOTION.
OKAY.
>> THANKS.
SO ORIGINALLY AS I RECALL YOU
THOUGHT THAT THE GLUCOCORTICOIDS
MIGHT BE THE MECHANISM OF DEATH
BY NEGLECT.
IS THAT STILL THE CASE?
DO YOU THINK THAT IT'S --
>> I CAN'T QUITE HEAR.
>> SO DO YOU KNOW IF THE
GLUCOCORTICOID -- ORIGINALLY YOU
THOUGHT THAT GLUCOCORTICOIDS
WERE THE INDUCERS OF DEATH BY
NEGLECT.
>> YES.
>> DO YOU STILL THINK THAT?
THAT THEY --
>> NO.
NO.
THAT WAS DISPROVED LONG AGO.
BACK IN THE EARLY DAYS, WE KNEW
NOTHING ABOUT, YOU KNOW, ALL THE
MECHANISMS WE KNOW TODAY AND WE
THOUGHT CONCEIVABLY THAT
GLUCOCORTICOIDS WOULD -- WERE
RESPONSIBLE FOR THE DEATH OF
CELLS, THE UNAN TAG NIETZED BY
ARE TCR REGULATION AND THE
ANSWER IS NO THE GR KNOCK-OUT
THYMOCYTES HAVE NO CHANGES IN
DEATH BY NEGLECT.
AND WE'RE NOT THE FIRST ONE TO
SHOW THAT.
LAST QUESTION?
>> SO YOU SHOWED THAT IT'S A
PROBLEM IN THE TCR REPERTOIRE IN
RESPONSE TO STIMULATION, SO
POST-SELECTION, I GUESS.
THE REPORTER MOUSE SHOWED THAT
THE CYP11B1 IS EXPRESSED IN
APPROXIMATE MINOR -- HOW DOES
THAT AFFECT POSITIVE SELECTION?
>> WE DON'T THINK IT HAS
ANYTHING TO DO WITH POSITIVE
SELECTION.
IN THE CORTEX, WE DON'T THINK
IT'S GOT TO DO WITH POSITIVE
SELECTION OF THE CORTEX.
WE THINK IT HAS TO DO WITH THE
RES RESCUE OF CELLS THAT WOULD
UNDERGO NEGATIVE SELECTION GOO
CELLS FOR ANTIGEN UNDERGO
NEGATIVE SELECTION IF THE
GLUCOCORTICOID SIGNALLING IS NOT
THERE?
>> THE IDEA IS THAT YOU EXPRESS
IT -- AND THAT T CELLS THAT
RECOGNIZE THOSE ANTIGENS, THAT
MADE IT THROUGH THE CORTEX,
THEY'RE STILL AROUND, THEY'RE
GOING TO BE DELETED BUT THEY'RE
RESCUED BY THE GLUCOCORTICOIDS.
AND THAT EXPANDS THE REPERTOIRE
TO INCLUDE ALL THOSE CELLS THAT
NOW SEE ALL THE DIFFERENT TRA'S,
THAT'S THE NOTION.
AND WORK DIDN'T HAVE A CHANCE TO
SHOW BUT AT THE TIME IS THAT
MATT ACTUALLY DEVELOPED AN ASSAY
WHERE WE COULD ACTUALLY MEASURE
GR INTERACTIONS WITH CHROMATIN,
SO THAT'S THE SIGNAL CELLS AND
WE SHOWED THE ONLY CELLS THAT
ARE RECEIVING THE SIGNAL ARE
THIS SIGNAL AMONG THE THYMIC
THYMOCYTES POPULATION ARE THE
TCR HIGH DOUBLE POSITIVES, THE
TRIGGER DOUBLE POSITIVES.
OKAY?
TO SOME EXTENT POST THAT.
SO THESE ARE VERY LOCALIZED
DELIVER RIFF GLUCOCORTICOIDS
FROM THE FROM THE PRESENTING
MTEC'S SO RESPONGE ON THE
SURFACE.
>> AND -- DOES ANYTHING THERE.
>> SORRY, WE'RE BEING TOLD IT'S
TIME FOR A BREAK.
>> OKAY.
>> OKAY, THANK YOU MPLETS.
[APPLAUSE]
>> THANK YOU.
[APPLAUSE]
>> OKAY RS WE'LL HAVE A 20
MINUTE BREAK AND WE'LL START
WITH THE NEXT TALK AT 10:50.
SO WE'LL GET STARTED IN THIS
SECOND PART OF THE MORNING
SESSION WITH DR. DINAH SINGER
FROM THE NCI TO TELL US ABOUT
THE DEVELOPMENT AND ROLE OF THE
FACTOR BRD4.
>> THANK YOU, STANLEY. SO IF I
RESIGN, DIE GET AN EXTRA FIVE
MINUTES?
[LAUGHTER]
OKAY.
IT'S A DEAL.
I WANT TO START BY THANKING THE
ORGANIZERS TOIN VOOTING ME TO
SPEAK, I'M DELIGHTED TO BE ABLE
TO TELL YOU ABOUT SOME OF OUR
RECENT STUDIES ON THE REGULATION
OF THYMIC DEVELOPMENT BY THE
TRANSCRIPTION FACTOR BRD4.
SO WHY BRD4?
BRD4 HAS BEEN STUDIED FOR SOME
TIME NOW AS AN ACTIVE REGULATOR
IN INFLAMMATORY DISEASE AS IN T
CELL FUNCTION, AND IT'S BEEN
PARTICULARLY STUDIED AS A DRIVER
IN A VARIETY OF DIFFERENT BOTH
LIQUID AND SOLID TUMORS, SO IT'S
GOTTEN A LOT OF ATTENTION AS A
POSSIBLE THERAPEUTIC TARGET.
FROM THE BIOCHEMICAL STANDPOINT,
BRD4 IS A DOMAIN PROTEIN WE'VE
SHOWN OVER THE YEARS HAS
PLEOTROPIC FUNCTIONS, REGULATES
CHROMATIN STRUCTURE THROUGH ITS
INTRINSIC TRANSFERASE ACTIVITY
HA REMODELS CHROMATIN, IT
REGULATES TRANSCRIPTION THROUGH
ITS KINASE ACTIVITY WHERE IT
PHOSPHORYLATES POLYMERASE 2 AND
NIC EXPREENTLY SHOWED THAT BRD4
REGULATES RNA SPLICING AND IN
SPITE OF ALL THE WORK THAT'S
BEEN DONE ON BRD4, VERY LITTLE
IS KNOWN ABOUT ITS ROLE IN
DEVELOPMENT AND DIFFERENTIATION
GENERALLY, BUT PARTICULARLY IN
THE THYMUS.
SO WE WERE INTERESTED IN ASKING
DOES BRD4 HAVE AN ACTIVE ROLE IN
THYMIC DIFFERENTIATION, AND I'M
GOING TO LEAVE YOU WITH THREE
TAKE-HOME MESSAGES IN CASE
STANLEY CUTS ME OFF.
ONE IS, THE FIRST ONE IS THAT IN
SINGLE POSITIVE THYMOCYTES --
IMMATURE POSITIVE THYMOCYTES ARE
UNIQUELY DEPENDENT ON BRD4 FOR
THEIR DIFFERENTIATION, THAT BRD4
REGULATES ALTERNATIVE RNA
SPLICING IN ALL THYMOCYTE
SUBSETS AND POSITIVE SELECTION
OF DOUBLE POSITIVE THYMOCYTES IS
UNIQUELY DEPENDENT ON BRD4
EXPRESSION IN THYMIC EPITHELIAL
CELLS.
SO WE'VE SEEN VARIOUS CARTOONS
OF THIS, THIS MORNING ALREADY,
OF THE SEQUENCE OF THYMIC
DIFFERENTIATION WHICH GOES IN
WELL-DEFINED SERIES OF STEPS,
STARTING WITH THE ARRIVAL OF THE
IMMATURE THYMOCYTES INTO THE
THYMUS THAT EXPRESS NEITHER SOAR
NOR CD8, THE DOUBLE-NEGATIVES
THAT THEN UNDERGO -- NEITHER CD4
NOR CD8, THE DOUBLE-NEGATIVES
THAT UNDERGO PROCEED LIFT UNITED
NATIONS DURING WHICH ARE
EXPRESSED, AND FINALLY THEIR
SIGNAL TO EXPRESS CD8 ON THE
SURFACE AND BECOME WHAT ARE
CALLED MAIM TOUR SINGLE
POSITIVES THAT EXPRESS CD8 ONLY.
THESE ARE TRANSITIONAL CELLS
BETWEEN THE VERY PROLIFERATIVE
DOUBLE-NEGATIVE AND THE
QUIESCENT DOUBLE POSITIVE, AND
THEY HAVE TO JUSHED GO --
UNDERGO ONE FINAL ROUND OF CELL
DIVISION IN ORDER TO BECOME
DOUBLE POSITIVES THAT THEN GO ON
TO MATURE TO BECOME EITHER THE
CD4 OR THE CD8 SINGLE POSITIVES.
SO THE QUESTION THAT WE WERE
INTERESTED IN ASKING IS, WHAT IS
THE ROLE OF BRD4, IF ANY, IN
EACH THESE STEPS MANY ONE OF THE
REASONS FOR ASKING THIS QUESTION
WAS THAT BRD4 IS ACTUALLY
EXPRESSED THROUGHOUT THYMIC
DIFFERENTIATION, SO WE WANTED TO
KNOW WHAT ROLE IT PLAYS IN EACH
THOSE STEPS.
TO DO THAT, WE CONDITIONED --
CONDITIONALLY DELETED AS EARLY
AS WE CAN TODAY, AND THAT IS
WITH LCK KCRE WHICH DELETES
AROUND 29 STAGE AND FOLLOW WHAT
HAPPENS TO BRD4 EXPRESSION AND
YOU CAN SEE IT'S ALREADY ACUTELY
DEPLETED IN THE DN STAGE
COMPLETELY DELETED BY THE ISP'S
AND CONTINUES TO BE COMPLETELY
DELETED FOR THE REST OF THE
DEVELOPMENTAL SEQUENCE.
SO WITH THAT IN MIND, WE ASKED,
WHAT IS THE EFFECT OF THIS
DELETION ON THYMIC
DIFFERENTIATION?
AND WHEN WE LOOKED AT CD4 CD8
PROFILES WHAT WE WERE ABLE TO
SEE WAS AN ACUTE DEPLETION IN
THE NUMBER OF DOUBLE POSITIVE
CELLS AND FREQUENCY OF DOUBLE
POSITIVE CELLS AND AN INCREASE
IN THE CD8 SINGLE POSITIVE
CELLS.
THE CELLS THAT ARE IN THIS
COMPARTMENT ARE BOTH THE MATURE
CD8 SINGLE POSITIVES BUT ALSO
THE IMMATURE CD8 SINGLE
POSITIVES, THOSE TRANSITIONAL
CELLS.
THEY CAN BE DISTINGUISHED BASED
ON THEIR EXPRESSION OF TCR BETA
ON THE SURFACE, AND WHEN YOU
LOOK AT TCR BETA EXPRESSION IN
THE CD8 QUADRANT, WHAT YOU SEE
HERE IN THE LIGHT GRAY IS THAT
THE WILD-TYPE CELLS
PREDOMINANTLY EXPRESS THE MATURE
CD8 SINGLE POSITIVES THAT ARE
EXPRESSED TCR BETA WHEREAS IN
THE KNOCK-OUT MOST OF THE CD8
CELLS THAT ARE IN THIS QUADRANT
DO NOT EXPRESS TCR BETA AND
THEREFORE ARE THE IMMATURE
SINGLE POSITIVES, THOSE
TRANSITIONAL CELLS.
WHEN WE QUANTITATE WE SEE THAT
THE DOUBLE-NEGATIVE POPULATIONS
ARE NOT AFFECTED BUT THERE IS A
HUGE INCREASE IN THE NUMBER OF
THIS ISP'S THAT ACCUMULATE IN
THE ABSENCE OF BRD4 AND OF
COURSE THAT THEN IS PROPAGATED
INTO THE DOUBLE POSITIVES AND
THE SINGLE POSITIVES BUT TELLS
US THAT BRD4 DELETION ARRESTS
THE TRANSITION OF ISP'S TO THE
DOUBLE POSITIVE STAGE.
I MENTIONED EARLIER THAT FOR AN
ISP TO MATURE TO A DP IT HAS TO
UNDERGO ONE LAST ROUND OF CELL
CYCLE AND ASKED IF PART OF THE
DEFECT WE'RE SEEING HERE IS THE
INABILITY OF THE CELLS TO
UNDERGO THAT LAST CELL CYCLE.
TO ANSWER THAT QUESTION, WE
TURNED ON AN IN VITRO SYSTEM IN
WHICH YOU CAN TAKE PURIFIED
ISP'S, CULTURE THEM IN VITRO
OVERNIGHT AND NOW THEY GO FROM
BEING ENTIRELY CD8 SINGLE
POSITIVE TO BECOMING DOUBLE
POSITIVE AND DOUBLE IN CELL
NUMBER.
WHEN YOU DO THAT NOW WITH THE
BRD4 KNOCK-OUT, TAKE PURIFIED
ISP'S, WHAT YOU SEE IS THAT THEY
HAVE REAL DIFFICULTY IN
UNDERGOING THAT MATURATION AND
THAT CELL DWIKS, SO THAT THE
DEFECT HERE IS IN
DIFFERENTIATION AND
PROLIFERATION DURING IN VITRO
CULTURE.
NOW, WE CAN LOOK AT THE
MOLECULAR BASIS FOR THIS DEFECT
BY DOING RNA SE E QUANL CIS --
SEQ ANALYSIS OF THE THYMOCYTES
AND WE DO THAT AND WE SEE
SURPRISINGLY IS VERY LITTLE
EFFECT ON RNA SEQ PROFILES OF
EITHER THE DN'S OR DP
POPULATIONS WHEN MSZ BRD4 IS
DELETED BUT YOU SEE ENORMOUS
CHANGE IN THE PROFILES WHEN YOU
DELETE BRD4 FROM THE ISP'S.
WHEN WE DO PATHWAY ANALYSIS,
MAJOR PATHWAYS AFFECTED ARE NIC
PATHWAYS AND GLYCOLYTIC PATHWAYS
AND WE'VE VALIDATED BOTH OF
THOSE PATHWAYS EXPERIMENTALLY
AND I'M SHOWING YOU HERE JUST
NIC IT'S AN IG -- MYC IT'S A
BROWSER VIEW LOOKING AT
EXPRESSION OF MYC IN WILD-TYPE
OR KNOCK-OUT ISP'S AND SEE THE
COMPLETE LOSS OF MYC WHEN YOU
LOSE BRD4.
WHAT'S REALLY SURPRISING THOUGH
IS YOU DON'T LOSE MYC IN THE
DOUBLE-NEGATIVES OR IN ANY OF
THE SINGLE POSITIVE.
SO BRD4 SELECTIVELY REGULATING
MYC IN THE IS P'S.
-- IN THE ISP'S.
FROM THESE STUDIES, WHAT WE
CONCLUDE IS THAT THE ISP
POPULATION IS UNIQUELY DEPENDENT
ON BRD4.
IN THE EARLY DN STAGES,
PROLIFERATION AND
DIFFERENTIATION OF BRD4
INDEPENDENT BASED ON THE RNA
PROFILING AND ALSO OTHER DATA
THAT I HAVEN'T SHOWN YOU, WE
KNOW THAT ONCE CELLS BECOME DP
AND BEYOND, THEY NOW BECOME BRD4
INDEPENDENT AND IT'S ONLY IN
THIS ISP STAGE THAT THE CELLS
ARE BRD4 DEPENDENT FOR BOTH CELL
CYCLE AND DIFFERENTIATION.
SO WE FOUND THAT TO BE A VERY
INTERESTING OBSERVATION, QUITE
SURPRISING, BUT WE WERE ALSO
PUZZLED BY THE FACT THAT BRD4 IS
NOW EXPRESSED IN ALL OF THE CELL
TYPES EVEN THOUGH WE DON'T SEE A
DEPENDENCE AT LEAST AS ASSESSED
BY RNA PROFILING.
SO WE WONDERED, IS IT POSSIBLE
THAT BRD4 IS REGULATING
ALTERNATIVE SPLICING IN THESE
THYMOCYTE POPULATIONS?
WHERE YOU WOULDN'T NECESSARILY
SEE A CHANGE IN THE OVERALL RNA
PROFILE, BUT YOU MIGHT SEE
CHANGES IN SPLICING.
SO WE WENT BACK AND REANALYZED
OUR DATA TO ASK THAT SPECIFIC
QUESTION AND IN FACT OF WE FIND
THAT BRD4 REGULATES ALTERNATIVE
SPLICING IN ALL LIKE SIGHT
SUBSETS AND WHAT THE PIE CHART
IS SHOWING YOU HERE IS THE
COMPARISON OF THE SPLICING
PATTERNS IN THE WILD-TYPE AND
BRD4 KNOCK-OUT ASKING WHAT
FRACTION DIFFER.
AND YOU CAN SEE IT'S A VERY
LARGE FRACTION, IT'S HIGHEST IN
ISP'S, MAYBE NOT SURPRISING
THERE BECAUSE -- YOU HAVE LARGE
SCALE DIFFERENCE IN THE PATTERNS
OF ALTERNATIVE SPLICING, WHICH
NOW ARE GOING TO BE REFLECTED IN
DIFFERENCES IN THE PROTEIN
PROFILE OF THOSE CELLS.
IN FACT, WHEN WE LOOK TO SEE
WHAT THE GENES MOST AFFECTED BY
ALTERNATIVE SPLICING IN BRD4
DEFICIENT THYMOCYTES H MANY OF
THOSE GENES AND THIS IS ONLY A
PARTIAL LIST ARE SOMEHOW RELATED
WITH IMMUNE RESPONSES.
EVERYTHING I'VE SHOWN YOU UP
UNTIL NOW IS BASED ON ANALYSIS
OF RNA SEQ DATA AND NEEDS TO BE
VALIDATED EXPERIMENTALLY.
SO I'M GOING TO SHOW YOU TWO
EXPERIMENTAL VALIDATIONS FOR CD
4R5 AND FOR THE COMMON GAMMA
CHAIN ALTHOUGH WE'VE VALIDATED
ALL OF THESE IN OTHER
EXPERIMENTS.
SO LET'S LOOK FIRST AT THE
ALTERNATIVE SPLICING OF CD45.
IT'S FAIRLY STRAIGHTFORWARD.
XR5 IS ALTERNATIVELY SPLICED
SUCH THAT IN THE ABSENCE OF BRD4
YOU HAVE GREATER RETENTION OF
AXON 5 AND WHAT THAT RESULTS IN
IS A LARGER TRANSCRIPT AND WE
CAN IDENTIFY IT BY SELECTIVE
RT PCR AND WOULD GENERATE AN
AMPLICON OF AROUND 265 BASE
PAIRS WHEN THE EXON IS EXCLUDED.
ININCLUDED, ONLY 118 BASE PAIRS
AND LOOKING AT THOSE TWO
PRODUCTS WE CAN SEE IF THERE ARE
CHANGES IN THE RATIO IS AS A
RESULT OF DELETING BRD4 AND IN
FACT THAT'S EXACT WHAT WILL WE
SEE.
THERE'S ABOUT A ONE TO ONE RATIO
IN THE WILD-TYPE BUT WHEN YOU
KNOCK OUT BRD4 YOU HAVE
INCREASED RETENTION OF EXON5
WHICH IS GOING TO LEAD YOU TO A
DIFFERENT PRODUCT THAN YOU GET
IN THE ABSENCE OF EXON5.
THEN YOU CAN LOOK AT THE COMMON
GAMMA CHAIN WHICH IS MUCH MORE
COMPLICATED BUT POSSIBLY MORE
INTERESTING EXAMPLE.
IN THIS CASE, THE COMMON GAMA
CHAIN UNDERGOES A LOT OF
DIFFERENT ALTERNATIVE SPLICING,
BUT THE ONE WE'RE PARTICULARLY
GOING ON LOOK AT IS EXON5 WHICH
SEEMS TO BE THE MOST AFFECTED BY
BRD4 DELETION, AND IN THIS CASE,
WHEN YOU DELETE BRD4 THE
PREDICTION IS YOU'LL GET
INCREASED EXON SKIPPING AND THE
REASON THAT'S PARTICULARLY
INTERESTING IS THAT EXON
SKIPPING INTRODUCES TGA STOP
CODON INTO THE PROTEIN SUCH THAT
YOU DON'T MAKE ANY COMMON GAMA
CHAIN. SO LET'S SEE WHAT
HAPPENS EXPERIMENTALLY WHEN WE
LOOK AT THE PARTICULAR
TRANSCRIPTS WE'RE LOOKING AT ARE
THOSE THAT HAVE SKIPPED EXON5
AND YOU CAN SEE THEM HERE,
LOOKING AT THE DIFFERENT STAGES,
KNOCK-OUT VERSUS WILD-TYPE, YOU
CAN SEE DISCRETE DIFFERENCES.
IT'S EASIER TO SEE IN THE
QUANTITATION WHERE WE'VE
CALCULATED 9 PERCENT OF EXON
SKIPPING ACROSS THE DIFFERENT
THYMOCYTE POPULATIONS, AND WHAT
YOU CAN SEE IS PRETTY EXTENSIVE
EXON SKIPPING IN THE
DOUBLE-NEGATIVES AND ISP'S IN
THE H WILD-TYPE THAT DROPS PRE
PRECIPITOUSLY WHEN CELLS BECOME
DP AND FURTHER DIFFERENTIATE.
IN CONTRAST, ALTHOUGH WE DO SEE
EXON SKIPPING IN THE KNOCK-OUT,
IT STAYS PRETTY CONSTANT, IT
DIMINISHES A LITTLE BUT IN FACT
YOU CAN SEE IT'S GREATER IN THE
DP'S AND IN THE SINGLE POSITIVES
IN THE KNOCK-OUT THAN IN THE
WILD-TYPE.
SO PUTTING ALL THIS TOGETHER, WE
CAN CONCLUDE THAT
ALTHOUGHTIVE -- THAT ALTERNATIVE
SPLICING IS REGULATED BY BRD4 IN
ALL THYMOCYTE SUBSETS LEADING
EITHER TO A LOSS OF PROTEIN OR A
DIVERSE SET OF PROTEIN PRODUCTS
IN THE DIFFERENT CONDITIONS.
OKAY.
SO IN THE LAST FEW MINIMUM
ITS -- SO IN THE LAST FEW
MINUTES I WOULD LIKE TO TURN TO
THE QUESTION OF WHAT IS THE ROLE
OF THE THYMIC EPITHELIUM IN THE
DEVELOPMENT OF THE THYMUS?
SO THYMIC DEVELOPMENT VALLEY A
REFLECTION OF THE CROSS-TALK
BETWEEN THYMOCYTES AND THYMIC
EPITHELIUM, SO WE WANTED TO KNOW
WHAT ROLE DOES BRD4 PLAY WHEN WE
DELETE OR IN THE THYMIC
EPITHELIUM WHAT HAPPENS WHEN YOU
DELETE IT.
SO AS WAS MENTIONED THIS
MORNING, THE TWO MAJOR THYMIC
EPITHELIAL SUB SUBSETS ARE THE
CT HE C'S WHICH REGULATE
POSITIVE SELECTION AND MTEC'S
WHICH REGULATE NEGATIVE
SELECTION, THE TWO CELL
POPULATIONS AS WE'VE HEARD HAVE
A VERY DIFFERENT EXPRESSION
PROFILE BUT WHAT THEY DO HAVE IN
COMMON IS THE EXPRESSION OF FOX
N1 THE TRANSCRIPTION FACTOR, SO
WE CAN DELETE BRD4 FROM THYMIC
EPITHELIAL CELLS WITH FOX N1 AND
CRE.
WE DO THAT AND SEE A DRAMATIC
LOSS IN CELLULARITY IN THE
TEC'S, YOU CAN SEE HERE IS THE
WILD-TYPE TE C'S, KNOCK-OUT
TEC'S, WHAT'S PARTICULARLY
INTERESTING IS WE'RE SELECT TISM
LOSING MTEC'S, THE TOTAL NUMBER
OF CT HE C'S DOESN'T CHANGE AND
YOU CAN SEE CHANGES IN THE
FREQUENCY THAT REFLECT THAT.
WE CAN NOW DRILL DOWN AND ASK
WHAT THOOPPED THE SINGLE CELL
LEVEL AND WHAT WE FIND WHEN WE
DO SINGLE CELL SEQUENCE SG THAT
WE HAVE FOUR CLUSTERS OF TEC'S,
THREE MTEC'S AND ONE CTEC, AND
THE THYMOCYTE POPULATION CD4 CD8
ARE COMPLETELY DISTINCT.
WHEN WE LOOK FURTHER AT THE CT
HE C'S WE CAN ASK WHAT HAPPENS
WHEN WE KNOCK OUT BRD4 AND WHAT
WE SEE IS QUITE DRAMATIC,
THERE'S A HUGE LOSS IN THOSE
THREE MTEC POPULATIONS AND
VIRTUALLY NO LOSS IN THE CTECS,
THAT'S QUANTITATED HERE.
SO IT REALLY LOOK AS THOUGH BRD4
SELECTIVELY AFFECTING MTEC
POPULATION.
WE CAN VISUALIZE THAT, INCLUDING
HISTOCHEMISTRY IF WE LOOK AT
AIRE EXPRESSION IN THE WILD-TYPE
WE SEE VERY NICE AIRE
EXPRESSION, MC HE T'S RUA1 IS
ALSO SHOWING VERY NICE STAINING
IN THE WILD-TYPE, YOU LOSE IT IN
THE KNOCK-OUT, IN CONTRAST BETA
5T MR FOR CTEC'S DOESN'T
INCREASE, IF ANYTHING WE MAY SEE
A RELATIVE INCREASE.
THAT'S ALSO REFLECT UNDERSTAND
IN THE SINGLE CELL EXPRESSION
LEVELS OF THE DIFFERENT MARKERS,
SO IF WE LOOK AT MTEC MARKERS IN
CLUSTER ZERO AND CLUSTER 4, WE
SEE AN ACUTE LOSS OF AIRE AND
RANK, IN THE ABSENCE OF BRD4
INDICATING THAT BRD4 IS
REGULATING EXPRESSION OF AIRE
AND RANK BUT WE DON'T SEE THOSE
CHANGES IN THE CTEC.
NOW IN MY LAST SLIDE, STANLEY,
WE'RE GOING TO ASK WHAT HAPPENS
AT THE LEVEL OF THE THYMOCYTES
DISRKS THE LOSS OF BIRD -- DOES
THE LOSS OF BRD4 IN THE T HE C'S
AFFECT THYMOCYTE
DIFFERENTIATION, AND THE ANSWER
IS YES.
WE DON'T SEE ANY CHANGE IN THE
DOUBLE-NEGATIVES OR THE ISP'S
BUT WE SEE A SIGNIFICANT
ACCUMULATION OF DOUBLE POSITIVES
AND THEN A SUBSEQUENT LOSS IN
THE SINGLE POSITIVE CD4 CD8
SUBSETS AS WELL AS IN T REGS
ALLOWING US TO INITIALLY
CONCLUDE THAT DELETION OF BRD4
IN C TEST CS BLOCKS THYMIC
SELECTION OF DOUBLE POSITIVE
THYMOCYTES, ALTHOUGH REMEMBER WE
SEE NO DIFFERENCE IN THE NUMBER
OF CT HE C'S, THERE APPEARS TO
BE A FUNCTIONAL DEFECT.
SO THE CONCLUSION FROM THIS
SECTION IS THAT MTEC DEVELOPMENT
DEPENDS ON BRD4 EXPRESSION BUT
POSITIVE SELECTION OF DOUBLE
POSITIVE THYMOCYTES DEPENDS ON
BRD4 EXPRESSION IN TEST C'S.
AND WE'VE HEARD ALL OF THIS, BUT
I DO TO WANT TAKE ONE MINUTE TO
GIVE A SPECIAL THANKS ON ALL THE
PEOPLE WHO WORKED ON THIS
PROJECT IN MY LAB, JAY MU, ANG I
GA --
IT ANNE, SHEETAL, DEVAIAH, AND
OUR COLLABORATORS.
THANK YOU.
[APPLAUSE]
>> HI.
I HAD A QUICK QUESTION.
WHEN YOU LOOK AT JUST H & E
HISTOLOGY DO YOU STILL HAVE
MEDULLARY ISLANDS OR IS THERE
JUST KNOCK-OUT --
>> NO.
>> THERE'S NO MEDULLARY ISLANDS
AT ALL?
>> RESIDUAL AT BEST.
>> THE RESIDUAL?
FOR YOUR SINGLE CELL, HAVE YOU
LOOKED AT THOSE MTEC CLUSTERS
AND IT SEEMED LIKE IT WAS
EQUALLY REDUCED FOR ALL OF THEM?
WERE THERE SPECIFIC CLUSTERS OF
MTEC?
OR --
>> SO THESE ARE REALLY NEW DATA,
SWREANT LOOKED AT THEM IN GREAT
DETAIL, BUT OVERALL, THE
REDUCTION SEEMS TO BE ABOUT
EQUIVALENT IN ALL THE THREE
SUBTYPES.
>> VERY INTERESTING.
THANK YOU.
>> OR SUB CLUSTERS.
>> CAN YOU HEAR ME?
OKAY.
SO I HAVE TWO QUESTIONS TO THE
THYMOCYTES, FIRST DO YOU SEE IF
THERE IS ANY CHANGE IN LIKE
SPLICING OF MACON?
>> NO.
EVERYTHING -- THE MYC GENE IS
NOT TRANSCRIBED.
>> OKAY.
>> SO NOTHING IS THERE.
>> NOTHING, OKAY.
>> ISP'S.
THE OTHERS ARE EXPRESSED
NORMALLY.
>> OKAY.
AND THEN DID YOU CHECK FOR HAD
THE I7 SUB EXPRESSION?
I KNOW THE COMMON GAMA CHAIN
WOULD AFFECT IT.
DO YOU THINK THAT THIS IS THE
REASON WHY YOU DON'T MOVE ON
INTO THE DIFFERENTIATION?
>> SO WE HAVEN'T LOOKED BEYOND
THE COMMON GAMA CHAIN IN TERMS
OF SPLICING.
SO YEAH, I DON'T KNOW.
GOOD QUESTION THOUGH.
THANK YOU.
>> HEY, DINAH, THAT WAS A GREAT
STORY.
MY QUESTION IS ACTUALLY A
CONTINUATION OF ROXANE'S.
SO YOU CAN ALWAYS GO ONE STEP
FURTHER.
YOU SHOWED THAT YOU HAD MUCH
MORE ALTERNATIVE SPLICING IN
ISP'S AS COMPARED TO OTHER
SUBSETS.
GIVEN SOME OF THE OTHER PEOPLE'S
WORK HERE, THE ISP'S ARE THE
MOST METABOLICALLY ACTIVE THYME
SIGHTED SUBSETS.
I WAS WONDERING IS IT BECAUSE
THAT THAT THE ISP'S HAVE MORE
ALTERNATIVE SPLICING?
WHAT IS YOUR HYPOTHESIS FOR WHY
YOU SEE MORE ALTERNATIVE SPLICE
NG ISP'S?
ONE THING I WAS TALKING ABOUT
WAS METABOLISM AND THE OTHER IS
IS THE DIVERSITY OF TRANSCRIPTS
IN AN ISP HIGHER THAN THE
DIVERSITY OF TRIPTSZ IN OTHER
SUBSETS?
-- TRANSCRIPTS IN OTHER SUBSETS?
>> LET ME TRY TO ANSWER ALL
PARTS OF THE QUESTION.
WE LOOKED AT GLYCOLYSIS AND IN
THE ABSENCE OF BRD4 YOU HAVE
EXTRAORDINARILY SUPPRESSED
GLYCOLYTIC FUNCTIONS.
IN TERMS OF ALTERNATIVE
SPLICING, THE TRIVIAL ANSWER IS
THAT THE RNA PROFILES OF THE
WILD-TYPE AND BRD4 ARE SO
DIFFERENT THAT ALMOST YOU HAVE
TO HAVE DIFFERENCES IN
ALTERNATIVE SPLICING BECAUSE YOU
HAVE DIFFERENT TRANSCRIPTS THAT
YOU'RE STARTING WITH.
THE TRANSCRIPTS OF THE ISP'S
COMPARED TO THE SINGLE POSITIVES
OR THE DOUBLE-NEGATIVES, THE
ALTERNATIVE SPLICING PATTERNS DO
NOT SIGNIFICANTLY OVERLAP.
THERE ARE IT HUGE GENES THAT
OVERLAP BUT LI AND LARGE THEY
DON'T.
>> GLYCOLYSIS FOR EXAMPLE, THE
SPLICING FOR RA VERSUS RO IS
CLEARLY ASSOCIATED WITH THE
ACTIVATION OF THOSE CELLS.
>> RIGHT Z SO IT MIGHT
ACTUALLY --
>> YEAH.
SO WE'RE SEEING THAT ALTERNATIVE
SPLICING IN ALL OF THE DIFFERENT
SUBSETS FOR TR CD45 -- SORRY,
WE'RE ONLY SEEING IT IN CD4'S
FOR CD45.
YEAH.
>> DO YOU THINK HOW THE BRD4
LIKE REGULATES THE SPLICING
INTERACTS WITH ANY SPLICEOSOME
COMPONENTS OR --
>> DIDN'T HAVE TIME TO SHOW YOU
THE DATA, BUT BRD4 ACTUALLY
PHYSICALLY INTERACTS WITH
SPLICING FACTORS AND IT APPEARS
TO BE A REGULATORY FACTOR OF
SPLICING.
>> DO YOU KNOW WHICH STEPS, LIKE
THE COMPLEX B OR C OR ANY STEPS
OF THE SPLICEOSOME OR IT'S JUST
LIKE --
>> SO ACTUALLY THE U1 RFP IT
INTERACTS WITH FUS, SO IT'S
INTERACTING WITH THE REGULATORY
FACTORS AS WELL AS WITH THE CORE
SPLICEOSOMAL SUBUNITS.
>> ANOTHER QUICK QUESTION IS
THAT YOU MENTIONED THE -- WHAT
ABOUT OTHER TYPES OF SPLICING
EVENTS -- HAVE YOU EVER CHECKED
THOSE ZOOR WE'VE ONLY LOOKED AT
THE ALTERNATIVE.
>> WE'VE ONLY LOOKED AT THE
ALTERNATIVE -- IF I UNDERSTOOD
YOUR QUESTION CORRECTLY, SWREANT
LOOKED AT OTHER KINDS OF
SPLICING.
>> THANK YOU.
>> ONE MORE QUICK QUESTION.
>> HEY, CARROLL.
>> I WAS JUST WONDERING, HOW
MUCH OF THESE ACTIVITIES FOR
YOUR LOSS OF BRD4 WOULD BE
RESCUED BY MYC AND THE OVERLAP?
>> YEAH, YEAH.
WE'VE TALKED ABOUT DOING THOSE
EXPERIMENTS BUT WE HAVEN'T DONE
THEM.
I THINK SOME OF THAT MIGHT BE
RESCUED BUT NOT ALL OF IT
BECAUSE OTHER PATHWAY RS AFFECT
AS WELL.
IN TERM OF THE SPLICING REMEMBER
MYC ISN'T AFFECTED IN ANY OF THE
OTHER SUBSETS.
>> I KNOW MYC CAN AFFECT
SPLICING TOO, SO LOSS OF MYC CA-
>> IN THE ISP'S, YEAH, BUT THOSE
ARE THE ONLY CELL TYPES WHERE
YOU SEE AN EFFECT.
>> YEAH.
>> THANK YOU.
[APPLAUSE]
>> OUR NEXT SPEAKER IS ROXANE
TUSSIWAND AND SHE WILL TALK
ABOUT NOTCH1 IN T CELL
DEVELOPMENT.
>> GOOD MORNING, EVERYONE.
SO THIS IS THE WORK OF AN
EXTREMELY TALENTED Ph.D.
STUDENT IN THE LAB DURING HIS
MASTER AND Ph.D.
SO WE'VE HEARD A LOT ABOUT T
CELL DEVELOPMENT, SO I WASN'T
HERE EARLIER, BUT SO NOTCH IS
DEFINITELY LIKE A KEY FACTOR IN
T CELL DEVELOPMENT, SO -- WHICH
WOULD INTERACTED WITH NOTCH ONE
SUPPRESSOR EXPRESSED BY -- THE
NEEDS OF THE ANY CD OR NOTCH
INTRACELLULAR DOMAIN AND WHAT IS
THOUGHT TO HAPPEN IS THAT THE
NOTCH WILL BIND TO RPJ
DISSOMEPLACE THE ARE -- DISPLACE
THE COMPLEX AND LEAD TO
INDUCTION OF TRANSCRIPTION.
SO WHAT WE'RE TRYING TO
UNDERSTAND IS REALLY LIKE HOW
THIS MECHANISM WORKS AND WHAT WE
CAN UNDERSTAND FROM T CELL
DEVELOPMENT.
SO WHAT WE KNOW IS THAT WHAT WAS
RECENTLY SHOWN IS THAT THE NOTCH
SIGNALLING ALREADY STARTED, IS
MULTIPLE PROGENITORS WHICH HAVE
THE CAPACITY TO DEVELOP INTO ALL
SUBSETS ARE WHICH RECEIVE THE
SIGNAL AND IT IS THE SIGNAL
CLEARLY THAT THIS IS THE SIGNAL
WHICH LEADS TO THE BONE MARROW
AND TWHEAN THE PROGENITORS WILL
SOMEHOW REACH THE THYMUS AS
EARLY THYMIC PROGENITORS,
RECEIVING AT THIS POINT AN
ACCIDENT TREEMLY STRONG NOTCH
SIGNALLING THROUGH THE
EXPRESSION OF CTEC'S LIKE
DELTALIKE 4.
IN THIS PROCESS THIS DEPENDENCY
OF NOTCH IS REALLY OCCURRING
THROUGHOUT ALL THIS EARLY
DIFFERENTIATION STAGES, UNTIL
THE T CELL RECEPTOR IS INDUCED,
WHICH THEN LEADS THEN TO THE
FURTHER MATURATION OF T CELLS AS
WE'VE HEARD JUST NOW.
SO WHEN WE LOOK EX-VIVO WE WERE
INTERESTED IN DOUBLE-NEGATIVE
SUBSETS WHICH IS STILL NEGATIVE
FOR CD4 AND CD8 AND THIS IS HOW
IT CAN BE SUBDIVIDED INTO DN1
WHICH IS EXPRESSED, DN2, DN3 AND
DN4 AND THIS IS BASED ON 44 AND
25 EXPRESSION.
SO THIS THINK THAT NOTCH IS
ESSENTIAL WAS SHOWN MANY YEARS
AGO BECAUSE DELETION OF NOTCH
ONE LEADS TO A COMPLETE BLOCK AT
THE DN1 STAGE WITH ECTOPIC BASAL
DEVELOPMENT WITHIN THE THYMUS.
OVERSPREFTION AN ICD LEADS TO T
CELL DEVELOPMENT IN THE BONE
MARROW WHERE NORMALLY THERE IS
NO NOTCH SIGNALLING OR NOT
ENOUGH NOTCH SIGNALLING TO DRIVE
T CELL DEVELOPMENT.
SO IT IS AN INSTRUCTIVE SIGNAL
WHICH SUPPRESSES THE OTHER FATE
AND LEADS TO T CELL COMMITMENT
AND SPECIFICAT I ON.
WE KNOW WITHIN ALL THOSE STAGES
OF T CELL DEVELOPMENT THERE IS A
CERTAIN DEGREE OF MULL
MULTI-POTENCY.
WHEN WE PUT THE CELLS UNDER
APPROPRIATE CONDITION WHICH CAN
GENERATE MACROPHAGES IN K CELLS
AND B CELLS BUT IT'S ONLY P AT
THIS STAGE THAT THE T CELL
SPECIFICA TIRKS ON HAS OCCURRED
SO THE PROGRESS INTO THE T CELL
FATE IS WITH DIENDZ.
NOW, BECAUSE OF THE DEFECT OR
THE BLOCK OF THE DN1 STAGE, WE
WERE WONDERING WHAT HAPPENS AND
HOW IS NOTCH IMPORTANT
THROUGHOUT ALL THOSE STAGES?
AND THIS IS -- TO ADDRESS THIS
QUESTION, WE WORKED A T CELL
CULTURE. SO WE USED NOTCH
LIGAND IS PROGENITORS IN THE
BRNS OF I7 AND STEM CELL FACTOR
AND WE CAN GENERATED A LARGE
NUMBER OF THE N2 CELLS, EXPRESS
24 AND 25, IN THE SUBSET BLOCKED
THIS STAGE, AND SO WHEN WE LOOK
AT EX-VIVO THYMOCYTES, MOST OF
THE CELLS PROGRESS VERY FAST
FROM N1 TO N2 AND WE HAVE MOSTLY
BN3 OR 4 CELLS.
SO WHEN WE PUT THE CELLS, THESE
PRO T CELLS IN VIVO, WE HAVE A
SINGLE WAVE OF T CELL
DEVELOPMENT AND HERE IT IS
SHOWN, CD4 AND CD8, THEY END UP
BEING PRESENT WITHIN THE THYMUS
AND THEN THEY CAN LEAVE THE
THYMUS AND PERFORM T CELL
FUNCTIONS.
SO THE CULTURE CELLS SEEMS TO BE
COMPLETELY FUNCTIONAL.
WHEN WE LOOK AT RNA EXPRESSION
AND COMPARE IT WITH SINGLE CELL
PROFILES OF THYMOCYTES, THEY
APPEAR TO CORRESPOND TO CYCLING
DN2 WHICH IS EXACT WHAT WILL WE
SEE IN CULTURES TOUR, THESE
CELLS ARE MOSTLY DN2 BY PROFILE
AND ACTIVELY PROLIFERATING.
WHEN WE LOOK AT THE MUCH MORE IN
DETAIL INTO THIS RNA SEQ DATA
WHAT WE SEE IS THAT THIS
PROGRESSION OF THE PRO T CELLS
THEY REALLY APPEAR TO BE BETWEEN
THE DN1 AND THE BACK EX-VIVO DN3
CELLS ALLOWING PC1 LINE WHICH
SEEMS TO BE THE DEVELOPMENTAL
PATHWAY WHICH DOES AN ESCAPE OR
IMMATURE CELLS INTO THE DOUBLE
POSITIVE THYMOCYTES.
SO WHAT WE SEE HERE IS REALLY
LIKE THERE IS AN UPREGULATION OF
WHAT THE TYPICAL T CELL
TRANSCRIPTION FACTORS ARE AND
DOWN REGULATION OF THE LINEAGES.
WHEN WE LOOK AT THE PATHWAY WE
REALLY HAVE LSK VERSUS PRO T
CELLS UPREGULATE ALL THE
PATHWAYS YOU WOULD EXPECT
INCLUDING T CELL DIFFERENTIATION
AND THEY ARE PRETTY MUCH
CORRESPONDING TO LSK TO DN2 AND
DN3 SUGGESTING THAT WHAT WE SEE
HAPPENING IF HAVE I TROA
CORRESPONDS MOACIALS TO WHAT WE
HAVE HAPPENING IN VIVO.
NOW, THE FACT THAT WE WERE ABLE
TO GENERATE THIS LARGE AMOUNT OF
CELLS GIVE US THE OPPORTUNITY TO
ADDRESS THE REQUIREMENTS FOR
NOTCH AND WHAT IS NOTCH DOING
BILY CRIP SEC AND MASS SPEC IT
WILL CHIP AS SPECTD AND HIGH C.
CHIP SEC WAS USED -- SEQ WAS
USED USING NOTCH TO CONFIRM ALL
THE NOTCH TARGETS ALSO HAVE THE
RBJ BINDING AND THE HISTOMARKS.
SO THE DATA WAS PRETTY GOOD
BECAUSE MOST OF THE PEAKS
CORRESPONDED TO NOTCH PEAK
RESPONDED TULS OUR RBJ BOOKS
WITH 99% OVERLAP AND THE OTHER
THING WAS THAT MOST OF THEM WERE
REFLECTING HAD ACTIVE CHROMATIN
ARE ACTIVE MARGE R RL BPJ VORKS
THEY CORRESPONDED TO SHIITES
WHERE WE EITHER HAD K27
ASSIMILATION OR K3 MUTATION.
SO AGAIN WHEN WE PLOTTED P, WHAT
WE SAW IS REALLY LIKE MOST OF
THE FLL THIS NRC SITES WERE
BINDING, ENHANCER SITES, RATHER
THAN PROMOTER SITES.
THE ONE WHICH WERE NOT ACTIVE SO
WHICH DID NOT OVERLAP WITH
EITHER HISTONE MARKS, ACTIVE
HISTONE MARKS WERE REALLY
DEPLETED BY PROMOTER SITES,
AGAIN AN ANSWER.
ONE THING WHICH BECAME VERY
OBVIOUS IS WHEN WE LOOKED AT ALL
THE TARGETS BY CHIP SEQ, WHAT WE
OBSERVED WAS THAT THE PATTERN OF
THE TARGETS, WHEN WE PROJECTED
THEM INTO A TIMELINE WAS VERY
DIFFERENT.
SO WE HAD TARGETS WHICH WERE
PROGRESSIVELY UPREGULATED,
UPREGULATED ONLY AT LATER
STAGES, OR TRANSIENTSLY INDUCED,
AS WELL AS DOWN REGULATED.
SO WHAT WAS CLEAR IS THAT THE T
CELL IDENTITY GENE WERE THE ONES
WHICH WERE MOSTLY UPREGULATED
AND MOSTLY UPREGULATED AT LATER
STAGES.
WHEREAS THE GENES, HERE ARE SOME
EXAMPLES, SO TCF ACTIVELY F
ELEVEN AND B AND OTHER COMPLEX
AND CD3.
SO ON THE OTHER SIDE, WHAT WAS
OBVIOUS THAT THE DOWN REGULATED
GENES WERE ASSOCIATED WITH
STEMNESS OR ALTERNATIVE
PATHWAYS, LIKE L1 OR I FORGOT
WHAT I HAVE HERE, SPI1.
SO WHAT WAS SURPRISING IS LIKE
NOTCH IS NORMALLY RELATED TO
INDUCTION OF TRANSCRIPTION AND
NOT REPRESSION OF TRANSCRIPTION.
SO WE WERE QUITE PUZZLED BY THE
FACT THAT WE OBSERVED
REPRESSION.
SO BASED ON OUR DATA, WE DECIDED
TO LOOK AT SUPER HYPHEN HANSERS
BECAUSE WE KNOW THAT SUPER
ENHANCER REGULATE IMAGE, SUPER
ENHANCER WE IDENTIFIED 680 SUPER
ENHANCER AND AGAIN WE SEE THE
SAME PATTERN, WE SEE GENES WHICH
ARE REGULATED BY SUPER ENHANCER
WHICH ARE ASSOCIATED WITH THE T
CELL PROGRAM AND GENES WHICH ARE
DOWN REGULATED WHICH AGAIN ARE
REGULATED BY SUPER ENHANCER
WHICH ARE LINKED TO STEMNESS AND
ALTERNATIVE LINEAGES.
SO WHEN WE LOOKED MORE CAREFULLY
AT THE FACTORS WHAT WE OBSERVE
IS THAT THERE IS A LATE NETWORK
OR TRANSCRIPTIONAL NETWORK WHICH
SEEMS TO BE RELATED TO THE T
CELL PROGRAM, AND THERE IS AN
EARLY NETWORK WHICH SEEMS TO BE
RELATED TOP THE DOWN REGULATION
OF ALTERNATIVE LINEAGES AND
STEMNESS.
AND THIS IS THE ONE WHICH STARTS
TO, SO IT'S EXPRESSED EARLIER,
AND THIS WILL BE DELAYED
NETWORK.
SO WE TOOK ADVANTAGE OF THE THIS
SEQUENCING DATA WHICH WAS
GENERATED BY ELLEN, AND SO WE
PLOTTED ALL THE, LIKE AGAIN N A
PSEUDOTIMELINE FROM PROGENITORS
DOWN TO N3, WE HAVE TARGETS
WHICH ARE DOWN REGULATED AND
TARGETS UPREGULATED IN TERMS OF
OR OPENED UP BY ACCESSIBILITY.
SO WHAT WE HYPOTHESIZED IS THAT
THERE IS SOMEHOW AN EARLY
NETWORK WHICH IS ACCESSIBLE AND
NEEDS TO BE LOCKED, THERE IS
NETWORKS FOR CLUSTER ONE WHICH
REMAINS OPEN ALL THE TIME, WHICH
IS LIKELY LINKED TO GENES WHICH
ARE REQUIRED ALL THE TIME, AND
HAD THERE ARE SITES OR TARGETS
WHICH TBLONG THE LATE NETWORK
WHICH ARE INCLUDED AT THESE
LATER POINTS AND THEN ARE OPENED
UP.
AND WHAT WAS SURPRISING IS WHEN
WE LOOKED AT WHAT ARE THE
MOTIVES FOR WHEN WE PERFORMED
THE MOTIVE ANALYSIS, IT'S REALLY
LIKE WE HAVE EDS FACTORS WHICH
ARE LINKED TO THEY RECALL
NETWORK OR CLUSTER TWO WHICH
GETS DOWN REGULATED AND WE HAVE
TCF AND START TARGETS WHICH ARE
RELATED TO THIS LATE NETWORK
WHICH BECOMES OPEN P AT LATER
TIME POINTS.
LY WE WANTED TO CON NATIVE
AMERICAN THE TARGETS -- WE
WANTED TO CONFIRM THAT THE
TARGETS OF THE TRANSCRIPTION
FACTOR WE IDENTIFIED WERE ALSO
PRESENT OF PART OF THE NOTCH
COMPLEX, WE PERFORMED MASS SPEC,
IT'S NOT HERE, HERE, MASS SPEC
ANALYSIS, AND WE INDEED
IDENTIFIED TCF7 FACTOR, AND WILD
RBPJ FACTORS SUGGESTING THAT EGS
FACTORS WERE LINKED TO THIS
EARLY NETWORK AND DOWN
REGULATION OF TARGETS WHICH ARE
RELATED TO STEM CELLS AND
PROLIFERATION AND TCF IF IN
RANKS GENE OR START GENES WERE
RELATED TO TARGETS THAT WERE
INDUCED UPON DIFFERENTIATION.
TO VALIDATE, AGAIN, BECAUSE WE
HAD THE ADVANTAGE OF LARGE
NUMBER OF CELLS WE PERFORMED A
CAS9 DELETION AND BECAUSE OF THE
RANKS GENES WE KNOW THAT RANKS
FUNCTION, WE THOUGHT ECF MALE
OVERLAPPING FUNCTIONS, WE
DECIDED TO DO EITHER SINGLY OR
DOUBLE KNOCKOUTS AND WE LOOKED
AT THE OUTPUT OF CELLS.
SO SURPRISINGLY, SO THIS IS THE
CONTROL, THIS IS WHAT WE EXPECT
AFTER THREE DAYS OF CULTURE,
LIKE JUST THE DN1 WHICH STARTED
USING DN2 AND THE NOTCH1 CONTROL
WHICH IS BLOCKED AT THE DN1
STAGE, SURPRISINGLY THE DELETION
OF THIS ETS FACTOR LED TO AN
INCREASE INTO P THE -- OR A
FASTER PROGRESSION INTO THIS
DEVELOPMENT.
AM ARE WE DONE?
SO SHOWED -- SO HE WILL -- SO
ELLEN SHOWED BEAUTIFULLY THAT IT
TAKES TIME TO GET THE ENTIRE
PROGRAM READY TO BECOME T CELLS,
AND SINCE TIME IS NOT PRESENT
THIS THOSE CELLS, AND SO LIKE
NOW LIKE I DON'T WANT TO GO,
BECAUSE WE HAVE ONLY ONE MINUTE,
SO THERE IS A LOT OF OTHER GENES
WHICH ARE SUPPOSED TO BE
EXPRESSED WHICH ARE DISTURBED OR
ALTERED IN THEIR EXPRESSION
PROFILES.
ONE THING WHICH WAS STRIKING, WE
PERFORMED INJECTION, INTRATHYMIC
INJECTION USING ULTRASOUND TO
LOOK AT THE CELLS, SO THIS IS
WHAT HAPPENS.
WE INJECT THEM H THE CAS9 AND WE
END UP HAVING DOUBLE POSITIVE
AND DOUBLE-NEGATIVE CELLS.
THE DOUBLE-NEGATIVE REALLY, WE
HAVE VERY FEW CELLS SURVIVING
THIS PROCESS, AND THEY ARE
REALLY DOUBLE-NEGATIVES.
WHEN WE TAKE OUR ETS DOUBLE
KNOCK-OUTS, WE SEE THE SAME
THING, THE CELLS WHICH ARE GUIDE
POSITIVE DO NOT SURVIVE.
NOW AND THIS IS LIKE QUANTIFIED
HERE.
SO WHEN YOU DELETE BOTH GENES,
IT'S NOT THAT THEY GROW INTO
ALTERNATIVE LINEAGES AS THEY
WOULD DO FOR NOTCH1, THEY REALLY
DON'T SURVIVE IN VIVO.
SO NOW WHAT HAPPENS IS THAT THEY
HAVE A DEFECT IN THE
PROLIFERATION, SO THE GENES, SO
THIS INCREASE IN THE PROGRESSION
SEEMS TO BE RELATED TO INCREASED
IN PROLIFERATION.
NOW, WE IDENTIFIED TWO OF THE
GENES WHICH ARE RESPONSIBLE FOR
THIS DEFECT AND THEY ARE REALLY
RELATED TO STEMNESS.
AND THIS IS KLF2 AND KLF4.
NOW, BECAUSE WE DON'T HAVE TIME,
I WILL JUST SKIP TO THIS SLIDE.
WHAT WE SEE LIKE WHEN WE PUT THE
CULTURES, THE PRO T CELLS IN
CULTURE, WE STIMULATE THEM ON
NOTCH AND THEN WE REMOVE NOTCH
FOR FOUR HOURS AND THEN WE LOOK
AT THE CHIP SEQ, IT'S THIS
DIRECT REGULATION OF NOTCH OF
THESE GENES WHICH ARE MEDIATED
THROUGH FREE DISMRG WE SEE HERE
THAT INDEED THE REMOVAL OF NOTCH
FROM NOTCH SIGNALLING LEADS TO
AN INDUCTION OF KLF2 AND ALSO
KLF4.
THERE'S ABOUT 25 FOLD INCREASE
OF THOSE GENES, AS EXPECTED
NOTCH TARGETS LIKE IL2 RECEPTOR
IS DOWN REGULATED.
AND THE ONLY GENE WHICH SEEMS TO
BE REMOVED IS REALLY NOTCH.
SO LBJ REMAINS ATOOCHED THE KF2
GENE AS FLE1 AND RFG, WE BELIEVE
THERE IS A COMPLEX FORMED
BETWEEN NOTCH RBPJ AND FLEET
FACTOR WHICH MEDIATES THE
REPRESSION OF KLF GENES AND
LEADS TO THIS DIFFERENTIATION OR
THIS FAST DIFFERENTIATION, AND
THIS IS, SO THIS IS THE MODEL
WHICH WE THINK IS LIKE, SO UNDER
NORMAL CONDITION, NOTCH RECRUITS
THE EARLY NETWORK, WHICH
INCLUDES ETS FACTORS, AND THIS
LEADS TO THE REPRESSION HAD OF
GENES WHICH ARE LINKED TO
PROLIFERATION AND STEMNESS.
AND THE EARLY T CELL PROGRAM CAN
START AND THIS LEADS TO THE
PROGRESSION.
WHEN WE HAVE LACK OF NOTCH
SIGNALLING, THIS T CELL PROGRAM
CM NEVER OCCUR SO WE HAVE
DIVERSION INTO MYELOID OR WHYLY
ALTERNATIVE LINEAGES.
BUT WHEN WE HAVE THE WSM LAC
FACTOR WE SEE BOTH PROGRAMS ARE
INDUCED, NOTCH IS ACTIVE, THEY
RECEIVE THE SIGNAL, BUT THE
STEMNESS AND ALTERNATIVE
PROGRAMS CAN NOT BE DOWN
REGULATED.
SO CAN SURVIVE IN VITRO WHERE WE
GIVE THEM EVERYTHING THEY NEED,
BUT AS SOON AS WE PUT THEM BACK
IN VIVO, THEY SOMEHOW CRASH
TRANSCRIPTIONALLY AND CANNOT
SURVIVE THIS PHENOMENON.
SO WITH THAT, AND I'M PROBABLY
OVER TIME AND I APOLOGIZE, I
WOULD LIKE TO ACKNOWLEDGE THE
PERSON WHO DID MOST OF THE WORK,
LADEN, WITH THE HELP OF GOLGI
FOR SOME OF THE INFORMATIC
ANALYSIS, PEOPLE IN LAB AND WILL
THERE ARE MORE NOW WHO HAVE
JOINED AND ALL THE OTHER
COLLABORATOR HIGH SCHOOL. -- AND
ALL THE OTHER COLLABORATORS.
I'M HAPPY TO TAKE QUESTIONS.
[APPLAUSE]
>> ROXANE, I THOUGHT YOU STUDIED
DEN DRITD I CAN CELLS.
>> I.
[LAUGHTER]
>> I HAVE A QUESTION ABOUT THE
MPP EXPRESSION.
>> SURE.
>> IS NOTCH EXPRESSION
STOCHASTIC AT THAT POINT?
DELTA LIGAND?
>> IN THE BONE MARROW?
GROO BONE MARROW, TO PUSH IT
MORE TOWARD T CELL AT THAT
POINT?
>> IT'S UNCLEAR TO ME YET.
I THINK IT'S STILL UNCLEAR.
LESS SHOWED THAT THERE IS A VERY
TRANSIENT INDUCTION OF NOTCH
SIGNALLING WHICH HAPPENS IN THE
BONE MARROW.
>> AND --
>> YEAH.
>> IS THAT STOCHASTIC OR
WHETHER, YOU KNOW, IT'S GOING TO
GO B OR --
>> IT MUST BE SOME STROMA WHICH
EXPRESSES IT, WHICH DRIVES IT,
THE DELTA.
SO YEAH, I DON'T KNOW.
YEAH.
>> SO VERY IMPRESSIVE TALK.
TWO QUESTIONS.
FIRST, THERE IS A IF FLY ONE IN
EARLY KNOCK-OUT TORQUES THEY
HAVE ANY DEFECT FRP ON THE
BLOCK?
>> SO LIKE THE PROBLEM IS LIKE
THE TIMING WHEN IT'S DELETED.
SO IN THE ONE, THE MOUSE WHICH
WAS GENERATED WAS A FLOX AND IT
WAS A CD4 SO IT'S TOO LATE.
>> BUT DOESN'T NIRS CK CRE OR --
INTLO YEAH, IT WAS KNOCKED DOWN
EARLIER.
>> VERY SIMILAR TO S1 AND S2,
TWO GENES ON --
>> YEAH.
>> SO S12 IS NECESSARY FOR CD4
DEVELOPMENT AND DOES THAT BLOCK
AT THE DOUBLE-NEGATIVE STAGE,
HAVE YOU LOOKED IN YOUR DOUBLE
DEFICIENT, ONE OR TWO STATE
OR --
>> SO BECAUSE I DIDN'T SHOW IT
BUT WE HAVE DONE THE RNA SEQ TO
TRY TO UNDERSTAND AND THAT'S HOW
WE IDENTIFY THELY FACTORS, SO WE
HAVE ALMOST COMPLETE DELETION OF
ERG BUT WE DON'T HAVE, WE HAVE
HALF PERCENT DELETION OF FLE ONE
SO IT'S A BIPOPULATION, SO WE
WERE WORK WITH BOC MAKES IT VERY
COMPLICATED TO ADDRESS YOUR
QUESTION PRECISELY.
>> SO YOU DEENTD DO IT AS A
COMPENSATORY --
>> WEBLES.
WEBLES BECAUSE LIKE EVERYTHING
WE SEE REQUIRES DELETION OF BOTH
SO I BELIEVE THERE IS SOME SORT
OF OVERLAP.
>> OKAY H.
>> AND PROBABLY, YEAH --
>> BETWEEN ONE AND TWO.
>> YEAH.
>> OKAY.
THANK YOU.
>> THANK YOU.
THANK YOU.
>> SO BEFORE ROXANE FINISHES,
ROXANE, YOU GET TO COME BACK,
BECAUSE I THINK THAT THIS IS P
THE -- ROXANE DESERVES A SPECIAL
AWARD AND WE ARE REALLY GRATEFUL
TO HER BECAUSE FOR THOSE OF YOU
WHO ARE NOT AWARE ACCIDENT SHE
HAD A BABY TWO AND A HALF WEEKS
AGO.
[APPLAUSE]
TWO AND A HALF WEEKS AGO!
[APPLAUSE]
>> WE'RE GOING TO HAVE THE LAST
SPEAKER FOR THIS SESSION, ELLEN
ROTHENBERG FROM CAL TECH, AND
TMS FOR BEING INVITED FROM CAL
TECH, YOU GET AN EXTRA FIVE
MINUTES TO TALK.
>> THANK YOU.
>> WELCOME.
>> THANK YOU SO MUCH.
[APPLAUSE]
I JUST WANT TO ALSO CONGRATULATE
ROXANE BECAUSE SHE DID A
BEAUTIFUL JOB.
SHE HAS MUCH MORE BEAUTIFUL
NOTCH CHIP SEQ DATA THAN WE DID
AND OF NOT BEING TOO TERRIBLE
OVER TIME.
THANK YOU SO MUCH FOR HAVING ME
HERE.
I WANT TO PICK UP ON SOME OF THE
SAME THINGS THAT ROXANE STARTED
TO TALK ABOUT AND REALLY DISCUSS
WHAT HAPPENS IN THE EARLIEST
PARTS OF T CELL DEVELOPMENT.
SO THIS IS JUST TOINT OUT THAT
ALL OF IMMUNOLOGY BEGINS TO
HAPPEN FROM THE TIME THAT T
CELLS BEGIN TO BE EXPRESSED IN T
CELLS WE'RE REALLY INTERESTED IN
THE FROM DEVELOPMENT OF T CELLS
IN STAGES LEADING COMMITMENT TO
BECOME T CELLS BEFORE T CELLS
ARE EVEN LOWED TO BEGIN TO DO
FULL REARRANGEMENTS AND AS
ROXANE JUST TOLD YOU, THIS IS
INTENSELY DEPENDENT ON NOTCH
SIGNALLING BOTH FOR THE
ACQUISITION OF T CELL PROPERTIES
AND FOR THE LOSS OF POTENTIAL
FOR B GRUN LOW SIGHT PREDICT
CELL ILC AND THEN K CELL
DEVELOPMENT.
SO WHAT'S HAPPENING IN THE CELLS
AT THIS TIME SM YOU GOT A SENSE
OF THIS FROM ROXANE, WE WANT TO
SIMPLIFY THIS A LITTLE BIT TO
SAY THERE'S THREE GROUPS OF
TRANSCRIPTION FACTORS IN TERMS
OF DEVELOPMENTAL EXPRESSION
PATTERNS THAT ARE ALL ACTUALLY
ESSENTIAL IN VIVO FOR MAKING T
CELLS, THAT INCLUDES THE OBVIOUS
SUSPECTS THAT ARE UPREGULATED
SPECIFICALLY IN THE THYMUS LIKE
BCL11B WHICH WE'LL TALK ABOUT A
FAIR BIT, GATA3 AND TCF7 WHICH
IS DRAMATICALLY UPREGULATED
EARLY ON AND ALSO DOWN
REGULATION OF STEM AND
PROGENITOR TRANSCRIPTION FACTORS
THAT YOU JUST HEARD INTRODUCED
BY ROXANE AND AT THE SAME TIME A
WHOLE SET OF TRANSCRIPTION
FACTORS INHERITED FROM THE STEM
AND PROGENITOR CELLS AND
CONTINUE TO BE EXPRESSED ALMOST
AT STABLE LEVELS BUT WHICH END
UP HAVING ABSOLUTELY ESSENTIAL
ROLES IN T CELL DEVELOPMENT.
ONE THING I'LL SAY ABOUT THIS
GROUP OF PROGENITOR FACTORS
RIGHT AT THE START IS THAT THEY
HAVE TO BE SILENCED AT LEAST BY
THE TIME OF T LINEAGE COMMITMENT
OR A LITTLE LATER AS YOU CAN SEE
FOR ERG AND IF THEY'RE NOT THEY
CAN ACTUALLY CAUSE A TALL SO THE
RE REPRESSION MECHANISMS HAVE TO
BE VERY, VERY GOOD.
AND THERE ARE ACTUALLY TWO
MOLECULAR LANDMARKS FOR THE
COMMITMENT PROCESS INSIDE THE
CELL.
ONE OF THEM IS A MAJOR LANDMARK
IS THE SILENCING OF PO1 ENCODED
BY THE SPE1 GENE, ITS EXPRESSION
PRETTY WELL COINCIDES WITH
COMMITMENT AND WE'VE BEEN STURD
DID IING THIS FOR QUITE A FEW
YEARS, WE KNOW QUITE A LOT ABOUT
THE DRIVERS OF REPRESSION,
INCLUDE GATA3 AND R YOU NX1
POTENTIALLY ADDITIONAL
MECHANISMS AND KNOW SOMETHING
ABOUT THE SITES INVOLVED BUT
IT'S STAGE SPECIFIC.
THE OTHER IS THE ACTIVATION OF
BCL11B EXPRESSION, WHICH IS
CRUCIAL TRANSCRIPTION FACTOR
WHICH IS GOING TO BE EXPRESSED
BY BASICALLY ALL T CELLS
AFTERWARDS BUT WHICH TAKES A
LONG TIME TO TURN ON IN TERMS OF
ABSOLUTE NUMBERS OF CELL CYCLES
THAT THE CELLS UNDERGO IN THE
THYMUS, AND WE'LL TALK A LITTLE
BIT ABOUT THIS, BUT WE'VE
STUDIED THIS AS WELL, IT'S
DRIVEN BY SPEED FORWARD NETWORK
OF TCF, GATA3, RUNX AND NOTCH
SIGNALS, ALL FOUR ARE NEEDED,
IT'S ALSO DEPENDENT ON A SLOW
CHROMATIN OPENING STEP WHICH IS
QUITE STOCHASTICALLY REGULATED
AND WE'VE DISCUSSED THIS IN THE
PAST.
NOW, BCL11B IS A LOVELY GENE IN
THIS PROCESS, AND BECAUSE OF ITS
SPECIFICITY OF FUNCTION AND THE
CLARITY OF ITS TIMING, WE WERE
ABLE TO MAKE A NONDISRUPTIVE
FLUORESCENT PROTEIN KNOCK ALLELE
FOR IT, WE MADE A YELLOW AND RED
VERSION AND THEY'RE BOTH VERY
USEFUL AND THE POINT IS THAT THE
BCL11B ONSET OF EXPRESSION
MARKED BY THESE REPORTERS
COINCIDES ALMOST PRECISELY AT
THE SINGLE CELL LEVEL WITH WHEN
THE CELLS LOSE THEIR ACCESS TO
THESE ALTERNATIVE LINEAGE FATES
SO WE CAN USE IT AS A ONE MARKER
APPROXIMATION FOR THE COMMITMENT
STATUS AT THE SINGLE CELL LEVEL.
WHEN YOU DO THAT, YOU CAN
ACTUALLY LOOK AT SINGLE CELL
CLONES OF ETP'S, EARLY T CELL
PRECURSOR DN1'S DEVELOPING AND
TRACK THEIR PROGRESSION IN
REALTIME ACROSS DIFFERENT
MILESTONES OF T CELL
DEVELOPMENT, MICROSCOPICALLY BY
LIVE IMAGING OVER A PERIOD OF A
WEEK OR SO.
WHAT YOU CAN SEE IS THAT THE
TIMING IS REALLY, REALLY, REALLY
NOISY.
NOW, WITH A STRONG NOTCH SIGNAL,
THE DESTINATION IS NOT IN DOUBT.
THE NOTCH SIGNALLING IS GOING TO
WHAM THOSE CELLS THROUGH INTO
THE T CELL PROGRAM, BUT THE
ACTUAL TIMING THROUGH WHICH OF
THEIR PASSAGE THROUGH THESE
DIFFERENT MILESTONES IS VERY
DIFFERENT CLONE TO CLONE AND
EVEN WITHIN A CLONE.
SO WE HAVE DIFFERENCES OF UP TO
FOUR DAYS IN THE LENGTH OF TIME
IT TAKES INDIVIDUAL ETP'S TO GO
TO THE DN2A STAGE MARKED BY THE
EXPRESSION ON THE SURFACE OF
CD25 BUT IT TAKES AN ADDITIONAL
ANYWHERE BETWEEN ZERO AND FOUR
DAYS FOR THOSE CELLS TO TURN ON
BCL11B AND THAT'S WITHIN THE
SAME THING, WHICH SAYS THAT IT'S
NOT JUST DIDN'T KNOW THE
STARTING POINT IN THE PIPELINE,
IT MEANS THAT THE PIPELINE
KINETICS THEMSELVES ARE
INTRINSICALLY STOCHASTIC.
WHAT IS ACTUALLY GOING ON?
NOW, I MADE AN OFF HAND COMMENT
ABOUT SOMETHING THAT I'VE TALKED
ABOUT IN THE PAST, BUT NOT
TODAY, ABOUT THE CHROMATIN
CONSTRAINTS ON THE BCL11B LOW
LOCUS AND WE KNOW THAT THE
OPENING OF THAT CHROMATIN IS
NOISY.
SO IT'S POSSIBLE THAT THAT'S
ACTUALLY ONE OF THE MAJOR
CONTRIBUTORS TO THE NOISE IN OUR
ABILITY TO DETECT THE BCL11B
BEING TURNED ON, SURPRISE,
SURPRISE.
BUT THERE COULD BE SOMETHING
ELSE AS WELL, AND WITH THE
ADVENT OF SINGLE CELL RNA SEQ --
IMAGING ALSO IN THE TEMPORALLY
STOCHASTIC WAY OR IF IT'S REALLY
JUST A ONE MARKER ISSUE.
AND THE ALTERNATIVE TO JUST THIS
SINGLE GENE EPIGENETIC BARRIER
IS THAT IN FACT THERE'S
SOMETHING ABOUT THE OVERLAP
BETWEEN THE EXPRESSION PATTERNS
OF THE STEM AND PROGENITOR
TRANSCRIPTION FACTORS AND THE
ONSET OF THE T CELL
TRANSCRIPTION FACTORS WITHIN THE
SAME CELLS WHICH WE'VE ACTUALLY
BEEN ABLE TO VALIDATE ON
MULTIPLEX SINGLE MOLECULE FISH.
WE KNOW THEY'RE OVERLAPPING IN
TIME.
THE QUESTION IS, HOW DO THEY
INTERACT WITH EACH OTHER IN THE
SAME CELLS?
AND ONE WAY TO LOOK AT IT IS
EXACTLY BY ADOPTING THE APPROACH
THAT ROXANE POINTED OUT TO YOU
ALREADY, WHERE YOU CAN ACTUALLY
DELETE SOME OF THESE REGULATORS
AND SEE WHAT EFFECT THAT HAS ON
THE PROGRESSION KINETICS OF T
CELL DEVELOPMENT.
SO FOR EXAMPLE H ONE OF THE
FIRST STEM AND PROGENITOR GENES
TO TURN OFF AS THE CELLS COME
UNDER THE INFLUENCE OF THE NOTCH
SIGNALLING FIELD IN THE THYMUS
IS LMO2, IT'S AN INCREDIBLY
IMPORTANT STEM AND PROGENITOR
CELL TRANSCRIPTION FACTOR.
FLING AM LI HAS DONE VERY GOOD
WORK ON THE LMO ONE COMPLEX
INSIDE GOMENT HEE MATT TOPO
EASES, SHOULD BE AN OLD FRIEND.
LM 12R-8 GOES OFF SO QUICKLY YOU
MIGHT THINK IT'S ONLY
REPRESENTING THE PRESENCE OF
CONTAMINANT CELL IN THE THYMUS
EARLY T CELL POPULATIONS BUT
THAT'S NOT TRUE BECAUSE IN FACT
IF YOU KNOCK OUT LMO2 THE CELLS
SHOOT FORWARD FASTER, AND THIS
IS AN EXPERIMENT BY A NEW GRAD
STUDENT IN THE LAB, SAMANTHA
CHANG USING POLYVINYL ALCOHOL
CULTURE SYSTEMS, SERUM FREE
CULTURE SYSTEM TO EXPAND THE
PROGENITOR SO WE CAN GET A GREAT
BIG WAVE OF SYNCHRONIZED
PROGENITORS AND THEN DOING THE
CAS9 DELETIONS ON THEM AND
TESTING THEIR SPEED OF
DIFFERENTIATION OVER THREE,
FIVE, SEVEN DAYS, AND YOU CAN
SEE THAT WHEN THE EMPTY VECTOR
CONTROLS ARE BASICALLY STILL IN
DN1 ETP STAGE, THE LMO2
KNOCKOUTS SPURT FORWARD, SPURT
FORWARD AND THEN ACTUALLY
ALREADY GOING INTO DN3 WHILE THE
CONTROLS ARE STILL IN DN1.
LMO2 IS ONLY ONE OF THIS GROUP
OF FACTORS.
ANOTHER ONE THAT TURNS OFF AT
ABOUT THE TIME OF COMMITMENT IS
HHEX DESCRENT A HUGE IT -- HHEX
DELETION DOESN'T ARE A HUGE
EFFECT ON DN2 TO DN3 PROGRESSION
JUST USING SURFACE MARKERS, IF
YOU ACTUALLY MONITOR ENDOGENOUS
BCL11B EXPRESSION, EXPRESSION TO
COMMITMENT OF THESE CELLS YOU
CAN SEE THERE'S ACTUALLY QUITE
ROBUST AND HIGHLY REPRODUCES I
BELIEVE UPREGULATION OF BCL11B
AND AT A MUCH EARLIER TIME THAN
IT WOULD NORMALLY GO UP IF YOU
TAKE AWAY HHEX SO WE THINK HHEX
IS ACTUALLY ACTING AS A RECEPTOR
REPRESSOR OF BCL1 <!0> B IN
ADDITION TO NEW YORK STOCK
EXCHANGE CLOSE CHROMATIN AND
THIS IS HOW MUCH DIFFERENT THE
BCL11B GEOMETRIC MEAN
FLUORESCENCE INTENSITIES IS
BETWEEN THE KNOCK-OUT IN RED AND
CONTROL IN GRAY HERE DONE BY A
POST-DOC HERE JIHYUN IRIZARRY.
HEAVY HITTERS IN THIS SYSTEM,
THEY WI THIS THE OPPOSITION
BETWEEN THE ONSET OF THE T CELL
FACTORS LIKE TCF7 AND GATA3 ON
THE ONE HAND AND THE MOST
DOMINANT PROGENITOR FACTOR WHICH
IS SPE1 IN OUR HANDS, AND I SAY
MOST DOMINANT BECAUSE SPE1 IS
REALLY EXPRESSED AND IT'S TBOWND
THE DNA, SOMETHING OVER 30,000
SITES IN THE EARLY -- AND IT'S
TBOWND THE DNA, SOMETHING OVER
30,000 SOWNDERS, IN THE EARLY --
CAN 30,000 SITES, IT ACCOUNTS
FOR A VERY LARGE FRACTION OF THE
OPEN CHROMATIN SITES AS CROSS
THE GENOME, YOU COULD HAVE SEEN
THAT IN SOME OF THE FIGURES THAT
ROXANE SHOWED AS WELL BECAUSE IT
HAS THE SAME MOTIF ALSO
IDENTIFIED AS SPEB AND IT'S
ACTUALLY BINDING THERE AND IT'S
IMPORTANT.
WE'VE SEEN IN REPEATED CASES
THAT PO1 KNOCK QULOWT ALSO
APPEARS -- PU1 KNOCK-OUT ALSO
MAKE THE CELLS APPEAR TO GO
FORWARD LOOKING AT THIS MORE
GLOBALLY AS CROSS THE GENOME
WHEN JO THE PERSON THIS MY LAB
WHO DID A LOT OF SINGLE CELL SEQ
ANALYSES BASICALLY DID A SMALL
SCALE PERTURBED SEQ EXPERIMENT
IN WHICH SOME OF THE THINGS WE
WERE PERTURBING WERE EITHER SPE1
OR TCF7 OR ERG OR SEVERAL OTHER
TRANSCRIPTION FACTORS, AND THESE
WERE ALL HASHTAGS AND THEN
SEQUENCED TOGETHER.
WHAT YOU'RE SEEING HERE IN GRAY
IS THE THE MAN FOLD OF ALL THE
SAMPLES IN THE EXPERIMENT, THE
DARK PURPLE DOTS ACTUALLY SHOW
YOU THE CONTROL CELLS WHICH ARE
SPREAD IN THIS RANGE OF STAGES,
THAT'S THE ASYNCHRONY I WAS
TELLING YOU ABOUT, BECAUSE THE
GENES ALTHOUGH THIS END, THIS
LOWER RIGHT END ARE ACTUALLY ALL
PROGENITOR GENE EXPRESSION
PATTERNS, THE ONES UP AT THIS
END ARE EARLY T CELL ENDS, THE
CONTROLS ARE SPREAD ALL ACROSS
HERE.
IF YOU KNOCK OUGHT TCF7 ENCODING
TCF1 THE CELLS DON'T EVEN GET
PAST THIS ENTRY GATE, THEY
BASICALLY DON'T LOOK LIKE
THEY'VE STARTED THE T CELL
PROGRAM AT ALL.
THIS IS CONSISTENT WITH MANY
OTHERS' WORK INCLUDING AZ
I NASHA'S BUT WITH P IS YOU GET
THE EXACT OPPOSITE PHENOTYPE,
KNOCKING OUT PU1 CELLS
FAST-FORWARD THROUGH THEIR
TRANSCRIPTOME TO THIS EXTREME
ADVANCE END AND SEE THAT PU1 IS
MOVING CELLS ACROSS THE SAME SET
OF STAGES BUT SIGNIFICANTLY
FASTER THAN THE CONTROLS.
SO THAT INFERS, IMPLIES THAT PU1
IS ACTUALLY NORMALLY A BREAK ON
THIS PROCESS.
AND BECAUSE OF THE SINGLE CELL
TRIPTDOMICS YOU CAN ACTUALLY SEE
WHICH GENES INDIVIDUALLY ARE UP
AND DOWN IN RESPONSE TO EACH
PERTUBATION SO WE CAN PUT
TOGETHER A LOT OF NODES IN AN
ACTUAL FUNCTIONALLY DEFINED T
CELL GENE REGULATORY NETWORK.
THIS IS NOT A MOTIF CORRELATED
WITH GENE EXPRESSION.
THIS IS ACTUALLY THESE ARE THE
GENES WHICH ACTUALLY CHANGE
EXPRESSION, BOOM, WHEN YOU TAKE
AWAY THIS TRANSCRIPTION FACTOR.
WHAT I WANT YOU TO TAKE AWAY
FROM THIS IS NOT ANY OF THE
DETAILS, BUT THESE COLORED
REGIONS REPRESENT SIMULTANEOUSLY
OPERATING NETWORKS IN THE SAME
CELLS.
THE BLUE NETWORK IS THE ONE
DRIVEN BY NOTCH DIRECTLY, AND
THAT'S INCLUDING THE FEED
FORWARD CIRCUITRY THAT GIVES YOU
THE T CELL PROGRAM COMING OUT.
TCF7, GATA3, TCF12 ALSO DRIVING
TOWARDS T CELL SPECIFICATION.
IN THIS ORANGE BOX, THIS IS THIS
ATTEMPTED SELF-MAINTAINING
PROGENITOR GENE REGULATORY
NETWORK WITH A LOT OF
CROSS-REGULATORY INTERACTIONS,
EVEN BEYOND THE ONES SHOWN HERE
IN WHICH SPE1 IS A MAJOR DRIVER
OF THE EXPRESSION OF OTHER
FACTORS LIKE LMO2, MEF2C AND
BCL11A TRYING TO HOLD BACK THE
CELLS FROM DIFFER HE BE YAITDING
TOO FAST SO YOU REALLY HAVE THE
CELLS FOR A WHILE ARE BALANCED
BETWEEN THE ACTIVITY OF TWO
DIFFERENT OPPOSING NETWORK ON
THE SAME CELLS WITH A LOT OF
CROSSES-REPRESSIVE INTERACTIONS
AT THE SINGLE GENE LEVEL.
NOW, HERE ARE THESE GENES IN
HERE, WHICH DON'T SEEM TO MAKE
SENSE.
RUNX1 AND 3.
THE REASON THEY DO PLAY A VERY
BIG ROLE, AND IF YOU FOLLOW THE
ARROWS, YOU CAN SEE THAT THEY'RE
BIASED, THEY'RE ACTUALLY HELPING
THE T CELL PROGRAM AND THEY'RE
ANTAGONIZING THE STEM AND
PROGENITOR PROGRAM.
WHAT ARE THEY DOING HERE?
ESSENTIAL A FLAT LEVEL OF
EXPRESSION ACROSS THESE STAGES.
THEY'RE NOT INCREASING WITH T
CELL DEVELOPMENT.
IN FACT, IF YOU WERE DOING
FUNCTIONAL GENOMICS AND YOU WERE
TRYING TO SAY WHICH
TRANSCRIPTION FACTORS ARE LIKELY
TO BE BORING IN THIS PROCESS,
YOU WOULD LOOK AT THIS FLAT
EXPRESSION OF RUNX AND SAY IT'S
OBVIOUSLY NOT DOING ANYTHING
INTERESTING.
BUT IN FACT WHAT IT IS DOING IN
TERMS OF DIRECT REGULATORY
EFFECT IS IT'S A MAJOR REPRESSOR
OF THE STEM AND PROGENITOR
PROGRAM GENES, SEVERAL OF THEM,
AND IT'S A MAJOR POSITIVE
REGULATOR OF EACH OF THESE THREE
MAJOR T CELL PROMOTING
TRANSCRIPTION FACTORS.
SO WHAT IS IT DOING THEN P IN
THE EARLY ETP STAGE WHEN THE
MAIN BUSINESS OF THIS IS TO
EXPRESS THESE PROGENITOR GENES?
WELL, WHAT HAPPENS AND SOME OF
YOU HAVE HEARD ME TALK ABOUT
THIS BEFORE, IS THAT RUNX
BINDING IS VERY, VERY STAGE
SPECIFIC.
AND IN THE CELLS THAT ARE STILL
EXPRESSING A LOT OF PU1, THE
RUNX IS BINDING TO DIFFERENT
GENOMIC SITES THAN IT IS AS THE
CELLS UNDERGO COMMITMENT HERE.
AND YOU CAN SEE IT'S REALLY,
THIS REALLY INVOLVES LARGE
FRACTIONS OF ALL OF THE SITES
THAT RUNX BINDS IN THE GENOME.
AND IF YOU'RE INTERESTED AND YOU
WANT TO LOOK, THIS IS RECENTLY
PUBLISHED WORK, SO YOU CAN
ACTUALLY LOOK UP, IT'S NOT JUST
FOLLOWING TAC ACCESSIBILITY.
IF ANYTHING SH IT'S ANTICIPATING
IT.
THE ATTACK ACCESSIBILITY IS
GENERALLY ENRICHED AT RUNX SITES
AND VICE VERSA BUT RUNX CAN GET
INTO SITES THAT HAVE REMAINED
TOLLINGLY IN TAC INACCESSIBLE OR
IT CAN DELAY GETTING INTO SITES
WHICH ARE JUST TOTALLY OPEN AND
JUST WAITING, IF THE TIME ISN'T
RIGHT.
SO SOMETHING ELSE IS ACTUALLY
CONTROLLING THE CHANGE IN RUNX
OCCUPANCY WHICH CORRELATES VERY,
VERY WELL WITH A SWITCHING OF
WHOLESALE OF RUNX FUNCTIONAL
EFFECTS ON THE CELLS.
SO WHY DO RUNX TBAK TORS BIND
DIFFERENT SITE AT DIFFERENT
STAINDLE?
-- RUNX FACTORS BIND DIFFERENT
SITES AT DIFFERENT STAGES?
RMS ACTUALLY IT HAS A SET OF
ALTERNATIVE INTERACTION PARTNERS
WHICH ARE IN COMPETITION WITH
EACH OTHER.
WE HAD PUBLISHED SOME YEARS AGO
THAT IF YOU TAKE A CELL, A T
LINEAGE CELL THAT'S ALREADY
TURNED OFF PU1, THAT PU1 CAN BE
ADDED BACK AGAIN AND IT WILL
TALL -- AND IT WILL ACTUALLY
CAUSE THE CELL TO LOSE ITS T
LINEAGE COMMITTED STATUS AND TRY
TO MAKE MYELOID CELLS AGAIN AND
THE WAY IT DOES THIS IN PART IS
BY SHUTTING OFF RUNX'S ABILITY
TO SERVE APPEARS THE T CELL
GENES THAT IT WAS SERVICING
BEFORE.
HOW?
BECAUSE THE PU1 THAT YOU ADD
BINDS TO SITES THAT WERE CLOSED
BEFORE HAD, PU1'S KNOWN PIONEER
FAX TORQUES HERE IT IS
PIONEERING, AND WHAT IT DOES IS
IT PULLS RUNX ALONG FOR THE
RIDE.
SO RUNX WAS NOT BINDING TO THESE
SITES BEFORE, YOU ADD PU1, RUNX
GOES THERE.
BUT THE INTERESTING THING TO US
IS THAT RUNX DOES THIS AT THE
EXPENSE OF ITS BINDING TO A
LARGE FRACTION OF ITS PREVIOUS
OCCUPANTS, WHAT DOES THIS
ACTUALLY MEAN?
COULD PU1 BE PUSHING IT OUT OF
THESE SITES?
WELL, NO, IT'S NOT PUSHING
BECAUSE PU1 ISN'T GOING THERE.
PU1 IS PULLING IS BUT IT'S NOT
PUSH IT GO OUT.
SO DOES THIS MEAN THERE'S NOT
ENOUGH RU NX TO GO AROUND?
ONE WAY TO ASK THAT QUESTION
MORE QUANTITATIVELY IS JUST TO
ADD MORE RUNX TO THE AND SEE HOW
THAT CHANGES THE RESULT.
WHEN YOU DO THAT, YOU GET A VERY
SATISFYING-LOOKING RESULT IN
THIS ARTIFICIAL T CELL LINE
SYSTEM WHERE YOU ADD PU1 NOW, IT
STILL GOES TO THE PU1 SITES THE
SAME WAY, BUT NOW IT NO LONGER
IS DEPLETED FROM THE PREVIOUS
SITES.
SO THE QUESTION IS, YOU KNOW,
AND WE KNOW SOMETHING ABOUT THE
PHYSICAL NATURE OF THESE SITES,
WE'VE BEEN VERY -- WE KNOW WHAT
PART IN OTHER WORDS ARE THERE
WHAT, MOTIFS ARE THERE, HOW HIGH
AFFINITY THEY ARE AND SO FORTH.
BUT THIS IS AN ARTIFICIAL
SYSTEM, AND THE QUESTION IS,
DOES THIS REALLY HAVE ANY IN
VIVO RELEVANCE?
JUST TO CLARIFY, WE'RE PROPOSING
THAT IN THE NATURAL STATES WHEN
THERE'S A LOT OF PU1 AROUND, THE
PU1 IS NOT JUST CO-BINDING WITH
THE RUNX BUT THAT IT'S ACTUALLY
SEQUESTERING THE RUNX AT THOSE
SITES AND WHEN THE PU1 GOES DOWN
THAT NOW ALLOWS THE RUNX TO GO
SOMEWHERE ELSE.
IF THAT WERE TRUE, THAT WOULD
MEAN THERE SHOULD BE A
DEVELOPMENTAL CONSEQUENCE TO
ADDING MORE RUNX, NOT TOO MUCH
RUNX BECAUSE TOO MUCH OF
ANYTHING IS BAD, BUT WHAT ABOUT
JUST A LITTLE MORE?
AND WE WERE VERY FORTUNATE TO BE
ABLE TO HAVE VECTORS WHICH
RESPONDED TO THEIR OWN USE
SUPPRESSED RUNX IN A VERY, VERY
MODERATE MEASURED WAY. SO WE
ESSENTIALLY JUST DOUBLED THE
AMOUNT OF RUNX IN THE CELL USING
THESE VECTORS, WHICH IS NOT
REALLY VERY MUCH OF AN
OVEREXPRESSION.
SO WE COULD THEN TEST THIS AT
THE PROTEIN LEVEL, VERIFY IT AND
LOOK AT THE CROSS-REGULATION
WITH OTHER RUNX FAMILY MEMBERS
AND WE COULD SEE THAT THEY
ACTUALLY WENT DOWN WHEN YOU
OVEREXPRESSED RUNX ONE SO YOU
END UP WITH NO MORE THAN ABOUT A
TWOFOLD INCREASE TOTAL.
THEN YOU COULD ASK, WHAT THOOPS
THE CELLS?
WE CAN LOOK IN TERMS OF SINGLY
CELL RNA SEQ, WE CAN ALSO LOOK
IN TERMS LONGER TERM T CELL
DEVELOPMENT.
THIS IS THE SCHEMA OF HOW WE
STUDY THIS, THIS WAS A MARVELOUS
POST-DOC WHO SOME OF YOU KNOW,
BOYOUNG SHIN WHO SET THIS UP AS
BOTH A GAIN EXPLOS OF FUNCTION
OF R YOU NX IN PARALLEL SETS OF
CELLS AT TWO DIFFERENT TIME
POINTS FOR EACH, SO TWO DAYS OR
FOUR DAYS POST ADDITION OF EXTRA
RUNX, THREE DAYS OR SIX DAYS
POST DELETION OF BOTH RUNX1 AND
3 TOGETHER IN THE SAME COHORTS
OF INPUT CELLS THAT WILL WE'RE
GETTING FROM EITHER CAS9 B CELL
2 TRANSGENIC MICE OR JUST B CELL
2 MICE AND ASKING WHAT THOOPS
THEIR DEVELOPMENT AND IF YOU
LOOK AT THE SINGLE CELL RNA SEQ
EFFECTS OF THESE PERTUBATIONS
HERE WHAT I'M SHOWING YOU IS THE
MANIFOLD OF ALL OF THE SAMPLES,
AGAIN, ALL OF THE TIME POINTS,
ALL OF THE GENOTYPES, ONE OF THE
VERY NICE THINGS ABOUT IT IS
THAT IF YOU MAP, YOU MAP ONE IS
ALMOST ALWAYS CELL CYCLE SO
THAT'S REANLTD AN ISSUE HERE.
BUT U MAP TWO AND U MAP THREE
ARE INCREDIBLY INFORMATIVE HERE.
U MAP 2 IS ALMOST PERFECT
REPRESENTATION OF DEVELOPMENTAL
PROGRESSION ALONG THE T CELL
PATHWAY, AND U MAP 3
MAGNIFICENTLY ENOUGH TURNS ON
THE TO BE A REFLECTION OF THE
RUNX GENOTYPE.
SO WE WERE ABLE TO SEPARATE RUNX
KNOCK-OUT FROM THE CONTROLS FROM
THE RUNX OVEREXPRESSION, AND YOU
CAN ACTUALLY SEE THEN VISUALLY
WHAT I'M GOING TO SHOW YOU HERE
CALCULATED FROM THE PSEUDOTIME
USING MONOCAL WHICH IS AT THAT
EVERY TIME POINT RUNX
OVEREXPRESSION MOVES THE CELLS
FASTER THROUGH THE T CELL
PATHWAY.
P AT EVERY TIME POINT THE RUNX
KNOCK-OUT MOVES THEM SLOWER.
IS THIS ACTUALLY REALLY RELEVANT
TO T CELL DEVELOPMENT OR IS IT
REALLY JUST SINGLE CELL RNA SEQ,
YOU KNOW, LABELING OF
TRAJECTORIES AND DOES THIS HAVE
ANYTHING TO DO WITH REAL T CELL
DEVELOPMENT?
WE WERE ABLE TO LOOK AT THIS IN
AN IN VITRO WAY.
RETROVIRAL VECTORS, EMPTY VECTOR
ONE COLOR, THE RUNX
OVEREXPRESSING ANOTHER COLOR
MIXED TOGETHER IN THE SAME
ARTIFICIAL ORGANOID CULTURE
SYSTEM WHICH IS VERY, VERY GOOD
FOR PUSH BEING THE CELLS PAST
THAT DM2 ARREST POINT THAT
ROXANE TOLD YOU ABOUT. SO THEY
GO ALL THE WAY INTO BETA
SELECTION.
WHAT I THINK YOU CAN SEE HERE IS
WITH THE RED DOTS BEING THE
OVEREXPRESSING ONES, YOU CAN SEE
THAT THE RED CELLS, THE
OVEREXPRESSING CELLS AT EACH
TIME POINT GET OUT OF THE
EARLIER STAGE FASTER AND INTO
THE LATER STAGE FASTER, OUT OF
THE EARLY STAGE FASTER, INTO THE
LATER STAGE FASTER ALL THE WAY
THROUGH BETA SELECTION, CELLS
RESPOND TO GO THIS RUNX
OVEREXPRESSION ACCELERATION
EFFECT ARE CELLS CAPABLE OF
GOING ON IN THE T CELL PROGRAM.
I JUST WANT TO SAY SOMETHING
ABOUT THE GENE EXPRESSION
DETAILS OF THIS PHENOTYPE
BECAUSE THEY'RE REALLY
INTERESTING FOR THE MECHANISM.
I ALLUDED TO THE FACT THAT ONE
OF THE THINGS ASILENCES
EXPRETION -- THAT SIGH LENS
EXPRESSION IS GATA3.
IF GATA3 WAS ALREADY THERE WHICH
IT WAS WERE WE SIMPLE JUST
TURNING OFF PU1, IN OTHER WORDS,
WERE WE SIMPLE REPEATING THE PU1
KNOCK-OUT EXPERIMENT AGAIN IN
THESE SPHERMTS?
OR WERE WE DOING SOMETHING NEW
THAT HAD TO DO WITH RUNX'S OWN
EFFECT?
SO TO ANSWER THAT, WHAT WE DID
WAS WE TOOK THE SINGLE CELL RNA
SEQ DATA AND WE BASICALLY SAID,
WE'RE ONLY INTERESTED IN THE
SLICES OF CELLS THAT ARE STILL
EXPRESSING PU1.
SO THESE ARE THE CELLS THAT ALL
HAVE STILL PU1 RNA OVER A
CERTAIN THRESHOLD LEVEL,
COMPARING THE CONTROLS WITH THE
RUNX OVEREXPRESSING.
NOW WE'LL LOOK AT THE OTHER
GENES THAT THOSE CELLS ARE
EXPRESSING.
DO THEY ACTIVATE THE T CELL
PROGRAM BEFORE THEY DOWN
REGULATE THE STEM AND PROGENITOR
GENES, OR DO THEY DO THIS STEM
AND PROGENITOR GENE DOWN
REGULATION FIRST?
AND THE RESULTS CAME OUT
UNBELIEVABLY CLEARLY, WHICH IS
THAT IF YOU PICK ON THE CELLS
THAT INDUSTRIAL PU1 THEY HAVE
ABSOLUTELY NO SIGNIFICANT
DIFFERENCE IN THEIR EXPRESSION
OF ANY OF THE STEM AND
PROGENITOR FACTORS THAT WE
LOOKED AT.
WITH AR WITHOUT THE EXTRA RUNX.
BUT THOSE SAME INDIVIDUAL CELLS
HAVE GREATLY UPREGULATED THEIR
EXPRESSION OF TCF7, LCK, GATA3,
TCF12 AND ONE THING VERY AMAZE
SG THEY UPREGULATE SOME ILC
GENES AND HAVE A TINY BIT OF
BCL11B VERY PREMATURELY UP
REGULATED, WHERE WEERVE VERY
INTERESTED, AGAIN, I'M SURE IF
WE HAVE ENOUGH TIME SAY THIS B
THE SITES THAT RUNX IS USING TO
MEDIATE THESE EFFECTS, IF ANY OF
YOU ARE INTERESTED, YOU CAN ASK
ME IN THE QUESTIONS, SO WE'VE
REALLY LOOKED AT WHAT ARE THE
SITES THAT ARE OCCUPIED IN THESE
EXACT EXPERIMENTAL CONDITIONS?
WE'VE LOOKED AT THE SITES WITH
REGARD TO THEIR AFFINITY, THEIR
MOTIF DENSITY, AND THE COFACTORS
THAT BIND THERE AND THERE ARE
SOME VERY INTERESTING PATTERNS
HERE THAT YOU'RE WELCOME TO ASK
ME ABOUT.
THE POINT IS THAT THESE ARE NOT
UNFORTUNATELY PURE RUNX TARGETS
IN THE SENSE THAT MOST
TRANSCRIPTION FACTORS WORK AS
MEMBERS OF ENSEMBLES ON
INDIVIDUAL ENHANCERS AND
UNFORTUNATELY A LOT OF THE EE
NEACTS WE'RE SEEING WITH RUNX
ARE COMPOSITES OF THE EFFECTS OF
RUNX ITSELF BINDING TO THESE NEW
SITES AND THE EFFECTS OF THE
OTHER TRANSCRIPTION FACTORS THAT
I JUST TOLD YOU THEY UPREGULATE.
OKAY.
SO STANLEY WANTS ME TO STOP.
SO FOR EXAMPLE, THE BCL11B
UPREGULATION DEPENDS ALSO ON THE
TCF7 THAT'S ALSO UP REGULATED.
SO I JUST WANT YOU TONED WITH
THIS PICTURE OF WHAT'S ACTUALLY
HAPPENING IN THESE CELLS.
THAT DURING ENTRY, TOWARDS THE T
CELL PROGRAM UNDER THE INFLUENCE
OF NOTCH SIGNALLING, THE CELLS
ARE ENTERING THIS PRO T CELL
STAGE WHICH IS VERY MUCH THE
STAGE, VERY CLOSE TO THE STAGE
THAT ROXANE WAS ABLE TO KEEP
SUSTAINED IN HER CULTURE SYSTEM.
BUT THAT THESE CELLS ARE REALLY
BALANCED BETWEEN POSSIBLY TRYING
TO GO BACK TO STEM AND
PROGENITOR, THEY HAVEN'T TOTALLY
LOST ALL OF THAT YET.
POSSIBLY GOING OFF SIDEWAYS TO
MYELOID AND DC WHICH IS ONE OF
THEIR FAVORITE OPTIONS IF YOU
TAKE AWAY THE NOTCH SIGNALLING,
OR GOING TO LYMPHOID CELL AND NK
AND THIS IS BASICALLY AN
EXPRESSION OF THE COMPLEX GENE
REGULATORY NETWORK RELATIONSHIPS
BETWEEN P THE DIFFERENT
REGULATORS THAT ARE
COINCIDENTALLY AT WAR IN THE
SAME CELLS.
OUR PICTURE OF T CELL
DEVELOPMENT THROUGH THESE EARLY
STAGES THEN COMES DOWN TO THESE
MAJOR POINTS.
THAT THE ENTRY INTO THE T CELL
PROGRAM DRIVEN BY NOTCH IS
REALLY A SLOW SUBVERSION OF AN
ESTABLISHED MULTITI POE NENLT
PROGENITOR REGULATORY STATE AND
THIS IS WHAT THE NOTCH
SIGNALLING CASCADE CASCADE IS
BUILT ON BUT THAT'S WHAT THE
NOTCH SIGNALLING CASCADE
UNDERMINES AND WE GET
ACQUISITION OF NEW
FUNCTIONALITIES OF TRANSCRIPTION
FACTORS LIKE RUNX AS A RESULT
BECAUSE OF THE CUMULATIVE
EXPRESSION OF OTHER PARTNERS,
WHICH HAS A NUMBER OF BEAUTIFUL
PIECES OF MOLECULAR BIOLOGY THAT
YOU CAN STUDY ABOUT HOW THE SITE
CHOICE WORKS AND HOW THAT
INTERFACES WITH THE EPIGENOME,
AND JUST THAT THIS WILL
ULTIMATELY NARROW OF THE SCOPE
OF THE RUNX ACTIVATED GENES TO
FAVOR T OVER INNAITD CELL
DEVELOPMENT ONCE YOU GET
DISAMBIGUATING FACTORS LIKE BCL
11B.
I WANT TO THANK IT MY LAB, THIS
IS MY LAB ABOUT A YEAR AK, THESE
ARE MARVELOUS PEOPLE, THEY
WORKED VERY, VERY HARD DURING
THE PANDEMIC EVEN THOUGH IT WAS
TOUGH TIMES FOR ALL.
AND I JUST WANT TO THANK THEM
AND I WANT TO THANK YOU FOR YOUR
ATTENTION.
AND IF STANLEY WILL BE KIND
ENOUGH TO LET ANY OF YOU ASK
QUESTIONS, EITHER INSIDE OR
OUTSIDE THE SESSION, I'M
DELIGHTED.
[APPLAUSE]
>> ELLEN, THAT WAS LOVELY.
CAN YOU HEAR ME?
>> YES, I CAN.
>> SO REALLY QUICK QUESTION.
I'LL ASK YOU ABOUT THIS AFTER,
BUT IN THE EXPERIMENTS WHERE YOU
OVEREXPRESSED RUNX SO MAYBE TAKE
AWAYLY PL ONE --
>> CAN YOU COME A LITTLE CLOSER?
YOU'RE TOO FAR AWAY FROM THE
MICROPHONE.
>> THE EXPERIMENTS WHERE THE TWO
ALLELES OF BCL11B ASYNC NIS
NUSLY EXPRESSED IN THE SAME
CELL, IS ITLESS --
>> THIS IS A BEAUTIFUL
EXPERIMENT ASKED ABOUT, I WANT
DESPERATELY TO DO THAT
EXPERIMENT H IT JUST HASN'T BEEN
DONE YET.
BUT IT IS A GREAT EXPERIMENT.
WE'RE A LITTLE SHORT OF
FLUORESCE ENTD PROTEIN COLORS,
BUT WE SHOULD BE ABLE TO DO IT
ANYWAY.
SO I VERY MUCH -- SO THE
QUESTION IS, DOES THE RUNX
OVEREXPRESSION COLLAPSE THE
EPIGENETIC BARRIER?
AND WE WOULD PREDICT THAT THAT
MIGHT BE THE CASE, AND I WOULD
LOVE TO DO THAT EXPERIMENT
SOONER RATHER THAN LATER.
ZOOS SO YOU SAID THE UF1 IS
MOSTLY PROLIFERATION GENES, SO
HOW MUCH IS THE CELL CYCLE?
I MEAN, YOU SHOWED IT WITH PU1 A
LONG TIME AGO, TRANSCRIPTION
FACTORS, LIKE HOW OFTEN IS THIS
INFLUENCING THE CONCENTRATION OR
THE AMOUNT OF PU1 AND FACTORS
AND INFLUENCING THE CHOICE OF
THE SITES THAT YOU HAVE?
>> WHERE THEY ARE IN THE CELL
CYCLE?
>> ZEECT WE HAVEN'T DONE THAT
EXACT SPRMT THE WAY YOU'RE
ASKING.
BUT PU1 IS A VERY STABLE
TRANSCRIPTION FACTOR IN THESE
CELLS, SO IT GETS CARRIED
THROUGH THE CELL CYCLE, BUT THAT
ALSO MEANS THAT ITS LEVEL DROPS
BY A FOK TORE OF TWO EVERY TIME
THE CELLS DIVIDE.
>> EXACT.
>> AND I REALLY DON'T KNOW THE
ANSWER TO THAT QUESTION,
LITERALLY.
THESE CELLS UNLIKE THE SITUATION
THAT YOU WERE HEARING ABOUT FROM
DINAH, THESE CELLS ARE REALLY
PROLIFERATING ALL THROUGH THIS
PERIOD, SO EVERY ONE OF THESE
DEVELOPMENTAL STAGES HAS THE
FULL RANGE AND THAT'S WHY IT
REALLY DOESN'T SEPARATE THINGS
VERY WELL UNTIL YOU GET TO DM3
STAGE.
BUT IT'S A REALLY COOL QUESTION,
AND IT SOUNDS LIKE IT MIGHT BE A
WINDOW OF OPPORTUNITY FOR GENE
EXPRESSION CHANGE TO OCCUR.
SO I REALLY LIKE YOUR QUESTION.
THANK YOU.
>> I HAVE A POTENTIALLY NAIVE
QUESTION.
SO THE RECRUITMENT OF RUNX TO
THESE ALTERNATIVE SITES WHEN YOU
HAVE PU1, HOW IMPORTANT, WHAT IS
THE CONTRIBUTION OF THAT EFFECT
BASED ON THE REPRESSIVE COME
PLEKS THAT RUNX CAN BRING AND
HOW THAT AFFECTS TRANSCRIPTION
OF THOSE GENES VERSUS JUST
DISTRACTING THE RUNX FROM
BINDING TO THE SITES.
>> IT'S I'VE GREAT QUESTION.
IT'S NOT AT ALL NAIVE.
THE PROBLEM IS RUNX ACTUALLY HAS
QUITE A LOT OF SITES AND IT
BINDS TO MULTIPLE KINDS OF SITES
AROUND EACH OF THE GENES, SO
WE'VE SPENT AN ENORMOUS AMOUNT
OF TIME TRYING ON ANALYZE THIS
GENOMEWIDE AND IT'S REALLY KIND
OF FRUSTRATING BECAUSE WHATEVER
THE GENE IS GOING TO DO IN
RESPONSE TO RUNX, THE MOST
ABUNDANT KIND OF SITES ARE THE
SITES THAT ARE ALWAYS THERE.
DOES IT REALLY EXPLAIN THE
DIFFERENTIAL ACTIVITY UP AND
DOWN, WILL BE THERE IN ADDITION
TO SITES THAT ARE JUST THERE ALL
THE TIME.
AND WITH SOMETHING ON THE ORDER
OF 25,000 SITES OCCUPIED IT'S
REALLY SCARY TO TRY TO COME UP
WITH A STRATEGY FOR KNOCKING OUT
INDIVIDUAL SITES F WE'RE REALLY
NICE TARGET GENES TO LOOK AT.
SO WE WOULD LOVE TO KNOW THE
ANSWER TO YOUR QUESTION.
WE'VE TRIED TO LOOK FOR MOTIF
ENRICHMENT THAT MIGHT BE MORE
ASSOCIATED WITH REPRESSIVE
VERSUS ACTIVATING INTERACTIONS
IN A STAGE-SPECIFIC WAY, BUT THE
SIGNAL YOU GET BACK FROM RUNX X
WHETHER IT'S RUNX AND PU1 IN
EARLIER STAGE OR RUNX AND X1 IS
EARLIER EXRRKS P PROTEIN IN
LATER STAGE, YOU JUST DON'T SEE
WHAT MIGHT BE SPECIFIC.
IT'S A GREAT QUESTION, WE HOPE
WE'LL COME UP WITH BETTER WAYS
OF ANSWERING IT.
>> THANK YOU.
>> ALL RIGHT.
THAT WAS GREAT.
I WAS WONDERING ABOUT THE ROLE
OF THE GENETIC BACKGROUND OF THE
MICE.
>> OH.
>> AND JUST BECAUSE LIKE WE'VE
BEEN LOOKING AT MACROPHAGES AND
SEE VAST DIFFERENCE INSIDE PU1
ACTIVITY BETWEEN DIFFERENT
STRAINS OF MICE.
>> IT IS REALLY A GREAT THING TO
DO.
I MEAN, WE DON'T HAVE THE
RESOURCES TO LOOK AT THE
DIFFERENT MOUSE STRAINS THE WAY
WE KNOW SHOULD BE DONE, AND IF
ANYONE WANTS TO DO THAT WITH A
GOOD LIBRARY OF MOUSE STRAINS,
WE WOULD BE HAPPY TO HELP
BECAUSE I THINK SCIENTIFICALLY
IT'S SUCH AN IMPORTANT QUESTION.
YOU KNOW, YOU HAVE
POLYMORPHISMLESS THAT AFFECT
THIS, AND IT'S SOMETHING WE WILL
NEVER BE ABLE TO DO IT AT CAL
TECH WITH OUR LITTLE MOUSE
COLONY BUT I WOULD LOVE TO SEE
SOMEBODY ELSE RUN WITH THAT.
>> SO IT'S VERY NICE.
TWO QUICK QUESTIONS.
FIRST TO GO BACK TO THE
QUESTION, DO YOU KNOW IF THE
BRAISH.
>> CLOSER TO THE MIKE ZEEN THE
MOTIF THAT BINDS -- AT THE END
IS NECESSARY TO SEE WHAT YOU
SEE, AND THAT COULD HELP YOU
PERHAPS DISTINGUISH BETWEEN
ACTIVATE AND GO REPRESSING
FUNCTIONS?
>> THERE'S VWRPY IS A FAMOUS
MOTIF AND A LOT OF WORK WAS DONE
IN JAPAN OVER TEN YEARS AGO
COMPARING THE EFFECTS OF RUNX1
WITHOUT VWRPY MOTIF.
S STILL DOING VERY ELEGANT WORK
ON THIS AS YOU KNOW BUT OTHERS
MAY NOT IN THE CD4, THE OLD DATA
MADE IT SEEM AS THOUGH THAT
WASN'T IMPORTANT FOR THE EARLY
STAGES THAT I WORK ON, BUT AT
THAT TIME IT REALLY WASN'T
APPRECIATED HOW EXTREMELY
PERVASIVE RUNX3 IS AT THE SAME
TIME.
AND RUNX3 IS BASICALLY EXPRESSED
THE AAN EVEN HIGHER LEVEL THAN
RUNX1 IN THE CELLS THAT ARE AT
THE ETP STAGE THEY'RE ABOUT
EQUAL IN DM2A AND THEN THE RUNX1
BECOMES DOMINANT IN THE NEXT
STAGES.
SO ESSENTIALLY THEY WOULDN'T
HAVE SEEN AN EFFECT BECAUSE THEY
WOULDN'T -- BY SIMPLY NOT IF
HANGING OUT OF R -- KNOCKING OUT
OF RUNX 1RBGS IT WOULD HAVE HAD
TO BE KNOCKED OUT OF BOTH AND I
DON'T KNOW IF THAT'S BEEN DONE.
>> YOU COULD DO THAT IS CORRECT
RIGHT, OVEREXPRESS THE --
>> YEAH, BUT YOU WOULD HAVE TO
WORRY ABOUT, WE MIGHT NOT BE AS
LUCKY WITH THE REGULATION OF THE
SELF-REGULATION IF YOU DIDN'T
HAVE THAT.
>> THE OTHER QUESTION, WHEN YOU
KNOCK OUT BOTH 1 AND 3, THE
CELLS DON'T CRASH?
THE CELLS DON'T CRASH?
EVEN DOUBLE POSITIVE THERM
SIGHTS, THEY DON'T --
THYMOCYTES, DHOANT GO VERY FAR?
>> WE'VE NEVER DONE THAT.
MAYBE OTHERS HAVE.
>> THE DOUBLE NEGATIVITY EARLY
PROGENITORS, THEY SURVIVE?
>> THEY COMPLETELY -- THEY LIVE.
>> OKAY.
>> THEY LIVE.
BUT THEY DON'T DEVELOP PAST THE
EARLY STAGE.
>> OKAY.
THANK YOU.
>> THANK YOU.
>> PERHAPS YOU ALREADY MENTIONED
THIS, BUT WHEN YOU OVEREXPRESS,
DO YOU SEE ANY PREFERENTIAL
BINDING TO SITES LIKE ENHANCERS
OR PROMOTERS?
>> SO THE PROMOTERS IN OUR
SYSTEM ARE ALMOST COMPLETELY
DEVOID OF USEFUL INFORMATION.
BASICALLY, YOU SEE IT BINDING TO
PROMOTERS AT LEAST AS MUCH OF
GENES THAT IT DOESN'T REGULATE
AT ALL AS THE GENES THAT IT DOES
REGULATE.
SO WE'VE REALLY FOCUSED ON
NONPROMOTER SITES WHERE MOST OF
THE BINDING IS AND BY USING CUT
AND RUN, WE'VE ALSO BIASED OUR
DETECTION TO NON-PROMOTER SITES.
SO THOSE ARE, IT'S MOSTLY THE
ADDED SITES, THE ECTOPIC SITES,
AS IT WERE, ARE, BEING SOME OF
THEM ARE BASICALLY SITES THAT
REALLY WERE JUST LOW LEVEL RUNX
SITES ANYWAY AND THEY WERE DOING
SOMETHING.
MANY OF THEM WERE IN CLOSE
CHROMATIN AND SEEM TO HAVE
ABSOLUTELY NO SIGNIFICANT
ASSOCIATION WITH GENES WHICH ARE
EXPRESSED UNDER RUNX CONTROL AND
OTHER THINGS FOR EXAMPLE LIKE
ILC'S, SO I THINK AGAIN THEY'RE
GOING MAINLY, WE'RE SEEING
MOSTLY THE INTERGENIC AND
INTRONIC SITES THAT ARE OCCUPIED
DIFFERENTIALLY.
>> AND DOES THE COMPLEX FORM, I
MEAN, DO YOU FORM A COMPLEX OF
THE PROTEINS OR IS IT A LARGE
MACROMOLECULAR COMPLEX WHEN YOU
OVEREXPRESS?
>> WE HAVEN'T DONE ANY
PROTEOMICS ON IT.
IT'S A TWOFOLD OVEREXPRESSION,
IT'S BASICALLY GOING TO HIGH
AFFINITY RUNX SITES THAT WERE
ENCLOSED CHROMATIN.
THANKS.
SORRY TO BE LONG.
THANKS.
[APPLAUSE]
>> I THINK WE ALL AGREE THAT WAS
A VERY STIMULATING SESSION THIS
MORNING.
SO PLEASE ON BEHALF, ANOTHER
ROUND OF APPLAUSE TO ALL THE
SPEAKERS THIS MORNING.
OUR FIRST SPEAKER IS CLAUDIA
KEMPER FROM NHLBI, TALKING TO US
ABOUT FUNCTIONS FOR
INTRACELLULAR COMPLEMENT WHICH
IS A FIELD THAT SHE DISCOVERED
AND ELABORATED AND I'M LOOKING
FORWARD TO HEARING ABOUT IT,
CLAUDIA.
>> CAN YOU HEAR ME ALL?
WONDERFUL.
THANK YOU.
I WOULD LIKE TO THANK THE
ORGANIZERS FOR INVITING ME.
IT'S A FANTASTIC MEETING.
I REALLY, REALLY ENJOY IT.
BUT WHAT I'M GOING TO DO NOW IS
I'M DOING SOMETHING SLIGHTLY
DIFFERENT, I'M NOT SHOWCASING
ONE STORY WE'RE WORKING ON I
HAVE PUBLISHED RECENTLY BUT I
WOULD RATHER TAKE YOU ONTO A
JOURNEY IN MY RESEARCH PROGRAM,
THE REASON BEING THAT I THINK
THERE WERE SOME CHANGES IN THE
COMPLEMENT HERE LATELY THAT ARE
PRETTY PROFOUND AND THAT MAYBE
NOT EVERYBODY HAS HEARD ABOUT
YET AND I THINK SOME OF THOSE
MAY BE REALLY IMPORTANT FOR WHAT
YOU'RE WORKING ON.
ONE OF THE REALLY MOST EXCITING
PARTS IN SCIENCE IS THAT I THINK
YOU NEVER TRAVEL ALONE, AND
NOTHING IS DONE IN ISOLATION.
SO WHAT I WANT TO DO IS ACTUALLY
I WANTED TO START FIRST WITH THE
MOST IMPORTANT SLIDE AND SHOW
YOU MY TEAM HERE AT THE NIH AND
ALSO THE COLLABORATORS ACROSS
THE NIH AND BEYOND THAT HAVE
HELPED DEVELOPING THESE NEW
PARADIGMS AND THE COMPLEMENT
FIELD THAT I'M SHARING WITH YOU
NOW.
I. HAD BEEN WHEN YOU HEAR
COMPLEMENT FIELD OR SOMETHING
LIKE THIS, OH, MY GOD, CASS
SCRAID, COMPLEMENT C3 AND C5.
JUST A BRIEF ONE.
WE ALL THAN THESE COMPLEMENT
COMPONENTS ARE SECRETED BY THE
LIVER, CIRCULATE NG YOUR BLOOD
AND YOU REALLY REALLY NEED THEM
BECAUSE C3 AND C5 ACTIVATION
FRAGMENTS PROTECT YOU AGAINST
INVADING PATHOGENS.
SO THAT'S A REALLY, REALLY GOOD
PART ABOUT THE COMPLEMENT
SYSTEM.
HOWEVER, THERE'S UNFORTUNATELY
ALSO REALLY A QUITE DARK SIDE
ABOUT THIS SYSTEM.
WHEN ACCOUNTS DYSREGULATED --
WHEN IT IS DYSREGULATED, AND
THAT UNFORTUNATELY IS BECAUSE
IT'S TYPICAL
INFLAMMATION-DRIVING SYSTEM AND
THESE ACUTE OR, YOU KNOW,
SUSTAINED CHRONIC COMPLEMENT
ACTIVATION PATHWAYS CAN THEN
CONTRIBUTE TO HUMAN DISEASES.
AND ACTUALLY THE LIST OF HUMAN
DISEASES, THE COMPLEMENT HAS TO
BE DEFINED AS A DEFINITIVE
EITHER INDIERS OR PATHOLOGY
DRIVER IS REALLY LONG.
-- EITHER INDIERS OR PATHOLOGY
DRIVER IS REALLY LONG.
THIS IS JUST A PART OF IT.
WE KNOW FOR ALL SIX DECADES THAT
COMPLEMENT AS SUPREME
THERAPEUTIC TARGET AND WE HAVE
WORKED ON TARGETING COMPLEMENT
IN RARE AND COMMON DISEASES FOR
A VERY, VERY LONG TIME, AND WHEN
I MEAN WE, IT'S ACTUALLY
CLINICIANS, SCIENTISTS AND THE
INDUSTRY.
AS YOU CAN SEE JUST THE NIH IN
THE LAST TEN YEARS SPENT ALMOST
1,000 MILLION DOLLARS, THE
OUTCOME SO FAR IS REALLY QUITE
DISAPPOINTING.
WE CURRENTLY HAVE ONLY TEN DRUGS
THAT ARE EITHER FDA APPROVED OR
APPROVED FOR CLINICAL USAGE OR
APPLICATION AT THE EUROPEAN OR
ASIAN MARKET.
AND WHAT IS REALLY IMPORTANT IS
ALL OF THESE DRUGS REALLY ONLY
TACKLE RARE DISEASES, NONE OF
THESE DRUGS TACKLE
ATHEROSCHOARSZ OR ARTHRITIS OR
IBD, WE ALL KNOW COMPLEMENT
PLAYS A MAJOR ROLE.
SO WE'RE NOT 100% SURE QULET AS
A FIELD WHY THAT IS -- YET AS A
FIELD WHY THAT IS, BUT WHAT I
WOULD LIKE TO SHARE WITH YOU
TODAY AND MAKE A CASE FOR IS
THAT MAYBE ONE OF THE REASONS
WHY WE HAVEN'T BEEN SO
SUCCESSFUL IS BECAUSE WE REALLY
HAVEN'T UNDERSTOOD THE SYSTEM
YET.
ALL OF KNEES DRUGS TRADITIONALLY
TARGET COMPLEMENT IN THE
EXTRACELLULAR SPACE BECAUSE WE
WOULD ARGUE MOST OF YOU PROBABLY
THINK COMPLEMENT IS JUST A SERUM
BLOOD LIVER SYSTEM.
NOW, WHAT OUR COLLABORATORS IN
THE LAB BASICALLY HAVE
DISCOVERED IS THAT EVERY SINGLE
CELL IN YOUR BODY HAS ITS OWN
CELL AUTONOMOUS COMPLEMENT
SYSTEM, AND THAT COMPLEMENT
SYSTEM QUITE OFTEN CAN BE EITHER
SECRETED OR ALSO WORKS IN
INTRACELLULARLY, AND WITH THIS
NEW LOCATION OF COMPLEMENT, ITS
INTRACELLULAR VASCULAR ALSO
COMES NEW FUNCTIONAL NICHE.
WHAT CELL AUTONOMOUS OR
INTRACELLULAR COMPLEMENT IS
CONTROL A SINGLE CELL METABOLISM
NORMAL CELL PHYSIOLOGY AND ALSO
CELL SURVIVAL HOMOEOSTASIS AND
CELL EFFECTOR FUNCTION.
STANDOFFS DISCOVERED BY A REALLY
TALENTED POST-DOC IN MY LAB
MARTIN COLAR WHO FOUND THAT
CIRCULATING CD4 POSITIVE T CELLS
EXPRESS C3 AND ACTIVATE C3 IN
THE LYSOSOMES.
THE C38 PORTION OF ACTIVATED C3
ENGAGES A LYSOSOMAL C3A RECEPTOR
WHICH IS REALLY IMPORTANT FOR
CO-ACTIVATION AND REQUIRED FOR
CD4 POSITIVE T CELL SURVIVAL.
T CELLS THAT CANNOT PRODUCE C --
C3 DIE.
WHAT MARTIN ALSO DELINEATED THAT
IN LINE WITH WHAT WE THINK ABOUT
COMPLEMENT TO BE A PROTECTOR
AGAINST INFECTIONS, THE SYSTEM
REALLY ALSO IS IMPORTANT FOR T
CELL RESPONSES.
DURING THE PRESENCE OF DANGER
HERE SIGNIFIED BY T CELL
RECEPTOR ENGAGEMENT THE C3B
PORTION GENERATED
INTRACELLULARLY FLOIPS THE
OUTSIDE OF THE DISPEL ENGAGES
ITS RECEPTOR C DP 46 AND SIGNAL
MEDIATED BY CD46 ABSOLUTELY
CRITICAL TO DRIVE GAMMA
PRODUCTION IN T CELLS AND THE UP
SELL SIGNAL IS NOT IS IMPORTANT
FOR TH1 OR TH17 RESPONSES.
MAY BE QUITE CRITICAL FOR
HEALTH, ALSO IMPORTANT FOR THE
TH1 SHUT DOWN PROGRAM WHICH IS
EQUALLY IMPORTANT FOR SURVIVAL
BECAUSE IF YOU DON'T HAVE TH1
RESPONSES YOU CAN'T --
INFECTIONS BUT IF YOU CAN'T SHUT
THEM DOWN YOU REALLY DEVELOP
PROFOUND TISSUE PATH OOLG.
CD46 ALSO CONTRIBUTES TO THE TH1
SHUT DOWN PROGRAM BY INDUCING
THE CORE EXPRESSION OF
IMMUNOSUPPRESSIVE IL10 AND TH1
CELLS INTO A TH1 SHUT DOWN AND
PRERM PROGRAM.
-- MEMORY PROGRAM.
WE HAVE SHOWN USING PATIENTS
THAT IF YOU HAVE -- IF YOU HAVE
TOO LITTLE OF THE SYSTEM, DON'T
HAVE SUFFICIENT CD46 YOU SUFFER
FROM RECURRENT MOST UPPER
RESPIRATORY TRACT INFECTIONS,
BUT IF YOUR SYSTEM IS
DYSREGULATED AND YOU PRODUCE TOO
MUCH C3 AND YOU HAVE EN
HADGESSED CD46 OR UNTIMELY CD46
SIGNALLING, TH1 HYPERACTIVITY
INDUCED BY THIS PATHWAY
CONTRIBUTES TO AUTOIMMUNITY AND
MOSTLY SO FAR IN ARTHRITIC
DISEASES.
NOW, WHERE THE REAL EXCITING
PART FOR MY LAB CAME IN IS WHEN
WE TRIED TO FIGURE OUT HOW
EXACTLY IS CD46 INDUCING TH1
RESPONSES.
WHAT THEY FOUND IS SO FAR UNTIL
TODAY THAT CD46 INDUCES THREE
KEY EVENTS.
IT INDUCES DIRECTLY AND WE HAVE
NOW TO THANK THE MECHANISM BUT
I'M TALKING ABOUT THIS TODAY,
THE EXPRESSION OF GENES THAT
ENCODE NUTRIENT TRANSPORTERS
THAT ALLOW GLUCOSE AND AMINO
ACIDS TO COME IN BASICALLY FOOD
FOR TH1 INDUCTION.
AT THE SAME TIME, CD46 SHUNS THE
COMPLEX LYSOSOME WHICH NOW
ALLOWS THE HIGH LEVELS OF
GLYCOLYSIS AND OXIDATIVE
PHOSPHORYLATION PLRSZ L AND
YEAH, THERE'S ALSO INTRACELLULAR
C5 SYSTEM, IT IS CONTROLLED BY
CD46 AND IT INDUCES VIA
MITOCHONDRIAL RECEPTOR ONE
SIGNALLING ECONOMICAL AND LP3 --
ILM LYNN DEDUCTION THAT DOES NOT
INDUCT -- RATHER SUSTAINS
RESPONSES AT MUCOSAL SITES.
SIMILARLY THE TH1 SHUT DOWN
PROGRAM IS ALSO APEX REGULATED
BY CD46 BY A METABOLIC
REPROGRAMING AND HERE INVOLVING
A LOT OF CHOLESTEROL METABOLISM.
SO WHEN I MOVED MY LAB WE HAD
DISCOVERED THIS SYSTEM IN T
CELLS AND WE STARTED WORKING ON
THIS SYSTEM MORE BRAWLD HERE AT
THE NIH.
ONE OF THE QUESTIONS THEN WAS
OKAY, WE HAVE FOUND IT IN T
CELLS H IT'S IMPORTANT FOR MODEL
T CELL BEHAVIOR.
IF IT MISBEHAVES IT CONTRIBUTES
TO TH1 MEDIATED AUTOIMMUNE
DISEASE OR OTHER DISEASES.
DIST SYSTEM ALSO EXIST -- DOES
THE SYSTEM ALSO EXIST IN OTHER
IMMUNE CELLS?
I'M JUST GIVING YOU A BROAD
OVERVIEW HERE, BUT YES, IT DOES,
IN THE VERY IMPORTANT MONOCYTE
MARKER NEUTROPHIL LINEAGE NUTD
NEUTROPHIL LINEAGES, REALLY
IMPORTANT FOR NORMAL FUNCTION.
IF THE SYSTEM IS HYPERACTIVE, A
LOT OF COLLABORATORS HERE AT THE
NIH HAVE CARVED THIS OUT,
HYPERACTIVE MACROPHAGES
CONTRIBUTE TO DISEASES AND WE
HAVE SHOWN IT CONTRIBUTES TO
ATHEROSCHER HOSES MR. TORE THE
INABILITY OF PERCEPTIVE PATIENTS
TO CLEAR CANDY DAVMENT THIS IS A
LITTLE BIT OF A TIDBIT HERE
TODAY FOR UNPUBLISHED DATD.
WHAT WE HAVE DONE IS ALSO UP
REGULATED C3 AND C5 IN HUMAN AND
MOUSE B CELLS AND WHAT WE
NOTICED UNEXPECTEDLY IS THAT THE
ANTIBODY PRODUCTION IN THESE
CELLS THAT CANNOT PRODUCE C5 IN
AN OTHERWISE C5 SUFFER HOST IS
INCREASED.
THAT IS REALLY, REALLY
UNEXPECTED, AND THEY'RE TRYING
TO FIGURE OUT NOW WHY THAT IS,
AND THE ANTIBODY RESPONSE IS
INCREASED IN T CELL AND
INDEPENDENT RESPONSES, AND
CURRENTLY SEEING RNA SEQ TOLL
FIND OUT HOW CELL AUTONOMOUS
FLRMSZ DRL C5.
THE INCOMES BIG QUESTION WE
TRIED TO TACKLE IN THE LAB WAS,
IS THE INTRACELLULAR COMPLEMENT
SYSTEM WHICH WE COINED THE
COMPLOSOME ALSO IMPORTANT FOR
NONIMMUNE CELLS, MEANING FOR
STRUCTURAL CELLS?
DURING COVID, A LOT OF NAIAD
PEOPLE HELPED US DEFINE THAT
CELL AUTONOMOUS C3 IN TYPE TWO
LUNG EPITHELIAL CELLS IS KICKED
ON BY SARS-COV-2 AND THEN
SECRETED AND THAT SECRETED C3A
LOCALLY REALLY CONTRIBUTES TO
THE LOCAL PROINFLAMMATORY
COMPLEMENT DURING RESPONSE THAT
PATIENTS THAT TRANSITION INTO
SEVERE COVID ARE EXPERIENCING.
WE'RE ACTUALLY WORKING RIGHT NOW
ON TACKLING THIS AXIS.
NOW, A LOT OF OTHER LABS NOW ARE
WORKING ON THIS AND WHAT WE NOW
KNOW IS THAT WHEREVER YOU LOOK,
BASICALLY EVERY CELL THAT GROUPS
ACROSS THE WORLD LOOKED AT
INTRACELLULAR COMPLEMENT IS
PRESENT.
HAD BETA CELLS, EPITHELIAL CELLS
AND HYDROBLASTS AND FOR EACH OF
THESE CELL TYPES CELL AUTONOMOUS
CELL TYPES IS REALLY FINANCIAL
CORRESPONDENT FOR HOMEOSTATIC
FUNCTION ASK ALSO CONTRIBUTES
DYSREGULATED LIKE T CELL
RESPONSES TO DISEASES,, THAT THE
COMBLOSOME IS PRESENT IN ALL THE
CELLS, REALLY WE LOOKED AT SO
FAR, AND THAT CELL AUTONOMOUS
COMPLEMENT IS ALSO A KEY FEATURE
WHEN NEW CELLS ENTER THE TISSUE.
I DON'T HAVE TIME TO SHOW YOU
THIS DATA, BUT WE HAVE SHOWN
THAT P THE INDUCTION OF THE CELL
AUTONOMOUS COMPLEMENT SYSTEM IS
AN ABSOLUTELY KEY CHARACTERISTIC
HAD OF TISSUE RESIDENT IMMUNE
CELLS.
NOW, JUST A LITTLE BENEFIT A
SUMMARY SLIDE.
WHENEVER WE OR OTHER GROUPS
CARVED AND LOOKED HOW A CELL
AUTONOMOUS COMPLEMENT CROAMG THE
FUNCTION ACROSS ALL OF THESE
CELLS, I'M COMING RIGHT BACK TO
THE TITLE OF MY TALK.
IT IS ALL SINGLE CELL
PHYSIOLOGY.
SO CONTRIBUTES TO EVERYTHING YOU
CAN THINK OF, CHOLESTEROL PRRKS
GENE DIRECT REGULATION SH
MITOCHONDRIAL ELECTRON TRANSPORT
CHAIN DIRECTION SO IT'S A REAL
NEW CONTROLLER OF CELL
PHYSIOLOGY.
AND BECAUSE WE'RE YOUR GUEST
TODAY FOR NCI, I WANTED TO JUST
GIVE A LITTLE CARROT AND SHARE
WITH YOU THAT WHAT WE HAVE FOUND
REALLY, AND THIS IS YOU SHOULD
UNDER SUBMISSION NOW, WE HAVE
FOUND THAT TONICALLY ACTIVE C3
HAVE I AUTO OM MYC SIGNALLING OF
THE C3A RECEPTOR IN HUMAN
EPITHELIAL CELLS IS ABSOLUTELY
CRITICAL TO STATIC TURN OF
KEEPING THESE CELLS GOING.
IF YOU TAKE THE C3 SYSTEM OUT,
THESE CELLS MOVE INTRAMALIGNANT
TRANCE FOR MAIPTION AND THE
PATHWAY IS SOME CONTROL OF SOME
REALLY INTERESTING ONCOGEN AND
RIBOSOMAL BIOGENESIS PATHWAYS.
SO I HOPE I HAVE CONVINCED YOU
THAT INTRACELLULAR COMPLEMENT
REALLY IS IMPORTANT.
WHAT I HAVEN'T TALKED WITH YOU
ABOUT YET IS THAT IT IS ALSO --
TO FORMALLY A PHARMACOLOGICAL
INTERVENTION.
WHILE WE'VE BEEN SUCCESSFUL IN
TAKING T CELLS OUT OR MONOCYTES
AND MACROPHAGES FROM SITES OF
HUMAN INFLAMMATION LIKE INFLAMED
JOINT FROM PATIENTS WITH
ARTHRORIGHTS AND WE HAVE BEEN
ABLE TO ANALYZE HYPERAK --
NORMALIZE RESPONSES DRIVEN BY C3
OR C5 ACTIVATION, EX-VIVO, WITH
A CELL COMPLEMENT INHIBITOR
ACCIDENT DOING THIS TOGETHER.
ARE WE ARE NOW ALSO WORKING ON
TESTING WITH APELLIS FIRST
CLINICAL MOUSE NODLE
INTRACELLULAR COMPLEMENT IN
ARTHRITIS AND IN CANCER.
SO WHAT I WOULD LIKE TO ARGUE
HERE ALSO IS THAT BECAUSE C3 AND
C5 HAS A FUNCTION IN SERUM, IN
THE CIRCULATION, AND IN CELLS,
AND THE FUNCTION IS DIFFERENT IN
CELL SPECIFIC, IF YOU ARE
INTERESTED IN STUDYING THIS
SYSTEM, I REALLY, REALLY URGE
YOU TO USE FLOXED AND CELL
SPECIFIC KNOCK-OUT MICE.
WE ARE MOVING AWAY IN OUR FIELD
FROM USE HAD GONE GLOBAL MICE
BECAUSE THEY MASK PHENOTYPES AND
DO NOT REALLY GIVE YOU AN ANSWER
WITH REGARDS TO MECHANISMS THAT
YOU MIGHT BE INTERESTED IN.
THE SECOND THING AI WOULD ALSO
SAY IS, I WOULD BET YOU A VERY
GOOD BOTTLE THAT IF YOU DIG INTO
YOUR SINGLE CELL RNA SEQ DATA
YOU WILL SEE THE COMPLEMENT
SYSTEM AND I ALSO BET YOU THAT
YOU PROBABLY IGNORED THAT
BECAUSE COMPLEMENT SHOULDN'T A,
BE THERE, AND SECOND, NOBODY
LIKES THE SYSTEM.
BUT IF YOU ARE INTERESTED, IT'S
IN YOUR TOP PATHWAYS, PLEASE
COME AND TALK TO US.
WE THINK IT'S A REALLY, REALLY
COOL SYSTEM, AND WE THINK IT'S
BY NO MEANS FULLY EXPLORED.
AND WE ALSO THINK THIS GIVES
U.S. THE OPPORTUNITY NOW TO
RETHINK THIS SYSTEM IN HUMAN
DISEASE BUT ALSO ULTIMATELY FOR
THERAPEUTIC INTERVENTION BECAUSE
WE THINK WE NOW NEED TO START
THINKING ABOUT TACKLING THE
INTRA AND EXTRACELLULAR
COMPLEMENT SYSTEM TO REALLY,
REALLY HAVE A A GO AT ARE
BROADENING DISEASES.
[APPLAUSE]
>> THANK YOU VERY MUCH.
THE LITTLE I KNOW ABOUT
COMPLEMENT I THINK THAT C3 HAS
TO BE CLEAVED BY SOMETHING, I
KNOW THERE'S SOME SPONTANEOUS
CLEAVAGE AND THEN C5 HAS TO BE
CLEAVED.
WHAT'S UPSTREAM OF C3 AND HOW DO
ACIDIC COMPARTMENTS IN THE CELLS
SOMEHOW PERHAPS CONTRIBUTE TO
SPONTANEOUS CLEAVAGE?
>> THAT'S A FANTASTIC QUESTION.
SO IN SERUM IT IS RELATIVELY
ESTABLISHED THAT YOU NEED IT A
COMPLEX OF THREE DIFFERENT
PROTEINS TO ACTIVATE C3.
WHAT WE HAVE SHOWN DPINLTD HAVE
TIME TO REALLY DISSECT HERE WAS
IN CELLS SINGLE AND EVOLUTIONARY
PROTEASES CAN ACTUALLY DLEEF C3
DIRECTLY.
NOW, 24-S REALLY INTERESTING
BECAUSE SPECIFICALLY KINAPSIN-L
CAN WORK IN ACIDIC AND IN
NEUTRAL PH SETTINGS SO THAT
ACTUALLY WORKS FOR US SO THAT
YOU HAVE DIFFERENT COMPARTMENTS
IN A CELL WHERE A PH AND AN OLD
PROTEASE IS CONDUCIVE TO CLEAVE
C3 NOW.
>> GREAT TALK, CLAUDIA.
SO HEAD TROA TRIMATO-G-PROTEINS
ARE THEY INVOLVED IN THE
INTRACELLULAR COMPLEMENT SYSTEM?
>> WHAT WAS THE WORD BEFORE
G-PROTEIN?
GROO HETRO --
>> HETRATROMA ATIC.
>> I THINK YOU'RE TROAMPG THE
G-PROTEIN SECTORS CAN PAIR AND
NOT JUST DIMERIC BUT ALSO
MULTI-MERIC.
>> ALL YOUR RECEPTORS AS YOU SAY
ARE RL COMPLEMENT SO INSIDE THE
RECEPTOR ARE THE BINDING
G-PROTEINS, BIENGSD SITES FOR
THOSE G-PROTEINS.
>> YES, THEY TORQUES THEY ACTUAL
EVEN BIND THEM INSPECT
MITOCHONDRIA, AND MITOCHONDRIAL
SPACE WE HAVE SHOWN AND
PUBLISHED THAT.
THEY ALSO PAIR WITH OTHER
G-PROTEIN COUPLED RECEPTORS
WHICH MAKES THE WHOLE SITUATION
REALLY QUITE COMPLEX.
SO YES AND YES.
>> SO IT'S THE G-PROTEINS THAT
ARE GRIEPTING THE SIGNAL THROUGH
CLASSIC MECHANISMS?
>> YEAH.
YES.
THROUGH ERG SIGNALLING AND
PHOSPHORYLATION IN THE
MITOCHONDRIA.
YEAH.
>> HI.
SO I HAVE TWO QUESTIONS THAT ARE
RELATED <!680> ONE IS WHAT IS AN
EXAMPLE OF A DISEASE THAT IS
CAUSED ONLY BY -- COMPLEMENT AND
THEN SECOND HOW WOULD YOU PLAN
TO TARGET IT WITHOUT EFFECTIVE
EXTRACELLULAR COMPLEMENT?
>> TWO GOOD QUESTIONS.
ONE DISEASE IS REALLY TRULY, IF
YOU CANNOT EXPRESS CD46 OR HAVE
ISSUES WITH C3 SECRETION, THESE
PATIENTS ARE REALLY PROFOUNDLY
DEFECTIVE IN TH1 RESPONSES, SO
YOU REALLY HAVE A CONNECTION
BETWEEN TH1 BIOLOGY AND THE
FAULTY CELL AUTONOMOUS
COMPLEMENT SYSTEM BECAUSE SERUM
C3 CANNOT COMPENSATE FOR CELL
AUTONOMOUS C3, ONE.
B, IT'S REALLY THE PAPER THAT WE
ARE SUBMITTING RIGHT NOW, VERY
SURPRISINGLY THAT IF YOU TAKE
OUT C3 FROM CERTAIN EPITHELIAL
CELLS, THESE CELLS MOST
SPONTANEOUSLY ENTER HYPERPLASIA
AND MA LIG NANLTD
TRANSFORMATION.
SO IT'S CANCER AND INFECTION --
MALIGNANT TRANSFORMATION.
SO IT'S CANCER AND INFECTION
RIGHT NOW.
>> SO IT'S NOT A DIRECT
TARGETING OF THE COMPLEMENT
IFTSZ ITSELF?
>> THIS VALLEY A BEAST.
EVERYBODY KNOWS THE STOP
INHIBITORS IN CANCER IS THE
PROBLEM, SO WHAT WE'RE TRYING TO
DO AS A PROGRAM IS TRYING TO
UNDERSTAND A LITTLE BET HOW THE
CELL AUTONOMOUS COMPLEMENT
SYSTEM CONTROLLED FROM THE CELL
SURVEYS SO WE MAYBE HAVE EVEN
EASIER TARGET.
WE'RE COMING HERE WITH A
HOLISTIC BRAM WE LOOK WHAT ARE
THE RECEPTORS THAT DRIVE THE
SYSTEM AND WE KNOW IT'S
INTEGRANS LFA1 WE HAVE PUBLISHED
THAT AND ALSO AT THE SAME TIME
WE ARE TRYING TO UNDERSTAND WHAE
DIFFICULT.
>> THANK YOU.
>> I ACKNOWLEDGE THAT.
>> THANK YOU.
>> THANK YOU VERY MUCH.
[APPLAUSE]
>> SO OUR NEXT SPEAKER IS THE
INCREASINGLY FAY FAMOUS SHRUTI
NAIK FROM NEW YORK UNIVERSITY,
HE'LL BE TELLING US ABOUT
IMMUNE-MEDIATED MECHANISMS OF
ADAPTIVE AND MALADAPTIVE TISSUE
RESPONSES.
LET ME ASK THAT WHEN WE ASK
REQUESTS AT THE END, PERHAPS IF
THE QUESTIONER WOULD JUST
INTRODUCE THEMSELVES, IT MIGHT
BE HELPFUL FOR ALL OF US, THANK
YOU.
WELCOME.
>> THANKS SO MUCH, AL.
REALLY WE CAN TALK ABOUT
ANYTHING AND IT'S GOING TO BE
KIND OF A GRAB BAG, YOU GET A
CHOCOLATE BOX AND YOU DON'T KNOW
WHAT YOU'RE GETTING, RIGHT?
ALL RIGHT.
SO LET'S SEE WHAT'S HAPPENING
HERE.
I HAVE TO FIND MY TALK.
ALL RIGHT.
>> FIRST I WANT TO REALLY THANK
THE ORGANIZER FOR HAVING ME HERE
HIST A SPECIAL MOMENT TO BE
HERE.
I LEFT THE NIH EXACTLY TEN YEARS
AND 28 DAYS AGO, NOT THAT ANYONE
IS COUNTING, AND BEING BACK HERE
IS JUST TRULY SPECIAL.
THIS IS A COMMUNITY THAT HAS
RAISED ME, I FEEL LIKE I'M A
CHILD OF THE NIH AND I'M REALLY
GRATEFUL FOR THE MENTORS THAT
I'VE HAD HERE AND THE NOW
COLLEAGUES AND FRIENDS THAT I
HAVE.
SO THANK YOU SO MUCH.
AS I WAS TELLING AL EARLIER, MY
TALK IS REALLY IMMUNOLOGY
ADJACENT SO I'M GOING TO TALK
ABOUT REALLY IMMUNE CELLS, BUT
ABOUT THEIR IMPACT ON TISSUES.
MY LAB TRIES TO BROADLY
UNDERSTAND HOW ENVIRONMENTAL
INPUTS ARE INTERPRETED BY OUR
BODY AT ESSENTIALLY BARRIER
TISSUES LIKE THE EXPINT GUT,
THESE ARE OUR WINDOWS TO THE
EXTERNAL ENVIRONMENT.
IN OUR ADMITTEDLY BIASED OPINION
BELIEVE THAT HOW OUR TISSUES
RESPOND TO THESE ENVIRONMENTAL
TRIGGERS REALLY DEPEND ON HOW
IMMUNE CELLS SENSE THESE
PERTUBATIONS OR ALTERATIONS, AND
COMMUNICATE WITH THE REST OF THE
TISSUE.
SO WE ARE INTERESTED IN
UNDERSTANDING THE MECHANISMS
UNDERLYING THESE COMMUNICATIONS
NOT JUST FROM THE PERSPECTIVE OF
IMMUNE CELL BUT ALSO FROM THE
PERSPECTIVE OF THE RECEIVER
CELLS AND THE CONSEQUENCES OF
THESE COMMUNICATIONS FOR BOTH
HEALTH AND DISEASE.
AS WAS BEAUTIFULLY BROUGHT UP BY
THE PREVIOUS TALK, THINGS THAT
ARE REALLY OPERATIVE IN HEALTH
ARE COOPTED PATHOLOGICALLY IN
DISEASE.
SO WE THINK THERE'S REALLY A
BEEN TIGHTED TO STUDYING HEALTH
TO UNDERSTAND NOVEL AVENUES OF
TARGETING DISEASE.
I'M GOING TO TELL YOU ONE SORT
OF NARRATIVE THAT'S BEEN EMERGE
FRG MY LAB OVER THE PAST FEW
YEARS THAT SORT OF EXEMPLIFIES
THIS.
SO WE STUDY IMMUNE TISSUE
CROSSTALK IN VARIOUS CONTEXT
TODAY I'LL TALK TO YOU ABOUT THE
CONTEXT OF TISSUE DAMAGE AND
REPAIR AND HOW OUR SYSTEMS COPE
WITH THIS.
WE'RE ADDRESS THIS QUESTION OF
THE SKIN WHICH IS OUR OF COURSE
PRIMARY BARRIER BUT ALSO HAS
REALLY SOPHISTICATED REPAIR
MECHANISMS.
EVERYONE HERE GETS CUTS AND
SCRAPES ALL THE TIME, AND EVERY
ONE HERE HAS IMMUNE CELLS AND
LYMPHOCYTES IN THEIR SKIN, SO WE
ARE REALLY INTERESTED IN
UNDERSTANDING WHAT DO THESE LIMB
FOAF SIGHTS DO WHEN YOU GET A
CUT OR SCRAPE IN PHYSIOLOGICAL
CONTEXT?
WHAT IS THEIR ROLE?
DO THEY PARTICIPATE?
AND HOW DO THEY REALLY MEDIATE
THEIR FUNCTION IN THIS CONTEXT
OF REPAIR?
IS IT BEYOND JUST KILLING THE
BUGS THAT GET IN?
SO THIS IS JUST AN IMAGE OF
HEALTHY HUMAN SKIN AND YOU SEE
THAT THERE ARE REALLY MANY, MANY
DIFFERENT TYPES OF LYMPHOCYTES
THAT HANG OUT AROUND THE HAIR
FOLLICLES AND AROUND THE
VASCULATURE, AND WE THINK THAT
UNDERSTANDING THIS QUESTION IS
REALLY IMPORTANT NOT JUST FOR
UNDERSTANDING THE FUNDAMENTALS
OF HOW TISSUES HEAL AND COPE
WITH DAMAGE BUT BECAUSE THESE
SAME FACTORS GO AWRY IN A PLET
AFTER INFLAMMATORY DISEASES.
DAMAGE PATHOLOGIES ARE COOPTED
IN MANY, MANY CONDITIONS, IN
FACT, I THINK THAT OFTEN WE
SUCCUMB TO DISEASE BECAUSE OF
TISSUE DAMAGE.
OUR ORGANS FAIL BECAUSE OF
TISSUE DAMAGE.
SO UNDERSTANDING HOW WE COPE
WITH THIS IS REALLY CENTRAL TO
ADVANCING HEALTH.
AND ALSO THESE PATHOLOGIES ARE
REEMERGED IN THE CONTEXT, SO
HERE I HAVE TWO EXAMPLES,
PSORIASIS WHICH IS REALLY A
PATHOLOGY OF OVERHEALING WHERE
YOU HAVE EPITHELIAL HYPERPLASIA
AND I AM UNIHYPERACTIVATION AND
CROHN'S DISEASE WHICH IS
PATHOLOGY WHERE YOU HAVE
REPEATED DAMAGE AND REPAIR
CYCLES AND INFLAMMATION THAT
CORRUPTS YOUR ENTIRE INTESTINE.
AGAIN, INCREASINGLY FOR THIS
TALK, BOTH OF THESE DISEASES ARE
IN SOME FORM OR ANOTHER MEDIATED
BY HYPERACTIVATED IMMUNE CELLS
AND PATHOLOGICAL SIGNALLING
EPITHELIA.
WHAT IS SUSTAINING THIS
CROSSTALK AND WHAT ARE THE
MEDIATORS OF THIS CROSSTALK?
SO WE RECENTLY PUBLISHED THIS
PAPER, I WANT TO ADVANCE TO
UNPUBLISHED FINDING, I'M
SUMMARIZING THIS, TWO FANTASTIC
POSTDOCTORAL FELL LOAPS IN THE
LAB UNCOVERED NEW MECHANISMS BY
WHICH IMMUNE CELLS CROPS THE
EPITHELIA 6789 SO WHEN YOU HAVE
A WOUND.
SO WHEN YOU HAVE A WOUND, AT THE
EDGE OF THE WOUND YOU HAVE STEM
CELLS THAT PROLIFERATE AND MIE
GRAIRT ACCIDENT EXPAND AND GO
MAKING BRAND-NEW TISSUE.
WHAT YUE AND PIOTRI FOUND IS LZ
TH17 WHICH TRIGGERS CELLS TO
EXPAND -- IL17 WHICH TRIGGERS
THE CELLS TO EX-BAND AND MOVE.
LYMPHOID CELLS PLAY CRITICAL
ROLES?
CROSSTALKING WITH STEM CELLS AND
PROMOTING THEIR SORT OF TISSUE
GROWTH POTENTIAL.
MORE SURPRISING WAS HOW THIS
HAPPENED.
SO WHAT THEY FOUND IS THAT IN
FACT IL-17A ENABLES THE
ADAPTATION OF THESE STEM CELLS
TO THE HARSH HYPOXIC ENVIRONMENT
OF A WOUND EDGE.
THIS IS SOME OF THE MECHANISTIC
EVIDENCE THAT SUPPORTS THIS.
ON THE TOP YOU HAVE CONTROL
WOUNDS AND THE RED REALLY MARKS
THE EPITHELIAL THAT IS RIGHT AT
THE EDGE OF THE WOUND AND THE
GREEN DOTS ARE AM AM HIF1 ALPHA,
MEDIATES HIGH POX YARKS BRIGHTLY
LIT UP AT THE WOUND'S EDGE
THERE.
IF YOU TAKE AWAY TYPE 17 CELLS
AND FOR THE CONNOISSEURS AS YOU
GUYS ARE, GAMA DELTA T CELLS,
DID ALL THE VARIOUS KNOCKOUTS,
YOU SEE THAT THAT HISTONE ALPHA
SIGNAL IS GONE, IT'S REMARKABLE
IT'S GONE EVEN THOUGH THERE IS
NO IFNLTS DIFFERENCE BETWEEN
HYPOXIA BETWEEN THESE TWO
SITUATIONS, THAT TOLD US REALLY
THAT IL17 WAS CRITICAL FOR THE
INDUCTION OF THIS TRANSCRIPTION
FACTOR THEAN ABLED ADAPTATION OF
THESE CELLS AT THE WOUND'S EDGE
TO THIS HYPOXIC CONDITION AND
THAT HYPOXIA WAS NOT SUFFICIENT
SO WE ESSENTIALLY SORT OF CAME
TO THIS CONCLUSION THAT, YOU
KNOW, -- LZ CONSISTENTLY
EXPRESSED OR DEGRADED, ACUTE
HYPOXIA, STABILIZED, GO TO
NUCLEUS AND ENABLE CELL TO
SURVIVE IN LOW OXYGEN CONDITION.
CONDITIONS OF CHRONIC HYPOXIA
YOU REALLY NEED SECONDARY
SIGNALS LIKE IL17A THAT SIGNAL
ML TO ENABLE THIS ADAPTATION AND
EPITHELIAL ORGANOID SYSTEM TO
DEMONSTRATE THIS.
SO IF YOU TAKE PRIMARY
EPITHELIAL ORG NOIDZ, CULTURE
THEM IN EITHER NORM OXIA OR
ACUTE OR CHRONIC HOX YARKS WHAT
YOU SEE IS THAT -- CAN YOU SEE
MY POINTER?
YEAH.
YOU SEE THAT IN ACUTE HYPOXIA
ALL SYSTEMS ARE G THRINGZ
WORKING AS THEY THEY SHOULD,
HIS TURNS ON FURTHER AUGMENTED
BY IL17.
IN CHRONIC HYPOXIA IT IS NOT THE
CASE.
IF YOU GIVE IL17 IT RESCUES
THIS.
AND THIS MODE UNTIL THEY GET
SIGNALS FROM THEIR SORT OF
IMMUNOLOGICAL BROTHER EN.
AND BROADLY THE SORT OF BROUGHT
UP THIS PICTURE THAT WHEN YOU
HAVE AN INJURY, YOU HAVE
COOPERATIVITY BETWEEN YOUR
IMMUNE CELLS AND YOUR WOUND EDGE
EPITHELIAL PROGENITORS AND THAT
REALLY THE 17 SIGNAL FROM YOUR
EU78 UNICELLS ENABLES THIS
ADAPTATION HYPOXIA WHICH THEN
ALLOWS THESE CELLS TO CHANGE
THEIR METABOLISM AND FUEL
REPAIR.
SO WE THINK THAT THIS IS IN FACT
AN EXEMPLAR OF MULTICELLULARITY.
HOW WE STUDY HYPOXIC RESPONSE
INSIDE A DISH IS REALLY STUDYING
ACUTE HYPOXIC RESPONSES WHICH
REALLY REPRESENT EITHER
UNICELLULAR ORGANISMS OR WHEN
YOU ARE FIRST ENCOUNTERING
HYPOXIA AND WHEN YOU THINK ABOUT
THE SORT OF PROLONGED TIME SCALE
OF WHAT IT TAKES TO HEAL A
TISSUE AND MAKE BRAND-NEW TISSUE
AND THE ENERGY INVOLVED AND
COMPLEXITY INVOLVED, WE THINK
THAT THERE IS ESSENTIALLY
CROSSTALK BETWEEN IMMUNE CELLS
AND THE EPITHELIA THAT ENABLE
THIS.
SO WHAT HAPPENS WHEN THIS
CROSSTALK GOES AWRY AND WHEN
THESE CONVERSATIONS ARE
UNCHECKED AND WHEN YOU HAVE
MAYBE TOO MUCH SECONDARY SIGNAL?
SO THIS IS WHERE I WANT TO COME
BACK TO PSORIASIS WHICH IS THIS
DISEASE OF OVERHEALING AND MANY
OF THE PATHOLOGICAL FEATURES YOU
SEE IN A WOUND ARE ESSENTIALLY
EXAGGERATED AND PSORIASIS WHERE
INSTEAD OF A WOUND HEALING AND
MAKING NEW TISSUE THIS WAY, IT'S
NOW 3W45EUBGING PLAQUES UPWARD
AND WHAT'S REMARKABLE SYNTHESIS
ONE OF THE DISEASES THAT'S
RESPONSIVE TO IL17 THERAPY SO IN
MANY CASES PATIENTS IN FACT THEY
RESOLVE THEIR DISEASE, THOUGH
THAT IS NOT CURATIVE.
SO WE THOUGHT THIS WOULD BE A
REALLY INTERESTING DISEASE TO
PROBE IF OUR NOVEL IL17 ALPHA
AXIS WAS OPERATIVE AND IF IT
PLAYED ANY ROLE IN DRIVING
PATHOLOGY OR WAS IT SIMPLY
FUELING THAT SORT OF EPITHELIAL
HYPERPROLIFERATION?
AND SO WE PAIRED UP WITH THE
DIRECTOR OF ARTHRITIS CENTER AND
TOGETHER JOSE AND I MENTORED A
WONDERFUL FELLOW NOW AT BRIGHAM
CONSTITUTED STUDYING MYOSITIS
AND LEVERAGED SPATIAL TO
UNDERSTAND HOW REPAIR MECHANISMS
MAY BE COOPTED IN THIS DISEASE.
HERE ICHT TO MAKE A POINT WE
DIDN'T JUST USE SPATIAL
SCRIPTOMICS OR PRETTY PICTURES
OR LOVE SPENDING MONEY WHICH I
DO NOT, BUT THIS ALLOWS ACCESS
TO CELL TYPES THAT ARE VERY HARD
TO GERKTS EITHER CELLS DHEENLTD
HAVE NUCLEI LIKE DIFFERENTIATED
EPITHELIAL CELLS OR SITES THAT
DIE OR EVEN WITH RNA SEQ YOU
DON'T REALLY GET THEIR
TRANSCRIPTIONAL LANDSCAPE.
SO ROCHELLE IS VERY INTERESTED
IN UNDERSTANDING SORE YAT I CAN
ARTHRITIS, THE MAN MANUSCRIPT IS
PUBLISHED.
WE WERE REALLY INTERESTED IN IS
OUR AXIS OPERATIVE HERE?
SO THESE ARE THE KIND OF
BEAUTIFUL PICTURES YOU GET, FULL
GENOME EXPRESSION ACROSS THE
TISH LUND SCAPE, EVERY DOT IS
YOU GUYS HAVE SEEN LOTS TODAY
HERE INSTEAD OF EVERY DOT BEING
A SINGLE CELL, EVERY DOT IS A 5S
ALREADY OUTDATED BECAUSE NOW
THERE'S MUCH HIGHER RESOLUTION
ST OUT THERE WE'VE MOVED TO BUT
YOU CAN ESSENTIALLY SEE WHAT
ECOSYSTEMS ARE EXPRESSING AND
WHAT'S DIFFERENT BETWEEN A
DISEASE STATE AND NOT AND THEN
DIG DEEPER INTO WHAT ARE THE
SPECIFIC GENES THAT ARE
DYSREGULATED IN THIS.
SO WE WANT TO DO ASK THIS
QUESTION, WE SPECIFICALLY WANTED
TO HONE IN ON THE ECOSYSTEM THAT
WAS DYSREGULATED IN THE SORE YAT
I CAN TISSUE -- PSORIATIC
TISSUE, MARKED IN RED, WITH BIG
RED ARROWS, LOOKING AT SPECIFIC
AREAS DYSREGULATED AND REALLY
EMERGE IN PSORIATIC SKIN AND
EXCITED TO FIND THAT IN FACT IL7
WERE UNIQUELY CO-ENRICHED IN
THESE AREAS, SO THIS IS
SEQUENCING AND WE MUST VALIDATE
AT A PROTEIN EXPLEFL A
FUNCTIONAL LEVEL, SO WE DID JUST
THAT -- LEVEL.
SOILY ANOTHER Ph.D. STUDENT IN
THE LAB AND PITRA TEAMED UP TO
TAKE THIS ON WITH OUR
BIOMATHEMATICIAN AND THEY FIRST
STARTED BY LOOKING AT IS HIS
OVEREXPRESSED IN -- IS HIF
OVEREXPRESSED IN INFLAMMATORY
DISEASES, IN HEALTHY SKIN, NO
HIF EXPRESSION IN EPIDERMIS OR
ELSEWHERE, GREEN IN THE
BACKGROUND IS JUST BACKGROUND.
THIS GOES UP LIKE GANG BUSTERS
IN PSORIASIS, ALWAYS TOPIC
DERMATITIS, AND RS IT CORRELATED
WITH THE PRESENCE OF IMMUNE
INFILTRATES AND IN AREAS WHERE
PATHOLOGY WAS EVEN WORSE AS
MEASURED BY EPIDERMAL
HYPERPLASIA AND REALLY
REMARKABLE ALMOST TO THE POINT
OF WHERE LIKE I DID NOT BELIEVE
THIS DARKTS I HAD THE LAB DO IT
OVER AND OVER AGAIN WAS THAT IF
YOU LOOKED AT RARE IL17 RECEPTOR
WAS EXPRESSED IN THE EPITHELIA,
WHERE, IN RED, AND IF YOU LOOK
AT WHERE HIF1 ALPHA EXPRESSED IT
WAS 100% CONCORD GNAT,
COMPLETELY CO-EXPRESSED IN HUMAN
TISSUE REALLY SUGGESTING THAT
HIF1 ACTIVATION IN EPITHELIA WAS
THIS RHEOSTAT STAT, THIS BROM
TEFER IL17 SIGNALLING.
FINALLY WE TOOK ADVANTAGE OF THE
FACT THAT WE COULD ESSENTIALLY
HAVE ACCESS TO PATIENTS THAT ARE
COMPLETELY THERAPY RESPONSIVE
AND LOOKED ABBOTT THEIR LESIONAL
SKIN PRE AND POST THERAPY AND
YOU SEE THAT PRETHERAPY THERE'S
TONS OF HIF1 ALPHA SIGNAL AND
POST THERAPY HIF1 ALPHA SIGNAL
GOES DOWN ON BASE LOON, NICE
CORRELATING DATA.
WHAT IS HIF ACTUAL DOOLG AND IS
IT ACTUALLY PLAYING A ROLE IN
THIS DISEASE?
IF SO, HOW?
TO DO THIS WE ESSENTIALLY SET UP
A QUOTE UNQUOTE TRIAL IN A DISH
SYSTEM BECAUSE IT'S A LOT EASIER
TO GET AN IRB AND THIS WOULD
REALLY ALLOW US TO REALLY START
PROBING WHAT HIF'S FUNCTION WAS
SIDE BY SIDE WITH STANDARD OF
CARE, WE ESSENTIALLY TOOK
BIOPSIES FROM PATIENTS WITH
PSORIASIS AND CULTURED THEM
EITHER WITH THE STANDARD OF CARE
OR WITH HIF1 ALPHA INHIBITORS,
DID ANALYSIS AND YOU SEE HIF1
AMFA HAS A REALLY DRAMATIC
EFFECT COMPARED TO STANDARD
WITHIN 24 HOURS, THESE ARE NOT
GENES KILLING OFF THE TISSUE OR
KILLING OFF THE CELLS.
IF YOU TAKE THESE GENES AND THEN
PROJECT THEM ONTO CLINICAL TRIAL
DATA THEY ESSENTIALLY PERFECTLY
MIRROR THE PRECISE GENES THAT
REFLECT FOLKS THAT HAVE RECEIVED
THERAPY FROM PLACEBO AND FURTHER
DISTINGUISH RESPONDERS FROM
NONRESPONDERS.
SO THIS IS REALLY TELLING US
THAT HIF MAY HAVE A FUNCTIONAL
ROLE.
BUT THIS IS PRETTY MUCH AS FAR
AS WE CAN GO WITH OUR HUMAN
DATA.
SO WE HAVE TO GO BACK TO OUR
MOUSE MODELS, WHICH MADE ME
VERY, VERY HAPPY BECAUSE THIS IS
KIND OF MY COMFORT ZONE, I
ALWAYS CALL MYSELF AN M-D A
MOUSE DOCTOR.
SO WE NEUFD A MOUSE MODEL OF
PSORIASIS, LZ TLR7 LIGAND, WELL
ESTABLISHED MOUSE MODEL IRVETION
GO FOR SIX DAYS, GET WELL
DEFINED COULD YOU TAKEN
CUTANEOUS PATHOLOGY.
FOR OUR PURPOSES HIF IS ALSO
HIGHLY HEIGHTENED IN THIS MODEL
AND ALSO DEPENDENT ON THE
EXPRESSION OF IL17 BECAUSE IF
YOU GOT RID OF THE 17 RECEPTOR
IN THE EPITHELIA YOU NO LONGER
HAVE THIS.
AND WHEN WE GIVE TOPICAL HIF1
ALPHA INHIBITOR, YOU NOW SEE
THAT IN FACT THESE MICE WERE
PROTECTED FROM DEVELOPMENT OF
DISEASE, SO WE STARTED TO WONDER
WHAT IS THE ROLE OF HIF IN THIS
EPITHELIUM AND HOW IS IT
CONTRIBUTING TO DAMPENING OF
THIS DISEASE?
SO WE GENERATED EPITHELIAL
SPECIFIC KNOCKOUTS AND IN FACT
THESE MICE JUST GETTING RID OF
HIF1 ALPHA IN THE EPITHELIA
COMPLETELY PROTECTED THEM FROM
DISEASE, ALSO IN THE SECOND
MODEL PSORIASIS.
SO WE DUG A LITTLE BIT DEEPER.
AND OF COURSE HIF IS KNOWN TO
CONTROL MANY, MANY DIFFERENT
ASPECTS OF DISEASE PATHOLOGY,
INCLUDING EXPRESSION OF VEGF
ALPHA, BUT WE SAW AWE KEY WAY IN
WHICH THINGS WERE CHANGING IN
THE HIF1 ALPHA DEFICIENT
EPITHELIAL DEFICIENT MICE WERE
GLYCOLYSIS, WONDERED IS TOGGLING
THIS METABOLIC PROGRAM GOING TO
ALTER DISEASE PATHOLOGY, IS THIS
A KEY MEANS BY WHICH THIS FACTOR
IS FUNCTIONING IN THIS CONTEXT?
SO FIRST WE NEEDED TO SEE, IS
THIS ACTUALLY HAPPENING IN VIVO
AND INDEED IN THE CONTEXT OF
SORT OF MOUSE MODEL YOU SEW SAW
THAT EXPRESSION OF GLUT1 HIGHLY
ENRICHED, HIGH RA INFINITY
TRANSPORTER FOR GLUCOSE, IF YOU
GET RID OF HIF1 ALPHA IN THE
EPITHELIA SPECIFICALLY HERE THE
ENTIRE IMMUNE COMPONENT IS
INTACTD YOU LOSE THIS GLUT1
EXPRESSION, REALLY COMPELLING
THAT IN FACT IT IS WORKING IN
THIS WAY.
SO THEN WE DECIDED TO GET RID OF
GLUT1 SPECIFICALLY IN THE
EPITHELIA AND JUST LIMITING THE
ABILITY OF THE EPITHELIA TO
UPTAKE GLUCOSE, NOT AT BASELINE,
BASELINE THESE MICE WERE FINE,
COMPLETELY PREKED THEM FROM THE
EPITHELIA PATHOLOGY, AND ALSO
THE VASCULAR AND NEURONAL
PATHOLOGY H SEVERAL FACETS OF
PATHOLOGY DOWNSTREAM OF THE
EPITHELIA WERE CONTROLLED BY
THIS.
AND OF COURSE MOST RELEVANT
HERE, REALLY CONTROLS THE IMMUNE
INFILTRATED AND T CELL
PATHOLOGY.
SO THIS WAS QUITE EXCITING TO US
BECAUSE BASICALLY LIMITING THE
METABOLISM OF ONE CELL TYPE WITH
NO PERTUBATION IN THE IMMUNE
SYSTEM COMPLETELY SHUT DOWN THIS
ENTIRE PATHOLOGICAL CIRCUIT H
AND REALLY SO WE STARTED TO
WONDER HOW THIS WAS HAPPENING
AND WHAT IS THIS METABOLIC
PROGRAM IN THE EPITHELIA REALLY
CONTROLLING.
SO WILL WE WENT BACK TO OUR
SEQUENCING DATA AND TYPE 17
IMMUNE CELLS, BOTH ALPHA DAY
THAT AND GAMMA DELTA INCLUDING
DJ'S GROUP WHO HAS BEEN A
PIONEER IN THIS SPACE BUT NONE
OF THOSE THINGS WERE REALLY
DIFFERENTIALLY EXPRESSED.
SO WHAT'S GOING ON?
WHY ARE WE NOT SEEING ANYTHING
THAT'S IMPORTANT FOR
IMMUNE-RELATED CONTROL IN THE
CONTEXT OF THIS GL YOU T1
DEFICIENT MOUSE AND WE WONDERED
MAYBE IT'S INNOCENT OUR GENE
EXPRESSION, MAYBE GENE
EXPRESSION DOESN'T CAPTURE WHAT
WE TBHEED TO SEE AND MAYBE
IT'S -- WHAT WE NEED TO SEE AND
MAYBE TS A METABOLITE THAT IS
REALLY AT THE CRUX OF THIS
CROSSTALK.
SO ONE OF THE TERMINAL
END-PRODUCTS OF GLYCOLYSIS IS
LACTATE.
THIS IS REALLY WELL-KNOWN
WHVMENT WE PUT IL17 INTO OUR
EPITHELIAL CULTURES WE GET DONS
TONS OF LACTATE.
ALSO WHEN YOU LOOK ATLESS IMQ
MOUSE THE PRESENCE OF THIS
INFLAMMATORY ENVIRONMENT
GENERATES A LOT OF LACTATE AND
DOES SO IN GLYCOLYSIS-DEPENDENT
MANNER.
WHEN YOU LOOK AT THE GLUT 1
DEFICIENT MICE, EPITHELIAL GLUTD
1 DEFICIENT MICE THEY DON'T MAKE
AS MUCH LACTATE, TELLING US
EPITHELIA ARE A PRIMARY SOURCE
OF LACTATE IN THIS CONTEXT AND
WONDERED IS LACTATE HERE
RESPONSIBLE FOR DRIVING THAT
PATHOLOGICAL IMMUNE RESPONSE?
SO WE INHIBITED HAD WITH EITHER
AN LDHLY INHIBITOR INHIBITS
LACTATE TO PYRUVATE OR INHIBITED
TRANSPORTERS THAT UPTAKE OR
RELEASE LACTATE BOTH ON
EPITHELIAL SIDE AND IMMUNE SIDE
AND IN BOTH CASES YOU'RE NOW
CUSHING THE IMMUNE RESPONSE AND
ESSENTIAL PHENOCOPYING GLUT 1
DEFICIENCY.
SO TO SUMMARIZE, I THINK WHAT
THAT TELLS US IS THAT DISEASE
STATES ARE MAINTAINED BY
METABOLIC CIRCUITS, AND WHEN WE
THINK ABOUT HOW DISEASE IS
FUELED AND THE ENERGY IT TAKES,
YOU NEED COOPERATIVITY.
SO I THINK ABOUT THIS A LOT, HOW
DO CELLS COMPETE FOR GROWTH
FACTORS.
BUT HOW DO THEY COMPETE FOR
NUTRIENTS AND WHY SHOULD THEY
COMPETE FOR NUTRIENTS WHEN THEY
COULD HAVE THIS SORT OF
COOPERATIVE ADAPTATION?
WE THINK THAT IN FACT AS THE
INFLAMMATORY CIRCUIT IS SET UP,
SO IS THE METABOLIC CIRCUIT AND
THAT ESSENTIALLY 17 SIGNALS
PATHOLOGICAL INTO THE EPITHELIA
WHICH THEN IN TURN PRODUCES A
NUMBER OF FACTORS THAT DRIVELY
17 CELLS BUT ALSO THERE'S THIS
METABOLIC CIRCUIT ESTABLISHED
AND SUSTAINED THAT SUSTAINS
THESE TWO CELLS, WHILE GLUCOSE
PROMOTES GLYCOLYSIS IN
EPITHELIA, LACTATE IS LIKELY
FEEDING INTO THE TCA CYCLE
ALTHOUGH WE'RE STILL WORKING ON
THOSE STUDIES AND THIS CIRCUIT
IS IN FACT UNDERPINNING ALL OF
THE OTHER PATHOLOGICAL FEATURES
OF THIS DISEASE AND COULD LIKELY
BE FUNCTIONING IN MANY, MANY
OTHER CHRONIC CONDITIONS WHERE
THE CIRCUIT IS LIKELY NOT GOING
TO BE BETWEEN EPITHELIA AND TYPE
17 BUT MANY OTHER TYPES OF
CELLS, FIBROBLASTS, ET CETERA,
AND IMMUNE CELLS.
SO I HOPE I'VE CONVINCED YOU AND
I TRIED TO STAY ON TIME, I CUT A
LOT OF SLIDES BECAUSE I'M AFRAID
OF AL, AND SO I HOPE I'VE
DEFENSED YOU THAT STUDYING
PHYSIOLOGY HAS A HUGE BENEFITED
IN UNDERSTANDING PATHOLOGY AND
CAN ACTUALLY TELL US
FUNDAMENTALS OF HOW OUR TISSUES
COPE WITH PERTUBATIONS AND THOSE
SAME FACTORS ARE COOPTED.
I DON'T THINK BIOLOGY IS
PARTICULARLY CREATIVE.
I THINK THEY JUST HIJACK WHAT'S
ALREADY THERE.
SO I'LL STOP THERE.
HAD AND THANK MY AMAZING LAB, IT
IS TRULY A PRIVILEGE AND
PLEASURE TO WORK WITH THEM, AND
I WILL TAKE ANY QUESTIONS IF
THERE ARE ANY.
[APPLAUSE]
>> VERY NICE TALK.
CAN YOU HEAR ME?
>> YES.
>> SO THE ARE TH17 CELLS DON'T
COME IN JUST ONE FLAVOR, RIGHT?
IN THEIR HOMEOSTATIC CONDITIONS
THAT YOU DESCRIBE, THE IL17
PRODUCING CELLS -- YOU STILL
MAINTAIN THE CONTEXT.
BUT IN THE PATHOLOGICAL
CONDITION LIKE PSORIASIS YOU
HAVE IL23 ACTING ON TL17 CELLS,
IT CHANGES THEIR METABOLIC
PROFILE AND FUNCTIONAL PROFILE
AS WELL.
SO IT MAY NOT BE JUST THAT THE
SAME CELL IS DOING BOTH BECAUSE
OF IL17 BECAUSE IF YOU LOOK AT
THE METABOLIC PROFILE USING
OTHERS, YOU WILL SEE THAT THE
METABOLISM OF HOMEOSTATIC TH17
CELLS IS VERY DIFFERENT FROM THE
PATHOGENIC TH17 CELLS.
AND HAVE YOU TRIED TO LOOK
WHETHER YOUR TH17 CELLS ACTUALLY
MAKE LACTATE AND IT GOES BACK
ONTO THE EPITHELIA?
>> YES.
SO FIRST, I TOTALLY AGREE WITH
YOU.
I THINK THAT WE'RE SO OBSESSED
WITH SUBSETTING CELLS AND I AM
MUCH MORE INTERESTED IN SORT OF
THE END FEEK TORE PROGRAMS LIKE
IL17.
IN THE CASE OF THE MOUSE MODELS
I'VE SHOWN YOU, GAMMA DELTA 17'S
ARE THE MOST QUOTE, UNQUOTE,
PREDOMINANT.
I THINK IN HUMAN TISSUES IT'S A
VERY DIFFERENT GAME, SO THE
SUBSET ISSUE IS I THINK MUCH
MORE AN ABERRATION OF HOW WE
WRITE PAPERS THAN BIOLOGY.
SO I 100% AGREE WITH YOU.
>> CAN I JUST SAY FOR JUST A
MINUTE -- CAN I JUST DISAGREE
WITH YOU FOR JUST A MINUTE?
>> WE'LL DISGREERKS THAT'S.
>> IF YOU REALLY LOOK AT THE
HOMEOSTATIC CONDITIONS EVEN IN
HUMANS IN THE GUT, YOU HAVE 17
AND TEN PRODUCING CELLS IN THE
GUT, THOSE ARE THE MAJORITY OF
THE CELLS IN THE GUT, UNLESS
THEY GET ACTED BOYNE IL23, THEY
DON'T BECOME PATHOGENIC.
THAT'S WHY ENTER IL23 WORKS SO
WELL IN PSORIASIS AND COLITIS,
IN FACT, THEY COULDN'T FIND THE
END DOSE, IL17 WORKS, BUTLY IL23
SITS OWN DATA, SO IT'S NOT JUST
ABERRATION OF ACTUALLY
SUBSETTING.
IN FACT, IF YOU LOOK AT OUR DATA
FROM THE MOUSE AND THOSE DATA
FROM HUMANS, PROFILE IS EXACTLY
THE SAME.
IN AND IN CASE OF CAN TI DA
ALBICANS YOU HAVE PATHOGENIC 17
CELLS N CASE OF STAPH AUREUS IN
HUMANS, YOU HAVE HOMEOSTATIC 17.
>> YES, I UNDERSTAND YOUR
QUESTION.
WHAT I'M SAYING IS THE SUBSETS
ARE VERY DIFFERENT DURING
BASELINE AND SO IL23 MAY BE
ENRICHING FOR DIFFERENT SUBSET
OF CELLS.
FOR INSTANCE, IN THE SKIN, THE
DOMINANT PRODUCERS OF IL17 ARE
NOT TH17 CELLS.
THEY ARE MATE CELLS, GAMMA
DELTA, THAT'S WHAT I'M SAYING,
THERE'S ALSO TISSUE-SPECIFIC
DIFFERENCES.
I DO THINK THAT WE HAVEN'T
TESTED FOR LACTATE PRODUCED FROM
TH17 CELLS AND AT BASELINE I
KNOW AND YOU'VE DONE BEAUTIFUL
WORK IN THIS WHERE THEY ARE
GLYCOLYTIC AND MAKING
GLYCOLYSIS, WHEREAS IN THIS
INFLAMMATORY CONDITION WE THINK
THEY'RE TURNING ON THE TCA
CYCLE, THAT MAY BE DEPENDENT ON
IL23.
>> I LOVED YOUR WORK.
>> NO, NO, I THINK WE'RE
AGREERKS RIGHT?
I THINK WE'RE AGREEING.
>> THANK YOU FOR THE ANSWER.
BUT ACTUALLY VERY NICE WORK.
>> WE'LL TALK LATER, BECAUSE YOU
CLEARLY DISAGREE WITH ME STILL.
[LAUGHTER]
>> DIDN'T INTRODUCE HIMSELF,
THAT'S VIJAY.
>> HI, SHRUTI.
NICE WORK.
I HAVE ONE QUESTION.
I'M R YOU TI, I'M A RESEARCH
FELLOW IN VANOSH'S LAB AND I
HAVE TWO QUESTIONS.
FIRST IS, SAY THAT IL17 ACTS
SECONDARY SIGNAL IN STABILIZING
HERE AND THEN DRIVING
GLYCOLYSIS, BUT DO YOU KNOW HOW
MUCH IS INDEPENDENT THAT CAN
IL17 DRIVE.
>> GLYCOLYSIS INDEPENDENT OF
HERE, FIRST?
AND CAN IL17 PRODUCE IT?
THE ROLE OF IL23'S TO COMPLETELY
RULE THEM OUT?
LIKE HAVE YOU TESTED IN RAG
KNOCK-OUT.
>> FIRST, I WILL SAY, SO WE
DON'T THINK THAT IN EPITHELIAL
CELLS WHEN YOU TREAT WITH IL17
YOU NEED HIF1 ALPHA.
THE HIF1 HAD ALPHA KNOCKOUTS
DON'T INDUCE.
I KNOW THERE'S OTHER GENES TO
TURN ON, LIGAND, THAT'S NOT
OPERATIVE IN THIS CONTEXT.
DISHEANLT MEAN IT NEVER HAPPENS.
IN THE ORIGINAL PAPER WE
ACTUALLY TESTED EVERY POSSIBLE
GAMMA T DEFICIENT MOUSE, GAMMA
DELTA'S MATES NTH17'S HAD, ILC'S
DWE WITH JUST RAG MICE AND SEEMS
TO COME DOWN TO GAMMA DELTAS IN
MATES.
AGAIN THIS COMES BACK TO THIS
ABERRATION OF MOUSE EXPIN HOW
IT'S VERY DIFFERENT THAN HUMAN
SKIN AND JUST THE CELLULAR.
PLAYER PLAYERS DRIRCHT.
I WOULD LIKE TO FOCUS -- PLAYERS
ARE DIFFERENT.
I WOULD LIKE TO REALLY FOCUS ON
THE END-PRODUCT.
>> THANK YOU.
I'M VALENTINA, ALSO WORKING ON
THE SKIN, I'M IN THE GROUP CR
WITH CHEN HERE AT NIH, I'M A
PH.D. STUDENT.
I ALSO WORK ON PSORIASIS AND I
HAVE TO CONFIRM THAT IN MICE
MODELS IT'S THE MAJOR 17
PRODUCER ARE GAMMA DELTA T
CELLS.
SO MY QUESTION WOULD RATHER BE
IN STEADY STATE CONDITION, SO
WHEN YOU WOUND THE MICE, IF ALSO
THEY ARE THE GAMMA DELTAS ARE
THE MAJOR IL17 PRODUCER, AND
THEN MY QUESTION WOULD BE, HOW
DOES IT COME HAD FOR THEM TO
PRODUCE 17?
IS IT BECAUSE BY GENERATING THE
WOUND YOU ALLOW THE BACTERIA TO
ENTER THE SYSTEM AND THEN YOU
HAVE THIS SORT OF REACTION, OR
DO YOU THINK IT'S MOSTLY BECAUSE
OF THE EPITHELIAL CELLS DID IN
INDEED GET DAMAGED AND THEN
START THIS CASCADE THAT THEN
PRODUCES OR INDUCES 17
PRODUCTION?
>> YEAH, I MEAN, I THINK THAT
THOSE THINGS ARE NOT MUTE
YOU'LLLY EXCLUSIVEMENT RIGHT
SNING THEY'RE PROBABLY ARE NOT
MUTUALLY EXCLUSIVE.
RIGHT?
I THINK THEY'RE PROBABLY
HAPPENING AT THE SAME TIME.
I THINK YOU'RE SPLITTING HAIRS.
IN MICE, GAMMA DELTAS, UNLESS AS
HAS SHOWN THEY RECEIVE THE RIGHT
MATE CELL DEVELOPING SIGNAL.
SO YOU CAN HAVE COMPENSATION
FROM MATE CELLS IF YOU GIVE MICE
THE RIGHT SIGNALS TO DEVELOP
MATE CELLS AS WE DO IN OUR SKIN
BECAUSE WE GET THE RIGHT SIGNALS
AT THE RIGHT TIME.
SO AGAIN SPF MICE JUST DIFFERENT
BEASTS.
>> THANK YOU.
>> THAT WAS GREAT.
THANK YOU VERY, VERY MUCH.
SORRY, I THINK IN THE BACK THEY
DON'T HEAR US IF YOU DON'T TALK
DIRECTLY INTO THE MIC.
SPECTACULAR TALK.
IN TERMS OF YOUR KNOCKOUTS OF
HIF1 ALPHA AND OF GLUT 1, DO
THEY HAVE SIMILAR EFFECTS ON THE
PROLIFERATIVE STATUS OF THE
CELLS, AND IN THAT CONTEXT, YOU
REALLY TALK ABOUT THE EFFECT OF
GLUT 1 IN TERMS OF LACTATE.
BUT DO YOU KNOW APPROXIMATE
THERE ARE DIFFERENCES IN TERMS
OF NUCLEOTIDE POOLS?
>> OH, WEFNLT LOOKED, WE HAVE
ACTUALLY NOT ASSESSED THAT THE .
THAT'S A REALLY IMPORTANT POINT
BECAUSE THERE IS A HUGE
PROLIFERATIVE EFFECT AS YOU
ALLUDED AND I DON'T KNOW HOW
MUCH OF IT IS BECAUSE IT'S
DEPLETED NUCLEOTIDE POOLS.
>> IS IT THE SAME IF YOU KNOCK
OUT HIF1 FAL OR GLUT 1?
>> IN TERMS OFLY PROLIFERATION,
YES.
>> IT'S EQUIVALENT AND.
>> IT'S QUIF ZEENT WE CAN TALK
LATER BUT IT WOULD BE
INTERESTING TO SEE WHAT THE
PERCENTAGE IMPACT IS OF LACK
TAILLIGHT VERSUS NUCLEOTIDE
POOLSES.
>> YEAH, WE JUST HAVEN'T EVEN
CONSIDERED T SO IT'S IT'S A
REALLY GOOD POINT.
>> JAY ZABROSKI FROM NCLI, I
HAVE MAY HAVE MISSED THIS, IS
THERE A ROLE FOR RGF1 IN THE
METABOLIC EFFECTS OF HIF1 ALPHA
THAT YOU DESCRIBE?
>> THAT'S NOT ONE OF THE FACTORS
WE SEE DIFFERENTLY EXPRESSED.
SO I REALLY CAN'T COMMENT ON IF
THERE IS OR ISN'T AS FAR AS --
BUT THAT'S WE DWHROOND BECAUSE
WE JUST HAVEN'T TESTED IT.
>> OKAY, BECAUSE IT HAS BEEN
ASSOCIATED WITH HIF1 EFFECTS SO
CAN OBVIOUSLY INFLUENCE GLUCOSE
METABOLISM AND OTHERS.
>> THAT'S A GOOD POINT.
ESSENTIALLY THE WAY WE DID THIS
IS WE TOOK A VERY LET THE DATA
TELL US WHERE TO GO, AND THE
GENES THAT WERE THE MOST
DIFFERENT WERE GLYCOLYSIS,
THAT'S KIND WHAF WE WENT AFTER.
WE DIDN'T SEE GENES THAT WERE
DIFFERENT IN IGF SIGNALLING OR
THE IGF RECEPTOR, BUT THAT
DOESN'T MEAN IT'S NOT -- I THINK
WE JUST HAVEN'T EXPLORED IT.
THANKS GROO THANK YOU.
>> THANK YOU.
[APPLAUSE]
>> SO I GUESS WE'RE STAYING WITH
OUR 17 PRODUCING CELLS FOR A B
OUR NEXT SPEAKER IS VAN
GENTLEMAN LAZ VICK FROM -- OUR
NEXT SPEAKER IS VAN GENTLEMAN
LAZAREVIC,.
>> THAT'S A PERFECT INTRODUCE
FOR MY TALK TODAY.
I'M DELIGHTED TO BE HERE AND
SHARE WITH YOU OUR MOST RECENT
STUDY ON THE ROLE OF THE
TRANSCRIPTION FACTOR ERRING 2 IN
CONTROLLING -- EGR2 IN
CONTROLLINGLESS TH17 CELLS IN P
THE CNS COMPARTMENT.
THEY SPECIALIZE INTO SUB SETSZ
UPON CYTOKINE STIMULATION, EACH
GUIDED BY THEIR LINEAGE DEFINING
TRANSCRIPTION FACTORS THAT
CONTROL UNIQUE GENE EXPRESSION
PROGRAMS.
WHILE THESE T HELPER CELLS ARE
INSTRUMENTAL IN CLEARING
PATHOGENS, SO THIS IS BEST
EXEMPLIFIED BY TH17 CELLS.
NOW, IN NORMAL TISSUES NORMAL
STEADY STATE FOUND HAD BARRIER
TISSUES WHERE THEY PROTECT US
FROM FUNGAL AND BACTERIAL
INFECTIONS, THEY ALSO MEDIATE
TISSUE HOMOEOSTASIS AND THEY CAN
ALSO PROMOTE WOUND HEALING.
THEY ARE -- THEIR PROTOTYPICAL
HAD FUNCTIONS MEDIATED BY
CYTOKINES, SUCH AS IL17 AND 22,
PATIENTS WHO HAVE IMPAIRED TL17
RESPONSES OFTEN SUFFER FROM
CHRONIC MUCOSAL CANDIDIASIS CIS
AND ALSO SKIN INFECTIONS, BUT
ALSO THEY HAVE BEEN IMPLICATED
AS KEY DRIVERS OF INFLAMMATORY
DISEASES AND AUTOIMMUNE DISEASES
AND DESPITE THIS EXTENSIVE
RESEARCH INTO TH17 CELL BIOLOGY
THERE'S SO MUCH TO BE LEARNED
ABOUT THE TRANSCRIPTIONAL
REGULATORS OF TH17 CELLS AND
ALSO THE MECHANISMS THAT
UNDERLIE TISSUE PATHOLOGY AND
DIFFERENT TYPES OF AUTOIMMUNE
DISEASES.
SO FOR EXAMPLE, IN PSORIASIS,
TH17 CELLS SECRETE IL22 THAT
TRIGGERS HYPERPLASIA CRLS BY
ACTIVATING STAT 3, WHILE IN
RHEUMATOID ARTHRITIS, INDUCE
RANK LIGAND AND MIE BRO BLAST
AND IS AGGRAVATE LOCAL
INFLAMMATION BY PRODUCING
INFLAMMATORY CYTOKINES N MOUSE
MODELS WHERE TH17 CELLS DRIVE
INFLAMMATORY BOWEL DISEASE
THERE'S A VERY IMPORTANT
TRANSITION OF TH17 CELLS INTO
TH1 LIKE CELLS CHARACTERIZED BY
EXPRESSION OF GAMMA AND THESE
CYTOKINES DRIVE PATHOLOGY.
JUST LIKE INFLAMMATORY BOWEL
DISEASE, THIS FUNCTIONAL
PLASTICITY OF TH17 CELLS ALSO
CONTRIBUTES TO NEUROPATHOLOGY IN
MS.
SO IN MY LAB WE TRY TO
UNDERSTAND HOW TH17 CELLS
PROMOTE NEUROINFLAMMATION,
SPECIFICALLY WE WANT TO IDENTIFY
TRANSCRIPTIONAL REGULATORS THAT
CONTROL DEVELOPMENT OF
PATHOGENIC TH17 CELLS IN THE CNS
COMPARTMENT.
SO IN LINE WITH THESE INQUIRIES
THERE ARE SEVERAL IMPORTANT
QUESTIONS THAT EMERGE.
FIRST K WE IDENTIFY
TRANSCRIPTIONAL REGULATORS OF
PATHOGENIC TH17 CELLS AND CAN WE
TARG THE PATHOGENIC EFFECTOR
PROGRAM OF TH17 CELLS WITHOUT
INTERFERING WITH THEIR
PROTECTIVE BARRIER FUNCTION IN
TISSUES?
SO TO TRY TO UNDERSTAND THE
TRANSCRIPTIONAL PROGRAM OF
PATHOGENIC TH17 CELLS, WE
COMPARED THE TRANSCRIPTIONAL
PROGRAM OF MEMORY CD4 T CELLS
THAT WERE CHARACTERIZED AS BEING
MILDLY REACTIVE OR AT THE TIME
MEASURE POSITIVE OR MYELIN
NONREACTIVE OR AT THE TIME TROA
MERE NEGATIVE, FROM HEALTHY
CONTROLS AND MS PATIENTS.
AS YOU CAN SEE HERE MYELIN
REACTIVE CD4 T CELLS WERE
CHARACTERIZED BY ELEVATED
SPREFTION EGR FAMILY OF
TRANSCRIPTION FACTORS, EGL1,
2-RBGS APPROXIMATE AND 4 DPIERD
THE MYELIN NONREACTIVE T CELLS
FROM THE SAME MS PATIENT.
WE CAN SEE HERE IF REALLY HIGHLY
UPREGULATED.
WHEN WE PERFORMED CLUSTERING
ANALYSIS INVOLVING PATHOGENICITY
ASSOCIATED GENES WITH EGR FAMILY
TRANSCRIPTION FACTORS, AN
INTERESTING PATTERN EMERGED.
SO THESE MYELIN REABLGHTTIVE CD4
T CELLS FROM MS PATIENTS THEY
CLUSTER SEPARATELY FROM MYELIN
NONREACTIVE CD4 T CELLS FROM MS
PATIENTS AND ALSO HEALTHY
CONTROL, SUGGESTING THAT PERHAPS
THERE IS ASSOCIATION BETWEEN EGR
FAMILY TRANSCRIPTION FACTORS AND
THE PATHOGENIC FUNCTIONS OF CD4
T CELLS IN MS.
WE ALSO DECIDED TO LOOK P AT --
INDUPSED BY MICROBES IN THE GUT,
SUCH AS BACTERIUM, SO WE WANTED
TO EXPLORE THIS DIFFERENTIAL
EXPRESSION OF EGR FAMILY MEMBERS
IN HOMEOSTATIC TH17 CELLS
ISOLATED FROM THE LAMINA PROPRIA
OF IL17 REPORTER MICE AS STEADY
STATE TO THAT OF PATHOGENIC CD4
T CELLS ISOLATED FRAWLS IL17 GFP
REPORTER MICE AT THE PEAK OF
NEUROINFLAMMATION USING EAE
MODEL.
UNLIKE EGL1 THERE WAS EXPRESSED
IN BOTH PROTACTIV AND -- CELLS
EGR2 SPECIFICALLY UP REGULATED
IN PATHOGENIC CD4 CELLS FROM THE
CNS OF EA TEST MICE, UNLIKE
HUMAN, MOUSE CELLS DO NOT
EXPRESS EGR3 AND 4 UNDER THESE
CONDITIONS.
SO WHAT ARE THE EGR FAMILY
TRANSCRIPTION FACTORS?
THEY STAND FOR EARLY GROWTH
RESPONSE FAMILY, THERE ARE FOUR
MEMBERS, ONE, TWO, THREE AND
FOUR.
IN MOUSE IMMUNE SYSTEM EGR ONE
TWORKS AND THREE ARE INDUCED BY
T CELL RECEPTOR SIGNALS.
THEY SHARE MORE THAN 80% OF HOME
OMG IN THE DNA BINDING DOMAIN
AND THEY BIND IDENTICAL DC RICH
SEQUENCES.
HOWEVER, IF YOU MAKE MICE THAT
LACK ONE OF THESE EGR FAMILY
MEMBERS, THEY HAVE VERY DISTINCT
FAMILY MEMBERS SUGGESTING
PERHAPS THEY ALSO HAVE UNIQUE
GENE REGULATION PROGRAMS.
IN IMMUNE SYSTEM, AS I MENTIONED
THEY'RE DOWNSTREAM OF T CELL
RECEPTOR SIGNALLING, SO NO NOT
SURPRISINGLY THEY PLAY AN
IMPORTANT ROLE AT DIFFERENT
STAGES OF T CELL DEVELOPMENT IN
THE THYMUS.
SO WE DECIDED TO LOOK AT THE
EXPRESSION OF EGR ONE AND EGR
TWO PROTEIN AT DIFFERENT STAGES
OF TH17 DIFFERENTIATION AND
FOUND THAT BOTH EGR ONE AND TWO
ARE CONSIDERED TO BE EARLY
INDUCED TRANSCRIPTION FACTORS,
THEY'RE UP WITHIN FOUR HOURS OF
ACTIVATION, AND THEN THEY'RE
CO-EXPRESSED IN ABOUT 30% OF
TH17 CELLS UP TO 48 HOURS POST
ACTIVATION AFTER WHICH THEIR
PROTEIN EXPRESSION IS COMPLETELY
DECLINED.
TO LOOK AT THEIR FUNCTION WE USE
RETRO VIAL ARE TRANS
TRANSDUCTION SYSTEM, ISOLATED
CD4 DISPELZ THROUGH RETRO VIAL
TRANSACTION OVEREXPRESS ERG1 OR
2 AND LOOK AT THE COMMITMENT
FIVE DAYS LATER.
AS YOU CAN SEE HERE, UNLIKE EGR
ONE, EGR IT TWO WAS ABLE TO
UPREGULATE EXPRESSION OF QUL RHO
GAMMA CODING GENE COMPARED TO
LSZ ANTI-VECTOR CONTROL AND ALSO
SIGNIFICANTED INCREASED THE
FREQUENCY OF IF IL17 PRODUCING
CD4 T CELLS SUGGESTING THAT EGR2
ACTS AS A POSITIVE REGULATOR OF
TH17 CELL DIFFERENTIATION
PROGRAM.
BUT IS EGR2 REQUIRED FOR TH17
LINEAGE COMMITMENT?
SO THIS QUESTION WAS RAISED
BECAUSE WE HAVE SEEN COMPENSATE
TO HER INCREASE IN EGR FAMILY
MEMBERS WHEN ONE OF THE MEMBERS
WAS DELETED.
SO EGR1 DEFICIENCY IN
SIGNIFICANT UPREGULATION OF EGR2
IN mRNA AND EGR2 DEFICIENCY
INCREASES EGR1 mRNA EXPRESSION
WHEREAS EGR1 AND 2 DOUBLE
INSUFFICIENCY RESULTS IN
ENHANCED EGR3 mRNA CR
TRANSCRIPTION, THAT WAS THE
CASE.
WE FOUND WE HAVE TO DELETE ALL
THREE EGR FAMILY MEMBERS TO
SIGNIFICANTLY INHIBIT CELL
COMMITMENT.
BASED ON THESE DATA WE CONCLUDED
THAT EGR2 IS EXPRESSED IN
PATHOGENIC TH17 CELLS IN MICE
AND HUMANS DURING INFLAMMATION
AND WHEN OVEREXPRESSED EGR --
REQUIRED FOR TH17 LINEAGE
COMMITMENT DUE TO FUNCTIONAL
REDUNDANCY AMONGST EGR FAMILY
MEMBERS.
DOES EGR TWO CONTROL DEVELOPMENT
HOMEOSTATIC OR INFECTION INDUCED
TH17 CELLS?
TO ADDRESS THIS QUESTION, WE
DECIDED TO LOOK AT EGR2 PROTEIN
EXPRESSION IN INTESTINAL
HOMEOSTATIC CD4 T CELLS ISOLATED
FROM THE LAMINA PROPRIA OF SMALL
EXPWEFN COLON OF COLONIZED MOUSE
IN STEADY STATE CONDITION AND
SURPRISINGLY WE FOUND THAT ONLY
A SMALL FRACTION OF CD4 T CELLS
EXPRESSED EGR2 AT STEADY STATE.
CYTOKINE PRODUCTION IN
PARTICULAR IL17 WAS COMPARABLE
BETWEEN EGR2 KNOCK-OUT AND
WILD-TYPE CD4 T CELLS IN BOTH
TISSUES, SUGGESTING THAT EGR2 IS
NOT REQUIRED FOR HOMEOSTATIC
TH17 CELL DEVELOPMENT VFT BUT TO
CLEAR INTESTINAL PATH JENS SUCH
AS REDEMPTION MOUSE AND HUMANS
DEPEND ON INFECTION INDUCED TH17
CELLS.
AND THIS CAN BE ASSOCIATED WITH
TISSUE INFLAMMATION AND
PATHOLOGY.
BUT SURPRISINGLY WHEN WE CHANGED
THE MICE IT FROMS SIT TROA BAC
TEST R, THERE WAS NO CHANGE IN
INTESTINAL CD4 CELLS, MOREOVER,
KNOCK OUT CELLS EXPRESSED
SIMILAR AMOUNTS OF GAMMA IN TH17
COMPARED TO WILD-TYPE CONTROLS.
ALSO WE FOUND THAT MICE THAT
LACK EGR2 EXPRESSION
SPECIFICALLY IN T CELLS
CONTROLLED BACTERIAL BURDEN AND
LIKE WILD-TYPE ANIMALS THEY
DIDN'T SHOW ANY SIGNS OF BODY
WEIGHT LOSS.
SO COLLECTIVELY THIS
DEMONSTRATES THAT EGR2 IS NOT
REQUIRED FOR EITHER HOMEOSTATIC
OR INFECTION INDUCED TH17 CELLS.
BUT THEN WE BECAME INTERESTED
ABOUT THE ROLE OF EGR2 IN
CONTROLLING PATHOGENIC CD4 T
CELLS DURING ITS UP REGULATION
DURING NEUROINFLAMMATION IN
HUMANS WITH MS AND ALSO IN MICE
WITH EAE.
USING ACTIVE IMMUNIZATION MODEL
WITH MOCK PROTEIN WE FOUND WITH
30% OF CNS INFILTRATING CD4
CELLS EXPRESSED EGR2, AND ALSO
MICE IN WHICH WE DELETED EGR2
EITHER ONLY IN T CELLS OR IN KIM
L17 PRODUCER CELLS WERE
SIGNIFICANTLY PROTECTED FROM
DISEASE DPIERTD WILD-TYPE
CONTROLS.
-- DPIERD THE WILD-TYPE
CONTROLS.
ERRING 1 DEFICIENCY HAD NO
IMPACT RS CONCLUDE THAT GO EGR2
BUNLT EGR1 PROMOTES PATHOGENIC
TH17 CELL DEVELOPMENT OF SOS
WHAT IS THE FUNCTION OF EGR2 IN
THIS PATHOGENIC TH17 CELLS?
WE REALLY WANTED TO GO DEEPER
INTO THE MOLECULAR MECHANISMS BY
WHICH EGR2 PROMOTE PATHOGENICITY
SO TO ANSWER THIS QUESTION WE
COMPARED THE GENE EXPRESSION
PROGRAM OF SPLENIC CD4 T CELLS
OR CNS INFILTRATING CELLS
BETWEEN THE WILD-TYPE AND
KNOCKOUTS AND YOU CAN SEE HERE
THAT EGR2 DEFICIENCY HAD MANY
MORE IMPACT ON GENE EXPRESSION
PROGRAM IN THE SPLEEN HOWEVER IN
THE CNS THERE WAS A HUGE
DYSREGULATION IN THE GENE
EXPRESSION IN THE CNS WHERE THEY
NORMALLY REENCOUNTER THEIR
ANTIGEN.
SO WE WANTED TO IDENTIFY
MOLECULAR PATHWAY THAT IS ARE
REGULATED BY EGR2 AND WE USED
EGR2 RETROVIRUS OVEREXPRESSION
AND WE FOUND THAT EGR2
OVEREXPRESSION NORMALLY IN
NONPATHOGENIC TH17 CELLS INDUCED
THE EXPRESSION OF TEN OUT OF
SIXTEEN PATHOGENICITY ASSOCIATED
GENES COMPARED TO THE EMPTY
VECTOR CONTROL.
PATHOGENICITY ASSOCIATED GENES
IN THE CNS DURING EAE.
AND THIS ALSO INCLUDED THE
TRANSCRIPTION FACTOR T-BET.
NOW, BECAUSE T-BET IS SO
IMPORTANT FOR PATHOGENICITY OF
TH17 CELLS WE WANTED TO VALIDATE
THESE RESULTS IN VITRO SO IN
THIS PARTICULAR EXPERIMENT, WE
USED CD4 CELLS THAT WERE
CULTURED UNDER NONPATHOGENIC
POLARIZING CONDITIONS IN THE
PRESENCE OF BETA ONE AND EITHER
TRANSDUCED THEM WITH EMPTY
VECTOR CONTROL OR EGR1 OR EGR2
OVEREXPRESSING RETROVIRUS AND
YOU CAN SEE THAT AS EXPECTED,
NONPATHOGENIC WILL LSES TH17
CELLS DO NOT EXPRESS T-BET ASK
IN CONTRAST TO ARE ERG-1, ERG
2-6S ABLE TO INDUCE T-BET
EXPRESSION IN THESE CELLS,
SUGGEST THAT GO PERHAPS SOME
PATHOGENICITY IS CONTROLLED BY
2ERG MEDIATED UPREGULATED
LAITION OF THE TRANSCRIPTION
FACTOR T-BET.
TO TEST THIS WE DID A PASSIVE
TRANSFER ADOPTED TRANSFER EAE
EXPERIMENTS IN WHICH THLSZ TH17
CELLS WERE CULTURED UNDER
NONPOLARIZING TH17 CONDITIONS IN
THE PRESENCE OF MS THETA,
RETROVIRUS.
ALSO INCLUDED PATHOGENIC IL23
STIMULATED CELLS AS A POSITIVE
CONTROL.
AND UNLIKE NONPATHOGENIC TH17
CELLS TRANSDUCED WITH THE
ANTI-VIRUS, THOSE THAT EXPRESS
EGR2 WERE NOW ABLE TO INDUCE
NEUROINFLAMMATION.
SO THESE DATA SUGGEST THAT EGR2
IS REALLY CONTROLLING THE
PATHOGENIC PROGRAM OF THESE
MYELIN REACTIVE PATHOGENIC TH17
CELLS.
IN ADDITION TO T-BET THERE WAS
ONE VERY INTERESTING PATHWAY
THAT STOOD OUT, AND IT INVOLVED
GENES INVOLVING T CELL MIGRATION
INCLUDING CHEMOKINE AND IS
CHEMOKINE RECEPTORS, SAME SUBSET
OF CHEMOKINES AND CHEMOKINE
RECEPTORS WAS ALSO UP REGULATED
IN PATHOGENIC IL23 STIMULATED
TH17 CELLS SO WE POSTULATED THAT
PERHAPS 2ERG CONTROLS IMMUNE
CELL MIGRATION TO THIS -- CNS,
INDEED THE CASE, COMPARTMENT WAS
NORMAL BETWEEN WILD-TYPE AND
EGR2 KNOCK-OUT MICE BUT IN THE
CNS THERE WAS A HUGE DECREASE IN
THE ACCUMULATION OF PATHOGENIC
CD4 T CELLS.
IN ADDITION, WE FOUND THAT THE
EXPRESSION OF MYELOCYTE KINES
L1, 3 AND 4 EXPRESSION, SINCE
THESE CHEMOKINES RECRUIT, WE
FOUND SIGNIFICANT REDUCTION IN
INFLAMMATORY MONOCYTES
SUMMARIZED HERE AND ALSO CAN
EXPRESSING DENDRITIC CELLS.
SO COLLECTIVELY WHAT I HAVE
SHOWN YOU HERE IS THAT WE HAVE
IDENTIFIED A NOVEL FUNCTION FOR
THE TRANSCRIPTION FACTOR EGR2 IN
DIFFERENTIATING PATHOGENIC TH17
CELLS THAT PROMOTE INFLAMMATORY
DISEASES SUCH AS MS, AND THOSE
THAT ARE PROTECTIVE AND
BENEFICIAL TH17 CELLS IN THE
GUT.
I HAVEN'T SHOWN YOU BUT WE ALSO
LOOKED AT CANDIDA ALBICANS AND
BACTERIUM AND RESPONSES, THESE
MUCOSAL TH17 CELLS ARE
COMPLETELY NORMAL WHEN WE KNOCK
OUT EGR2.
BUT IN THE CONTEXT OF THE CNS
AND AUTOIMMUNE INFLAM ARE
INFLAMMATION, THIS --
PATHOGENICITY ASSOCIATED GENES
AND ALSO CHEMOKINES AND CELL
RECRUITMENT TO THE CNS.
I SLEANLT A CHANCE TO SHOW YOU
THE DATA, BUT THIS CONTEXTUAL
REQUIREMENT FOR EGR2 WAS
REGULATED BY THE AFFINITY AND
THE STRENGTH OF T CELL RECEPTOR
SIGNAL SIGNALLING AS WELL AS
TISSUE SPECIFIC CUES.
AND FINALLY, I WOULD LIKE TO
THANK THE MEBLES OF MY TEAMLY.
-- THE MEKS OF MY TEAM.
-- AND FINAL HL, I WOULD LIKE TO
THANK THE MEMBERS OF MY TEAM.
SO I WOULD LIKE TO THANK MY NIH
COLLABORATORS AND MY COLLEAGUES
AT EXPERIMENTAL IMMUNOLOGY
BRANCH.
THANK YOU VERY MUCH FOR YOUR
ATTENTION.
I'M HAPPY TO ANSWER ANY
QUESTIONS.
[APPLAUSE]
SO THIS IS DR. NICKI --
>> YES, A WONDERFUL TALK.
CAN YOU HEAR ME?
>> YES.
>> SO THE WAY YOU PRESENTED
THIS, IT SEEMS LIKE EGR2 IS VERY
SPECIFIC TO YOUR INFLAMMATION
FROM WHAT BEUF SEEN SO FAR.
CAN YOU ELABORATE A LITTLE MORE
ON THE TISSUE SPECIFIC CUES
REGULATED IN THE BRAIN OR
CONTEXT OF INFLAMMATION AND
BECAUSE SINCE YOU'RE SHOWING IT
MIGRATION, SOME OF IT MIGHT BE
UPREGULATED BEFORE THEY GET
THERE OR HOW ARE YOU THINKING OF
THAT?
>> SO THE SIGNAL THAT DEFINITELY
CROAMS THE LEVEL OF EGR2
EXPRESSION EVEN IN MONOCLONAL
POPULATION CELLS IS THE STRENGTH
AND THE AFFINITY OF T CELL
RECEPTOR SIGNALLING.
ONE POSSIBILITY IS THIS AUTO
REACTIVE T CELLS THAT EXPRESS
HIGH AFFINITY TCR'S MAY HAVE
HIGH LEVELS OF EGR2 THAN
POLYFUNCTIONAL MICROBIAL
SPECIFIC T CELLS.
SO ONE OF THE THINGS THAT CAN BE
MAKING THE DIFFERENCES ARE THOSE
TISSUE --
>> IMMUNITY AREA, ARE THERE
SETTINGS BETWEEN IMMUNITY YOU
COULD HAVE THAT AS WELL
POTENTIALLY?
>> SO ONE THING WE LOOKED AT WAS
COLITIS FOR EXAMPLE, T CELL
DRIVEN COA LIGHTS MODEL.
THERE JUST LIKE WITH CITRO
BACTER RETENTION WE FOUND NO
CHANGE IN DR.-- EXPRESSION, DOWN
REGULATED.
THERE'S SOMETHING ABOUT THE GUT
THAT IS SUPPRESSING EGR2.
NOW, TGF BETA SOMEBODY
MENTIONED, YES, WE LOOKED AT IT,
DEFINITELY HAS A DOSE DEPENDENT
INHIBITION OF ALL EGR FAMILY
MEMBERS BUT THE CNS ALSO HAS
HIGH LEVELS OF TGF BETA AS WELL
SO WE'RE NOT QUITE SURE WHETHER
TGF BETA IS THE ONLY SIGHT DHIEN
SUPPRESSES IT.
>> SECOND SIDE OF TISSUE
COMPARTMENT WITH THE HUMAN ATLAS
DATA OUT I DON'T KNOW IF YOU HAD
AN OPPORTUNITY TO LOOK THE ADATA
PUBLISHED IN HUMANS AND MICE AT
DIFFERENT SETTINGS TO SEE
WHETHER T CELLS IN DIFFERENT
PATHOLOGIES HAVE 2ERG OR NOT.
>> WE DID LOOK AT, YEAH, DIDN'T
LOOK AT OTHER PSORIASIS FOR
EXAMPLE, WE HAVEN'T LOOKED IN
THAT PARTICULAR CASE.
BUT WE WILL DEFINITELY LOOK AT
THAT IN THE FUTURE.
>> WONDERFUL TALK.
>> THANK YOU.
THANKS.
>> HELLO.
I CAN JUST SPEAK LOUD.
CAN YOU HEAR ME?
>> YES.
>> SO IT WAS A VERY NICE TALK.
I HAD A QUESTION ON THE
CHEMOKINE PART.
SO KIND OF THE OTHER SIDE OF THE
COIN, ON THE RESIDENTIAL PART,
DO YOU THINK THERE'S A LOSS OF
RESIDENCY AND MORE MIGRATION AND
CIRCULATION OF THOSE CELLS?
ARE.
>> IN THE ABSENCE OF --
>> YEAH.
>> THAT'S ACTUALLY A GREAT
QUESTION.
WE DO NOT LOOK, FOR EXAMPLE, AT
S1P1 RECEPTOR EXPRESSION BECAUSE
YOU NEED TO DOWN REGULATED IT TO
STAY IN THE CNS COMPARTMENT.
>> EXACTLY.
>> NORMALLY EVEN IN THE
WILD-TYPE SETTING WHEN WE LOOK
AT THE TH17 CELLS THAT GOT TO
THE CNS WE DIDN'T SEE MUCH AS
RECEPTOR SO WE CAN DEFINITELY
LOOK AT LEAST AT THE mRNA
LEVEL WHETHER WE SEE ANY DIMPS
THERE.
IT DIDN'T POP OUT AS ONE OF THE
PATHWAYS THAT IS DIFFERENTIALLY
REGULATED GROO BECAUSE EVEN THE
OTHER MEMBERS, ALL THE RETENTION
MODULES REGULATED BY THE T CELLS
BECAUSE MAYBE THEY'RE JUST, I
DON'T KNOW, MIGRATING MORE
BETWEEN THE TISSUE AND HAVING A
MORE --
>> OTHER TISSUE RETENTION ARE WE
LOOKED AT WERE CD69 AND 103 AND
THERE WERE NO DIFFERENCES IN
PROTEIN EXPRESSION.
>> OKAY.
THANK YOU.
>> THANK YOU VERY MUCH FOR YOUR
TALK.
MY NAME IS -- I WORK IN NIAD ON
VIRUSLESS RECEPTION, SO SINCE
THOSE EGR TRANSCRIPTION FACTORS
ARE INDUCERS OF TH17 CELLS HAVE
YOU LOOKED TO SEE WHETHER THEY
AFFECT EITHER DIRECTLY OR
INDIRECTLY ROR OR STAT OF THE
TRANSCRIPTION FACTORS?
>> THEY DEFINITELY WELL EGR2 NOT
OTHER EGR FAMILY MEMBERS,
REGULATE GAMMA T EXPRESSION, WE
DIDN'T SEE ANY DIFFERENCES IN
HIF1 OR STAT 3 EXPRESSION WHEN
WE KNOCK OUT EGR2 BUT DEFINITELY
EGR2 UP REGULAR LAILTS FOR GAMMA
T SO PROMOTES TH17
PROLIFERATION, LOSES EFFECT WITH
IF WE INCOME OUT GAMMA T.
IT'S DEFINITELY GAMMA T
DEPENDENT.
>> THANK YOU.
>> THANK YOU FOR THE NICE TALK.
I HAVE A BASIC QUESTION.
-- TH17 ARE LIKE RESIDENT CELLS,
BUT EAE MODEL ARE THEY COMING
FROM LIKE NOT THE RNTD OR THEIR
RECRUIT, GET RECRUITED TO THE
SITE?
-- ARE NOT OR THEIR RECRUIT?
IS THAT POSSIBLE THAT THAT
SITUATION MAKES A DIFFERENCE?
>> THANK YOU FOR THE QUESTION.
DEFINITELY EAE IS DRIVEN BY THE
RECRUITMENT OF THESE PATHOGENIC
TH17 CELLS SO RETENTION WOULD BE
ONE OF POSSIBLE MECHANISM THAT
IF IT'S DYSFUNCTIONAL COULD LEAD
IN THEIR CIRCULATION BACK OUT OF
THE CNS COMPARTMENT BUT THAT'S
WHATNOT WE SEE.
USUALLY WE DON'T SEE ENOUGH
RECRUITMENT TO THE CNS SO THE
MICE REMAIN PROTECTED.
THANK YOU.
THANKS A LOT FOR YOUR QUESTION.
THANK YOU.
[APPLAUSE]
>> OUR NEXT SPEAKER AND FINAL
SPEAKER BEFORE THE BREAK IS
VIJAY KUCHROO, FROM HARVARD
MEDICAL SCHOOL, NEURO IMMUNE
INTERACTIONS IN ENTERIC NERVOUS
SYSTEM.
THANK YOU FOR COMING, VIJAY.
>> AFTER SHRUTI AND VAN
GENTLEMAN'S TALK, I THOUGHT I
SHOULD ALSO GIVE A TH17 TALK.
-- AND VANJA'S TALK, I THOUGHT I
SHOULD ALSO GIVE A TH17 TALK.
S ARE.
[PAUSE] THIS IS NOT THE MAIN TOM
TOOP -- THIS IS NOT THE MAIN --
THIS IS NOT THE MAIN TOPIC OF MY
LAB BUT I THOUGHT I WILL TALK
ABOUT SOMETHING I NEVER TALK B
I'LL TALK BTD NEURO IMMUNE
INTERACTIONS AND AND HOW
IMPORTANT THEY MAY BE IN HOW THE
IMMUNE SYSTEM SEES THE NERVOUS
SYSTEM AND NERVOUS SYSTEM SEES
IMMUNE SYSTEM AND HOW THEY
CROSSTALK WITH EACH OTHER.
CAN YOU GUYS HEAR ME AT THE
BACK?
>> YES.
>> OKAY.
>> SOY OFTEN SEEM TO RUSH AT THE
END, AND IN FACT, THE PEOPLE
FROM IT WAS A COLLABORATION
BETWEEN THREE LABS, MY LAB AND
THESE OTHER LABS, SAM AND (?)
DID MOST OF THE WORK AND HELPED
US WITH SOME OF THE DATASETS.
AND WE GOT LOTS OF -- FROM THE
CHILDREN'S HOSPITAL AND GAVE US
R13 RECEPTOR, AND WITH ISAAC
CHLU WE DID SOME OF THE WORK AND
DAVID ARTIS I DON'T KNOW IF IS
IN THE AUDIENCE HERE OR NOT BUT
ALSO PROVIDED US WITH SOME HELP.
SO MY TALK I'LL DO IN TWO PARTS.
THE VERY FIRST PART OF THE TALK
IS SOME HISTORIC DATA ON ON HOW
WE IDENTIFIED NEUROPEPTIDE
RECEPTORS ON INNATE LYMPHOID
CELLS AND HOW THEY MAY FB
CROSSTALK INNATE LYMPH CELLS ARE
GETTING CUES FROM THE NERVOUS
SYSTEM.
THE SECOND PART OF THE TALK, I
EVER NOT SPOKEN ABOUT IT BEFORE,
SO YOU ARE MY FIRST GUINEA PIGS.
SO LET ME GET THE FIRST PART OF
THE TALK.
IN FACT, THE STORY STARTS ABOUT
30 YEARS AGO WHEN MY SON WAS
LITTLE, AND EFS FOUND TO BE
ALLERGIC TO ALMOST EVERYTHING.
HAD TO MILK, TO EKSZ HR TO NUTS,
AND -- TO EGGS, TO NUTS, THIS
WAS A TIME WHEN THERE WERE NO
PEANUT-FREE ZONES IN KINDER GARR
TENSE OR WHATEVER AND IT OFTEN
SO HAPPENED THAT ONCE HE HAD A
BOUT OF FOOD ALLERGIES, HE WILL
ACTUALLY WE CAN GIVE HIM
EPI-PEN, WE CAN GIVE HIM
ANTIHISTAMINES, EVERYTHING WAS
NORMAL OUT OF DANGER BUT IF HE
STARTED THROWING UP, IT WOULD
NOT STOP.
IN FACT, WE HAD TO TAKE HIM TO
THE BOSTON CHILDREN'S HOSPITAL
TO GET SEDATED.
THIS APPARENTLY HAPPENS IN ABOUT
20% OF THE YOUNG KIDS THAT HAVE
VERY SERIOUS FOOD ALLERGIES.
SO AT THAT TIME, IN FACT, I CAME
WITH THIS IDEA THAT MAYBE THEIR
IMMUNE SYSTEM AND NERVOUS SYSTEM
ARE INTIMATELY COMMUNICATING
WITH EACH OTHER, AND IT'S
POSSIBLE THAT THE IMMUNE SYSTEM
IS MAKING CYTOKINES AND
CYTOKINES ACTIVATE THE NERVOUS
SYSTEM WITH NEUROPEPTIDES AND
THAT ACT ON THEIR IMMUNE CELLS
AND THIS IS LIKE A FEED-FORWARD
LOOP AMPLIFYING AND YOU GET
ACTIVATION AND IRRITATION OF
NERVES HAD.
AND IRRITATION MUCH NERVES.
HAD AND THE IDEA THAT YOU SEE.
SO ONE OF THE EXPERIMENTS WE DID
MANY QULIERTZ IS THAT WE
LOOKED -- MANY YEARS LATER IS WE
DID SIMILAR ILC2'S IN THE GUT
AND LUNG, THIS IS FROM THE LUNG,
YOU CAN SEE, IL 25RBGS IL
23-RBGS ABOUT 80,000 SINGLE
CELLS YOU CAN SEE THEY CLUSTER
DIFFERENT AND WILL WE IDENTIFIED
THIS RECEPTOR NEUROPEPTIDE
RECEPTOR FOR NMUR1 WHICH IS
GENERALLY PRESENT AND HAD BEEN
IDENTIFIED BEFORE ON SMOOTH
MUSCLES AND WHEN AK TI VASE
NMUR1 SMOOTH MUSCLE IT CAUSES
BRONCHIAL SPASM AND IS DIARRHEA
AND VOMITING, INTERESTING THAT
THIS NEUROPEPTIDE RECEPTOR
EXPRESSED ON INNATE LYMPHOID
CELL CLEARLY INVOLVED IN TYPE 2
IMMUNITY AND ALLERGIES AND
ASTHMAS, AND THE QUESTION WAS
HAD, WHAT'S THE ROLE OF THIS
NEUROPEPTIDE COMING FROM NEURONS
ACTING ON INNATE LYMPHOID CELLS?
AND WHAT HAPPENS TO INNATE
LYMPHOID CELLS?
AND THE DATA IS PUBLISHED.
AND IT WAS VERY CLEAR THAT --
WILL EXPAND INNATE LYMPHOID
CELLS UNDER MUCOSAL SURFACE BUT
WILL NOT EN DO YOU SAY DISEASE
OR ALLERGIES UNLESS THEY GET A
SECOND SIGNAL DPROMG NEURONS AND
FALL OFF NEUROPEPTIDE CALLED NMU
AND THE TWO TOGETHER WILL GIVE
YOU CHANGE THIS INNATD LYMPHOID
CELL FROM BEING HOMEOSTATIC INTO
INFLAMMATORY ILC2'S AND IF YOU
INTERRUPTED THIS NMU, NMUR1
PATHWAY YOU CAN ESSENTIALLY
INHIBIT THE DEVELOPMENT OF THIS
ALLERGY BOUT AT LEAST IN THE
MOUSE.
SO EVERY TIME I PUBLISH A GOOD
PAPER THERE ARE MANY OTHERS
RIGHT WITH T THREE PAPERS COMING
UP TOGETHER SHOWING NMU AND
NMUR1 IN TRACTIONS ARE CRITICAL
FOR ACTIVATE INNATE LYMPHOID
CELLS AND IN FACT THE YES IS,
WHO PRODUCES NMU AND WHEN?
AND WE BELIEVE THAT IT IS
ACTUALLY IN THE GUT, THE PSM2
NEURONS, SENSORY NEURONS THAT
ARE COMING FROM MI ONTR --
MYENTERIC PLEXUS, MUSCLES THAT
GO AROUND THE GUT, AND THEY FORM
THESE GANGLIA AND WILL YOU CAN
SEE THE SUB MUCOSAL PLEXUS AND
THESE ARE YOUR VILII AND WE
THINK ESSENTIALLY WE HAVE WE CAN
IDENTIFY THESE NEURONS THAT
PRODUCE NMU.
IN FACT, THEY START FROM THIS
MYENTERIC PLEXUS AND GO ALL THE
WAY THROUGH INTO THEVILLE LIE
AND THAT'S -- THE VILLI, AND
THEY INTERACT WITH IMMUNE CELLS
AND REGULATE THEIR FUNCTION BOTH
IN TERMS OF IMMUNE CELLS
REGULATING THE NERVOUS SYSTEM
FUNCTIONS AND NEURONS AND
NEURONS ACTUALLY REGULATING
IMMUNE FUNCTION.
THEY ACTUALLY HAVE MANY OTHER
FUNCTIONS IN WHICH YOU WILL SEE
IN A MINUTE.
SO IT'S VERY CLEAR THAT THESE
HAD LONG DISTANCE RUNNERS COMING
FROM THE MYENTERIC PLEXUS ARE
COMING FROM THE VILLI AND
INTERACTING WITH IMMUNE CELLS.
S THE FIRST PART OF THE TALK
WHAT I TOLD YOU, TO SUM UP, BY
DOING SINGLE CELL RNA SEQ OF
ILC'S AND WE IDENTIFIED
RECEPTORS FOR NEUROPEPTIDES AND
NMUR1.
AND ILC2'S.
WHAT I ALSO TODAY YOU IS THAT
THE NMU NEUROPEPTIDE WAS
SYNERGIZED WITH THESE ALARMENS
TO MAKE THESE ILC'S
INFLAMMATORY, AND TOGETHER THEY
WILL IN FACT PROMOTE P THE
ALLERGIC REACTION AND THIS IS
WHAT WE SAW AND I THINK DAVID
ART I S' LAB AND HENRIK'S LAB IN
FACT IDENTIFIED THE SAME
INTERACTION IN THE GUT.
SO I TOLD YOU THE FIRST PART OF
THE TALK IS THAIN DEED
NEUROPEPTIDE RECEPTORS EXPRESSED
ON THEIR INNATE LYMPHOID CELLS
THAT INDUCE ALLERGIC REACTION,
AMPLIFY ALLERGIC REACTION.
BUT THE QUESTION IS THAT WE
SHOWED YOU THAT THE NEURONS OR
NEUROENDOCRINE CELL WILL MAKE
NMU AND ACT ON INNATE LYMPHOID
CELLS BUT WHAT ARE THE CYTOKINE
RECEPTORS ON THE NEURONS THAT
WILL RESPOND TO THESE CYTOKINE
SIGNALS COMING FROM IMMUNE
SYSTEM?
AND IF SO, WHAT DO THEY DO?
AND THE QUESTION IS WHAT
CYTOKINE RECEPTORS ARE EXPRESSED
ON THESE NEURONS?
AND FORTUNATELY FOR US,S THERE
WAS A BIG DATASET BOTH FROM
HUMAN AND MOUSE, WHERE THEY HAD
LOOKED AT ALL THE RECEPTORS
EXPRESSED ON NEURONS.
IN FACT, WE TOOK THREE DIFFERENT
DATASETS THAT WERE PUBLISHED AND
TRIED TO MAKE AN ATLAS OR A
CATALOG OF ALL THE RECEPTORS
THAT ARE EXPRESSED ON THESE
ENTERIC NERVOUS SYSTEM, ENTERIC
NEURONS.
AND HERE IT IS ACTUALLY I HAVE
CUT A LOT OF DATA DOWN, AND
ANYBODY WHO HAS QUESTIONS CAN
COME AND TALK TO ME A LITTLE
LATER.
THESE ARE YOUR NMU POSITIVE
NEURONS, ALSO CALLED PSM2,
SENSORY NEURO NEURONS, IPAN'S T
THESE ARE ALL THE CYTOKINE
RECEPTORS WE COULD IDENTIFY IN
THESE THREE DATASETS AND WE
CATALOGED THEM TOGETHER.
AND THEN YOU CAN FIND IF YOU
LOOK AT TYPE 2 CYTOKINES IL4 AND
IL13 CYTOKINE RECEPTORS YOU CAN
CLEARLY SEE THEM ON THESE NMU
NEURONS, YOU CAN SEE THAT
THERE'S IL4 RECEPTOR AND IL13
RECEPTOR EXPRESSED.
SO IT'S NOT JUST IMAGINATION.
THAT INDEED, THE NEUROPEPTIDE
RECEPTORS ARE PRESENT ON INNATE
LYMPHOID CELLS, AND INNATE
LYMPHOID CELLS WOULD MAKE
CYTOKINES LIKE IL4, 5 AND IL13
AND NOW WE ARE FINDING FROM THIS
DATASET THAT IL13 AND IL4
RECEPTORS ARE ACTUALLY PRESENT
ON ENTERIC NEURONS, IPAN'S OR
PSM2 CLUSTER OF SENSORY NEURONS.
YOU GUYS ARE ALL IN IMMUNOLOGY,
YOU KNOW THAT IL13 AND IL4 FORM
DIFFERENT TYPES OF RECEPTORS, IN
FACT, IL4 RECEPTOR IMMUNE SYSTEM
IS MADE BY IL4 RECEPTOR TOGETHER
WITH COMMON GAMMA CHAIN AND THE
PATHWAY, AND THE JAK PATHWAY.
IN OTHER CELL TYPES INCLUDING
SMOOTH MUSCLES IT'S A DIFFERENT
TYPE OF RECEPTOR AND TRACK TIL4
AND IL13 FORM THESE HETERODIMER
RICK RECEPTORS ACCIDENT I TOOK A
BUNCH OF SLIDES OUT, THAT'S THE
RECEPTOR WE ARE FIENG ON THE
NEURONS THAT IT IS A COMBINATION
OF IL4 RECEPTOR WITH IL13
RECEPTOR ALPHA CHAIN.
SO WE ACTUALLY STRUGGLE A LOT TO
REALLY FIND WHAT ROLE DOES
CYTOKINES HAVE, AND A MASTER
STUDENT IN FACT STRUCK HE WOULD
TO FORM THESE IN VITRO
NEUROSPEAR CULL CULTURES AND
FINALLY WE FOUND THE CONDITIONS
WHERE WE CAN ACTUALLY GENERATE
THESE SENSORY NEURONS IN THE
CULTURE DISH.
WHAT THEY DID IS THAT THEY PUT
ON IL13 OR IL4 ON THEM, AND WE
CAN IMMEDIATELY SEE THAT WHEN
YOU PUT THE IL13 RECOMBINANT
CYTOKINE IN VITRO CULTURE YOU
CAN SEE AN INCREASE IN THE
EXPRESSION OF THESE NEURO
NEUROPEPTIDES NMUU THIS CGRP AND
GRP.
AND YOU CAN LOOK AT THIS HERE
AGAIN IS THAT YOU CAN SEE THAT
IF YOU HUS A COMBINATION OF
CYTOKINES AS YOU KNOW THAT ENTER
ON GAMMA GENERALLY INHIBITS TH
TO CYTOKINE PRODUCTION AND THE
FUNCTIONS, AND SO WE STUDY USING
THE COMBINATIONS OF IL4 AND IL13
AND GAMMA ON IL4 AND IL13
TOGETHER AND LOOKING AT THE
NEUROPEPTIDE FLUKS IN VITRO
CULTURE AND IT'S CLEAR THAT THE
IL4 AND IL13 WILL BOTH INDUCE
NMU, CGR ARE P DATA AND
NEUROPEPTIDES, INHAVE I TROAT
NEUROSPEAR CULTURES.
IT'S NOT FOR EVERYTHING.
IN FACT, THERE ARE NEUROPEPTIDES
LIKE VIP THAT THE CYTOKINE
RECEPTOR TRIGGERING WOONTD
INDUCE THEM.
SO IT LOOKS LIKE IT'S SOMEHOW
SPECIFIC HOW WE ARE DOING WITH
THE TIME?
YEAH?
OKAY.
SO AT LEAST IN VITRO, WHEN YOU
USE A NEUROSPHERE AND ACTIVATE
THE RECOMBINANT CYTOKINES YOU
CAN SEE THAT YOU ARE FULFILLING
THIS POSTULATE THAT THE CYTOKINE
RECEPTOR SIGNALLING IS ACTUALLY
INDUCING THESE NEUROPEPTIDES,
IT'S NOT TRUE FOR ALL OF THEM.
IN FACT, NMU AND CGRP ARE THE
TWO NEUROPEPTIDES THAT WE AND
OTHERS HAVE PUBLISHED TO HELP
IMMUNOMODULATORY OR
IMMUNOREGULATORY PROPERTIES.
BUT ET IN VITRO IS WELL AND GOOD
BUT WE WANTED TO SEE WHAT
HAPPENS IN IN VIVO, BECAUSE
CYTOKINES HAVE SUCH SHORT
HALF-LIFE.
WE USED IL4 COME PLEKS AND GAVE
THEM IN VIVO INTO THE MICE AND
THEN LOOKED AT THE ENTERIC
NERVOUS SYSTEM TO SEE WHETHER
THIS RECOME BY NANTD IL4 GRIFN
IN VIVO FOR ANTIBODY CYTOKINE
COMPLEX WILL INDUPS THE SIGHT
EXIENS IS THIS IS DIFFERENT
PARTS OF THE GUT, DUODENUM ILEUM
AND COLON AND YOU CAN SEE THAT
IL4 COMPLEX WILL INDUCE NMU FROM
ALL THESE REGIONS, ALSO INDUCE
CGRP AND IT WILL ALSO INDUCE GRP
FROM THESE NEURONS.
AND AGAIN, AND IT HAD NO EFFECT
ON VIP.
SO WHAT I TOLD YOU IS THAT THE
CYTOKINE RECEPTORS INDEED ARE
PRESENT ON ENTERIC NEURONS.
AND I GAVE YOU EXAMPLE OF PSN2
OR IPAN THEY'RE CALLED AND THEY
EXPRESS HETEROIL4 AND IL13
RECEPTOR, CYTOKINES COMING FROM
LY THE CELLS WILL INDUCE
EXPRESSION OF THESE
NEUROPEPTIDES NMU AND CPRG FROM
THEM.
SO THE QUESTION IS THAT IN
ACTUALLY I TEACH THIS COURSE AT
HARVARD ABOUT THE BEFORE T CELL
SUBSETS AND THE FAVORITE LINE IS
THAT P THE TH2 CELLS MAKE THE
CYTOKINES IL2, 5 AND IL13 AND
IL13 ACTS ON SMOOTH MUSCLES AND
SMOOTH MUSCLES THEN CONTRACT AND
THAT GIVES YOU BRONCHOSPASMS,
THAT GIVES YOU -- WE HAVE A QUAN
DAIRY FINDING IL13 RECEPTORS,
THEY TOOK THE SMOOTH MUSCLES AND
IN FACT FROM ORGAN THEY PUT IL13
ON AND THEY SHOWED THAT INDUCES
SMOOTH MUSCLE CONTRACTION.
SO THE QUESTION, IS IT DIRECTLY
ACTING ON SMOOTH MUSCLES OR WHAT
IS THE ROLE OF THE IL13 RECEPTOR
THAT WE ARE FINDING ON THE
ENTERIC NERVOUS SYSTEM?
SO THIS IS WHAT WE TELL
STUDENTS, THAT THE ELEMENTS AND
ALLERGENS WILL ACT WITH
EPITHELIA AT THE BARRIER SITE
AND THEY WILL PRODUCE THESE
ALARMING, IL 25RBGS TSLP, IL33
WHICH WILL INDUCE ILC 2Z WHICH
WILL AMPLIFY TH2 RESPONSES AND
THEN IL13 WILL ACT ON THE SMOOTH
MUSCLES AND MAST CELLS AND
GOBLET CELLS AND EOSINOPHILS AND
INDUCE ALLERGIC REACTION, WHAT
IS CALLED WEEP AND SWEEP
PHENOMENON.
WHICH IS WHAT YOU SEE THERE'S A
LOT OF MUCOUS SECRETION AND
BRONCHOSPASMS AND DIARRHEA AND
VOMITING, ALL OF IT ASSOCIATED
WITH THE IL4, IL5, TH2
RESPONSES.
BUT THEN HERE IS THE PROBLEM.
THE PROBLEM IS THAT WE ARE
FINDING THAT THE IL13 RECEPTORS
AND IL4 RECEPTOR INDEED PRESENT
ON THE ENTERIC NERVOUS SYSTEM
AND OTHER NERVOUS SYSTEMS,
NEURONS AS WELL, AND WHAT'S THE
ROLE OF THESE CYTOKINE RECEPTORS
ON NERVOUS SYSTEM?
AND WE GOT THIS IL13 RECEPTOR
KNOCK-OUT MOUSE AND IN FACT THE
BLACK C I S MICE RNLT VERY GOOD
AT RESPONDING AND MOUNTING
ALLERGIC REACTION, SO WE HAVE TO
ACTUALLY USE THIS MODEL WHERE
THE PARASITE GOES IN AND BURROWS
IN THE GUT INTO THE MUSCULARIS,
I HAD A VERY NICE PICTURE, IT
GOES VERY CLOSE TO THE MYENTERIC
PLEXUS IN THE MUSCLE AND COMES?
VERY CLOSE TO THESE NEURONS THAT
PRODUCE MMU -- PRODUCE NMU AND
MAKES A BUNDLE.
AND WE WANTED TO DIG THIS IL13
RECEPTOR FLOCKS ALLELE AND
WANTED TO GET RID OF ONLY ON THE
ENTERIC NEURONS.
BUT THE PROBLEM IS THERE IS NOT
A SPECIFIC CRE DRIVER THAT ONLY
DELETES IT FROM ENTERIC NERVOUS
SYSTEM.
IN FACT, ROCKY TOOK THE IL13
FLOCKS FLOCKS ALLELE AND STARTED
CROSSING TO EVERY CRE HE COULD
FIND AND THERE WAS A WHOLE
ANIMAL HOUSE BREEDING WITH ONE
JUST FLOCKS ONE FLOX ALLELE.
HE COULD FIND A COUPLE OF CRE
DRIVERS THAT ARE DELETING IL13
REACCEPT NOR A CELL INTRINSIC
MANNER IN THE ENTERIC NERVES.
AND WHAT HE DID THEN IS THAT HE
USED A COUPLE OF THESE DRIVERS,
THIS IS CRE CROSSED ON IL
RECEPTOR FLOX-FLOX AEMPLOY LEGAL
AND INJECTED THEM AND FIRST
LOOKED WHAT THOOPS THE EGG
BURDEN AND IT WAS VERY CLEAR
THAT IF YOU DELETE THE IL13
RECEPTOR JUST FROM ENTERIC
NERVOUS SYSTEM AND ENTERIC
NEUROHONS, YOU HAVE INCREASED
FOCUNDITY, TECHNICAL WORD FOR
INCREASED EGG BURDEN.
I HAD TO LOOK IT UP.
AND THEN IF YOU CROSS IT ONTO
INDUCIBLE I BELIEVE CGRP CRE
DRIVER AND YOU CAN'T EXPEL WORMS
AS WELL AS THE WILD-TYPE MICE.
BUT IT IS NOT THAT EVERY CRE
DRIVER DOES IT, IN FACT -- WHICH
DELET'S FROMS VAIOUS NERVE ASK
OTHER NERVES AS WELL, NO EFFECT
ON FOCUNDITY OR ACTUALLY
EXPELLING WORMTZ, SO IT LOOKS
LIKE ONE OF THE FUNCTIONS OF
THESE NEURONS, CYTOKINE
RECEPTORS OR NEURONS IS IN FACT
PROMOTE THE GUT MOTILITY, IN
FACT, THAT'S WHAT THE MYENTERIC
PLEXUS DOES, IN FACT, THIS PSM2
GOES ALL THE WAY AND IN FACT
INCREASE THE MUSCULAR
CONTRACTIONS, THAT'S WHAT THEIR
JOB IS.
AND WILL WHAT YOU HAVE IS THAT
IT CAN'T HAVE AS MUCH, THEY
CAN'T EXPEL WORMS AND THERE'S AN
INCREASE FOCUNDITY IN THOSE
MICE.
WHAT'S ALSO INTERESTING IS THAT
THESE LARVAES BURROW INSIDE AND
FORM THESE LARGE WOUNDS AND YOU
CAN SEE HEMORRHAGIC WOUNDS, IF
YOU DELETE THE IL13 RECEPTOR
FROM THE NEURONS.
AND IT'S ACTUALLY VERY
SIGNIFICANT, ONLY A COUPLE OF
EXPERIMENTS YOU CAN SEE THERE
ARE LARGER WOUNDS, THEY DON'T
HEAL AND THEY BECOME VERY
HEMORRHAGIC.
THEN THE QUESTION IS THAT IF
IL13 RECEPTOR, IL4 RECEPTOR IS
EXPRESSED ON ENTERIC NEUROTONS,
SENSORY NEURONS, WHAT IS ITS
NORMAL JOB?
BECAUSE AS YOU REMEMBER THAT WE
HAVE HOMEOSTATIC ILC 2Z UNDER
THE MUCOSA THAT ARE PRODUCING
IL4, IL13 AT LOW LEVELS ALL THE
TIME.
AND WE STARTED LOOKING AT WHAT
HAPPENS TO NERVES THEMSELVES AT
THE HOMEOSTATIC LEVEL AND
FOLLOWING INFECTION WITH -- AND
ONE OF THE VERY FIRST THINGS
THAT YOU SEE -- AND ACTUALLY IT
DROPS HAD AND YOU ACTUALLY START
LOSING NUMBER OF NEURONS FROM
THE MYENTERIC PLEXUS INNERVATING
THE V I LLI.
THAT'S THE FIRST THING YOU SEE.
SECOND THING YOU SEE IS THAT
THEY START PRODUCING THE
NEUROPEPTIDES THAT I TALKED TO
YOU ABOUT THAT MODULATE OR
REGULATE IMMUNE SYSTEM.
SO WHAT DO THEY TELL YOU IN THE
SUMMARY NUMBER TWO IS THAT IF
YOU INFECT THESE MICE WITH
H POLYGYRUS AND NMU PRODUCING
NEURONS IN THE WILD-TYPE MICE
YOU EXPEL WORMS AND YOU ALSO
DON'T HAVE AS MUCH EGG BURDEN,
THE MOMENT YOU GET RID OF IL13
RECEPTOR ON ENTERIC NERVOUS
SYSTEM YOU DON'T HAVE AS MANY
PSM2 NEURONS GOING THROUGH INTO
THE VILLI, THE NEURON ACTUALLY
COLLAPSES AND YOU MAINTAIN A
LARGER NUMBER OF THESE WORMS AND
FOCUNDITY IN THE GUT AND YOU
CAN'T EXPEL WORMS AS MUCH.
OKAY?
NOW, THE QUESTION IS, AND HOW
DOES IT DO IT?
AND IN FACT, WE HAVE STHEEN DATA
FROM EVERY WHICH WAY YOU CAN
THINK OF AND I'M GOING ON BORE
YOU WITH ALL OF THAT.
SO WE TOOK ALL THE RECALL CELLS
ANDLY SEQ AND LOOKED AT
MUSCULARIS CELLS IN MUSCULARIS
AND NEURONS IN AND LOOKED AT
EPITHELIAL CELLS AND I'LL SHOW
YOU ONE SLIDE AND ANYBODY WHO
HAS QUESTIONS CAN TALK TO ME.
SO WE TOOK THIS WILD-TYPE AND
KNOCK-OUT MOUSE AND INFECTED
THEM WITH THE H PHYSICAL POLYAND
YOU CAN MAKE ALL THE SEQ YOU
WANT, MAKE ALL THESE NEW MAPS
AND ONE OF THE FIRST THING YOU
SEE IS THESE MICE IN WHICH YOU
HAVE DELETED IL13 RECEPTOR FROM
THE NEURONS, THERE'S A DEFECT IN
BOTH THE FREQUENCY AND THE
AMOUNT OF ILC2'S IN SUBMUCOSA.
THAT'S THE FIRST THING THAT YOU
SEE.
OKAY?
WHAT YOU ALSO SEE IS MAXIMUM
NUMBER OF INFECT CHANGED IN THE
MYELOID CELLS -- CHANGES IN THE
MYELOID CELLS, AND LOOKING AT
WHAT ARE THE CHANGES AND THE
CHANGES ARE ASSOCIATED WITH I
CAN'T READ IT FROM HERE, IS THAT
THERE'S A DEFECT IN INTERACTION
BETWEEN SPECIES, THAT MEANS THE
WORM IS BURROWING INTO A MAMMAL
AND THAT PATHWAY IS DEFECTIVE.
THERE'S A DEFECT IN MOUNTING
INFLAMMATION, AND SOME OF THE
HEALING PROCESSES ARE LOST IN
THE IL13 RECEPTOR DELETION ON
ENTERIC NEURONS.
THERE'S ALSO SOMETHING THAT I
HAVE TAKEN OUT OF THOSE SLIDES
COMPLETELY, THERE'S A CHANGE IN
THE NUMBER OF PANA CELLS ARE
DECREASING, THERE'S A REMODELING
OF GUT EPITHELIALIUM.
IMAGINE YOU ONLY DELETED IL
RECEPTOR FROM THE NEUROHON, YOU
ARE CHANGING THE GUT BARRIER HAS
CHANGED AND THERE'S A CHANGE IN
EPITHELIA IN THE GUT, YOU CAN'T
MAKE AS MANICALLY PEPTIDES TRKTS
ENTEROMICROPEPTIDES GO DOWN AND
THE HEALING, WHAT YOU SEE
NORMALLY UPON INFECTION IS
ACTUALLY REGULATED BY THESE IL13
RECEPTOR OR NEURONS SOMETHING,
THAT I AM NO GISTS IT'S HARD TO
BELIEVE BUT THAT'S WHAT YOU SEE.
SO LET'S MAKE SURE WE SUM IT UP.
RECEPTORS ON ENTERIC NEURONS,
AND I GAVE YOU EXAMPLE OF IL4,
IL13 HEAD TROA DIMER AND PSM2
SENSORY NEURONS, AND WHEN YOU
DELETE IT AND YOU ARE NOT ABLE
TO EXPEL WORMS AND THIS INCREASE
FOCUNDITY, AND THEN WHAT YOU DO
ALSO IS THERE'S ACTIVATION
PATHWAY BUT IL13 ACTING ON THE
NEURONS MAKING NEUROPEPTIDES AND
ALSO WE'RE FINDING A WHOLE
SERIES OF NEW NEUROPEPTIDES THAT
THE NEUROSCIENTISTS HAVE NOT
ACTUALLY SEEN BEFORE.
AND BECAUSE THEY ARE ALWAYS
USING NEURONAL TRIGGERS, HERE WE
ARE USING CYTOKINE AS A TRIGGER,
AND THESE NEUROPEPTIDES ARE
ACTIVATING THE MYELOID CELLS.
AND ALSO, SOME INTERACTION WE
DON'T KNOW WHAT THAT IS
GENERATED BY NEURONS TO REGULATE
THE EPITHELIAL BARRIER WHEN YOU
LOSE IT, YOUR EPITHELIAL BARRIER
IS AFFECTED BY JUST CHANGING THE
EFFECT ON NEURONS.
LET ME SUM IT UP.
THIS IS THE LAST PART OF MY
TALK.
SO THAT I SHOWED YOU THAT THE
CYTOKINE RECEPTORS ARE ON
ENTERIC NEURONS, AND ACTIVATION
WITH IL4 AND IL13 OF THIS
HETERODIMER WILL INDUCE
NEUROPEPTIDE AND IF YOU DELETE
IL13 RECEPTORS YOU CAN'T IN FACT
EXPEL WORMS AND YOU HAVE
INCREASED FOCUNDITY AND LOSS OF
IL13 RE RECEPTOR AND NEURONS
WILL ALSO DECREASE THE NUMBER
AND FREQUENCY AND THE
INNERVATION OF THE NM -- NMU
POSITIVE NEURONS.
I SHOWED YOU THAT NEURONS
REMODEL THE GUT EPITHELIUM AND
PRODUCTION OF MANY OF THE
ANTIMICROBIAL PEPTIDES.
JUST WHAT I TOLD YOU FROM THE
VERY BEGINNING, 30 YEARS AGO
WHEN I SAID THAT MAYBE THE
NERVOUS SYSTEM HAS SOMETHING TO
DO WITH THE IMMUNE SYSTEM AND IN
FACT FROM MY SON'S ALLERGY
BOUTS, AND THIS IS PROBABLY ONE
OF THE THINGS AT THAT CAME OUT
OF IT, IS THAT YOU INDEED
THERE'S INDEED INTERACTION
BETWEEN THE TWO SYSTEMS, WE
DON'T LIVE IN SILOS, AND
IMMUNOLOGY IS NOT WORKING ON
IMMUNE SYSTEM ALONE.
IT'S WORKING WITH EVERYBODY ELSE
THAT'S IN THE SYSTEM.
SO I SHOWED YOU THE CYTOKINES
PRODUCED BY AT LOW LEVELS
GENERALLY ARE PRODUCED BY THE
INNATE LYMPHOID CELLS AND THE
CYTOKINE RECEPTORS ARE ON THE
NEURONS AND THE NEURONS PRODUCE
NEUROPEPTIDES AND THAT WILL
BECOME A SELF-AMPLIFYING LOOP.
THIS MUST BE ESSENTIAL, IN FACT,
THE NEUROPEPTIDES THAT WE ARE
SEEING BY CYTOKINE ACTIVATION OF
NEURONS, THESE NOVEL
NEUROPEPTIDES HAVE VERY POTENT
ANTIMICROBIAL ACTIVITY AND MAYBE
THE NERVOUS SYSTEM IS PROBABLY
THE FIRST INNATE IMMUNE SYSTEM
THAT EVER EXISTED.
AND IN FACT, THEY DIRECTLY
AFFECT THE MICROBIOME AND
MICROBIAL SPECIES, IN FACT, IT
CHANGES THE MICROBIOTA.
ACTUALLY I DON'T WANT TO BLABBER
TOO MUCH AND I WANT TO INTRODUCE
YOU TO MY LAB, AND WE HAVE A
SOCCER TEAM, IT'S CALLED CT
KUCHROO IT LIONS, WE HAVE LOST
EVERY GAME WE HAVE PLAYED.
[LAUGHTER]
FINALLY BEFORE COVID WE DID WIN
A GAME, AND THEN WE HAD ALL
THESE POSTERS AND MAGNETS THAT
WE BECAME THE CHAMPIONS OF
HARVARD MEDICAL SCHOOL, AND THIS
IS ROCKY BARELLA WHO DID MOST OF
THE WORK.
I'M HAPPY TO TAKE ANY QUESTIONS
YOU MAY HAVE.
THANK YOU FOR YOUR ATTENTION.
[APPLAUSE]
>> THANK YOU.
MITCHELL CORN BELLE BERG --
INSTITUTE.
I'M SUPPOSED TO INTRODUCE --
ILC2 CAN MAKE OTHER THINGS, CAN
MAKE REGULAR LYNN, MAYBE IL9 AND
CAPULENS.
DO YOU SEE RECEPTORS FOR OTHER
ILC2 PRODUCTS ON THE NERVES?
>> I THINK THIS IS GOING TO BE A
BE -- AND WE ARE FINDING THINGS
THAT WE NEVER THOUGHT EXISTED.
AND THERE ARE LOTS OF CYTOKINE
RECEPTORS AND OTHER MOLECULAR
RECEPTORS ON THESE NEURONS AS
YOU GO THROUGH THE ATLAS.
AND WE THINK THE EXPRESSION OF
AMPHOREGLAN BY THE ILC2'S IS
VERY IMPORTANT FOR MAINTAINING
THE NEURONS AND NEURON BARRIER
FUNCTIONS AND ALSO MAINTAINING
HOMOEOSTASIS.
SO ACTUALLY ONCE WE STARTED
HAVING THE CROSSTALK BETWEEN THE
TWO SYSTEMS, ACTUALLY IT'S A
WHOLE NEW WORLD.
>> CL (AWAY FROM MICROPHONE).
>> CORRECT.
CORRECT.
>> JAY --
>> JAY FROM NCI.
VIJAY, THAT'S A FASCINATING
TALK.
SINCE BOTH IL4 AND IL13 CAN ACT
ON THAT SAME RECEPTOR THAT
INVOLVES IL4 ALPHA AND IL13
ALPHA IS THERE SOME PREFERENCE
FOR IL13 AS YOU WERE DESCRIBING
IT, IS IT BECAUSE IL13 IS MORE
PREVALENT IN THAT ENVIRONMENT?
THEY BOTH INDUCE PHOSPHORYLATION
DOWNSTREAM OF THE RECEPTOR.
>> SO I TOOK ALL THAT DATA OUT
AND IN FACT, WE CAN CLEARLY SEE
THAT UPON ACTIVATION WITH EITHER
IL4 OR IL13 YOU GET
PHOSPHORYLATION OF THE RECEPTOR
TAIL, AND YOU ALSO INDUCE STAT 6
IN THESE NEURONS.
AND EVERYTHING LOOKS NORMAL.
THE REASON WE WENT THROUGH IL13
IS SIMPLY BECAUSE OF I THOUGHT
IL13 ACTS ON SMOOTH MUSCLES TO
INDUCE THE VOMITING AND DIARRHEA
AND BRONCHOSPOOSM WENT AFTER
IL13 RECEPTOR DELETION.
BUT WE COULD HAVE EASILY DONE IT
WITH IL4 RECEPTOR BECAUSE BOTH
IL4 RECEPTOR AND IL13 RECEPTOR
ARE FORM RNG THE HETERODIMER AND
IL4, IL13 ACTIVATION WILL INDUCE
BOTH THOSE NEUROPEPTIDES.
>> IN 234 AL WE STUDIED ONE OF
THE REASONS WHY IN THAT CASE
IL13 WORKED AND IL BE 4 DIDN'T
WAS THAT YOU ALSO NEEDED TO
ACTIVATE THE IL13 OR ALPHA 2.
>> YEAH.
>> THAT IT RESPONDS ONLY TO IL13
AND THAT UP REGULATE THE OTHER
RECEPTORS.
>> CORRECT.
BUT IN THE NEURONS IT'S
SPECIFICALLY THESE TWO RECEPTORS
FORM THE HETERODIMER THAT ARE
RESPONDING.
>> THANK YOU.
>> HI.
IN.
REALLY BEAUTIFUL WORK YOU D I
WAS REALLY STRUCK BY THE LOSS OF
INNER VAIPTION IN THE IL13
DEFICIENT AND HOW THAT RELATED
TO THE FAILURE TO SORT OF
REEPITHELIAL EYES.
WHAT DO YOU THINK THE NEURONS
ARE FUNCTIONING IN THAT SYSTEM?
THEY DON'T SEEM TO BE AT THE
BOTTOM, UNLESS I JUST COULDN'T
SEE IN THE BACK R THEY AFFECTING
STEM CELLS OR DIRECTLY AFFECTING
THE DIFFERENTIATED CELLS?
>> IT'S A GOOD QUESTION.
I DON'T HAVE A DIRECT ANSWER FOR
YOU.
WE SEE THE FREQUENCY OF NEURONS,
NMU POSITIVE NEURONS ACTUALLY
NUMBERS GO DOWN AND INNERVATION
GOES DOWN AND THEY ARE NOT GOING
DOWN FROM THE MYENTERIC PLEXUS
INTO THE VILLI.
SOILY ARE NEURONAL THAT GOES
INTO THE VILLI.
ALTHOUGH IF YOU TUK A
NEUROSCIENTIST, THEY -- IF YOU
TALK ON A NEUROSCIENTIST THEY
WILL ALWAYS SAY THAT ONCE YOU
HAVE THE NEURONS, NOTHING ELSE
CHANGES, WHICH IS PROBABLY NOT
TRUE.
BUT WE DO FIND THAT IT'S
ESSENTIAL FOR MAINTAINING THAT
CLUSTER OF NEURONS, THE PSM2 AND
NMU POSITIVE, CRG POSITIVE
NEURONS, BUT WE DO NOT KNOW AT
WHAT STAGE IS IT NECESSARY, IS
IT STEM CELLS MAKING THEM GROW
OR IS IT MAKING NEW NEURONS OR
IS IT MAINTAINING THE NEURONS
THAT YOU ALREADY HAVE, WE DO NOT
KNOW.
>> COOL.
THANK YOU.
>> SO I HAVE TWO QUICK SLIGHTLY
NAIVE QUESTIONS.
>> HOLD ON -- AM OKAY.
SO SAME STORY, SO TOUGH CELLS
AND BECAUSE AS IL33 PRODUCERS
AND IS THIS THE SAME PATHWAY
THAT WORKS IN IT CUTANEOUS
ALLERGIES WITH MAST CELL
PERIPHERAL NEURON CIRCUITS?
>> DO NOT KNOW BUT WE SUSPECT
SO.
>> TOUGH CELLS DO THEY HAVE A
ROLE IN THIS GUT SITUATION?
ARE THEY ACTUALLY --
>> IN FACT, WE DO SEE WHEN YOU
DELETE ALPHA 13 RECEPTORS FROM
THE NEURONS YOU DO SEE CHANGE IN
BOTH DEPENDENT CELLS AND TOUGH
CELL IN THE GUT.
>> THANK YOU.
>> OKAY.
DAVID?
>> HELLO.
DAVID DID -- HELLO, VIJAY.
GREAT TALK.
I CAME IN A TINY BIT LATE.
I JUST WANTED TO REMIND MYSELF
AND THE AWD YEN, YOU KNOW, NOT
ALL WRORMS THE SAME, AS YOU KNOE
EXPAIM HLY POLIGYRUS IS UNUSUAL
BECAUSE IT LIVES IN THE
MUSCULARIS, IT'S NOT A MODEL FOR
STUDYING NEURAL HE THEY'LL YOLG
BIOLOGY NECESSARILY, IT'S A
BRILLIANT MODEL IT STUDY NEURO
IMMUNE INTERACTIONS WITH THE
MUSCULARIS PERHAPS.
BUT YOUR CONCLUSIONS ABOUT NEURO
EPITHELIAL STUFF MIGHT BE AN
EXAGGERATION S THAT FAIR?
>> FOR THIS ZOODLE OR HAVE YOU
DONE ALL THELY ALL THE OTHER
SYSTEMS?
SO WE DON'T LEAVE THE AUDIENCE
THINKING EVERY WORM IS THE SAME.
H POLYGYRUS IS THE LEAST USED GI
NEMATODE SYSTEM AND ITS UNUSUAL
BECAUSE ITS BIOLOGY LIVES IN THE
MUSCLE S THAT FAIR?
>> SO I THINK I DIDN'T QUITE GET
THE FULL QUESTION.
>> THE QUESTION IS.
>> BUT --
>> IS IT THE BEST MODEL SYSTEM
WHEN IT LIVES IN THE MUSCULARIS
TO TALK ABOUT NEURO EPITHELIAL
BIOLOGY?
BECAUSE PROBABLY NIPO OR
TRICHINELLA OR TRICURUS ARE MORE
APPROPRIATE SYSTEMS TO
INTERROGATE BIOLOGY.
THAT'S WHAT --
WE LOOK AT IMMUNITY, THERE IS
CHANGE IN THE FUNCTIONS AND HOW
MUCH OF THAT IS DIRECT OR
INDIRECT WE DO NOT KNOW.
BUT WHAT HE SAID IS ARE THERE
OTHER MODELS WE KNOW COULD
ACTUALLY ADDRESS THIS BETTER.
>> YEAH I'M JUST ASKING IF IT
TRAVERSE, IF THE BIOLOGY YOU'RE
PROPOSING TRAVERSES OTHER MODEL
SYSTEMS.
>> I THINK YOU AND I SHOULD TALK
OVER A BEER.
>> TONIGHT, DINNER, SEE YOU
THEN.
>> DINNER TION TONIGHT.
>> YOU OWE ME A BEER.
>> RACHEL?
>> SO THANK YOU FOR A GREAT
TALK.
THIS MAY BE A LITTLE BET RELATED
TO WHAT DAVID WAS ASKING.
THIS MAY BE A LITTLE BIT RELATED
TO WHAT DAVID WAS ASKING.
YOU TALK A LOT ABOUT EFFECT OF
EPITHELIAL CELLS BUT I WAS
WONDERING DO YOU SEE EFFECTS ON
PERISTALSIS, FOR EXAMPLE SH.
>> OH, YEAH.
YEAH.
SO I SPENT $40,000 TO GET THE
MACHINE, AND TO DO EXACTLY THAT.
AND THE DATA IS NOT QUITE --
SOME DAYS IT WORKSZ AND SOME
DAYS IT LOOKS LIKE IT AFFECTS
AND SOME DAYS IT DOESN'T.
SO SWREANT COME TO ANY
PARTICULAR CONCLUSION THAT BY
LOSS OF IL IF CU RECEPTORS ARE
AUTO EFFECT EFFECTING THE NORMAL
PERISTALSIS MOMENT AND
EXPULSION.
THE JURY IS STILL OUT.
IT MAY BE ASKED TOO BECAUSE WE
DO NOT KNOW HOW TO DO THESE
THINGS.
>> JIM DID HE SANTO -- JIM DE
SANTO.
WHAT DO YOU THINK TURNS HAD OFF?
YOU MENTIONED IT'S AN AMPLIFYING
LOOP AND WE ALL PROBABLY
EXPERIENCE ACTIVATION OF THESE
CELLS AT SOME POINT IN OUR LIVES
BUT DON'T END UP ALL MAYBE
HAVING THE KIND OF CONSEQUENCE
THAT IS YOUR SON HAD.
WHAT DO YOU THINK IS REGULATING
THIS?
>> AND HOW TO INTERRUPT IT.
IN FACT, I WAS TALKING TO -- AT
MASS GENERAL AND APPARENTLY THIS
IRRITATION AND VOMITING
CONTINUOUS VOMITING ONCE IT
STARTS IS THERE IN 20% OF THE
PATIENTS THAT GET THE FOOD
ALLERGIES, AND ALL THEY DO IS TO
TAKE THEM IN AND SEDATE THEM.
SO I DO NOT KNOW WHAT WOULD BE
NORMAL WAY TO INTERRUPT IT, IS
THERE A NORMAL BIOLOGICAL
PROCESS BY WHICH TO TURN IT OFF.
I DO NOT KNOW.
OKAY?
THANK YOU VERY MUCH.
[APPLAUSE]
>> I HAVE A QUESTION FOR YOU.
[APPLAUSE]
>> SO REMODELING THAT YOU SEE,
IS THAT WORM DEPENDENT?
DOES THIS HAPPEN IN THE IL13
RECEPTOR KNOCK-OUT, FOR EXAMPLE,
IF YOU DON'T HAVE THE WORMS?
>> YEAH, IT DOES.
>> IT DOES, OKAY, GOOD.
SO THE NEXT SESSION, SESSION WE
HAVE A COFFEE BREAK UNTIL
3:15 SO LET'S REASSEMBLE AT 34
1R5 AND WE'LL FINISH IT OFF.
>> THANK YOU ACTUAL THE SPEAKERS
HELLO, I THINK GET STARTED.
I'M PAM SCHWARTZBERG FROM N I
AD, OUR FIRST SPEAKER.
>> I WOULD LIKE TO THANK OF
COURSE THE ORGANIZER AND I HAVE
TO IMMEDIATELY CORRECT YOU
BECAUSE WITHIN A FEW WEEKS I
WILL BE MOVING FROM EAST COAST
TO WEST COAST.
BUT FOR NOW, IT'S STILL NEW
YORK.
AND THAT IS WHERE ALL OF THIS
WORK WAS DONE.
I DO HAVE A CONFLICT OF
INTEREST, SOME OF THE WORK I
WILL BE SHOWING YOU WAS ACTUALLY
SPONSORED BY A CCH SIRRUS AND
THEY'VE ALSO HECHED US TO DO A
TRIAL WHICH I WILL GET TO
TOWARDS THE END.
SO ANYWAY TO GET MICROPHONE
HIGHER?
BECAUSE I'M SLUMPING OVER HERE.
I'M NOT THAT TALL.
CAN YOU HEAR ME?
OKAY, GOOD.
SO FOR THIS PURPOSE, SINCE I'M
GOING TO TELL YOU MOSTLY ABOUT
GRAFT VERSUS HOST VERY QUICKLY
FOR THOSE OF YOU WHO DOAN KNOW
OR DON'T DEAL OFTEN WITH AN
ALLOGENEIC TRANCE PLNT, DOING
ABOUT 40 THOWCH TRANSPLANTS DONE
SPECIFICALLY FOR -- 40,000
TRANSPLANTS DONE SPECIFICALLY
FOR PATIENTS WITH BLOOD
TRANSPLANTS, ONE OF THE FEW
CURATIVE THERAPIES WE HAVE,
MEANING WE'RE WILLING TO TAKE
ENORMOUS RISKS, ONE OF THE MAJOR
RISKS IS GRAFT VERSUS HOST.
TESTING ONE TWORKS, THREE, GOOD,
THANK YOU VERY MUCH.
SORRY, GUYS.
SO GRAFT VERSUS HOST IS A
DISEASE THAT LEADS TO OFTEN
MAJOR COMPLICATIONS.
SOME OF THEM CAN ACTUALLY BE
LETHAL.
AND ALL YOU NEED TO KNOW FOR
THIS TALK IS THAT WHAT IS
CRITICAL THERE IS THAT IT'S ALL
DEPENDENT ON IT TELLS WITHIN THE
ALLO, WE'LL SEE VARIOUS ALLO,
MOSTLY IN GUT, LIVER AND SKIN
AND IT'S REALLY GULT GRAFT
VERSUS HOST WHICH LEADS TO MOST
OF THE PROBLEMS.
SO TRANSPLANT INTERESTED IN GUT
FLORA MICROBIOME?
STUDIES IN THE 1970 INDICATED
THAT GERM FREE MICE HAD LESS
GRAFT THAN HOST AND THAT
ACTUALLY LED FOR DECADES TO TO
EVERY CENTER DOING THEIR OWN
THING.
PATIENTS BEING PUT ON DIFFERENT
TYPES OF ANTIBIOTICS, SOMETIMES
INTO A BUBBLE SETTING REALLY AND
DOING ALL KINDS OF THINGS TO DO
SOMETHING WITH THE GUT FLORA.
BUT IT WAS NEVER COMPLETELY
CLEAR WHAT WAS ACTUALLY
NECESSARY AND WHAT ACTUALLY
WORKED.
SO THAT WAS ONE OF THE REASONS
WHY IN 2009 OR SO WE TOOK A
FRESH LOOK AT THAT AND OVER THE
YEARS WE'VE LEARNED SOMETHING
ABOUT THIS.
I'M TRYING TO SUMMARIZE A LOT OF
WORK HERE NOW FROM SPECIFICALLY
MY COLLEAGUE ERYK BA AMER AND
WORK FROM MY GROUP FROM THIS
GRAM WHICH INDICATES IN BLUE THE
VARIOUS TEXTS THAT WE HAVE SHOWN
BOTH IN MOUSE AND MEN MEN TO BE
OF SOME GA FAVORABLE OUTCOME AND
THOSE THAT HAVE BEEN IN RED
LINKED TO WORSE OUTCOME, I'M
SHOWING YOU HERE HOW THESE ARE
LINKED TO GRAFT VERSUS HOST.
BASICALLY ALL OF THE STUDIES WE
HAVE DONE OVER THE LAST DECADE
OR SO WE HAVE SHOWN THAT THESE
VARIOUS TECHS CAN BE IMPLICATED
IN ALMOST EVERY SERIOUS
COMPLICATION OR OUTCOME AFTER
ALLOGENIC TRANSPLANT.
SOME OF THE LESSONS WE HAVE
LEARNED IN BODIES RELEVANT FOR
THE ONE STORY THAT I WOULD LIKE
TO FOCUS ON IS THAT THE LOSS OF
THE DIVERSITY WITHIN THE GUT
FLORA HAPPENS IN ALMOST ALL OF
THESE PATIENTS AND IF IT'S MORE
SERIOUS THEN IT'S ACTUALLY ALSO
LINKED TO LETHAL GRAFT VERSUS
HOST, COMMENSAL ANAEROBES
TYPICALLY LOST IN THE FIRST DAY
OR WEEK AFTER TRANSPLANT AND KEY
FOCUS BACTERIA WE'VE FOCUSESSED
ON IS BLAUTIA, ALMOST ALL
SPRAISHTS A COURSE OF DOMINANCE
DURING THE COURSE OF THEIR
TRANSPLANT IN SOME CASES MEANING
THEIR FLORA IS TAKING OVER BY A
SINGLE REMEMBER, MORE FREQUENTLY
THAN TR ANY OTHERS IS
ENTEROCOCCUS WITHIN THE MOUSE
ENTEROCOCCUSLY -- AND WITHIN THE
HUMAN ENTEROCOCCUS SOME OF THE
THINGS IT DOES AND HOW IT MAKES
GRAFT VERSUS HOST WORSE.
LESS ANOTHER STRIKING FEATURE IS
OF COURSE ALL OF THESE PATIENT
GOING THROUGH ANNAL LOWGENIC
TRANSPLANT, GOING THROUGH
PERIODS OF NEUTROPENA WILL BE
TREATED AT ONE POINT WITH
ANTIBIOTICS, SPECIFICALLY THE
EXPOSURE TO BROAD-SPECTRUM
ANTIBIOTICS HAS BEEN LINKED TO
AN INCREASED RISK FOR GRAFT
VERSUS HOST.
BUT THERE ARE MANY FACTORS THAT
CAN HAVE IMPACT OF COURSE ON
CHANGES WITHIN THE GUT FLORA,
BUT WE'RE LOOKING AT CURRENTLY
AND I'M ONLY SHOWING ONE VIN NET
HERE IS DIET -- VIGNETTE HERE IS
DIET VERSUS MOUSE AND MAN,
PATIENT CAN TAKE PHOTOS OF THEIR
MEALS AND USE THAT FOR
DOCUMENTATION OF THEIR DIET, AN
APP, WHICH IS WORKING VERY, VERY
NICELY.
WHEEF LEARNED SO FAR IS THAT
CALORIE INTAKE AND FIBER INTAKE
IS POSITIVELY CORRELATED WITH A
DIVERSITY AND WITH BLAUTIA SO
THAT ONE KEYSTONE AND NEGLECT
TWIFL ENTEROCOCCUS WHICH IS THE
ULTIMATE BAD BUG IN ALL OF OUR
STUDIES.
HAD.
STUDY WE RECENTLY PUBLISHED HAS
BEEN LOOKING ACTUAL AT ALL OF
THE OTHER DRUGS THAN THESE
TYPICAL ANTIBIOTICS THAT HAVE
BEEN IMPLICATED BY MANY AND MANY
DIFFERENT SETTINGS TO LEAD TO
CHANGES IN THE GUT FLORA.
SO WHAT WE DID IS WE TOOK ALL OF
THE PATIENTS THAT WE HAD AT THAT
POINT AND ALL OF THE SAMPLES, SO
THERE WAS MORE THAN 1,000
PATIENTS AND ALMOST 9,000
SAMPLES.
WE PUT THEM TOGETHER INTO A U
MAP CLUSTERED THEM IN TEN
DIFFERENT CLUSTERS AND SINCE WE
HAD IN MANY CASES SEQUENTIAL
SAMPLES AND VERY DETAILED DATA
OF ALL OF THE DRUGS THAT THEY
WERE ON, ALL OF THESE PATIENTS
AT ANY MOMENT ARE ON DIFFERENT
TYPES OF DRUGS FOR PAIN, FOR
NAUSEA, FOR WHATEVER, AND SO
LOOK P AT THESE CLUSTERS AND
THEN LOOK NG A SEQUENTIAL WAY AT
WHAT DRUGS MIGHT DO TO HAVE
PATIENTS MOVE FROM ONE CLUSTER
TO THE OTHER OR STAY WHERE THEY
ARE, GAVE US SOME INDICATORS
ABOUT WHAT ALL OF THESE OTHER
DRUGS CAN ACTUALLY DO IN TERMS
OF CHANGES IN THE LEVELS OF THE
FREQUENCY OF THESE BACTERIA.
AGAIN, FOR THESE TWO ULTIMATE
GOOD AND BAD BUGS, BLAUITY A AND
ENTEROCOCCUS THAT ALL KINDS OF
DRUGS LIKE NAUSEA MEDICATIONS,
PAIN MEDICATIONS CAN HAVE IMPACT
ALSO, SO THIS OACHES THE DOOR IT
MAYBE START IN A DIFFERENT WAY
ABOUT THE CHOICES FOR NAUSEA
MEDICATIONS, PAIN MEDICATIONS,
CHOICES THAT WE MAKE DAILY FOR
ALL OF OUR PATIENTS.
ONE THING THAT I WANT TO
HIGHLIGHT ALSO SPECIFICALLY
DRUGS THAT CAN MAYBE LEAD TO
LOWER LEVELS OF ENTEROCOCCUS,
ONE DRUG I WANT TO HIGHLIGHT IS
OTCA BECAUSE THAT IS SORT OF A
BRIDGE TO THE NEXT STORY THAT I
WOULD LIKE TO TELL YOU.
THIS IS A SECONDARY BOWEL ACID
WHICH IS COMMONLY USED IN MANY
TRANSPLANT CENTERS AT THE MOMENT
FOR THREE MONTHS OR SO DURING
THE WHOLE COURSE OF A
TRANSPLANT.
AND PERFORMANCE THOUGHT ALWAYS
TO BE BENEFICIAL FOR LIVER
PROBLEMS, VOD, SOS SYNDROME AND
WE WON'T GO INTO ALL OF THE
DETAILS NOW ABOUT THAT.
SO I TOLD YOU ABOUT THIS
SECONDARY BOWEL ACID BECAUSE
THAT'S THE ONE STORY THAT IS
GOING TO COME OUT SOON, AND I
APOLOGIZE TO ALAN AND TO ELLA
BECAUSE THEY'VE ACTUALLY HEARD
PART OF THAT STORY ALREADY
ALREADY ON BEAUTIFUL ISLANDS IN
THE GREAT BARRIER REEF, RIGHT?
BUT NOW YOU'RE GOING TO HEAR THE
FULLER STORY BECAUSE WE ANSWER
TO ALL OF THE REVIEWERS'
QUESTIONS.
SO WHAT WE WERE LOOKING AT WAS
THE IMPACT OF CHANGES WITHIN THE
BOWEL ACIDS ON OUTCOMES BOTH IN
MOUSE AND MEN AFTER ALLOGENIC
TRANSPLANT. SO FOR THOSE OF YOU
WHO DON'T OFTEN THINK ABOUT
BOWEL ACIDS BUT MAYBE YOU STILL
KNOW IT FROM HIGH SCHOOL OR
SOMETHING, BOWEL ACIDS ARE OF
COURSE MADE WITHIN THE LIVER,
PRIMARY BOWEL ACIDS SPECIFICALLY
ARE CONJUGATED TO GLYCINE AND TO
TOUR IN-- TAURINE, GHET INTO THE
GUT THROUGH THE BLADDER AND OF
COURSE THEIR PRIMARY FUNCTION IS
FOR LIPID UPTAKE AND A FEW OTHER
THINGS.
BUT ONCE THEY GET FURTHER INTO
IT AND GET INTO THE TERMINAL
ILEUM OR WITHIN THE LARGE BOWEL,
THAT IS WHERE THEY COME INTO
CONTACT WITH ALL THE VARIOUS
BACTERIA AND BACTERIA ACTUALLY
CARRY A PLETHORA OF ENZYMES TO
TURN PRIMARY BOWEL ACIDS INTO
SECONDARY BOWEL ACIDS.
WE ARE VERY FORTUNATE THAT ONE
OF THE PEOPLE WHO WORKED WITH
JUSTIN CROSS TOOK A SPECIAL
INTEREST AND MADE ALL OF THESE
ASSAYS TO LOOK AT ALL THESE
DIFFERENT PRIMARY AND ALSO
SECONDARY BOWEL ACIDS THAT WE
COULD TAKE A MORE DETAILED LOOK
AT WHAT WAS HAPPENING BOTH IN
MOUSE AND MEN.
THE OTHER THING THAT YOU NEED TO
KNOW IS THAT THESE SECONDARY
BOWEL ACIDS SPECIFICALLY BUT
ALSO SOME OF THE PRIMARY BOWEL
ACIDS CAN BIND TO AGAIN A WHOLE
SERIES OF RECEPTORS AND THE ONE
THAT I'M GOING TO FOCUS ON IS
FXR, A FINAL THING ON HIGHLIGHT
ON THIS SLIDE IS THAT ONE OF THE
FIRST THINGS THAT ALL OF THESE
BACTERIA DO IS USE AN ENZYME
BOWEL SALT HYDROLASE TO DEACON
JEW GAIRKTS TAKE OFF THE TAURINE
FROM BOWEL ACIDS.
SECONDARY ARE ACIDS HAVE BEEN
STUDIED BY MANY PEOPLE IN THIS
AWD EXPWRENS AUTHORS IN -- AND
AWRD YENS AND AUTHOR INSIDE SOME
OF THESE PAPERS, JUST
SUMMARIZING, SOME OF THESE
PAPERS INDICATING THAT SECONDARY
BOWEL ACIDS CAN BE RELEVANT FOR
TH17 AND FOR TREX SPECIFICALLY
LOWER LEVELS OF TH17 AND
INCREASED LEVELS OF TREGS AND
FOR REASONS THAT I DON'T KNOW
ALL OF THESE PAPERS ARE
PUBLISHED IN NATURE, I GUESS
NATURE LIKES BOWEL ACIDS.
WHAT WAS KNOWN WITHIN THE
CONTEXT OF THE GRAFT VERSUS HOST
NRKSZ IF YOU GIVE THIS STEKD RI
BOWEL ACID THAT THAT COULD LEAD
TO LOWER LEVELS OF GRAFT VERSUS
HOST, OLDER STUDIES TWO YEARS
PLUS OLD FROM SWEDEN ACTUALLY
THAT INDICATED THAT IF YOU GIVE
UDCA TO PATIENTS THAT THAT COULD
LEAD TO OVERALL BETTER OUTCOMES
AND LOWER LEVELS OF GRAFT VERSUS
HOST, BUT THERE NEVER HAD BEEN
ANY FURTHER STUDIES OR ANY
FURTHER FOWP ON THOSE EARLY
CLINICAL DATASETS.
SO WE TOOK A FRESH LOOK AND WE
STARTED WITH MOUSE MODEL SO WE
OFTEN USED MOUSE MODELS FOR
GRAFT VERSUS HOST.
FIRST THANK WE NOTICED WAS THAT
IF WE LOOKED ABOUT A WEEK OR SO
OUT FROM TRANSPLANT, THAT IN
THOSE MICE DEVELOPING GRAFT
VERSUS HOST THE LEVELS TOTAL
BOWEL ACIDS POOL WITHIN THE GUT
AND ALSO WITHIN THE SERUM WITHIN
THE PLASMA ACTUALLY WAS LOWER.
SO THINKING THAT MIGHT BE
BECAUSE THERE'S LIVER DAMAGE,
RIGHT, MENTIONED THAT THERE'S
LIVER GRAFT VERSUS HOST, SO
MAYBE THAT'S WHY.
SO WE DID SOME SEQUENCING OF THE
LIVER OF THESE MICE WITH A GRAFT
VERSUS HOST AND INDEED NOTICED
THAT ALL OF THE PATHWAYS, ALL OF
THE ENZYMES THAT WERE RELEVANT
FOR THE SYNTHESIS OF BOWEL ACIDS
WERE PRESENT AT LOWER LEVELS AND
OF COURSE YOU SEE EVIDENCE OF
THE INFLAMMATION.
BUT WHEN WE LOOKED INTO MORE
DETAIL AT THE DROP IN THE BOWEL
ACID WE SAW -- POOL WE SAW THAT
MOST OF THE LOSS WAS ACTUALLY
WITHIN THE SECONDARY BOWEL ACIDS
AND DEACON JEW GATED BOWEL --
DECONJUGATEB BOWEL ACIDS, COULD
BE RELEVANT.
WHEN WE LOOKED AT MORE DETAIL AT
STIEPS OF DOWEL ACIDS AND SAW
ALL THE ONES IMPLICATED TO
REGULAR LATE TH17 AND TREG ALL
OF THOSE WERE PRESENT IN MUCH
LOWER LEVELS IN THOSE MICE
DEVELOPING GRAFT VERSUS HOST.
WE THEN DID SOME SHUT BEGUN
SEQUENCES -- SHUT GUN SEQUENCING
SO WE COULD BETTER ANALYZE GENES
OF INTEREST AND ONLY THING TO
HIGHLIGHT HERE IS THE LOWER
LEVEL OF THIS DISIEM THAT I
MENTIONED EARLIER WAS RELEVANT
IN THE FIRST STEP BY TBOORKT
DECONJUGATE PRIMARY BOWEL ACIDS
AND SPECIFICALLY BMH IN GRAFT
VERSUS HOST WAS PRESENT AT A
SIGNIFICANTLY LOWER LEVEL --
BSH.
I TOLD YOU ALREADY THAT WE HAD A
SPECIAL INTEREST IN FXR, ONE OF
THE MOST RELEVANT PATHWAYS HERE,
SPECIFICALLY FOR BOWEL ACID
SYNTHESIS.
BUT FXR CAN BE FOUND ON MANY
OTHER CELL TYPES AND HAVE BEEN
IMPLICATED IN A NUMBER OF OTHER
PROCESSES AND OF COURSE OUR
INTEREST WAS SPECIFICALLY IF IT
COULD REGULATE IMMUNE CELLS.
WHEN WE STARTED OUT WITH WAS
DEVELOPING AN ASSAY SO THAT WE
COULD MEASURE THE ACTIVITY ON
FXR OR THROUGH FXR OF A PANEL OF
BOWEL ACIDS, BOTH PRIMARY AND
SECOND BY BOWEL ACIDS AND WHAT
WAS VERY STRIKELY IN THIS ASSAY
WAS THAT ONLY ONE PRIMARY BOWEL
ACIDS CDCA WAS ABLE TO FUNCTION
AS AN AGONIST, ALL OF THE OTHERS
WERE VERY WEAK OR PARTIAL
AGONIST AND ACTUALLY IF YOU MIX
IT, THEY COULD FUNCTION SOMEWHAT
AS AN ANTAGONIST ALSO.
WE THEN ZOOMED IN SPECIFICALLY
ON T CELLS FOR TWO REASONS.
FIRST OF ALL, BECAUSE I TOLD YOU
ALREADY THAT T CELLS WITHIN THE
CON ESKS IT A GRAFT VERSUS HOST
ARE THE MOST CRITICAL CELLS THAT
WILL GET YOU TO A GRAFT VERSUS
HOST, AND THE SECOND THING WAS
THAT ONE OF THE FOLKS WHO WERE
WORKING WITH US ON THIS CLARISSA
CAMPBELL WHO NOW HAS HER OWN LAB
IN AUSTRIA, THAT SHE HAD ALREADY
DEMONSTRATED THAT IN ACTIVATED T
CELLS YOU CAN SEE IN TIME AN
UP-REGULATION OF THE EXPRESSION
OF FXR.
SO AFTER THEY HAD BEEN
ACTIVATED, I'M ONLY SHOWING YOU
HERE THE CD4 DATA AND SHOWING
INDEED THAT CDCA, PRIME PRIMARY
BOWEL ACID THAT COULD FUNCTION
AS AN AGONIST COULD READ TO
UPREGULATION OF INFLAMMATORY
GENES WHERE UDCA SEKSD RI BOWEL
ACIDS THAT CAN FUNCTION AS A
VERY WEAK AGONIST OR MAYBE EVEN
AN ANTAGONIST DOESN'T DO THAT.
AND THESE ARE THE CONTROLS BOTH
DRUG THAT CAN FUNCTION AS AN FXR
AGONIST OR AN ANTAGONIST.
SO ACTUALLY PRETTY STRONG
AGNOSTIC FUNCTION FOR CDCA.
WE THEN TOOK THAT TO SOME
FUNCTIONAL ASSAYS AND SHOWED IN
AN ASSAY THAT IF YOU LOOK AT THE
BASELINE PROLIFERATION OF
ACTIVATED T CELLS IF YOU ADD
AGAIN THAT PRIMARY BOWEL ACID
THAT CAN FUNCTION AS AN AGONIST
TO CDCA THAT YOU CAN BETTER
PROLIFERATION WHEREAS UDCA LEADS
TO WORSE PROLIFERATION.
FINALLY LOOKING AT ACTIVATION
MEASURED HERE BY CD25
EXPRESSION, WE SAW AGAIN THE
SAME THING, HERE ARE FXR
WILD-TYPE T CELLS SHOWING THAT
THE PRIMARY BOWEL ACID CDCA CAN
LEAD TO HIGHER LEVELS IN UDCA TO
LOWER LEVELS, WE ALSO DID THIS
WITH AN FXR KNOCK-OUT EXPHOWS
THERE WE SAW NO ACTIVITY AT ALL
OF CDCA AND YOU DCA.
SO THEN WE WENT BACK TO MOUSE
MODELS FOR GRAFT VERSUS HOST AND
STARTED BY SIMPLY GIVING THESE
MICE AN AGONIST AND SO
OVERALL -- AND SAW OVERALL WORST
OUTCOMES, WORST GRAFT VERSUS
HOST, THAT DOESN'T TELL YOU YET
THROUGH WHICH PATHWAY, WHICH
CELL TYPE.
WE GENERATED CONDITIONAL
KNOCK-OUT MOUSE AND IN THESE
MODELS YOU CAN SPECIFICALLY TAKE
ONLY T CELLS THAT ARE DEFICIENT
FOR FXR.
AND IN TWO DIFFERENT MODELS WE
SAW LESS GRAFT VERSUS HOST IF WE
USE FXR DEFICIENT DONOR T CELLS.
WE ANALYZED THAT ALSO UPSIDE
DOWN AS WE ALWAYS DO OF ALL OF
THE TISSUES, THE CYTOKINES, THE
INFILTRATION OF T CELLS AND SO
ON AND SO ON, WILL SPARE YOU
THAT AND JUST SAY THAT OVERALL
WE SAW LESS GRAFT VERSUS HOST
SPECIFICALLY WE SAW LESS GRAFT
VERSUS HOST WITHIN THE LARGE
BOWEL.
SO THEN WE TOOK ALL OF THIS TO A
SETTING AND SELECTED 60 PLUS
PATIENTS THAT WERE MATCHED, ONE
GROUP WITH WITH, ONE GROUP
WITHOUT GRAFT VERSUS HOST AND
I'LL TELL YOU NOW THAT THAT
BASICALLY MIRRORED WHAT WE FOWN
IN THESE MOUSE MODEL -- FOUND IN
THESE MOUSE MODELS.
SO STARTING WITH HAVING INDEED
IN PATIENTS WITH GRAFT VERSUS
HOST, LOWER LEVELS OF BOWEL
ACIDS OVERALL.
AGAIN, SMESKLY LOWER LEVELS OF
THESE SECONDARY BOWEL ACIDS AND
DECONJUGAT HE D BOWEL ACIDS AND
SHOTGUN SEQUENCING WE SAW AGAIN
SPECIFICALLY A LOWER EXPRESSION
OF BOWEL SALTS, HYDROLASE IN
BACTERIA FROM PATIENTS WITH
GRAFT VERSUS HOST.
AND AGAIN, THESE SECONDARY BOWEL
ACIDSES THAT HAD BEEN IMPLICATED
TO BE RELEVANT FOR TH167 AND FOR
REGULATORY -- TH17 AND FOR
REGULATORY T CELLS WERE FOUND IN
MUCH LOWER LEVELS IN PATIENTS
WITH GRAFT VERSUS HOST.
NOW, INTERESTING AND I GUESS
FORTUNATE FOR US, THERE WAS A
TIME HAD OUR CENTER WAS NOT
GIVING PATIENTS IRSODIOL,
SECONDARY BOWEL ACIDS I
MENTIONED ALREADY SO WE LOOKED
BACK AT PATIENTS THAT WERE NOT
GETTING URS DIOL AND AFTERWARDS
PATIENTS WHO DID AND SAW INDEED
A STRIKING DIFFERENCE IN THE
INCIDENCE HF ENTEROCOCCUS, I
TOLD YOU EARLY ERB IN THAT NAIRP
WE RECENTLY PUBLISHED THAT
SPECIFICALLYILY OTICA COULD BE
ONE OF THE DRUGS ASSOCIATED WITH
LOWER LEVELS OF ENTEROCOCCUS,
SEEMED TO BE HOLDING UP ALSO IN
THESE PATIENTS.
SO I DON'T KNOW HOW I'M DOING ON
TIME.
IT SEEMS LIKE I VINYL FEW
MINUTE, SO I'M GOING TO READ THE
WHOLE SUMMARY HERE.
JUST TO TELL YOU THAT WHERE WE
ARE AT THE MOMENT IS OF COURSE
TRYING TO TAKE THIS INTO THE
CLINIC, AS YOU KNOW, MANY
SETTINGS AT THE MOMENT IS WHERE
PEOPLE ARE TESTING FECAL
TRANSPLANTS, BUT WE ARE
INTERESTED IN AND PARTNERING
WITH THIS COMPANY TO GIVE REALLY
DEFINED CON SORE -- CONSORTIA.
WE ARE GIVING 16 STRAINS
SELECTED FOR VARIOUS FUNCTIONS
THAT WE THINK WOULD BE RELEVANT
FOR THE OUTCOME AFTER ALLOGENIC
TRANSPLANT FROM GRAFT VERSUS
HOST TO IMPROVED BARRIER TO
REMEMBER COLONY REZIS SENTENCE
AGAINST PATHOGEN -- RESISTANCE
AGAINST PATHOGENS, THIS TRIAL IS
ONGOING AT THE MOMENT AND
ENTERING PRESS RELEASE ACTUALLY
SHOWS THAT SULFUR IT -- THAT SO
FAR IT SEEMS TO BE WORKING
ACCORDING TO PLAN AND THAT
SPECIFIC WHAT WILL WE'RE SEEING
IS LESS DOMINATION WITH
PATHOGENS IN PATIENTS GETTING
BEFORE AND AT A CERTAIN TIME
POINT AFTER THE ALLOGENIC
TRANSPLANT A COUPLE OF DAYS OF
THIS DEFINED CONSORTIUM.
SO THAT'S ALL THAT -- I MEAN, I
HAVE A SECOND STORY, BUT I KNEW
ALREADY THAT I WOULDN'T GET TO
THAT.
[LAUGHTER]
THAT'S WHY I PUT MY END SLIDE
HERE.
OF COURSE I'M GOING TO THANK MY
FUNDING AND OF COURSE HERE IN
THIS ROOM I SPECIFICALLY WANT TO
THANK THE NIH THAT HAS BEEN VERY
GENEROUS TO US.
AND THE VARIOUS GRANTS TO THE
PEOPLE WITHIN MY LAB AND OF
COURSE FIRST AND MOST OF AWFUL
THE PEOPLE IN MY LAB WHO WERE
DOING ALL OF THIS WORK.
THANK YOU VERY MUCH.
[APPLAUSE]
>> THANK YOU.
QUESTIONS?
>> I HAVE ONE L BEAUTIFUL TALK,
AS ALWAYS.
THERE WILL NCI, FOR THOSE THAT
ME.
ACTUALLY, I WAS CAUGHT WITH THE
LIST OF DRUGS YOU HAD THERE THAT
THE PATIENTS RECEIVE AND THEY
ACTUALLY HAVE A REALLY HARD TIME
WITHOUT THEM, SO IT'S HARD TO
TAKE THOSE AWAY.
SO MY QUESTION WAS A BIT BROADER
AND WHAT IS THERE THAT ONE CAN
DO ABOUT IT?
BECAUSE THIS IS A QUESTION THAT
I KEEP ASKING, YOU KNOW, THE
ANSWER WE USUALLY GET FROM
ONCOLOGISTS IS THAT'S THE
PROTOCOL.
DO YOU WANT TO TAKE YOUR
CHANCES?
YOU KNOW, NOT THE ANSWER WE LIKE
TO HEAR.
BUT DO YOU SEE ANY POSSIBILITY
OF REALLY DOING, YOU KNOW,
MEANINGFUL STUDIES TO ADDRESS
THE IMPACT OF ALL THESE DRUGS IN
MIKE BOAM AND THEREFORE --
MICROBIOME AND THEREFORE
RESPONSE TO SAY THERAPIES?
>> YES, FOR SURE.
A STUDY IS OPEN NOW THAT I
DIDN'T MENTION STARTED WITH OF
COURSE THE FIRST CATEGORY OF
DRUGS THAT WE'RE INTERESTED IN
WHICH IS BROAD-SPECTRUM
ANTIBIOTICS.
EVERY CENTER FOR REASONS THAT
ARE OFTEN NOT COMPLETELY CLEAR
HAVE THEIR OWN FAVORITE, BUT
THERE ARE PHASE III STUDIES THAT
HAVE DEMONSTRATED THAT ABOUT
FIVE, SIX DIFFERENT ONES HAVE
EQUAL OUTCOMES.
SO YOU HAVE A CHOICE.
YOU CAN MAKE A CHOICE DL.
AND JUST TO BE VERY SPECIFIC,
BACTIN, BANT, CEPHAPIME MUCH
BETTER SO WE ARE DOING A
RANDOMIZED STUDY AT THE MOMENT
WHERE PATIENTS WHO AFTER ANNAL
LOWGENIC TRANSPLANT HAVE FEVER
AND NEUTROPOENA ARE BEING
RANDOMIZED TO EITHER GETTING --
FOR NAUSEA MEDICATIONS WHERE WE
CHOOSE FROM AT LEAST FIVE OR SIX
DIFFERENT ONES, OFTEN THEY'RE
EQUALLY BENEFICIAL, AND PAIN
MEDICINES.
SO CERTAINLY IN THOSE TWO
CATEGORIES, WE HAVE CHOICES, SO
WE CAN TEST THAT.
>> HEY, MARCEL.
>> HEY, DAVID.
>> HELLO.
>> HI THERE NEIGHBOR.
>> WE MUST STOP DATING LIKE THIS
IN BETHESDA.
WE DO LIVE NEXT-DOOR TO EACH
OTHER.
GREAT PRESENTATION, AS ALWAYS.
I'LL ASK YOU A SLIGHTLY BROADER
QUESTION THAT RELATES TO A
CONVERSATION I HAD EARLIER THIS
WEEK ABOUT THE NIH STRATEGIC
MISSION ON NUTRITION AND HUMAN
HEALTH, 2030 DOCUMENT I'M SURE
EVERYONE IS AWARE OF.
SO BEAUTIFUL WHAT YOU'RE TALKING
ABOUT IN TERMS OF FIBER AND CDCA
IN PARTICULAR AND REGULATING
INFLAMMATORY T CELL RESPONSES.
HOW DOES IT RELATE TO PRECISION
NUTRITION?
WHAT DO WE BRING THIS TO REAL
LIFE?
BECAUSE YOU DO THE GREAT BASIC
SCIENCE COOL EXPERIMENTS IN
MICE, RIGHT, ALL THE COOL STUFF.
BUT YOU ALSO DO THE HARD STUFF
IN REAL-LIFE CLINICAL TRIALS.
SO HOW CAN WE BRING THE
NUTRITION PIECE PAST, YOU KNOW,
AM BUY BAD PRODUCTS OR BADLY
LABELED IN THE SUPER MARKETED?
HOW DO WE BRING IT TO REAL LIFE?
>> RECALL SO TELL ME C WITH THE
HEALTH FOOD STORIES FINALLY HAVE
TO END, AND FOR THOSE OF YOU WHO
ARE NOT CLINICALLY ACTIVE BUT
EVERYBODY WHO IS CLINICALLY
ACTIVE AND IS TREATING A CANCER
PATIENT AT ONE POINT WILL GET
THE QUESTION, WHAT SHOULD I EAT,
DOCTOR?
IT'S ON EVERY PATIENT'S MIND
ALWAYS.
SO DIET STUDIES CAN BE DONE
BETTER, I'M SURE YOU KNOW ABOUT
SOME COMPANIES THAT ARE ACTUALLY
DOING THAT BASED UPON MICROBIOME
PROFILING, WHAT WE ARE DOING AT
THE MOMENT IS IN TWO WAYS
ANALYZING IN GREAT DETAIL WHAT
PEOPLE ARE EATING.
FIRST OF ALL, WHEN THEY ARE
INPATIENTS WE KNOW EXACTLY WHAT
FOOD WE ARE SERVING AND WE KNOW
EXACTLY ALL OF THE DETAILS OF
EVERY FIBER CONTENT AND
WHATEVER, AND I DIDN'T WANT TO
GO, I MEAN, THAT IS A WHOLE TALK
THAT I DIDN'T WANT TO GIVE NOW.
SO THERE WE CAN GET IN MUCH MORE
DETAIL ABOUT WHAT TYPE OF
NUTRIENTS.
BUT THERE'S A SECOND WAY OF
DOING IT WHICH IS A TAXONOMY OF
FOOD.
YOU ARE MIGHT HAVE SEEN SOME OF
THE TALKS BY FORMER POST-DOC WHO
NOW HAS HIS OWN LAB, SONY PALLET
WHERE HE FOR INSTANCE SHOWS HA
CERTAIN FRUITS AND JUICES
SPECIFICALLY PROVIDE SUGAR THAT
IS CAN BE BAD FOR THE OUTGROWTH
OF THESE PATD GENERALS THAT CAN
LEAD TO DOMINANCE.
SO THERE ARE TWO WAYS --
PATHOGENS.
TWO WAYS OF ANALYZING, SIMPLY
THE CONTENTS OF THE NUTRIENTS
AND SECOND THE FOOD GROUPS.
BASED UPON THAT, WE SHOULD BE
ABLE TO DEFINE STUDIES.
WE'RE DOING SOME OF THAT ALREADY
WITHIN MYELOMA, DIFFERENT
SEGHTD, WHERE WE HAVE POSITIVE
OUTCOMES WHEN WE JUST INCREASE
FIBER, WHICH IS A VERY OBVIOUS
ONE AND I DON'T HAVE TO TELL YOU
WHY, RIGHT?
SHORT CHAIN FATTY ACID, HEALING
OF THE GUT, ET CETERA.
>> MY QUESTION IS A COMPLEMENT,
NOT A CRITICISM I THINK WHERE
CANCER IS IN TERMS OF
UNDERSTANDING NEW THRITION AL
INTERVENTION INSIDE --
NUTRITIONAL INTERVENTIONS IN
HUMAN HEALTH AND DISEASE,LESS
SOME OF THE BEST STUDIES BEING
DONE IN HUMAN AND MOUSE RELATE
AROUND HOW NUTRITION IS
AFFECTING.
ONE OF THE STUDIES YOU WERE
INVOLVED IN I THINK HIGHLIGHTS
IT THAT, YOU KNOW, HIGH FIBER
DIETS CAN BE BENEFICIAL,
PROBIOTICS CAN BE DETRIMENTAL IN
CERTAIN CIRCUMSTANCES.
>> I THINK --
>> AND I HAVE TO STOP TALKING.
BUT I THINK YOU WERE PROVIDING
US WITH -- SO THANK YOU.
>> THANK YOU RK DAVID WE'LL TALK
LATER.
>> SHORT QUESTIONS ZEES SURE.
I WILL MAKE OUT WITH THIS
MICROPHONE LIKE NOBODY'S
BUSINESS.
>> THANK YOU.
>> DAVID ALREADY ASKED A BIG
PART OF MY QUESTION.
YOU KNOW, RELATED TO PRECISION
MEDICINE AND HOW YOUR BEAUTIFUL
PHASE I THRIEL YOU'RE -- THAT
YOU SPOKE ABOUT AT THE END,
YOU'RE ACTUALLY TAKING CONSORTIA
AND APPLYING IT TO EVERYONE BUT
IT'S THE SAME CONSORTIA SHA
WHICH I'M WONDERING HOW DO
DIFFERENT PEOPLE REACT TO THAT
SAME CONSORTIA AND I WAS REALLY
IMPRESSED THAT THERE WERE NO
ADVERSE EVENTS IN CONTRAST TO
THE LAST TRIAL, SO I'M CURIOUS
WHAT LESSONS HAVE YOU LEARNED
AND IMPROVED UPON THAT HAVE
ALLOWED YOU TO REACH THIS
REMARKABLE MILESTONE?
>> YEAH 6789.
SO YOU'RE HINTING, PART OF AN
NIH PROJECT AND THAT IS TO
UPFRONT MEASURE WHAT'S THE
DEFICIENT SIZ THAT MIGHT BE IN
SOMEBODY'S MICROBIOME.
BASED UPON THAT PROFILE, DEFINE
A SPECIFIC CONSORTIUM.
SO PRECISION MEDICINE IN TERMS
OF THE CONSORTIA THAT WE WOULD
WANT TO GIVE.
THINK ONE OF THE REASONS WHY OUR
STRATEGY WAS RELATIVELY SAFE WAS
BECAUSE OF THE BACTERIA ONLY
BEING GIVEN BEFORE THE
CONDITIONING REGIMEN.
AT THAT POINT WE DIDN'T GIVE
ANYTHING ANYMORE UNTIL THEY WERE
OFF ANTIBIOTICS AND HAD THEIR
NEUTROPHILS BACK, SO THEY WERE
ABOUT THREE WEEKS OUT FROM
ALLOGENIC TRANSPLANT, THEN THEY
GOT A SECOND COURSE, MAYBE
THAT'S WHY IT WAS RELATIVELY
ZIEF VERY IMPRESSIVE.
>> THANK YOU.
>> IS IT OKAY IF I USE THIS?
>> THE OTHER ONE.
HAD.
>> DOES THIS WORK?
OKAY, GOOD.
OH, THAT'S NOT MY TALK.
OKAY.
R MY TALK WITHOUT GLASSES, NOT
EASY.
I WANT TO THANK THE ORGANIZER
FOR INVITING ME TO PRESENT HERE
TODAY.
OKAY.
THERE WE ARE.
I'M REALLY HAPPY AND ACTUALLY
HAD, THIS COULD HAVE NOT BEEN
BETTER BACK TO BACK
PRESENTATIONS AND ALL THE
QUESTIONS HAVE REALLY KIND OF
INTRODUCED WHAT I'M GOING TO
TALK ABOUT, AND YOU'LL SEE WHY
I'M SAYING THIS IN A MOMENT, BUT
I'M GOING TO FOCUS ON WHAT WE'RE
DOING ON TRY TO UNDERSTAND HOW
MICROBIOTA ACTUALLY REGULATE
THESE ANTI-TUMOR RESPONSES.
I DON'T NEED TO TELL YOU THAT
TUMORS DEVELOP ALONGSIDE A
HIGHLY COMPLEX AND DYNAMIC TUMOR
MICROENVIRONMENT AND ALL THE
RESULTS WE CAN FIND IN IMMUNE
CELLS PRESENT IN THESE TUMOR
MICROENVIRONMENT, THOSE FAMILIAR
WITH MACK FAJS, MONOCYTES AND
DENDRITIC CELLS MAJOR COMPONENTS
OF THESE TME.
THESE CELLS ARE CRITICAL
INITIATORS AND REGULATORS OF
IMMUNITY AND PLAY A VERY
IMPORTANT ROLE BOTH IN
PATROLMANNING OR CONTROLLING
TUMOR PROGRESSION AS WELL AS
RESPONSE TO THERAPY.
PROBABLY FOR THE MOST PART WHEN
PEOPLE THINK ABOUT MACROFADGES
IN THE CONTEXT OF THE TME THEY
TONED THINK ABOUT A PROTEOGENIC
CELL TYPE WHILE ON THE OTHER
HAND WHEN THEY THINK ABOUT
DENDRITIC CELLS, THEIR UNIQUE
ABILITY TO PRIME OR ACTIVATE 19
CELLS WE THINK ABOUT THEM AS
ANTI-TUMORGENIC, BUT THE
REALITIES THAT NOTHING IS SO
BLACK AND WHITE AND THESE CELLS
ARE VERY PLASTIC AND THEY CAN
ALLLY OBSERVE -- AND
ANTI-TUMORGENIC.
WHAT ARE THE CUE FOR THESE --
THAT REGULATE IN THE TME?
I DON'T NEED TOLL YOU NOW WE
KNOW THAT MICROBUY ON THE
MICROBIOTA PLAYS A IMPORTANT
ROLE IN ANTICANCER TREATMENT.
FOR THE MOST PART THE STUDIES
THAT NOW ARE MANY THAT HAVE BEEN
TAKING PLACE IN THE PAST DECADE
OR SO HAVE MOSTLY FOCUSED ON THE
ADAPTING IMMUNE RESPONSE.
SO WHAT THESE STUDIES HAVE SHOWN
IS THAT MICROBIOTA INFLUENCES
RESPONSE TO ANTICANCER THERAPIES
AND THIS IS EITHER BY DIRECTLY
OR INDIRECTLY REGULATING
ANTI-TUMOR T CELL FUNCTION.
HOWEVER, EVEN THE STUDIES THAT
IDENTIFY SPECIFIC MICROBIOTA
METABOLITES, T CELL INFECTION,
THEY SHOW THESE BENEFICIAL
EFFECTS OF MICROBOT AT THAT
STILL RELIES ON THE PRESENCE OF
MONO THUK LAR.
OUR PARTICULAR INTEREST IN THESE
CELLS TO ASK WHETHER MICROBIOTA
REGULATE THE FUNCTIONAL STATE OF
MONONUCLEAR PHAGOCYTES AND IF SO
HOW.
I WANT TO MAKE A VERY LONG STORY
VERY SHORT AND GIVE YOU THE
SUMMARY BUT BASICALLY THIS IS
THE WORK, THIS IS PUBLISHED SO
I'M GOING TO GO THROUGH ALL THE
DETAILS, THIS WORK WAS LED BY
TWO FELLOWS IN THE LAB, AND WHAT
THEY SHOWED WAS THAT MICROBIOTA
AGO -- STING AGONISTS, ANTI-TOMB
MONO SIETLE IN TERM ACTIVATING
NK CELLS THAT PRODUCE XCL1 AND
CCL5 AND AT THE SAME TIME TYPE 1
INTERFERON HAS A MONOCYTE THAT
DIFFERENTIATES INTO MACROPHAGES
WITH A MORE ANTI-TUMORGENIC
PROFILE.
IN THE ABSENCE OF MICROBIOTA,
THIS CASCADE IS ALTERED AND
MONOCYTES INSTEAD DEFAULT ON A
MORE PROTEINGENIC MACROPHAGE
PROFILE.
WE WERE ABLE TO SHOW THAT
ACTUALLY HAD SIMPLY
ADMINISTERING CDR, STING, WE
COULD COMPLETELY RESTORE THE
SYSTEM INDUCE TYPE I IN K AXIS
AND RESTORE THE REPERTOIRE OF
MONONUCLEAR PHAGOCYTES.
SO WE BASICALLY ESTABLISHED THAT
ACTUALLY INDEED MICROBIOTA
REGULATE ANTI-TUMOR IMMUNITY VIA
THE REGULATION OF MONONUCLEAR
PHAGOCYTES IN TME AND THEIR VERY
COMPLEX INTERPLAY WITH OTHER
CELLS AND SHOWED YOU CDM COULD
RESCUE FUNCTIONING IN TME BUT
THE QUESTION WAS IS THERE
ANYTHING WE COULD DO TO
MANIPULATE MICROBIOTA PRIOR SO
THAT WE CAN SET THE STAGE FOR A
MORE PERMISSIVE ANTI-TUMOR
RESPONSE AND BETTER RESPONSES TO
THERAPY?
AND AS MENTIONED AMONG THE MANY
APPROACHES THAT WE CAN USE TO
CHANGE OR ALTER MICROBIOTA
COMPOSITION, WE HAVE THE FMT'S,
WE HAVE DIET INTERVENTIONS, WE
KNOW THE USE OF ANTIBIOTICS OR
SINGLE AGENT OR COMBINED
PROBIOTICS.
SO WE DECIDED TO START WITH DIET
BECAUSE WE FIGURE IT'S PRETTY
EASY TO TRANSLATE CLINICAL LI
AND PROBABLY A LITTLE BIT MORE
PLEASANT THAN SOME OF THE OTHER
APPROACHES.
AND INDEED IN THIS CASE WAS WE
PUT ANIMALS ON DIFFERENT DIETS
AND THEN HE LOOKED AT THE EFFECT
OF THESE DIETS ON TUMOR GROWTH.
WHAT YOU'RE LOOKING AT IN THIS
GRAPH HERE IS THE EFFECT OF THE
DIET OF EACH OF THESE DIET
COMPARED TO THE STANDARD CHOW
THAT WE FEED THE MICE AND THE
OVERALL TUMOR GROWTH.
SO ANYTHING THAT IS TO THE
RIGHT, SO ABOVE C MEANS IT'S A
DETRIMENTAL EFFECT THE TUMORS
WERE BIGGER AND ANYTHING BELOW
ZERO SHOWS A BENEFICIAL EFFECT
OF THE DIET.
AND YOU CAN SEE THAT DIFFERENT
DIETS MAY HAVE DIFFERENT
EFFECTS.
SOME ARE ACTUALLY WORSE, SOME
DON'T MAKE ANY DIFFERENCE AND
SOME CAN BE VERY POWERFUL AT
IMPROVING TUMOR GROWTH CONTROL
AS IS THE CASE OF HIGH FIBER
DIETS.
NOW, I JUST WANT TO POINT OUT
HERE BECAUSE WE WERE TALKING AND
THE QUESTION CAME UP ABOUT, YOU
KNOW, WHAT DO WE EAT, WHAT
FIBERS, ET CETERA, NOT EVERY
FIBER HAS THE SAME EFFECT. SO
THAT'S SOMETHING TO ADD TO THE
DISCUSSION THAT WAS HAPPENING
EARLIER.
BUT WE DECIDED TO FOCUS ON THESE
FIBER IN PARTICULAR THAT HAD THE
MOST STRIKING EFFECT AND WE DID
ALL OUR WORK DOWNSTREAM ANALYSIS
WITH THAT AND WE SHOWED THAT
INDEED NOT ONLY IT IMPROVES
TUMOR GROWTH CONTROL, IT
ACTUALLY HAS A SYNERGISTIC
EFFECT WHEN WE TREAT THE ANIMALS
WITH ANTI-P PDL1.
DIS YOU MAY BE WONDERING DOES
THIS HAVE ANYTHING TO DO WITH
THE MECHANISM I SHOWED YOU THERE
WITH AXIS AND THE SHORT ANSWER
IS YES, IT DOES.
ACTUALLY WHAT WE FOUND WAS THAT
HAD THE ANIMALS THAT WERE ON
HIGH FIBER DIET WHEN WE LOOKED
AT THE TUMOR IN THE MIKE
MICROBIOTA ENVIRONMENT, FOUND IT
INCREASED PRODUCTION -- COMPARED
TO CONTROL.
FURTHERMORE AND IF WE THINK
ABOUT A PROBIOTICS APPROACH, WE
LOOKED AT THE MICROBIOME CHANGES
IN THESE ANIMALS AND IDENTIFY.
A BACTERIUM THAT WAS ACTUALLY
ENRICHED BY THE HIGH FIBER DIET
THAT WAS CAPABLE OF COMPLETELY
RECAPITULATING THE FIBER DIET
BENEFICIAL EFFECT.
TRIGGERING EXACTLY ALL THE SAME
INNATE MECHANISMS.
SO HOW IS THIS RELEVANT TO
PATIENTS?
WE JUST HEARD A LOT OF THESE
TYPE OF APPROACHES ARE ACTUALLY
BEING USE IN THE CLINIC. SO WE
ASKED THE SAME QUESTION, AND HAD
WHAT WE FOUND WAS THAT ACTUALLY
FMT, SO MICROBIOTA FROM IMMUNE
BLOCKADE WAS IN A PATIENT,
TRANSFERRED INTO ANIMALS,
CAPABLE OF COMPLETELY
RERECAPITULATE THE SAME
PHENOTYPE, TRIGGEREDLY TYPE 1
AND K PATHWAY, REMODEL THE
REPERTOIRE AND LED TO IMPROVED
TUMOR GROWTH CONTROL, NOT ONLY
THAT, WHEN WE ANALYZED DATA,
WHEN WE ANALYZED TUMORS, RNA
SEQUENCING FROM TUMOR SAMPLES OF
THE HUMAN TRIALS THAT HAVE BEEN
RECENTLY PUBLISHED, WE FOUND
THAT ACTUALLY THE HUMAN TO HUMAN
FMT RESULTED IN THE SAME,
INCREASING ALL THESE INGNAT
IMMUNE SIGNATURES THAT WERE
ASSOCIATED WITH BETTER RESPONSE
TO IMMUNE BLOCKADE.
SO ALL OF THESE IS CURRENTLY
ACTUALLY BEING DONE IN THE
CLINIC.
NOW, THE REALITY IS THAT AND
THAT WAS RELATED TO ONE OF THE
QUESTIONS, NOT ALL PATIENTS ARE
RESPONDING THE SAME WAY TO THE
SAME TYPE OF MICROBIOTA TARGETED
APPROACH, THAT WAS VERY CLEAR IN
THE CASE OF FMT INITIAL TRIALS
THAT MAISHTS IT WE SEE, SAME
DONOR, MICROBIOTA, SOME
RESPONDED BUT SOME DIDN'T.
THE PATIENTS THAT WE SEE.
THAT REALLY TRIGGERED US TO
WONDER, DO YOU ATUMORS RESPOND
EQUALLY TO THE SAME MICROBIOTA
TARGETED THERAPY?
TO ADDRESS THIS QUESTION, WHAT
KIM IS CURRENTLY DOING IN THE
LAB IS HE WENT BACK TO THESE
FIBER DIET THAT HAD SUCH A
STRONG RESPONSE IN THE MODELS WE
HAD LOOKED AT AT THE TIME AND
TESTED 16 DIFFERENT MODELS,
INCLUDING IMPLANTED
SUBCUTANEOUSLY IMPLANTED AND
SPONTANEOUS TUMOR MODELS AND YOU
CAN SEE IN THE GRAPHS AGAIN IF
WE GO ABOVE ZERO, DETRIMENTAL
EFFECT, BELOW ZERO -- CERTAIN
TUMORS MAMMARY CARCINOMA SRKSZ
HAVE ACTUALLY NO RESPONSE TO THE
DIET OR THEY ACTUALLY MAKE IT
WORSE, WHILE THE VAST MAJORITY
OF MELANOMAS ACTUALLY DO
RESPOND.
BUT THEY DO SO AT VARYING
DEGREES.
SO WE CAN STRATIFY THESE TUMOR
AS NONRESPONDERS, PARTIAL
RESPONDERS OR EXCEPTIONAL
RESPONDERS.
WHAT IS THANK OR WHAT IS
AFFECTING ALL OF THESE AND ONE
OF THE MAIN THINGS THAT COME TO
MIND ARE HOST FACTORS, BUT WHAT
KIM DID TO ADDRESS THAT QUESTION
WAS HE TOOK ANIMALS THAT FED THE
DIET AND THEN HE IMPLANTED IN
THE SAME ANIMAL CONTRALATERALLY
EITHER AN EXCEPTIONAL RESPONDER
MELANOMA OR IN THE OPPOSITE SIDE
A MAMMARY CARCINOMA IN THAT
DOESN'T RESPOND.
WHAT YOU'RE LOOKING AT IN THIS
GRAPH HERE IS THAT EVEN WHEN
IMPLANTED INTO THE SAME HOST,
THE EFFECT OF FIBER IS
MAINTAINED, RESPONDERS ARE STILL
RESPONDER AND NONRESPONDERS ARE
NONRESPONDERS, WHICH MEANS THERE
IS CLEARLY A TUMOR INTRINSIC
EFFECT THAT SOMEHOW IS
CONTROLLING -- SO WHAT ARE THESE
FACTORS?
THESE ARE THE THINGS WE ARE
ADDRESSING.
I DON'T HAVE THE ANSWERS YET.
THIS IS WHAT WE'RE TRYING TO
FIGURE OUT.
BUT WE KNOW TUMOR TYPE MAKES A
DIFFERENCE.
WE KNOW THAT TUMOR IN
MICROENVIRONMENT IS VERY
IMPORTANT, WE SEE CHANGES IN
RESPONDER TUMORS THAT DO NOT
HAPPEN IN THE NONRESPONDER
TUMORS.
WE HAVE NOW PRIMARILY DATA THAT
SUGGESTS TUMOR METABOLISM MAY BE
A FACTOR AS WELL AND TUMORLY
IMMUNOGENICITY.
I'LL SHOW YOU A LITTLE BIT OF
WHAT WE HAVE WITH TUMOR
IMMUNOGENICITY, BECAUSE I THINK
IT'S IMPORTANT.
IT FAVORS THE RESPONSE, SO IT
ENCOMPASSES THE RESPONSE OF NOT
ALREADY RESPONDER TUMOR WHAVMENT
I MEAN WI THAT IS IF WE TAKE ONE
OF THE MELANOMAS THAT WE
CLASSIFY AS A PARTIAL RESPONDER
AND NOW WE ARTIFICIALLY INCREASE
THE IMMUNOGENICITY BY FOR
EXAMPLE OVEREXPRESSING VITAMIN
HIGHLILY -- IMMUNOGENIC TO MICE,
IF WE DO THAT WITH MAMMARY
CARCINOMAS THAT DON'TS
RERESPONSIBILITY, NOTHING
CHANGES.
THIS MAKES SENSE THAT INCREASING
IMMUNOGENICITY CONTRIBUTES
BECAUSE SOMETHING I DIDN'T SHOW
YOU IS THAT -- SORRY, THE
CHANGES THAT WE SEE IN THE TUMOR
MICROENVIRONMENT OF THOSE TUMORS
THAT BENEFIT OF THE FIBER EE
EFFECT ARE THAT DOWNSTREAM ALL
THESE INNATE PATHWAY THAT I
SHOWED YOU EARLIER AND OF COURSE
RELATED TO THE DENDRITIC CELLS
THAT WE SEE, WE SEE CHANGE IN
THE T CELL COMPARTMENT PARTMENT
AS WELL WHICH ARE A REDUCTION IN
RECALL RM T CELLS AND UP
REGULATION AND ENHANCED, SEE
REDUCTION IN T REGULATORY CELLS
AND MORE CD4 ACTIVATED T CELLS
WHICH SETS REALLY A VERY NICE
STAGE FOR A GOOD RESPONSE TO AN
IMMUNE CHECK BLOCKADE SUCH AS
EPITHELIAL 1 THAT I SHOWED YOU
EARLY HAD SYNERGISTIC EFFECT AND
WHAT'S BEING SHOWN IN THE CLINIC
IN OBSERVATION STUDIES WITH
FIBER AND BLOCKADE.
WHAT ABOUT OTHER THERAPIES?
NOW WE GO BACK TO THE PREVIOUS
CONVERSATION.
WHAT HAPPENS TO PATIENTS THAT
ARE RECEIVING CHEMOTHERAPY?
THESE PATIENTS I ALWAYS ALMOST
INEVITABLY TREATED WITH --
THESE PATIENTS ARE ALMOST
INEVITABLY TREATED WITH
ANTIBIOTICS.
CHECKPOINT, I APOLOGIZE, IT'S A
REALITY WHAVMENT WE SHOWED IN
OUR ORIGINAL STUDIES TEN YEARS
AGO WAS THAT MICROBIOTA ALSO
INFLUENCES RESPONSE TO CHEMO,
FOR EXAMPLE, PLATINUM BASED I AM
FLOW THERAPIES, SO FOR EXAMPLE
ANIMALS THAT LACK MICROBIOTA DO
NOT RESPOND TO OXALIPLATIN,
THESE DRUGS AND MANY OTHER
CHEMOS NEED IS HIGH ACTIVATION
OF REACTIVATION SPECIES AND WE
SHOWED AT THE TIME THAT IN
ABSENCE OF MICROBIOTA THERE WAS
AN IMPAIRED PRODUCTION OF ROS IN
THE TUMOR AND HOST IMMUNE CELLS
WERE NEEDED FOR THE RESPONSE F
WE GO BACK TO THE TME AND THE
MINOR COMPARTMENT WHO ARE
AMAZING HOST PRODUCERS WE CAN
SEE IN THE TME NEUTROPHILS.
ROS PRODUCERS.
SO THE ROLE OF NEUTROPHILS IN
RESPONSE TO CHEMOTHERAPY, WORK
LED BY ROMEO R RUSSIA IN THE LAB
AND SHOWED THAT THE NEUTROPHIL
IS INDEED THE MAIN SOURCE OF ROS
IN RESPONSE AND THEY'RE ACTUALLY
NECESSARY FOR THERAPEUTIC
EFFICACY.
BUT FURTHERMORE, WHAT WE FOUND
WAS THAT MICROBIOTA REGULATES
THEIR QUANTITY AND THEIR QUALITY
IN THE TME AND THAT THE CHANGES
THAT ARE INDUCED BY CHEMO, WHICH
IS A REPROGRAMING OF THE
NEUTROPHILS THAT BECOME MORE
ANTI-TUMORGENIC AND PRODUCE
BUCKETS OF ROS LEADING TO AN
EFFECTIVE RESPONSE IS IMPAIRED
WHEN WE DON'T HAVE MICROBIOTA.
SO WE'RE GOING BACK TO THE
BIGGEST QUESTIONS, HOW, AND IS
THAT THE SAME MECHANISM AT THAT
I SHOWED BEFORE, THE OTHER ONE
WAS VERY -- AND AGAIN, I'M GOING
TO MAKE IT SHORT, BUT WE KNEW
THAT MICROBIOTA REGULATES
NEUTROPHIL ANTIMICROBIAL
FUNCTIONS.
WE ALSO KNEW THAT IT REGULATES
ROS PRODUCTION BY MACROPHAGES
AND THIS WAS THE ACTIVATION OF
THESE NOD1 RECEPTOR, WE HAD A
SIMPLE HYPOTHESIS, DIST SAME
PATHWAY REGULATE ROS PRODUCTION
IN NEUTROPHILS IN TUMOR
MICROENVIRONMENT, WE TESTED
THAT, AS I TOLD YOU EARLIER IF
THERE'S NO MICROBIOTA THE ROS
PRODUCTION STLINLT, WE GAVE
LIGAND AND THERE WAS SLEURTLY NO
EFFECT.
HOWEVER, WHEN WE GAVE THEM A
DIFFERENT NDP THAT BINDS TO A
DIFFERENT RECEPTOR NOT TO
COMPLETELY RESTORE NEUTROPHIL
FUNCTION IT WASN'T JUST A
FUNCTION, IT'S RESTORED THE
WHOLE DYNAMICS OF NEUTROPHILS
AND WAS ABSENCE OF MICROBIAL BUT
MOST IMPORTANTLY IT RESTORED THE
ABILITY OF THESE ANIMALS TO
RESPOND TO TREATMENT.
SO BASICALLY WE HAVE A SCENARIO
WHERE NOW WE HAVE CHEMOTHERAPY,
IT ENGAGES ALTHOUGH VERY
DIFFERENT CELLULAR SEQUENCE,
NEUTROPHILS ARE IMPORTANT AND IF
WE HAVE MICROBIAL SIGNALS THESE
NEUTROPHILS ARE GOING TO RESPOND
PRODUCE ROS AND LEAD TO TUMOR
ERADICATION.
IF NOT, THEY WILL BECOME --
FNLT, WE'LL FAIL TO TREAT THESE
TUMORS, BUT I JUST SHOWED YOU
THAT WE IDENTIFIED MDP THAT CAN
REVERT THIS SCENARIO.
AND THIS IS VERY IMPORTANT IN
THE CLINIC FOR PATIENTS
RECEIVING CHEMO BECAUSE AS
MARCEL JUST MENTIONED, THESE
PATIENTS BECOME NEUTROPOENIC,
DEVELOP FEVERS AND THERE THEY GO
TO RECEIVE THEIR ANTIBIOTIC
COCKTAIL.
SO AS NEUTROPHILS ARE COMING
BACK, THEY ARE DOING SO IN AN
ENVIRONMENT WITH AN ALTERED
MICROBIOTA BECAUSE OF THE
ANTIBIOTIC TREATMENT.
SO IF WE HAVE A MOLECULE THAT WE
CAN USE TO REEDUCATE THESE
NEUTROPHILS AS THEY COME BACK
AND GET READY FOR THE NEXT DOSE
OF CHEMO, THAT CAN MAKE A BIG
DIFFERENCE IN TERMS OF
THERAPEUTIC RESPONSE.
AND WHAT IS EVEN BETTER ABOUT
THIS IS THAT THERE ARE MANY MVP
SYNTHETIC AN ANALOGS THAT HAVE
ALREADY BEEN DEEMED SAFE IN THE
CLINIC AND ARE USING DIFFERENT
SETTINGS, SOME INCLUDING
ACTUALLY CANCER.
SO I THINK THAT IT SETS REALLY
GIVES THE RATIONALE TO START
THINKING ABOUT CLINICAL TRIALS
OR SOMETHING LIKE THIS WHERE IT
COULD BE DONE FOR CHEMO.
I'M FINISHING WITH THE LAST
SLIDE.
WHAT I WANT TO YOU TAKE HOME AS
A BIG MESSAGE FROM THIS IS, YES,
MICROBIOTA PROGRAMS THE TME FOR
A GOOD RESPONSE, BUT AND IT'S A
BIG BUT, NOT ALL TUMORS RESPOND
EQUALLY TO THE SAME MICROBIOTA
TARGETED THERAPY, BOTH HOST AND
TUMOR INTRINSIC FACTORS
INFLUENCE THIS RESPONSE, AND
DIFFERENT THERAPIES, DIFFERENT
CELLULAR CIRCUITS AND DIFFERENT
MICROBIOTA MEDIATED MECHANISMS.
THE ONLY WAY WE CAN COME UP WITH
MEANINGFUL ACTIONABLE APPROACHES
IS IF WE REALLY UNDERSTAND HOW
ALL OF THIS IS HAPPENING, SO
THAT WE CAN BYPASS A LOT OF WHAT
IS UPSTREAM AND HAVE SOMETHING
WE CAN USE AS A DRIVER DIRECTLY
AND I CAN'T EMPHASIZE THAT
ENOUGH.
AND I'M GOING TO FINISH, I
ALREADY MENTIONED KIM, I WANT TO
THANK EVERYBODY IN THE LAB,
SMALL GROUP, EVERYBODY
CONTRIBUTES RG ALL THE
COLLABORATORS WITHIN AND OUTSIDE
NIH AND MANY COURSE AND
FACILITIES THAT WE HAVE ACCESS
HERE THAT MAKE ALL OF THIS
POSSIBLE.
I THANK YOU FOR YOUR ATTENTION
AND I LOOK FORWARD TO YOUR
QUESTIONS.
[APPLAUSE]
>> VERY NICE TALK.
SO YOUR HIGH FIBER DIET MODEL,
SO YOU MENTIONED THAT XL IS 1 IS
INCREASED, RIGHT?
SO JUST WONDERING, THE OTHER
CHEMOKINE FUNCTION YOUR MODELS
CCR5 HAVE YOU ALSO CHECKED THAT?
AND I'M JUST WONDERING IF THAT
IS SPECIFICALLY.
>> YES, ALSO INCREASED AND IT'S
ALSO PRIMARILY PRODUCED BY
ENCASING THE TME ALSO T CELLS
ALSO PRODUCE IT BUT IT'S
INCREASED IN ALL.
>> OKAY, THANK YOU.
>> YOUNG PEOPLE FIRST.
>> GREAT TALK.
I APPRECIATE THAT.
I'M A POST-DOC IN -- LAB.
MICROBIOME AFFECTING NEUTROPHILS
IN TUMOR ENVIRONMENT, WHY IS IT
HAPPENING ONLY IN THE TUMOR
ENVIRONMENT?
GROO THAT'S A VERY GOOD
QUESTION, AND ONE THAT I DON'T
HAVE AN ANSWER TO YES.
BUT WE DON'T KNOW WHERE EXACTLY
P THE EDUCATION ACTIVATION
WHATEVER NAME YOU'RE GOING TO
GIVE IT OF COURSE WE'RE LOOK
INTUG T I'M HOPING ONE DAY WE'LL
KNOW.
BUT I DON'T THINK, IT MIGHT BE
MORE THAN ONE PLACE.
LET'S LEAVE IT LIKE THAT.
>> IN YOUR STUDIES EVER SEE
AFTER ADDING THE DRUG THAT
ACTIVATES, LIKE MDV IS THERE
ANNUITY FILLS THAT COULD
POTENTIALLY CAUSE DAMAGE TO THE
BODY?
>> I DON'T THINK THAT WE LOOKED
IN PARTICULAR.
I BELIEVE WITH MDP IT WAS MOSTLY
LOOKED AT IN THE TUMOR, ROMEO, I
DON'T KNOW IF EUROPE IN THE
AUDIENCE, IF YOU LOOKED IN
CIRCULATION.
I THINK TELL YOU SHE'S SAYING NO
FROM THE BACK, I CAN TELL YOU
THAT THE CHANGES WE SEE IN THE
TUMOR IN THE NEUTROPHILS AFTER
TREATMENT FOR EXAMPLE WE DO NOT
SEE THOSE IN BLOOD.
WHAT HAPPENS IN THE TUMOR
HAPPENS IN THE TUMOR, IS NOT
REFLECTED IN CIRCULATION.
THAT'S ANOTHER --
>> GREAT TALK.
MY NAME SUSPECT NATALIE.
CAN YOU HEAR ME?
>> YEAH.
>> GREAT.
>> MY NAME IS NATALIE.
HE ASKED MY FIRST QUESTION WHICH
IS ABOUT WHERE THAT PRIMING IS
ACTUALLY TAKING PLACE.
BUT THEN THITION SORT -- BUT
THEN I GUESS SORT OF SIMILARLY
TO THAT QUERKS I WANTED TO ASK,
YOU LOOKED AT MAMMARY GLAND AND
MELANOMA AND OTHER MODELS AS
WELL BUT I WANTED TO ASK IF
YOU'VE LOOKED AT OTHER MODEL
SYSTEMS THAT HAD A LARGER SORT
OF LIKE INTERFACE WITH
MICROBIOTA IN GENERAL, LIKE
INTESTINAL OR LIVER FOR EXAMPLE.
>> YEAH, NO, WE IT USED
CANCER -- CANCER BUT THAT WAS
SUBCUTANEOUSLY IMPLANTED, WE
HAVE NOT LOOKED IN THE GUT, MAIN
FOCUS IS LOOKING AT SYSTEM
SITES, WE'RE NOT REALLY LOOKING
AT LOCAL EFFECT.
BUT SOMETHING TO BE DONE.
>> THANK YOU.
>> IN THE BACK.
>> YOUNG PEOPLE FIRST.
>> YOUNG PEOPLE FIRST?
>> YEAH.
[LAUGHTER]
>> ONE QUESTION APIECE.
SOMEBODY START.
>> IN THE BACK.
>> GREAT TALK,
SUPER-INTERESTING.
I WAS WONDERING ABOUT GERM-FREE
EXPERIMENTS THAT WERE DONE AND
THE RESPONSE THAT YOU GOT.
HOW EARLY WOULD YOU NEED TO
TRANSFER THE -- OR TRANSFER THE
MICROBIOME TO THE GERM FREE MICE
IN ORDER TO GET THE ANTI-TUMOR
RESPONSE?
>> SORRY, I MISSED THE FIRST
PART.
HOW WHAT?
>> HOW EARLY WOULD YOU ZAISH ON
HOW EARLY.
>> YEAH.
>> I BELIEVE WE DID TWO WEEKS
PRIOR.
>> SO TWO WEEKS IS ENOUGH FOR
THE MICE TO THEN DEVELOP THE
ANTI-TUMOR RESPONSE?
>> YEAH, AND PROBABLY LESS.
GERM FREE MICE GET COL NIELTZED
VERY EASILY.
DON'T HAVE ANYBODY TO COMPETE
WITH.
>> I'M SORRY, I'M BEING TOLD
THAT WE NEED TO CUT OFF JUST
BECAUSE WE'RE LOSING THE ROOM
SOON.
I'M REALLY SORRY.
>> I'LL BE AROUND FOR MORE
QUESTIONS.
>> I'M FROM NCI, I'LL CHAIR THE
REMAINING SESSION, MY IMPORTANT
JOB THAT WE'LL GET OUT OF THE
ROOM BY 5, OTHERWISE WE'LL GET
KICKED OUT.
AND YOU CAN THINK THE SCREEN IS
FLICKERING.
IT IS NOT YOUR EYES.
(AWAY FROM MICROPHONE).
FROM NCI.
THE LONGEST THIETLE OF THE
CONFERENCE FOR THE TALK WILL --
TITLE.
EXCELLENT SPEAKER.
OF.
>> ALL RIGHT.
CAN EVERYONE HEAR ME?
>> YEAH.
>> ALL RIGHT.
SO I WOULD REALLY LIKE TO THANK
THE ORGANIZERS THE OPPORTUNITY
TO PRESENTED VERY NEW FLERCH MY
LAB.
I AM SOMEONE PASSIONATE ABOUT
UNDERSTANDING HOW HERPESVIRUS
SYN EFFECT FOR LIFE AND I'M VERY
INTERESTED IN CANCER CAUSING
HERPESVIRUSES.
SO HERE I'M TAKING KNOWLEDGE IN
THE FIELD ON HOW THESE VIRUSES
REPLICATE AND HOW THEY ESTABLISH
LATENCY TO NOW MAKE MUTANT
VIRUSES THAT AREN'T ABLE TO DO
SO AND SEE IF THOSE ARE
EFFECTIVE VACCINES IN VIVO.
SO MANY OF YOU ARE AWARE THAT
THERE ARE MANY VIRUSES THAT ARE
ASSOCIATED WITH HUMAN CANCERS,
IN TWO OF THESE ARE CAPIC THY
HERPESVIRUS AND EPSTEIN-BARR
VIRUS, GAMMA HERPESVIRUS.
CAPISARCOMA, NEOPLASIA TYPICALLY
IN SKIN, CAN BE IN THE LUNG AND
GI TRACT AND EPSTEIN-BARR VIRUS
ASSOCIATED WITH CAUSATIVE AGENT
PNEUMATIC CLEEOSIS ALSO
ASSOCIATED WITH MANY LYMPHOMAS
IN ADDITION TO CANCERS OF THE
EPITHELIUM.
SO THESE VIRUSES ARE ESPECIALLY
IMPORTANT IN PEOPLE LIVING WITH
HIV WHICH WE HAVE A LARGE
POPULATION WORLDWIDE AND PEOPLE
WHO ARE NEWLY INFECTED EVERY
DAY, EVEN IN THOSE THAT HAVE
GOOD CD4 SCROWNTS A HIGHER
PREVALENCE OF GAMMA HERPESVIRUS
CANCERS.
A LITTLE BIT OF INFORMATION
ABOUT HERPESVIRUSES, THERE ARE
NINE OF THEM, THEY'RE BIG
VIRUSES, DNA VIRUSES AND PART OF
THE COMPLEXITY IS THEY HAVE A
LOT OF GENES THAT MANIPULATE THE
HOST, THEY ALSO HAVE NONCODING
RNA AND IS THEY ACTUALLY BOTH
REPLICATE TO MAKE NEW INFECTIOUS
PARTICLES, GETTING THIS STRAIGHT
HERE, AND THEY CAN ESTABLISH
LATENCY WHERE THEY'RE ACTUALLY
NOT EXPRESSING MANY GENES AND
THAT'S A GREAT WAY TO EVADE
IMMUNE CONTROL.
WE HAVE ANTI-VIRAL DRUGS BUT
THOSE ARE ONLY EFFECTIVE AGAINST
THE LYTIC PHASE NOT THE LATE
LATENT FADES OF HERPESVIRUS
INFECTIONS ASK WE HAVE VACCINES
AVAILABLE ONLY AGAINST ONE OF
THE HERPESVIRUSES, VARI TELL LA
ZOSTER.
I'VE UNDERLINED THE ONES
ACTUALLY BEING USED IN PEOPLE
RIGHT NOW, SHIN GREKS USED IN
OLDER INDIVIDUALS TO PREVENT
SHINGLES, REACTIVATION AND WE
HAVE ATTENUATED VIRUS ZOSTIVAX
USE #-D IN CHILDREN FREERCHT
CHICKENPOX, PRIMARY INFECTION.
ATTENUATED VIRUS THAT DOES
ESTABLISH LATENCY, DOES NOT
PREVENTED STERILIZING IMMUNITY
BUT IT DOES PREVENT REALLY
DETRIMENTAL CHICKENPOX
INFECTIONS.
I'M VERY EXCITED WITH A mRNA
BASED ARE VACCINE H ITD TO
MENTION THAT JEFF COHEN AT
NIANIAD AT NIH, INSTRUMENTAL IN
VACCINES IN COLLABORATION ALSO
IN N I AD, THOSE ARE ALSO IN
CLINICAL TRIALS.
WHAT YOU DO NOT SEE ON THIS LIST
IS KSHV, NOILY VACCINE IN
PROGRESS FOR HERPESVIRUS.
WHY IS THAT?
IT IS A LITTLE BIT BEHIND, IT'S
A MORE NEWLY DISCOVERED VIRUS,
MORE RECENTLY DISCOVERED THAN
EBV, WE HAVE THE PROBLEM WAWFLT
HERPESVIRUSES IS THERE'S NOT
JUST ONE RECEPTOR, THERE'S NOT
JUST ONE GLYCOPROTEIN THERE ARE
MULTIPLE GLYCOPROTEINS THAT FORM
COME PLEKS AND THESE
GLYCOPROTEINS CAN ENGAGE
MULTIPLE RECEPTORS FOR BOTH
ATTACHMENT AND ENTRY.
SO IT IS A COMPLICATED SCENARIO
TO DEVELOP NEUTRALIZING
ANTIBODIES AGAINST.
KSHD AND THE -- KSHV AND THE
MOUSE PATH I'LL TELL YOU ABOUT
TODAY SHARELY KINASES FOR ENTRY,
A POST DMOK MY LAB HAS DONE
AMAZING WORK THAT, THERE IS
OVERLAP THERE.
SO ANOTHER ASPECT OF KSHV IS UN
-- IS IT DOES NOT TAPE A
LARGE PARTED OF OUR T CELL
REPERTOIRE, THOSE INFECTED WE
DON'T SEE ANY CLEAR PATTERNS FOR
IMMUNE DOMINANT EPITOPES AND
DON'T HAVE DEFINED IMMUNOCAR
LATS OF PROTECTION.
HOW CAN WE GENERATE A SAFE
VACCINE WITH BROAD
IMMUNOGENICITY THAT CAN BLOCK OR
CONTROL KSHV INFECTION?
I USE A HERPESVIRUS PATHOGEN,
NATURAL PATHOGEN OF RODENTS AND
GENOME COLINEAR WITH KSHV, VIRUS
STUDIED FOR ABOUT 30 YEARS NOW
AND WE HAVE A NICE GENETICALLY
TRACKABLE SYSTEM WHERE WE'VE
USED MUTANT VIRUSES AND MUTANT
MICE AND LEARNED A LOT ABOUT THE
SPATIAL TEMPORAL KINETICS.
LAB ACTUALLY WHEN HE WAS AT
SAINT JUDZ A LOT OF THE
FOUNDATION -- SAINT JUD HE'S, WE
KNOW A LOT ABOUT TYPES OF IMMUNE
CELLS THAT RESPOND TO CONTROL,
REPLICATION AND I LA TENDENCY.
TAKEN TOGETHER THIS IS A GREAT
SYSTEM TO EXPLORE POSSIBLE
VACCINE PLATFORMS AND BLEARNT
IMMUNE CORRELATES OF PROTECTION.
SO MY COLLABORATOR CRAIG FOREST,
UNIVERSITY OF ARKANSAS MEDICAL
SIENLSES I FORGOT TO MENTION ON
THE FIRST SLIDE, WE'VE
COLLABORATED AND DECIDED TO DO A
RATIONAL APPROACH WHERE WE'VE
KNOCKED OUT DETERMINANTS OFLYTIC
REPLICATION HERE I'M FOCUSED ON
RTA ENCODE BY OR50, NECESSARY
AND SUFFICIENT TO TRIGGERLYTIC
GENE EXPRESSION AND THEN WE'VE
ALSO TARGETED LATENCY
DETERMINIST LEFT IN THE VIRUS
THAT PROMOTE LATENCY AND AGAIN
THIS IS MHV68 COLINEAR WITH KHSV
ON THE -- KSHV ON THE BOTTOM.
DECIDE TO DO MAKE A REPLICATION
DEAD VIRUS.
HOW DO YOU GROW SUCH A VIRUS IN
WE CAME WUP A STRATEGY WHERE WE
CAN PRODUCE IT IN A CELL LINE
WITH RESPECT TO THE
COMPLIMENTING GENE IS CODON
SHUFFLED, PREVENTS RECOMBINATION
SO WE DON'T GET REVERT ENS.
WE END UP WITH A VIRUS THAT
BASICALLY ENTERS A CELL AND
UNDERGOES AN ABORTIVE INFECTION,
SINGLE ROUND OF INFECTION WITH
NO INFECTIOUS PARTICLES ON THE
BACK END.
WE'VE TAKEN THESE VIRUS STOCKS
AND INFECTED IMMUNODEFICIENT
MICE AND IN THIS SYSTEM WE CAN
ONE PFU A WILD-TYPE IMAGE V68
CAN LEAD TO 100% MORTALITY, WE
PUT IN A MILLION PFU OF OUR
VIRUS WHICH I'LL REFER TO AAS
THE RDV ORF50 STOP CODON, 100%
SURVIVAL.
SO WE ENVISION THIS VIRUS AS
ENTERING A CELL, IT'S BASICALLY
SORT OF LIKE A VLP BECAUSE THE
VIRUS IS INTACT.
BUT IT HAS A GENOME, IT DELIVERS
THAT TO THE NUCLEUS AND YOU HAVE
NEW GENE EXPRESSION AND
DEPENDING ON THE CELL TYPE YOU
CAN GET MHC1 AND 2 PRESENTATION.
WE LOOKED AT THE RNA SEQ OF
INFECTED CELLS WITH THE 50 STOP
VIRUS HERE OR THE WILD-TYPE
VIRUS ON THE TOP AND YOU CAN SEE
THERE'S GREATLY DIMINISHED GENE
EXPRESSION, THESE ARE THE ORF'S
OF MHV68 MUCH DEPRESSED GENE
EXPRESSION WRVMENT WE SEE GENE
EXPRESSION IT'S INTERESTING
BECAUSE SOME OF THESE ARE
LATENCY DETERMINE ABILITY,
SOMETHING TO KEEP IN MIND AND WE
ALSO HAVE WHAT WE USE AS
IMMUNODOMINANT EPITOPES TO
FOLLOW VIRUS SPECIFIC T CELL
RESPONSES IN BLACK SIX MICE,
EXPRESSED INDEPENDENTLY OF RTA.
OKAY.
SO HE WITH TAKE THIS VIRUS AND
NOW WE'RE INFECTING BLACK SIX
MICE.
WE DO A PRIME WITH A MOIL YON
PFU IP, BOOST ONCE OR TWICE, AND
THEN FOLLOW ANTIBODY AND VIRUS
SPECIFIC T CELL RESPONSES.
AND THIS IS A BLOT WHERE WE'VE
TAKEN SERUM FROM MICE EITHER
INFECTED LIKE THE NORMAL
WILD-TYPE VIRUS OR VACCINATED
AND IT'S PRETTY OBVIOUS WE DON'T
SEE MUCH SIGNAL REACTING AGAINST
VIRALLY INFECTED LYSATES WHERE
50 STOP AS COMPARED TO WILD-TYPE
BY SECOND BOOST RER SEEING SOME
REACTIVITY.
THIS WAS ALSO CONFIRMED BY OUR
VIRUS SPECIFIC IGG ELISA, WE
HAVE WILD-TYPE PREDOMINANT, LOTS
OF GOOD ANTIBODIES BEING
GENERATED, WE HAVE LESS SO FOR
THE RTA50 STOP.
INTERESTINGLY THOUGH, WE DO SEE
THAT NEUTRALIZING ANTIBODIES BY
THIS PLAQUE REDUCTION
NEUTRALIZATION ASSAY WE DO SEE
NEUTRALIZING ANTIBODIES THAT
APPROACH WILD-TYPE INFECTION.
SO WE ARE GENERATING
NEUTRALIZING ANTIBODIES WITH
THIS VACCINE.
SO I'LL SHOW YOU DATA NOW, WE
ALSO DID T CELL WORK-UP BUT I'LL
SHOW YOU THAT T CELL DATA NOW IN
THE NEXT SET OF DATA.
NOW WE'RE PRIME BOOSTING THESE
MICE AND THEN WE'RE CHALLENGING
WITH WILD-TYPE MHD68 BOOSTING IP
CHALLENGING INTRANASALLY.
WHAT WE GET HERE IS REPLICATION
IN THE LUNGS FOR THE ACUTE PHASE
AND THEN WE FOLLOW LATENCY
ESTABLISHMENT IN THE SPLEEN FOR
LATENCY.
LUNGS OF THESE VAC NAITD MICE,
HERE WE HAVE SHA AM VACCINATOR
IN RED AND VACCINATED WITH RDV50
STOP VIRUS IN BLUE.
YOU CAN APPRECIATE WE HAVE A
FOUR LOG REDUCTION IN THE LEVELS
OF VIRUS REPLICATION IN THE
LUNGS OF THESE MICE AND IT LOOKS
A LITTLE BIT BETTER WITH TWO
BOOST HERE ALTHOUGH THE MICE
NUMBERS ARE LIMITED.
I'M SHOWING YOU WE ALSO SAW
NEUTRALIZING ANTIBODIES AS I
SHOWED YOU BEFORE THAT ARE
COMPARABLE TO WILD-TYPE.
WHAT ABOUT THE T CELL RESPONSE?
SO WHAT ARE THOSE IMMUNE
CORRELATES OF PROTECTION?
HERE WE'RE LOOKING AT THE LUNGS
AT THE SAME DAY, DAY SEVEN POST
CHALLENGE AND I HAVE FOUR
GROUPS, WE HAVE -- SORRY -- WE
HAVE THE SHAM VACCINATED, NOT
CHALLENGED OR WILD-TYPE
CHALLENGE, WE HAVE OUR
VACCINATED ANIMALS NOT
CHALLENGED AND VACCINATED MICE
CHAL HE COULD WITH WILD-TYPE
VIRUS -- CHALLENGED WITH
WILD-TYPE VIRUS, LOOKING AT P79
AND 56, LOOKING BIND TO
RECOGNIZE THESE TWO EPITOPES.
AND WE FIND THAT IN THE
VACCINATED ANIMALS UPON
WILD-TYPE CHALLENGE WE SEE A
STRONG VIRUS SPECIFIC T CELL
RESPONSE IN THE LUNGS, AND THAT
IS WITH BOTH P 1K3-79 P56 AT THE
TIME MEASURES.
-- TETRAMERS.
IN THOSE VACCINATED ANIMALS
SEEING T CELL PRESENT WHICH
CORRELATES WITH REPLICATION.
WE ALL LOOKED IN THE SPLEEN AND
WE CAN ACTUALLY SEE A POPULATION
OF VIRUS SPECIFIC T CELLS IN THE
VACCINATED ANIMALS WITHOUT
CHALLENGE.
SHOWN HERE.
WE SEE SIMILAR LEVELS WITH AND
WITHOUT CHALLENGE.
THOSE VACCINATED MICE THAT THE
VIRUS SPECIFIC T CELLS HAVE A
SHORT LIVED EFFECTOR PHENOTYPE
AND OF THE IMPACTS WE ALSO SEE
THAT THEY ARE CD62L LOW
INDICATING THEY HAVE EFFECTOR
SERVICE PHENOTYPES CONSISTENT
WITH EFFECTOR T CELLS.
THEN WE LOOKED AT THEIR ABILITY
TO PRODUCE EFFECTOR CYTOKINES.
SO WE STIMULATED THE T CELLS
AGAIN FROM THESE SAME SETS OF
ANIMALS AND THIS COMPARISON WITH
EITHER P79 OR P56 AND WE'RE
LOOKING FOR TNF ALPHA AND
INTERFERON GAMMA PRODUCTION
UPPER RIGHT QUADRANT AND SEE
GHEAN THE VACCINATED ANIMALS
DOING LESS VIRUS SPECIFIC T
CELLS THAT CAN RAPIDLY RESPOND
TO PEPTIDE STIMULATION.
WHAT ABOUT LATENCY?
IMPORTANT PART OF GAMMA
HERPESVIRUS AND IS WE SEE THAT
ONE INDICATOR FOR MHD68
COLONIZATION OF THE SPLEEN,
SPLEN NOTICEMEGALY, SIMILAR TO
LYMPH NODE ENLARGEMENT ONE MIGHT
SEE WITH PATIENTS WITH EBD IM,
INFECTIOUS MONO NUCLEOSIS, THESE
MICE DEVELOP THREEFOLD
SPLENOMEGALY IN THE SHAM
VACCINATED CONDITIONS, IF
VACCINATED, WE SEE NO SPLEN
NOTICEMEGALY.
TWO LAW REDUCTION IN THE
FREQUENCY OF SITES THAT HARBOR
THE GENOME AND WE DO THREE WEEK
EX-PLANT REACTIVATION ASSAY,
SENSITIVE AND QUANTITATIVE
MEASUREMENT OF REACTIVATION FROM
LATENCY, WE SEE NEAR COMPLETE
ABLAIFTION REACTIVATION IN THOSE
VACCINATED ANIMALS.
NOT GOING ON SHOW YOU THE DATA.
BUT WE WAITED UNTIL THREE MONTHS
POST BOOST AND WE SEE SIMILAR
PROTECTION, SIMILAR IMMUNE
RESPONSES.
SO IT IS DURABLE PROTECTION.
SO THEN WE MOVED TO KIND OF HOW
COULD WE PUSH THE SYSTEM TO
REALLY CHECK EFFICACY.
HERE WE'RE LOOKING AT TYPE ONE
INTERFERON RECEPTOR KNOCK-OUT
MICE, WE'RE USING A DOSE ABOUT
TWO MILLION PFEYM WITH WILD-TYPE
LEADING TO AREA OF ROBUST
LETHALITY IN THESE ANIMALS,
THESE ANIMALS WERE PRIME BOOSTED
WITH R ARE BT STOP VIRUS AND
LOOKING AT THE RED LINE
UNVACCINATED ANIMALS DPIERD THE
BLACK LINES THAT, INDICATES THE
VACCINATED ANIMALS, WE SEE NO
WEIGHT LOSS OR SEE VERY LIMITED
WEIGHT LOSS IN THE -- NOW, ONE
ASPECT OF THIS IS WE LOOKED OF
THE MICE THAT SURVIVED, WE
LOOKED AT WHAT TYPE OF VIRUS IS
PRESENT IN THOSE SPLEN NOTICE
SIGHTS, WHAT IS THE VIRAL GENOME
GONE LATE ENTD.
INTERESTING TO FIND, WE DO SEE
LATEN GENE EXPRESSION IN 50 STOP
VIRUS AND WE DO SEE EVIDENCE
THAT THIS IS THE GENOTYPING
ASSAY LOOKING FOR THE 50 STOP
VIRUS USING GENOME TYPE SPECIFIC
PRIMERS AND THEN JUST A PAN
DEFECTION FOR ALL OF MRV68 AND
GET A COUNT OF ABOUT 16% OF THE
VIRUS OUR VACCINE VIRUS, CAIGHTD
THAT VACCINE VIRUS CAN ESTABLISH
LATENCY AND ALSO INDICATES WE'RE
NOT BEING STERILIZING IMMUNITY
BECAUSE WE'RE DETECHING SOME
WILD-TYPE VIRUS IN THOSE
SPLENOCYTES.
RECENTLY WE DECIDED TO TOWARDS
KNOCKING OUT LATENCY, HERE
ATTACKING LEFT MH68 WHICH HAS M
GENES UNIQUE TO 68 THAT PROMOTE
LATENCY, USED SIMPLE COMPARISON,
THIS REPLICATION DEAD VIRUS TO
NOW REPLICATION DEAD VIRUS
LAYERED ON THAT WE'VE KNOCKED
OUT GENES NEEDED FOR LATENCY.
AND WE FOUND THAT IF YOU COMPARE
THE BLACK TO THE BLUE SQUARES IN
ALL OF OUR COMPARISONS, BOTH ARE
PROTECTIVE.
WE HAVE LOSS OF SPLENO
MEGMEGALY, WE DON'T SEE LATENCY
AND EVEN LESS WITH DELTA 4
MUTANT VIRUS AND DON'T SEE
REACTIVATION.
WE ALSO SEE COMPARABLE LEVELS OF
VIRUS SPECIFIC T CELLS IN THE
LUNGSZ, THE SPLEENS AND THEY
HAVE AN EFFECTOR PHENOTYPE AND
PRODUCE ANTI-VIRAL CYTOKINES.
SO I'VE SHOWN YOU WE CAN PRIME
BOOST WITH REPLICATION DEAD
VIRUS AND A VIRUS THAT'S
DEFECTIVE FOR LATENCY AND
PROTECT AGAINST WILD-TYPE
CHALLENGE USING MANY ASPECTS OF
PATHOGENESIS IN ANIMAL MODEL,
REDUCE REPLICATIONS, SPLEN
NOTICEMEGALY AND PROTECT FROM
LETHAL DISEASE, AND I'VE SHOWN
YOU SOME IMMUNE CORRELATES OF
PROTECTION WHICH ARE STARTING TO
GIVE US SOME IN ROADS INTO HOW
THIS MAY BE WORKING.
THERE'S A LOT MORE WORK TO DO,
WE'RE VERY EXCITED BECAUSE NOW
WE'RE ACTUALLY GOING TO TRY TO
COMPLETELY AN LATE ALL LATENCY
BY LOCKING OUT LANA NEED TO DO
TETHER THE VIRAL EPISOME TO THE
HOST AND PUSH BACK THAT BLOCK OF
REPLICATION FROM EARLY TO
ACTUALLY VERY LATE IN THE LYTIC
LIFE CYCLE BY TARGETING
DIFFERENT VIRAL PROTEIN AND WE
LIKE TO COMPARE IT THIS TO HEAT
AND ACTIVATION S THERE BENEFIT
FROM ADJUVANT AND I'M SETTING UP
A COLLABORATION WITH JEFF COHEN
AND NASARO TO USE THAT APPROACH
WITH EBV USING SERATIN NANO
PARTICLES THAT WE CAN SEE IF WE
CAN DO A SUBUNIT BOOST TO
FURTHER INCREASE PROTECTION.
AND I APPRECIATE ANY FEEDBACK
FROM THE I AM NO GISTS IN THE
ROOM BECAUSE WHAT WE NEED TO DO
IS ACTUALLY TAKE THIS A STEP
FURTHER.
ARE WE GETTING SECRETORY IGA, IS
THIS GENERATING NEUTRALIZATION
AGAINST SPECIFIC PROTEINS AND
HOW ARE THE ANTIBODIES WORKING
BEYOND NEUTRALIZING ANTIBODIES,
WHAT IS THE ROLE OF CD4 T CELLS
AND CAN WE USE THIS TO A MODEL
THAT PSES KSHV DISEASE AND
EFFECTIVE VACCINE THERE.
COLLABORATORS CRAIG FOREST, I'VE
GREAT INSIGHT FROM BRIAN
SHERIDAN T CELL I AM NO GIST
FROM STONY BROOK UNIVERSITY
AND -- IN MY GROUP ANNA GROSCOFF
AND DIPA HAVE GENERATED DATA TO
THIS PROJECT AND PREVIOUS HADLY
DPAISH IS I'LL STOP THERE AND
TAKE ANY QUESTIONS.
[APPLAUSE]
>> I DON'T KNOW WHO IS OLDER,
REMY, YOU OR ME, SO I'LL GO
FIRST.
THAT WAS GREARKTS LAURIE, THANK
YOU.
-- THAT WAS GREAT, LAURIE, THANK
YOU.
A QUICK QUESTION, BECAUSE IT
LOOKED TO ME, MAYBE I MISSED IT,
THAT THE REDUCTION OF YOUR
LATENCY WAS LESS THAN YOUR
REACTIVATION FROM LATENCY.
SO IN THE CONTEXT OF SOME OF THE
TRANSFORMATIONS WHICH ARE LATENT
WHAT DO YOU THINK WOULD HAPPEN
THEN?
>> SO IN GENERAL, WILD-TYPE
VIRUS ABOUT TEN PERCENT OF THE
VIRUS THAT ESTABLISHES LATENCY
REACTIVATES, SO THAT'S ONE
DIFFERENCE THAT MAY HAVE LOOKED
DIFFERENT IN THOSE GRAPHS.
WE ACTUAL HAVE A BETTER BLOCK
WITH REACTIVATION THAN LATENCY.
>> THAT'S WHAT I WAS SAYING.
YES.
>> SO AT THIS POINT WE DO NOT
HAVE STERILIZING IMMUNITY, VIRUS
IS STILL ESTABLISHING LATENCY, I
THINK WHAT I FORESEE SYNC WE
WOULD BE REDUCING, WE CAN TEST
THIS VIRAL LOAD OVERALL BUT I
THINK THE IMPORTANT PART IS
BLOCKERRING REACTIVATION BECAUSE
A LOT OF GAMMA HERPESVIRUS
DISEASES ARE WHEN YOU LOSE
IMMUNE CONTROL, THESE VIRUSES
COULD REACTIVATE, INFECT NEW
CELLS AND DRIVE
LYMPHOPROLIFERATIVE EXPANSION OF
THOSE TARGETED CELLS.
>> I GUESS YOU COULD ARGUE BOTH.
I AGREE WITH YOU WE WOULD
PREDICT IT WOULD BE HARDER TO
CONTROL LATENCY BECAUSE THERE'S
SO MANY FEWER OPEN READING FRAME
EXPRESSED IN TERMS OF THE
IMMUNITY AS WELL.
>> RIGHT.
SO AGAIN, IT'S ACTUALLY ALSO NOT
WELL DEFINED IN HERPESVIRUSES
WHEN YOU HAVE A LATENT
INFECTION, THERE IS A
REANIMATION, THE VIRUS IS
CONSTANTLY REACTIVATING TO SOME
DEGREE SO ANTIGENS ARE BEING
EXPRESSED SO WE'RE HOPING WE
WOULD GENERATE MORE OF A
RESPONSE TO THOSE LYTIC ANTIGENS
THAT MAY CLEAR INFECTED CELLS.
>> SO THIS IS A VERY NAIVE AND
SIMPLISTIC QUESTION, I DON'T
KNOW IF YOU CAN HEAR ME.
WE'RE HEARING MANY GREAT THINGS
ON BACTERIA, FUNGI AND THE LIKE.
>> YOU NEED TO KISS THE MIKE
ZEEN I NEED TO WHAT?
TO DO THIS?
OKAY.
SO WE ARE HEARING MANY GREAT
THINGS -- IS THERE -- IS IT
POSSIBLE THAT THEY ACT AS
COMMENSURATE FOR US TOO SO THAT
WE USE THE RESPONSES, DID
ANYBODY EVER LOOK AT THIS?
>> I'M NOT SURE I CAUGHT THAT.
COULD YOU REWORD THAT AGAIN?
I'M SORRY, IT KEPT ZIESH YOU
DON'T UNDERSTAND FRENCH.
>> I'M SORRY.
[LAUGHTER]
>> THE QUESTION IS, IS IT
POSSIBLE, I SUPPOSE THESE
>> I THINK THERE IS A PAPER A
FEW YEARS AGO FROM SKIP BURGES
LAB THAT HERPES IS GOOD FOR YOU.
>> I GINT THAT FAR SCWHRO THAT
IF YOU HAD A HERPESVIRUS
INFECTION IT COULD PROVIDE SOME
PROTECTION AGAINST SOME OTHER,
IT COULD ENHANCE CYTOKINE
RESPONSES AND SOME OTHER
EFFECTS.
>> ACTIVITY WITH SOME OTHER
ANTIGEN OR --
>> I THINK THERE WAS A GOOD TALK
ON VCN THAT I WASN'T REALLY
AWARE OF.
SO IT WAS PRETTY SURPRISING.
KSHV IS UNLIKE EBV IT'S NOT
SEROPREVALENT IN THIS COUNTRY,
IT'S ENDEMIC IN CERTAIN AREAS
SUCH AS SUB-SAHARAN AFRICA AND
THOSE POPULATIONS CERTAINLY
SUFFER FROM MORE KSHV ASSOCIATED
DISEASE -- DISEASE DISEASES THAN
CANCER.
I THINK THE JURY OUT ON EBV AND
CANCER AND THAT.
>> THANK YOU.
>> CAN YOU HEAR ME?
SO MY JOB IS TO GET EVERYBODY
OUT BY 5:00.
SO I WILL TRY TO DO THAT,
BECAUSE THIS WORK, SO I'M GOING
TO TELL YOU ABOUT, SWITCH GEARS
A BIT.
BY THE WAY, I'M -- IN CASE YOU
NOTICED.
SO I'M GOING TO TALK ABOUT A NEW
APPROACH FOR CANCER
IMMUNOTHERAPY THAT INVOLVES WHAT
WE THINK IS AN INTERESTING
EPIGENETIC ENZYME CALLED COM1.
SO WE DISCOVERED COM1 IN A T
CELL CRISPR SCREEN SO WE WANTED
TO IDENTIFY NOVEL REGULATORS OF
T CELL FUNCTION, MAYBE I'LL USE
THE POINTER HERE.
SO THE WAY THE SCREEN WORKED WAS
THAT WE INJECTED EDITED T CELLS
INTO MICE WITH SUBCUTANEOUS
MELANOMAS THAT EXPRESSED THE
OVER TUMOR ANTIGEN AND THE IDEA
WAS TO IDENTIFY GUIDE RNAS THAT
WOULD ENHANCE T CELL EXPANSION
SELECTIVELY IN THE TUMOR BUT NOT
IN THE CONTROL ORGAN LIKE THE
SPLEEN.
SO THE TOP HINT IN THE SCREEN
WAS COM1 WHICH IS AN EPIGENETIC
ENZYME, THE NUMBER OF POSITIVE
CONTROLS HERE FOR EXAMPLE PDC1
IS PD1, SMAT2 IS A KEY
TRANSCRIPTION FACTOR IN TGF BETA
PATHWAY SO WE ARE REALLY
INTERESTED IN WHAT THIS ENZYME
DOES.
SO CARM1 IS A CO-TRANSCRIPTIONAL
ACTIVATOR.
IT IS ACTUALLY INITIALLY
DISCOVERED AS A CO-ACTIVATOR OF
NUCLEAR HORMONE RECEPTORS AND
THEREFORE PLAYS A VERY IMPORTANT
ROLE IN THE PATHOGENESIS OF BOTH
BREAST AND PROS STATED CANCERS
BUT IT -- PROSTATE CANCERS BUT
IT ALSO METH LATES, SO WHAT
CARM1 DOES IS IT METH LATES --
METHYLATES AND SPECIFICALLY THE
BATH ARE 55 SUBUNIT.
BAF155 SUB UBT.
WE DID TO VALIDATE THAT CARM1
HAS A ROLE IN T CELLS AND KNOCK
OUT CARM1 GENE IN T CELLS IN
VITRO AND THEN TRANSLATE THESE
CELLS TO MICE WITH OVER TUMORS
WE FIND THAT THE T CELL NUMBERS
IN THE TUMORS ARE GREATLY
INCREASED, THE T CELLS HAVE
GREATLY INCREASED FUNCTIONALITY,
AS READ OUT HERE BY GAMMA
INTERFERON EXPRESSION, AND
THERE'S ALSO RETENTION OF A
GREATER POPULATION OF MEMORY T
CELLS THAT EXPRESS TCF7.
AND INDUCTIVE TRANSFER APPROACH
WE SHOWED THAT CARM1 DEFICIENCY
CELLS HAVE ENHANCED INTERTUMOR
IMMUNITY, SHOWING HERE AGAIN THE
B16 MELANOMA MODEL WITHOUT T
CELL TRANSFER THE TUMORS GROW
VERY RAPIDLY.
THE OT1 TLZ HAVE SOME ACTIVITY
IN SLOWING TUMOR GROWTH BUT THEN
KNOCKING OUT CARM1 SIGNIFICANTLY
SLOWS TUMOR GROWTH AND PROLONGS
SURVIVAL IN THESE MICE.
NOW, THE MOST INTERESTING
FINDING HERE WAS THAT IN
ACTIVATING CARM1 SUBSTANTIALLY
REDUCED THE EXPRESSION OF
INHIBITORY RECEPTORS, SO BOTH
PD1 AND TIM3, COMPARING HERE THE
CONTROL KNOCK-OUT MICE THAT HAVE
A SUBSTANTIAL POPULATION OF BOTH
PD1 AND TM3 POSITIVELY T CELLS
COMPARED TO CARM1 KNOCK-OUT T
CELLS WHERE EXPRESSION OF BOTH
OF THESE INHIBITORY RECEPTORS
WAS SUBSTANTIALLY DIMINISHED,
ALSO EXTENDED TO EXPRESSION OF
CD39 INHIBITORY RECEPTOR.
SO AS I MENTIONED EARLIER, IT
CARM1 KNOCK-OUT ACTUALLY
ENHANCED MAINTENANCE OF
RESERVOIR OF MEMORY-LIKE T CELLS
WITHIN TUMORS THAT IS SHOWN HERE
BASED ON STAINING WITH TCF7 AND
BCL2 DPIERD THE CONTROL
KNOCK-OUT T CELLS AND QUANTIFIED
HERE ON THE RIGHT-HAND SIDE.
SO WHEN WE LOOK AT GENE
EXPRESSION DATA, SO THIS IS
BLOCK RNA SEQ DATA, YOU CAN SEE
OF COURSE THAT CARM1 IS DOWN AND
THE CARM1 KNOCK-OUT T CELLS AS
EXPECTED, BUT THEN THERE'S UP
REGULATION OF MANY GENES THAT
ARE INVOLVED IN T CELL MEMORY,
FOR EXAMPLE TCF7, MYC, CD27 AND
IGE AND ALSO VALIDATED THESE BY
QPCR.
NOW, WHAT WE'RE DOING RIGHT NOW
IS SO THIS IS ALL ONGOING.
SO WE'RE RIGHT NOW PERFORMING
ATAXIC AND CUT AND WRUN THE
CARM1 ANTIBODY TO DEFINE THE
PRECISE MECHANISM OF ACTION IN
THE T CELLS.
HOWEVER, WHAT WE ALSO NOTICED IS
THAT CARM1 IS BROADLY EXPRESSED
IN MANY MANY DIFFERENT HUMAN
CANCERS AND IT'S ALSO AMPLIFIED
IN SOME CANCERS AND
OVEREXPRESSED.
FURTHERMORE, HIGH LEVELS OF
CARM1 NEGATIVELY CORRELATE WITH
SURVIVAL IN A NUMBER OF HUMAN
CANCER TYPES, FOR EXAMPLE, IN
MELANOMA, BLADDER CANCER,
SARCOMAS, KIDNEY CANCER AND
MESOTHELIOMA.
WHEN YOU LOOK AT THE KEY
PATHWAYS THAT CORRELATE WITH
CARM1 EXPRESSION PATTERNS, THE
PATHWAYS ARE MYC TARKTS AND
PATHWAYS RELATED TO
PROLIFERATION AND GLYCOLYSIS AND
IMPORTANTLY THE PATHWAYS THAT --
THE INTERFERON RESPONSE,
ALLOGRAPH WHICH ACTUAL SL A TERM
FOR T CELL RESPONSE AND THEN
TNRF SIGNALLING AND THIS IS TRUE
FOR MULTIPLE CANCER TYPES.
SO WHAT WE DID THEN WAS ACTUALLY
KNOCK OUT CARM1 IN THE TUMOR
CELLS AND WE GOT A VERY
SURPRISING RESULT.
SO THE CONTROL KNOCK YOWTD
TUMORS ARE OF COURSE --
KNOCK-OUT TUMORS OF COURSE GROW
RAPIDLY IN THESE MICE BUT CARM1
KNOCK-OUT TUMORS ACTUALLY GROW
VERY SLOWLY, WE ASKED IF THIS T
CELL TYPE IS -- CANCER TYPE IS T
CELL MEDIATED, WE DEPLETE IN
CARM1 KNOCK-OUT DISPURMS YOU CAN
SEE THEY GROW AGAIN VERY RAPIDLY
SO CARM1 KNOCK-OUT IN THE TUMOR
CELLS ACTUALLY IT ELICIT A T
CELL MEDIATED RESPONSE.
IN VITRO WE SEE SIMILAR
PHENOTYPE, THIS IS A T CELL
CYTOTOXICITY ASSAY FROM LEFT TO
RIGHT, WE ARE INCREASING THE
RATIO OF T CELLS TO TUMOR CELLS,
YOU CAN SEE THAT THE CONTROL
KNOCK-OUT CELLS CAN BE KILLED BY
T CELLS BUT YOU NEED SUBSTANTIAL
NUMBER OF T CELLS FOR EFFICIENT
KILLING, WHILE THE CARM1
KNOCK-OUT TUMOR CELLS ARE
ACTUALLY VERY SENSITIVE TO
CYTOTOXIC T CELLS.
NOW, IN 41 BREAST CANCER MODEL
WE FOUND THAT NOT ONLY GROWTH OF
THE PRIMARY TUMORS WAS REDUCED,
BUT ALSO THE NUMBER OF LUNG MET
AS CITY SEES WAS
SIGNIFICANTLY -- METASTESES WAS
SIGNIFICANTLY DIMINISHED.
WE OF COURSE ASKED WHETHER CARM1
KNOCK-OUT WOULD IMMUNIZE, THIS
IS RESISTANT TO ANTI-CTLA4,
ANTI-PD1 OR EVEN THE COMBINATION
OF PD1 ANDLY CTLA4 ANTIBODIES
AND WE FOUND THERE WAS PROFOUND
SYNERGY WITH ANTI-CTLA4 SO THESE
TUMORS GREW VERY SLOWLY AND
THERE WAS A VERY SUBSTANTIAL
SURVIVAL BENEFIT WHEN THE CARM1
KNOCK-OUT WAS COMBINED WITH
ANTI-CTLA4 COMPARED WITH THE
CARM1 KNOCK-OUT WITH ISOTOPE
CONTROL ANTIBODY.
WE OF COURSE WANTED TO KNOW HOW
THIS WAS WORKING AT A
MECHANISTIC LEVEL, AND SO THERE
WAS A VERY STRIKING PHENOTYPE IN
THIS RNA SEQ ANALYSIS WHERE WE
GREW CARM1 KNOCK-OUT OR CONTROL
TUMOR CELLS IN VITRO. SO AS A
STRIKING UP REGULATION OF MANY
GENES IN INTERFERON SIGNALLING
IN THE CARM1 KNOCK-OUT CELLS
DPIERTD CONTROL KNOCK-OUT AND
ALSO UP REGULATION OF TNF
SIGNALLING.
SO I SHOWED YOU THE LEFT PART OF
THE CYTOTOXICITY ASSAY EARLIER,
BUT THEN WHAT WE DID HERE WAS TO
KNOCK OUT CGAS BECAUSE WE
THOUGHT THAT THE CGAS THINK
PATHWAY COULD BE IMPORTANT IN
THE TYPE ONE PATHWAY INTERFERON
RESPONSE I JUST ILLUSTRATED, SO
WHEN WE KNOCK OUT CGAS, THESE
TUMOR CELLS ACTUALLY BECOME
HIGHLY, THE CARM1 KNOCK-OUT
TUMOR CELLS BECOME HIGHLY
RESISTANT TO CYTOTOXIC T CELLS.
NOW, FORTUNATELY, THERE IS A
VERY SELECTIVE IMPORTANT CARM1
INHIBITOR AVAILABLE THAT WAS
DEVELOPED BY EPIZYME, STILL IN
PRECLINICAL DEVELOPMENT BUT
EFFORT TO ADVANCE THIS TO THE
CLINIC.
SO THIS INHIBITOR HAS AN IC50 OF
6 UNANIMOUS MOW MOLAR AND IS
SELECT -- NANOMOLAR AND
SELECTIVE AS COMPARED TO RELATED
ENZYMES.
THIS INHIBITOR ACTUALLY INDUCES
SIGNIFICANT UP REGULATION OF
MULL PELL INTERFERONS BOTH
INTERFERON ALPHAS AND -- THIS
INHIBITOR WOULD ACTUALLY SHOW
IMMUNOCHECKPOINT AND I'M SHOWING
THIS HERE WITH EF4, SIMILAR DATA
ALSO WITH ANTI-PD1 AND WE FIND
THAT THE INHIBITOR ALONE
ACTUALLY HAS SIGNIFICANT
THERAPEUTIC ACTIVITY, BUT THERE
IS AGAIN PROFOUND ACTIVITY WHEN
WE COMBINE THIS INHIBITOR WITH
ANTI-CDLA4 WITH A VERY
SUBSTANTIAL SURVIVAL BENEFIT.
AGAIN, THIS IS A MODEL THAT IS
RESISTANT IN CHECKPOINT.
WE WANTED TO KNOW WHETHER THIS
INHIBITOR HAD TOXICITY, SO AT
THE SAME DOSE, AT THE SAME
DURATION OF TREATMENT, WE
TREATED MICE WITHOUT TUMORS AND
THEN DID A COMPLETE PATH
ANALYSIS AND I'M SHOWING YOU A
SELECTED ORGANS, THERE WERE NO
INFLAMMATORY CHANGES, AND THERE
WAS ALSO NO CHANGE IN THE BODY
WEIGHT REGARDLESS OF WHETHER THE
MICE GOT VEHICLE OR CARM1
INHIBITOR.
WHAT WAS PARTICULARLY STRIKING
WAS THE DEGREE OF T CELL
INFILTRATION.
TEN IS RESISTANT TO THE BLOCKADE
BECAUSE AT BASELINE VERY LIMITED
INFILTRATION BY CDA T CELLS, AS
YOU'RE PROBABLY AWARE,
ANTI-CTLA4 AND ANTI-PD1 DOES NOT
REALLY INCREASE INFILTRATION
SUBSTANTIALLY HOWEVER WHEN WE
LOOK AT THE CARM1 MONOTHERAPY
TREATED MICE YOU CAN SEE THAT
MORE THAN HALF ALL OF THE IMMUNE
CELLS IN THE TUMOR SO THIS
INCLUDES ALL OF THE MYELOID
CELLS ACTUALLY BECOME CD8 T
CELLS AND THIS INCREASES FURTHER
WHEN WE TREAT THE MICE ALSO WITH
ANTI-CTLA4 AND ANTI-PD1 AND THIS
IS QUANTIFIED HERE ON THE
RIGHT-HAND SIDE.
FURTHERMORE, THERE'S SUBSTANTIAL
UP REGULATION OF GRAHAM ENZYME B
WHICH AGAIN IN THE ISOTOPE
CONTROL MICE IS VERY LOW IN THE
CD8 T CELLS BUT BECOME
SUBSTANTIAL IN THE CARM1
INHIBITOR MONOTHERAPY OR IN THE
COMBINATION THERAPY.
WHAT IS ALSO INTERESTING IS THAT
CARM1 INHIBITOR TREATMENT
INDUCES INFLUX OF NK CELLS WHICH
ARE RARE IN THE BASELINE TUMORS
AND INDUCES RECRUITMENT OF
DENDRITIC CELLS AND SO DEN DRITD
I CAN CELL ACTUALLY PURSUING
RIGHT NOW AND AS A CONSEQUENCE
OF THESE CHANGES IN THE IMMUNE
MICROENVIRONMENT, THE STRIKING
CHANGE IN THE CD8 TO FOX P RATIO
WHICH USUALLY IS LOW BUT IN
THESE INHIBITOR TREATED MICE
IT'S AT APPROXIMATELY 200 OR
HIGHER.
SO THE CD8 EFFECTOR CELLS VASTLY
OUTNUMBER THE REGULATORY T
CELLS.
SO WHAT I'VE SHOWN YOU, I'VE
TRIED TO BE BRIEF, IS THAT CARM1
AND ACTIVATION AND CD8 CELLS
GREATLY ENHANCES TUMOR -- AND
INHIBITORY RECEPTORS AND IT
HELPS INTRATUMORAL RESERVOIR OF
MEMORY-LIKE CELLS.
WE'RE CURRENTLY WORKING ON A
MECHANISM, AND THAT IN THE TUMOR
CELLS, WE ALSO HAVE A STRIKING
EFFECT AS AN INDUCTION OF A TYPE
1 INTERFERON RESPONSE THAT IS
INDUCED BY CGAS SIGNALLING AND
THAT SENSITIZES THE TUMOR CELLS
TO T CELLS AND CARM1 SELECTIVE
CARM1 INHIBITOR WE SEE A
STRIKELY INFILTRATION OF TUMORS
LY BY CD8 DISPELZ NK
EXPRELZ CDC1 DENDRITIC CELLS.
THE POINT HERE IS THAT THIS
INHIBITOR ACTUALLY HAS DUAL
ACTION ON BOTH TUMOR CELLS AND T
CELLS, AND ONE OF THE MAJOR
ACTIVITIES IN THE LAB RIGHT NOW
IS TO ACTUALLY ALSO LOOK AT THE
MOLECULAR PATHWAYS THAT ARE
INDUCED IN TUMOR CELLS.
I CAN TELL YOU THAT THE R
RNA SEQ DATA IN TUMOR CELLS AND
T CELLS ACTUALLY VERY DIFFERENT
SO IT'S NOT THE SAME MOLECULAR
PATHWAY THAT'S RESPONSIBLE FOR
THIS DUAL EFFECT.
AND THE CGAS THINK PATHWAYS ALSO
NOT RELEVANT FOR THE IMPROVED T
CELL RESPONSE.
SO THERE ARE MANY PEOPLE I NEED
TO THANK HERE, SOILY SUSHUA MOST
OF THE WORK TOGETHER WITH CC
ANDLY WAHAN, DID INITIAL GENETIC
SCREENING, WE HAD A GREAT
COLLABORATION WITH SHIRLEY LIU'S
LAB AND COMPUTATIONAL ANALYSIS I
SHOWED YOU, THE GENETIC
LIBRARIES AND THE T CELL SCREEN
DONE IN COLLABORATION WITH JOHN
DE -- WITH JOHN D DOENCH AND
STEVE ELLEDGE AND ALSO A GREAT
COLLABORATION WITH DAVID MOONEY.
I THANK YOU FOR YOUR ATTENTION.
[APPLAUSE]
>> HI.
BACK HERE.
>> OKAY.
>> I WILL KISS THE MIC AGAIN.
REALLY BEAUTIFUL WORK.
I'M CURIOUS ABOUT YOUR CARM1
DATA IN THE TUMOR CELLS
THEMSELVES.
>> YES.
>> AND ESSENTIALLY ACTIVATING
THE C GAS PATHWAY.
DOES IT INDUCE CHROMOSOME
INSTABILITY IN MICRONUCLEI IF
YOU HAVE THE INHIBITOR IS THAT
WHAT'S GOING OR WHAT IS
MECHANISTICALLY GOING ON?
>> LIKE VIJAY I CUT OUT A LOT OF
SLIDES.
I COULD HAVE TALKED FOR A LONG
TIME.
YEAH.
SO THERE IS EXACTLY, THERE IS
DNA DAMAGE AND YOU KNOW WITH
GAMMA H2X WE CAN SEE DNA DAMAGE
AND WE ALSO CAN VISUALIZE
CYTOPLASMIC MIE YOA NUCLEI AND
THAT ACTIVATES THE C GAS
PATHWAY.
THAT DOES NOT HAPPEN IN THE T
CELLS, WE LOOKED AT IT VERY
CAREFULLY.
THERE IS NO DNA DAMAGE MT T
CELLS.
SO THE MECHANISM OF ACTION IN T
CELLS AND TUMOR CELLS IS THE
SAME.
>> I'M ELLEN ROTHENBURG.
ONE OF THE THINGS INTERESTING
ABOUT THIS IS WHICH NORMAL CELLS
EXPRESS CARM1 AT REASONABLE
LEVELS, I WAS CHECK MGEN AND
IT'S PRETTY LOW IN T CELLS
ACCORDING TO THE RNA.
SO WHERE ELM IN THE NORMAL BODY
IS THIS INHIBITOR GOING TO BE
HAVING AN UNSCHEDULED EFFECT?
>> SO THAT'S PRETTY BROADLY
EXPRESSED BECAUSE IT'S A
TRANSCRIPTIONAL ACTIVATOR FOR A
NUMBER OF TRANSCRIPTION FACTORS.
IN T CELLS IT'S ACTUALLY
STRONGLY UP REGULATED, DIDN'T
SHOW YOU THAT, IT'S STRONGLY UP
REGULATED FOLLOWING ACTIVATION,
ACTUALLY VERY EARLY FOLLOWING
ACTIVATION.
AND THEN WE REACTIVATE THE T
CELLS IT GOES UP FURTHER SO WE
THINK IT ACCUMULATES SORT OF
WITH REPETITIVE T CELL
STIMULATION.
>> I GUESS I'M WONDERING ABOUT
GETTING RID OF SOME OF THESE
PROTECTIVE MECHANISMS THAT ARE
NORMALLY WORK AGAINST
AUTOIMMUNITY.
IS THERE ANY POSSIBILITY THAT
IT'S GOING TO SENSITIZE TO
AUTOIMMUNE ATTACK?
>> IN THE MICE, AS I SAID, THE
TOXICITY EXPERIMENT WAS DONE FOR
THE SAME DURATION AND AT THE
SAME DOSE, AND WE DIDN'T SEE ANY
INFLAMMATORY LESIONS ANYWAY.
SO BUT YOU BRING UP A GREAT
POINT.
SO, YOU KNOW, HOW DO YOU -- I
MEAN, WITH ALL IMMUNOTHERAPY
DRUGS THERE IS IMMUNE RELATED
TOXICITY HAD, AND THE QUESTION S
HOW TO MANAGE IT.
SO WITH THIS TYPE OF DRUG, WE
MAY ACTUALLY NOT WANT TO GIVE IT
CONTINUOUSLY FOR A LONG PERIOD
OF TIME, BUT YOU MAY WANT TO
GIVE IT AT ENTER VAMS BECAUSE
ACTUALLY IN ACUTE INTERFERON
RESPONSE MAY BE MORE BENEFICIAL
THAN SOME OF LIKE A CHRONIC
RESPONSE WHICH CAN INDUCE
NEGATIVE.
>> THANKS VERY MUCH.
>> GREAT TALK.
I HAVE TWO QUESTIONS.
ONE WAS A LITTLE BIT OF A
FOLLOW-UP FROM WHAT YOUR
RESPONSE WAS TO ALAN.
SO IS CARM1 UP REGULATED IN
EXHAUSTED CELLS AND THEREFORE
IT'S A BARRIER?
IS THAT PART OF THE EXHAUSTION
PROCESS AND IF SO, WHAT'S
DRIVING THAT?
SECONDLY, WHAT IS CARM1 ACTUALLY
DOING THAT MOD LAITSZ T CELL
FUNCTION -- MODULATES T CELL
FUNCTION ASK MIGRATION?
IS IT SORT OF GENERAL RELEASING
THE BRAKES SO EVERYTHING GOES
UP, METABOLISM, ACTIVATION, ET
CETERA, OR IS IT MORE SELECTIVE
IN TERMS OF WHAT IT'S TARGETING?
>> SO THE MOLECULAR MECHANISM
WITHIN THE ACTIVE INVESTIGATION
SO WE HAVE SOME ATAXIC DATA.
SO IN P THE KNOCK-OUT CELLS WE
SEE GREATER ACCESSIBILITY AT
GENES THAT ARE -- AT SITES WHERE
AP1 TRANSCRIPTION FACTORS BIND.
SO WE THINK THE KNOCK-OUT
ENHANCES ACTIVATION.
BUT SO, YOU KNOW, WHAT WE'RE
STILL WORKING ON, THE CUT AND
RUN DATA, I MEAN WE HAVE SOME
INITIAL -- WE HAVE SOME SPECIFIC
HYPOTHESES AS TO WHAT THE TARGET
GENES ARE, BUT THOSE EXPERIMENTS
STILL NEED TO BE DONE.
SO I REALLY DON'T KNOW.
YEAH.
AT THIS POINTED.
>> FIRST QUESTION IS IS THIS
SPECIFICALLY UP REGULATED IN
EXHAUSTION?
>> BUT THE SPREKS IS CLEARLY
RELATED TO T CELL ACTIVATION,
AND AS I MENTIONED IN MY
RESPONSE TO ELLEN, SO WITH
REPETITIVE STIMULATION, THE
EXPRESSION GOES UP.
SO WE THINK IT'S ONE OF THE
ENZYMES THAT'S RELATED TO T CELL
EXHAUSTION, SO IN A BROADER
SENSE.
>> THANKS.
>> OKAY.
>> HI.
IS THIS ON?
HELLO?
CAN YOU HEAR ME?
>> YES.
>> OKAY.
>> GO AHEAD.
>> FROM NAID.
WONDERFUL TALK.
THINKING ABOUT MAYBE THERE IS A
COMMON MECHANISM IN TUMOR VERSUS
IN CD8'S, HAVE YOU CAREFULLY
CHECKED THE CARM1 KNOCKOUTS CD8S
WHETHER THEY HAVE ANY
PROLIFERATION, I WAS WONDERING
IF SLOWING DOWN THE
PROLIFERATION OF CD8 THAT THEY
MAY RE RETAIN SOME STEM LIKE
FEATURE SO THEY CAN ACTUALLY
RESIST TO EXHAUSTION AND
EX-ZOOND I CAN TELL YOU THAT --
>> I CAN TELL YOU THAT IN VIVO
TUMOR CELLS AND T CELLS ISN'T
RELATED TO PROLIFERATION.
SO IF YOU SORT OF RECALL HOW WE
INITIALLY FOUND IT WAS ACTUALLY
BY LOOKING AT T CELLS THAT
ACCUMULATED TUMORS, RIGHT?
SO IT'S ACTUALLY THE T CELLS
ACCUMULATE BETTER IN TUMORS WHEN
WE INACTIVATE CARM1 AND WE'VE
LOOKED VERY CAREFUL AT TUMOR
CELL PROLIFERATION IN VITRO AND
THERE'S ABSOLUTELY NO DIFFERENCE
IN TUMOR CELL PROLIFERATION, SO
THEANT THE MECHANISM.
SO WE THINK THAT -- SO THAT'S
NOT THE MECHANISM. SO WE THINK
THAT TUMOR CELLS HAVE DEFECTS IN
DNA REPAIR PATHWAYS, AND WE
THINK THAT CARM1 IS INVOLVED AS
A CO-TRANSCRIPTIONAL ACTIVATOR
OF DNA REPAIR PATHWAYS, BUT THE
DETAILS NEED TO BE WORKED OUT.
>> LAST QUESTION.
>> SO YOU MENTIONED MOST LIKELY
LTS RELATED IN THE TUMORS -- IT
IS RELATED, IN THE TUMORS IT IS
RELATED TO DNA DAMAGE REPAIR,
BUT HAVE YOU EXPLORED THE
POSSIBILITY OF A CHANGE IN THE
EXPRESSION OF MHC ONE BECAUSE I
KNOW MANY TIMES FREQUENTLY IN
BASIC CELLS THERE IS DOWN
REGULATION OF MHC ONE SO I'M
WONDERING IF IN THE KNOCK-OUT
YOU CAUSE THE BE -- THERE ARE
MORE DETECTABLE BY THE T CELLS?
>> ACTUALLY WHAT HAPPENS SUSPECT
THAT THE CARM1 KNOCK-OUT CELLS
HAVE THIS ENDOGENOUS TYPE 1LY
INTERFERON RESPONSE AND WE'VE
SHOWN THAT THE KNOCK-OUT CELLS
ARE VERY SENSITIVE TO GAMG MA
INTERFERON DUE TO THIS
ENDOGENOUS TYPE OF INTERFERON
SIGNALLING.
SO ENDOGENOUS TYPE 1 INTERFERON
SIGNALLING SENSITIZES THEM TO
GAMMA INTERFERON AND UP
REGULATED EVEN WITH LOW LEVEL OF
GAM GAMMA INTERFERON, THAT'S HOW
IT WORKS.
THAT EXPLAINS SORT OF THE
HEIGHTENED SENSITIVITY IN T CELL
KILLING HAD.
>> GREAT.
THAT'S REALLY COOL.
THANKS.
>> OKAY.
[APPLAUSE]
>> THANK YOU.
>> SO THANK YOU ALL FOR STAYING
UNTIL THE VERY END, ALSO FOR THE
SPEEKS, JUST SOME CLOSING
REMARKS.
TOMORROW WE ARE GOING TO START
AT 8:30 WITH A BANG, JIM DE
SANTO WILL BE OUR FIRST SPEAKER,
AMAZING SPEAKER, PLEASE COME
BIVMENT OUR SESSION CHAIRS FOR
THE FIRST SESSION, WE START AT
8:30.
IF YOU ARE LATE, YOU ARE MIRING
A LOT OF THINGS, SO PLEASE BE
HERE AT 8:30 AND WE'LL SEE YOU.
THANK YOU VERY MUCH.
SEE YOU TOMORROW.