THANKS VERY MUCH FOR THE
INVITATION.
FANTASTIC MEETING.
I AM ALREADY ENJOYING THE
FIRST DAY AND LOOKING FORWARD
TO THE DAY.
IT IS ALSO A PLEASURE TO BE
BACK IN BETHESDA, GREAT
SCIENCE, GREAT DISCUSSIONS,
GREAT FRIENDSHIP SO THANKS
AGAIN FOR GIVING ME THIS
OPPORTUNITY TO COME AND VISIT
AND I AM HAPPY TO SHARE WITH
YOU WHAT WE HAVE BEEN DOING IN
THE FIELD OF INNATE LYMPHOID
CELLS.
I THINK EVERYBODY IN THE
AUDIENCE KNOWS ABOUT LYMPHOID
CELLS, PROBABLY HEARD ABOUT
THEM SO HOW DO WE FRAME THESE
CELLS IN THE CONTEXT OF
ILLNESS AND THEY ARE THE --
SIMILAR TO THE T CELLS WE ALSO
KNOW.
WE HAVE PARALLELS BETWEEN
THESE DIFFERENT HEALTH
PARIASES, T1, T2, T3 AND SO WE
HAVE ASKED ABOUT THEM, WHY WE
HAVE THESE CELLS THAT ARE MORE
ANTIGENT AND OTHERS ARE MORE
OPEN IN THE WAY THEY CAN
REACT, WHO DOES WHAT, WHEN AND
WHERE AND ALSO SOME
PHILOSOPHICAL QUESTIONS AS
WELL WITH THE T CELLS AND
DETERMINING FACTORS WITH
SULPHATE AND NOW WE HAVE T
CELLS THAT DON'T REACT.
SO A LOT OF QUESTIONS.
WE HAVE LEARNED HOW THEY REACT
TO INFECTION, AN INSTANT WAY
TO START THINKING ABOUT THEM
ESPECIALLY IN T CELL HEALTH
AND SO WE THINK OF THEM HOW
THEY FIT IN IN THE KIND OF
FASHION PEOPLE CAN GRAB ON TO
AND ACCEPT IN TERMS OF THE
INDICATE SKATERS OUTPUT OF THE
CELLS AND THEIR IMMUNE
RESPONSES.
BUT WHEN YOU LOOK A LITTLE
DEEPER, YOU SEE THERE ARE SOME
ASPECTS OF THE BIOLOGY OF THE
CELLS WHICH ASK NOT
NECESSARILY -- DOES NOT
NECESSARILY MATCH WITH
INFECTION.
YOU HAVE SOME THAT ARE RELATED
TO THE HOMEOSTASIS AT THE
TISSUE LEVEL AND THERE MAY BE
FUNCTIONS JUST BEYOND HAVING
ANOTHER SET OF CELLS IMPORTANT
IN DEFENDING US AGAINST
DIFFERENT TYPES OF PATHOGENS.
AND THAT HAS COME TO BE TRUE
AND WE HAVE LEARNED A LOT
ABOUT HOW NOW ILCS ARE
INVOLVED IN ACTIVITIES ON THE
LEVEL THAT ARE NOT RELATED TO
THOSE FUNCTIONS AND WE HAVE
LEARNED HOW THE CELLS MAY BE
INVOLVED IN OPERATING AT THE
SYSTEMIC LEVEL.
TISSUE REPAIR IS ANOTHER AREA
THAT IS EXTREMELY INTERESTING
AND A LOT OF PURCHASE --
PURSUAL IN THIS AREA, TRYING
TO UNDERSTAND HOW THEY ARE
IMPORTANT FOR TISSUE REPAIR
AND REGENERATION FOLLOWING
INJURY AND THEN WE LEARNED
ABOUT HOW THEY OPERATE WITH
THE NERVOUS SYSTEM, DAVID WILL
HAVE THINGS TO SAY WITH --
ABOUT THAT HERE AND THERE HAVE
BEEN A NUMBER OF WAYS THAT THE
I OC ACTIVATION ARE TAKING
PLACE AND I THINK WE'RE AT THE
TIP OF THE ICEBERG WITH HOW
THE CELLS ARE WORKING.
SO WE WERE INTERESTED FROM A
DEVELOPMENTAL STANDPOINT TO
TRY TO UNDERSTAND HOW THE
CELLS ARE MADE AND WE LEARNED
A LOT FROM OTHER GROUPS AND
ESPECIALLY A NUMBER OF
IMPORTANT CONTRIBUTIONS BY
ALBERT VILLAGE DELAC AND
SEVERAL IN THE AUDIENCE OF THE
PREOCCURS SORES ABLE TO GIVE
RISE TO ILCS AND THOSE CELLS
ARE VERY IMPORTANT BECAUSE
THEY ALLOW US TO MAP THE
DEVELOPMENTAL PATHWAY AND OF
COURSE MANY OTHER GROUPS
CONTRIBUTED TO THIS BY LOOKING
AT INDIVIDUAL TRANSCRIPT
SCRIPTION FACTORS AND ALLOWS
US TO LOOK AT OTHER THINGS AND
WE HAVE THIS MAP.
SO WE WERE INTERESTED IN
REPORTER MICE AND LOOKED AT
THE WORK OF ILC2 THAT GAVE US
SOME REHAB BILL TORE PATHWAYS
AND SOME PEOPLE WORKING IN
THIS AREA ABOUT HOW NK CELLS
AND ILCS MAY BE INDEPENDENTLY
WORKING OR ARE RELATED AND
SHOWN HERE, POSTDOC WITH
AYING, MANY OF YOU KNOW HER,
AND SHE HOOKED UP WITH AN
INDIVIDUAL WHO HAS MADE A
CAREER OF GROWING T CELLS IN
CULTURE AND THEY SET UP
CONDITIONS TO GROW HUMAN ILCS
IN THE LABORATORY AND THAT LED
TO DISCOVERING THIS POPULATION
IN THE BLOOD WHICH ACTUALLY
HAS ILC PRECURSOR ACTIVITY.
SO HUMANS HAVE IN THEIR
CIRCULATION THE PREOCCURS
SORES FOR ALL THE DIFFERENT
ILCS AND IF YOU LOOK AT THE
ONES WE FOUND IN MICE AND
FOUND IN BLOOD YOU CAN SEE
TRANSCRIPTIONALLY THEY ARE
VERY CLOSE AND ABLE TO GIVE
RISE IN VITRO AND IN VIVO TO
SOME ILK CELLS.
THEY ARE NOT COMMITTED TO ANY
FATE, THEY ARE MULTIPOTENT AND
THEIR SULPHATE IS DETERMINED
ENVIRONMENTALLY.
SO IT SETS UP AN INTERESTING
SCENARIO WHERE YOU HAVE THESE
CELLS AND YOU HAVE TO NOW
DISCOVER HOW THEY ARE GOING
ABOUT THEIR DIFFERENT
CONSIDERATION PROCESS.
SO WE HAVE AN ENTRY POINT IN
HUMANS, A VERY ENTRY POINT FOR
DISEASE ARE IN THE BLOOD.
HAVING THEM IN CIRCUMSTANCE
CIRCULATION LED US TO PROPOSE
HOW THEY MIGHT DEVELOP, THIS
IS ILC SITUATION IN SITU AND
WHAT IS HAPPENING IS THEY ARE
ABLE TO ENTER THE BODY
ANYWHERE WHERE THEY CAN
RECEIVE THE NECESSARY SIGNALS
THAT HAVE EFFECTIVE FUNCTION
RELATIVE TO THAT TISSUE.
SO IT IS A MATTER OF MAKING
ILCS ON DEMAND AND IN THE
RIGHT PLACE AND THE RIGHT
TIME.
SO THERE IS SOME EVIDENCE OF
THIS IN MOUSE STUDIES.
WE'RE STILL TRYING TO
UNDERSTAND EXACTLY THE SIGNALS
THAT ALLOW THESE MICE TO GET
IN THE TISSUES AND IT WAS
STRIKING TO HAVE IT KIND OF
IDEA GOING BECAUSE IT REMINDED
US OF COURSE HOW T CELLS
DIFFERENTIATE AND THIS HAPPENS
IN LATIN AMERICA -- LYMPHOID
TISSUES AND WE KNOW THIS VERY
WELL SO WE LOOKED AT THE
TISSUE WITH THE ILC BUT THIS
TIME AT THE TISSUE LEVEL.
SO THE IDEA YOU HAVE THE ILC
AND TISSUE AND ENTERING
APPROPRIATELY IN PROGENY IS
THE MODEL WE'RE WORKING ON.
SO WHAT KIND OF SITUATION DO
WE HAVE WITH THE ILCS THAT ARE
KNIFE EVE?
THEY ARE LYMPHOID CELLS, THEY
DON'T HAVE MUCH CD7, THEY
DON'T HAVE ANY CLEAR METABOLIC
STATUS AND WHEN YOU START
USING THEM, YOU FIND THE
ILCP'S HAVE VERY LITTLE
ACTIVITY ALONG THE MAJOR
METABOLIC PATHWAYS.
SO HERE YOU HAVE THE T CELLS
TRANSCRIPTIONALLY IN TERMS OF
THE DIFFERENT PATHWAYS AND
FATTY ACIDS AND YOU CAN SEE
THEY ARE PRETTY MUCH BLACK
ACROSS THE BOARD AND LAURA
SIRACHE WHO DID VERY NICE WORK
AND YOU CAN SEE THEY ARE
STAINED FOR MIGHT MITOCHONDRIA
THAT ARE HIGHLY ACTIVE AND
POLARIZED BUT NONCONSISTENT.
SO THESE CELLS ARE WAITING TO
GET SIGNALS AND WAITING TO
TURN INTO MATURE ILC'S SO OUR
MODEL IS AGAIN BUILT AROUND
THIS T CELL PERSPECTIVE.
WE LIKE TO THINK WE MIGHT USE
THE SAME RULES T CELLS USE TO
DEVELOP INTO A PROGENY SO WE
POSTULATE THEY MAY HAVE A
NAIVE CELL, KNOW THERE IS A
REPOSSIBLE TORE CELL THEY NEED
TO STAY ALIVE, WE CAN IDENTIFY
THEM AND THEY RECEIVE SIGNAL 1
AND SIGNAL 2 THROUGH THIS ILC
RECEPTOR, COSTIMULATION, THAT
WOULD GENERATE THE EXPRESSION
AND WE KNOW THAT HAPPENS
ACTUALLY, WHEN WE TRIGGER THE
CELLS, THEY PRIVILEGE RATE
CLONALLLY.
NOW THE QUESTION HAPPENS WHAT
HAPPENS WHEN THEY ARE INVOLVED
IN THE REGENERATION PROCESS
AND HOPE TO GET THAT IN A
HUMAN SETTING AND SO WE LOOK
AT THE STUDY BY JOHN WHO HAS A
ROW -- ROBUST CULTURE SYSTEM
WITH THE CELLS AND TRY TO
PROCESS THEM AT A MOLECULAR
LEVEL.
SO I WILL SHIFT HERE AND TALK
ABOUT SOME WORK WE HAVE DONE
IN THE MOUSE AND THIS IS IN
THE GUT.
WE HAVE BEEN INTERESTED IN
TRYING TO UNDERSTAND THE ROLE
OF MATURE ILCS WITH THEIR
PARTICIPATION IN DEFENSE
MECHANISMS.
WE KNOW THIS IS A VERY COMPLEX
ECOSYSTEM.
THERE ARE MANY CELL TYPES IN
THE BARRIERS.
ILC IS PLACED IN CONNECTION TO
THE PHAGOCYTES AND DIFFERENT
KINDS OF CYTOKINES MAPPED OUT.
BUT THIS PICTURE CAN BE
MISLEADING PAUSE YOU SEE --
BECAUSE YOU SEE THE ARROWS AND
YOU THINK THESE THINGS ARE
MOVING AROUND IN A SECTIONAL
WAY BUT YOU NEED PROOF THAT
THAT IS ACTUALLY HAPPENING.
SO WE WERE INTERESTED IN THESE
PICTOGRAMS AND WE HAVE AN
EXPERT NEARBY, MANY OF YOU
KNOW HIM, AND HE HOOKED UP
WITH INDIVIDUALS IN THE LAB
INCLUDING A PH.D. STUDENT, AND
ONE HELPING OUT FROM FELIPE'S
LAB, THEY LOOKED AT THESE
CELLS IN MICE IN THE GUT.
NOW, THIS IS COMPLICATED BUT
THEY WERE ABLE TO GET
BEAUTIFUL MOVIES AND I WILL
SHARE THEM WITH YOU.
THE QUESTION IS DO THESE CELLS
MOVE AND WE MIGHT IMAGINE THEY
DO BECAUSE IN THE END, WE
THINK OF ILCS MOVING AROUND
AND BEING PART OF THE DEFENSE
MECHANISM BUT IF THEY DO MOVE,
HOW IS IT REGULATED AND CHANGE
MAYBE WHEN YOU HAVE DIFFERENT
TYPES OF CHALLENGES?
SO HERE IS SOME IMAGES.
I WON'T GET EXACTLY INTO THE
MODEL BUT IF YOU HAVE
QUESTIONS THAT IS BUILT AROUND
THIS, THOSE THEN ALLOW YOU TO
TRACK THE ILC'S IN THE GUT.
SO IF YOU LOOK, YOU CAN SEE
THESE CELLS AND THEY TO MOVE
AROUND ALTHOUGH COMPARED TO
THE BLUE CELLS WHICH ARE T
CELLS THAT WE MARKED
INDEPENDENTLY, THEY ARE REALLY
NOT MOVING AS MUCH.
SO THEY ARE NOT AS MOTILE AS
WE HAVE IN T CELLS.
NOW, IF YOU LOOK IN THE CRIPS
WHERE YOU HAVE THE PATCHES,
THEY ARE NOTED BY THE GROUPS
BUT THESE ARE APART AND NOT
MOVING AT ALL.
SO THE ILCS THAT ARE PRESENT
IN TWO PARTS IN THE GUT HAVE
TWO DIFFERENT TYPES OF
ACTIVITY AND THAT CAN BE
QUANTITATED.
THE T CELLS ARE IN THIS GROUP
AND YOU CAN SEE THEY ARE
MOVING AROUND IN DIFFERENT
DIRECTIONS.
THE I WILL HAVE LOUSE ILCS ARE
HERE, NOT DOING MUCH BUT DOING
SOME AND THESE ARE THE ONE IN
THE CRIP.
SO IF YOU LOOK AT THIS
BEHAVIOR, YOU CAN SEE MOST OF
THE ILCS ARE ARRESTED AND THE
T CELLS ARE MOVING AROUND.
SO THIS IS A BIT OF SURPRISE,
T CELLS MOVING ILC'S STUFF.
AND WHY WOULD THAT BE?
WE DON'T REALLY HAVE AN ANSWER
BUT WE HAVE SOME THOUGHTS AND
WILL SHARE THEM WITH YOU.
CAN THE ILC'S MOVE?
WELL, WE SET THIS UP AND IF WE
USE THIS MODEL, WE CAN SEE
AGAIN THE CELLS ARE NOT REALLY
MOVING IN THE I WILL HAVE
LOUSE BUT IF WE ACTIVATE THEM
WITH INFLAMMATION AND THAT WE
CAN DO BY PUTTING FLAGO.J.
LLIN -- FLAGELLIN IN THE
SYSTEM AND YOU CAN DOCUMENT IT
AND SEE THEY HAVE CHANGED FROM
ARRESTED TO MOVING AND
INCREASED VELOCITY AND
PLACEMENT.
SO ILCS CAN MOVE AROUND, IT IS
JUST UNDER HOMEO STATIC
CONDITIONS THAT THEY ARE NOT
AND WHAT DOES IT TAKE TO GET
THEM GOING.
SO WHY WOULD THEY BE
RESTRICTED IN HOMEOSTASIS AND
WHY WOULD YOU NEED THEM TO BE
MOVING?
SO IF YOU TAKE AWAY THE CELLS,
LOOK AT ILC3'S, THEY ARE
MOTILE AND MAKING IMMUNITY.
SO THESE ARE CHARACTERISTIC ON
ADAPTING IMMUNITY IN THE
SYSTEM.
SO WHAT IS INVOLVED?
IT IS INTERESTING, YOU CAN SET
UP TYPES OF OTHER MUTANTS AND
HERE WE HAVE THE T CELL AND
WHEN YOU PUT THE T CELL IN THE
SYSTEM, NOW YOU TURN THE
MOBILE ILC3'S AND THE T CELLS
SHUT THEM DOWN.
SO WHY IS THIS HAPPENING?
WE LOOKED AT THE INTERACTION
AND CAME UPON A MATCH AND THAT
WAS EXACTLY CCR9 WHICH IS
EXPRESSED ON ILC3 AND ON T
CELLS IN THE GUT ARE ACTUALLY
RELEVANT RANT AND IT IS THE
CCR9 EXPRESSION IN THE GUT
THAT IS REGULATING THE ILC3'S
SO WE EXPRESSED THAT BY
PUTTING ABELATED T CELLS IN
THE SYSTEM AND THOSE ILC3'S
ARE NOW ABLE TO MOVE AROUND
WHERE IF YOU PUT BACK IN THE
SYSTEM NORMAL T CELLS, YOU
SHUT THEM DOWN.
SO THIS STORY IS BASICALLY
SUMMARIZED HERE.
IT BUILDS ON PREVIOUS
OBSERVATIONS MADE HERE AT NIH
CONCERNING THE ACTIVE ILC3'S
IN THE EARLY NATAL PERIOD
WHERE THEY ARE COMING INTO
PLAY BEFORE THE T CELLS ARE
FOUND IN THE GUT.
THESE ARE HIGHLY ACTIVE,
CYTOKINE STIMULATED AND ACTING
VERY STRONGLY WITH THE
EPITHELIAL BARRIER.
THEN THE T CELLS COME IN AND
SHUT THAT DOWN, AND THEY SHUT
THAT DOWN APPARENTLY THROUGH
THEIR ABILITY TO COMPETE WITH
THE ILC'S WITH CCL25 WHICH
THEN TURNS THEM INTO ILC3'S
THAT ARE IMMOBILE AND NOT
ANYMORE CONTROLLING IN THE
INTESTINAL BARRIER.
NOW THAT IS NOT A PERMANENT
STATE.
THAT STATE CAN BE ACTIVATED
AND THEY CAN TURN INTO ILC3'S
AFTER BEING ACTIVATED BY THE
RELEVANT SIGNAL.
SO WE CAN TALK ABOUT WHY THEY
MIGHT NOT BE MOVING AROUND AT
THIS STAGE BUT I WILL LEAVE
THAT FOR QUESTIONS AND IN THE
LAST MINUTE OR TWO, SOME
QUESTIONS RELATED TO
ADAPTATION FOR ILC3'S AND THIS
IS WHERE THE ILC3'S GET
ACTIVATED AT MULTIPLE TIMES
AND WE KNOW THAT HAPPENS IN
THE GUT, ILC3'S MAY BE
RECEIVING SIGNALS THROUGH THE
LIFETIME OF THE ORGANISM.
WE KNOW THAT CAN ACTIVATE T
CELLS HAVING MULTIPLE
ENCOUNTERS WITH INFECTIONS BUT
THE QUESTION IS WHAT HAPPENS
WITH THE ILC3'S IN THIS, DO
THEY HAVE ANY LONG-TERM IMPACT
OVER A LIFETIME, IS THERE AN
IMPACT ON THEIR CAPACITY TO
DEFEND THE BARRIER AND IF THEY
DO, WHAT IS INVOLVED?
SO NOT VERY SIMPLE TO TO THIS.
WE USED THIS MODEL TO LOOK AT
ILC3 PRODUCTIVITY AND ACTIVATE
IL22 PRODUCTION WHICH IS
IMPORTANT IN THE MOUSE MODEL,
THAT THEN ACTIVATES A SYSTEM
THAT IS CHALLENGED AND A LOT
OF THINGS GOING ON.
SO WE SET UP A SYSTEM TO MAKE
THIS TARGETED THROUGH ILC AND
ILC3'S IN PARTICULAR.
WE INFECT THE MICE WITH
CITROBACTER, THEN TREAT THEM A
FEW DAYS LATER WITH AN
ANTIBIOTIC, AND WE LOOK AT
WHAT HAPPENS LATER IF YOU
EXPOSE THOSE MICE TO
CITROBACTIN, SO HERE IS THE
CONTROL, WE COLONIZE THE MICE,
GET THE CITROBACTIN IN THERE,
GET SOME ACTIVATION OF THE
ILC, PARTICULARLY ILC3'S, NOW
YOU CLEAR IT, ACTIVATE THE
CELLS WITH A SECOND EXPOSURE
AND NOW THE ILC3'S HAVE THIS
INCREDIBLE ADDITIONAL
FUNCTIONAL CAPACITY IN THESE
MATTERS.
WE CALL THEM TRAINED ILC'S SO
SOMEHOW THEY HAVE WORKED OUT
AND HAVE SOME MUSCLE AND THEY
CHARACTERIZE IN DIFFERENT
LEVELS, I WILL SHOW THAT HERE,
THEY HAVE A LOT MORE
PRODUCTION, THEY PERSIST IN
THE TISSUES, WE HAVE DONE THAT
IN MAPPING.
THEY CAN EVEN BE GENERATED IN
RAG MICE AND THEY ALLOW FOR
PROTECTION AGAINST OTHER KINDS
OF PATHOGENS SO THEY ALLOW
CROSS PROTECTION AND WE CAN
TRANSFER THAT.
SO THIS IS THE END OF THE
STORY, MAYBE THE BEGINNING FOR
SOME OTHER STORIES BUT IT
LOOKS LIKE THIS ENCOUNTER WITH
CITROBACTER REPROGRAMS THE
CELLS CERTAINLY AT THE LEVEL
OF THEIR METABOLISM, THE OX
PLUS PRO LIVE RATES -- OX PH
OS PROLIFERATES AND THAT
ALLOWS THEM TO HAVE A BETTER
AND PERHAPS FASTER RESPONSE
DURING THE LIFETIME FOR
PROTECTION OF THE INDIVIDUAL.
SO THIS IS THE SUMMARY AND I
DON'T THINK I NEED TO GO
THROUGH THIS IN DETAIL.
THERE ARE STILL SOME AREAS
THAT ARE IMPORTANT TO
UNDERSTAND.
THE PLASTICITY OF THE ILC3'S
ARE SOMETHING WE STILL HAVE TO
WORK ON, WE HAVE LEARNED A LOT
HOW THEY ARE INVOLVED IN THE
STRUCTURES.
SO I WILL STOP HERE AND
ACKNOWLEDGE PEOPLE WHO DID
THIS WORK.
AND OUR FUNDERS, AND I WILL BE
HAPPY TO TAKE QUESTIONS.
[APPLAUSE]
>> LOOKING FOR SOMEONE
YOUNGER.
[LAUGHTER]
>> HI, VERY GREAT TALK AND
USEFUL INFORMATION.
SO I HAVE COUPLE OF QUESTIONS
BUT SO YOU SHOW THAT THE MOUSE
SEASON WHERE THEY EXIST AND
YOU SIGNAL 1, 2, 3 AS YOU ARE
IDENTIFYING TO BECOME
ACTIVATED ILC.
BUT I WAS WONDERING IN TERMS
OF METABOLISM, THEY ARE QUITE
DISSIMILAR TO WHAT WE THINK OF
ABOUT METABOLISM.
SO YOU SHOW THEY HAVE DURING
THE PRIMARY FUNCTION,
GLYCOLISIS AND IN THE
SECONDARY INFECTION, THEY
SWITCH TO OXPHOS.
>> THEY HAVE THE OXPHOS IN THE
PRIMARY AND THAT IS
PERSISTENT.
THE ACTIVATION IN THIS CONTEXT
IS NOT VERY STRONG.
WE LOOKED AT THAT AND THEY
DON'T HAVE A DRAMATIC INCREASE
IN GLYCOLISIS.
NOW HUMANS DO AND WHEN THEY
GET ACTIVATED WITH ILC2'S THEY
GET VERY ACTIVE.
SO EVERY TISSUE IS DIFFERENT
AND ACTIVATE DIFFERENTLY.
>> SHORT T QUESTION.
T CELLS, IF THEY ARE ENOUGH TO
KNOCK AROUND, PROLIFERATE ILC.
HOW DID THAT HAPPEN IN THE
CONTROL?
>> THEY DO GET PROLIFERATION
BUT NOWHERE NEAR THE AMOUNT OF
T CELLS.
>> BUT I WAS ASKING ABOUT THE
MEMORIES THAT YOU WERE --
>> THE MEMORY CELLS DO CREATE
A LITTLE BIT MORE AND IN THAT
CONTEXT, YOU KNOW, WE DON'T
KNOW EXACTLY WHAT DRIVES THAT.
>> SO I HAVE A PHILOSOPHICAL
QUESTION AND THE QUESTION IS
WE HAVE IL C1, ILC2, ILC3 AND
WHY DON'T WE HAVE PHOS3 TO
ILC?
>> GOOD QUESTION.
ACTUALLY THERE WAS ONE REPORT
ON A ILC RAG THAT WAS
PUBLISHED BUT LOOKS MORE LIKE
A ILC2 THAT HAS A BIT OF HYPER
ACTIVATION.
SO THE QUESTION IS WHAT
REGULATES THEM AND WHAT IS
NEEDED TO REGULATE THEM AND SO
COMPARED TO T CELLS WHICH HAVE
THIS AUTONOMOUS IL3 PRODUCTION
AND SOME WAY OF PROLIFERATING
IN SOME WAY, OBVIOUSLY YOU
HAVE TO HAVE A REGULATORY
MECHANISM.
ILCS DON'T HAVE THAT SO THEY
MAY NOT NEED THAT EXTRA LAYER
VIA A DEDICATED CELL AND I
THINK SOME OF THE OTHER
FACTORS WHICH ARE PART OF
THEIR HOMEOSTASIS ARE LIMITED
AND THE SYSTEM IN GENERAL IS A
LITTLE MORE COMPACT AS WELL.
>> SO WONDERFUL TALK AND I
HOPE YOU CAN HEAR ME, I AM
TRYING TO EAT THE MICROPHONE.
SO I WAS WONDERING IF YOU
COULD SAY A LITTLE BIT MORE
ABOUT THE TISSUE SPECIFICITY
WHEN IT COMES TO GENE PROFILES
THAT ARE SPECIAL IN ILC3'S
GOING TO A CERTAIN TISSUE BUT
ONLY FOR ACTIVATION, FOR
REPAIR FUNCTIONS BECAUSE FOR A
NUMBER OF CELLS, THEY HAVE
BEEN SHOWN TO BE BASED ON THE
TISSUE --
>> ABSOLUTELY.
I THINK THERE HAVE BEEN A
NUMBER OF OTHER GROUPS THAT
HAVE CON BOOTED A LOT MORE
THAN US IN THAT DOMAIN AND
DON'T HAVE MUCH MORE TO SAY IN
ADDING TO THAT KNOWLEDGE BASE.
RICHARD LOXIE HAD A HUGE
IMPACT IN THAT AREA AND AGAIN
IT IS VERY TISSUE-SPECIFIC.
SO THE KINDS OF QUESTIONS
RELATED TO WHAT IS KNOWN IN
THE MOUSE AND TRANSLATING THAT
IN THE HUMAN IS AN IMPORTANT
AREA TO GO TO AND I THINK
PEOPLE SHOULD FOCUS IN ON
TRYING TO HAVE A DEEPER
UNDERSTANDING HOW THE CELLS
ARE TISSUE-SPECIFIC IN TERMS
OF PROPERTIES.
>> THE T CELLS STOPPING THE
MOVEMENT OF THE ILC SUGGESTS
THAT THE T CELLS WORK SLOWLY.
DOES THE ILC HAVE THE ABILITY
TO PRODUCE THEIR OWN SITE
CANES --
[INDISCERNIBLE]
>> WE INTRODUCED THEM TO THE
TARGET AND DON'T UNDERSTAND
WHY.
YOU WOULD THINK IT WOULD BE
DETRIMENTAL TO HAVE THEM
CONTROLLING ALL THE TIME AND
THAT IS THE CASE IN THE RAG.
THESE CELLS ARE MOVING AROUND
AND THEN THE BORDER IS GETTING
BOMBARDED BASICALLY WITH 22
WHICH CAN, IF IT IS NOT IN A
CONTROLLED WAY, GENERATE THEN
TRANSFORMATION.
THAT HAS BEEN SHOWN IF YOU
TAKE AWAY THE BINDING PROTEIN
FOR INSTANCE.
SO MAYBE THE REASON THEY ARE
NOT MOVING IS TO PREVENT THAT.
BUT WHEN YOU HAVE THE CONTEXT
OF REPAIR AND YOU WANT TO HAVE
MORE AND YOU HAVE BASICALLY
THE RESERVE, THEN THE CELLS
MOVE AROUND DELIBERATE.
AND I HAVE DATA I DIDN'T SHOW
BUT IF YOU STOP THIS IN RAGS,
YOU ACTUALLY DO DECREASE THE
PERMEABILITY IN THE MEMBRANE
SO IT IS HAVING A POSITIVE
EFFECT IN THE MEMBRANE IN THE
RAG PATROL.
>> I HAVE A QUESTION ON THE
ILCS.
IN BONE MARROW TRANSPLANTS,
THEY ARE NOT LARGELY
REGENERATED.
[INDISCERNIBLE]
>> HE DID LOOK AT THAT IN THE
PAPER WITH ALLEN, THIS IS A
PAPER IN IMMUNOLOGY, ALLEN
FISCHER COLLABORATION AND THAT
IS A GREAT QUESTION AND HE
LOOKED AT THAT.
THAT LOOKED AT SYSTEMIC
RESPONSES, THE PATIENTS WERE
OUT OF THE HOSPITAL BUT STILL
HAD CLINICAL SYMPTOMS AND
PROBLEMS, A LOT OF THEM WERE
ON IBIG FOR INSTANCE.
AND WE WERE LUCKY ENOUGH TO DO
NASAL SWABS ON ALL THOSE
PATIENTS AND STUDIED THEIR
MUCOSAL RESPONSES, PUBLISHED
THAT IN A PAPER IN BLOOD THIS
YEAR SO IF YOU ARE INTERESTED,
YOU CAN TAKE A LOOK AT THAT.
ALL THOSE PATIENTS HAVE ILCS
IN THEIR NOSE.
THEY ARE NOT NORMAL AND THEY
HAD SOME ISSUES AND THEY HAVE
CARRIAGE OF STRENGTHO --
STREPTOMONIA THAT IS NOT
NORMAL.
SO I THINK WHAT THAT IS
TELLING US IS THE ILCS ARE
IMPORTANT FOR THE IGA
PRODUCTION AND EVERYBODY WHO
UNDERGOES THOSE TRANSPLANTS SO
PROBABLY DO IT THROUGH
INTRAVENOUS GLOBULARITY.
[APPLAUSE]
>> THANK YOU.
IF YOU CAN HEAR ME, OUR NEXT
SPEAKER IS AL HE IS FROM
SINGER WHO WILL TALK ABOUT
TRANSSCRIPTIONAL DETERMINATION
OF T LINEAGE FATE.
>> CAN YOU HEAR ME?
I AM MIC'D AND NOBODY CAN HEAR
ME.
SO THANK YOU FOR COMING TO
THIS MEETING, THANK YOU FOR
COMING TO THE SESSION.
REALLY ENJOYING IT.
SO SO THIS IS MY GROUP, THAT
IS NOT MY BOSS, HE WORKS FOR
ME, AND I HAVE TO ACKNOWLEDGE
THEM.
SO WE ALL KNOW ILCS ARE
COMPLICATED T CELLS AND I WILL
CAN TALKING ABOUT K CELLS --
YOU KNOW WHAT?
I AM NOT GOING TO DO THIS.
SO I AM GOING TO BE TALKING
ABOUT NK CELLS TODAY AND THESE
ARE THE INNATE COUNTERPART TO
T CELLS BECAUSE THEY CAN KILL
IN THE CASE OF LOOKING FOR
VIRUSES.
AND ELIMINATE TUMOR CELLS.
SO INTERESTING CELLS.
AND THIS IS THE PATHWAY OF
EARLY DEVELOPMENT ILCS IN
ADULT MICE, WE KNOW THEY ARE
EARLY NK PRECURSORS.
WE THINK OF THE IL C1'S, 2'S
AND 3'S THAT WE THINK OF AS
HELPER CELLS.
THE DEVELOPMENT OF NK CELLS IS
MUCH LESS UNDERSTOOD,
ESPECIALLY WITH NK CELLS.
SO I THINK MOST RECENTLY AND
CLEARLY, THEY HELP ILCS BUT
HOW DO THEY DEVELOP THESE
CONSEQUENCES?
DO THEY MAKE ANOTHER CELL TO
WORK ON THE CELLS?
SO THIS IS THE SEQUENCING AND
WE MAKE A NUMBER OF
ASSUMPTIONS THAT I DON'T HAVE
TIME TO GO INTO.
BUT MOST OF THESE CELLS ARE NK
CELLS.
SO YOU HAVE THE ENKP HERE AND
OTHER CELLS WE KNEW ABOUT AND
ONE SMALL GROUP OF CELLS WE
DIDN'T KNOW ABOUT.
THE HIGHLY EXPRESSED ILCCELLS
WE DIDN'T KNOW ABOUT, THEY
HAVE OTHER MARKERS AND WE
IDENTIFIED THEM AND PUT THOSE
DOWN HERE.
THEY HAVE BEEN DESCRIBED WITH
AVERAGE EXPRESSION, BUT A TINY
POPULATION HERE, I HAD NOT
KNOWN THAT.
SO HERE WE ARE ISOLATING THE
PROCESS, TRANSFERRING THEM
INTO MICE AND THE FUNCTION
THEY HAVE WITH REGARD TO LIVER
AND THESE CELLS WOULD BE ILCP1
AND JUST TO CONFIRM WHAT
PEOPLE KNOW, THEY ALSO MAKE T
CELLS.
SO WHEN WE TRANSFER THESE GUYS
USING ILC CELLS, THEY DON'T
MAKE ANY T CELLS.
SO WE HAVE TWO GROUPS THAT CAN
MAKE T CELLS AND WE WANT TO
UNDERSTAND THE RELATIONSHIP
BETWEEN THEM.
AND THIS IS A COMPLICATE SLIDE
SO IF I GET THROUGH THIS, I AM
DOING WELL, OKAY?
THE TOP PART, JUST REPEATING
AN EXPERIMENT THAT ALLEN
PUBLISHED, IT IS A LITTLE
COMPLICATED BECAUSE IT IS NOT
AS LEAKY SO WE HAVE TO
DEMONSTRATE THE CELLS AND THAT
IS WHAT WE HAVE DONE.
NK CELLS HAVE SOME LEVEL OF
LINEAGE TRACING, IT IS NOT
ZERO BUT IT IS NOT 20 PERCENT.
ILC'S ARE HEAVILY TRACED.
THIS NEW ACTIVITY WE ARE
DESCRIBING IS INCREASED BY
INKP.
AND DOWNSTREAM, YOU CAN SEE
THEY ARE STARTING TO EXPRESS
THE ROSA STOP, IS WHAT THEY
ARE DOING SO WHAT HE WAS DOING
WAS TRANSFERRING THIS OUT OF
THE MICE.
AND I WILL START OVER HERE.
IF YOU TRANSFER THEM, THEY
MAKE ILC'S.
LOOKING AT THE LIVER HERE AT
IL C1'S, THESE ARE HEAVILY
LINEAGE TRACED.
AND THESE ENKP CELLS ARE
HEAVILY LINEAGE TRACED.
IF WE TRANSFER ENKP CELLS,
THEY MAKE ILC CELLS AND THEY
ARE NOT HEAVILY TRACED.
SO IF WE TRANSFER THE ENKP-NK
CELLS, THEY ARE VERY HEAVILY
TRACED.
SO ON AVERAGE, 70 PERCENT
ACROSS THE EXPERIMENT.
THESE MAKE NK CELLS THAT ARE
MARKED BY PLZF.
MAYBE 10 PERCENT OR LESS.
IT IS NOT ZERO.
AND YOU MIX THESE TWO, YOU GET
WHAT PEOPLE NORMALLY SEE WHICH
IS 20 PERCENT ON AVERAGE
LINEAGE TRACING AND IF YOU
LOOK AT THAT, YOU CAN MAP IT
AND YOU CAN CONCLUDE FROM JUST
THOSE NUMBERS, IT -- IF YOU DO
THE MATH THAT 80 PERCENT ARE
COMING FROM THIS NEW CELL THAT
I TOLD YOU ABOUT AND ACTUALLY
A SMALLER FRACTION BUT A
SUBSTANTIAL FRACTION,
20 PERCENT OR MAYBE A LITTLE
LESS ARE ACTUALLY COMING FROM
ILC'S.
SO TWO PATHWAYS.
OKAY, ARE THESE DIFFERENT
CELLS OR THE SAME CELLS?
SO WE SORT THEM OUT AND PUT
THEM IN MICE AND LOOK AT
THOSE.
AND IN THIS CASE THE
SEQUENCING, ALL CONTROLLED
TOGETHER AND THE SHORT ANSWER
IS THEY ARE NOT
TRANSCRIPTIONAL CELLS.
BUT IF YOU LOOK AT THE NK
CELLS, THESE ARE THE GENES
THEY HIGHLY EXPRESS.
SO THESE ARE THE TRANSCRIPTION
FACTORS I AM SHOWING YOU IN
DEVELOPMENT AND THEN A BUNCH
OF GENES ACTIVATING THE
RECEPTORS.
IF YOU LOOK AT THESE ILCP'S,
THEY ENGAGE OTHER
TRANSCRIPTION FACTORS.
THIS IS THE ZEB2 AND THEY
EXPRESS OTHER MODELS.
YOU KNOW ONE OF THESE SCALES,
I WANT TO TALK ABOUT THAT NOW,
THIS IS THE MOST DIFFERENTLY
EXPRESSED GENE BETWEEN THESE
TWO AND I AM SHOWING YOU THAT
HERE.
SO THE TWO OF THEM, WHAT THEY
DID WAS PRETTY MUCH SOMETHING
IN THE MIDDLE SO IT IS A
MIXTURE OF NK CELLS.
SO IF YOU LOOK AT THIS, IT IS
EXPRESSED BY THESE GUYS AND
BARELY BY THESE GUYS.
SO I AM NOT TALKING ABOUT I
C1'S OR SOMETHING, THESE ARE
NK CELLS AND ALL EXPRESSED IN
WAYS THAT PEOPLE CONSIDER IN
THE MOUSE.
SO THESE CELLS ARE EXPRESSED
IN THE RECEPTOR OF -- MYMV.
AND IF YOU GET THIS, IT IS
HUGELY EXPRESSED AND THE ONE
EXPRESSED HERE ARE THE BULK OF
THE EXPRESSION.
AND THIS IS IMPORTANT BECAUSE
IF YOU USE THE ANTIBODIES, THE
MICE DIE.
SO MICE NEED THIS TO SURVIVE,
MOUSE CMV.
SO OF COURSE THIS WAS AN
OPTION BETWEEN THESE TWO
GROUPS OF NK CELLS, ARE THEY
FUNCTIONALLY DISTINCT, CAN WE
SHOW THAT.
AND WHAT WE ARE DOING NOW IS
JUST GOING BACK AND CHECKING
AND WHEN YOU CROSS THEM, THEY
ABSOLUTELY DO EXPRESS THE
LINEAGE.
THE ONES COMING FROM ILCS,
VERY LITTLE AND AGAIN THIS IS
WHAT YOU SEE IN THE MOUSE, YOU
END UP GETTING SOMETHING LIKE
THAT.
BUT NOW WITH SIMON'S HELP,
PICKING UP MICE, WAITING FIVE
WEEKS, COME BACK AND LOOK AT
ILC3'S.
SO THESE WOULD BE THE ENKP
DERIVED CELLS.
BUT IF YOU LOOK AT THE MOUSE,
THIS IS VERY DIFFERENT THAN
WHAT HAPPENED BECAUSE THESE
ARE BEING EXPRESSED
ENORMOUSLY.
NORMALLY MICE WE DON'T GIVE
CMV TO, EVEN ON A PER CELL
BASIS, MAKE THREE OR FOUR
TIMES AS MANY NK CELLS BUT
THEY ONLY RECOVER A FEW
THOUSAND CELLS FROM THIS.
IN MICE GIVEN MCMV, IT IS
MILLIONS OF CELLS, SEVERAL
HUNDRED FOLD EXPANSION.
BUT ONLY IN THOSE MICE BUT
NOTHING HAPPENS IN THE MICE
WHOSE NK CELLS WERE NOT
EXPRESSED.
SO NK CELLS ARE KNOWN TO BE
HETEROGENEOUS AND IT IS NOT
JUST IN ACTIVATION STATES, IT
IS DEVELOPMENTALLY.
THERE IS TWO STATES AND THEY
AREN'T THE SAME CELLS AND
REALLY THE COMING FROM THE NEW
SET I SHOWED YOU, PEOPLE DON'T
THINK OF THESE AS NK CELLS BUT
THEY MAKE A DIFFERENT CELL AND
IT IS UNKNOWN WHAT THEIR
FUNCTIONS MIGHT BE.
IT IS SOMETHING WE HAVE YET TO
UNDERSTAND.
SO NOW I WANT TO ADDRESS HUMAN
POPULATIONS THAT ARE VERY
HETEROGENEOUS AND ONE COMMON
WAY OF LOOKING AT IT IS TO
LOOK IN RELATION TO SMALLER
MARKERS.
AND THE HUMAN NK SUBSETS IN A
MAJOR POPULATION SHOW THE NK
CELLS.
YOU HAVE THIS MINOR POPULATION
AND THEN THE LARGER POPULATION
OF CD16 CELLS AND THESE ARE
THE CELLS THAT RESPOND TO CMV
AND PEOPLE GIVEN CMV OR WITH
THE HISTORY OF CMV HAVE THE
PRESERVATION OF OTHER MARKERS
ON THE CELLS INCLUDING CD56.
SO WHAT WE DID IS WE PULLED A
BUNCH OF SINGLE CELL DATA FROM
OTHER PEOPLE, THESE DATA HERE,
AND BECAUSE THEY ARE
SITE-SEQ'D HERE, THESE ARE
CD56 AND 57 AND WE TAKE THESE
EXPRESSED GENES AND APPLY THEM
TO GENERATE A TRANSCRIPTIONAL
SCORE ACROSS THESE CLUSTERS,
WHAT YOU SEE IS THESE TWO
CLUSTERS HIGH LIE EXPRESS THE
GENES THAT ARE CHARACTERISTIC
OF THE ENK CELLS.
AND CONVERSELY IF YOU LOOK AT
THE GENES THAT ARE HIGH LIE
EXPRESSED IN THE ILC CELLS,
THOSE ARE HIGHLY EXPRESSED IN
THE 56 CELLS BUT NOT THE 57.
SO THAT IS ALL THE TIME I
HAVE.
WHAT I HAVE SHOWN YOU IS THAT
NK CELLS ARE HETEROGENEOUS BUT
NOT TRANSCRIPTOMIC, THIS MAKES
THE BULK OF CELLS AND GENERATE
A SMALLER POPULATION OF
UNCONVENTIONAL NK CELLS THAT
WE DON'T YET UNDERSTAND AND
THE PATHWAYS MAY BE CONSERVED
IN HUMANS.
THANK YOU.
[APPLAUSE]
[ OFF MIC ]
>> WELL, IT IS IMMUNODEFICIENT
MICE.
THE NK IS FROM EFFICIENT MICE
BUT YOU SEE THE SAME
TRANSCRIPTIONAL PROFILES IF
YOU LOOK FOR THOSE SETS OF
GENES I SHARED WITH YOU THAT
ARE DIFFERENTIALLY EXPRESSED
YOU GET 80 PERCENT THAT
EXPRESS ONE SET OF GENES AND
20 PERCENT THAT EXPRESS THE
OTHER GENES.
[ OFF MIC ]
>> NO, NO, AND THEY -- OH, I
AM SORRY, YOU WANT TO --
>> MY QUESTION WAS WITH THE
GENE EXPRESSION YOU SHOWED
WITH NK COMING FROM THE ILC'S,
WHETHER THAT WAS IN TISSUE
VERY CLEAN AND IT WOULD BE
DIFFERENT IF WE WERE THINKING
ABOUT TUMORS BECAUSE NK CELLS
ARE EXPRESSED DIFFERENTLY IN
TUMORS.
>> SO YOU HAVE STARTED TO LOOK
AT THAT?
>> YES.
>> ABSOLUTELY.
SO WE HAVE LOOKED AT THE NK
CELLS, CD56 AND SEEN MORE OF
THOSE IN MICE SO THEY SEEM TO
BE DOING SIMILAR THINGS IN
OTHER TISSUES.
>> THANK YOU FOR YOUR TALK.
WHAT FACTOR OR EFFECT DO YOU
BELIEVE IS CAUSING THE SPLIT
OF ILC'S IN PRO GENUINE
TORES -- PROGENITORS --
>> I WOULD LIKE TO ADDRESS
THAT.
SO WE HAVE BEEN DOING THE WORK
THAT SHOW THESE ARE SIGNAL
TRANSCRIPTIONAL FACTORS BUT I
DON'T KNOW THAT FOR SURE.
>> THANKS FOR THE TALK, VERY
NICE.
SO THE MCMV INFECTION, DID YOU
LOOK IN THE LIVER?
BECAUSE THE SPLEEN IS ONE AREA
YOU START TO SEE THIS BUT
THERE IS A LOT MORE GOING ON
IN THE LIVER BECAUSE THINGS
TEND TO BE MORE RICH THERE AND
SO I AM WONDERING IF YOU
LOOKED IN THE LIVER.
>> SO WE LOOKED AT LIVER AND
SKIN BOTH AND WHAT I SHOWED
YOU IS TRUE IN BOTH SITES.
THEY ARE NOT RESPONDING TO
CMV.
>> REMARKABLE THE
UNCONVENTIONAL ONES ARE REALLY
LACKING THE IL49'S AND IT
MAKES THEY THINK ABOUT THE
SCRIPT FROM KUMAR WHO WOULD
PUT THEM IN VITRO AND EXPAND
THEM WITH IL13 AND BASICALLY
YOU GET THESE CYTOTOXIC NK
CELLS BUT NEVER DEVELOPED INTO
IL9'S SO I WONDER IF THE
PATHWAY IS PROHIBITING THEM
FROM --
[INDISCERNIBLE]
>> SO WE GENERATE NK
POPULATIONS IN VITRO BUT WE
DON'T SEE THE CYTODYNAMIC
POPULATIONS SO WE THINK THAT
MIGHT BE A LATE MATURATION
EVENT.
THANK YOU, IT IS GOOD TO KNOW
ABOUT THIS EXPERIMENT.
THANK YOU.
[APPLAUSE]
>> MY GREAT PLEASURE TO
INTRODUCE A FRIEND AND
COLLEAGUE SHAWN PARK AND
STUDYING ILC CELLS.
>> GOOD MORNING, MY NAME IS
SHAWN PARK AND TODAY I AM
GOING TO SHARE WITH YOU DATA
ON THE INNATE POPULATION OF NK
CELLS.
SO AS YOU KNOW, THEY ARE
INVARIANT NKT CELLS, THEY ARE
LINEAGE CELLS, SELECTED BY
NONCLASSICAL MHC-1 AND REWRACK
TO GLYCO LIPIDS, NOT PEPTIDES.
SO THE INVARIANT CELLS ARE
VARIABLE AND THIS RAISES THE
QUESTION OF HOW THEY ARE
RELATED TO T CELLS.
SO HERE YOU SEE THE VARIABLE
CHAIN WHERE THE T CELL IS
EXPRESSING MHC AND YOU SEE THE
MAJORITY OF CELLS ARE COMING
FROM THE MHC AND IN THE DATA
BINDS TO THE GLYCOLIPID FOR
THE SIGNALING.
AND THE VITAL LIKE IN COLIPID
-- GLYCO LIPIDS AND THIS
STIMULATES NKT CELLS OVER HERE
AND SHOWS A STRONGER
ACTIVATION OF THE MHC65 AND
69.
SO YOU CAN USE THIS TO LOOK AT
THE INNATE T CELLS AND YOU CAN
SEE THESE IN A NORMAL MOUSE
WITH THE ILC CELLS AND MORE
IMPORTANTLY, THE NKT CELLS
DOING ALL THE WORK WITH THE
MOLECULAR, IF YOU LOOK AT THE
TRANSCRIPT ALONE, IT IS MAKING
NKT CELLS.
SO IN THE MOUSE, YOU CAN SEE
10 TO 24 INCREASE IN T CELLS
SO THE NKT CELLS IS A GOOD
MODEL FOR THE STUDIES AND WHAT
WE WANT TO UNDERSTAND
INITIALLY IS THE MOUSE LINK
WITH GENERAL ACTIVITIES AND
THIS IS THE EXPERIMENT WE DID
INITIALLY.
SO WE TAKE THE MOUSE AND
INJECT INTO THE NKT CELLS AND
THEN WITH THE EXPERIMENT LOOK
AT THE NKT CELLS SO HERE IS
WHAT IT LOOKS LIKE.
YOU SEE THE CELLS IN THE
SPLEEN AND WITH NKT CELLS, YOU
CAN SEE WITH BINDING AND
ACTIVATION HERE GENERATES
PRETTY MUCH THE SAME BUT WHAT
WE KNOW IS THEY ARE HIGHLY
ACTIVATED WHEN YOU EXPRESS A
HIGH AMOUNT OF CELLS YOU GET T
CELLS BUT DON'T REALLY EXPAND.
SO YOU THINK MAYBE THERE IS
SOMETHING MISSING IN VIVO
WHERE YOU CAN ACTIVATE THE NKT
CELLS BUT DON'T DRIVE TO
PROLIFERATION SO MAYBE THERE
IS NOT ENOUGH NKT CELLS HERE
OR MAYBE THIS IS MISSING A
SIGNAL, A SECOND SIGNAL THAT
CAN ACTUALLY DRIVE THEM TO
PROLIFERATION.
AND THE IDEA WE HAD, THE
GALCER SIGNAL THAT CAN DRIVE
THEM TO T CELLS, THE NKT CELLS
TO ACTIVATION WITH
COSTIMULATION.
SO WHAT EVERYBODY THINKS, IS
THERE COSTIMULATORY MOLECULE
FOR NKT CELLS AND IS
COSTIMULATION REQUIRED FOR
FULL ACTIVATION OF EXPANSION
OF NKT CELLS?
SO WE COMBINE THE T CELLS IN
CD28 AND SEE IF IT GETS
ACTIVATED AND CAN EXPAND AND
THE RESULTS WERE PRETTY
DISAPPOINTING COMPARED TO
ALONE, THE ANTI-CD28 DIDN'T
REALLY WORK OUT VERY WELL.
SO CONSISTENT WITH THE
LITERATURE ALSO DOESN'T REALLY
DO A LOT, THE CD28 WITH ENK
CELLS.
SO WHEN YOU ARE LOOKING AT VA
14, IT DIDN'T REALLY EXPRESS
THAT.
SO WE PUBLISHED A PAPER
EARLIER THIS YEAR AND WHAT WE
FOUND IS THE VL3 -- DR3, WHAT
WE FOUND IS THEY PRODUCEDDR3
AND LIVE DR3 LEADS TO
ACTIVATION.
SO LO AND BE HOLD WHAT THEY
FOUND IN THE T CELLS IS DR3 IS
EXPRESSED IN ALL T CELLS.
SO WHAT IS DR3?
IT IS CALLED DEFENDANT
RECEPTOR, A PRIMER, IT IS A
TNF RECEPTOR IN THE SUPER
CYTOKINE FAMILY AND CAN
TRIGGER LIKE ANY MOLECULE
INTERACTION.
T L1A IS CLEAN.
AND IN THE MOUSE MODEL, THERE
IS A DR3 ANTIBODY SO WHAT WE
DID WAS SAY OKAY LET'S TEST
THIS IF DR3 CAN DO SIGNALING
THROUGH ACTIVATION AND
EXPANSION.
SO THE IDEA IS LET'S INJECT
THIS WITH ANTI-DR3 AND SEE IF
WE CAN GET A EXPANSION OF NKT
CELLS.
AND I REMEMBER COMING TO THE
LAB AND SAYING THERE IS A
PROBLEM.
THE MICE INJECTED DIED
OVERNIGHT AND I MUST HAVE
INJECTED SOMEWHERE THAT WAS
CRITICALLY IMPORTANT.
SO I INJECT AGAIN AND THE MICE
DIED AGAIN.
SO THREE TIMES, WE INJECT AND
WITHIN A COUPLE OF HOURS, THE
MOUSE IS SHIVERING.
I TOUCH THE MOUSE AND SAY THE
MOUSE IS COLD AND WITHIN 24
HOURS, THE MOUSE DIED.
SO WE INJECT THE MOUSE AND
WHEN INJECTED WITH DR3, THEY
DIED WITHIN 24-48 HOURS AND
NONE OF THE MICE SURVIVED OVER
48 HOURS SO THAT WAS PRETTY
MUCH STRIKING AND THEN THE
QUESTION IS OF COURSE WHY DID
THEY DIE?
SO WE INJECT THE MOUSE WHO WAS
BARELY ALIVE AND HE HAD A
TREMENDOUS AMOUNT OF ALC AND
TEST THE BLOOD AND BASED ON
THIS, THIS LOOKS LIKE COMPLETE
LIVER FAILURE AS THE MOUSE IS
DYING.
SO WE LOOKING FOR A SECTION
AND THIS IS WHAT YOU FIND.
WITHIN FIVE TO SIX HOURS, YOU
SEE THIS HUMONGOUS INTILTATION
OF THE CELLS IN THE LIVER AND
SO THIS IS SOMETHING WE DON'T
KNOW ABOUT WITH THE DR3 CELLS
IN THE LIVER SO WE THOUGHT
MAYBE THEY ARE ACTIVATING IT
TO SUCH EXTENT THAT IT CAN
CAUSE FULMINANT HEPATITIS.
SO IF YOU TAKE OUT THE NKT
CELLS BY THEMSELVES, WE COULD
ACTUALLY RESCUE IT.
SO THE GENERATION OF THE CELLS
DEPENDS ON DR3 SO THE KNOCKOUT
MICE WITH THIS, THE MOUSE IS
NOT DYING.
SO WHAT WE ARE SHOWING YOU IS
THE COINTRODUCTION OF THE ANT
TIE DR3 STIMULATION INDUCES
FULMINANT HEPATITIS AND LETHAL
TO VA 14 MICE.
BUT ALONE IT DOESN'T DO IT SO
WE THINK THE RESULTS SUGGEST
THAT DR3 PROVIDES POTENT
COSTIMULATORY SIGNALS TO NKT
CELLS.
BUT WE WANT TO KNOW WHAT IS
GOING ON WITH THE MOUSE MODEL,
SO WE TEST THE COSTIMULATORY
EFFECT OF DR3 IN WILD TYPE NKT
CELLS SO WE INJECT THE ANTIDR3
AND THEY SURVIVED.
ARE THEY HAPPY?
THEY ARE NOT.
SO YOU CAN SEE THE INDICATION
OF THE STRESS SIGNAL IN THE
MOUSE BUT AFTER FOUR DAYS, OFF
HEALTHY RESULT.
BUT THERE IS A TINY AMOUNT AND
WE LOOK INTO THE CELLS AND
WHAT THEY ARE DISAPPEARING,
HERE YOU SEE THE CD4 AND 5
PROFILE, YOU FIND DR3 DOESN'T
DO MUCH BUT THERE IS A
CYTOKINE SOLUTION COMING UP.
SO EVEN IN A NORMAL MOUSE, YOU
HAVE A HEAVILY ACTIVATED
PHENOTYPE IN THE MOUSE AND THE
MICE ARE NOT HAPPY.
SO WE WANT TO DO THIS IN
NORMAL MOUSE WITH ACTIVATION
OF NKT CELLS.
SO HOW CAN WE TEST IT?
SO THERE IS A MOUSE THAT IS
COMPLETELY NORMAL EXCEPT FOR
THE ABSENCE OF A NKT CELL.
SO WE INJECTED THESE MICE AND
WANT TO SEE IF WE CAN RESCUE
THE PHENOTYPE.
SO WITH THE WILD TYPE MICE,
AGAIN, WE INJECT AND YOU SEE
THESE NUMBERS AND AFTER FOUR
DAYS, YOU DEPLETE THE
POPULATION BUT IN THE MOUSE
THAT HAS NO NKT CELLS, THE
MOUSE IS NOT AFFECTED, NUMBERS
REMAIN SO WHAT THIS TELLS YOU
IS THIS NKT ACTIVATION CAUSED
HAVOC IN THESE MICE CREATING
CYTOKINE.
SO WHAT DOES IT?
WE LOOK AT THESE MOUSE IN
LIVER, OTHER ORGANS AND SPLEEN
AND WHEN YOU LOOK AT THE FOLKS
SEE OF THE NKT CELLS, THE
A-GALCER AND ANTIDR3 PRODUCE
ACTIVATION OF NKT CELLS IN
ENORMOUS AMOUNT AND HERE THIS
HAS BEEN DOCUMENTED WITH
LITERATURE.
BUT WHAT WE FOUND IS IF YOU
COINJECT DR3, YOU GET ABOUT 24
INCREASE IN NKT CELL NUMBERS
AND IN SOME CASES, WE DETECT
24 TIMES IN A SPLEEN, STRONGER
EXPANSION OF CELLS.
BUT THE QUESTION IS ARE THESE
CELLS FUNCTIONAL, ARE THEY
DOING ANYTHING AND TO TEST IT,
WE WANT TO LOOK AT CYTOKINE
PRODUCTION AND THIS IS WHAT
YOU SEE NORMALLY LOOKING AT
THE NKT CELLS, WE INJECT IL-4
AND YOU SEE IT IS MUCH
CLASSIC.
SO NOW WE WANT TO EXPAND T
CELLS, MAKE THEM ACTIVATED AND
THE LAST DATA SLIDE FOR THIS
ONE AND SAY WHAT CAN WE DO
WITH THIS ONE AND CAN WE
UTILIZE THIS ACTIVATED NKT
CELL FOR THERAPEUTIC PURPOSES.
SO WE TAKE THE LUNG MELANOMA
MODEL, WE INJECT THE ANTIDR3
AND THEN THE TWO TOGETHER AND
YOU SEE AN IMPROVED SITUATION.
SO IT GETS ACTIVATED, THE NK
CELL AND IT COULD BE EITHER
THE MOUSE CELL OR SOMETHING
ELSE, WE DON'T KNOW AND SO WE
SEE THEY CAN DIRECTLY TARGET
CANCER CELLS.
SO WE IDENTIFY A NEW CELL
WHICH IS DR3, AND ALONE, IT
CANNOT DO IT, IT AUGMENTS IT,
BUT PROMOTING NKT CELL
EXPANSION, CYTOKINE PRODUCTION
AND ANTITUMOR ACTIVITY.
SO I WANT TO CLOSE WITH THE
PEOPLE WHO DID THE WORK, MOST
OF THE WORK IN THE LAB AND
MOST OF THE WORK DONE BY
NURCIN LIMAN WHO HAD A POSTER
HERE WHERE HE IS DAY BUT NOW
YOU CANNOT SEE IT BUT SHE IS
HERE AND I WILL TAKE
QUESTIONS.
>> CAN YOU SEE ALL THE
EXPANSIONS OF NK17 AND DR3?
YOU MENTIONED YOU COULD SEE IT
IN THE THYMUS BUT ONLY THERE?
>> YES, WE NORMALIZE THE CELLS
AND ACQUIRE PHENOTYPE WHICH WE
DON'T KNOW.
THE EXPANSION IS FOR ALL THREE
SUBSETS, THEY ALL EXPAND.
>> THANK YOU, LOVELY TALK.
SO ARE THESE CELLS LIKELY TO
HAVE NK AGENTS WHEN THEY
RECOGNIZE THE LIGAND?
>> WE THINK SO, YES.
>> SO THE LIGAND IS --
>> NO, THOSE ARE CONSIDERED
MACRO CELLS.
SOME PEOPLE TOGETHER WITH THE
GLYCOGENUINE ARE GIVEN A
BOOST.
>> SO THIS CAN HAVE THE
RESPONSE OF PRIMARY AND
SECONDARY AND IN ONE
SITUATION, THE NORMAL MOUSE
WILL DIE BUT THE WILD TYPE
WILL NOT SO IF YOU HAVE
EXPANSION OF NKT
POPULATIONS -- WILL THEY STILL
LIVE?
>> CAN YOU REPHRASE THE
QUESTION?
>> SO YOU INJECT NKT AND --
[INDISCERNIBLE]
>> THAT IS A REALLY GOOD
EXPERIMENT.
I DIDN'T THINK ABOUT IT BUT WE
SHOULD DO IT, YEAH.
YOU CAN DO REPEATED INJECTION,
THE DR3 LEVEL GOES DOWN
BECAUSE IT IS LIGATES SO I AM
NOT SURE HOW MUCH DR3.
THE REASON WE DID FOUR DAYS,
IF WE WAIT ONE WEEK, THE DR3
COMES UP BUT EXPRESSED THE
LIBA -- LIGAND.
SO WE NEED TO STUDY THIS
QUESTION.
>> HOW DO YOU THINK DR3 IS
BEING ACTIVATED AND COULD IT
NOT BE ACTIVATED TO KILL YOUR
MICE?
AND CONVERSELY, THE DR3
KNOCKOUT AND ENKS, WHAT IT DID
TO YOUR MICE?
>> THE AWARD EXPERT IN DR3, HE
USED KNOCKOUT MICE AND NOW IN
SWITZERLAND AND HE LEFT SO NOW
WE ARE BREEDING THIS MOUSE.
THE CONDITION IS THE DR3 IN
THE BREEDING, THAT IS ONE
QUESTION.
BUT WE THINK THE BR3 ON
DENDRITIC CELLS WILL PRODUCE
THE SIGNAL.
WE ARE NOT SURE ABOUT TR1A.
I THINK THE CELL THAT PREVENTS
THE GLYCOGEN --
[INDISCERNIBLE]
GREAT QUESTION.
>> TWO QUESTIONS.
IF I RECALL DR3 MADE THE
BIGGEST DIFFERENCE IN T L1 A
IN EXPANSION OF T CELLS IN THE
PERIPHERY IN INFLAMED ORGAN.
>> YES.
>> SAVES WONDERING DO YOU SEE
A DIFFERENCE IN THE SPLEEN
VERSUS IN PERIPHERAL ORGANS
AND, TWO, DO YOU KNOW ANYTHING
MUCH ABOUT WHAT SIGNALING
PATHWAYS ARE ACTIVATED
DOWNSTREAM OF DR3.
>> SO THE SECOND QUESTION,
WHAT IS DOWNSTREAM, THE DR3
HAS THREE PATHWAYS AND THE
MOST IMPORTANT THING IS WE
COULD INHIBIT THIS EFFECT
USING CHYNASE CONTRIBUTOR.
THE SECOND THING IS ABOUT T
CELL ACTIVATION.
DR3 IS ACTUALLY EXPRESSED IN
HIGHER AMOUNT OF T CELLS AND
THOSE ARE THE THINGS WE ALSO
DID BUT THE DIFFERENCE IS IN T
CELLS, MOST OF THEM EXPAND AND
THEY ARE NOT FUNCTIONAL.
THEY ACTUALLY CANNOT DO
ANYTHING.
OUR ENK CELLS CAN EXPAND AND
DO SOMETHING SO I THINK THAT
IS THE DIFFERENCE.
[APPLAUSE]
>> SO IT IS A PLEASURE TO
INTRODUCE OUR NEXT SPEAKER
COMING ALL THE WAY FROM THE
WEST COAST AND GOING TO TALK
ABOUT MEMORY RESPONSES.
>>> OKAY, THANK YOU VERY MUCH,
THANK YOU FOR THE INVITATION.
THE MEETING HAS BEEN GREAT,
WONDERFUL TO BE HERE AND I AM
VERY IMPRESSED WITH THE OTHER
SPEAKERS.
I WILL TRY NOT TO DISAPPOINT
YOU.
THIS WORK IS DONE BY A NUMBER
OF PEOPLE AND I WILL MENTION
SOME NATION QUICKLY, GABRIEL
ASCUI-GAC, MALL REMURRAY, AND
OF COURSE FUNDING.
I CONSULT FOR A COMPANY THAT
WANTS TO DO NKT CELL THERAPY
SO THAT IS MY DISCLOSURE.
THIS TOPIC HAS BEEN INTRODUCED
AND WE HAVE GONE THROUGH THE
SUBSETS THAT SEEM TO, IN SOME
PEOPLE'S MINDS, IN BETWEEN
INNATE AND ADAPTIVE IMMUNITY
AND THAT INCLUDES THE GAMMA
AND BETA CELLS AND TWO TYPES
OF NKT CELLS.
I WILL TALK ABOUT NKT CELLS
AND WHAT ARE CALLED MUCOSAL
INVARY UNITED T CELLS.
ONE OF THE STRIKING THINGS
ABOUT THESE CELLS WHICH HAS
MOTIVATED OUR WORK OVER MANY
YEARS IS THE CONSERVATION
WHICH I WILL SAY MY LAB FOUND
MANY YEARS AGO.
SO IN OTHER WORDS MOUSE AND
HUMAN NKT CELLS ARE NEARLY
IDENTICAL.
IN FACT, THERE'S INTEREST
INTERSPECIES CROSS REACTIVITY
SO THESE HAVE BEEN PRESERVED
FOR TENS OF MILLIONS OF YEARS
IN EVOLUTION.
BECAUSE OF THE CONSERVATION,
THESE WILL NOT CAUSE DISEASE
AND THERE ARE A LOT OF
ATTEMPTS TO USE THESE KIND OF
CELLS IN VARIOUS CANCER IMMUNE
THERAPIES, IN OTHER WORDS
THERAPIES WITH ANTIGENS OR
CELL-BASED OR VAC ACT
VARIETIES -- ACTIV ATORS.
SO WE LOOK AT THE PROPERTIES
OF NKT AND MAIT CELLS, THE
MICROBIAL REACTIVITY TO
NONPEPTIDE ANTIGENS, THEY MAKE
RAPID RESPONSES AND CAN BE
ACTIVATED BY CYTOKINES SUCH AS
IL18, VERY MUCH LIKE ILC OR
ENK CELLS.
SO A FEW YEARS AGO WE LOOKED
AT NKT CELLS WITH SURFACE
MARKERS THAT WOULD
DIFFERENTIATE THEM AND FOR
EXAMPLE WE HAVE A SUBSET OF
NKT CELLS AND A ROW OF T CELLS
AND WE INPUT SINGLE SELL
RNASEQ AND IN TRIP KATE, WHAT
IS THE TONE?
OKAY, NEITHER DO I.
IT IS MY TIMER, I THINK.
SO ALL OF THESE ARE DIFFERENT
AND HIGHLY POSITIVE CELLS
WITHIN THE MICE WITH
TRANSCRIPTOMIC RELATIVITY.
SO THEN WHAT MALL REMURRAY AND
SCOTT BROWN AND OTHERS DID, WE
LOOKED AT THE NKT CELLS IN
DIFFERENT ORGANS, LIVER, LUNG,
SPLEEN AND THYMUS AND SORTED
THEM AND YOU CAN SEE HERE BY
ATAC-SEQ AND RNA-SEQ, THE DATA
BELOW.
BUT SOME CELLS CLUSTER, NOT
WHERE THEY ARE BUT
PREDOMINANTLY, PREDOMINANTLY
BY THE SUBSET.
SO WHETHER IT IS LUNG, SPLEEN,
THYMUS, NKT1 IS MORE SIMILAR
THAN NKT2 AND SOME OF THE
SPLEEN DATA IS SHOWN BELOW.
BY TRANSCRIPTION, THE LUNG
CELLS TEND TO BE SOMEWHAT
DIFFERENT IN THIS DIMENSION AS
SEEN HERE.
BUT NEVERTHELESS, THE
FUNCTIONAL THING IS THE SUBSET
AND THAT MIGHT LEAD TO A MODEL
THAT MAYBE THESE CELLS ARE
PREFIXED.
THERE ARE SOME SIGNALS THAT
HAVE BEEN DEFINED SUCH AS
CYTOKINES AND THEN AFTER THAT,
THEY GO AFTER TISSUE, THEY SIT
IN THE TISSUES AND THEY DON'T
CHANGE.
ARE THEY FIXED?
AND THE FACT OF THE MATTER IS
THEY ARE NOT FIXED.
AND, OKAY, I WILL JUST GO.
SO THIS IS WHAT NKT CELLS LOOK
LIKE IN UNIMMUNIZED MICE, AND
YOU SEE PD1, YOU HAVE A HUGE
POPULATION KNOWN AS NKT HELPER
CELLS AND IN FACT A NUMBER OF
GROUPS HAVE CHARACTERIZED
THESE CELLS PREVIOUSLY AND
THEY ACTUALLY DO FUNCTION AS
HELPERS FOR B CELLS.
AND THEN THERE'S ANOTHER GROUP
DOWN HERE THAT DIDN'T HAVE
THOSE MARKERS SPECIAL THOSE
CELLS, AS I WILL TELL YOU,
ALSO TURN OUT TO BE DIFFERENT
AND THIS IS A 6-DAY ANALYSIS.
SO IF YOU LOOK AT THESE
ANTIGENIC EXPERIENCED CELLS
AND ATAC-SEQ, YOU SEE THEY ARE
HIGHLY DIFFERENTIATED AND ARE
DIFFERENT FROM ANYTHING THAT
EXISTED BEFORE, NKT1, 2 OR 17.
THIS OTHER POPULATION REALLY
EXPRESSES A LOT OF THE MARKERS
OF AN ACTIVATED K CELL, HIGH
LEVEL OF EXPRESSION OF NK
RECEPTORS AND SO ON.
SO IT LOOKED LIKE THE ANTIGEN
MADE TWO POPULATIONS, A HELPER
POPULATION AND AN EFFECTIVE
POPULATION AFTER THE
STIMULATION EVENT AND THIS WAS
DONE AT 6 DAYS AND WE NOW HAVE
SINGLE CELL DATA FROM 30 DAYS
AND I WON'T GO OVER ALL THAT
TODAY BUT WHAT I CAN SHOW YOU
IS THAT THESE POPULATIONS
PERSIST.
SO AT SIX DAYS IN THE SPLEEN,
WE HAVE THE POPULATION OF NKT
EFFECTORS, AND THE SAME THING
FOR THE HELPER.
THE ANTIGEN HAS A HALF LIFE
AROUND 18 HOURS SO WE BELIEVE
THEN THESE ARE TRANSCRIPTIONAL
AND GENOMIC CHANGES THAT
PERSIST AND GIVE RISE TO
FUNCTIONAL CHANGES AND NEW
SUBSETS OF CELLS.
SO WE MIGHT CALL THESE CELLS
MEMORY, MIGHT CALL THEM
ANTIGEN ADAPTED, WE MIGHT CALL
THEM TRAINED, NOT SURE WHAT
THE BEST WORD IS BUT THE POINT
IS YOU HAVE A POPULATION THAT
DEVELOPS IN THE THYMUS WHICH
IS A KIND OF EFFECTOR CELL AND
YET THEY ARE CAPABLE OF
LONG-TERM CHANGES.
SO I WILL SPEND THE REST OF
THE TALK ON MAIT CELLS AND
THESE ARE QUITE SIMILAR AND
THIS WORK IS DONE BY GABRIEL
ASCUI-GAC AND SHILPI CHANDRA.
SO THE MOUTH MAIT CELLS ARE
BORN EFFECTOR CELLS, YOU HAVE
THE MAIT1 AND 17 AND YOU CAN
SEE WE HAVE THE IL17A AND IFNY
AND LOOKING AT FOUR TISSUES,
LIVER, LUNG, SPLEEN AND THYMUS
AND YOU CAN SEE THERE ARE TWO
SUBSETS, A MAIT1 AND MAIT17
AND THE THIGH MUSCLE IS CONTRI
-- THYMUS IS SHOWING THE
SIGNATURE WITH THE MAIT1 OR
MAIT17 TRANSCRIPTOME.
SO THE QUESTION IS ARE THESE
CELLS PREFIXED OR NOT?
AND HERE WE ADOPTED THROUGH
THE WORK OF ALEXANDER CORBETT
AND MELVIN, THIS IS A MODEL
WHERE WE GIVE SOME MON ELLA IN
THE LUNG AND THIS CAUSES A
HUGE NUMBER OF MAIT CELLS
WHICH STAY GREATLY ELEVATED
FOR MONTHS.
WE KEPT USING THIS SYSTEM
BECAUSE IT IS VERY EFFECTIVE
BUT WE CAN DO THE SAME THING
WITH THE F.TULARENSIS AND IT
DOESN'T HAVE TO STAY IN THE
LUNG BUT PERSISTS AND SO SAM
MON ELLA HAS AN ANTIGEN AND
YOU CAN SEE AFTER NINE WEEKS,
IF WE RE INFECT, THE CFU IN
THE LUNG ARE MUCH LOWER
COMPARED TO THE SO-CALLED
NAIVE MADE CELLS AND IF WE
EXPERIMENT HERE, USE A
DIFFERENT BACTERIAL,
S.PNEUMONIA AND YOU CAN SEE
THE MAIT CELLS.
SO WE KNOW THE BACTERIA ARE
CLEARED, WE USE A QUORUM
SENSING BACTERIA AND YOU STILL
GET THE EXPANSION SO IT IS NOT
A QUESTION AND WE HAVE DONE
OTHER EXPERIMENTS AS WELL.
SO WE DID THE RNA-SEQ AFTER 40
DAYS OF THE SALMONELLA
INFECTION, YOU CAN SEE THE
CELLS CLUSTERED AND ONCE AGAIN
THEY FIT INTO TH1 AND TH17
TRANSCRIPTOMES AS YOU SEE
HERE.
AND THESE TWO RECEPTORS HAVE
GOOD EXPRESSION.
THESE ARE FAIRLY GOOD SURFACE
MARKERS THAT ALLOW US TO
SEPARATE THE MAIT17'S WHICH IS
WHAT WE CALL THEM FROM THE
MAIT1'S.
SO THEY ARRIVE LATE FROM THE
INFECTION AND A FAIRLY POP
LIESD SUBSET.
BY DAY 67, SEEMS LIKE THEY ARE
TURNING OVER AND IF YOU DO A
CD INJECTION, MANY OF THEM ARE
IN THE BLOOD WHILE IL17 ARE IN
THE BLOOD.
SO THIS IS AN IL17 FATE
KNOCKOUT MOUSE IF YOU WILL AND
THIS IS THE ININFECTED MAIT
CELLS IN THE MOUSE AND MOST
ARE INACTIVE MEANING THEY HAVE
MADE ENOUGH CELLS IN THE
BLOOD.
IF WE LOOK 40 DAYS AFTER
RECEPTION, THE EFFECTOR
POSITIVE CELLS -- I THINK IT
IS MY OWN MOUSE ACTUALLY.
LET ME SEE.
YES, YOU SHOULD HAVE
INTERRUPTED ME.
IT TOOK ME 20 MINUTES TO
FIGURE IT OUT.
ALL RIGHT, SO THESE STAYED
POSITIVE, THE ILG1 CELLS, THEY
NEVER MADE IL17, OKAY?
SO THESE CELLS HAVE DIFFERENT
ABILITIES TO PROTECT AND SO
WHAT WE DID IS PUT THESE
EXPANDED CELLS IN THE KLRG1 OR
CD127 -- I AM JUST GOING TO GO
WITH IT.
THANK YOU FOR PUTTING UP WITH
ME.
WHERE AM I WITH TIME, BY THE
WAY?
OKAY, GOOD.
SO THIS IS A TRANSFER
EXPERIMENT AND WE'RE LOOKING
AT PROTECTION HERE AND THE
IL17 RECEPTOR PROTECT AGAINST
PNEUMONIA.
THIS IS MEASURED AFTER TWO
DAYS AND THE IL CELLS DID NOT.
WE ALSO DID AN INFLUENZA VIRUS
AND YOU SEE THAT EXPRESSED
THERE.
IT TURNS OUT IT IS NOT UNUSUAL
THAT THEY ACT DIFFERENTLY
BECAUSE THEY ARE DIFFERENT IN
THEIR METABOLISM.
MAIT17 CELLS TAKE UP FATTY
ACIDS AND HAVE MITOCHONDRIAL
ACTIVITY WHICH YOU SEE HERE.
YOU HAVE THE CD127 AND THIS IS
STAYING LOWER WITH THE
RECEPTIVE POSITIVE ONES
STAYING HIGHER.
AND THEN IN THIS MICROSCOPY,
THAT IS NOT MINE -- YEAH.
[ OFF MIC ]
OKAY, THANK YOU.
[LAUGHTER]
SO IN ANY CASE, THIS IS THE
FATTY AS SIT UPTAKE AND THIS
IS THE MITOCHONDRIAL COUNT.
SO OF COURSE THE MAIT17 CELLS
HAVE THE FATTY ACID UPTAKE AND
MIGHT DON'T -- MITOCHONDRIAL
ACTIVITY AND THESE CELLS HAVE
A HIGHER CONCENTRATION RATE
AND THEY HAVE MORE NPK.
SO BASICALLY THEY HAVE MORE
ACTIVE MIGHT DON'T --
MITOCHONDRIA AND WHEN WE PUT
THESE IN ARE A VERY LOW
NUTRIENT MATERIAL, IT WILL
ELEVATE THEIR SURVIVAL WHILE
THE MAIT1 CELLS, WHEN WE ARE
GAVE THEM THE NUTRIENT
SUPPLEMENT, IT WAS NOT AS
DRAMATIC.
AND THEN YOU HAVE THESE
TRANSGENIC MICE AND YOU CAN
SEE THE MAIT17 CELLS HAVE
HELPED TO MAINTAIN
MITOCHONDRIA.
ADDITIONALLY IF WE LOOK IN
VITRO AT THEIR ABILITY TO
RESPOND, THE MAIT17 CELLS
RESPOND ON FATTY ACIDS AND YOU
CAN SEE HERE IT IS HIGHLY
SUBJECTED TO THE INHIBITOR AND
THE INTERFEREON RESPONSE WAS
HIGHLY DEPENDENT ON THE
GLUCOSE.
SO FINALLY DOES METABOLISM
MATTER AND WE LOOKED AT
KNOCKOUTS TO LOOK AT THE HIF1A
WHICH WILL STABILIZE AND YOU
CAN SEE THE MAIT17 CELLS AND
THEIR ABILITY TO MAKE MAIT17
BUT NOT SO MUCH IN THE MAIT1.
SO WE LOOKED AT THE TISSUE
SPECIFIC TRANSCRIPTION
SIGNATURES FOR HUMAN MAIT
CELLS, YOU CAN SEE, WE HAVE
THE DIFFERENT CLUSTERS AND
SOME APPEAR TO BE PROLIFERATED
IN THE BLOOD.
SO ARE THEY LIKE THE MICE MAIT
CELLS OR MAYBE THEY ARE NOT?
SO HERE WE LOOK AT THE LUNG,
THE RESULTS ARE THE SAME FROM
THE BLOOD AND THE MAIT17 CELLS
DO TAKE UP FATTY ACIDS, THEY
HAVE A BIT OF TISSUE MEMORY,
DIFFERENT FROM THE NAIVE CELLS
BUT THE MITOCHONDRIAL
POTENTIAL IS NOT ELEVATED LIKE
IT WAS IN THE MOUSE SO IT
LOOKS LIKE IT HAS A DIFFERENT
METABOLIC PROFILE AND THEN
WHEN WE LOOK AT THE
TRANSCRIPTOME OF THE AGGREGATE
OF THE FOUR DIFFERENT ORGANS,
YOU DON'T SEE THE CLEAR MAIT1
OR 17 PHENOTYPE AND I THINK
THE QUESTION REMAINS WHETHER
THESE SUBSETS EXIST IN HUMANS.
HOWEVER THIS IS THIS RECENT
PAPER THAT LOOKED AT PNEUMONIA
IN ADULTS AND YOUNGER CHILDREN
AND MOST OF THE MAIT CELLS HAD
THE CAPACITY TO MAKE IL AND
THIS FOES DOWN WITH AGE AND IN
A CERTAIN CONTEXT, YOU SEE THE
MAIT17 CELLS.
SO TO SUMMARIZE, THERE ARE
ANTIGEN OR MEMORY-LIKE T
CELLS, THEY REQUIRE EFFECTOR
PROGRAMS IN THE THYMUS, CAN
GIVE RISE TO NEW CELLS.
AND FOR THE MAIT CELLS, THE
RESPONSES ARE PREVALENT AT
LEAST IN THE MOUSE AND THE
HUMAN APPEARS SO FAR TO BE
INTRAVENOUS BUT WE DON'T
REALLY HAVE THE EQUIVALENT OF
THE KIND OF INFECTION THAT I
TOLD YOU ABOUT IN THE MOUSE SO
THAT LEAVES AN OPEN QUESTION
AND THANK YOU FOR YOUR
ATTENTION.
[APPLAUSE]
>> HELLO.
I AM WONDERING ABOUT THE
INKTFH CELL.
IF YOU CHECK ON THE FUNCTION
OF THEM, BECAUSE SINCE THEY
ARE EXPRESSING, GIVING THE
SAME MARKERS OF THE TSH'S, IT
MORE OR LESS COULD ALSO MAIT
FUNCTION IF YOU TESTED THEM,
LIKE YOU SEE IN OTHER ORGANS
FOR EXAMPLE THE LYMPH NODES
AND ALSO THE SPLEEN?
>> YEAH, SO I MENTIONED WORK
FROM OTHER PAPERS THAT CON
SADD THAT AND PEOPLE HAVE
SHOWN THEY ARE CAPABLE OF
HELPING T CELLS IN BOTH A
PROGNATE AND NONPROGNATE
FASHION, THEY EXPAND QUICKLY
SO WE DIDN'T DO A LOT OF THAT
WORK.
I CAN TELL YOU THERE ARE MANY
MORE OF THEM IN THE SPLEEN
THAN THE LIVER AND THE LUNG
BUT CAN ALSO FIND THEM IN THE
LYMPH CELLS SO THEY ARE AS YOU
WOULD EXPECT THEM TO BE.
>> THANK YOU.
>> MITCH, VERY NICE TALK.
ACTUALLY I HAD A QUESTION.
DO THE MAIT17 CELLS EXPRESS
IL17 RECEPTOR AND DO THEY PLAY
ANY ROLE IN OUGHT OUGHT --
AUTO IMMUNITY RECEPTOR?
>> THEY DO PLAY A ROLE IN AUTO
IMMUNITY RECEPTORS AND THERE
WAS A PAPER RECENTLY THAT SAID
THEY CONTROL ROT IN THE BRAIN
AND IN THE ABSENCE OF MAIT
CELLS, YOU DEVELOP ROT IN THE
MENINGES AND YOU GET
INFILTRATION, TAKES SIX MONTHS
AND MIGHT REPORT ON THE
DEFECT.
I BROUGHT THAT UP BECAUSE I
KNOW YOU ARE INTERESTED IN
NEUROINFLAMMATION.
WE ONLY HAVE WORKED IN
INFECTIOUS MODELS SO WE
OURSELVES HAVE NOT DONE
ANYTHING ON THAT.
>> THANK YOU.
>> I HAVE A QUICK QUESTION.
YOU SAID THE --
[INDISCERNIBLE]
>> IS THE MEMORY SYSTEMIC?
IT IS.
SO WE DID AN INHALATION MODEL
BUT SAW SOME MEMORY IN THE
LIVER AND WE DID LESS SO IN
OTHER ORGANS AND WE HAVE ALSO
LOOKED AT FRANSISLLA WHERE YOU
COULD SEE MEMORY MADE CELLS IN
DIFFERENT ORGANS.
>> OKAY.
[ OFF MIC ]
>> THE COMPUTER IS OFF, OKAY.
>> SO I WILL INTRODUCE OUR
LAST SPEAKER FOR THE MORNING
SESSION FROM CORNELL.
>> OKAY, THANK YOU VERY MUCH,
HI EVERYONE MY NAME IS DAVID
FROM WALL GROUND NEW YORK,
GREAT PLEASURE TO BE HERE.
I WAS GETTING READY IT TH-RPBG
AND WAS THINKING EVERY TIME I
COME TO THE D.C. AREA, CRAZY
STUFF HAPPENS.
YOU KNOW, GOVERNMENT SHUTDOWN,
CRAZY DEBATE THAT I DIDN'T
SEE, JUST SAW THE EDITED
HIGHLIGHTS AND THEN I REALIZED
NO, IT IS NOT EVERY TIME I
COME, IT IS JUST EVERY DAY IN
THE D.C. AREA SO FOR OUR NIH
FOLKS, THANK YOU FOR
ORGANIZING THE EVENT AND
PUTTING UP WITH THE CRAZINESS.
AND SO I THANK YOU ALL, I KNOW
THIS IS A TREMENDOUS AMOUNT OF
WORK BEHIND THE SCENES.
SO I WILL GIVE YOU A QUICK
HIGHLIGHT OF RECENT STUDIES IN
THE LAB THAT DEAL WITH
REGULATION AND I WAS BEING
AMBITIOUS TO SHARE SOME OF THE
INFORMATION BUT THANKS TO THE
OTHER SPEAKERS, I CAN MAKE MY
SHORT.
SO I WILL TALK ABOUT THE
EPITHELIAL INTERACTIONS, THE
NEUROIMMUNE CIRCUITS IN THE
GUT AND THEN DIET AND INNATE
IMMUNITY.
HOPEFULLY MY COMPUTER BEHAVES
ITSELF.
THE EARLY WORK WE DID AND MANY
OTHER LABS DID THE SAME
ANALYSIS TO LOOK AT
TRANSCRIPTIONAL PROFILES OF
CELLS AND IF I GO BACK TO THE
INTRODUCTION, WHEN I STARTED
IN MY PH.D. MANY YEARS AGO,
MUCOSAL IMMUNOLOGY WAS ON A
VERY RESTRICTED NUMBER OF CELL
TYPES AND WE ARE JUST NOW
BEGINNING TO UNDERSTAND THE
CROSS TALK BETWEEN THE
ESPECIALLY TEAL CRIMINAL AND
NEUROIMMUNE INTERACTIONS WHICH
WE CAN TALK ABOUT.
SO THE EARLY WORK WE DID
LOOKING AT ILCS, PURIFIED FROM
TISSUE AND LOOKING AT THE
TRANSCRIPTIONAL PROFILES.
THIS IS BULK RNA-SEQ THAT WE
DID TO SORT THE ILC2'S AND
THIS IS FROM EITHER THE LUNG,
SMALL INTESTINE, LARGE
INTESTINE, HAD AIP -- AND
ADIPOSE AND YOU CAN SEE IT IS
QUITE DIFFERENT DEPENDING ON
WHICH ILC2'S WE LOOK IN.
COLON ILC2 EXHIBITS GREENS
MOST DIFFERENTIALLY EXPRESSED
IN THE COLON ASSOCIATED WITH
WOUND HEALING AND REPAIR TO
HOMEOSTASIS.
LUNG ILC2'S PARTICULARLY
ENRICHED IN GENES REGULATED BY
IL-33 AND GROWTH FACTOR
SIGNALING AND THEN THE ADIPOSE
TISSUE ILC2'S SPECIFICALLY
EXPRESSED IN METABOLIC-RELATED
GENES.
SO AS WAS POINTED OUT, THESE
COME FROM DIFFERENT SITES OF
THE BODY AND TISSUE-SPECIFIC
IN THE MICRO BIOME IN WHICH
THEY RESIDE.
SO LOOKING AT THE ILC2'S AND
ADOPTIVE TRANSFER, WE LOOKED
SIMILARLY TO PROTECTIVE TYPE
RESPONSES.
WE ALSO KNOW THEY ARE
IMPORTANT IN THE TISSUE REPAIR
AND HOMEOSTASIS.
SO THIS IS IN A MICE MODEL OF
DSS, TOXIC IN DRINKING WATER
AND IS A GREAT MODEL FOR ACUTE
INTESTINAL DAMAGE,
INFLAMMATION AND REPAIR AND
HERE IN THE INTEREST OF TIME,
I WILL SHOW YOU THE TISSUE
THAT SHOWS THE WHOLE STORY.
THE BLUE-PURPLE STAIN YOU SEE
HERE, THE WILD TYPE
ABNORMALITIES -- ANIMALS
EXPOSED TO DSS ARE MUCH MORE
DRAMATIC IN THIS TISSUE REPAIR
FOR THE ILC2.
IF WE RESTORE TO AREGULIN, WE
CAN SEE THE REPAIR AND IF WE
TRANSFER THE AREGULIN WITH THE
ILC2, IT DOESN'T WORK.
SO THE WORK EMERGING FROM MANY
FIELD IN THE LAST DECADE, WE
BEGIN TO RECOGNIZE THE
EXTERNAL FACTORS LIKE
CYTOKINES, GROWTH FACTORS,
PROSTAGLANDINS THAT CAN BE
ACTIVE EFFECTORS AND IN THE
LOWER PANEL, THEY CAN BE
DEPEND ENT ON THE FUNCTION OF
THE TISSUE IN WHICH THEY ARE
FOUND.
SO YOU SEE IMMUNOGLOBULIN, THE
CONTROL OF MET TAB LYNN SO
THESE CELLS ALL DRIVE FROM THE
COMMON REGENERAL ENITOR BUT
ALL THE TISSUES THEY ARE IN.
SO I WAS DISCUSSING WITH
ANOTHER SCIENTIST,
CHRISITOPH KLOSE, HOW DO THESE
REGULATE IN THEIR FUNCTIONS
AND PERHAPS IT IS THEIR
COUNTERPARTS BUT AS JIM SAID,
IT IS MORE THE PATHWAY.
SO I WILL SHOW YOU A LITTLE
IMAGING TO CUT TO THE CHASE TO
IDENTIFY BE WITH THE GUT AND
OTHER BARRIER SURFACES, ILC2'S
ARE PARTICULARLY ASSOCIATED
WITH THE INNERVATION.
IN THE GUT AS YOU KNOW, THIS
IS THE INTRINSIC AND ENTERIC
CIRCUITS.
NEUROSCIENTISTS TELL ME THERE
ARE MORE INDIVIDUAL NEURONS IN
THE CELL BODIES THAN THERE ARE
IN THE GUT OR SENSORY SYSTEM.
I DON'T KNOW IF THAT IS TRUE
OR SOMETHING THEY JUST LIKE TO
TELL GRADUATES BUT EITHER WAY,
YOU CAN SEE THEY ARE HIGHLY
EXPRESS ANT HERE.
THIS IS JUST CLARITY OF GUT
TISSUE AND THEN WE'RE
VISUALIZING THE CHOLINERGIC
NEURONS IN RED.
SO THE CENTRAL PART IS THE
LUMEN AND THE EXTERNAL PART IS
THE CHOLINERGIC NEURONS AND
YOU CAN SEE IT IS ALL THE WAY
THROUGH THE SUBMUCOSA AND INTO
THE INTESTINE ITSELF.
AND ON THE RIGHT, YOU CAN SEE
THE COLLOCALIZATION OF THE LIN
AND KLRG1 AND CLOSELY
ASSOCIATED WITH THE NEURONS.
SO WE ASKED THEN IS THERE ANY
FUNCTIONAL SIGNIFICANCE TO
THIS, SO DO NEUROPEPTIDES
DERIVE FROM THE NEURONS AND SO
HERE IS AN ILC C2 WITH IL13
AND HOPEFULLY A SHORT EXPOSURE
PROMOTES THE EXPANSION OF IL2
CELLS AND IF WE COMPARE THAT
TO THE CLASSIC STIMULATION OF
IL2, 7 AND 33 OR WITH
BIOLOGICAL STIMULATION WITH
THE MOUSING, YOU SEE IN A
SHORT PERIOD OF TIME, THE
EFFECT ON INFECTION.
WE CAN DO THIS ON OTHER TYPES
OF INFECTION AND I AM SHOWING
YOU THE GLOB LET CELL RESPONSE
HERE ON THE LEFT TO VISUALIZE
THAT AND THEN THE PARASITES
USED TO ACTIVATE THE ILC2'S.
THIS IS RATHER ILC2 DEPEND ENT
AND AN NNMU ROW ZEST TO BE IS
CRITICAL -- A NMU RECEPTION IS
REQUIRED.
SO THEN THERE ARE A FEW PAPERS
ON THE SELECTIVE EXPRESSION
AND REQUIREMENT OF NMUR1 ON
ILC2'S IN MULTIPLE TISSUES FOR
THE GUT AND LYMPHOID CELLS.
SO WE SEE THE INNATE AND
ADAPTIVE IMMUNITY, WOULD WE
FIND IT IS VERY MICROBIO AND
ENVIRONMENTAL STIMULATION.
SO WE ASK IF THERE ARE A
REQUIREMENT FOR ILC2 CELLS OR
IF THERE IS A HANGOVER PRIOR
TO THE DEVELOPMENT OF ANTIGEN
RECEPTORS IN ADAPTIVE
POPULATIONS AND CERTAINLY ONE
COULD MAKE A COMPARISON IN THE
ILC2 CELLS, THE ONLY
DIFFERENCE BEING THE PRESENCE
OF ANTIGEN RECEPTORS.
SO A BUNCH OF WAYS TO ASK WITH
BONE TRANSFERS AND CHIMERAS,
ALL DONE OVER THE YEARS BEFORE
BUT SOMEWHAT LIMITED BECAUSE
THEY ARE IMPERFECT.
SO THERE HAS ALWAYS BEEN THIS
STRIDE OF NOT PERFECT BUT
CLOSER TO PERFECT GENETIC TOOL
TO SHOW THESE.
SO THIS IS WORK DONE IN THE
LAB AND HERE WE WERE LOOKING
AT EXPRESSION OF THE RECEPTOR
FOR NMU, THE UP WITH I JUST
TOLD -- THE ONE I JUST TOLD
YOU ABOUT AND HERE THE NMU
RECEPTOR ARE LOOKING ALMOST
IDENTICAL AND WHERE THEY ARE
MOST ACTIVE APPEARANCE PEERS
TO BE THE ILC2 POPULATION.
SO REINSERT A ILC2 RECEPTOR
AND IT SEEMS TO WORK FAIRLY
WELL.
ON THE RIGHT THE FLAVORS AND
THEN THE RECEPTOR, EGFP
APPEARS TO NICELY BE COLLECTED
BUT THEN IF YOU LOOK AT THE
OTHER CELLS, SHOWING YOU THE
COLON ON THE LOWER RIGHT,
APPEARS TO BE RECEPTIVE HERE
AS WELL.
SO THIS MAY BE EASIER TO DO A
HEAD TO HEAD COMPARISON
BETWEEN THE ILC2'S SO WE WENT
TO THE AMPHIREGULIN LIGAND, A
MEMBER OF A LARGE FAMILY AND
WE KNOW ILC2'S ARE NOT THE
ONLY PRODUCERS FOR
AMPHIREGULIN AND MANY TISSUE
T-REGS ARE HIGH FOR
EXPRESSION.
SO IT IS INTERESTING WE CAN GO
BACK WITH THIS SELECTIVE THING
THAT EXISTS IS THERE ANY
ADAPTIVE AND COMPARATIVE
MODELS.
SO WE LOOK AT THE AREG WITH
THE TRICHURIS MURIA INFECTION,
THERE IS TARGETED DELEASE OF
AREG IN ILC2'S.
HERE WE SHOW THE PROXIMAL
COLON GOBLET CELL AND THIS
HISTOLOGY TELLS THE STORY THAT
THE CYTOKINE RESPONSE IS
ALMOST COMPLETELY GONE AND THE
ILC2 SPECIFIC REGULATION AND
THOSE ANIMALS HAVE A DEFECT IN
THEIR BIOLOGY TO EXPRESS THIS
PARTICULAR PARASITE.
SO GOING BACK TO DSS FOR A
SECOND, WE ALSO SEE THE NMU IN
THE COLON TISSUE AND THIS
APPEARS TO BE A RESPONSE TO
AREGULATE INFECTION.
HERE WE GAVE THE MICE
AMPHIREGULIN IN TYPE 2
IMMUNITY AND YOU CAN SEE IT
AFFECTS ALMOST ALL OF THEM
WITHIN 24 HOURS.
SO WHAT HAPPENS IN THE CONTEXT
OF DAMAGE SOBERRIVE WITH THAT,
YOU SEE WEIGHT LOSS AND OTHER
SYMPTOMS, AND ON THE RIGHT,
YOU CAN SEE THE WEIGHT LOSS
AND THIS TELLS THE WHOLE STORY
HERE.
SO IS THAT DAMAGE A RESULT OF
THE AREG FOR THE ILC2.
SO IF WE GET THE WILD TYPES
THERAPEUTIC NMU, YOU CAN SEE
THERE IS A RESTORATION OF THE
EPITHELIAL BARRIER TENDENCY
AND OVER ON THE FAR RIGHT,
THAT MEDIATED TISSUE IS ABSENT
AND THE ABSENCE OF ILC2
DERIVED AREGULIN SO THIS SHOWS
THE NEURON DERIVED DRIVING A
TISSUE CELL AREGULA.
IT IS MAKING A FAIR AMOUNT OF
AREGULA BUT IT IS SOMETHING
ABOUT THE ENVIRONMENT THAT
AFFORDS THEM A TISSUE
PROTECTIVE FUNCTION THAT
CANNOT BE COMPENSATED FOR BY
ADAPTIVE LINKEDINO --
LYMPHOCYTES.
SO LOOKING AT THE PATIENT,
THIS IS A LIVE TISSUE BANK AT
CORNELL, WE GET LIVE TISSUES
EVERY DAY AND YOU SEE ENOUGH
WHERE THE CHOLINERGIC NERVES
ARE CAN A ACTIVATED, THESE ARE
PATIENTS WITH ACTIVE DISEASE,
NOT IN REMISSION AND THEN YOU
CAN SEE WHERE WE ACTIVATE THEM
AND THE ILC2 ALSO PRODUCES
AREGULIN.
SO IN THE LAST MINUTE OR TWO I
WILL SWITCH TO OTHER STUDIES
IN THE CONTEXT OF THE KNOW
RESEVENTH TORSION OR PAIN
RECEPTOR FAMILY.
AND AS YOU KNOW THIS WAS A
NOBEL PRIZE A COUPLE OF YEARS
AGO.
I AM AN IMFOLLOW GIFT AND WE
THINK ABOUT WHAT IS IN TISSUE
PROTECTION, WHETHER IT BE
ITCHING, PSORIASIS, PATIENTS
DON'T COMPLAIN ABOUT ON
CYTOKINE STORM.
PATIENTS WITH CHRONIC
INFLAMMATION COMPLAIN ABOUT
PAIN.
THAT IS THE SYSTEM TOM.
SO THERE MUST BE SOME
COMMUNICATION BETWEEN THESE
IMMUNE COMPLEX THAT RESULT IN
THAT PAIN PHENOTYPE AND CAUSES
A BUNCH OF INFLAMMATORY
DISEASES.
SO ANOTHER COMPETENT POST-DOC
IN THE LAB LOOKING AT THE GUT
INFLAMMATION AND THE PRESENCE
OF ILC2'S IN A HEALTHY GUT
VERSUS ONE WITH INFLAMMATION
SO THIS IS A HEALTHY GUT AND
YOU CAN SEE A REMARKABLE
CHANGE IN THE INNERVATION IN
THE CONTEXT OF THIS ACTIVE
INFLAMMATORY DISEASE.
AND THEN WE ASK WHAT, IF
ANYTHING, IS THE CAUSE OF
SENSE SENSATION OF PAIN AND
MICROBIOSA AND I WILL SHOW YOU
THERE ARE MANY TOOLS WE CAN
USE TO ACTIVATE THE NEURONS,
OR DELETE NEURONS AND HERE WE
CAN SILENCE THE NEURONS AND
YOU SEE THE ANIMALS GET A LOT
SICKER, GETTING NOTABLY WORSE.
SO WE ARE NOT CHANGING THEIR
IMMUNE SYSTEM, JUST THE
INNERVATION, WE CHANGE THE
COMPONENTS OF THE MICROBIOTA
AND THEN YOU CAN LOOK AT THE
CARTOON THAT THERE IS A
TRANSMISSIBLE TRANSBIOSIS
ELICITED WHEN WE DELETE THE
NEURONS IN THE GUT AND THAT IS
AN IMPORTANT DRIVER OF THE
INFLAMMATION.
SO I WANT TO PRESENT SOME WORK
WE'RE DOING IN NUTRITION AND I
WILL TAKE JUST A SECOND TO
INTRODUCE ANOTHER IMPORTANT
PERSON IN THE LAB, ANTOINE
GUZMAN WHO HAS BEEN STUDYING
TIE VET -- DIET AND A SYSTEM
WHERE WE CAN SELECT CHANGES IN
THE DIET AND HOW THAT CHANGES
THE MICROBIOTA, THE
METABOLITES AND HOW THOSE ARE
SHOWING IMMUNE RESPONSES.
A VERY NICE PAPER PUBLISHED
NOT LONG AGO THAT ONE
PARTICULAR FIBER, NOT ALL
FIBERS ARE THE SAME BUT
INNULIN FIBER IS A VERY
IMPORTANT DRIVER OF PART 2
INFLAMMATION, MICROBIOTIC
DEPENDENT, SPECIFICALLY
DEPENDENT ON FOLIC ACID AND
THERE'S CRISPR TARGETING, A
SINGLE ENZYME ENCODED IN A
SINGLE BACTERIAL SPECIES THAT
IS IMPORTANT IN MEDIATING THIS
DIET INDUCED INFLAMMATORY
RESPONSE SO HAVING THESE ALL
IN ONE STUDY WILL BE VERY
POWERFUL AS WE THINK ABOUT HOW
TO HARNESS DIET AND NUTRITION,
WHETHER IT IS IN THE CONTEXT
OF CANCER TO IMPROVE
CHECKPOINT BLOCK CADE OR
INFLAMMATION OR BIOLODGICS FOR
INFLAMMATORY DISEASE WHICH ARE
ONLY 60 PERCENT EFFICACIOUS
AND WHY IS THAT AND IS IT
BECAUSE PERHAPS WE CAN TWEAK
THAT PERCENTAGE JUST WITH
DIET.
SO IN THE END THERE, THE THREE
PEOPLE I TRIED TO EMPHASIZE,
HIROSHI AND OTHERS, THANK OUR
FUNDERS AND SPONSORS.
>> BEAUTIFUL TALK.
I WAS WONDERING SINCE THE
AREGULIN IS SAID TO GO HAND IN
HAND WITH IL C1, I WAS
WONDERING IF ILC2'S MIGHT
PRODUCE ALL THE SPONSORING S-R
IF THIS IS ONLY WITH RESPECT
TO T1?
>> GREAT QUESTION.
SO FAR MOST SYSTEMS THAT
PIERCE ILC2'S ARE NOT MAJOR, I
THINK THERE ARE INFLAM
INFLAMMATORY CIRCUMSTANCES
THAT ARE VERY PREDICTIVE WHEN
YOU CONSIDER IMPACTS BUT THERE
IS SOME FUNCTION WITH THE ILC2
AND THE BIGGEST CROSSOVER
BETWEEN TRG, PERHAPS THEY
SHARE AREGULIN PRODUCTION.
IT APPEARS DIFFERENT
LOCALIZATION OF ILC2'S AND
TREGS EVEN THOUGH THERE ARE
VERY DIFFERENT CONSEQUENCES ON
WHAT THE EFFECTS ARE.
>> THANK YOU.
>> I AM A POST DOC AND
INTERESTED IN WHETHER UNION IS
IMPORTANT FOR TREATMENT OR IS
IT THE MMU-R1 THAT IS
SOMETHING SECRETING TO BE
IMPORTANT.
SO WHEN YOU SHOWED IN THE
FIRST PART OF YOUR SLIDE WHERE
YOU TRANSFERRED THE ILC IN
HOST MICE, DOES THE SOURCE OF
ILC MATTER?
>> THE VERY EARLY EXPERIMENTS
YOU MENTIONED WERE ONES THAT
WE DID USING ADOPTIVE TRANSFER
AND ALL OF THAT STUFF BECAUSE
WE DIDN'T HAVE THE GENETIC
TARGETING FOR SELECTIVE
MANIPULATION OF ILC2'S.
BUT THE TISSUE PROTECTIVE
EFFECTS ARE -- CAN OCCUR
BOTH -- I DIDN'T SHOW YOU IN
THE INTEREST OF TIME BUT IN
THE GUT AND THE SKIN AND
USUALLY WHAT WE DO AS A
NUMBERS GAME MORE THAN
ANYTHING ELSE, ELICIT A LARGE
POPULATION OF DONOR TISSUES,
TAKE THE DONOR AND ACTIVATE
THEM WITH IL33 FOR A COUPLE OF
DAYS PRIOR TO INVIVO TRANSFER.
SO MOST OF THEM ARE PREACT
'SAID THE.
MOST OF THEM IN OUR HANDS ARE
POSITIVE.
THE SYSTEMS WE HAVE WORKED ON
SO FAR ARE ALPHAREGULIN DEPEND
ENT AND PART OF THE EARLY WORK
WAS KNOWN TO BE DEPENDENT WITH
ILU SO IT IS KIND OF A CIRCLE.
>> GREAT TALK -- DISCERN.
[INDISCERNIBLE]
YOU SHOWED THE BARRIER CELLS
AND EPITHELIAL FUNCTION, I WAS
CURIOUS WHAT SIGNALS THEY ARE
GETTING, WHAT THEY ARE SENDING
FOR THE TISSUE THEY ARE IN?
>> A GREAT QUESTION AND I
THINK IF I HAD TIME I WOULD
HAVE GIVEN YOU A SNEAK PREVIEW
OF SOMEONE WHOSE WORK IS
COMING ALONG VERY INTERESTING.
CERTAIN WE KNOW EPITHELIAL AND
BARRIER SURFACES THROUGH THE
ALARMS OF ILCS AND CYTOKINES
ARE VERY IMPORTANT AND WE KNOW
INNERVATION IS VERY IMPORTANT
AS WELL.
I CAN GIVE YOU ONE EXAMPLE BUT
THERE ARE MANY, MANY OTHERS.
I THINK YOU RAISED THE POINT
OF IS THERE A TRIFECTA HERE IN
BIOLOGY AND I THINK THE ANSWER
IS DEFINITELY YES.
AND IT WOULD MAKE SENSE IN --
YOU KNOW, I THINK THE IMMUNE
SYSTEM IS THE MOST
DEVELOPMENTALLY DISTINCT BUT
SHARE COMMON FUNCTIONAL
TRAITS, SENSE THE INSIDE AND
OUTSIDE WORLD, EVOLVE TO FORM
COMPLEX SIGNALS AND EVOLVE TO
FORM --
[INDISCERNIBLE]
SO I THINK THAT IS A GREAT
QUESTION.
>> DAVID --
>> YOU ARE STILL YOUNG TO ME
JEFF.
>> I HAVE GOT A QUESTION
WHETHER THE UNIQUE OR
RESOUNDANT FUNCTION OF T
CELLS, THE MOST FAIR
COMPARISON IS WHEN THEY ARE
BOTH PRESENT, BUT YES AT
CERTAIN POINT WHEN YOU LOOK AT
THE INFECTION, THERE MAY BE NO
REDONE DANCE BECAUSE T --
REDONE DANCE BECAUSE T CELLS
HAVE HAD NO TIME TO WORK ON
INFECTION.
BUT IS IT FAIR TO SAY THE T
CELLS ARE REDUNDANT BECAUSE
ILC CELLS MAY EXPERIENCE DELAY
AND WORK OVER TIME AND WE HAVE
DATA FOR THAT.
>> GREAT QUESTION.
AN EXPERIMENT CAN BE DONE IN A
MUCH MORE RIGOROUS AND
CONTROLLED WAY TO EXPRESS THE
CONTEXT AND THE ANSWER IS, I
CAN GIVE YOU A LONG OR SHORT
ANSWER AND THE SHORT ANSWER IS
WE DON'T KNOW BUT THE LONG
ANSWER IS WITH EXPOSURE AND
DEVELOPMENT OF ANTIGEN
SPECIFIC TH2 MEMORY CELLS AND
THEN USE SOME OF THOSE TO
TARGET THE PRESENCE OF AN
ESTABLISHED T CELL POPULATION
AND ARE THEY REDUNDANT.
PERHAPS IT IS YES, PERHAPS IT
IS NO, PERHAPS IT COMES BACK
TO THE TISSUE THEY ARE IN,
CERTAINLY A GREAT EXPERIMENT
BUT HAVEN'T DONE IT.
>> THANK YOU SO MUCH FOR THE
FASCINATING TALK.
I WAS STUCK ON THE FIRST SLIDE
AND THOUGHT IT SHED QUESTIONS
ON THE REST OF IT.
I WAS SHOCKED BY THE AMOUNT OF
TRANSCRIPTIONAL DIVERSITY
BETWEEN THE ILC POPULATIONS
INCLUDING DEFINING LINEAGE AND
TRANSCRIPTOR FACTORS.
SO ONE OF THE QUESTIONS IS TO
WHAT EXTENT IS INVOLVED WITH
FUNCTION OF IL22 SEPARATELY IN
INDIVIDUAL TISSUE, ARE THEY
REALLY THE SAME CELL TYPES AT
ALL OR IS THAT A REFLATION OF
THE DIFFERENT CELLS AND
DIFFERENT TYPES.
IS THE RECEPTOR PHYSICALLY
USED BY ILC2 AT OTHER SITES?
>> THAT IS A GREAT QUESTION.
AND I THINK THE TISSUE
SPECIFICITY OF ENRICHMENT OF
GENES IS INTERESTING BECAUSE
IT TELLS US THAT ALTHOUGH
DERIVED FROM COMMON SELLS,
THEY HAVE REMARKABLE
PLASTICITY IN TERMS OF SHAPING
THEIR PREDOMINANT THAN THE
FUNCTION BASED ON THE MICRO
ENVIRONMENT THEY FIND
THEMSELVES IN.
CAN YOU TAKE THE ILC FROM ONE
TISSUE AND DELIVER IT EX
EXCLUSIVELY TO ANOTHER TISSUE
AND ASK IT DOES IT REQUIRE A
SECOND SIGNATURE FUNCTION
DEPENDING ON THAT IF IT IS
THAT, WHETHER A NEW HE IS DENS
OR COMMUNITY OR WHATEVER.
CERTAINLY SOME OF THESE
EXPERIMENTS HAVE BEEN DONE.
THEY ARE NOT THE BEST BECAUSE
THEY ARE A LITTLE HARD TO
INTERPRET AT SOME TIMES BUT
THERE APPEAR TO BE SOME
ASPECTS THAT APPEAR TO BE AT
SOME POINT BUT ANOTHER PART
ISN'T RESPOND TO WHERE THEY
FIND THEMSELVES.
SO THE TISSUES WE SEE THAT THE
MOST, ILC2'S ARE THE RECEPTORS
OF THE INFLAMMATORY RESPONSE.
NOW CAN THEY BE
INTERDEPENDENT?
ABSOLUTELY.
WE HAVEN'T SEEN IT YET BUT I
WOULDN'T BE AMAZED IF THAT
WERE THE CASE.
SO I DON'T THINK THAT IS THE
END OF IT, I THINK THAT IS
JUST ONE INTERESTING ASPECT.
I THINK THAT IS SOMETHING THAT
SPOTLIGHTS THE IL-IMMUNE
BIOLOGY AND A GOOD ROAD MAP TO
INVESTIGATE FURTHER.
>> GREAT TALK BUT AS YOU
MENTIONED, THE CHRONIC
INFLAMMATION IS NOT ABOUT
MEDICATION, COMPLAINING ABOUT
PAIN, THE FIRST THING WE DO IS
TAKE MEDICATIONS.
WHAT HAPPENS WHEN YOU TAKE
RAND RANDOM PAIN PILLS ONE
AFTER ANOTHER?
>> I DON'T KNOW THE ANSWER TO
THAT BUT WE HAVE BEEN TALKING
ABOUT THIS BECAUSE CERTAINLY
MANIPULATION, NOT USE I THINK
OVER THE COUNTER PAIN MEDS, AN
EASIER EXPERIMENT WE CAN DO AT
THE MOMENT BUT WHERE WE CAN
ALTER THE STATE OF THE
CIRCUITS AND MAP THAT TISSUE
TO MICRO ENVIRONMENTS AND WHEN
WE DO ALL OF THAT, YOU SEE
REMARKABLE CHANGES AND I DON'T
HAVE TIME TO SHOW BUT THE
COMBINATION COMPETITION OF THE
MICROBIOTA AND ALSO IN THE
COMPLEX, EVEN ACUTE PAIN
KILLERS CAN HAVE A DRAMATIC
EFFECT ON T CELL FUNCTION AND
I THINK WE DON'T UNDERSTAND IT
YET.
VERY GOOD POINT.
OKAY, THANK YOU.
[APPLAUSE]
>> I WOULD LIKE TO THANK ALL
THE SPEAKERS FOR THIS VERY
EXCITING AND ENGAGING SESSION.
PLEASE JOIN US FOR A COFFEE
BREAK FOR MORE DISCUSSION AND
WE WILL RECONVENE AT
11:05 A.M.
PLEASE VISIT THE POSTER
SECTION TO SUPPORT OUR NIH
TRAINEES DURING THE COFFEE
BREAK AND THE LUNCH BREAK. THANK YOU.
WELCOME BACK, HOPE YOU HAD
A NICE COFFEE AND POSTER
SESSION INTERACTION.
IT IS MY PLEASURE TO LAUNCH
SESSION 5 ON INNATE CELL
SIGNALING BY JULIA GRIFFIN WHO
IS JOINING US FROM FAR AWAY
AND WILL TALK ABOUT DYNAMIC
CHANGES AND I THINK THERE ARE
SOME COOL VIDEOS.
>> OKAY, THANKS VERY MUCH, A
PLEASURE TO JOIN NIH, I WILL
START THE TIMER AND MY TALK,
THESE ARE NOT ONLY IMPORTANT
CELLS IN FIGHTING OFF
INFECTION BUT ALSO HAVE
INCREDIBLY COOL BIOLOGY
OVERLYING IT AND THIS IS WHAT
DREW ME INTO STUDYING THE
CELLS AND WHAT GOES ON INSIDE
THEM IN TERMS OF THE REAT THE
PRESENT TIMEOR -- OF THE
RECEPTORS.
SO THEY ARE MADE UP OF THESE
SYNAPSES AND INSIDE THEM THEY
HAVE SHE IS SECRETORY
LYSOSOMES CONTAINING PERFORIN
AND GRANZYMES.
THIS IS AN UNUSUAL AMOUNT OF
TOLL LARRIZING SECRETION.
SO YOU SEE THE BACK OF THE
CELL WITH AN ACCUMULATION OF
TARGETS OFFICIALLY AND THEN
YOU WILL SEE IT DEPLETES AND
AT THAT POINT, THE CENTROSOME
COMES FORWARD BRINGING THE
GRANZYMES WHICH THEN CAN KILL.
SO THIS WAS BROUGHT FORWARD AT
THE NIH BETWEEN A STUDENT AND
MYSELF AND YOU CAN SEE THE
BODIES, THIS IS THE ACTIN
ACROSS THE CENTER AND THE
SECRETORY SIGNALS AND YOU CAN
SEE IT GOES POP.
IT IS TERMINATED BY A VERY
ACTIVE ACTIN RECOVERY AND SO
WE WANT TO KNOW HOW DOES IT
WORK.
SO SOME WORK WE DID IN 2018
AND WHAT WE DISCOVERED IS
BECAUSE OF THE DYNAMIC
MEMBRANE CHANGES, THE
COMPOSITION OF THE MEMBRANE
CHANGES AND THAT IS WHAT
CONTROLS WHETHER THE ACTIN IS
THERE OR NOT.
AND JUST VERY BRIEFLY, DURING
T CELERY SEPTEMBEROR
SIGNALING, THERE IS
SPECIALIZATION AT THE SYNAPSE
AND THAT GENERATES GLYCEROL SO
THERE IS A PARTICULAR MEMBRANE
SECRETION REQUIRED TO DEPLETE
THE ACTIN.
SO THEN WHAT WE LOOKED INTO
NEXT IS WHAT HAPPENS AT THE T
CELERY SEPTEMBERO -- THE T
CELL CE -- RECEPTOR AND YOU
CAN SEE THIS PIP 2 HERE
RECRUITING THE ACTIN AND IT IS
IMMEDIATELY KILLED.
SO THERE IS A SEAMLESS
RECOGNITION, ACTIVATION AND
THEN NEEDS TERMINATION IN
ORDER TO RELEASE THE T CELLS
SO YOU CAN HAVE SERIAL
KILLERS.
SO HOW DOES THAT WORK?
TO TRY TO UNDERSTAND AT A VERY
HIGH RESOLUTION, WE TOOK
ADVANTAGE OF A T CELL
ACTIVATION AND THAT WAY THE
EVENT CAN BE REVEALED BY
ELECTRON MICROSCOPY.
SO IT ALLOWS TO YOU SEE THE
VERY FIRST CONTACT IS MADE
FROM THE TINY TIPS WHERE THE T
CELLS ARE CONCENTRATED AND
THEN YOU CAN SEE IT THEN
CLUSTERS INTO WHAT IS KNOWN AS
THE SUPER ACTIVATION COMPLEX.
THIS IS ALL VERY WELL BUT WE
WANT TO SEE THE WHOLE BIG
ASSIGNMENT.
SO WE BUILT THIS IN 3D UNDER A
MICROSCOPE TO CONSTRUCT WHAT
IS GOING ON.
THIS ALLOWS US A 3 DIMENSIONAL
STRUCTURE ACROSS A RESOLUTION
AND THIS IS WHAT WE FOUND.
THE BOTTOM HERE IS THE T CELL,
THIS IS THE TARGET CELL AND
YOU CAN SEE THE T CELLS IN A
VERY TIGHTEN CLOSED MEMBRANE
SO WE LABELED WHERE THERE IS
JUST T CELL MEMBRANES AND THEN
THE T CELL APEX LABELED IN
GREEN.
SO WHAT YOU SEE IS NICE
ENDEMIC STRUCTURES FORMING,
AND ACTIN IN T CELL AND WHERE
IS THE STRUCTURE OF THE T
CELLS?
IT IS IN THIS MEMBRANE OUTSIDE
THE CELL.
SO THEY ARE FROM 225 TO 200
NANOMETERS AND I LEARNED
IMMUNOLOGY IN THE CELL
BIOINSTITUTE AND SO I
DESCRIBED THIS TO MY COLLEAGUE
AND HE SAID OH, WE CALL THESE
ECTOCITES WHICH DESCRIBES THE
FREQUENT SHEDDING OF RIGHT
SIDE OUT, A PITCHING OUT FROM
THE MEMBRANE AND WHAT WAS
INTERESTING IS THEY SAW
SORTING OF THE MEMBRANE BY
THESE T CELLS.
SO OF COURSE THESE OUTWARD
BUDDING OF THE NEUTRO PHILS
WHICH WAS SHOWN A NUMBER OF
YEARS AGO, THEY COULD SEE SOME
BUDDING BUT IT WAS DIFFICULT
TO SEE ON THE SECTION AND THEY
WERE JUST USING CORRELATED
LIGHT MICROSCOPY WHERE IT IS
NOT VERY CLEAR.
SO WHAT THIS SIGNALS IS THE T
CELLS ARE ACTIVATING OTHER
CELLS AND THEY THOUGHT THIS
WAS A MECHANISM FOUND IN DOWN
REGULATION.
AND THEY ALSO THOUGHT THE T
CELLS ARE NOT ACTIVATED
BECAUSE THEY WERE DEVOID OF
PHOSPHORYLATION.
SO THESE WERE THE CONCLUSIONS
AT THE TIME BECAUSE IT FITTED
WITH WHAT WE THOUGHT WE KNEW
ABOUT THE T CELL DOWN
REGULATION WHERE IT GOES
ACROSS THE MEMBRANE AND
THOUGHT TO UNDERGO ACTIVATION
END OCYTOSIS --
[INDISCERNIBLE]
SO WHEN WE LOOKED AT OUR
IMAGES, WE COULD SEE THAT YES
INDEED WE COULD SEE THE
ENDOCYTOSIS, THE FACTOR AT THE
BOTTOM AND THE ECTOCYTOSIS.
SO THROUGH A LOT OF WORK, WE
THOUGHT THERE ARE NOT MA MANY
ENDOCYTOSIS STRUCTURES.
SO A GOOD FRIEND AND COLLEAGUE
RIGHT DOWN THE STAIRS FROM ME,
DAVID AHERN HAS DONE ENORMOUS
ECTOCYTOSIS AND END OCYTOSIS
WORK AND WHAT HE HAS SHOWN IS
IT IS THIS MOTIF THAT ALLOWS
IT TO BE EXPOSED AND WHERE YOU
GOT T CELL ACTIN REGULATION,
THERE IS VERY LITTLE T CELL
ACTIVATION.
SO WHAT THIS MEANS, I DON'T
KNOW.
SO WHAT THIS SHOWS, THERE IS
ACTIN REGULATION BUT YOU ARE
NOT GETTING THE ENDOCYTOSIS.
SO WHAT IS ENDOC --
ECTOCYTOSIS?
THIS WILL SHED THE T CELERY
SEPTEMBER TORE AND THEY ALL
SHED ON THIS VERY TINY PATCH
ON THE MEMBRANE HERE AND WE
SAW MORE AS WE GET MORE AND
MORE TINY PATCHES.
AND THE OTHER THING WE FOUND
IS AS THE ECTOCYTES ARE
DETACHED, THERE IS A BINDING
THAT APPEARS TO BE GOING ON
AND AS THEY SHED, THEY PULL
OFF AND YOU GET AN AREA OF
SEPARATION.
THIS ENABLES THE T CELLS TO
LET GO OF THE TARGET AND MOVE
ON TO SKILLING SO WE THINK
THAT IS THE IMPORTANT ROLE.
HOW DOES THAT WORK?
IN THE EARLIER PAPER, Dr.
JACKSON SUGGESTED IT IS REALLY
LIKE A VIRAL BUDDING AND THE
ECTOCYTES INVOLVED IN THAT
BUDDING AND THIS COULD BE THE
SAME THING THAT IS HAPPENING
WITH THE T CELERY SEPTEMBER
TORE COMING OUT IN VIVO.
BUT ACTUALLY AS PREVIOUSLY
REPORTED IN ECTOCYTOSIS, IT
MESSES UP THE SYNAPSE AND IS
COMPLICATED SO THAT IS WHY
NOTHING HAPPENS.
SO IT IS AN OPEN QUESTION.
WE DON'T KNOW EXACTLY THE
MATRIX AND THAT IS ONE OF THE
THINGS WE'RE LOOKING INTO NOW.
SO WHAT HAPPENS THEN TO THE T
CELERY SEPTEMBEROR --
RECEPTOR ECTODERMS THAT ARE
SHED?
YOU CAN SEE HERE THEY ARE
INTERNALIZED AND TAKEN INTO
THE TARGET CELLS.
SO HERE YOU CAN SEE IT IS
OCCURRING, TAKEN INTO THE
TARGET CELLS AND OVER TIME IS
KILLING IT.
SO THE SAYING EAT MY DIRT
COULD BE THE RESPONSE FROM THE
T CELL BUT ESSENTIALLY IT IS A
VERY CLEVER WAY OF GETTING RID
OF YOUR ECTOSOMES.
SO THE PRIME TARGETS ARE THE
ECTOSOMES THEMSELVES AND SO
WHERE DID THIS IDEA COME FROM
THAT THE T CELL RECEPTOR IS
LOST?
THERE IS A PAPER THAT SHOWS IT
IS LOST AS CALCIUM INCREASES
AND IS DEPEND ENT ON A SIGNAL.
SO WE LOOKED AT THE
ECTOCYTOSIS, LOOKING FOR A
SIGNAL AND YOU CAN SEE IT
INCREASES WITH THE SIGNAL AND
HAS THE TCELL RECEPTOR
DECREASES AND THE ECTOCYTOSIS
INCREASES.
SO HOW CAN THIS BE?
YOU CAN SEE LOTS OF T CELLS
AND WHERE YOU HAVE A STRONG
SIGNAL, THE ECTOCYTOSIS IS
HERE, THEY ARE ALL
INTERNALIZED.
SO T CELLS OUTSIDE WHERE THE
CELLS ARE PRESENT BUT THE
SYNAPSE IS ECTOCYTOSED.
SO I CAN'T SAY HOW THAT MAKES
ANY SENSE, THAT IS AN
INTERESTING QUESTION BUT I CAN
TELL YOU WHY IT WOULD MAKE
SENSE AND THAT IS BECAUSE WHEN
YOU NEED TO DELIVER MORE T
CELLS, YOU HAVE LOT OFF YOUR
TCR, SHED IN ECTOCYTOSIS AND
THEN WHEN YOU NEED MORE, THE
ENDOCYTOSIS CLUSTERS WITHIN
THREE MINUTES OF CONTACT AND
THAT IS BEING CLUSTED JUST
WITHIN THE SURFACE BUT THEN
YOU SEE THE POOLS OF TCRS ARE
DELIVERED LATER SO THEY ARE
REPLENISHED FROM THE VEHICLE
AS SHOWN BY MY MOVIE HERE.
SO WHAT IS IN THE T CELL
RECEPTOR?
WHAT DO WE SEE?
WE LOOKED TO SEE ARE THEY
ACTIVATED OR NOT AND WHAT WE
SEE VERY CLEARLY IS THEY ARE
ACTIVATED AND WITHIN THE T
CELLS YOU NOT ONLY GET THE
FOUR LEGGED FORMS AND THE T
CELL RECEPTORS CHANGE, THE
ECTOSOMES PICK UP THE
ACTIVATED T CELLS.
SO QUANTITATING THIS, WE FOUND
THE VARIOUS CHANGES TO THE T
CELL THERE AND ALSO FOUND
TKPWHREUS ROLL THERE.
SO WHY?
WELL, MY COLLEAGUE WHO SHOWED
ME SOME FANTASTIC PAPERS FROM
THE 70s WHERE THEY WERE
LOOKING AT THIS IN TERMS OF
SHEDDING BUT ESSENTIALLY
LOOKING FOR ECTOCYTOSIS IN THE
CELLS.
CHEMICALLY THEY COULD SHOW IT
WAS ENRICHED WITH TKPWHREUS
ROLL WHICH IS A CONE SHAPED
DISEASE, HAD ITS HEAD CHOPPED
OFF AND IT WOULD CAUSE AN
OUTWARD BUDDING OF THE
MEMBRANE.
SO THIS RAISES THE REALLY GOOD
QUESTION THAT THE SHEDDING
COULD BE INVOLVED IN
ECTOCYTOSIS.
THE REAL QUESTION IS HOW DO WE
VISUALIZE THAT?
SO WE INJECT THE CELLS WITH A
PRO TKPWHREUS ROLL, HERE IS
THE TARGET CELL AND THIS IS
WHAT HAPPENS.
IT IS LIKE A LITTLE BIOPROBE
PINNING MACHINE WHERE THEY BUD
OFF WITH THE T CELL INSIDE
THOSE STRUCTURES.
SO THIS BRINGS US TO A MODEL
WHICH IS THAT UPON
RECOGNITION, SO THIS IS THE T
CELL ON THE BOTTOM, THE
BIOPROBE ON THE TOP, TARGETING
BY THE T CELL RECEPTOR AND YOU
GET ACTIVATION WITH TKPWHREUS
ROLL.
AS IT BUILDS UP, IT INITIATES
THE OUTWARD BUDDING THAT STAYS
IN CONTACT WITH THE BUDDING
CELL AND ACTIVATES THE T CELL
AND THEN SHEDS.
SO IN THIS WAY, WHAT THE MODEL
SHOWS IS THE T CELL ACTIVATION
SEAMLESSLY ACTIVATES TO
TERMINATION AND RELEASE OF
MORE T CELLS.
WHAT WE HAVE FOUND IS THIS IS
WIDELY USED IN MANY BIOLOGICAL
SYSTEMS OTHER THAN THE ONE I
JUST SHOWED YOU.
THE SAME THING IS HAPPENING IN
SILIA, THEY SHED THE ACTIN
ACTIVATED RECEPTORS AND SO I
WOULD LIKE TO THANK THE MANY
CONTRIBUTORS TO MY WORK AND
CAN TAKE ANY QUESTIONS.
>> THANK YOU FOR THE
WONDERFUL TALK.
IS THERE ANY CONTEXT TO THE
TARGET CELL, IS THERE A
CONSEQUENCE TO THE CELL AND
SECONDLY WHEN YOU MENTION IT
DOES NOT GO IN THE TARGET CELL
IS THAT BECAUSE IT DOESN'T OR
DROPS OFF AND --
>> WELL, FIRST OF ALL, THE
CD8 DOES GO IN.
WE HAVE NOT LOOKED AT THAT IN
DETAIL BUT I KEEP TELLING JANE
LET'S LOOK AT CD4'S WHICH ARE
VERY INTERESTING BECAUSE THEY
ARE NOT BEING KILLED.
PERSONALLY I DON'T THINK THEY
INFECT, I THINK THIS IS A WAY
TO GET RID OF YOUR ACTIVATED
RECEPTOR.
BUT I READ YOUR PAPERS WHEN I
WAS WRITING THIS.
>> MY NAME IS NATALIE AND I AM
A PH.D. STUDENT VISITING FROM
MOUNT SINAI.
I LOVED YOUR TALK.
TWO QUESTIONS, JUMPING OFF OF
THAT ONE, DO YOU THINK THE
IMPORTANCE OF THIS PROCESS IS
MORE TO CREATE THE T CELL
FURTHER DISCONNECTING FROM
THAT SYNAPSE OR THE CARGO THAT
IS TRANSFERRING AND PERHAPS
BOTH?
SO IT IS TOTALLY NOT, IT IS
LAGGING BEHIND SO IT TAKES SIX
MINUTES TO GET THE LISOSOMES
OUT SO I THINK IT IS REQUIRED
FOR THE KILLING AND CERTAINLY
IN CTL WHICH ARE RECOGNIZING
TARGETS.
I MEAN THE TARGETS, THEY DO GO
TO HELL.
>> I HAVE ANOTHER QUESTION --
>> WE CAN TALK AFTER.
>> THANKS, JILLIAN, THAT WAS
GORGEOUS.
SO I HAVE A QUESTION.
WE CAN CONSIDER THIS IS A
MECHANISM THAT ENABLES TO
KILL, WHAT IS THE IMPORTANCE
OF THE T CELL THAT ENABLES THE
SYNAPSE?
>> GREAT QUESTION.
I DON'T KNOW THE ANSWER.
MAYBE IT IS THE T CELLS
THEMSELVES.
>> GREAT TALK.
HAVE YOU IDENTIFY WHETHER THE
PHENOMENON OCCURS DEPENDENTLY
OR INDEPENDENT PENDLY OF THE T
CELL FORMATION?
>> OH, INTERESTING QUESTION.
NO, WE DIDN'T DO IT.
>> IS IT WHEN THE T CELL IS
ACTIVATED OR ONLY THE
RELEASE --
>> IT IS MORE DIFFICULT TO
IMAGE BECAUSE YOU DON'T HAVE
THE LEVEL ON THESE THINGS BUT
AGAIN, I WILL NEED TO DO THIS.
>> HOW DOES THE TARGET CELL
TAKE A CLASS FORM WHICH
DOESN'T HAVE THE SIGNAL FOR
ENDOCYTOSIS?
OR HOW DOES IT GET TAKEN OUT
OR DOES IT MATTER?
THERE MUST BE CELLS THAT DO IT
BETTER THAN OTHERS.
>> A NUMBER OF TARGET CELLS
KILL VERY WELL AND THEY ALL
SEEM TO TAKE IT UP.
ARE YOU SURE THERE IS NOT
ANY --
>> ANY MOW TEASE, MOST OF --
MOTIFS, MOST OF THEM DON'T.
>> YEAH, I WILL HAVE TO GET
BACK TO YOU ON THAT ONE.
>> GIVEN THE DIFFERENCES THAT
HAVE BEEN CHARACTERIZED IN THE
IMMUNE SYNAPSE BETWEEN
STANDARD T CELL AND A CARD T
CELL, YOU THINK THIS TEN NONE
NONE WILL OCCUR FOR CARD T?
>> YEAH, THAT'S WHAT I REALLY
WANT TO DO NEXT.
>> GREAT TALK.
ARE THE T CELLS ALSO GETTING
POPULATED --
>> YEAH, WE'RE TRYING TO LOOK
AT THAT.
IT IS DIFFICULT TO LOOK AT
BECAUSE THESE ARE THINGS THAT
PEOPLE LIKE GET EXTRA CELLULAR
VEHICLES AND SPIN THEM DOWN
AND IT IS VERY, VERY DIRTY.
>> SO FOR EXAMPLE THE C28.
>> YEAH, WE HAVEN'T LISTED
THAT YET BUT IT IS ON THE
TO-DO LIST.
IT IS A LONG TO-DO LIST, JUST
AS WELL IF I AM WRITING A
GRAPH.
>> ENJOYED YOUR TALK.
MY QUESTION IS CAN YOU USE
THIS MECHANISM TO DELIVER
SOMETHING OTHER THAN THE TCR
TO YOUR TARGET CELL?
>> YEAH, THERE IS A PIECE IN
NATURE THIS WEEK ABOUT USING
EXTRA CELLULAR VEHICLES AS A
DELIVERY SYSTEM.
I DON'T THINK THIS IS -- THEY
ARE VERY SMALL, MANY OF THEM.
I DON'T KNOW THAT YOU COULD
DIRECT SECRETION BECAUSE THEN
YOU STILL HAVE A MEMBRANE
AROUND IT.
SO I'M SITTING ON THE FENCE
WITH THAT ONE.
I PROBABLY AM ON THE NEGATIVE
SIDE.
>> HAVE YOU TRIED MANIPULATING
DIAZEGLYCEROL TO SEE WHETHER
THAT WOULD BE EFFECTIVE OR
NOT?
>> YEAH, WE HAVE A GREAT
COLLABORATION WITH A CHEMIST
AND ARE DOING THIS EXPERIMENT.
>> AND WHAT WOULD BE THE
ADVANTAGE OF THIS FOR OTHER
CELL TYPES?
YES, THIS WILL ALOU SERIAL
KILLING BUT IF YOU THINK IT IS
MORE BROAD A PHENOMENA, I AM
JUST CURIOUS IF IT WORKS --
>> WE CAN DISCUSS THAT --
>> FOR EXAMPLE, SYLVIA WANT%
CILIA WANT TO SHED THEIR
ACTIVE RESEPARATETO --
RECEPTORS AND MAYBE THAT IS
WHY WE SEE SO MANY OF THEM.
>> LOVELY TALK.
YOU ALSO HAVE A PAPER THAT
SHOWS YOU CAN'T DISENGAGE
TARGETS IF YOU DON'T DELIVER A
LETHAL HIT --
>> I DISAGREE.
>> AND AS IN PERFERO
INEFFICIENT T CELLS.
HOW DO YOU FIT THAT WITH YOUR
DATA?
>> THAT IS VERY DIFFERENT
WHERE YOU HAVE PERFERO
INEFFICIENT GOING ON.
BASICALLY THEY DON'T RECOGNIZE
SO THEY CONSTANTLY MANUFACTURE
MORE T CELL RECEPTORS AND
CONSTANTLY ARE ENGAGED AND
ACTIVATED.
SO WHILE SOME DO AND OTHERS
DON'T AND WHY?
WE DON'T QUITE KNOW.
>> THANK YOU.
[APPLAUSE]
>> ALL RIGHT, IT IS MY
PLEASURE TO INTRODUCE
CHRISTIAN MEYER WHO IS AN
ADMIN INVESTIGATOR HERE AT NCI
AND WILL TALK ABOUT T CELLS
AND REPERTOIRE SELECTION.
>> THANK YOU VERY MUCH.
SO FIRST I WOULD LIKE TO THANK
THE ORGANIZERS FOR THE
INVITATION TO PRESENT AND ALSO
FOR PUTTING TOGETHER THIS
FANTASTIC CONTENT.
SO TODAY I WOULD LIKE TO SHARE
UNPUBLISHED DATA FROM MY LAB
AND SOMETHING ABOUT WHERE AND
WHEN THESE T CELLS CLUSTER AND
THE IMPORTANCE IN THE UNITS.
SO AS WE ALL KNOW, B CELLS
UNDER GO CONCENTRATION IN THE
BONE MARROW AND THIS RESULTS
HIGHLY IN UNIQUE B CELL ON THE
SURFACE.
WHEN IT RECEIVES ANTIGEN, IT
BECOMES ACTIVATED AND THEN
DIFFERENT THINGS CAN HAPPEN.
ONE OF THE RESPONSES TO
PROTEIN ANTIGEN, BUT ADULTS
ALSO HAVE B CELLS IN THE ORGAN
AND THEY CAN BE SEGREGATED IN
TWO ANATOMIC DEPARTMENTS AND
THESE ARE CALLED LIGHT ZONES
AND DARK ZONES AND WE KNOW THE
PROCESS GOING ON IN EACH OF
THE CELLS.
THE LIGHT ZONE IS THE AFFINITY
BASED SELECTION OF THE B CELL
AND THE DARK ZONE ARE THE
HELPER CELLS REACTIVE TO THE
SAME ANT BEN.
IN CONTRAST, IN THE DARK ZONE
WHICH IS THE SECOND MECHANISM
BY WHICH THEY GENERATE
DIVERSITY.
THIS IS DRIVEN BY THE VDJ AND
CREATES VARIANTS OF THE B
CELERY SEPTEMBER TORE AND THIS
CAUSES SOMATIC HYPER MUTATION.
SO IT IS TOUT TO BE THE
MECHANISM OF --
[INDISCERNIBLE]
AND THEN FINALLY THE B ELSE
AND PLASMA CELLS CAN BE
RELEASED IN THE CENTER AND
THIS IS ANOTHER EXAMPLE OF
CROSS REGENERATION SO THERE IS
THIS QUESTION OF WHETHER THE B
CELLS CAN BE GENERATED DURING
BCR.
SO THERE ARE TWO TYPES OF B
CELL AND WHEN WE THINK OF AN
AUTO REACTIVE B CELL THAT
FEEDS A RECEPTOR, FOR EXAMPLE,
ONE OUGHT TO RECOGNIZE ONE
ANTIGENUINE AND NOT ANOTHER
ANTIGENUINE BUT WE ALSO KNOW
THERE ARE POLY REACTIVE B
CELLS THAT ARE AUTO REACTIVE
FOR ANTIGEN AND NO ANTIGEN AND
BOTH OF THESE HAVE TO BE
CONTROLLED BECAUSE OTHERWISE
IT CAUSES A RISK TO CREATE
AUTO IMMUNITY.
AND WE KNOW OF THESE
MECHANISMS AND THIS IS FROM MY
LAB.
HOWEVER, B CELL DEVELOPING IN
THE BONE MARROW WHICH
CONTINUES EDITING FOR THE
ARRANGEMENT FOR HEALTHY
ACCESS.
SOMETHING SIMILAR IS GOING ON
IN THE MECHANISM RECEPTOR.
IN A NORMAL REPERTOIRE, IT IS
NOT FULLY UNDERSTOOD IF
GERMINATION OCCURS AND IF IT
IS TO CREATE HEALTHY IMMUNITY.
SO HOW DO WE SOLVE THIS
PROBLEM AND IDENTIFY THE
MECHANISM?
WE DOES THE CLONAL DELETION
OCCUR PHYSICAL LOGICALLY?
SO WE LOOK AT THE ROSA 26
APOPTOSIS IN INDICATOR MICE
WITH FRET AND WITH APOPTOSIS,
YOU CAN SEE THE LOSS OF FRET.
FROM THE DONOR TO THE
RECEPTOR, YOU CAN SEE THE FRET
LOSS IN TWO DISTINCT
POPULATIONS, ONE CORRESPONDING
WHERE THEY HAVE LOST THE
SIGNAL AND THE OTHER ONE
RESPONDING TO THE FRET
NEGATIVELY.
SO WE TO WANT HIGHLIGHT THE
FRET BECAUSE IT CAN ACTIVATE
MUCH EASIER THEN ILC3 AND IS
IMPORTANT IN IMAGING BUT WHAT
I WANT TO SHOW YOU TODAY IS
THAT THE DETECTION OF ACASP3
IS ALMOST IMPOSSIBLE TO SEE.
THERE IS HIGH SENSE SIFT AND
WE WOULD EXPECT THAT THEY
WOULD BE HIGHLY ENRICHED IN
THE LIVE CELL.
SO FIRST WE QUANTITATED THIS
ACROSS PHYSICAL LOGIC
DEVELOPMENT AND I WILL SHOW
YOU THE DATA AND DEVELOPMENTAL
SCHEME AND THEN SHOW THE
RESULT DIRECTLY BELOW THE
RESPECTIVE DEVELOPMENTAL
STAGE.
IS IN THE BONE MARROW, THE
EARLIEST B CELL EXPRESSES ON
THE SURFACE IS THE FRET B CELL
AND WE WERE VERY SURPRISED
THAT A VERY SMALL FRACTION OF
B CELLS THAT WERE IMAGED IS
ONLY SLIDE -- SLIGHTLY INSIDE
THE BONE MARROW.
AS IT FINDS ITS WAY OUT OF THE
BONE MARROW, THERE IS ALSO A
VERY LOW FRACTION OF MATURE
CELLS.
AS THEY ARE RELEASED IN THE
BLOOD CIRCULATION, WE CALL
THESE TYPE 1B CELLS, WE FIND
THERE ARE A QUITE NEGATIVE
NUMBER OF B CELLS AND AS
DEVELOPMENT CONTINUES, YOU CAN
SEE THE LETTER LEVEL OF
TRANSITIONAL CELLS AND
DISTINGUISHED BY FOLLICULAL
PROCESS.
SO WE PERFORMED THIS
EXPERIMENT AND THERE WERE HIGH
LEVELS OF APOPOSIS IN
TRANSITIONAL1 B CELLS.
FOR SELFLY YACHTIVITY AND
APOPTOSIS DURING B CELL
DEVELOPMENT, WE TOOK THE ROSA
26-INDIA MOUSE AND SUBJECTED
IT TO SINGLE CELL SORTING WITH
IMMATURE AND EARLY IMMATURE
CELLS AND THIS ALLOWED THEM TO
REDUCE IN POLY CELLS.
SO WE DON'T HAVE THE TIME TO
GO THROUGH ALL OF THIS BUT WE
SHOW THE AUTO AND POLY
REACTIVITY AND RECOGNIZE THE
ACTIVE PART OF THE B CELLS AND
FOR THE WIDE TIME THAT THEY
WERE GOING OFF, WE RECOGNIZE
THE B CELL DEVELOPMENT.
SO WE FOUND THAT ABOUT
30 PERCENT OF THE B CELLS WERE
SELF-REACTIVE.
AND THEN WE FOUND THAT THEY
SIGNIFICANTLY REDUCE WITHIN
THE BONE MARROW AND THERE ARE
OTHER FACTORS WITHIN THE FRET
COMPARTMENT SO WE WOULD EXPECT
IT TO BE HIGHLY ENRICHED WITH
POLY CELLS, HOWEVER THIS IS
NOT THE CASE AT ALL AND YOU
CAN SEE THERE IS NO POLY
REACTION ACTIVITY.
SO THERE WASN'T A REDUCTION IN
B CELL DEVELOPMENT WITHIN THE
BONE MARROW BUT THIS WAS NOT
YET ASSOCIATED WITH INCREASED
IMAGING OF AUTO AND POLY
CELLS.
SO YOU CAN SEE THE IMAGING WAS
ALMOST STABLE BUT IT WAS
DIFFICULT TO IMAGE IN THE
OTHER DEPARTMENT BECAUSE THIS
IS THE B CELL AT ITS PEAK AND
IT IS POSSIBLE THEY MIGHT GO
THROUGH APOPTIC OR
TRANSITIONAL PHASE AND YOU CAN
SEE 90 PERCENT OF THE B CELLS
ARE DYING FOR DIFFERENT
REASONS.
SO TO SUMMARIZE WHAT I HAVE
SHOWN YOU IN THIS GRAPH, THE
RED SHADED AREA WAS THE B CELL
SELF REACTIVITY.
THE GREEN IS POLY RECEPTOR
CELLS AND BASED ON THESE
FINDINGS, WE THINK THIS
REDUCTION IN THE BONE MARROW
IS NOT DUE TO DELETION BUT
OTHER MECHANISMS SUCH AS
EDITING.
HERE IN THE PERIPHERY IS THIS
MAPPED SECTION OF B CELL BUT
EDITING REMOVES MOST OF THE
SELF-REWRACKIVE B CELLS IN THE
MARROW.
SO BECAUSE OF THAT, WE THINK
THEY ARE NOT SIGNALED TO
SURVIVE.
SO NOW THIS BRINGS UP THE
QUESTION, IF B CELLS ARE AT
ALL IMPORTANT FOR CREATING
AUTO IMMUNITY?
AND THE QUESTION IS YES.
WE KNOW EVEN IN MOUSE MODELS
THAT B CELLS HAVE DIFFICULTY
AND SO WE ASK ROLE OF B CELL
APOPTOSIS IN AUTO IMMUNITY.
THE ORIGINS OF THIS DISEASE IN
THE MOUSE MODEL IS NOT ENGAGED
WELL SO WE USE SPONTANEOUS
AUTO ANTIBODY PRODUCTION THAT
ALLOWS US TO BE EXPRESSED IN
DIFFERENT COMPARTMENTS AND I
AM SUMMARIZING THE RESULTS
HERE.
FIRST WE SCREEN AFTER THESE
MICE AGE WITH THE CONTROL
MICE, WE HAVE HIGH BODIES AND
FOUND ALL THREE GROUPS
DEVELOPED HIGH TITERS OF AUTO
ANTIBODIES AND COULDN'T
MEASURE ANY DIFFERENCE IN THE
THREE GROUPS SO THIS SUGGESTS
THAT THE AUTO IMMUNITY IS
ACTIVATED AFTER DEACTIVATION.
BUT IN THE EARLY FINDINGS,
WHERE WE LOOK AT APOPTOSIS,
YOU CAN SEE THE MICE WHERE IT
WAS ONLY AFTER IT WAS FOUND IN
DISEASE.
AND MOREOVER, THE DISEASE
FACTOR IN THE MB1 MICE, THESE
MICE STARTED DYING ONCE THEY
REACHED A CERTAIN AGE.
SO I AM SHOWING YOU THE
PATHOLOGY AND YOU CAN SEE HERE
THE MB3 MICE HAD VERY
DIFFERENT CONTROLS THAN THE
MB1 MICE SO FROM THIS IT WOULD
SUGGEST THE RESULT IS IT
INHIBITS DISEASE BUT HAS A
VERY SURPRISING MECHANISM.
SO TO SUMMARIZE THIS, WE --
[INDISCERNIBLE]
WHERE APOPTOSIS IS DEVELOPED
AND THESE ARE USEFUL TOOLS TO
FURTHER DETECT THE EVOLUTION
OF THE DISEASE AND ASSOCIATED
THERAPY.
SO TO SUMMARIZE WHAT I HAVE
SHOWN YOU TODAY, WHAT IS THE
ROLE IN THE NEPHRITIS
DEVELOPMENT, THE B CELLS
PROTECT FROM THE DISEASE BUT
WE THINK THIS IS NOT BECAUSE
OF RECOMBINATION OCCURS BUT BY
DIFFERENT MECHANISM, WHEREAS
WE THINK HOST ACTIVATION AND
MEMORY B CELLS IN PLASMA
CELLS, I DIDN'T HAVE THE DATA
TO SHOW YOU THIS TIME BUT WE
THINK THIS IS THE PLACE WHERE
WE GET THE MEMORY B CELL
PRODUCTION AND BOTH ACT
TOGETHER.
AND THESE ARE VERY CENTRALIZED
FOR THE AUTO ANTIBODY
PRODUCTION.
SO A SUMMARY OF MY WORK,
EVERYONE HAS DON'T BOOTED TO
THIS WORK, COLLABORATORS AND
THANK YOU FOR YOUR ATTENTION.
[APPLAUSE]
>> HI, GREAT TALK.
HOW OLD ARE THE MICE WHEN YOU
TRIED FOR THE NEPHRITIS.
>> 40 WEEKS OLD.
>> 14?
>> 40.
>> SO MY SECOND QUESTION, IF
YOU WAIT LONGER FOR THE AID,
YOU WOULD ALSO DEVELOP
NEPHRITIS IN 40 WEEKS?
>> GOOD QUESTION.
WE HAVEN'T SPACED THEM LONGER
BECAUSE 40 WEEKS TAKES A VERY
LONG TIME TO GET THE DATA BUT
IT IS INTERESTING POINT.
IT WOULD BE INTERESTING TO SEE
IF THEY CATCH UP EVENTUALLY
BUT WE HAVEN'T DONE IT SO FAR.
>> OKAY, GREAT TALK, CHRIS.
I WAS WONDERING, WE DID THE
CORNELL EXPERIMENT ON
CONDITIONS AND WONDERING HOW
THIS IS AFFECTED BY INFECTION
WHERE YOU HAVE MASS
INEFFECTION, WOULD IT BE
SIMILAR TO THE ROLE OF
APOPTOSIS?
>> YOU MEAN IN DEVELOPMENT OR
THE --
>> RIGHT, WHEN YOU CLEANED OUT
THE B CELLS TO SEE IF THEY ARE
AUTO REACTIVE, THAT WAS ON THE
BASELINE CONDITION?
>> YES, ON THE BASELINE
CONDITIONS, YES.
>> SO IF YOU TAKE THE MOUSE
WHICH HAS MASSIVE EXPANSION
DUE TO INFECTION, WHAT WOULD
IT SHOW?
>> WELL, WE HAVEN'T LOOKED AT
THE MOUSE WITH LYNN EFFECT --
INFECTION BUT HAVE LOOKED AT
IT AFTER VACCINATION AND --
[INDISCERNIBLE]
SO IT IS NOT A VERY FREQUENT
EVENT EITHER IN VACCINATION OR
INFECTION.
>> BECAUSE I WAS WONDERING
SINCE YOU HAVE THE AUTO BODY
REACTI THINK VE.
>> MY QUESTION IS SIMILAR TO
WHAT HE IS ASKING, GERMA
CENTERS, HAVE YOU EVER TRIED
TO COMPARE THE GERMA CENTERS
WITH APOPTOSIS CENTERS AND
WOULD THEY SHOW A SIMILAR
SIGNATURE.
AND WHERE DO YOU PUT THE T
CELL IN THIS MODEL --
[INDISCERNIBLE]
>> GOOD QUESTION.
WE DIDN'T LOOK AT T CELL, ONLY
AT THE --
[INDISCERNIBLE]
AND TR, THAT IS ALSO A GREAT
QUESTION, YEAH, BUT THEY
DEFINITELY REGULATE SOMETHING
BUT NO.
MAYBE NOT SELF REACTIVE BUT I
AM NOT SURE.
THERE IS A LOT OF WORK YET TO
BE DONE.
>> THANK YOU SO MUCH.
>> SO FOR THE FIRST PART OF
THE TALK, THE ASSUMPTION IS IF
YOU SEE ACTIVATION OF CAT 3,
THE CELLS ARE IN APOPTOSIS AND
I ASK ARE YOU SURE?
AND I ASK THE QUESTION BECAUSE
THE LITERATURE SHOWS THAT WITH
T CELL APOPTOSIS, THEY CLEARLY
SURVIVE SO HAVE YOU CLONED THE
B CELLS OR LOOKED AT THE CELLS
AND IDENTIFIED THEY ARE IN
APOPTOSIS?
>> THEY DEFINITELY ARE AND I
DIDN'T HAVE TIME TO SHOW ALL
THE DATA BUT FOR EXAMPLE, WE
DID THE SIDE STAINING THAT
OVERLAPS.
AND ACTUALLY I WOULD SAY PART
OF THE MAIT SPACE, COME FROM
MAIT AND THE EARLIEST PROCESS
AND WE ALSO COULD DO IT IN VET
VITRO AND IN VIVO AND EVERY
CELL WOULD APPARENTLY DIE.
>> THANK YOU.
[APPLAUSE]
[.
[ WITH OFF MIC ]
>> THANK YOU AND THANKS TO THE
ORGANIZERS AND FOR THE
INVITATION.
JUST TRYING TO GET MY BEARINGS
HERE.
OKAY.
SO TODAY I AM GOING TO BE
TALKING ABOUT SOME WORK THAT
WE HAVE BEEN DOING THAT REALLY
CAME OUT OF OUR EARLIER STUDY
THAT DEMONSTRATED THAT LDK
OCCURS WITH ASSOCIATION AS
WELL AS T CELL SILLALLING IN
CONVENTIONAL DOCKING AND
NONCONVENTIONAL DOCKING.
SO THERE ARE TWO PHASES OF THE
T CELL LIFE WHERE IT MUST BE
LIGATED OR RECOGNIZED TO
RECEIVE A SURVIVOR SIGNAL AND
ALSO NOT TOO STRONG AN
AFFINITY IN ORDER TO SURVIVE
NEGATIVE SELECTION AND OF
COURSE IN THE PERIPHERY, THE
ENGAGEMENT OF THE TCR IS
CRITICAL FOR T CELL
ACTIVATION.
SO WHAT IS REALLY INVOLVED IN
THIS INTERACTION, OKAY, SO WE
KNOW THAT HERE WE HAVE PMHC IN
THE T CELL THAT RECOGNIZES
VIRAL PEPTIDE FRAGMENTS IN THE
CONTEXT OF MHC.
THIS IS A PRIMARY RECOGNITION
INTERACTION AND THEN THE THIS
THEN INITIATES THE WHOLE
SIGNAL AS WE KNOW IT AND THAT
HAS BEEN RESULTING,
PROLIFERATION OF THE T CELL
AND ACTIVATION OF THE EFFECT
TORE FUNCTION SO REALLY GOING
BACK TO WHAT I AM FOCUSING ON
IN THE TALK TODAY IS THIS
INTERACTION BETWEEN T CELL AND
NTK.
SO TODAY THERE HAS BEEN TWO
CHARACTERISTICS OF T CELLS
THAT HAVE WITHIN THOUGHT TO
BE -- BEEN THOUGHT TO BE
IMPOSED WITH THE NTK AND THERE
IS SOME EARLY SUGGESTIONS IN A
PAPER THAT IT IS RESTRICTED
WITH THE INFECTED T CELL AND
WHAT BRINGS LCK INTO THE
COMPLEX OF THE LAB AND
CORRELATION, THAT SIGNAL IS
NOT GENERATED WHEN THE T CELL
IS RECOGNIZED BY THE LIGAND.
AND THEN EARLY LAST YEAR WE
WERE LOOKING AT THE
ASSOCIATION OF POLICY LATER
AND WHAT WE KNOW IS THAT IN
ALMOST ALL CASES, HERE SHOWING
THE CLASS 1 RECOGNITION, THIS
IS A BIRDS EYE VIEW OF SORT OF
INTERACTION WHERE CRYSTAL
STRUCTURES HAVE BEEN
DETERMINED AND WHAT YOU CAN
SEE IS ALMOST INVARIABLY, THE
T CELL IS ON TOP OF THE ALPHA
1 HELIX AND THE T CELL SITS
OVER THE TOP OF THE MHC ALPHA
2 HELIX.
SO WHAT WE SHOWED A COUPLE OF
YEARS AGO IS THE CURRENT
ASSOCIATION WITH LCK WAS
REALLY CRITICAL FOR PREVENTING
THE UNCONVENTIONAL ACTION FROM
GENERATING A SIGNAL SO WE KNOW
THOSE T CELL RECEPTORS COULD
NOT BE DRAWN INTO THE IMMUNE
RESPONSE AND WE SHOWED -- SO I
WILL SHOW YOU THIS BRIEFLY HOW
WE SHOWED THAT.
SO THIS IS JUST THE SYSTEM SET
UP WHERE WOULD YOU HAVE THE
DATA WITH THE FUSION PROTEIN
AND WE GET A CLEAN THREAT
SIGNAL AND WHAT WE DID WAS
LOOK AT SORT OF THE GENERATION
OF SIGNALING ACROSS A RANGE OF
I FINNY SHOWN HERE AS A PLUS
SIGN AND QUICKLY TO SUMMARIZE,
THIS IS NO PEPTIDE ON THE LEFT
AND THE RIGHT AND WHAT WE
FOUND IS WE ARE SIGNALING
THREAT WHEN THE T CELL IS
DOCKED IN A CERTAIN LOCATION
AND NOT A REVERSED LOCATION
AND COULD NOT BRING THE LCK IN
THE PROCESS.
SO THEN IF WE BRING IN THE LCK
WE CAN GET IT IN
UNCONVENTIONAL FORMATS AND
THAT IS WHAT IS SHOWN HERE IN
THE BLACK HERE.
SO IF WE STIMULATE WITH
PEPTIDES AND AS I MENTIONED,
WE CAN GET SIGNALING IN THE
REVERSION AND NOT THE TCI SO
OF COURSE WE HURT THE
CANONICAL T CELERY SEPTEMBER
TORE AND IF WE LEAVE THE T8 IN
THESE CELLS SO THEY CAN STILL
LIVE UP TO THE INTERACTION, WE
SEE ROBUST SIGNALING BY THE
TCR.
SO THESE ARE TWO SORT OF
PARADIGMS THAT ARE TO BE
IMPOSED WITH LCK.
SO I BELIEVE THEY HAVE BEEN
ASSESSED IN BOTH CASES USING
INDIVIDUAL TCRS OR IN VITRO
SYSTEMS WITH A LIMITED NUMBER
OF TCRS SO THAT REALLY RAISES
THE QUESTION FOR US HOW DOES
THIS ASSOCIATION IMPACT ON THE
DOCKING POLE LATER OF INFECTED
T CELL RESPONSES.
SO WE HAVE THIS IMPACT IN THE
MEMBRANE OF ASSOCIATION.
AND QUICKLY TO GO OVER THIS
BECAUSE IT HAS BEEN SHOWN
BEFORE, WHAT WE FOUND IN THE
THYMUS IS A SIGNIFICANT CD4
BUT REAL KNOW NO CHANGE IN CD8
NUMBERS AND JUST AS AN ASIDE,
SORRY, THIS PHENOTYPE IS SHOWN
IN THE PERIPHERY AND CD4 IS NO
DIFFERENT THAN CD8T CELL
NUMBERS AND IN TRUE NAIVE,
THERE IS A VIRTUAL INCREASE IN
THE POPULATION, THIS IS AN
ANTIGEN INCREASE IN POPULATION
OF T CELLS FORMED IN THE
THYMUS AND DEVELOPMENT
COMPLETED IN THE PERIPHERY.
SO WE FOUND THAT WAS DUE TO AN
INCREASE IN IL4 AND PROBABLY
BECAUSE OF INCREASE IN T CELL
NUMBERS AND I WILL COME BACK
TO THIS A LITTLE LATER IN THE
TALK.
SO THE FIRST THING WE WANTED
TO DO WAS ACTUALLY CHALLENGE
THESE THAT ARE REDUCED AND
THIS IS WHAT WE FOUND.
YOU CAN SEE HERE THERE IS
ABSOLUTELY NO DIFFERENCE IN
THE MAG TODAY TO -- MAGNITUDE
OF THE RESPONSE BETWEEN THE
LCK FREE MICE IN CD8T CELL
RECEPTORS AS FAR AS WE COULD
MEASURE AND THERE WERE ONLY
MINOR DIFFERENCES SO LOOKING
AT POSSIBLY A SLIGHT DROP IN
CELLS THAT REDUCE
MITOCHONDRIAL RESPONSE BUT
ROBUST RESTRICTED RESPONSE TO
INFECTION.
AND BY CONTRAST LOOKING AT THE
TCRAVIDITY AND T CELL
RESPONSE, THIS IS THE CYTOKINE
PRODUCTION AND YOU CAN SEE
HERE YOU HAVE THE T CELL ABLE
TO RESPOND IN THE LCK AND THIS
IS OBSERVED FOR A SECOND HERE
WITH IHA211 AND THIS IS JUST
INTRODUCED AFTER STIMULATION.
CYTOKINE PRODUCTION WAS
RELATIVELY NORMAL SO IT COULD
RESPOND GIVEN THE RESPECT TOY,
RESPONDED -- REPERTOIRE,
RESPONDED REASONABLY WELL.
SO THEN WE INFECTED THE MICE
AT DAY 10 AND FOUND THE
TETRAMER WITH THE LCK CELLS
AND DID TARGETED SCRNA AND THE
SEQUENCING SHOWS VERY MINIMAL
CHANGES IN THE EFFECTOR T CELL
RESPONSE.
THE BIGGEST DIFFERENCE YOU CAN
SEE HERE, THAT WAS A
SUBSTANTIAL DROP IN A
SUBPOPULATION IN THE VIRTUAL
MEMORY OF T CELLS AND THAT IS
QUANTITATED OVER HERE SO
ESSENTIALLY THE VIRTUAL MEMORY
T CELLS SHOWN IN THE RED, DARK
GREEN AND LIGHT GREEN AND WHAT
WE FOUND IS THEY WERE MISSING
OR RATHER MIDDLE EAST --
DEFICIENT IN THESE SMALL
MEMORY RESPONSES FOR LCK
RECEPTORS.
OTHERWISE, THERE WAS LITTLE
DIFFERENCE, ONLY A SLIGHT
REDUCTION HERE CLASSIFIED AS A
TH1 SUBSET BUT IN FACT AN
INSPECTION OF THE EXPRESSED
EXODE, WE SAW K2 EXPRESSED IN
THESE MICE SO VERY MINIMAL
ACTIVATION IN THESE T CELL
POPULATION.
SO THEN WE LOOKED AT THE CDA
POSITIVE REPERTOIRE AND HERE
SHOWING THE IMMUNE REPERTOIRE
AND THAT IS SHOWING THE ALPHA
B AND ALPHA J SO THESE WILD
TYPES HAVE BEEN REALLY WELL
CHARACTERIZED BY US AND OTHERS
OVER THE YEARS AND WHAT WE
SHOW IS A DBNP DOMINANCE
PAIRED WITH IL16 AND THESE
CELLS HAVE SELECTIVELY
EXPANDED AFTER INFECTION WITH
AFFINITY.
AND WHAT YOU CAN SEE IF YOU
COMPARE THE WILD TYPES WITH
LC3'S, THE LC3'S REALLY SHOW
THE HIGH RESPONSE.
THESE TALES OF MORE TCI
ALGORITHM TO QUOTE THE
SIMILARITY BETWEEN THE
PHENOTYPES, WHAT IS SHOWN HERE
IS THE CLUSTER REALLY DEFINED
BY THESE SO WE KIND OF PULLED
ON THE HEADS OF THESE AND THEN
THE TAILS HAVE MORE RECEPTIVE
T CELL RECEPTORS AND YOU CAN
SEE THEY ARE REALLY FOCUSED ON
THE CLUSTERS.
SO ON THE LEFT OF THE MORE
CLUSTER TCR, YOU CAN SEE A LOT
MORE OF THE REPERTOIRE IS
DOMINATED BY THE CLUSTERS AND
THAT IS LESS TRUE OF THE WILD
CARD THAT HAS MORE OF THESE
TALES.
SO WE WANTED TO COMPARE IS
THIS A SHIFT THAT IS OCCURRING
UPON SELECTION OF THE
REPERTOIRE OR IS THERE AN
ALTERATION IN THE NAIVE
REPERTOIRE?
SO WE LOOKED AT NAIVE, NP
CELLS FROM THE REPERTOIRE AND
THIS IS TRICKY BECAUSE THEY
ONLY GET AROUND 5-20 CELLS IN
THE INDIVIDUAL MOUSE AND WHAT
YOU CAN SEE IS THIS
PREFERENCE, THIS SORT OF
DOMINANCE IS PREEXISTENT IN
THESE NAIVE REPERTOIRE.
SO HERE WE HAVE THE WILD FIVE
REPERTOIRE WHICH IS MUCH MORE
DIVERSE AND THE NAIVE
REPERTOIRE WHICH IS ALREADY
MORE RESTRICTIVE.
AND YOU CAN SEE THAT SORT OF
STRIKE IN HERE WHERE WE'RE
ALREADY SEEING CLUSTER SORT OF
DOMINATING IN THE NAIVE
REPRESENT TORE -- REPERTOIRE.
SO THIS IS ALSO TRUE, THIS IS
THE PA REPERTOIRE, WE SEE THIS
DOMINANCE IN THE WILD TYPE AND
NOT QUITE THAT SENSE IN THE
NIGH NAIVE REPERTOIRE WHICH IS
MUCH MORE DIVERSE ALTHOUGH THE
DBPA IS MORE PRESENT BUT
EXACERBATED IN THE FREE
REPERTOIRE AND THAT IS THE
CASE HERE WHERE THIS CLUSTER
IS SHOWN HERE AND RATHER
DEVOID OF THESE TALES.
SO AS A CONSEQUENCE OF THESE
WHAT I WANT TO POINT OUT IS WE
SEE A NARROWING OF THE
REPERTOIRE.
SO WE GO FROM A MORE DIVERSE
REPERTOIRE TO A MORE NARROW
REPERTOIRE AND THAT IS TRUE OF
THE NP AND NAIVE REPERTOIRE
AND WE SEE THE PATH FOR
SELECTION IS REDUCED CAPACITY
FOR IMMUNE SELECTION AND THESE
ARE INDIVIDUAL SELECTIONS OF
INDIVIDUAL CLIENTS SO YOU CAN
SEE WHILE WE ARE STILL GETTING
SOME SELECTION, THE CAPACITY
IS LESS DOMINANT IN THE NAIVE
VERSUS THE PA.
SO THIS IS THE COLONIZATION OF
THE NUMBER OF CELLS IN THE
REPERTOIRE WHICH WE MIGHT
EXPECT IS UNCHANGED BECAUSE
THE TOTAL NUMBER IN THE CD8 IS
UNCHANGED BUT THERE APPEARS TO
BE AN AFFINITY ALREADY IN THE
LCK FREE VERSUS THE TCRMHC.
SO THIS IS THE DISTANCE
MEASUREMENT SHOWING THE
DISTANCE FROM THE NAIVE TO THE
IMMUNE REPERTOIRE BECAUSE
WE'RE SEEING SELECTION OF THIS
DOMINANT CLUSTER OF TCR SO
THIS REDUCES TYPICALLY AND
THAT IS TRUE FOR BOTH N & PA
BUT WE CAN SEE WE ARE NOT
SEEING THAT NARROWING OR THAT
SORT OF REDUCTION IN DISTANCE
IN THE LCK BECAUSE IT HAS THIS
LIMITED REPERTOIRE.
SO THIS IS A TERRIBLE SLIDE
BUT I WANTED TO USE IT TO
RAISE THE QUESTION BECAUSE
THIS IS THE QUESTION WE WENT
INTO THESE EXPERIMENTS FOR AND
STILL HAVEN'T QUITE ANSWERED
IT.
AND THE HYPOTHESIS WAS T CELLS
WITH DOCKING ORIENTATION
SHOULD BE ABLE TO PREVENT A
RESPONSE WITH LCK ESPECIALLY
WHAT WE SHOWED IN SREUT VITRO
SO IT MAY HAVE AROUND A
QUARTER OF THE NAIVE
REPERTOIRE AND VERY INFREQUENT
WITH THE IMMUNE REPERTOIRE
BECAUSE THEY CAN'T GET INTO
THE REPERTOIRE.
SO IN THE DNP, I AM NOT
SHOWING THIS HERE BUT WE KNOW
THEY HAVE A CANONICAL
ORIENTATION SO THE DOCKING IS
IRRELEVANT IN THE THY MUS SO
WE SEE THE SIMULATION OF THE T
CELLS IN THE NAIVE REPERTOIRE
BUT IN THE IMMUNE REPERTOIRE,
WE ARE NOT SEEING RECORRUPT IN
THE TCR OR THE NAIVE LCK AND
IT IS POSSIBLE THAT THEY ARE
ACTIVATING THE IMMUNE PROCESS
AND WHAT WE'RE DOING IS
EXPRESSING THESE DOCKERS IN
THE CONTEXT OF A LCK
BACKGROUND IN ORDER TO ANSWER
THE QUESTION WHERE THE
ASSOCIATION WILL OCCUR THAT
CAN ALLOW THESE
UNCONVENTIONALT TTCRS TO BE
RECRUITED IN THE IMMUNE
RESPONSE.
AND SO THE ONE FINAL THING I
WANTED TO MENTION IS IN THIS
STIMULATION WHEN WE ARE
INFECTING THE T CELLS WITH
NEUROPEPTIDES, THEY CLUSTER
AND THAT IS SHOWN HERE FOR THE
TWO DIFFERENT CLASS 2 PEPTIDES
AND THESE WERE.
>> Genny: WIN CLASS 2
RESPONSES, LOW FREQUENT SEE OF
COURSE BUT ENRICHED WITH CLASS
2TETRAMINE, YOU CAN SEE THIS
IS GENUINE CLASS 2TETRAMINE
STAINING, IT IS IMPORTANT TO
NOTE THAT THEY CLEARLY
EXPANDED AFTER INFECTION, WE
REEXPRESSED THEM AND THEY CAN
BE IMAGED WITH THE HELP OF PR
IYANKA CHAURASIA AND OTHERS.
BUT THEY DID HAVE A HIGH
AFFINITY.
INTERESTINGLY, TWO TCRS FROM
TWO DIFFERENT MICE HAD VERY
SIMILAR CHARACTERISTICS FOR
COR5 AND CDR -- TCR2 AND WE
ARE NOT SURE THE
CHARACTERISTICS THAT ALLOW
THAT.
THE T CELLS ARE GENUINE TD 8T
CELLS, THEY EXPRESS HIGHS AND
LOWS OF TH LCK AND ALSO
ENZYMES WHICH I AM NOT SHOWING
HERE AND APPEARS WHEN WE LOOK
AT THE WILD TYPE REPERTOIRE,
WE THOUGHT THIS WAS A
REDIRECTION FROM THE CD4
LINEAGE TO THE CD8 LINEAGE
RATHER THAN AN EXPRESSION OF A
REALLY ODD LINEAGE.
BUT YOU CAN SEE IN THE
BACKGROUND WHEN EXPRESSED ON A
WILD TYPE BACKGROUND, IT
EXPRESSED T CELLS BUT ON THE
LCK FREE BACKGROUND, THEY ARE
ABLE TO FAVOR A LINEATION FOR
MHCH RESTRICTED.
SO THIS RESULTS IN A LOSS OF T
CELLS EXPRESSING MODERATE MHC
SPECIFIC CD4T CELLS, RESULTS
IN LOSS OF T CELLS EXPRESSING
LOW AND MODERATE AFFINITY OF
TCRS AND IT REDUCES THE
CAPACITY FOR IMMUNE SELECTION
AND SELECTIVE RECRUITMENT OF
HIGH AFFINITY T CELLS AND
ALLOWS FOR THE LINEAGE
REDIRECTION OF MHCII
RESTRICTED CELLS TO CD8T
CELLS.
SO WE KNOW FROM BUSH'S LOVELY
PAPER THAT IT IS THIS TALE OF
T CELL RECEPTORS THAT ARE NOT
CRITICAL FOR THE VIRAL
INFECTION OR ANT GENT BUT ARE
CRITICAL FOR RECOGNITION OF
VIRAL EXPRESSION.
SO THIS IS JUST AN OBSERVATION
TO SHOW THE MODEL OF T CELL
EXPRESSION, WHETHER CLASS 1 OR
CLASS 2 AFTER POSITIVE
SELECTION WHEN THEY RECEIVE A
SIGNAL, BOTH WILL TRANSIENTLY
POPULATE TO CD8 AND IN THE
ABSENCE OF CD8, IT WILL BECOME
CD4 POSITIVE CELLS AND
OTHERWISE IF THE SIGNAL IS
INDUCED OR INTERRUPTED, IT
WILL BECOME CD8 POSITIVE
CELLS.
SO WHAT I SHOULD HAVE
MENTIONED IS WHERE WE KNOW AT
CD4, IT MAKES SENSE DURING
POSITIVE LINEATION THE CD8 HAS
INTERMEDIATE T CELLS AND ALSO
LCK BINDS CD4 WITH HIGHER
AFFINITY THAN CD8 AND I THINK
IT ALLOWS T CELLS THAT
OTHERWISE SHOULD HAVE GOTTEN A
STRONGER SIGNAL BE DIRECTED
DOWN THE PATHWAY TO CD8
POSITIVE CELLS.
SO THANK THE PEOPLE INVOLVED
IN THIS WORK, THOSE INVOLVED
WITH THE DOCKING STUDY, THE
STUDENTS IN THE LAB SHOWN HERE
HAVE DONE MUCH OF THIS WORK
AND THIS HAS BEEN A GREAT
COLLABORATION WITH STRUCTURAL
BIOLOGIST JAMIE ROSSJOHN,
THANK YOU AND I AM HAPPY TO
TAKE ANY QUESTIONS.
>> SO A VERY NICE TALK AND I
THINK I HAVE SIGNAL FOR THIS
SO TO COME OUT OF THIS, THIS
FITS SUM PHAEUGSZ FROM DEVIN
SEVERAL YEARS AGO IN WILD TYPE
MOUSE --
[INDISCERNIBLE]
SO I THINK THIS IS -- THE IDEA
IS FOR SOME REASON THIS ECO IS
NOT AS STRONG AS IT SHOULD BE
AND CAN BE SELF --
[INDISCERNIBLE]
AND ALSO SEVERAL YEARS CD4
EXPRESSED IN CD8 AND YOU CAN
SEE THIS VERY WELL.
AND THE OTHER THING I WOULD
LIKE TO SAY IS IT APPEARS TO
ME THAT THIS IS CONSISTENT
WITH THE MODEL BECAUSE IN A
SENSE --
[INDISCERNIBLE]
THERE IS NO CD4 AND CANNOT
KEEP THE BARRIER FOR THIS
IMAGE, JUST MY 2 CENTS.
>> THANK YOU.
>> I WOULD NOT VARY THAT FAR.
>> REALLY INTERESTING, THANK
YOU.
I AM CURIOUS WHETHER IN THE
LCK3 MICE DOES IT STOP THE
INTERPRETATION IN LCK45 AND
WHAT DOES IT DO TO THEM?
>> NOT THAT I AM AWARE.
IIT ONLY AFFECTS THE
ASSOCIATION OF CD4 AND CD8.
I WILL HAVE A LOOK BUT I AM
PRETTY SURE IT DOESN'T.
>> SO WOULD YOU -- SO THE WAY
YOU -- AS I UNDERSTAND THE
STORY, YOU HAVE THE FOUR ROWS
NOW YOU THINK THE RESERIOUS
ORIENTED T CELL RECEPTORS ARE
ACTUALLY SELECTED BY COLLECTOR
ORIENTATION.
AND IF YOU THINK THE PRELCK
WOULD ALLOW SIGNALING BY THOSE
REVERSE OR KWREPBD TCR WOULD
WE NOT HAVE EXPECTED TO SEE
MORE OF THEM WITH THE PRELCK?
I DON'T KNOW IF I MADE MYSELF
CLEAR BUT --
>> YEAH, I THINK SO.
SO YOU ARE SUGGESTING NOW THAT
WE HAVE LIB RATED LCK, THAT
MAY BE THEY CAN BE SHOWN IN
THE REVERSE ORIENTATION.
>> YES, AND MAYBE MANY OTHERS
IN THE SELECT REVERSE
ORIENTATION --
>> YES, I THINK IT MIGHT BE
TWO DIFFERENT LCKS.
I THINK MAYBE IS THERE EVEN
THE PEPTIDES THAT CAN SELECT
THESE TCRS WHEN THEY ARE IN A
REVERSE ORIENTATION?
>> RIGHT.
>> I DON'T KNOW THAT THAT IS
EVEN, YOU KNOW, WHY WOULD WE
NECESSARILY EXPECT THAT?
AND SO I WASN'T SURPRISED TO
SEE THAT THEY ARE EQUIVALENTLY
REPRESENTED.
SO JUST FOR CLARITY, WE KNOW
OR WE THINK THEY ARE SELECTED
IN AN ECONOMIC ORIENTATION
BECAUSE WE MADE A MUTATION
ACROSS A MOUSE THAT HAD A A89E
AND THAT IS UNION UNIQUELY
IMPORTANT FOR THE REVERSE
DOCKING RECOGNITION AND NOT A
CANONICAL RECOGNITION AND WE
SAW NO IMPACT ON THOSE TO COME
OUT.
SO I THINK THOSE ITSELF ARE AN
ACCIDENT BUT AN ACCIDENT THAT
IS IDEOLOGICAL.
I MEAN WE FOUND THEM IN MICE
AND NOW IN HUMANS SO THE
HYPOTHESIS IS THIS
CENTRALIZATION HAS A WAY OF
STOPPING IT FROM GETTING INTO
THE MICE BECAUSE THEY
SHOULDN'T BE THERE.
>> OKAY, GREAT TALK.
A RANDOM THOUGHT.
I AM WONDERING WHETHER IN
SITUATIONS WHERE CORECEPTOR
AND LCK CAN'T BE UTILIZED,
WHETHER AS ANOTHER CONSEQUENCE
IS THE LACK OF MEDIATED
TERMINATION OF THE SIGNAL FOR
THE L.A. X WOULD BE RESISTANT
AND THAT WOULD MEAN THE SIGNAL
WOULD GO ON A LITTLE LONGER
AND WHETHER THAT WOULD BE THE
CASE AND IF IT HAS ANY
DOWNSTREAM CONSEQUENCES?
>> MAYBE.
SO YOU ARE TALKING ABOUT JUST
IN RECOGNITION OR ACTIVATION
THAT OCCURS INDEPENDENT?
>> OBVIOUSLY THERE ARE EARLIER
EVENTS BUT THERE ARE LATER
EVENTS WHERE THEY ARE SO THERE
IS A TEMPORAL TIME PERIOD.
IF THOSE SIGNALS OF INHIBITOR
RECEPTOR ARE INDEPENDENT?
[INDISCERNIBLE]
>> LAG 3 FUNCTIONS BY KNOCKING
OFF LCK AND THE INDEPENDENCE
OF LCK WON'T BE ABLE TO
TERMINATE -- .
>> I MEAN IT IS ENTIRELY
POSSIBLE.
BUT LIKE THE CORECEPTORS, WHAT
IS HAPPENING WITH THEM AND IS
THIS WHY WE'RE FINDING OUGHT
THESE WEIRD INNATE PCR'S THAT
ARE CLUSTERED IN STRANGE WAYS
BECAUSE THEY CAN DO WHATEVER
THEY LIKE SO I AM LOOKING AT
THAT.
>> I AM BEING TOLD WE HAVE TO
MOVE ON TO GET LUNCH SO I WILL
DISCUSS THAT DURING LUNCH.
THANK YOU.
SO THE FINAL FIGURE AND
PRELUNCH SPEAKER IS LARRY
TALKING ABOUT T CELLS.
>> I NOTICED ON YOUR ID THERE
IS A PICTURE FROM MY LONG TIME
COLLEAGUE LAKSHMI AND THAT
WILL BE THE EXTENT THAT I
SHOW.
SO WE HAVE BEEN RUNNING THIS
FOR YEARS AND TALKING ABOUT
THE PERSPECTIVE, IS PHOSPORUS
SERVE AS A BINDING FOR SH2
PROTEINS AND MULTIPLE BREATHE
TONES -- PROTEINS ARE THUS
FORMED SO HERE YOU HAVE THE
WORK AND WE'RE GOING TO FOCUS
ON THIS COMPLEX BUT THERE ARE
LITERALLY HUNDREDS OF THEM AND
THIS IS HOW THE CRITICAL
EVENTS OF TLC ACTIVATION IS
INUSE ITD, IT IS RECRUITEDDED
BUT DOESN'T COME ALONE TO THE
LCG RECEPTOR SO THAT COMPLEX
IS RESPONSIBLE FOR CALCIUM AND
IB3 GENERATION AND BAD
PRODUCTION, ACTIVATION OF GLS
AND MANY EVENTS FOLLOW WHICH
WE WON'T GO INTO BECAUSE WE
KNOW THEM.
SO BACK TO THIS COMPLEX, AS
LONG AS WE HAVE BEEN DOING I
AM IMAGING, 20, 25 YEARS AGO,
WE START TO TAKE APART THIS
COMPLEX AND SHOW THIS
PRECIPITATION STUDY THAT IT
EXISTED.
SO WE STARTED BY PURIFYING IT
AND MAKE PEPTIDES CONTAINING
L.A. T AND ABLE TO SHOW ALL
THESE INTERACTIONS OCCUR AT
THAT LEVEL AND WE COULD
MEASURE THE AFFIN TEASE USING
ISOTHERMALCALOMETRY AND WILL
SHOW THIS SH2 MEDIATOR ACTION
HAS AN AFFINITY AS WELL AS
THIS SH2 MEDIATOR AND THESE
SH3 MEDIATORS ARE A LITTLE
DIFFERENT, DOMAINS ACTING WITH
EVERYBODY ENRICHED FOLINE
CONVENIENCES AND THIS IS J THE
JOHN HOPKINS CHART WHERE THEY
GENERATED THE SEQUENCE IN THE
LAB AND THIS IS USING A CELL
LEAN RICH PEPTIDE WITH THE SH3
PROTEIN OF GASSES, IT IS
ACTUALLY THE HIGHEST AFFINITY
OF THESE INTERACTIONS AND THE
SHOCK OF THIS IS THIS
INTERACTION THAT HAD BEEN
PREDICTED BY THE CORPORATE
SOLICITATION IS THE
INTERACTION OF PLC, SH3 DOMAIN
WITH ANOTHER POLICILY OR PLC
DOMAIN.
HOW CAN THAT BE?
SO WE CAME BACK TO THIS IN THE
LAB AND FIGURED THESE LITTLE
REGIONS THEMSELVES ARE NOT THE
WHOLE STORY AND YOU NEED THEM
IN THE CONTEXT OF LARGER, PURE
PROTEINS IF YOU WILL SO AGAIN
WE'RE GOING TO WORK WITH A
LITTLE DOUBLEYQUAT COMPLEX,
YOU HAVE THE L.A. T CONSTRUCT,
THE GADS CONSTRUCT AND PRETTY
LARGE CHART OF SLP76 AND ON
THREE OF THESE YOU WILL NOTICE
THEY HAVE UNIQUE FLUORESCENT
TAGS ATTACHED.
SO THE 383 ON LAT AND SH3 ON
GADS WITH 64 OF AND THERE WE
COLLABORATED WITH PETER SHOOK
WHO IS THIS BIOENGINEERING
WIZARD HERE ON CAMPUS.
SO WITH LIMITED TIME, I AM
GOING TO TRY TO ZIP THROUGH A
SLIDE HERE THAT IS SORT OF
COMPLICATED AND THIS REQUIRES
A LITTLE BALANCE, A LITTLE
COORDINATION.
SO YOU SEE THIS AFFINITY IS PL
C1 AND THIS GRAPH REPRESENTS A
RUNNING MIX WITH DIFFERENT
SPECTOMETRY SO THE GADS AND
SLP ARE ONE-ON-ONE AND THEN
FOR SOME REASON, THE PLC AND
FOR REASONS I WILL EXPLAIN IN
A SECOND WE HAVE INCREASING
CONCENTRATIONS OF PLC WITH A
ONE TO ONE CONCENTRATION WITH
THE OTHER.
AND THE REASON FOR THAT IS IF
YOU LOOK AT THE GREEN LINES,
THAT IS ONE TO ONE TO ONE, ALL
OF THEM AND YOU SEE THIS PEAK
HERE AND THEN YOU SEE THIS
PEAK HERE WHICH HAS TO BE THE
TETRAMER, THE COMPLEX, BUT
PRESUMABLY THE COMPLEX IS
FALLING APART AND IT FALLS
APART AS IT GOES THROUGH THE
UNITED CURRENT AND AS IT
LEAVES THE STATION, IT CAN'T
GET BACK ON.
SO YOU SEE THE SEPARATION
THERE.
SO WE FIGURE THERE IS A LOW
AFFINITY FOR THE PLC, CAN WE
JUST TITER THAT UP, RAISE THE
RATIO OF PLC AND THAT IS WHAT
YOU SEE HAPPENING HERE.
SO AS IT TITERS UP, YOU HAVE
THIS MOVEMENT HERE, SAME
MECHANISM, THE TETRAMER STAYS
AROUND LONGER AND THE PLC
FALLS OFF.
SO THERE IS INCREASING PLC BUT
AS YOU WILL NOTICE, IT IS
CONSUMED AS WE GO TO THE BLUE
AND THE RED.
SO THIS MIGRATION REFLECTS
THIS LOW AFFINITY INTERACTION
OF PLC BUT THIS IS THE
PREDICTED MOLECULAR WEIGHT OF
THE MASS OF THE TETRAMER SO WE
HAVE BEEN ABLE TO PROVE FOES
FOUR MOLECULES COME TOGETHER
AND WE CAN DEMONSTRATE THAT
HERE.
NOW IS THERE ANY CONSEQUENCE
IN TERMS OF FUNCTION -- WELL
HERE IS THE PICTURE AGAIN.
WE HAVE THESE FOUR
INTERACTIONS, FOUR INTERACTING
SITES, SH2 WITH TYROSINE AND
THEN CHOLINE ENRICHED HERE
WITH THIS BEING THE WEAK LINK.
DOES IT HAVE FUNCTIONAL
IMPACT?
STILL WORKING WITH FOLATE
PROTEINS TURNING TO THE WORK
OF JUNE WADDI ITSTILL IN THE
LAB AND THE SL LP AND A LARGE
CHUNK OF SLP AND FULL LENGTH
GADS AND WHAT WE WANTED TO SEE
IS IF THIS COULD BE LINKED TO
THE MOLECULES.
SO WE ATTACH UNILAMELLAR
VESICLES, WE HAVE PROLINE AND
THAT ALLOWS IT TO DOCK ON TO
THE MEMBRANES AND THEN WE WILL
SEE IF THESE OTHER GUYS CAN
FORM A COMPLEX AND WHAT
HAPPENS THEN.
SO NEED TO TELL YOU TWO OTHER
THINGS.
THOSE IN THE AUDIENCE OF MY
AGE WILL REMEMBER THAT YOU
USED TO BE ABLE TO BUY
TRITIALE-- TRITIATED LIPIDS IN
CHEMICAL CONCENTRATIONS BUT
THE COMPANIES DON'T MAKE THEM
ANYMORE SO THERE IS THIS
COMPOUND WHERE YOU GENERATE A
VERY RED FLUORESCENT WHICH YOU
CAN MEASURE.
IN THE PRESENCE OF IP3 WHICH
IS THE PRODUCT WE'RE GOING TO
BE MEASURING, THE IT IS BOUND
TO THE ITRIUM AND NOW YOU LOSE
THE RED SIGNAL SO YOU SEE A
NEGATIVE PEAK.
THIS IS THE ABSORB BENZ OF THE
RED AND IT MEASURES A NEGATIVE
IP3.
HERE IS AN INITIAL EXPERIMENT,
I NEED TO ADD ONE ONE POINT --
WELL, NOT YET.
WE HAVE THE LAST WHICH IS FOSS
NORTHERN-- PHOSPHO LAT WILL
BIND PLC IN THIS EXPERIMENT
BUT NOT THE NONPHOSPHO LAT AND
IF IT BINDS YOU CAN MEASURE A
CLEAVE VANS SO THAT IS
BRINGING PLC TO THE MEMBRANE
ARTIFICIALLY.
I NEED TO TELL YOU THAT YOU
NEED PLC TO PHOSPHORYLATE IT
AND IN OUR LAB WE WILL INTRIPS
INTRINSICALLY PHOSPHORYLATE IT
AND WITH THAT, WE NEED TO DO
THE BIG EXPERIMENT.
OOPS, GIVING AWAY THE PUNCH
LINE.
SO ON THE LEFT HERE, NOW WE
CAN JUST ADD PLC BY ITSELF AND
YOU GET A LITTLE BACKGROUND
ACTIVITY BUT IF YOU ADD IT TO
THE PHOSPHORYLATED LAT, YOU
GET A LOT OF BINDING AND YOU
CAN ADD IT TO THE GAD.
SO YOU GET A LITTLE ACTIVITY,
BOUNCING OFF THE MEMBRANES AND
THERE IS MORE ACTIVITY IN THE
FOSS FOR LATE FIELD.
SO HERE IS WHAT WE HAVE SHOWN
YOU BEFORE, ATTACH PLC TO THE
LAT AND DO BETTER AND THIS IS
THE INTERESTING RESULT.
THE ADDITION OF GAD AND L.A. T
GIVES YOU MORE OBJECTIVITY.
SO HOW DOES THIS WORK?
THIS IS FROM THE LAB AT PLC
WHICH IS AN ACTIVE ENZYME WITH
PHOSPHORYLATE AND THEY COVER
UP THE ACTIVE SITE WHICH IS
BLOCKED AND PHOSPHORYLATION OF
PLC WOULD BE OCCURRING HERE
AND THAT HAS BEEN SHOWN TO
INDUCE AN OPENING OF THIS
COMPLEX AND MAKE IT MORE
ACCESSIBLE TO THE SUBSTRATE
AND WE WOULD PROPOSE THE
FORMATION OF THE BINDING OF
THE SH2 AND SH3'S IN THIS
COMPLEX WILL KICK IT OUT
FURTHER AND INCREASE THE
ACTIVATION STILL FURTHER.
OKAY, SO SOME OF THE MAPPING
OF THESE -- HERE AGAIN IS THIS
IDEA THAT WE HAVE THIS
INTERACTION HERE WHICH IS VERY
WEAK.
WE CAN DEMONSTRATE IT IN THE
FORMATION OF TETRAMER BUT IT
WILL HAVE AN OPEN AND CLOSED
REACTION AS WELL AND PERHAPS
WHEN THAT HAPPENS, THE WHOLE
COMPLEX FALLS APART.
SO LONG AGO IN THE EARLY DAYS
OF SH3 STUDIES WHEN PEOPLE
DON'T KNOW WHAT SA3 DID, THIS
GROUP DID A FACE DISPLAY
ANALYSIS TO FIND THE OPTIMAL
SH3 CONSENSUS -- BINDING SITE
CONSENSUS SEQUENCE AND THAT IS
WHAT IS SHOWN HERE IN THE
MIDDLE LINE.
HERE IS THE PAROLE LEAN RICH
REGION WHICH WE PRESUME IS
ACTING WITH THE SL & HERE IS A
BETTER BINDING DOMAIN AND ONE
CAN ACTUALLY KILL THESE CYTES
WHICH HE DID.
IT IS NOT AN ALL OR NONE
ALTHOUGH WHEN YOU LOOK AT THIS
DEAD ONE, YOU CAN SEE THE
DIFFERENCE, IT IS ABOUT
20 PERCENT BETTER SO WHAT ARE
THE CONSEQUENCES THERE WHEN
THEY ADDED AND MADE THE
COMPLEX OF PROTEINS COMPARING
THE WILD CYTE AND THE ACTIVE
CYTE AND THIS IS WHAT I HAVE
JUST SHOWN YOU, A LITTLE PLC
ALONE GIVES YOU REACTIVITY,
BRING IT TO THE L.A. T, IT
GETS BETTER AND THE GADS,
BECOMES ACTIVE STILL.
WHEN YOU ADD THE SEQUENCE, YOU
DO BETTER STILL.
THE FACT YOU SEE SOMETHING
WITH THE DEAD THING IN, YOU
HAVE TO THINK OF ALL THESE
CYTES OF OPENING AND CLOSING,
THAT IS HOW THE BINDING WORKS
BUT IN A SITUATION WHERE YOU
HAVE THE ACTIVE BINDING WHERE
THE TETRAMER IS MORE STABLE,
THE REST OF THE COMPLEX IS
FORMED AND YOU WILL GET A
LITTLE ACTIVITY.
NOW WE'RE GOING TO SWITCH TO
OLD FASHIONED DEVELOPMENT AND
IMMUNOLOGY.
DOES THIS MUTATION SO NOW WE
HAVE THE T CELL SPECIFIC
CONDITIONAL SLP76 AND PUT IN
THE KI MUTATION AND ROSA YFP
SO THE BOTTOM LINE, YOU
INTRODUCE THIS CONSTRUCT, YOU
KNOCK THIS SOUTH AND ARE LEFT
WITH JUST THE MUTATED HIGH
AFFINITY SITE AND YOU WILL
ALSO HAVE RED CELLS.
GREEN CELLS.
THERE YOU GO.
SO THAT'S WHAT HAPPENED.
AND HERE IS SOME OF THE --
JUST GOING TO SHOW A LITTLE
BIT OF THE STUDY SO YOU HAVE
THESE VERY COMPLEX GENOTYPES
AND MARKER ANALYSIS BUT THE
BOTTOM LINE IS HERE IS THE
WILD TYPE MOUSE AND HERE IS
THE SLP TYPE MOUSE AND YOU
TAKE THE T CELLS FROM THAT AND
THEY ARE RECEPTOR POSITIVES IN
BOTH CASES, YOU MIX THESE ON
TO PEPTIDE LOADED DEPLETED T
CELLS AND STIMULATE WITH WHAT
YOU ALREADY HEARD ABOUT IN
EARLIER TALKS, THE LT1 SERIES,
THESE WOULD BE THE WILD TYPE
WITH DECREASING AFFIN TEASE
AND GIVING TO INCREASING
RESPONSE AND HERE ARE THE
RESULT.
YOU CAN'T SEE A DIFFERENCE
WHEN YOU USE THE WILD TYPE ODA
BUT AS YOU SORT FTITER DOWN
YOUR AFFINITY PEPTIDES, Q T4
AND Q4H7 YOU SEE THOSE HAVE A
SLIGHTLY BETTER RESPONSE TO
THE TITER AND EVEN IF YOU GET
THE SATURATION, YOU ARE
GETTING THIS SHIFT SO YOU ARE
GETTING THIS ENHANCED RESPONSE
BY INCREASING THE AFFINITY OF
THAT PEPTIDE AND PRESUMABLY
THE KINETICS OF THE DURATION
OF THAT PERSISTENCE OF THE AT
THE TIME -- TETRAMER.
SOMEBODY PAUSE IN --
[ PAUSE IN CAPTIONING ]
>> OKAY, LET'S EVERYONE, PLEASE
TAKE YOUR SEATS.
WE'RE GOING TO GET STARTED WITH
THIS AFTERNOON'S SESSION.
SO WE STAY ON TIME.
I THINK WE ONLY HAVE THE ROOM UL
5:00.
SO, WE DON'T WANT TO GET THROWN
OUT.
WE HAVE TO WAIT FOR AL TO SIT D.
OKAY, SO WELCOME TO SESSION 6,
WE'RE GOING TO TALK ABOUT IMMUNE
CELL FUNCTION AND OUR FIRST SPER
IS PAMELA SCHWARTZBERG -- I THIK
MAYBE SOME PEOPLE KNOW YOU?
[ LAUGHS ]
IF THERE IS ANYONE IN HERE WHO
DOESN'T KNOW PAM, THEY NEED TO
LEAVE RIGHT NOW.
AND PAM IS GOING TO TELL US ABOT
HER WORK ON PI3 KINASE AND
AMBULANCING T-CELL ACTIVATION AD
EXHAUSTION.
>> THANK YOU VERY MUCH.
AND I WANT TO THANK THE ORGANIZS
FOR AN INCREDIBLE MEETING.
IT'S A MEETING WHERE YOU JUST FL
GREAT ABOUT BEING AT NIH, WHICH
PROBABLY OVER THE WEEKEND, WE WT
FEEL SO GREAT ABOUT, BUT THAT'S
OKAY.
TODAY I THOUGHT I'D TELL YOU A
SHORT STORY ABOUT PI3 KINASE ONS
EFFECT ON EXHAUSTION.
SO, T-CELL EXHAUSTION, WHICH
SURPRISED ME WE HAVEN'T DISCUSSE
YET, IS AN ADAPTIVE STATE OF
HYPERRESPONSIVENESS TO -- WHICH
RESULTS IN RESPONSE TO CHRONIC R
PERSISTENT ANTIGEN STIMULATION.
WE TEND TO THINK ABOUT THIS IN
TERMS OF LOSS OF CONTROL AGAINST
VIRUSES AND TUMORS.
IT'S IMPORTANT TO REALIZE THAT
EXHAUSTION IS AN ADAPTIVE PHENOE
AND HELPS PREVENT IMMUNOPATHOLOY
UNDER CONDITIONS OF CONTINUAL
STIMULATION.
AND SO AS WE ASK THE QUESTION OF
HOW CAN WE REINVIGORATE EFFECTOR
T-CELL FUNCTION UNDER CONDITIONF
EXHAUSTION, WE HAVE TO KEEP THOE
ISSUES OF BALANCE IN MIND.
AND SO WE GOT INTO THIS THROUGHR
STUDIES OF A PRIMARY
IMMUNODEFICIENCY ACTIVATED PI3
KINASE DELTA SYNDROME.
THIS IS AN IMMUNODEFICIENCY
CHARACTERED RIGHTS BY CMD,
RECURRENT RESPIRATORY AND GUT
INFECTIONS AND ALSO IMMUNE DIGS
REGULATION.
SO THE PATIENTS HAVE LYMPHOPENIA
YET AND ALTERED T AND C CELL
POPULATIONS AND A FEATURE OF THE
MUCOSAL LYMPHOID NODULES.
SEQUENCING OF THE GENOMES OF THE
EXOMES OF THESE PATIENTS REVEALD
THAT THEY HARBORED HETEROZYGOTE
ACTIVATING MUTATIONS AFFECTING 0
DELTA, THE SUBUNIT OF A
HEMATOPOIETIC SPECIFIC CLASS 1A3
KINASE DELTA.
AND SO PI3 KINASE IS THE CLASS ,
ACTIVATED DOWNSTREAM OF MANY
SIGNALING.
THEY WILL PHOSPHORYLATE TO GENEE
PIP 3 AND THAT WILL CREATE --
RESULT IN THE RECRUITMENT OF
PROTEINS THAT HAVE HOMOLOGY OR
OTHER PIP 3 BINDING DOMAINS WHEE
THEY GET ACTIVATED.
AND THE BEST KNOWN ARE THE AKT
KINASES.
AKT WILL PHOSPHORYLATE A RANGE F
DOWNSTREAM EFFECTORS LEADING TO
ACTIVATION OF mTOR REGULATION OF
PROTEINS INVOLVED IN CELL SURVIL
AS WELL AS PHOSPHORYLATION OF
TRANSCRIPTION FACTORS SUCH AS TE
FOXO FACTORS AND PHOSPHORYLATIOF
THEM LEADS TO SEQUESTRATION OUTE
OF THE NUCLEUS AND IN ACTIVATIO.
SO TOGETHER, THOSE EFFECTS CONSE
TO REGULATE METABOLISM, GROWTH,
PROLIFERATION, SURVIVAL, MIGRATN
AND DIFFERENTIATION WHICH WILL E
KEY FOR T-CELL FUNCTION.
THE QUESTION WE HAD IS WHY DO TE
PATIENTS WITH ACTIVATED PI3 KINE
SYNDROME, WHY DON'T THEY CLEAR
CERTAIN VIRAL INFECTIONS?
TOW TRY TO ADDRESS THESE QUESTIS
WE GENERATED MICE OR KNOCKED INE
MOST COMMON MUTATION ACTIVATING
MUTATION INTO PI3 KINASE DELTA D
THEY RECAPITULATED MANY FEATUREF
THE PATIENTS.
SO THEY WERE LYMPHOPENIC IN THE
BLOOD.
THEY HAD LIMP KNOP THEE AND
INCREASED ACTIVATED T-CELLS AND
THEY ALSO GOT THOSE MUCOSAL
FOLLICLE.
THEY NICELY MODELED THE PATIENTF
WE STIMULATED THEM WITH ANTI-CD3
AND CD TREAT, WHAT WE SAW IS THT
THESE CELLS UNDERWENT MASSIVE
APOPTOSIS.
AND WE LINKED THIS TO INCREASEDD
HIGH LEVELS OF EXPRESSION OF FAS
LIGAND STIMULATING THESE CELLS O
UNDERGO APOPTOSIS THROUGH FAST
MEDIATED PATHWAYS.
WHEN WE LOOKED AT THE SURVIVING
CELLS, WE REALIZED THAT THESE CS
HAD AN ACCELERATED EFFECTOR CELL
PROGRAM AND SO THEY PRODUCED EAY
AND INCREASED INTERFERON GAMMA,
TNF -- AND ELEVATED mTOR ONE AND
MYC AND DRIVEN SIGNATURES ALONG
WITH ALTERED TRANSCRIPTION AND
EPIGENETIC LANDSCAPES.
WHAT THESE CELLS LOOKED LIKE IS
THAT THEY HAD A PUSH FROM A NAIE
PHENOTYPE TO A TERMINAL EFFECTOR
PROGRAM AND THIS IS AT THE EXPEE
OF THE FORMATION OF CENTRAL MEM.
AND WE CAN SEE THAT HERE IF YOU
LOOK IN FLOW PLOT POST INFECTIO,
THIS IS TWO WEEKS POST INFECTION
WITH ACUTE LCMV, WE CAN SEE THAT
WILDTYPE CELLS A GRAND ZYME P HH
POPULATION REPRESENTING THE
EFFECTOR CELLS AS WELL AS THIS 1
HIGH POPULATION THAT REPRESENTSE
PRECURSORS TO CENTRAL MEMORY.
IN THE PI3 KINASE MUTANT CELLS,U
CAN SEE A LARGE AMOUNT OF THESE
EFFECTOR CELLS BUT YOU LOSE
EXPRESSION OF THE TCF ONE POSITE
POPULATION.
AND THIS IS REALLY OF INTEREST O
US BECAUSE CHUAN WU HAD DONE INR
LAB, ALONG WITH SEVERAL OTHER L,
A NUMBER OF YEARS AGO, THAT SHON
THAT TCF7, WHICH ENCODES TCF1,
MARKED A SMALL POPULATION OF CES
THAT YOU COULD FIND IN CHRONIC
INFECTION AND CANCER.
AND THAT -- EVEN THOUGH A SMALL
NUMBER OF CELLS EXPRESSED TCF7,
WHEN YOU INFECTED MICE, WHAT YOU
SAW IS OVER TIME, TCF1 DEFICIENT
CELLS -- SORRY.
WOULD LOSE THEIR ANTIGEN-SPECIFC
CELLS AND THEN BE UNABLE TO CLER
VIRUS.
AND WHAT BECAME CLEAR IS THAT T7
MARKED A SMALL POPULATION OF CES
THAT HAD A PROGENITOR OR STEM-LE
PHENOTYPE THAT COULD SELF RENEWD
ALSO DIFFERENTIATE INTO MORE
EXHAUSTED CELLS AND THIS IS WHAT
ALLOWED CELLS TO BE MAINTAINED
DURING CONDITIONS OF EXHAUSTION.
NOW, IT'S NOW RECOGNIZED FROM WK
BY MANY PEOPLE THAT TCF1 POSITIE
CELLS, THEY RESPOND TO PD-1
BLOCKADE DURING EXHAUSTION.
WORK FROM ANOTHER LAB SHOWED THT
TCF7 DEFICIENT CELLS LOOKED LIKE
TERMINAL EFFECTORS.
SO WHAT LOOKED LIKE WHAT WE SAWN
THE ACTIVATED PI3 KINASE MICE.
WE ASKED, DOES ACTIVATEED PI3
KINASE DELTA EFFECT THE DEVELOPT
OF THIS TCF1 PROGENITOR CELL
POPULATION IN RESPONSE TO CHRONC
INFECTION AND COULD THIS CONTRIE
TO THE ALTERED RESPONSES TO CHRC
INFECTION IN THESE MICE?
SO TO LOOK AT THAT, WE TOOK A
CHRONIC INFECTION MODEL WITH LCV
CLONE 13 WHERE WE COULD LOOK ATE
PROGENITOR CELLS, THE EXPRESSIOF
TCF1 AND THEN LOOK AT THE TERMIL
EXHAUSTED CELLS THAT DIDN'T EXPS
TCF1 BUT HIGHER LEVELS OF
EXHAUSTION MARKERS OR INHIBITORY
RADIOY SEPTEMBERORS SUCH AS TEM.
AND WHAT WE SAW IS WHEN WE INFED
THE MICE, YOU COULD SEE JUST LIE
IN THE ACUTE INFECTION, THAT THE
GENERATION OF TCF1 POSITIVE
POPULATION WAS SERIOUSLY IMPAIR.
IFFY WOO LOOKED BY GENE EXPRESS,
WHAT WE COULD SEE IS A LOSS OF E
PROGENITOR-LIKE SIGNATURE, MANYF
THE GENES THAT WERE ASSOCIATED H
PROGENITOR TCF1 POSITIVE CELLS.
HOWEVER, WHEN WE LOOKED AT THE L
LATERY AND THE RESPONSE, UNLIKE
TCF1 DEFICIENT MICE OR DEFICIENT
T-CELLS, WE DID NOT LOSE T-CELL.
THIS WAS SOMETHING DIFFERENT, A
RESPONSE LOOKS DIFFERENT.
YOU COULD TELL THAT EVEN JUST
LOOKING AT THE MICE BECAUSE UNLE
TCF1 DEFICIENT MEAS, THESE MICE
ACTUALLY DID NOT DO WELL.
MANY OF THEM DIED WITHIN THE FIT
TWO WEEKS OF INFECTION IF THEY
SURVIVED THEN THEY MANAGED TO
CONTINUE ON.
THEY LOOKED SICKER IN THE FIRST
WEEK.
HOWEVER, WHEN WE LET THEM, THE
SURVIVORS, THEY DID BETTER AND
GAINED WEIGHT FASTER.
YOU COULD SEE WITHIN THE FIRST K
THAT THERE WAS HIGHER VIRAL TITS
BUT AGAIN THE SURVIVORS WERE ABE
TO CLEAR THE VIRUS, MUCH BETTER
THAN WILDTYPE.
SO SOMETHING ELSE IS GOING ON.
SO WHAT COULD THAT BE?
SO THERE IS A HETEROGENEITY OF
CELLS DURING EXHAUSTION AND THEE
IS A TCF1 PROGENITOR POPULATIOND
YOUR MORE TERMINALLY DIFFERENTID
EXHAUSTED CELLS.
HOWEVER, IT'S NOW RECOGNIZED FRM
WORK FROM MULTIPLE GROUPS,
PARTICULARLY [ INAUDIBLE ]
THAT THERE IS ALSO THE PRESENCEF
A KLRG1 POSITIVE CELL POPULATION
THAT LOOKS MORE LIKE EFFECTOR-LE
CELL POPULATION, EVEN DURING
EXHAUSTION.
AND YOU CAN LOOK AT THOSE CELLSY
EXPRESSION OF THE MARKER CX3CR1,
POSITIVE FOR THAT, WHEREAS THE 1
POSITIVE POPULATION IS IT ALSO
POSITIVE FOR LIE 108 AND EXHAUSD
CELLS ARE NEGATIVE FOR BOTH.
THIS PROVIDES A NICE SYSTEM OF
MARKERS TO LOOK AT THESE.
AND SO WHEN WE LOOK AT OUR PI3
KINASE ACTIVATED MICE, WHAT YOUN
SEE IS THAT THERE IS A REAL DIRH
OF THESE PRECURSORS OR PROGENITR
STEM-LIKE CELLS AND YOU CAN SEE
THAT MARKED DROP IN THOSE CELLS,
PARTICULARLY OVER TIME N CONTRA,
IF YOU LOOK, WE DO GET EXHAUSTED
CELLS BUT WHAT WE REALLY SEE IS
THIS LARGE INCREASE IN
EFFECTOR-LIKE CELL POPULATION TT
INCREASES OVER TIME.
SO ACTIVATED PI3 KINASE SEEMS TE
DRIVING THIS EFFECTOR-LIKE
POPULATION.
WE CAN SEE THIS ALSO IF WE LOOKN
SINGLE CELL RNA-SEQ WHERE THERES
MANY MORE DIFFERENT POPULATIONS.
AND WHAT CAN YOU SEE IS THAT YOU
GET DECREASE IN THESE PROGENITOR
POPULATIONS WITH INCREASE AND
EXPANSION OF THESE EFFECTOR CELS
BUT ALSO AN INCREASE OF MANY OFE
CELL POPULATIONS THAT ARE EN ROE
TO TAKING THAT ROUTE TOWARDS THE
EFFECTOR CELL POPULATION.
IF WE LOOK SPECIFICALLY AT MARKS
FOR THESE DIFFERENT CELL
POPULATIONS, WHAT WE CAN SEE IN
BOTH OF THE EXHAUSTIVE CELL
POPULATION AND THE EFFECTOR-LIKE
POPULATION, IS THAT THEY EXPRESS
LOWER LEVELS OF MARKERS OF
EXHAUSTION SO MANY INHIBITORY
RECEPTORS AS WELL AS THE
TRANSCRIPTION FACTOR TOX, WHERES
THEY EXPRESS HIGHER LEVELS OF
MARKERS FOR EFFECTOR-LIKE CELLSF
CX.
THERE CR1 AND R3 AND T BAT AND
KLRG1.
WHEN WE STIMULATE THE CELLS, EAH
THE EXHAUSTED CELLS PRODUCE MORE
CYTOKINES.
SO THEY LOOK LIKE THESE CELLS TT
ARE SUPPOSEDLY EXHAUSTED LOOK LE
THEY ARE ACTUALLY NOW FUNCTIONAN
THESE MICE.
SO ARE THESE PHENOTYPES CD8 T-CL
INTRINSIC OR DUE TO INTERACTIONS
FROM OTHER CELLS?
TO ASK THAT QUESTION, WE TOOK
ADVANTAGE OF THE P14 TRANSGENIC
SYSTEM WHICH WAS SPECIFIC FOR TE
MAJOR EPITOPE PLUS ONE RESTRICTD
EPITOPE ON LCMV.
WE COULD TAKE P14 CELLS MUTANT R
PI3 KINASE OR WILDTYPE, MIX THEM
ONE-TO-ONE AND WITHOUT THEM INTA
HOST SO WE ARE LOOKING AT THE
RESPONSES IN THE SAME BACKGROUN.
SO WE ARE NOT BEING CONFOUNDED Y
DIFFERENCES IN VIRAL TITER.
AND WHAT CAN YOU SEE IS, CLEARLY
HAVE THE CELL INTRINSIC LOSS INE
PRECURSOR OR PROGENITOR EXHAUSTD
CELLS, VERY CLEARLY MARKED HERE.
WE DO SEE SOME INCREASE IN THE
EFFECTOR-LIKE CELLS ALTHOUGH ITS
NOT AS DRAMATIC.
SO WHY ARE WE LOSING THOSE CELL?
SO TCF7 EXPRESSION IS DRIVEN IN
EXHAUSTED CELLS IN LARGE PART
THROUGH THE TRANSCRIPTION FACTOR
FOX 01 AND THAT TRANSCRIPTION FT
ON ARE IS UPACTIVATED DOWNSTREAF
PI3 KINASE -- INACTIVATED.
IN WE TAKE CELLS, WE CAN TAKE
WILDTYPE P14 CELLS AND WE CAN UE
CHRIS PER TO KNOCKOUT -- CRISPR-
FOX 01 IN NAIVE CD4 CELLS, TRANR
THEM INTO A MOUSE AND THEN INFET
THEM WITH LCMV13.
YOU CAN SEE THAT FOX 01 KNOCKOUS
VERY NICE HERE AND YOU CAN SEE T
LEADS TO A LOSS OF EXPRESSION OF
MOST OF THE TCF1.
WHEN WE DO THAT, WHAT YOU SEE IS
THAT YOU CAN RECAPITULATE VERY
NICELY THAT DRAMATIC LOSS IN THE
PROGENITOR CELL POPULATION.
HOWEVER, WE DON'T RECAPITULATE E
INCREASE IN EFFECTOR CELLS.
SO FOX 01 INACTIVATION IS
RESPONSIBLE FOR THE LOSS OF THE
TCF1 POSITIVE POPULATION.
PROGENITOR POPULATION, BUT NOT R
THE INCREASE IN EFFECTORS.
NOW WE ALSO LOOK TO SEE IF THERE
WERE CELL EXTRINSIC EFFECTS.
TO DO THAT, WE HAD TO TAKE WILDE
P14CD8 CELLS AND THANS FER THEM
INTO WILDTYPE OR MUTANT HOSTS.
AND THEN WHAT WE COULD SEE IS T,
THERE WERE ALSO CELL EXTRINSIC
ASPECTS TO THIS PHENOTYPE.
SO THEY ARE NOT ALL CD8 CELL
INTRINSIC.
WE SAW SOME REDUCTION IN THE
PROGENITORS BUT WE SAW THIS LARE
INCREASE IN THE EFFECTOR-LIKE
CELLS.
AND SO WHY IS THAT?
WHAT ARE THE POTENTIAL CELL
EXTRINSIC EFFECTS?
COULD IT BE OTHER T-CELLS, T
REGULARS?
IT'S BEEN SHOWN THAT INHIBITING
THESE CELLS BLOCKS T-REG FUNCTI.
THAT'S THE OPPOSITE.
WE FOCUSED ON WHAT CYTOKINES MIT
BE PRODUCED IN THESE MICE.
WHAT WE SAW IS THAT IN FACT IN
RESPONSE TO LCMV, WE COULD SEE
LARGE INCREASES IN A NUMBER OF
CYTOKINES AND PARTICULARLY WE HD
INTO IL21.
AND PREVIOUS DATA SHOWN THAT IL1
IS VERY POTENT IN INDUCING EFFER
CELL CD8 CELL FUNCTION AND
SURVIVAL.
AND MORE RECENT WORK FROM ANOTHR
LAB HAD SHOWN THAT TFH CELLS ARE
THAT SOURCE OF IL21 THAT CAN LED
TO THE GENERATION OF THESE EFFER
CD8 CELLS UNDER CONDITIONS OF
EXHAUSTION.
AND SO IF WE LOOK AT OUR MICE, T
WE SEE IS THERE ARE -- THERE ISN
INCREASE IN TFH CELLS OR ANNUAL
JEN-SPECIFIC TFH CELLS.
SO TO SEE IF THAT WAS IMPORTANTE
DID THE SAME INFECTION WITH LCMN
THE PRESENCE OF BLOCKING ANTI-I1
RECEPTOR ANTIBODIES OR DEPLETING
ANTI-CD4 MONOCLONAL ANTIBODIES.
WHAT WE COULD CLEARLY SEE IS THT
IF YOU LOOK AT THAT LARGE INCREE
IN EFFECTOR CELLS IN THE MUTANT
MICE, THAT WAS LARGELY ABROGATED
WHEN WE BLOCKED IL21 RECEPTOR, O
PRETTY WELL BLOCKED BY ANTI-CD4.
SO, WHAT ABOUT THE RESPONSE TO
IL21?
SO WE TOOK ISOLATED CD8 CELLS FM
THESE MICE AND STIMULATED THEM N
THE PRESENCE OF IL21 OR STIMULAD
THEM AND ADDED IL21.
WE SAW THAT THE MUTANTS IN YELLW
SHOWED HIGHER LEVELS OF PHOSPHOT
3, DOWNSTREAM OF IL21 RECEPTOR D
ALSO SHOWED A GREATER INDUCTIONF
CX3CR1.
SO YOU WOULD THINK THIS IS A CEL
INTRINSIC EFFECT OF THIS.
AND SO, DOES THAT ARGUE -- IS TE
AN ALTER DEVELOPMENTAL PATHWAY?
ARE THESE CELLS INTRINSICALLY
PRONE?
SO TO DO THAT, WE INFECTED THE
MYSELF, SORTED OUT PROGENITOR
CELLS, TRANSFERRED THEM INTO
INFECTION MATCHED HOSTS AND LOOD
AT THE PROGENY A WEEK LATER.
THE WILDTYPE PRECURSOR CELLS, MT
STAYED AS PROGENITOR OR PRECURSR
BUT A LARGE FRACTION OF THEM ALO
BECAME TERMINALLY EXHAUSTED CEL.
THE MUTANT PROGENITOR CELLS, VEY
FEW OF THEM STAYED AS PROGENITOR
AND MAINLY BECAME EFFECTOR-LIKE
CELLS.
SO ACTIVATED PI3 KINASES IS
PROPELLING THESE CELLS TO BECOME
EFFECTOR-LIKE CELLS.
SO IN WILDTYPE BACKGROUND YOU SE
MULTIPLE CD8 CELL POPULATIONS
DURING CHRONIC INFECTION BUT THY
ARE ALTERED IN ACTIVATED PI3
KINASE.
SO FEWER PROGENITOR CELLS
PRESUMABLY YOU WOULD GET POORER
RESPONSES TO ICB.
BUT AT THE SAME TIME YOU GET
INCREASED VIRAL AND TUMOR -- SO.
INCREASED EFFECTOR CELLS WHICH
PRESUMABLY WILL LEAD TO INCREASD
VIRAL AND TUMOR CLEARANCE.
SO IS PI3 KINASE GOOD OR BAD FOR
EXHAUSTION?
I THINK THE JURY IS OUT AND THAS
BECAUSE IT EFFECTS BOTH CELL
POPULATIONS.
SO IF YOU HAVE LITTLE PI3 KINASE
ACTIVITY, IF YOU INHIBIT IT, YOU
PROMOTE THOSE PROGENITOR CELLS T
YOU WANT FOR CHECKPOINT BLOCKAD.
IT ALSO IS VERY GOOD FOR CURBING
T-REG ACTIVITY HOWEVER, ACTIVATN
OF PI3 KINASE IS REALLY IMPORTAT
FOR PROMOTING EFFECTOR-LIKE CELS
AND THAT IS PART OF WHAT PD-1 BK
AID DOES, IS TO ACTIVATE PI3 KIE
PATHWAYS.
AND SO, IT'S VERY INTERESTING TN
THE LAST YEAR, A PAPER CAME OUT
THAT INTERMITTENT PI3 KINASE
INHIBITION WAS VERY GOOD FOR
SUSTAINING ANTI-TUMOR IMMUNITY.
THAT WAS FOCUSED ON T-REGS BUT
PERHAPS ALSO EFFECTS ON CD8 CELS
AND CERTAINLY SUGGESTS THAT YOU
NEED TO THINK ABOUT THESE BALANS
AS YOU'RE TRYING TO BALANCE BOTH
THE PROTECTED AND THE DETRIMENTL
EFFECTS OF EXHAUSTION.
AND THIS WAS RELATED TO THE WORF
A TALENTED FELLOW WHO IS IN THE
AUDIENCE WITH HELP FROM OTHERS.
THANK YOU.
[ APPLAUSE ]
>> THANK YOU, QUESTION?
>> BEAUTIFUL TALK.
I HAVE A VERY NICE QUESTION.
WOULD YOU EXPECT THE SAME TO HAN
ALSO IN A TUMOR MODEL IN THESE
MICE?
>> SO WE HAVE BEEN STARTING TO K
AT IT AND ALSO OTHER GROUPS HAVE
TOO, AND IT DEPENDS ON THE TUMOR
MODEL AND IT MAY DEPEND ON WHAT
YOU'RE DOING.
AND SO SOME OF THIS -- TREATINGR
NOT TREATING MAY -- FOR EXAMPLEN
ADOPTED CELL THERAPY-TYPE MODEL,
PERHAPS YOU WANT TO INHIBIT PI3
KINASE BUT YOU DON'T WANT TO DO
THAT IN-VIVO.
SO IT MAY DEPEND ON THE TISSUE D
THE TYPE OF TUMOR.
>> I READ SOMEWHERE THE SIZE WAS
EXPRESSED IN THE BRAIN.
EITHER PATIENTS OR MICE SHOW ANY
NEURONAL EFFECTS?
>> YES, SO MANY THINGS ARE
EXPRESSED IN THE BRAIN.
AND WE HAVE NOT DETECTED IT BUTE
HAVE NOT PUT MICE THROUGH VARIOS
CHALLENGES TO ACTUALLY FIND OUTF
THEY ARE SMART OR DUMB.
BUT WHETHER THE INHIBITORS EFFET
THE BRAIN -- INHIBITORS HAVE BEN
USED IN TUMORS IN PATIENTS AND I
DON'T THINK THAT'S BEEN LOOKED .
THERE HAS BEEN OTHER PROBLEMS WH
ALTERNATE IMMUNITY AND T-REGS.
>> THERE IS NO KIND OF -- [
INAUDIBLE ]
>> I'M SORRY.
THAT'S A GREAT QUESTION.
THE PATIENTS ARE COGNITIVELY ARE
FINE.
>> GREAT TALK.
SORRY IF YOU SAID THIS AND I MID
IT.
IS THERE ANY CHANGE IN
FOXEXPRESSION?
>> FOXEXPRESSION IS DOWN.
-- TOX EXPRESSION IS DOWN.
IT GOES ALONG WITH THEM BEING LS
EXHAUSTIVE.
>> THANK YOU.
>> SO [ INAUDIBLE ]
AGREED TO STAND IN WITH US ANDS
GOING TO INTRODUCE --
>> IT GIVES ME A LOT OF PLEASURO
BE ABLE TO INTRODUCE STANLEY ADO
WHO IS A HOMETOWN HERO FROM THE
CENTER FOR CANCER RESEARCH HERET
NCI.
HE IS GOING TO BE TALKING TO US
ABOUT PROTEOSTASIS AND ITS LINKO
T-CELL AUTONOMOUS DYSFUNCTION.
THANK YOU, STANLEY.
>> I THINK EVERYONE CAN HEAR ME,
RIGHT?
I WOULD LIKE TO THANK THE
ORGANIZERS FOR THE CHANCE TO
PRESENT THE WORK FROM MY LAB.
AND I ALSO WANT TO THANK THEM FR
GETTING STARTED IN THE EXHAUSTIN
CONVERSATION.
TODAY I'D LIKE TO SHARE WITH YOU
SOME UNPUBLISHED WORK FROM A
TALENTED GRAD STUDENT FROM MY L.
WE THINK THE KEY -- MAY PLAY A Y
IMPORTANT ROLE IN THE PROGRAM TT
MAY CONTRIBUTE TO CELL EXHAUSTI.
SO, YOU HAD ENOUGH BACKGROUND ON
EXHAUSTION SO I WANT TO GET YOU
INTO REALLY THE PROGRAM I WANT O
TALK ABOUT TODAY, THE -- MY LABS
INTERESTED IN HOW -- CONTRIBUTEO
IMMUNE CELL DIFFERENTIATION AND
FUNCTION.
THERE ARE MANY OF THESE PATHWAYS
BUT ISLAND LIKE TO FOCUS ON THE
PROTEIN RESPONSE.
SO FOR THIS, IF WE DON'T THINK
ABOUT UPR ALL THE TIME.
THERE ARE THREE ENZYMES.
THEY ARE PLASMA MEMBRANE ENZYMEO
THESE ENZYMES ARE THOUGHT TO BE
ENACTED AND THE ACTIVATION IS
DRIVEN BY THE ASSOCIATION WITH
THE -- THEY GET ACTIVATED, THE
CURRENT MODEL IS WHEN THE PROTES
ARE IN THE LUMEN AND THERE --
HIGHER ASSOCIATION, AFFINITY FOR
MISFOLDED PROTEINS SUCH THAT ONE
YOU HAVE -- THESENCE ZYME, THEY
LEAD TO ACTIVATION.
-- THESE ENZYMES.
YOU DON'T HAVE TO REMEMBER WHATS
ON THIS SLIDE BUT THE TAKE-HOME
MESSAGE IS THOSE THREE ENZYMES A
SET OF FACTORS THAT ACTIVATE
DOWNSTREAM PROGRAM.
SO [ INAUDIBLE ]
ATF6 IS PROCESSED IS ACTUALLY
TRANSCRIPTION FACTOR.
SO THESE THREE ENZYMES -- DRIVE
DOWNSTREAM FUNCTION.
I WANT TO POINT OUT HERE THAT TE
THREE FACTORS ARE ALL -- PROTEI.
AND IN MY TALK, THAT IS ACTUALL-
DECISION.
CLASSICALLY THE EPR IS KNOWN TO
TURN ON PROGRAMS THAT WILL PROME
PROTEIN FOLDING BECAUSE THAT'S T
IT'S EVOLVED TO DO.
SOME OF THESE TARGETS EXTEND BED
CLASSICAL PROTEIN HOMEOSTASIS
PROGRAM.
SO WE GOT INTERESTED ON WHAT WE-
QUESTION HOW IT MAY -- T-CELL
DIFFERENTIATION.
SO T-CELLS ARE ACTIVATED AND THY
ARE TURNED -- VERY EARLY.
HOWEVER, THE LITERATURE OUT THEE
IS AT BEST, CONTRADICTORY IN THE
SENSE THAT THERE IS SOME STUDIES
USING INDIVIDUAL SINGLE KNOCKOUS
OF THESE ENZYMES IN T-CELLS THAT
SUGGEST THEY ARE DISPENSABLE FOR
T-CELL DIFFERENTIATION.
OR SURPRISINGLY THESE ENZYMES CD
PROMOTE IMMUNOSUPPRESSION BY
T-CELLS.
ONE THING THAT HAS BEEN OVERLOO,
YOU HAVE THREE ENZYMES, IS IT
POSSIBLE THEY FUNCTION REDUNDAN?
THE QUESTION I WANTED TO ADDRESS
WAS, IS THERE REDUNDANCY IN THER
SIGNALING PROGRAM?
IF SO, WHAT IS THE CONSEQUENCE F
UPR DEFICIENCY?
SO TO ADDRESS THAT, THE MOUSE ML
IN WHICH WE USE CD4 TO REMOVE TE
ENZYMES, THESE MICE LACK ALL THE
SENSORS.
WE CALL THEM UPR KNOCKOUT ANIMA.
WHEN WE KNOCK IT OUT FROM T-CEL,
T-CELL DEVELOPMENT IS FINE --
SO THE QUESTION WE WANTED TO GET
IS, HOW DOES THIS IMPACT T-CELL
EFFECTOR DIFFERENTIATION?
CONTRARY TO -- I'M GOING TO BE
USING THE ARMSTRONG MODEL.
THAT MODEL IS AN ACTIVE INFECTIN
MODEL THAT IS RESOLVED AROUND T[
INAUDIBLE ]
AND MAMMARY CELLS.
WE ASKED THE QUESTION, WHAT HAPS
WHEN WE INFECT WILDTYPE AND UPR
KNOCKOUT ANIMALS WITH ARMSTRONG?
WHAT CAN YOU APPRECIATE HERE IS
THAT AT THE AGE WHEN MOST OF THE
WILDTYPE ANIMALS HAVE SIGNIFICAY
CLEARED THE VIRUS, THERE IS A HE
AMOUNT OF VIRUS IN THE KNOCKOUTN
THE T-CELLS -- UNABLE TO RESOLVE
THIS ACUTE INFECTION.
WHY IS THAT?
SO WE LOOKED AT THE MODEL CLOSEY
AND YOU SEE -- GENERATE SPECIFIC
T-CELLS BUT IN NUMBERS AND
FREQUENCY -- NUMBERS SIGNIFICANY
ARE DEPLETED.
AND WHAT WAS MOST STRIKING IN
THIS -- AMONG THESE T-CELLS, YOU
ARE MISSING THE KRG1 -- CELL
POPULATION, INDICATING THAT NOT
ABLE TO CLEAR VIRUS, NO EFFECTOR
CELLS.
IT ALSO TELLS YOU THE UPR
CONTRIBUTES TO THIS PROCESS THAT
GENERATES EFFECTOR CELLS.
SO THE QUESTION WE WANTED TO GET
AT, WHAT ARE THEY BECOMING?
SO TO GET AT THAT, WE PERFORMED
RNA-SEQ ON T-CELLS FROM THIS ACE
MODEL AND CONSISTENTLY NOT CLEAG
VIRUS, THEY LACK A LOT OF THE
CLASSICAL EFFECTOR PROGRAM.
WHAT WAS SURPRISING IS WHEN WE
LOOKED AT THE -- IN CONTRAST TOE
DEFICIENCY PROGRAM THEY COMPLETY
EXPRESSED A LARGE SET OF CLASSIL
T-CELL EXPRESSION MARKERS.
SO YOUR USUAL SUSPECTS -- THAT S
ALSO TRUE AT THE PROTEIN LEVEL.
SO LACT EFFECTOR GENES AND ENZYE
AND THEY HAVE VERY HIGH LEVELS F
PD-1 AND TOX 1.
SO SO FAR, THE UPR MAY NOT BE
IMPORTANT FOR THE DEVELOPMENT OF
T-CELLS BUT WE THINK THAT WE FID
THAT IT IS ESSENTIAL TO GIVE YOU
EFFECTOR T-CELLS.
AND JUST IF YOU WERE CURIOUS,
PHENOTYPE OF THESE T-CELLS ARE
TERMINAL.
SO VERY HIGH FOR PD-1.
SO BECAUSE THIS WAS A VERY
STRIKING -- WE WANTED TO GET AT
BASIS OF HOW THIS WAS HAPPENINGD
ONE -- THE QUESTION WE ASKED, DS
ANIMAL STILL HAVE VIRUS BECAUSE
THEY EXPRESS VIRUS THAT WAS TRAD
IN THE EXOZOME PROGRAM?
SO WE ENVISION A SCENARIO WHEREE
CLEAR THE VIRUS, AND PROVIDE THM
WITH CELLS THAT ARE ABLE TO CLER
THE VIRUS, ARE THE CELLS ABLE TO
DIFFERENTIATE INTO EFFECTORS?
WE DID THAT BY TRANSFERRING P14.
SO WILDTYPE P14 INTO OUR WILDTYE
ANIMALS AND KNOCKOUT ANIMALS.
SO NOW EFFECTOR T-CELLS.
AS WONG EXPECT, THESE WILDTYPE
CELLS WE PROVIDED THEM, WHICH IA
BLUE GRAPH HERE, THEY
DIFFERENTIATED SIMILARLY ACROSS
YOUR WILDTYPE AND YOUR KNOCKOUT
HOST.
SO IF YOU LOOK AT PD-1 AND TOX ,
THEY ARE EQUALLY EXPRESSED TO TE
SAME DEGREE ACROSS THE WILDTYPED
THE UPR KNOCKOUT HOST.
WHAT HAPPENS TO THE WILDTYPE CES
AND THE UPR KNOCKOUT?
SO IF YOU LOOK AGAIN THE ENDOGES
CELLS IN BOTH -- EVEN IN THE ANL
THAT HAS BEEN ABLE TO RESOLVE
VIRUS, THEY STILL EXPRESS VERY H
VIRUS OF TOX, SUGGESTING THAT TS
PROGRAM CONTROLS THE EXHAUSTION
PHENOTYPE -- SO WHAT DOES THAT L
US?
THE SET OF EXPERIMENTS I SHOWEDU
IS THAT EXHAUSTION IS ASSOCIATEE
WITH LOW UPR ACTIVITY AND THE
PROGRAMMED THAT DRIVES THE EFFET
OF T-CELL GENERATION REQUIRES
ACTIVATION OF THE PROTEIN RESPO.
SO IN THE -- ABSENCE OF UPR WILL
PROMOTE CELLS AND CAUSE THEM TO
BECOME EXHAUSTED.
SO THIS WAS GENERALIZABLE TO
INFECTION MODELS -- LOOK AT THIS
NOW IN HUMAN AND DATASETS IN HUN
INFECTION.
HERE THIS IS DATA FROM OUR HIV.
SO T-CELLS -- WE LOOKED AT THE
DIFFERENT CLUSTERS FOCUS ON THE
CLUSTERS THAT REPRESENT THE
EXHAUSTED POPULATION AND EFFECTR
POPULATION.
I WANT TO JUST POINT OUT HERE TT
AS WE SEE PREVIOUSLY, THERE IS L
THE -- FOR UPR PROGRAMS TURN ONN
EFFECTOR CELLS AND IN THE TEAR,
EXHAUSTED CLUSTER, THESE PROGRAS
ARE VERY LIMITED.
THEY ARE ALMOST ABSENT.
THIS SUGGESTS THAT THIS MECHANIM
INVOLVING UPR EXHAUSTION IS
POTENTIALLY HAPPENING IN ALL
DISEASE IN NORMAL HUMAN CONTEXT.
THIS IS ALSO TRUE FOR COVID.
SO IN MY FINAL FEW MINUTES, I WD
LIKE TO GET AT THE MECHANISM WHH
WE THINK THIS IS HAPPENING.
SO HOW IS THE UPR PROMOTING
EFFECTOR?
DIFFERENTIATION AT THE SAME TIME
EXHAUSTION.
SO THERE ARE TWO REAL SPECIFIC
PROBLEMS HERE.
WHAT IS THE BASIS OF REDUNDANCY?
THIS IS ACTING REDUNDANCY AND WT
I DIDN'T MENTION IS THAT THE SAE
SOURCE OF THE UPR IS SIMILAR
PROTEINS.
DOWNSTREAM THEY TURNED ON BASICY
PROTEINS.
THEN SECOND QUESTION I DON'T THK
I'LL HAVE TIME TO GET TO TODAY,
SPECIFIC EFFECTOR PROGRAM KNOWNS
UPR TURNING ON, THAT IS DRIVINGE
EFFECTOR PROGRAM.
SO, THE FIRST QUESTION WAS, WHAS
THE BASIS OF THIS REDUNDANCY?
SO DOWNSTREAM OF THESE ENZYMES E
BASICALLY TRANSCRIPTION FACTORSD
WE HAVE THESE PROTEINS MAY FUNCN
REDUNDANTLY AND INTERCHANGEABLY
SUCH THAT WHEN ONE ENZYME IS
MISSING, CAN BUFFER THE PROGRAM.
SO TO TEST THAT, WE TOOK THE UPR
KNOCKOUT ANIMAL AND
TRANSGENERICALLY INTRODUCED IN A
CONDITIONAL MANNER THE -- TURNED
ON.
THIS ANIMAL LACKED THREE SENSORS
AND ONLY EXPRESSED JUST THIS
DOWNSTREAM -- SO THE QUESTION WE
ASKED WAS WHAT HAPPENS WHEN WE
INFECT THESE ANIMALS AND SBP1 WE
PUT BACK IN SUFFICIENT TO RESTOE
NOW THE -- SO I'M SHOWING HERE
CLASSIC DATA JUST TO GIVE A MORE
DEPTH INTO WHAT WE FOUND.
SO IN OUR KNOCKOUT AT DAY 8, INE
ABSENCE OF UPR YOU SEE MASSIVE
CHANGES IN THE LANDSCAPE SHOWING
HERE BY THIS.
WHEN WE INTRODUCED OUR XBP1, WE
LARGELY -- RESCUED -- MORE
IMPORTANTLY, KNOCKOUT WE DON'T
GENERATE EFFECTOR CELLS.
BUT WHEN WE PUT BACK XBP1, YOU E
THAT WE ALSO ARE RESTORE THE
NUMBERS OF EFFECTOR T-CELL
POPULATION.
AND THOSE EFFECTOR T-CELLS NOW E
LAUER LEVELS OF PD-1 AND TOX.
SO IN CONCLUSION WHAT I HAVE SHN
YOU TODAY, WE THINK THAT UPR PLS
IMPORTANT ROLE IN EFFECTOR T-CEL
DIFFERENTIATION AND THE UPR MAYE
A MECHANISM TO PREVENT THIS
EXHAUSTION OF T-CELL PROGRAM.
I DON'T KNOW IF I HAVE TIME BUTI
WILL PROBABLY END IT HERE.
WE NOW HAVE A CLUE -- MORE INSIT
IN SOME OF THESE DOWNSTREAM WHAM
AND ONE OF THE WAYS THAT LOOKEDT
THE DEPENDENT ON THE SIGNALING S
THE -- SUCH THAT WHEN THERE IS O
UPR, YOU DISRUPT OR DOWN REGULAE
ID2 PROGRAM.
THAT IS INTERESTING BECAUSE WORY
OTHERS YEARS AGO SHOWED THAT ADD
TO ANIMALS -- EFFECTOR CELLS SO
CONSISTENT WITH WHAT WE FIND.
AND THEN WE ALSO HAVE -- I'M NOT
GOING TO GO INTO THAT.
I'M GOING TO STOP HERE AND TAKEY
QUESTIONS.
[ APPLAUSE ]
>> THANK YOU.
I GUESS MAYBE YOUR LAST POINT
ADDRESSED THIS SOMEWHAT BUT I'M
WONDERING WHETHER THE EXHAUSTIOS
AN INDIRECT CONSEQUENCE OF JUSTA
REALLY STRONG HIT THAT THEY'RE
GETTING OR ARE THEY COMPLETELY Y
PASSING THE EFFECTOR STAGE AND
GOING STRAIGHT TO EXHAUSTION?
>> SO GREAT QUESTION.
SO WE LOOKED HARD AND NOT SEEN Y
OF THE -- IT LOOKS LIKE REALLY
BYPASSING.
WE DON'T SEE ENOUGH OF THE -- AS
EARLY AS DAY 3 WE SEE THIS VERYW
KLGR1 LOAD.
SO IT LOOKS LIKE THEY ARE BYPASG
THAT STAGE.
SO THAT IS PROBABLY WHAT I WOULD
SAY.
>> CLARIFY -- ARMSTRONG --
[ LOW AUDIO ]
>> THAT'S THE EXPERIMENT WE ARE
SETTING UP TO DO NOW.
THE QUESTION IS WHETHER WE CAN
NOW -- CAN WE REVERT OR RESCUE
EXHAUSTION IN OUR MODEL?
WE ARE LOOKING AT THAT NOW.
GOOD QUESTION.
>> GREAT TALK.
SO EFFECTOR CD8S SHOW A HIGH UPR
SIGNATURE.
WHY -- CAN YOU SPECULATE ON WHYU
THINK WHY IT'S SO SYNTHETIC FOR
GRANULES AND INTERFERON GAMMA?
IT'S INTERESTING BECAUSE IT KINF
PARALLELS A PLASMA CELL BUT NOT
QUITE AS DRAMATIC.
>> THAT'S A VERY INTERESTING
QUESTION.
ALSO WHY HAS THE SYSTEM EVOLVED
TO -- UPR INTO A PROGRAM -- INTO
THE EFFECTOR CELL PROGRAM?
ON THE ONE HAND, YOU ARE CORRECT
THAT THE ACTIVATED CD8 T-CELLS E
EXPANDING TO MAKE MORE CYTOKINEO
MAKE EFFECTORS.
BUT IT TURNS OUT THAT IN THE
ABSENCE OF THE -- WE HAVE TRIEDO
RESCUE THESE ANIMALS -- ONE
POSSIBLY IF WE GIVE THEM CHEMICL
CHAPERONES THAT CAN RESTORE THET
IS TRUE THIS PATHWAY IS POTENTIL
TOW HELP PROTEIN SYNTHESIS OR
T-CELLS: BUT THE FACTORS ARE ALO
SOMEHOW COUPLED TO THE -- WHICH
COULD BE POTENTIALLY TO --
CHECKPOINT TO ENSURE THAT THE CS
ARE PREPARED -- MAKING EFFECTOR-
ALSO BETTER ABLE TO --
>> SO THANK YOU.
SO WHEN P14 TRANSGENIC EXPERIME,
THE CELLS YOU TRANSFERRED WERE L
WILDTYPE.
YOU DIDN'T TRANSFER THE CURE CES
INTO WILDTYPE MOUSE.
>> SIGH THAT AGAIN.
>> DID YOU TRANSFER P14 KNOCKOUT
INTO WILDTYPE MICE?
>> WE HAVE DONE THAT AS WELL.
WE HAVE DONE TWO MODELS WHERE WE
HAVE THE P14 KNOCKOUT INTO THE E
HOST.
YOU ALSO FIND THAT THE KNOCKOUT
CELLS DON'T MAKE EFFECTOR CELL.
>> EVEN IN WILDTYPE HOST?
>> EVEN IN WILDTYPE HOST.
>> SO YOU HAVE INDEED --
>> EVEN IN WILDTYPE HOST THEY DT
MAKE -- YES.
>> THANK YOU.
>> NICE TALK, I'M CURIOUS ABOUTE
HIV DATASET YOU SHOWED.
WERE THE PATIENTS ON THERAPY AND
WAS THIS FROM A CHRONIC INFECTIN
TIME POINT?
>> SO THE PATIENTS ARE -- YES,
PATIENTS WERE ON THERAPY.
THIS WAS IS PUBLISHED -- I'M SO,
YES THEY WERE ON SOME THERAPY.
[ INAUDIBLE ]
>> SO THESE ARE QUESTIONS -- IT
WASN'T CLARIFIED.
IT WAS NOT DATA FROM US BUT IT
WASN'T CLARIFIED IN THAT DATASET
BUT -- I MEAN, THE REASON I WAS
SHOWING IS TO AVOID THE FACT THT
WITHIN THE PROCESS AFFECTED BY V
AND THAT POTENTIALLY IMPACT THE
UPR.
SO THAT WAS FOCUSED ON THE CD8
POPULATION.
>> THANK YOU VERY MUCH.
FASCINATING TALK.
[ APPLAUSE ]
>> IT IS MY PLEASURE TO ANNOUNCE
THE LAST SPEAKER OF THIS SESSIO,
WHICH IS SHORTENED.
THIS IS JAGAN MUPPIDI WHO WILL L
US ABOUT NUTRIENT DRIVEN
PROLIFERATION IN MUCOSAL GERMINL
CENTERS.
>> CAN YOU HEAR ME?
SO, CHRISTIAN ALREADY DID A NICE
JOB OF INTRODUCING THE GERMINAL
CERTAINLY BUT THE ONGOING
MUTATIONAL PROGRAM THAT LEADS TO
HIGH AFFINITY ANTIBODIES CAN ALO
GENERATE MUTATIONS THAT CAN LEAO
GENERATION OF MALIGNANCIES.
I'M INTERESTED IN TWO AGGRESSIVE
MALIGNANCIES THAT ARE DERIVED FM
GERMINAL CENTER B CELLS, LYMPHOA
AND BURKITT'S LYMPHOMA.
SO THE GERMINAL CENTERS DIVIDED
INTO TWO ZONES.
IN THE LIGHT ZONE SELECTED EVENS
HAPPEN THAT ARE T-CELL MEDIATED
WHEREAS IN THE DARK ZONE SOMATIC
HYPERMEITATION.
AND THE INITIAL SELECTION OF RAE
CLONES IN THE LIGHT ZONE IS
MEDIATED PRIMARILY BY T-CELL HEP
WHICH LEADS TO INCREASE mTOR 1
SIGNALING AND INCREASED MYC
ABUNDANCE INSIDE
POSITIVELY-SELECTED CELLS.
SO THE BALANCE BETWEEN LIGHT ZOE
AND TACKER ZONE IS MEDIATED BY E
DIFFERENCES IN THE ABUNDANCE OF
ACTIVE ADA AND FOX O1.
SO INCREASED FOX FORELATED AKT
WHICH LEADS TO PHOSPHORYLATION D
DEGRADATION OF FOX O1 AND IN THE
DARK ZONE IT IS THOUGHT THAT IS
REVERSED AND IT ALLOWS THE DARK
ZONE PROGRAMS TO TAKE PLACE.
SO, GERMINAL CENTERS FORM AT THE
CENTER OF THE B-CELL FOLLICLE AD
THIS CONFINEMENT TO THE GC NISHS
MEDIATED PRIMARILY BY TWO PROTEN
COUPLED RECEPTORS, WHICH ACT
THROUGH THE G PROTEIN G ALPHA 1O
INHIBIT MIGRATION.
SO THAT CONFINEMENT OCCURS IN TE
CONTEXT OF A DECAYING GRADIENT F
THE LIGANDS FOR THESE TWO
RECEPTORS.
SO, IN THE ABSENCE OF G ALPHA 1E
SHOWED A NUMBER OF YEARS AGO THT
YOU CAN SEE SPREAD OF I CAN'T
REMEMBERINAL CENTER B CELLS
THROUGHOUT THE FOLLICLE AND YOUN
FIND THEM IN CIRCULATION WHEREAS
NORMAL GERMINAL B CELLS ARE NON
RECIRCULATORY.
AND WE WERE PARTICULARLY INTERED
IN THIS PATHWAY BECAUSE MUTATIOS
IN EITHER THE RECEPTORS OR G ALA
13 ARE AMONG THE MOST COMMON
GENETIC EVENTS THAT OCCUR IN BOH
GCB AND B-CELL LYMPHOMA AND BURT
LYMPHOMA.
WHEN YOU LOOK AT ANIMALS THAT LK
G ALPHA 13 IN MATURE B CELLS, YU
CAN SEE THAT AS WE AGE THESE
ANIMALS, THEY DEVELOP TUMORS THT
INTERESTINGLY FORM IN THE -- YOU
CAN SEE HERE THAT OVER TIME ALLE
ANIMALS DEVELOP TUMORS THAT LACG
ALPHA 13.
IF WE LOOK AT THE DISTRIBUTION F
TUMORS, THE VAST MAJORITY OF MIE
THAT HAVE TUMORS HAVE MESS ENTEC
LYMPH NODE INVOLVEMENT SUGGESTIG
THAT IS THE SITE OF ORIGIN.
WHEN WE LOOK AT YOUNGER MICE AND
MIXED BONE MARROW CAME ERAS WHEE
WE GENERATED USING G ALPHA 13
DEFICIENT BONE MARROW MARKED WIH
CD45.2, THERE IS A VERY STRONG L
INTRINSIC OUTGROWTH THAT HAPPENN
THE GERMINAL CERTAINTIES IN MESS
ENTERIC LYMPH NODES BUT THERE IS
BASICALLY NO OUT GROWTH THAT
HAPPENS IN THE BLOOD CELL IMMUND
SETTINGS.
SO WE WERE INTERESTED IN TRYINGO
EVALUATE THE BASIS OF THIS.
SO, WHEN WE ORIGINALLY WERE LOOG
AT THESE MICE WE PROPOSED THAT S
OUTGROWTH AND PROPENSITY TO DEVP
TUMORS WAS DRIVEN BY INCREASED T
BECAUSE WHEN WE LOOKED AT SIGNAG
IN CELLS DIRECTLY EX-VIVO, WE FD
THE STIMULATION COULD INHIBIT A.
TO THE WASN'T CLEAR BASED ON THE
INITIAL STUDIES WHETHER OR NOT T
WAS THE PRIMARY MECHANISM THAT D
TO TUMOR FORMATION.
AND SO, IN DATA THEY DON'T HAVE
TIME TO SHOW YOU, WE COMPARED
ANIMALS IN WHICH B CELLS EXPRESS
HIGHER LEVELS OF AKT.
AND WHAT WE KNOW FROM PREVIOUS K
IS THAT IN THOSE SITUATIONS, YOU
HAVE A BIAS TOWARDS THE LIGHT ZE
AND RESULTING DECREASE IN
PROLIFERATION IN THE GERMINAL
CENTER.
BUT, IN CONTRAST, IN THE ABSENCF
G ALPHA 13, WE DIDN'T SEE ANY
CHANGE IN THE LIGHT ZONE-DARK ZE
DISTRIBUTION.
INSTEAD WHAT WE FOUND IS THERE S
INCREASED PROLIFERATION.
SO TO TRY TO ADDRESS WHAT THE OR
MECHANISMS WERE THAT WERE PROMOG
TUMORIGENESIS IN THE MESS ENTERC
LYMPH NODE, WE PERFORMED A SINGE
CELL LINE OF SEQUENCING EXPERIMT
AND SO WE SORTED GERMINAL CENTEB
CELLS FROM WILDTYPE AND KNOCKOUT
ANIMALS FROM POLICE OFFICER LYMH
NODE THAT HAD BEEN IMMUNIZED FOR
MESS ENTERIC LYMPH NODE.
LIGHT ZONE AND DARK ZONE CLUSTES
ARE HIGHLIGHTED HERE AND THE MES
ENTERIC LYMPH NODE FROM OUR
KNOCKOUT ANIMALS, ENRICHMENT IN
THREE CLUSTERS THAT WERE OUTSIDF
THE LIGHT ZONE AND DARK ZONE.
WHEN WE LOOKED AT GENE SIGNATURS
ENRICHED IN EACH OF THESE CLUST,
WHAT WE FOUND WAS THAT THERE WAS
STRIKING INCREASE IN MULTIPLE
SIGNATURES OF POSITIVE SELECTION
INCLUDING MYC AS WELL AS mTOR 1.
SO, TO VALIDATE THESE GENE
EXPRESSION SIGNATURES, WE STAIND
MYC CHIMERA FOR MYC AND THE LIGT
ZONE GERMINAL CENTER B CELLS, TE
IS INCREASED ABUNDANCE OF MYC
PROTEIN IN THE KNOCKOUTS FROM OG
ALPHA 13 DEFICIENT CELLS.
AND YOU CAN SEE THAT OVER A NUMR
OF MICE HERE.
BUT THERE IS NO CHANGE IN THE
ABUNDANCE OF MYC IN THE POLICE
OFFICER LYMPH NODES SUGGESTING S
IS A SPECIFIC PHENOMENA HAPPENIG
IN THE SITE WHERE THE MICE GET L
THE TUMORS.
SO, TO GET AT A A LITTLE BIT MOE
MECHANISTICALLY OF WHAT THE
MECHANISM IS THAT IS LEADING TO
INCREASED MYC IN THE ABSENCE OFG
ALPHA 13, WE DEVELOPED A CELL LE
MODEL I'M NOT GOING TO GET INTOE
SPECIFIC DETAILS BUT SUFFICE ITO
SAY, WE PUT IN A RECEPTOR THAT S
NOT NORMALLY EXPRESSED IN OUR
GERMINAL CENTER CELL LINE AND WE
CAN ADD A LIGAND.
AND WHEN YOU DO THAT, OVER TIME
THERE IS A DROPOUT OF TRANSDUCED
CELLS WHEN LIGAND IS ADDED AND E
CAN SEE THAT THERE IS A STRONG S
OF MYC AND THEN WHEN WE KNOCKOUG
ALPHA 13 WITH CRISPR/CAS 9 YOU
CAN'T SEE A REDUCTION IN MYC.
SO TO SUMMARIZE SOME DISK WORK I
DON'T HAVE TIME TO SHOW YOU, WE
CAME TO THE CONCLUSION THE PRIMY
REASON WHY THERE WAS A DRAMATIC
LOSS IN MYC WAS DUE TO DECREASED
TRANSLATION OF MYC.
AND THAT DECREASED TRANSLATION S
DUE TO IN ADDITION OF mTOR 1.
SO WHEN WE DID OUR MIXED CULTURE
EXPERIMENT WHERE WE FOUND IF WE
TREATED THESE CELLS WITH RAPAMYN
TO INHIBIT mTOR 1, IN THE
PRESENCE OF STIMULATION OF G ALA
13, OUR KNOCKOUT CELLS CAN OUT W
WILDTYPE COMPETITORS ROBUSTLY AD
THAT IS DECREASED IN THE PRESENE
OF mTOR 1 INHIBITION SUGGESTING
EXAGGERATED mTOR ONE IS
RESPONSIBLE, IN PART, FOR THE
OPERATIVE IN VITRO.
WHEN WE LOOKED AT MYC BONE MARRW
CHIMERA THAT HAS BEEN TREATED WH
RAPAMYCIN, THERE WAS DECREASE IN
THE AMOUNT OF MYC SPECIFICALLY N
OUR KNOCKOUT CELLS SUGGESTING IT
WAS EXAGGERATED mTOR ONE IN THE
MESS TEARIC LYMPH NODE.
SO AS IMMENSED, IN THE
INTRODUCTION -- MENTIONS, THE
CURRENT THINKING IS M TORQUE 1 D
MYC ARE INDUCED AS A RESULT OF
T-CELL DEPENDENT INTERACTIONS.
WE WANTED TO KNOW WHETHER OR NOT
T-CELL HELPS SOMEHOW COULD SUPPT
THE EXPANSION OF G ALPHA 13
DEFICIENT CELLS IN THE MESENTERC
LYMPH NODE.
TO DO THAT, WE TOOK OUR MIXED CE
ERAS AND DEPLETED THEM OF CD4
CELLS.
YOU CAN SEE HERE THAT IN THE
WILDTYPE CELLS IN THE MIXED
CHIMERA, THERE IS A LOSS OF
GERMINAL CENTER B CELLS WHEN WE
DEPLETE CD4 CELLS BUT THERE IS H
LESS OF A LOSS IN THE ABSENCE OG
ALPHA 13, SUGGESTING THAT THEREE
OTHER CUES OTHER THAN T-CELLS
SUPPORTING THEIR SURVIVAL AND/OR
EXPANSION IN THE MESENTERIC LYMH
NODE.
SO, AS I MENTIONED, IN THE ABSEE
OF G ALPHA 13 SIGNALING, CELLS N
EXPAND THROUGHOUT THE FOLLICLE D
ACTUALLY ENTER LYMPHATICS AND WE
WANTED TO KNOW IF THIS EXPANSION
COULD ALLOW THEM TO ACCESS
DIFFERENT ENVIRONMENTAL QUEUES T
WILDTYPE CELLS WOULD NORMALLY NT
SEE.
AND SO, WE INITIALLY DID
IMMUNOHISTOCHEMISTRY EXPERIMENT
WHERE WE STAINED GERMINAL CENTES
FROM WILDTYPE ANIMALS FOR MYC AS
WELL AS LYMPHATIC MARKERS.
SO THERE IS RARE AND POSITIVE CS
IN THE WILDTYPE THAT ARE POSITIE
FOR MYC BUT THERE IS A GREAT
INCREASE IN THE AMOUNT OF MYC
POSITIVE CELLS IN THE KNOCKOUT S
WE SEE, BUT STRIKINGLY OR
INTERESTINGLY WHAT WE ALSO FOUNS
THAT THERE WERE RARE GERMINAL
CENTER B CELLS IN LYMPHATICS THT
ALSO EXPRESSED MYC AND THAT IS
SHOWN IN THE INSETS THERE.
SO PREVIOUS WORK THAT WE HAD DOE
HAD SHOWN THAT A PRO MIGRATORY P
RECEPTOR, MEDIATES PART OF THIS
MIGRATION INTO THE OUTER FOLLICE
AS WELL AS THE LIMB AND WE WANTD
TO KNOW IF THAT COULD BE TARGETD
TO SPECIFICALLY REDUCE THE AMOUT
OF GERMINAL CENTER EXPANSION INE
ABSENCE OF G ALPHA 13.
SO TO DO THAT, WE GENERATED BONE
MARROW CHIMERAS IN WHICH THE BOE
MARROW WAS EITHER WILDTYPE BONE
MARROW THAT HAD A CONDITIONAL
EXPRESSION OF CAS9 IN GERMINAL
CENTER B CELLS WILDTYPE OR KNOCT
FOR G ALPHA 13 AND TARGETED THAT
WITH RETROVIRUS EXPRESSING GUIDE
RNAs.
SO YOU CAN SEE HERE THAT IN OUR
WILDTYPE BONE MARROW CHIMERAS, O
LOSS OF ABUNDANCE IN GENERALINAL
CENTER B CELLS COME S1PR3 IS
TARGETED BUT A STRIKING LOSS INE
MESENTERIC LYMPH NODES IN ANIMAS
THAT -- UNIQUE SURVIVAL FACTOR N
OUR G ALPHA 13 KNOCKOUTS.
ALSO INTERESTINGLY IN THE OTHER
ORGANS IN GERMINAL CENTERS OR
IMMUNIZED PERIPHERAL GERMINAL
CENTERS, THERE WAS NO DEPENDENC,
SUGGESTING THIS IS SOMETHING THT
WAS SPECIFICALLY HAPPENING IN TE
MESENTERIC LYMPH NODE.
SO TO GET AT WHAT OTHER MESENTEC
LYMPH NODE SPECIFIC QUEUES, WE
WANTED TO KNOW IF ANY OTHER GUT
DERIVED QUEUES MIGHT BE IMPORTAT
FOR PROMOTING THESE OUTGROWTHS.
SO THE FIRST QUESTION IS WHETHER
NOT MICROBIOTA IN THE SMALL
INTESTINE COULD BE SUPPORTING TE
EXPANSIONS OF THESE GERM.
SO WE WENT TO THE EFFORT OF
GENERATING GERM-FREE ANIMALS THT
LACKED G ALPHA 13.
AND WHAT WE FOUND IS THAT YOU CN
STILL SEE EXPANSION OF GERMINAL
CENTER B CELLS.
THESE ARE NOT MYC SETTINGS.
SO INCREASE IN GERMINAL CENTERSS
SIMILAR TO WHAT WE SEE IN THE SF
ANIMALS.
ADDITIONALLY, IF WE STAINED THEE
GERMINAL CENTER B CELLS FOR MYC,
YOU CAN SEE THERE WAS ALSO
INCREASED MYC IN THE ABSENCE OF
MICROBIOTA.
SO, WE WANTED TO GET AT OTHER
QUEUES THAT MIGHT BE SUPPORTING
THIS SURVIVAL AND SIMPLISTICALLY
WHAT WE DECIDED TO DO IS FAST ME
FOR 24 HOURS.
SO WHEN WE DID THIS, WHAT WE FOD
IS THAT WHEREAS OUR WILDTYPE CES
WERE SOMEWHAT DECREASED, THERE S
A MUCH LARGER DECREASE IN THE
AMOUNT OF LOSS OF G ALPHA 13 CES
SUGGESTING SOMETHING IN THE DIET
WAS SPECIFICALLY SUPPORTING THE
EXPANSION OF G ALPHA 13 DEFICIET
CELLS.
SO, TO SUMMARIZE A LOT OF WORK,E
FOUND THAT DIETARY FAT OR DIETAY
GLUCOSE COULD NOT DIFFERENTIALLY
SUPPORT THESE CELLS IN VITRO OR
IN-VIVO.
AND WE LOOKED AT ALSO REGULATOR
DEPENDENT AMINO ACID SENSING ANE
DIDN'T THINK THAT WAS INVOLVED.
BUT WE DECIDED TO LOOK AT
GLUTAMINE.
SO GLUTAMINE IS NOT AN ESSENTIAL
AMINO ACID BUT IT CAN DRAMATICAY
SUPPORT mTOR ONE SIGNALING AS
WELL AS MYC EXPRESSION.
AND IT'S SENSING IS NOT CURRENTY
CLEAR WHAT IT'S INTRACELLULAR
SENSORS ARE BUT THEY ARE
INDEPENDENT OF THE REGULATOR
COMPLEX.
SO IN VITRO WE GREW CELLS THAT E
G ALPHA 13 DEFICIENT IN THE
PRESENCE OR ALPHENCE OF STIMULAN
AND ABSENCE OF GLUTAMINE YOU CAN
SEE THAT SIMILAR TO OUR RAPAMYCN
TREATMENT, WHEN WE LOSE GLUTAMIE
THERE IS DECREASED OUT GROWTHS G
ALPHA 13 DEFICIENT CELLS.
WE THEN DECIDED TO PUT MICE ON
DIETS IN WHICH ALL THE PROTEIN S
IN THE FORM OF AMINO ACIDS AND A
CONTROLLED DIET HAD GLUTAMINE AS
WELL AS BHUTANIC ACID.
OUR EXPERIMENTAL DIET LACKED
GLUTAMINE AND GLUTAMINE.
YOU CAN SEE HERE OUR CONTROL
ANIMALS, STRIKING INCREASE IN
AMOUNT OF MYC EXPRESSION IN OURG
ALPHA 13 DEFICIENT CELLS AND THT
IS REDUCED IN MICE THAT ARE ON R
QE DEFICIENT DIET.
ADDITIONALLY WHEN WE LOOK AT
PROLIFERATION, AS ASSESSED BY A
30-MINUTE PULSE, WE CAN SEE HERE
THAT THERE IS A SELECTIVE LOSS F
PROLIFERATION IN OUR KNOCKOUT
ANIMALS.
AND WE ALSO DID THE CONVERSE
EXPERIMENT WHERE WE PUT ANIMALSN
ACCESS GLUTAMINE IN THEIR WATER.
THE OUTGROWTHS OF G ALPHA 13 CES
WERE EXAGGERATED WHEN WE DID THT
FOR THREE WEEKS.
SO YOU CAN SEE HERE WHEN WE DO T
THERE IS INCREASE IN THE ABUNDAE
OF G ALPHA 13 CELLS AND SLIGHT
REDUCTION IN WILDTYPE CELLS.
SO TO SUMMARIZE, WE THINK THAT G
ALPHA 13 LIMITS NUTRIENT-DRIVEN
EXPANSION AND MEAS TAKER LYMPH E
GERMINAL CENTER B CELLS VIA THE
SUPPRESSION OF M TORQUE 1 AND MC
IN THE MESENTERIC LYMPH NODE AND
THIS GREATER ACCESS TO NUTRIENTN
THE MESENTERIC LYMPH NODE COULD
CONTRIBUTE TO THE GUT TROPISM O-
OR THE TUMOR SUPPRESSIVE CAPACIY
OF G ALPHA 13.
SO THIS WORK WAS PRIMARILY DONEY
A VERY TALENTED POSTDOC IN MY LB
AND WE HAD A LOT OF HELP FROM TE
NCI AND OTHERS.
WITH THAT, I WILL TAKE ANY
QUESTIONS.
[ APPLAUSE ]
>> THANK YOU.
GREAT PRESENTATION.
MYC PLAYS A ESSENTIAL ROLE IN TE
HUGE METABOLIC -- BECAUSE OF
T-CELLS ASSUMING THIS IS THE PHE
OF THE B CELLS SO WHAT IS THE
CONSEQUENCES FOR THESE GERMINAL
CENTER B CELLS?
DO THEY HAVE ANY MODULATION OF
METABOLIC PROFILE AND WHAT IS TE
FUNCTIONAL CONSEQUENCES?
>> SO, WE HAVEN'T BEEN ABLE TO O
METABOLOMICS OR ANYTHING BECAUSF
JUST THE NUMBER OF CELLS THAT IS
REQUIRED BUT WE THINK THAT THERS
ALTER METABOLISM.
SO THE FIRST PIECE OF EVIDENCE E
HAVE IS -- SO, MOST GERMINAL CER
B CELLS REQUIRE FATTY ACID
OXIDATION TO SUPPORT ENERGY.
WILDTYPE CELLS DO BUT G ALPHA 13
CELLS DON'T AND WE HAVE DONE THT
IN A NUMBER OF DIFFERENT WAYS.
WE GENERATED CAS9 CHIMERAS, WHEE
WE KNOCKOUT THE ENZYME RESPONSIE
FOR TRANSPORT.
AND SO ADDITIONALLY THEY DON'T M
TO DEPEND UPON GLUCOSE AS MUCH.
WHEN WE KNOCKOUT GLUTE 1, WE SEE
DEPENDENCE IN WILDTYPE BUT NOT
KNOCKOUT.
I CAN'T GIVE YOU SPECIFIC EXAMPS
OF WHAT OTHER THINGS --
>> BEAUTIFUL TALK.
I HAD A QUESTION REGARDING -- YU
SHOWED IN YOUR SLIDE THERE WAS
REALLY NO DIFFERENCE BUT I WONDD
PERHAPS HAVE YOU LOOKED AT PROXL
TO DISTAL IN TERMS OF SMALL
INTESTINE IF THERE IS ANY GRADIT
OF --
>> SO THERE ARE OUTGROWTHS THAT
HAPPEN IN THE MYCS CHIMERA.
I DIDN'T SHOW THAT.
IT'S AN AIR MEDIATED EFFECT.
THE REASON -- AND NONE OF THE ME
DEVELOPED TUMORS IN THEIR BATCH.
AS YOU PROBABLY KNOW, THE AN ATE
OF THE PAIR PATCH IS UNIQUE IN W
THE LIGHT ZONE AND DARK ZONE ARE
FLIPPED AND ADDITIONALLY THERE S
NO LYMPHATICS IN THE PAIRS PATC.
SO THE DISTANCE A METABOLITE WOD
HAVE TO TRAVEL IN THE PAIRS PATH
IS LARGER THAN IN THE MEAS TEARC
LYMPH NODE IN TERMS OF ACCESS.
I THINK THAT MIGHT BE THE REASON
BUT WE HAVEN'T EXPLICITLY SHOWN
THAT.
>> AND SECOND QUESTION.
REGARDING JOURNALS AND REACTION
HAVE YOU LOOKED AT CELL CONTENTN
BONE MARROW, LET'S SAY, AFTER DT
AND INTERVENTIONS --
>> SO I DIDN'T SHOW THAT HERE BT
WE DON'T THINK THAT THERE IS
GREATER GENERATION OF EITHER PLA
CELLS OR MEMORY CELLS IN THE
ABSENCE OF G ALPHA 13.
SO WHEN WE KNOCK IT OUT IN A FAE
MAPPING APPROACH WE SEE GREATER
CLONAL LONGEVITY WITHOUT ANY
INCREASE IN THE AMOUNT OF MEMORY
CELLS AND LIKELY PLASMA CELLS
PRODUCED FROM THOSE GERMINAL
CENTERS.
>> THANK YOU, REALLY COOL.
>> LOVELY TALK.
SO I WAS WONDERING WHEN YOU STAE
THE WHOLE THING, DEPRIVE OF
NUTRITION, OR WHEN YOU'RE ADDING
RAPAMYCIN, ONE OF THE PATHWAYS,
LIKE MOST -- ARTIFICIAL INDUCTI.
HOW DOES THAT EFFECT THIS WHOLE
THING PARTICULARLY SHOWN TO BE
RECENT MEMORY AND -- HOW IS THAT
WORKING HERE?
>> SO, ARE YOU ASKING HOW WE'RE
MAKING SURE THAT WE'RE NOT INDUG
MULTI.
>> STRONG EFFECT IN PHASIA --
>> IN VITRO WHEN WE STIMULATE WH
G ALPHA 13 IN OUR CELL LINES, WE
DON'T INDUCE ANY AUTOPHAGY THROH
G ALPHA 13.
I DON'T KNOW WHY THAT IS, IF WEE
INHIBITING mTOR 1 BUT WE DON'T
SEE ANY EVIDENCE.
WE VIOLENT DONE AS MUCH IN-VIVO.
>> VERY NICE.
DID YOU LOOK AT EXPRESSION OF
NUTRIENT TRANSPORTERS IN THE
KNOCKOUT?
>> SO, WE'VE TRIED TO TAKE THE
OPPOSITE APPROACH AND KNOCKING T
CANDIDATE GLUTAMINE TRANSPORTERN
OUR BONE MARROW CHIMERAS AND WE
HAVEN'T BEEN ABLE TO SEE A SPECC
EFFECT WITH SINGLE TRANSPORTERS.
BUT THERE IS A LOT OF -- AT LEAT
SIX EXPRESSED IN GERMINAL CENTE.
>> DO YOU HAVE ANY IDEA OF THE
DOWNSTREAM SIGNALING PATHWAYS TT
MAYBE EFFECTING --
>> BETWEEN mTOR 1?
THAT'S BEEN REALLY TOUGH.
WE DON'T KNOW.
>> COULD I ASK A QUESTION.
COULD YOU REMIND US WHAT ARE THE
EFFECTS OF THE G ALPHA 13 EFFECS
IN THE KNOCKOUTS --
>> THIS IS IT A CONDITIONAL
KNOCKOUT SO I HAVE NEVER DONE
ANYTHING OUTSIDE OF B CELLS.
SO, OFF THE TOP OF MY -- I'M
DRAWING A BLANK.
>> NO PROBLEM.
>> SO WE THINK T ALPHA 13 IS
IMPORTANT IN -- SO S1PR2 IS THE
RECEPTOR EXPRESSED ON GIRLINAL
CENTER AND WHEN YOU KNOCK IT OUN
T-CELLS, WE THINK THAT THEY CANT
LOCALIZE TO THE GERMINAL CENTER.
IT SHOULDN'T HAVE AN EFFECT.
>> THANK YOU VERY MUCH.
AND THANKS TO ALL THE SPEAKERS N
THIS SESSION.
AND I THINK WE HAVE A BREAK.
DO YOU KNOW -- SO WE RETURN AT 5
SHARP FOR THE TRIUMPHANT CONCLUN
OF THIS AMAZING, MARVELOUS
SYMPOSIUM.
THANK YOU.
>> AS YOU ALL KNOW -- IF YOU DOT
WE'LL BE HAPPY TO INTRODUCE YOU
THE CONCEPT.
BUT ORGANIZING A MEETING DOES CE
WITH SOME WONDERFUL CHANCES,
RESPONSIBILITIES, E-MAILS AND
ORGANIZATION.
ANDULE OF US ARE REALLY LUCKY TO
HAVE THIS WONDERFUL TEAM OF PEOE
WHO ARE HELPING US.
SO THAT MEANT WE DIDN'T HAVE TOO
LOTS AND LOTS OF DIFFERENT THIN.
THEY REALLY MANAGED TO GET THISL
OFF AND WE OWE YOU A HUGE GRATIE
OF THANKS AND I'D LIKE TO THANK
JULIA AND HER TEAM AND OUR
WONDERFUL SUPPORT.
SO WE HAVE SOMETHING FOR YOU ANE
ARE REALLY HAPPY IF YOU CAN COME
CELEBRATE TOGETHER.
[ APPLAUSE ]
>> WOW!
THANK YOU!
[ APPLAUSE ]
FOR THOSE WHO ARE SPEAKERS, THEY
KNOW JUST HOW MUCH WE WERE ABLEO
RELY ON THIS WONDERFUL TEAM ANDO
HAVE EVERYTHING ORGANIZED.
SO AGAIN, A HUGE THANKS FOR ALLU
DO.
WE KNOW THAT BASICALLY WHAT HAPS
IS EVERY TIME THERE IS A PROBLE,
EVERY TIME THE SLIDE FLICKED ORA
NOISE OR SOMETHING -- JULIA I'MN
TROUBLE!
SO WE BITCH A LOT BUT WE DO WANO
ALSO SAY THANK YOU.
SO THANK YOU VERY MUCH FOR ALL R
HELP.
[ APPLAUSE ]
>> WELCOME TO THE LAST SESSION F
THIS REALLY TERRIFIC MEETING ON
CANCER IMMUNOLOGY PART II.
OUR FIRST SPEAKER IS DARIO VIGN.
I'M AFRAID WE DON'T HAVE ANY
FLOWERS FOR YOU BUT --
>> SHOULD I LEAVE NOW?
>> WE ARE LOOKING FORWARD TO
HEARING ABOUT LAG3.
SO DARIO.
>> THANK YOU.
THANK YOU VERY MUCH.
FIRSTLY, THANKS SO MUCH TO THE
ORGANIZERS, REALLY A PLEASURE TE
HERE TO HAVE THIS OPPORTUNITY TO
TALK TO YOU.
IT'S BEEN A FANTASTIC MEETING.
HOPEFULLY IT DOESN'T GO DOWNHILL
FROM HERE.
I'M GOING TO TALK TO YOU ABOUT
LAG3.
I'M GOING TO TAKE A LITTLE BIT A
LEAF OUT OF CLAUDIA'S BOOK AND T
TALK DEEPLY ABOUT ONE QUESTION R
ONE PROJECT, BUT RATHER START WH
AN OVERVIEW OF WHAT LAG3 DOES, R
THOSE WHO DON'T KNOW MUCH ABOUT,
TALK A LITTLE BIT ABOUT A RECENT
PAPER THAT CAME OUT, VERY, VERY
BRIEFLY, AND THEN MOST OF THE RT
WILL BE TALKING ABOUT TWO
UNPUBLISHED STORIES, HOPEFULLY Y
WON'T BE UNPUBLISHED FOR TOO MUH
LONGER.
AND TRY AND THROW IN A LITTLE BT
OF PROVOCATIVE COMMENTS AND
STATEMENTS TO INDUCE SOME THOUGT
PROVOKING CONSEQUENCES AND MAYBE
GET YOU A LITTLE BIT EXCITED ABT
LAG3 AND HOW IT MIGHT BE PLAYING
ROLLS IN YOUR FAVORITE PROJECTS.
THESE ARE MY DISCLOSURES.
OBVIOUSLY I'LL TALK ABOUT LAG3 O
IF I SEEM OVERLY ENTHUSIASTIC OR
CONFLICTED, YOU'LL UNDERSTAND W.
SO LAG3 IS IN MANY WAYS LIKE OTR
INHIBITORY RECEPTORS, LIMITS T-L
FUNCTION, HOMEOSTASIS AND A VARY
OF ACTIVITIES T HAS ROLLS IN OTR
CELL TYPES BUT I'M REALLY GOINGO
FOCUS ON T-CELLS TODAY.
IMPACTS TCR SIGNALING LIKE PD-1T
UNTIL RECENTLY AN UNKNOWN MODE F
FUNCTION, WHICH I'LL TALK ABOUTN
A LITTLE BIT.
IT HAS A VARIETY OF LIGANDS WITH
SOME CONTROVERSIES WHICH I WON'O
INTO, BUT CONSIDERED CANONICAL
LIGANDS.
AND RAPIDLY SHED BY PROTEASES AD
I'LL TALK ABOUT THAT BRIEFLY, WH
IS SORT OF UNUSUAL COMPARED TO E
OTHER INHIBITORY RECEPTORS.
IT IS, AS YOU HAVE HEARD,
ESPECIALLY NICELY FROM PAM AND
TANLY'S PRESENTATIONS AND OTHER,
INHIBITORY RECEPTOR AS PART OF A
COHORT OF INHIBITORY RECEPTORS T
ARE UPREGULATED ON CHRONICALLY
EXHAUSTED T-CELLS IN CANCER AND
VIRAL INFECTIONS.
WE SHOWED RECENTLY OR A NUMBER F
YEARS AGO, FIRST IN COLLABORATIN
WITH JOHN WEARY IN CHRONIC LCMV
CLONED 13 MODELS AND SUBSEQUENTY
IN TUMOR MODELS, THE COMBINATORL
BLOCKADE OF LAG3 IN PD-1 CAN GIE
RISE TO SYNERGISTIC ACTIVITY.
TO GIVE YOU A SENSE WHAT HAVE TT
ACCIDENT LOO LIKE, THESE ARE
CLASSIC TUMOR GROWTH MODELS EACH
LINES AND INDIVIDUALS MOUSE.
AND YOU CAN SEE WITH THESE TUMOR
TYPES THE TUMORS GROW OUT.
SINGLE MODALITY ANTI-LAG3 OR PD1
GIVES RISE TO SOME TUMOR REDUCTS
BUT RELATIVELY LIMITED TO A
CLEARANCE.
AND THE IT'S ONLY REALLY WHEN YU
SEE THE COMBINATION THAT YOU'RE
GETTING SIGNIFICANT TUMOR
CLEARANCE.
SO THIS COMBINATORIAL SYNERGISTC
ACTIVITY INDUCED A LOT OF INTERT
IN DEVELOPING CLINICAL ANTIBODI[
INAUDIBLE ]
TO ENTER THE CLINIC, THERE ARE W
OVER 16 DIFFERENT ANTIBODIES INE
CLINIC EITHER MONOSPECIFIC LAG3
ANTIBODIES, SPIKE SPECIFICS, THE
MOST ADVANCED IN TERMS OF
REGISTRATIONAL CLINICAL ACTIVITY
WAS RELATIVITY 47 -- [ INAUDIBL]
PHASE III TRIAL IN TREATMENT NAE
METASTATIC MELANOMA PATIENTS THT
MET ENDPOINT WITH PROGRESSION-FE
SURVIVAL IT WAS REPORTED BY THEW
ENGLAND JOURNAL OF MEDICINE THED
OF LAST YEAR.
AND THEN SUBSEQUENT DATA SHOWING
THAT IN THE NEW ADVENT SETTING T
HAD EVEN A GREATER EFFICACY, AT
LEAST IN TERMS OF PATHOLOGICAL
RESPONSE IN RECEPTIBLE MELANOMA.
THIS LED TO FDA APPROVAL OF FIXD
DOSE COMBINATION OF DUO LAG FORE
TREATMENT OF UNRECEPTAL AND
METASTATIC MELANOMA APPROVED ATE
BEGINNING OF LAST YEAR.
SO WITH ALL OF THIS EXCITEMENT D
CLINICAL ACTIVITY, I THINK MOSTF
US APPRECIATE THAT WE STILL HAVN
MANY WAYS A LONG WAY TO GO, MANF
THE CHALLENGES THAT FACE LAG3 AE
THE SAME AS FACED WITH PD-1, AND
THAT IS THE REALITY IS WE STILL
DON'T REALLY UNDERSTAND HOW IT
WORKS.
AND YOU CAN THINK ABOUT, WHAT DS
THAT MEAN?
THINK ABOUT HOW SOMETHING WORKS
EITHER INSIDE THE CELL OR OUTWAD
CONSEQUENCES OF THAT.
SO I'LL TALK A LITTLE BIT ABOUT
THAT TODAY.
SO FIRSTLY, VERY BRIEFLY ABOUT A
PAPER THAT IS ALREADY PUBLISHEDN
THE INHIBITORY SIGNALS BRAS IT S
RELEVANCE TO WHAT I'M GOING TO K
ABOUT, AND THEN TALK A LITTLE BT
MORE -- AGAIN ONLY RELATIVELY
BRIEFLY WITH THE TIME AVAILABLE,
WITH SOME MOUSE STUDIES AND THET
THE END WITH SOME HUMAN STUDIES.
SO, HOW DOES LAG3 WORK?
AND THIS IS AN UNFORTUNATELY VEY
LONG STORY THAT TOOK ALMOST 10
YEARS TO COMPLETE.
PERHAPS YOU'LL SEE A LITTLE BIT
WHY.
DRIVEN BY TWO FANTASTIC PEOPLE N
THE LAB, POSTDOC IN THE LAB, ATE
TAIL END EVER MY TIME AT ACCEPT
JUDE, CRAIG, AND A LOT OF BENEFT
AND A LOT OF PRAISE FROM A LOT F
THE LAG3 STUFF THAT WE HAVE DON.
HE HADS BEEN IN THE LAB FOR 25
YEARS WORKING ON LAG3 AND SENIOR
AUTHOR ON THIS PAPER.
AND WE ASKED TWO BASIC QUESTION.
HOW DOES LAG3 MEDIATE INHIBITORY
ACTIVITY?
AND ALSO TRYING TO UNDERSTAND
WHETHER THE MHC CLASS TWO WAS
REALLY REQUIRED FOR LAG3 FUNCTI?
AND THIS WAS DRIVEN BY THREE
CURIOUS OBSERVATIONS.
THE FIRST IS AN ANTI-LAG3 ANTIBY
THAT IF ANY OF YOU DO ANYTHING 3
BLOCKING ANTIBODY EXPERIMENTS, U
LIKELY USE WE MADE MANY DECADES
AGO, DOES NOT BLOCK MHC PLUS 2
LIGAND INTERACTION.
RAISING THE QUESTION OF HOW CANT
WORK IF IT DOESN'T BLOCK THAT
INTERACTION?
THE INTERACTION IS BLOCKED BY AL
THE CLINICAL ANTIBODIES TO THE
EXTENT WE KNOW.
THE SECOND POINT IS LAG3 INHIBIY
ACTIVITY UNLIKE CROSSLINKING, IT
DOESN'T DRIVE AN INHIBITORY SIG,
WHICH WAS ALSO CONFUSING IN TERS
OF THINKING ABOUT HOW IT MIGHT K
INTRACELLULARLY.
AND THEN THE THIRD INTERESTING
THING IS A LONG TIME AGO IN
HYPERBAKER EXPERIMENTS WE SHOWN
THAT LAG3 ONLY HAS INHIBITORY
ACTIVITY IN THE PRESENCE OF
CORECEPTORS, CD4 AND 8.
WE SPECULATED EARLY ON THAT MAYE
THAT IS WHAT LAG3 IS TARGETING
FUNCTIONALLY.
THIS IS JUST TO INDICATE THAT TS
IS THE SIGHT'S PLASMIC DOMAIN OF
LAG3 AND A PHYLOGENETIC
RELATIONSHIP TO OTHER SPECIES.
AND I APPROXIMATE UT THIS UP HEE
TO KIND OF STILL STRAIGHT TWO
THINGS.
THE FIRST IS, THERE ARE NO OBVIS
MOTIFS THAT MIGHT ASCRIBE FUNCT.
NO ITEMS OR TYROSINES.
AND IT WASN'T AS IF WE COULD LOK
AT THIS THE AND SAY HA HA!
THIS IS HOW POTENTIALLY IT MIGHT
WORK.
THE ONE KEY FEATURE AS YOU CAN E
HERE, IS AN EXTENSIVE NUMBER OF
BHUTANIC ACIDS.
SO LOOKING THROUGH THE LITERATU,
WE IDENTIFIED A COUPLE OF THOUGS
THAT DROVE A LOT OF THE PROJECT,
WHICH I WON'T GO INTO DETAIL IN
TERMS OF THE DATA BUT SUMMARIZEN
THE NEXT SLIDE AMOUNT COUPLE OF
CURIOUS FEATURES.
THE FIRST IS WITH ALL THE BLEW
TANNIC ACIDS, THE CYTOPLASMIC
DOMAIN HAS ACIDIC PI.
THE PICD1 AND CD8 CYTOPLASMIC
DOMAINS IS ALSO VERY BASIC AND E
DID A LOT OF EXPERIMENTS TO SHOW
THIS SUBSTANTIAL INTERACTIONS
BETWEEN THOSE TWO SIGHT'S PLASMC
DOMAINS.
ELECTROSTATIC INTERACTIONS WHICE
COULD DUPLICATE IN A SIMPLIFIED
SYSTEM.
GIVEN THIS HIGH NUMBER OF ACIDSE
WONDERED IF IT MIGHT HAVE LOCAL
PERTURBATION OF THE MEMBRANE WHH
WE DID SHOW IN A NUMBER OF SUPER
RESOLUTION MICROSCOPY EXPERIMEN.
AND THEN THIRDLY, FOUND A RATHER
OBSCURE STUDY WHICH SUGGESTED TT
IN THE SAME WAY THAT DICYSTEINSN
COLLATE METAL IONS, ALTERNATING
BLEW TANNIC ACIDS COULD DO THAT.
THE REASON IT WAS OF INTEREST IS
BECAUSE RATHER UNUSUALLY, THE
CORECEPTORS INTERACT WITH THE
DOWNSTREAM KINASE BY THIS
INTRIGUING DIOCESESTEIN ZINC CLS
THAT WAS ELUCIDATED BY STEVE
HARRISON AT HARVARD A NUMBER OF
YEARS AGO.
AND SO WE WONDERED WHETHER THISS
ACTUALLY THE TARGET OF LAG3.
SO IN THE INTEREST OF TIME, I'M
GOING TO BASICALLY SUMMARIZE WHT
THIS PAPER SHOWED.
AS MANY OF YOU KNOW, THE STANDAD
MECHANISM OF TCR SIGNALING IS
ENGAGEMENT WITH PEPTIDE COMPLEXS
AND BRINGS IN CD4 AND 8 AS WE HA
NUMBER OF PEOPLE TALK ABOUT.
LCK BROUGHT CLOSE TO THE T-CELL
RECEPTOR AND THEN MEDIATES THE Y
EARLIEST DOWNSTREAM SIGNALING
EVENTS AND PHOSPHORYLATIONS OF -
IN THE PRESENCE OF LAG3, WE SHOD
THAT LAG3 CAN PHYSICALLY INTERAT
WITH THE T-CELL RECEPTOR BY COIP
AND SUPER RESOLUTION MICROSCOPY
APPROACHES.
THIS PUTS IT INTO CLOSE PROXIMIY
WITH CD4 AND CD8.
THESE OPPOSING CHARGES IN THE
SILENT'S PLASMIC DOMAINS ALLOW R
ELECTROSTATIC INTERACTIONS AND O
WE CAN SHOW YOU USING A CASE
SENSITIVE DYE THAT LOCAL PH OF E
IMMUNOLOGICAL SYNAPSE IS REDUCEY
ONE PH, WHICH DOESN'T SEEM VERY
MUCH, BUT WAS SUFFICIENT AS
DEMONSTRATED BY SOME NMR
SPECTROSCOPY EXPERIMENTS, TO WEN
THE ASSOCIATION OF LCK WITH THE
CORECEPTOR AND WE COULD SHOW BY
COIP EXPERIMENTS THAT THAT RESUD
IN ESSENTIALLY A LOSS OF LCK
ASSOCIATION WITH THE CORECEPTOR,
HENCE MIDE QUESTION.
SO BASICALLY, LAG3 IS NOT AS
SIGNALING MOLECULE.
IT'S A SIGNAL DISRUPTOR.
IT MEDIATES THE INHIBITORY ACTIY
BY ASSOCIATING WITH THE T-CELL
RECEPTOR REDUCING CORECEPTOR
DISASSOCIATION AS A CONSEQUENCEF
LOWERING THE LOCAL PH BINDING TO
ZINC AND THEREFORE INHIBITING
DOWNSTREAM SIGNALING.
AND YOU COULD ASK THE QUESTION,Y
HAVE SUCH A COMPLICATED, ODD
MECHANISM OF ACTION?
WE THINK THE REASON IS BECAUSE S
PRO EVOLVED WITH WHAT IS ALSO AN
UNUSUAL INTERACTION OF CORECEPTR
WITH LCK BY AS A CONSEQUENCE OF
THIS DICYSTINE.
SO THESE TWO MECHANISMS EVOLVED
TOGETHER AND THEREFORE UNIQUELY
SPECIFIC.
THIS OPENS QUESTIONS AS TO WHETR
OR NOT LAG3 HAS ACTIVITIES IN CS
THAT DON'T EXPRESS CD4 AND CD8.
THIS WAS RAISING QUESTIONS AND T
IS OBVIOUSLY STILL TO BE
DETERMINED.
THE OTHER THING I WANT TO TOUCHN
IS WE KNOW FROM OUR OLD IMMUNOLY
TEXTBOOKS THAT SIGNAL ONE IS
DOMINANT AND SIGNAL TWO IS REQUD
BUT SECONDARILY OF IMPORTANCE IN
TERMS OF T-CELL ACTIVATION.
AND WHAT I SHOWED YOU IS THAT L3
IS REALLY TARGETING SIGNAL ONE D
SOME BEAUTIFUL WORK FROM OTHERSS
SHOWN THAT WHILE PD-1 COULD EFFT
SIGNAL 1 IT HAS A DOMINANT EFFET
ON CD28.
SO YOU THINK THE LAG3 SHOULD BEA
DOMINANT INHIBITORY RECEPTOR OVR
PD-1 BUT WE CAN SEE THAT'S NOT E
CASE IN THE CLINIC.
SO WHY?
SO ONE POSSIBILITY IS SIMPLY
BECAUSE PD-1 IS MORE BROADLY
EXPRESSED AND SO MAYBE THAT'S WY
IT'S DOMINANT.
BUT THE ONE THING I'D LIKE TO
PROPOSE IS MAYBE THE REASON THAT
LAG3 IS NOT MORE DOMINANT IS
BECAUSE IT'S RAPIDLY SHED BY
METALLIC PROTEASES AND THAT PROS
IS CRITICALLY IMPORTANT IN
MITIGATING INHIBITORY ACTIVITY.
WE DID ONE STUDY A NUMBER OF YES
AGO BY LAWRENCE ANDREWS, TALENTD
POSTDOC IN THE LAB WHOSE IN THE
AUDIENCE.
AND WHAT HE DID WAS TO MAKE A
NON-SHEDABLE CONDITIONAL KNOCK-N
MOUSE.
SO WE COULD BASICALLY IN A CELL
DEPENDENT MANNER, EXPRESSING
NON-SHEDABLE LAG3 AND ASK HOW TT
INFLUENCED T-CELL BIOLOGY.
AND THIS IS JUST A SLIDE TO
SUMMARIZE THIS ON WHAT HE SHOWE.
WHEN T-CELLS EXPRESS A NON-SHEDE
LAG3, AND EVALUATING IN A MODEL
THAT IS PD-1 SENSITIVE, THOSE ME
BECOME THEN PD-1 RESISTANT.
SO LAG3 BECOMES DOMINANT OVER P1
AND BLOCKING PD-1 IS INSUFFICIET
TO DRIVE AN EFFECTIVE ANTI-TUMOR
IMMUNITY RESPONSE, AT LEAST IN S
MODEL.
THIS WAS ALSO SORT OF FOLLOWED P
WITH SOME HUMAN STUDIES LOOKINGT
COHORTS OF PATIENTS WITH EITHER
HEAD AND NECK CANCER OR METASTAC
MELANOMA AND SHOWED A VERY TIGHT
INVERSE RELATIONSHIP BETWEEN LA3
EXPRESSION AND ADAM 10 SO WHEN T
WAS HIGH, VERY LITTLE LAG3 ON TE
CELL SURFACE AND VICE VERSA.
AND ALSO THE PATIENTS THAT HAD H
ADAM 10 AND THEREFORE LOW LAG3 E
MUCH MORE RESPONSIVE TO ANTI-PD1
THAN THE CONVERSE.
SO PULLING THIS TOGETHER, LAG3
INHIBITORY ACTIVITY IS TIGHTLY
REGULATED BY CELL SURFACE SHEDD,
SOMETHING THAT NEEDS TO BE
CONSIDERED IN EVALUATING,
ESPECIALLY IN THE CLINIC LAG3
THERAPEUTICS.
NOW TO TALK A LITTLE BIT MORE AT
THE FUNCTIONAL SIDE OF THINGS.
WE HAVE BEEN SORT OF LOOKING AT
THIS IN PARALLEL BOTH IN MOUSE
STUDIES AND IN CLINICAL TRIALS H
THE HOPE OF LEARNING █FROM BOTHF
THESE IN A CROSS DIRECTIONAL
MANNER.
I'M GOING TO TALK REALLY JUST AT
CD8 TODAY BUT WE HAVE ONGOING
STUDIES TRYING TO LOOK AT THE
IMPACTS OF LAG3 PD-1 AND THE ENE
TUMOR MICROENVIRONMENT.
SO WITH REGARD TO ITS ACTIVITY N
CD8, WE LOOKED AT THIS IN TWO
DIFFERENT MODEL SYSTEMS, EITHER
USING CONDITIONAL FLUXED MOUSE
MODEL SYSTEMS, WHICH I WON'T TAK
ABOUT, AND IN PARALLEL, USING
ADOPTED TRANSFER SYSTEMS OF A TR
REACTED T-CELL POPULATION, P MA,
TO RECOGNIZE GP100.
AND YOU CAN DO THESE EXPERIMENT.
IF YOU ADOPTIVELY TRANSFER A LAE
NUMBER OF CELLS, T-CELLS ARE
SUFFICIENT TO CLEAR GP100
EXPRESSING B16.
HOWEVER IF YOU REDUCE THE NUMBEO
10,000, IT REALLY HAS NO EFFECT
ALLOWING US TO LOOK AT THE
CONSEQUENCE OF PD-1 LAG3 OR THE
COMBO DELETION.
WHAT YOU CAN SEE HERE IS
ESSENTIALLY A RECAPITULATION OF
WHAT YOU WOULD SEE WITH ANTIBODY
TREATMENT THAT LAG3 ALONE HAS VY
LITTLE EFFECT.
PD1 HAS AN EFFECT BUT IT'S ONLY
WITH A DOUBLE DELETION THAT YOUE
A BIG REDUCTION IN TUMOR GROWTHD
YOU CAN SEE THIS BOTH IN THE
PROPHYLACTIC AND THE THERAPEUTIC
SETTING.
THIS ALLOWED US TO INTERROGATE E
INDIVIDUAL ROLES OF PD-1, LAG3 D
THE COMBINATION TO SORT OF
HIGHLIGHT JUST A SNIPIT OF DATA
FROM WHAT IS A VERY LARGE DATAS.
ONE OF THE BIGGEST PHENOTYPES IA
SUBSTANTIAL INCREASE IN T-CELL
INFILTRATION.
WHEN YOU DELETE BOTH PD-1 AND L3
AS INDICATED HERE.
ONE EXPERIMENTAL APPROACH WE USD
TO ASK THIS QUESTION IN A
COMPETITIVE ENVIRONMENT BECAUSEE
TUMOR IS SMALLER, IS IT THE THE
TUMOR IS GETTING SMALLER OR CHAE
IN T-CELL ACTIVITY THAT IS
INFLUENCING THIS?
IT'S TO DO A QUADTRANSFER.
SO THESE ARE CONGENICALLY MARKED
CELLS THAT WERE TRANSFERRED AT E
SAME TIME IN EQUAL RATIOS OF 25%
EACH AND THEN ASK, WHAT IS THE
REPRESENTATION OF THESE FOUR
GENOTYPES IN THE TUMOR
MICROENVIRONMENT?
WHAT CAN YOU NICELY SEE IS THE
DELETION OF PD-1 LAG3 RESULTS IA
SUBSTANTIAL COMPETITIVE ADVANTAE
OVER THE WILDTYPE CELLS BUT WHAS
INTERESTING IS THE LAG3 ALONE DS
HAVE SOME EFFECT IN TERMS OF HAG
A PHENOTYPE EVEN THOUGH THAT'S
INSUFFICIENT TO HAVE AN EFFECT N
THE THERAPEUTIC OUTCOME.
AND AS EXPECTED, PD-1 ALSO HAS A
COMPETITIVE ADVANTAGE WHEN DELE.
IN TERMS OF ADDITIONAL FEATURES
THAT ARE SPECIFIC TO LAG3, LAG3
SEEMED TO HAVE A VERY KEY ROLE N
MODULATING TOX EXPRESSION.
WHEN YOU DELETE LAG3 ALONE, EVEN
THOUGH THERE ISN'T MUCH EFFECT N
TERMS OF INCREASED ANTI-TUMOR
ACTIVITY, YOU CAN SEE A SUBSTANL
REDUCTION OF TOX EXPRESSION
FOLLOWING LAG3 DELETION.
AND OF COURSE THE GREATEST
REDUCTION OCCURS WITH THE DOUBLE
KNOCKOUTS.
IN TERMS OF PD-1, IT'S DOM
PERIGNONINANT TYPE WAS INCREASEN
EFFECTOR ACTIVITY.
GAMMA INTERFERON PRODUCTION BUTU
CAN SEE THAT THERE IS A SUBSTANL
INCREASE WITH THE COMBINATION AS
INDICATED HERE.
THIS MADE US ASK THE QUESTION, T
IS THE ROLE OF GAMMA INTERFERON?
MOST OF THE FOCUS HAS BEEN
INFLUENCE OF THE TUMOR
MICROENVIRONMENT ITSELF BOTH THE
CELLS AND ALSO THE TUMOR.
BUT SOME EXTENSIVE SINGLE CELL
RNA-SEQ AND OTHER STUDIES MADE S
SUSPICIOUS THE PRIMARY ACTIVITYY
NOT ACTUALLY BE OTHER CELLS IN E
TUMOR MICROENVIRONMENT USING OTR
GENETIC APPROACHES AND ANTIBODY
BLOCKADE.
MADE US FOCUS ON THE ROLE OF GAA
SIGNALING INTRINSICALLY INTO THE
CD8 AND THE DEFINITIVELY EXPERIT
BUT -- THAT LAWRENCE AND SMOTHES
IN THE LAB DID WAS TO DO A CRISR
MEDIATED -- NOT CRISPR MEDIATED.
A KNOCK DOWN OF THE GAMMA
INTERFERON RECEPTOR IN THESE
ADOPTIVELY TRANSFERRED P MALL
CELLS.
SO WE TOOK THE DOUBLE DEFICIENT
CELLS AND THE WILDTYPES AND KNOD
DOWN GAMMA INTERFERON RECEPTOR D
ASKED WHETHER OR NOT THAT IMPACD
THE ENHANCED ANTI-TUMOR IMMUNITE
SEEN.
AS YOU CAN SEE, THAT INCREASED
ANTI-TUMOR IMMUNITY WAS COMPLETY
LOST WHEN YOU REMOVED THE GAMMA
RECEPTOR, SUGGESTING THE KEY ROE
OF GAMMA IN THIS SETTING WAS CEL
INTRINSIC RATHER THAN CELL
EXTRINSIC.
SO MOVING ON TO THE HUMAN STUDI.
WE INITIATED THIS IN COLLABORATN
WITH OUR CLINICAL COLLEAGUES --O
USED TO BE A RESEARCH ASSISTANT
PROFESSOR IN THE LAB WHEN WE
STARTED THESE STUDIES.
NO YOU SHE HAS HER OWN LAB FOCUG
ON B CELLS AND TLS.
WE GOT TOGETHER TO INITIATE A
BIOMARKER FOCUSED PHASE II CLINL
TRIAL IN TREATMENT NAIVE.
SO THESE PATIENTS HADN'T RECEIVD
ANY OTHER PRIOR TREATMENTS.
THESE ARE METASTATIC NON RESECTE
MELANOMA PATIENTS.
SO IT WAS REALLY AN ENORMOUS AMT
OF FIRST FROM THE TEAM TO EXTRAT
THE HIGH QUALITY DATA WITH
RELATIVELY FEW CORE BIOPSIES WE
COULD OBTAIN FROM THESE PATIENT.
WHAT WAS PARTICULARLY UNIQUE ABT
THIS TRIAL IS IN ADDITION TO A O
AND COMBO ARM WHICH WOULD BE
EXPECTED, WE DID GET APPROVAL FA
LAG3 ONLY ARM, WHICH WAS
CHALLENGING TO GET THROUGH THE B
BECAUSE IT HAS NO MONOEFFICACY.
BUT THEY ALLOWED US TO ENROLL
PATIENTS AS LONG AS WE RESTRICTD
TO TWO CYCLES.
SO PATIENTS WERE ENROLLED.
THERE WERE TWO BIOPSY AND BLOOD
DRAW PRIOR TO TREATMENT.
THEY GET TWO CYCLES TWO WEEKS EH
AND THEN AFTER 4 WEEKS ALL THE
PATIENTS GO ON THE DOUBLE LET.
THAT'S THE BENEFIT TO THE PATIE.
I'M NOT GOING TO TALK ABOUT DATE
EVALUATED POST THAT TIME POINT D
FOCUS JUST ON PRE-POST FOUR-WEEK
WINDOW.
WE FOCUSED ON THAT BECAUSE A LOF
PRIOR STUDIES HAVE SHOWN THAT MH
OF THE ACTIVITY DOWNSTREAM OF
ANTI-PD-1 IS EVIDENT WITHIN THE
FIRST 2-4 WEEKS.
SO WE FELT CONFIDENT IF THESE
THINGS WERE GOING TO HAVE SOME
EFFECT ON THE FUNCTION AND IMMUE
TRANSCRIPTOME, WE WOULD SEE IT N
THIS WINDOW.
AGAIN, WE FOCUSED VERY HEAVILY N
TRANSCRIPTOMIC APPROACHES.
WE HAVE DONE SOME SPACIAL ANALYS
AND FLOW BUT I WON'T TALK ABOUT
THAT TODAY.
AND I'M GOING TO FOCUS ON CD8, I
SAID.
PARTLY BECAUSE COMPUTATIONAL
ANALYSIS CLEARLY SHOWED THAT THE
HIGHEST LEVELS OF PD-1 AND LAG3
WERE EVIDENT IN CD8s AND WE SAW
HUGE CHANGES IN OTHER CELL
POPULATIONS BUT AS I SAID I'M NT
GOING TO FOCUS ON THAT TODAY.
SO JUST TO HIGHLIGHT SOME OF THE
KEY OBSERVATIONS.
WE USED A WHOLE SERIES OF
COMPUTATIONS APPROACHES.
I'M HAPPY TO GO INTO THIS LATER,
USING BOTH CONVENTIONAL LIGHT AD
THE BASED CLUSTERING ANALYSIS AD
ALSO RNA VELOCITY PSEUDOTIME
ANALYSIS, TO ASK WHAT GENE PROGS
WERE SELECTIVELY MODULATED BY TE
IMMUNOTHERAPIES WITH THE NOTION
THAT WE WOULD AT LEAST AS A FIRT
MEASURE, EXPECT TO SEE WHAT OTHS
HAVE SEEN FOLLOWING TREATMENT WH
PD-1 BLOCKERS.
SO, ONE OF THE KEY THINGS THAT S
EXPECTED IS BOTH AN INCREASE TO
SOME EXTENT, OF EFFECTOR PROGRAS
AND A REDUCTION OF EXHAUSTION
SIGNATURES AT LEAST WITH NEVO.
AS MANY MAY KNOW, THE PRIMARY
TARGET OF PD-1 IMMUNOTHERAPIES S
THE PRE EXHAUSTIVE POPULATION TT
STAN WAS TALKING ABOUT, WITH
REDUCTION OF EXHAUSTION.
AND SO, AS EXPECTED, WE SAW THIS
REDUCTION OF EXHAUSTION PHONOTYE
WITH AN INCREASED TO SOME EXTENF
EFFECTOR SIGNATURES BUT WHAT WE
WERE SURPRISED TO SEE IS A
SUBSTANTIAL INCREASE IN THE DOUE
LET BUT SURPRISINGLY A RETENTIOF
EXHAUSTION SIGNATURES WITH THE
DOUBLE LET.
WE DID NOT SEE A REDUCTION OF
EXHAUSTION SIGNATURES WHICH MIGT
HAVE BEEN ANTICIPATED.
AND THE THIS IS THE MOST EVIDENE
IF WE DO AN X WIDE PLOT, THE
MAJORITY OF CD8s ARE BOTH
INCREASING EFFECTOR SIGNATURES,
BOTH AT THE LEVEL OF GAMMA
INTERFERON SIGNALING AND TCR
SIGNALING, BUT RETAINING AN
EXHAUSTION SIGNATURE.
THIS WAS CONFUSING BECAUSE WE WD
EXPECT THIS TO RESULT IN A
LIMITATION OF ANTI-TUMOR ACTIVIY
NOT THE ENHANCED ANTI-TUMOR
ACTIVITY, WHICH IS SEEN IN THE
CLINIC.
SO LASTLY, WHAT WE WANTED TO DOS
UNDERSTAND THE TRANSCRIPTIONAL
UNDERPINNING OF THIS PROCESS.
SO WE USED A PROGRAM CALLED SEE
NICK WHICH INTEGRATES BOTH
TRANSGRESSION FACTOR MODULATIOND
THE GENES DOWNSTREAM OF THAT, TE
REG LON SYSTEM AND WAS SURPRISEO
SEE NOT ONLY IN REGULATION OF
TRANSCRIPTION FACTOR FUNCTION, H
AS BLIMP AND BAT F, BUT ALSO A
RETENTION AND CENTRAL ROLE FOR
MAINTENANCE OF TOX EXPRESSION.
SO YOU MIGHT ASK THE QUESTION, Y
IS TOX BEING MAINTAINED?
WHAT I'D LIKE TO KIND OF THROW T
AS A FEW IDEAS IS THERE HAS BEEA
NUMBER OF STUDIES WHICH HAVE
SUGGESTED THAT TOX IS ALSO
IMPORTANT FOR T-CELL LONGEVITY.
WE SHOWED THIS IN A STUDY LAST R
LOOKING AT THE QUESTION OF WHY L
IMMUNE T-CELLS DO NOT BECOME
EXHAUSTED EVEN THOUGH THEY RECEE
CONTINUOUS TCR STIMULATION?
IN THIS SETTING THEY DO MAINTAIN
TOX EXPRESSION.
A LOT OF EXHAUSTION PHENOTYPES D
YET ARE OBVIOUSLY DRIVING THE
DISEASE.
AND ALSO CORATIVELY, YOU MAY REL
SOMEWHAT CRAZY PAPER THAT DAVE
PUBLISHED RECENTLY WHERE THEY DA
10-YEAR SERIAL TRANSFER OF CELLS
AND SHOWED THAT THESE CELLS GET
HIGH LEVELS OF TOX EXPRESSION AD
YET MAINTAIN A MEMORY TYPE.
SUGGESTING THAT TOX MAY BE
PREDOMINANTLY DRIVING T-CELL
LONGEVITY.
EXHAUSTED CELLS ARE NOT DEAD.
AND SO IT MAY BE IMPORTANT TO
MAINTAIN TOX TO ENSURE THAT YOUT
A DURABLE ANTI-TUMOR RESPONSE.
LAG3 IS A THIRD CHECKPOINT BECAE
IT'S THE THIRD ONE TO BE APPROVD
FOLLOWING PD, APPROXIMATE.
DL1 AND CTLA4.
IMPORTANT FOR LAG3 AND THE FIRST
NEW FDA APPROVAL FOR -- LAG3 ISA
SIGNAL DISRUPTOR THAT MEDIATES
ACTIVITY BY ASSOCIATING WITH THE
T-CELL RECEPTOR.
I SHOWN YOU A MOUSE MODEL SYSTEM
WITH THESE MOLECULES COMBINE TO
ENHANCE T-CELLS PUNCTION.
WHAT SEEMS TO BE PARTICULARLY
UNIQUE AND IMPORTANT IS THE CELL
INTRINSIC ROLE FOR GAMMA INTERFN
AND THEN LASTLY, IN A PHASE II
CLINICAL TRIAL, WHAT WAS
PARTICULARLY SURPRISING IN TERMF
TWO MESSAGES IS FIRSTLY, ALONE S
NOT SILENCED.
IT DOES CREATE HUGE TRANSCRIPTIL
CHANGES BUT FOR REASONS WE ARE
STILL TRYING TO UNDERSTAND, THAT
DOES NOT GIVE RISE TO CLINICAL
EFFICACY.
BUT IT RAISES THE POSSIBILITY TT
COMBINING LAG3 IMMUNOTHERAPIES H
OTHER THERAPIES MAY BE EFFICACI.
AND THEN LASTLY, THE COMBO SEEMO
GIVE RISE TO A COMPLETELY DIFFET
TRANSCRIPTIONAL AND FUNCTIONAL
OUTPUT THAT IS NOT EITHER INCRED
IN TERMS OF WHAT YOU WOULD SEE H
PD-1 OR ADDITIVE OF THE TWO
SIGNATURES SEPARATELY.
SO I'LL STOP THERE AND ACKNOWLEE
THE PEOPLE THAT CONTRIBUTED TO S
WORK, PARTICULARLY THE SORT OF
EARLY STUFF ON LAG3 SIGNALING FM
CLIFF GUY AND CRAIG WORK MAN, SE
TERRIFIC MOUSE STUDIES, NON
CLEAVABLE STUFF FROM LAWRENCE
ANDREWS AND THEN THE MORE RECENT
STUFF THAT I SHOWED YOU HERE AND
THEN TONY DRIVES ALL THE
COMPUTATIONAL ANALYSIS OF SAMPLS
FROM PATIENTS AND THEN LAST BUTT
LEAST, WE WOULDN'T HAVE BEEN ABE
TO DO THIS WITHOUT OUR CLINICAL
COLLEAGUES THAT ARE IN THE MEL
GNOME ALD PROJECTS AND LASTLY,
PATIENTS AND FAMILIES FOR WILLIG
TO CONSENT.
SO I'LL STOP THERE AND HAPPY TO
TAKE QUESTIONS, THANK YOU.
[ APPLAUSE ]
>> THANKS.
I THINK WE HAVE TIME FOR ONE ORO
QUESTIONS.
>> SO THE ANTI-LAG3 ANTIBODIES
WHICH BLOCKS CD4, BUT WHAT YOU D
ABOUT MECHANISM IMPLIED THAT THT
INTERACTION WASN'T CRITICAL, THT
THERE IS MORE OF A CELL INTRINSC
MECHANISM.
HOW WOULD YOU EXPECT THOSE
ANTIBODIES TO PROVIDE FUNCTION?
>> SO OBVIOUSLY IN THE INTERESTF
TIME I COULDN'T GO INTO THAT IN
GREAT DETAIL.
WHAT WE HAVE SHOWN IN THAT NATUE
IMMUNOLOGY PAPER, IS THAT THE
ANTIBODY BLOCKS LAG3 ASSOCIATION
WITH TCR.
SO IF LAG3 CAN'T -- TCR, IT CANT
INHIBIT.
THAT'S PARTICULARLY IMPORTANT.
BUT WE ALSO THINK THERE MAY BE E
OTHER ACTIVITIES OF THAT ANTIBOY
THAT ARE ALSO INDEPENDENT OF THE
LIGAND WHICH COULD BE IMPORTANT.
>> SO THIS MAY BE TOO EXPLORATOY
FOR THIS SO YOU CAN CUT ME OFF F
I'M OUT OF LINE.
I'M INTERESTED IN TWO CONNECTIOS
OF LAG3.
ONE IS CD4 THAT IT REMAINS IN TE
GENOME SO CLOSELY.
THE OTHER IS WITH THE ASSOCIATIN
WITH NOTCH WHERE IT HAS THE 10 M
17 CLEAVAGE OF NOTCH AND
EXTRACELLULAR DOMAINS -- I'M
WONDERING AND THEN YOU SHOWED AN
RVPJ REG LON BEING ENRICHED IN S
SIGNATURE AT THE END.
IS IT POSSIBLE THAT THERE ARE KD
OF LATERAL ASSOCIATIONS OF THE 3
ON THE T-CELL SURFACE IN SOME OF
THESE OTHER FAMILY MEMBERS
POTENTIALLY NOTCH, POTENTIALLY 4
THAT ARE PROVIDING THE COMPOSITE
SIGNAL TRANSDUCTION CASCADE?
>> IT'S A GOOD QUESTION.
WE HAVEN'T SPECIFICALLY LOOKED T
THAT.
YOU MAY RECALL SOME TYPE AGO WED
A NATURE IMMUNOLOGY PAPER THAT
TALKED ABOUT THE IMPORTANCE OF
NOTCH MODULATION DOWNSTREAM OF .
AND SO THERE MAY WELL BE A TRIFA
HERE WE JUST HAVEN'T LOOKED INT.
>> THANK YOU VERY MUCH.
>> THANK YOU.
[ APPLAUSE ]
>> OUR NEXT SPEAKER ALL THE WAY
FROM FREDRICK, WHICH IS PART OF
NCI, IS DANIEL MCVICAR WHO WILL
TELL US ABOUT A POD 1 MEDIATED -
I'M NOT EVEN GOING TO TRY --
>> VERY GOOD.
THANK YOU VERY MUCH.
SO THANKS TO THE ORGANIZERS FOR
GIVING ME AN OPPORTUNITY TO TALA
LITTLE BIT TO YOU TODAY ABOUT WT
IS RAPIDLY BECOMING ONE OF OUR
FAVORITE METABOLITES.
THIS METABOLITE KNOWN ASIT CONE.
WE HAD A INTEREST IN THE PERITOL
CAVITY FOR A NUMBER OF REASONS.
FROM A CANCER STANDPOINT IT'S VY
RELEVANT, COLORECTAL AND GYNECOG
CALL METASTASES, RARELY RECALL
CANCERS CAN METASTASIZE TO THE
PARTINIUM.
THERE ARE A PRIMARY MALIGNANT MS
THEME OHM AS IN THE PARTINIUM AD
HGCs OFTEN CAN METASTASIZE AND
SPREAD IN THE PARTINIUM FOR A VY
BAD OUTCOME.
FROM AN IMMUNOLOGIC STANDPOINT W
THIS IS THE SITE OF UNIQUE
MACROPHAGE POPULATIONS.
SO THE RESIDENT MACROPHAGES ARE
REARRIVED FROM THE YOKE SACK.
THEY ARE DEPENDENT ON GABA 6
INDUCED BY RETINOIC ACID.
AND THEY ARE INVOLVED IN DETECTG
AND REMOVING MICROORGANISMS FROM
THE PERITONEAL CAFIT AND
PARTICIPATE IN WOUND REPAIR.
THEY ACTUALLY MOVE INTO INJURED
ORGANS FOR REPAIR.
FROM THE METABOLIC STANDPOINT WE
DID A COMPARISON OF PERITONEAL
FLUID SHOWN HERE WITH SERUM.
AND THIS WAS PART OF A STUDY WHE
WE ARE THINKING ABOUT UNIQUE
METABOLIC NICHES AND CONDITIONS.
WHAT WE FOUND IS THAT THE
PERITONEAL SPACE HAD UNIQUE
METABOLIC MILL YOU.
IT WAS RICH IN AMINO ACID FUELSD
RELATIVELY POOR IN LIPID FUELS.
THIS CONTRASTED TO THE SERUM.
AND IN THIS PAPER, WE SHOWED THT
THAT RESIDENT PERITONEAL MACROPE
POPULATION WAS POISED TO TAKE
ADVANTAGE OF THAT ENVIRONMENT AD
PREFERENTIALLY USE THOSE AMINO D
FUELS.
SO WE SAID LET'S START TO PERTUB
THIS ENVIRONMENT WITH TUMOR.
SO THIS IS THE EXPERIMENTAL SETP
HE DID A COUPLE OF YEARS AGO.
WE IMPLANT A TUMOR INTO THE
PERITONEAL SPACE AND WAIT A COUE
OF DAYS AND THEN INJECT -- TO CR
OUT THE RESIDENT MACROPHAGE
POPULATION.
AND THEN WE LET IT GO FOR A COUE
OF DAYS.
WHAT CAN YOU SEE IS THAT IN ALLF
THESE CASES, THE CLEARANCE OF TE
RESIDENT CELLS REDUCES TUMOR
BURDEN.
SO IMMUNOLOGISTS MIGHT SAY THOSE
ARE MYELOID CELLS INHIBITING T-L
FUNCTION.
IT'S NOT THAT.
THESE ARE RESIDENT PERITONEAL ME
THAT ARE DIRECTLY INFLUENCING TR
BURDEN IN THE PERITONEAL SPACE.
AND ALTHOUGH THIS RELATIVELY
QUICKLY BECAUSE WE PUBLISHED A
COUPLE OF YEARS AGO, BUT THEN WE
PULLED THESE RESIDENT MAGS AND
CHARACTERIZE THEM METABOLICALLY.
AND WHAT YOU FIND IS THAT RESIDT
MAGS COME FROM A PERITONEAL SPAE
AND HAVE VERY HIGH OXIDATIVE RA,
HIGH BASAL AND HIGH MAXIMUM
OXIDATIVE RATES T MATTERS WHEREE
TUMOR IS.
SO THE BLUE HERE IS AN IP TUMOR.
THE GREEN IS A SUB TUMOR.
SO NOT JUST A TUMOR BEARING HOST
HAS TO BE IN THE PARTINIUM.
WE DO B16s IT'S EASY.
THEY ARE EASY TO SEE.
WE CAN SEE THE SAME EFFECTS WITH
MORE TRADITIONAL MODELS OF OVARN
CANCER.
SO WE WENT ON TO THEN CHARACTERE
THESE FURTHER T TURNS OUT THAT T
INCREASE IN METABOLIC ACTIVITY D
BASAL SPOTS IS INCREASE IN FATTY
ACID OXIDATION.
SO YOU CAN KNOCK IT BACK DOWN BY
TREATING THOSE CELLS AND BRING T
BACK TO THE CONTROLLED LEVEL.
WHEN WE DID SOME TARGETED
METABOLOMICS ON THOSE CELLS, WEW
SEVERAL THINGS THAT WERE UP
REGULATED IN TUMOR-BEARING, BUTE
THAT JUMPED OUT WAS THIS
METABOLITE.
IT WAS ABOUT 15-FOLD HIGHER IN E
RESIDENT MAGS FROM TUMOR-BEARING
ANIMALS.
SO YOU MAYBE HEARD ABOUTIT CON8Y
THIS POINT, ALTHOUGH BACK WHEN E
WERE DOING THIS, THERE WAS VERY,
VERY LITTLE OUT THERE.
SO LET ME TELL YOU ABOUT IT.
BECAUSE I HOPE TO GET YOU
INTERESTED THIS AFTERNOON.
IT'S PROT DUCT OF IMMUNE RESPONE
GENE 1 OFTEN KNOWN IN THE HUMANS
A COD 1.
THOSE OF US THAT ARE VERY OLD TT
DID MICROARRAYS YEARS AND YEARS
AGO, IRG1 WAS NEAR THE TOP WHENU
DID STIMULATED MAGS.
EVERYONE IGNORED IT.
SO IT IS A DECARBOXYLASE.
THE INTERMEDIATE IN THE CONVERSN
OF CITRATE TO THE ISOCITRATE WIN
THE TCA.
SO DECARBOXYLATED -- THE CELESTL
DESIGN COMMITTEE GAVE US THIS
LIKELY BECAUSE IT IS A GOOD
INHIBITORS OF THE GLYOX LATE CYE
AND WILL BACTERIA.
THIS IS A BACTERIA THAT THEY INE
WHEN THEIR CARBON SOURCES ARE
LIMITED.
IT ALSO HAS BEEN SHOWN TO BE
INHIBITOR OF COMPLEX 2 OF THE T.
AND MANY OF THESE YOU CAN SEE WE
SHOWN SOMETIME AGO ALTHOUGH THEY
HAVE BEEN REDISCOVERED MORE
RECENTLY.
THERE WAS SOME COMPELLING EVIDEE
AT THE TIME IT WAS INVOLVED IN N
DOE TOXIN CLEARS BUT WHAT CAUGHT
OUR EYE WAS A ZEBRAFISH STUDY TT
SHOWS THAT IT PROMOTED FATTY ACD
OXIDATION THROUGH MITOCHONDRIAL
LOSS.
WE SAW THIS INCREASE, WE PURSUED
THIS LINE OF STUDY.
BUT SO THROUGH THESE MACROPHAGE,
THEY HAVE IRG MESSAGE AND THEN N
WE GO IN AND START TO MAKE THAT
CONNECTION TO FATTY ACID OXIDAT,
WE CAN USE SI ARE.
IN.
As, KNOCK DOWN IRG1 AND KNOCK
THAT ACTIVITY DOWN.
IF YOU TREAT YOU KNOCK IT DOWN O
THE SAME LEVEL SO IT'S THAT FATY
ACID OXIDATION PART THAT IS
DEPENDENT.
AND THAT ALSO IS CONNECTED TO
GENERAL ROSS LOVES IN THESE CELS
SO THEY HAVE HIGH ROSS LEVELS
EXPOSED IN THE TUMOR.
SO IT ALL LOOKED GOOD.
AND THE KEY PART IS THAT IF YOU
KNOCKED DOWN IRG1 YOU CAN ALSO
REDUCE THE TUMOR BURDEN.
WE CAN SHOW THAT BY siRNAs AND
ALSO BY KNOCKING DOWN ALL THE CS
AND THEN REPLACING THEM WITH
WILDTYPE CELLS AND THE BURDEN GS
BACK UP.
OR REPLACING WITH NULL CELLS AND
THE BURDEN STAYS LOW.
I HAVEN'T SHOWN YOU ALL THE DATA
BECAUSE WE SHOWED IT A COUPLE OF
YEARS AGO BUT THE GENERAL MODEL
THAT RESULTED WAS THE TUMORS WEE
DRIVING EXPRESSION OF IRG1 IN TE
MACROPHAGE POPULATION AND IT WAS
DRIVING FATTY ACID MITOCHONDRIAL
FUEL ROSS IN THE MACROPHAGES WHH
RESULTED IN INCREASED LEVELS OF
ROSS SIGNALING IN THE TUMORS.
WE COULD SHOW THAT BIOCHEMICALLY
THAT THEY WERE SHOWING EXPOSUREO
ROSS AND ULTIMATELY THEN THAT
DRIVES TUMOR BURDEN AND PROMOTES
ACTIVITY.
THERE REALLY WAS VERY LITTLE-KNN
ABOUTIT CON8.
NOW IT'S VERY, VERY POPULAR.
THERE IS A VARIETY OF PAPERS THT
HAVE COME OUT, MANY IN HIGH PROE
JOURNALS, SHOWING THAT IT CAN DE
AN EFFECT TRANSCRIPTIONAL
REGULATION.
I'M NOT GOING TO GO INTO ALL OF
THESE.
I WANT TO GIVE YOU A WORD OF
CAUTION THAT MAINTENANCE OF THEE
PAPERS ARE DONE WITH BIOCHEMICAL
MODIFICATIONS TO THEIT CON8.
SO EITHER DIMETHYL OR -- [
INAUDIBLE ]
WHICH I'LL MENTION IN A MOMENT.
THESE MODIFICATIONS WERE MADE TO
FACILITATE GETTING IT INTO THE
CELLS BUT IT'S NOT REALLY NECESY
BECAUSE IT GETS IN JUST FINE.
UNFORTUNATELY, THOSE MOLECULES E
CHEMISTRY AND BIOLOGY THAT YOU O
NOT GET FROM NATIVEITA CON8.
SO PAY ATTENTION TO WHETHER THE
REAL IS USED OR A DERIVATIVE.
WE WERE MORE INTERESTED IN SOMEF
THE ACTIVITIES THAT HAD COME OUA
LITTLE BIT EARLIER ABOUT THIS.
IT HAS MULTIPLE METABOLIC FUNCTS
THAT HAVE BEEN REPORTED.
INHIBITOR OF FDP PRODUCTION.
IT CAN INHIBIT SBH.
COMPELLING DATA WITH INTERFERENE
WITH VITAMIN B12 SIGNALING.
WE LIKED THESE LAST TWO ON THE T
WHICH HAVE BEEN SHOWN TO INHIBIT
SUBSTRATE LEVEL PHOSPHORYLATIOND
I'LL SHOW YOU ABOUT THAT IN A
MOMENT.
AND OF COURSE AS I MENTIONED, IT
CAN PROMOTE LIPID UTILIZATION AD
FATTY ACID OXIDATION.
BECAUSE THAT WAS AN ACTIVITY THT
SEEMED TO BE SHOWING UP IN THIS
TUMOR MODEL AND IN OTHER MODELSF
FUNCTION, JOHN DECIDED TO ASK TE
STRAIGHTFORWARD QUESTION OF WHAT
HAPPENS IF WE THEN STRESS IRG KN
MICE BY GIVING THEM HIGH-FAT DIS
AND FORCING THEIR LIVER AND THER
PHYSIOLOGY TO ADAPT TO THAT
HIGH-FAT ENVIRONMENT?
WE THOUGHT THIS WAS A WORTHY
EXPERIMENT BECAUSE WE HAD SHOWN
THAT IT CAN BE EXPRESSED IN
HEPATOCYTES DURING ISCHEMIA AND
REPERFUSION.
MAYBE THERE IS A INDICATION ANDT
WILL SHOW A PHENOTYPE.
IT DOES.
WHEN YOU PUT THEM ON HIGH FAT
DIETS, SERUM FATTY ACID LEVELS E
UNCHANGED BUT MESENTERIC FAT MAS
HIGH AND OVER ALL WEIGHT IS HIGH
AND WHEN YOU LOOK AT THIS UNDERE
MICROSCOPE, YOU CAN SEE WHY.
THEY HAVE LOADED TREMENDOUS AMOT
OF LIPID IN THE IRG NULLS.
SO, THE IRG MICE ARE LOADING A T
OF LIPID.
WHAT IS GOING ON?
IS IT THE HEPATOCYTES DOING THI?
WE DON'T THINK IT IS.
SO HERE WE ARE LOOKING AT THE IG
MESSAGE LEVELS IN WHOLE LIVER FM
A WILDTYPE MOUSE ON HIGH FAT DI.
YOU SEE IT COME UP NICELY.
ALBUMIN CRE KNOCKOUTS YOU DO.
IT'S THE IMMUNE SYSTEM NOT THE
HEPATOCYTES.
SO IN THE LAB WE WENT BACK TO A
PUBLISHED DATASET OF SINGLE CELL
RNA-SEQ OVER THE COURSE OF 36 WS
OF HIGH FAT DIET AND WHAT HE FOD
WAS THAT THERE ARE SEVERAL IRG1
EXPRESSING POPULATIONS IN HERE.
THESE ARE THE NEUTROPHILS.
NEUTROPHILS EXPRESS QUITE A BITF
IRG1 BUT YOU ALSO NOTICE THEY AE
NOT VERY REACTIVE DURING THE
HIGH-FAT DIET.
IN CONTRAST, THESE MACROPHAGES E
VERY RESPONSIVE TO THE HIGH-FAT
DIET AND INDUCING A LOT OF IRG1.
THESE ARE THE INFLAMMATORY
MACROPHAGES ENTERING THE LIVER
DURING THE HIGH-FAT DIET.
SO THAT SUGGESTED IT WAS REALLYE
MACROPHAGE POPULATION THAT WAS
DOING IT.
SO WE COULD THEN GO IN AND SAY,
OKAY, IS THERE SUFFICIENT LIVERT
CON8, AND IT SHOWS YOU THE ROUGY
LEVELS IN ANIMALS PER GRAM.
HERE IS WILDTYPE MICE AND OF COE
IN THE KNOCKOUTS ON HIGH-FAT DIT
THAT IS ABLATED.
IF WE GIVE A VERY STRONG STIMULS
TO THE ANIMAL FORIT CON8
PRODUCTION, YOU CAN SEE THAT NOW
YOU GET TREMENDOUS LEVELS OFIT 8
IN THE LIVER.
ALBUMIN CRE IS INTACT AND THE LE
IN CRE IS LOW.
IT'S NOT QUITE ZERO.
SO THERE IS SOME EVIDENCE THAT
OTHER POPULATIONS CAN DO IT.
BUT IN THESE INSTANCES, IT'S THE
MACROPHAGES THAT ARE DOING IT.
SO WE WANTED TO KNOW IF THIS MIT
BE RELEVANT TO HUMAN DISEASE.
SO WE COLLABORATED WITH THE
CLINICAL BIOSPECIMEN REPOSITORYD
WE GOT SAMPLES OF BLOOD AND LIVR
FROM MASH PATIENTS OR NON-MASH
CONTROLS.
-- NASH.
WHEN YOU START TO LOOK AT THIS
MECHANISM THEN WHAT CAN YOU SEES
THAT THEY HAVE HIGH LEVELS OF I1
AND THOSE LIVERS ARE HIGH FORIT
CON8 PRODUCTION.
BUT NOT IN THE BLOOD.
AND WE HAD SEEN THAT EVEN IN OUR
HIGH FAT DIET MICE.
EVEN THOUGH IT ACCUMULATES IN TE
LIVER T DOES NOT ACCUMULATE OR W
IN THE PERIPHERAL BLOOD.
SO, WHAT'S THE RESULT OF THIS LD
LOADING?
YOU CAN IMAGINE IT MIGHT BE
METABOLIC DISORDER AND THAT IS N
FACT WHAT IT IS.
THESE MICE DON'T HAVE DIFFERENCS
IN OVERALL WEIGHT BUT HERE IS TE
TRACES FROM GLUCOSE TOLERANCE,
QUANTIFIED OVER HERE.
THE I ARE RG NULL MICE ON THE HH
FAT DIETED HAVE WORSE GLUCOSE
TOLERANCE.
FOR THAT SORT OF SITUATION, IF I
CAN FIGURE OUT THIS POINTER, THE
BLOOD INSULIN LEVELS ARE ALSO
SUBSTANTIALLY HIGHER IN THE IRG
NULLS ON WILDTYPE -- SORRY ON
WESTERN DIETS.
AND HERE IS INSULIN TOLERANCE TT
THAT SHOWS THEY HAVE EXACERBATED
METABOLIC SYNDROME FOR THE IRGs
IN WESTERN DIET.
WHY?
WHY ARE THE LIVERS LOADING WITH
LIPID AND DRIVING THIS DISEASE?
SO TO TRY TO GET AT THE MECHANI,
WE USED ISOLATED HEPATOCYTES INE
ROW AND THIS SHOWS YOU WHY YOU
DON'T NEED THOSE MODIFIEDIT CON8
COMPOUNDS.
THIS IS THE INTRACELLULAR
ACCUMULATING AS WE BATHE THE CES
IN VITRO.
SO USE THIS AS A MODEL OF EXPOS.
AND JOHN CAME UP WITH A LIPID
LOADING ASSAY WHERE WE CAN STAIN
THE HEPATOCYTES AND SUPPLY THEM
WITH ACCESS LIPID AND THEY LOADE
LIPID VERY NICELY BUT IF YOU PRE
TREAT THEM WITHIT CON8 YOU CAN
SUPPRESS THAT.
IT IS DIRECTLY AFFECTING THE
ABILITY OF THE PEPAS SITES TO
ACCUMULATE LIPIDS WHEN THE LIPID
CONCENTRATIONS ARE HIGH.
IT'S A METABOLIC MECHANISM AS YU
MIGHT IMAGINE.
HERE IS ANALYSIS SO NOW WE TREAD
THEMSELVES EITHER WITH LIPID,IT
CON8 ALONE OR BOTH HERE AT THE .
AND WHAT YOU CAN SEE IS WHEN YOU
TREAT THESE HEPATOCYTES WITHIT
CONEIGHT BASAL PHOSPHORYLATION S
UP AND THAT SHOWS WHETHER THEY E
IN THE PRESENCE OF LIPID OR NOT.
SO THEIT CON8 IS GEARING UP THER
METABOLISM AND CAUSING THEM TO N
MORE LIPID.
SO INTERESTINGLY BY USING C13 PM
TRACING, WE DON'T SEE ANY TRACIG
OF THE OF THE CELLS TO UPITATE
LIPID.
WHEN YOU TRACE THE METABOLITES N
INTO THE TCA CYCLE, YOU SEE A
GREATER INCORPORATION OF
LIPID-DERIVED CARBON INTO THE LR
METABOLITES SHOWING THAT THEY AE
USING MORE LIPID EVEN THOUGH THE
RATE OF UPTAKE IS THE SAME AND
AGAIN THAT WOULD BE CONSISTENT H
THEM BEING ABLE TO REDUCE THEIR
OVERALL LIPID BURDEN.
THE BIOCHEMICAL MECHANISMS FORIT
CON8 FUNCTION ARE STILL VERY
CONTROVERSIAL BUT ONE THAT SEEMO
BE DEVELOPING IN THE LITERATURES
THE ABILITY OFIT CON8 TO INTERFE
WITH [ INAUDIBLE ]
IT'S WHAT CHANGES SUCKS WITHIN E
TCA CYCLE AND CARRIES OUT
PHOSPHORYLATION.
AND IT'S NOW BEEN SHOWN IN VITRO
THATIT CON8 IS A DIRECT INHIBITR
OF THIS ENZYME AND THAT IT IS A
DIRECT SUBSTRATE OF THE TRANSFEE
WHICH CONVERTS WHILE MOVING IT.
SO THIS MECHANISM WAS WORKING IN
OUR CELLS WE SHOULD BE ABLE TO E
THE FROM TIME TO TIME AND IT LOS
LIKE WE CAN.
SO WHEN WE TREAT WITHIT CON8 OR
LIPID, YOU GET INCREASES IN SUCS
NO COA AND PERHAPS MOST
IMPORTANTLY, YOU GET A DEPRESSIN
IN ATP LEVELS AND INCREASE IN AP
LEVELS SUGGESTING THESE CELLS AE
STRUGGLING TO MAINTAIN THEIR
ENERGETICS AND RAMPING UP THEIR
METABOLISM TO TRY TO DO THAT.
MAYBE WE COULD SUPPLYIT CON8 BAK
AND HERE WE USED ONE OF THE
DERIVATIVES.
HERE IS THE HIGH FAT DIET AND TE
NICE JOB OF KNOCKING DOWN THE LD
LEVELS.
HERE IS MANY, MANY MICE
CHARACTERIZED UP HERE AND YOU CN
SEE THAT THE 4OI HAS AN EFFECT N
LIPID DROP WILL THE SIZE AND AR.
WHEN WE TREAT WITH ROI.
THE EFFECT ON WILDTYPE MICE IS H
MORE MODERATE.
IT DOESN'T REACH SIGNIFICANCE BI
THINK YOU CAN SEE THAT IS IT TRG
TO KNOCK IT DOWN A LITTLE BIT.
PROBABLY NOT SIGNIFICANT BECAUSE
THEIR LEVELS ARE ALREADY LOWER.
THE 4OI CORRECTS THE MESENTERICT
MAS PHENOTYPE AND IT ALSO REVERS
THOSE METABOLIC DISEASE PARAMET.
SO I'LL LEAVE WITH YOU THIS WHIH
IS OUR WORKING MODEL FOR WHAT IS
HAPPENING.
SO WE THINK THAT DURING HIGH FAT
DIET THE RESIDENT MACROPHAGES
WITHIN THE LIVER ARE BECOMING
ACTIVATED.
THEY ARE PRODUCINGITE CONE 8.
AND WITHIN THE MITOCHONDRIA IT
INTERFERES WITH SUBSTRATE LEVEL
PHOSPHORYLATION, DRIVING DOWN TE
ATP LEVELS AND CAUSING CELLS TO
RAMP UP THEIR METABOLISM TO TRYO
PRESERVE THEIR ENERGETICS.
THE LAST THING I WANT TO LEAVE U
WITH BECAUSEIT CON8 IS BEING TAD
ABOUT A LOT AS THIS
IMMUNOMODULATORY METABOLITE.
AND ALTHOUGH IT MAY BE IN SOME
RESPECTS, I REALLY WANT TO KEEPU
FOCUSED A LITTLE BIT ON WHAT I
THINK IS REALLY HAPPENING.
AND THAT THESE CELLS ARE NOT
SUPPOSED TO BE EXPOSED TOIT CON.
IT IS WHAT IS GENERATED WHEN A L
DETECTIVES A BACTERIAL INFECTION
AND TRIES TO MAINTAIN THAT.
IT'S THAT ACCESSIT CON8 PRODUCEY
THE EFFECTOR CELLS THAT IS WORKG
IN TRANS, AND FORCING THESE OTHR
CELLS TO DEAL WITH A METABOLITE
THEY ARE NOT USED TO SEEING AND
THEY HAVE TO DETOXIFY IT.
THEY WILL DETOXIFY IT BY LOSING
THEIR SUBSTRATE LEVEL
PHOSPHORYLATION.
THEY'LL LOUIS THEIR SDH ACTIVIT.
DEPLETION OF KOA AND B12 SO YOUE
NOT REALLY SUPPOSED TO SEE IT.
SO DON'T THINK OF IT AS A
REGULATOR.
IT'S A TOXIN THAT THE CELLS HAVO
DEAL WITH.
SO WITH THAT, I'LL THANK THE PEE
THAT DID THE WORK.
THE VAST MAJORITY OF OUR WORK IS
DONE BY JOHN WISE, SHOWN HERE.
ALL OF OUR METABOLISM INCLUDINGE
STORY AND OTHERS IS DONE BY ERIC
CAPONE HERE.
THANK YOU VERY MUCH.
[ APPLAUSE ]
>> WE DO HAVE TIME FOR A FEW
QUESTIONS.
>> LOVELY TALK.
JUST A QUICK QUESTION.
WHEN THE MACROPHAGES ARE SECRET,
THEY ALSO EXPOSE THEMSELVES TO E
METABOLITE.
WHAT HAPPENS THERE?
DO THEY M1 AND M2 SWITCHING
PHENOTYPE OR SEPARATION OF CYTOE
SECRETION OR --
>> YES.
SO YOU CAN SEE ALL OF THE
PARAMETERS THAT I'M SHOWING HERN
HEPATOCYTES, YOU CAN SEE IN THE
MAGS.
SO YOU CAN SEE AS I SHOWED YOU,E
SAW IT INITIALLY IN THOSE
MACROPHAGES THAT CAME OUT OF
TUMOR-BEARING MICE SO THAT HIGH
BASAL OXIDATIVE PHOSPHORYLATION,
THAT HIGH UTILIZATION OF FATTY
ACIDS, THAT'S WITHIN THE
MACROPHAGES THAT ARE MAKING IT.
SO JUST THE WAY THEIR INFLUENCIG
CELLS IN TRANS, THEY ARE SUBJECO
ALL OF THIS IN CYSAS WELL.
>> THANK YOU FOR YOUR TALK.
I APOLOGIZE IF I MISSED IT.
BUT M1 AND M2 MACROPHAGES HAVE
DIFFERENTIAL METABOLISM.
IS A HETEROGENOUS POPULATION YOU
WERE STUDYING?
>> SO IN THE RESIDENT COMPARTME,
IT DOESN'T REALLY FALL INTO A CN
M1 AND M2.
SO YOU PROBABLY SEEN PEOPLE THAT
WILL KIND OF TELL THESE STORIES
MORE FARINGLY AND SAY IF YOU TAE
OUT THE RESIDENT MAGS, THEY'LL E
SOME M1 GENES BUT IF YOU LOOK FR
THE M2 GENES THEY'LL HAVE THOSE
TOO.
IT'S NOT CLEAR-CUT.
THIS EFFECT SEEMS TO BE MOSTLY D
TO THIS M1 RESPONSE WHICH DRIVEA
LOT OFIT CON8.
A LAB SHOWED THAT THERE CAN BE
DIRECT INHIBITION OF M2 DEVELOPT
BYIT CON8.
SOME OF THAT IS DONE WITH THE
MODIFIED METAP LIGHTS.
SO YOU SHOULD TAKE A LOOK AT IT.
BUT IT'S POSSIBLE THAT NOT ONLYS
IT MOSTLY ASSOCIATED WITH ONE BT
IT'S AN ACTIVE REPRESSOR OF 2.
>> VERY COOL TALK.
I KNOW THATIT CON8 IS OR CAN BE
DEGRADED BY CERTAIN MICROBES.
I DON'T KNOW IF YOU LOOKED INTOE
ROLE OF WIDE RANGING IF IT'S
VARIABLE -- SPREAD THROUGHOUT TE
GUT TO THE PARTINIUM OR SOMETHIG
LIKE THAT --
>> YES.
SO VERY GOOD POINTS.
YOU'RE RIGHT.
WHAT HE'S REFERRING TO IS IN THE
BATTLE THAT IS GOING ON WHERE A
MACROPHAGE WILL EAT A BUG, THE G
CHANGES THE METABOLISM TO SURVIE
AND THE BUG MAKESIT CON8 TO POIN
THAT.
MANY MICROORGANISMS CAN
METABOLIZEIT CON8.
WE LOOKED AT THE MICROBIOTA IN E
RESTING STATE.
WE SEE LITTLE IF ANY CHANGE THE.
WHETHER SOMETHING MAY BE HAPPENG
IN HIGH FAT DIET, WE DON'T KNOW.
WE HAVE TO LOOK.
OVER ALL, I THINK THAT THERE ISA
GOOD LIKELIHOOD THAT SOME OF THE
BACTERIAL SPECIES ARE CHANGING,
HOWEVER, IT'S DIFFICULT TO DO
BECAUSE YOU CAN GO IN AND YOU CN
DO THESE EXPERIMENTS WHERE YOU
TITRATE INIT CON8 INTO A GIVEN
BACTERIAL STRAIN AND THEN SUPPRS
IT AND NOW IT FEEDS ON IT.
SO I'M NOT SURE IF WE'RE GOING O
SEE IT PROMOTE SPECIES.
WE ARE JUST TRYING TO SORT THAT
OUT.
>> GREAT POINT.
THANK YOU.
>> ALWAYS A PLEASURE.
IT WAS REALLY GREAT, THANK YOU.
SO THERE IS A LOT OF STUFF ON TE
ALPHA SUCKS NAT RATIO IN TERMS F
BEING A MARKER OF DIFFERENTIATI.
IN YOUR CONTEXT WHERE YOU MIGHTE
CHANGING SUCKS INHALATION BECAUE
OF YOURIT CON8 LEVELS.
SO ONE IS, DO YOU KNOW IF THEREE
DIFFERENCES IN SUCKS INHALATIOND
IN YOUR IRG KNOCKOUT AND CAN YOU
PLAY AROUND WITH IT AS A RATIO F
SUCKSINATE?
>> SO WE HAVEN'T LOOKED DIRECTLT
PROTEIN SUCKS INHALATION.
THE RATIOS ARE DEFINITELY CHANGG
IN THE SENSE THAT IF YOU DO BROD
METABOLOMICS ON CELLS EXPRESSINA
LOT OFIT CON8 VERSUS THOSE THAT
AREN'T, ONE OF THE FEW THINGS TT
SHOWS UP IS THE ACCUMULATION OF
SUCKSINATE THAT NORMALLY OCCURS
DURING ACTIVATION IS GONE IN THE
ABSENCE OFIT CON8.
THERE IS ONE OF THE MODELS THATS
BEEN PUT OUT FOR THE
ANTI-INFLAMMATORY ACTIVITY IS
INTERFERENCE WITH TET 2.
WHICH IS A SUCKSINATE APP INDICD
GLUTEERATE-DEPENDENT ENZYME.
WHAT THEY THINK IS HAPPENING THE
IS DIRECT COMPETITION FOR THE
BINDING SITES ON THE ENZYME BYIT
CON8 BECAUSE IT'S SO SIMILAR TO
ALPHA GLUTE RATE.
IT'S NOT EVEN THE RATIO, IT'S A
DIRECT INHIBITION.
THAT'S WHAT THEY SUGGEST.
THANK YOU.
[ APPLAUSE ]
>> GREGOIRE ALTAN-BONNET WILL GO
FIRST AND THE DR. TAYLOR WILL GO
SECOND AND WE'LL TAKE QUESTIONS
AFTER THEY ARE BOTH DONE SPEAKI.
>> WE ARE VERY EXCITED ABOUT THE
LAST TALKS.
-- REALLY NICE TO BE ABLE TO MOL
T-CELL ACTIVATION AND USE THAT O
MAKE BETTER DESIGNS.
I'M GOING TO STOP WITH THE 3
MUSKETEERS WHO DID MOST OF THE
WORK.
SO A TIGHT COLLABORATION WITH TE
LABS.
[ INAUDIBLE ]
DOING A LOT OF THE WORK AND MY B
HAS BEEN DEVELOPING A LOT.
VERY TIGHT INTEGRATION OF SYSTES
ENERGY AND COLLABORATION -- [
INAUDIBLE ]
SO WE ARE INTERESTED IN MODEL
CANCER -- CAN COLLECT THE BLOOD
FROM THE PATIENTS AND COLLECT SE
LEUKOCYTE FROM TUMORS AND TRY TO
EXPAND THEM AND MODIFY THEM AS E
DRUG -- THE IDEA IS TO DEVELOP A
GOOD POPULATION OF T-CELL IN CA.
WE CAN -- WE MUST BE AWARE OF TE
THINGS AND SOME SUCCESS AND
CONTEXT -- IN WOB CLINICAL TRIA-
SO WE ARE VERY INTERESTED IN BEG
ABLE TO CHARACTERIZE TO REALLY
UNDERSTAND WHAT DRIVES RESPONSE
AGAINST CANCER.
AND TO DEVELOP THAT, I'M GOING O
GO QUICKLY.
WE DEVELOPED A PLATFORM WHICH I-
IT'S GEARED FOR -- ROBOTIC PLATM
EXPERIMENTS AUTOMATICALLY.
WHAT THIS PLATFORM DOES COLLECT
TUMOR MODELS AND LOAD IT UP AND
REALLY GO AND COLLECT THE TIME
POINTS AND LOOK AT THE CYTOKINE,
COLLECT THE CELLS TO CHARACTERIE
THEM.
I'M SHOWING YOU A -- T-CELLS ANB
CELLS RESPONDING TO A MOUSE MODL
WE USED TO BUILD THE MODEL.
SO I WOULD LOVE TO SHOW YOU A LE
VIDEO BUT IT NEVER WORKS SO I'M
GOING TO SHOW YOU A VIDEO OF WHT
THE ROBOT LOOKS LIKE.
IT DOES EXACTLY WHAT WE WILL BE
DOING IN THE LAB AT THE BENCH.
COLLECTS PLATE FROM THE INCUBAT,
GRAB IT, REMOVE IT.
YOU CAN PROGRAM IT AND OF COURSE
IT'S STILL GOING TO KEEP WORKING
WHILE YOU GUYS WILL BE AT HOME.
AND WE HAVE DONE A LOT OF WORK O
DEVELOP -- [ INAUDIBLE ]
WE HAVE A WAY TO COLLECT QUICKLO
USE FOR MACHINE LEARNING.
SO STRONGLY RECOMMEND YOU CHECKT
THE PAPER LAST YEAR.
IT'S REALLY A GOOD WAY TO RUN AT
OF IMAGES VERY QUICKLY ON A FEW
SAMPLES.
WE CAN UPDATE THAT.
SO THIS IS -- TEST RUN TO CHECKE
PLATFORM WAS WORKING.
WE -- SIX HEALTHY DONORS.
I'M SHOWING THAT TO GIVE YOU A
SENSE OF THE RICHNESS.
THESE ARE OVER THREE DAYS AND WE
ARE JUST GOING -- RATIOS AND D
DONORS -- MULTIPLY CONDITIONS.
VERY INTERESTING TO MODEL.
WE CAN REALLY USE THAT TO -- VEY
RICH AND WE WANTED TO USE THAT O
REALLY BUILD MODELS TO UNDERSTAD
THE WAY WE CAN TWEAK TO MAKE THM
BETTER.
AND IT GOES BACK TO VERY OLD
PROGRAM I HAVE BEEN INTERESTED N
IN MANY CONTEXT OVER THE YEARS
WHICH IS WHEN WE LOOK AT T-CELL
ACTIVATION WE CAN USE DIFFERENT
CYTOKINE AND MARKERS AS READ OUT
ACTIVATION.
OLD PAPER LOOKING AT RESPONSE
AGAINST PEPTIDE AND DIFFERENT
INTERFERON GAMMA, IL-2 AND 3, TY
ARE REALLY DIFFERENT RANGES TO E
TO -- ACTIVATION.
THIS GOES BACK TO CLASSICAL MODL
WE DEVELOPED AND THE WAY WE THIK
OF T-CELL ACTIVATION AND STAGGED
SET OF INTERACTIONS WHERE EACH
STEPS OF PCR MIGHT CONNECT TO
DIFFERENT READ OUTS AND WE MIGHE
ABLE TO REVEAL DIFFERENT -- CONT
OF PCR, WE SEE -- YOU'RE
CONNECTING -- SO MOVING FORWARD
OVER 20 YEARS, WE CAN'T HAVE
DATASETS COMING OUT OF THE DATAS
WITH JUST DATA.
SO IF YOU DO LIKE THE -- PROCESG
YOU SEE THERE IS NO RHYME OR REN
TO THIS DATA.
VERY HARD TO COMPRESS.
SO I'M SHOWING YOU SEVEN CYTOKIS
AND 12 T-CELL TYPES.
FOUR ACTIVATIONS POINTS --
DIFFERENT QUANTITY AND TIME POI.
YOU SEE IF YOU JUST DO SPLASH OF
DATA, THERE IS NO RHYME OF REAS.
I'M GOING TO MAKE ANALOGY THAT I
MADE MANY TIMES AND MAYBE GETTIG
BORED OF IT BUT IT IS SO NICE -A
LOT OF NEW TOOLS IN THE FIELD OF
MACHINE LEARNING WHICH ENABLES U
TO LOOK AT THIS DATA AND
AUTOMATICALLY OBTAIN A MODEL AND
COMPRESS THE DATA AND REALLY
INTERPRET THE DATA IN A NICE WA.
SO JUST TO DO THE ANALOGY, THISS
SOMETHING WE DO IN THE CONTEXT F
ASTROPHYSICS.
THIS THE IS WHAT THE SYSTEM LOOD
LIKE BEFORE.
PEOPLE MEASURE ANGLES OF PLANETS
BUT THE MODEL THEY HAD IN TERMSF
ASTRONOMY WAS THE EARTH WAS THE
CENTER.
SO YOU GET VERY BEAUTIFUL ARRAY,
VERY DIFFICULT GEE NET RE.
BUT IT'S NOT A VERY USEFUL MODEL
UNLESS YOU WANT TO SHOOT A ROCKT
AND GO TO THE MOON.
IF YOU HAVE THE RIGHT MODEL, ITS
EASIER TO MODEL AND UNDERSTAND.
MACHINE LEARNING REDEVELOPED A T
OF METHODOLOGY.
SO A PAPER FROM 2018 AND
INSPIRATION FOR EXAMPLE A LOT OF
WHAT WE DO FOR FORMULAS THAT WE-
MACHINE LEARNING TOOLS AND VERY
COMPLEX DATASETS AND COMPRESSEDD
WE WANT TO LEARN VERY --
REPRESENTATION OF THE DATA TO
RECAPTURE ALL THE COMPLEX SYSTEM
AND PREDICTABILITY.
SO YOU SEE THE ANGLE IN COLORS D
THE BEST WAY TO COMPRESS THE DAA
AND REPRESENT IS TO PUT THE SUNN
THE MILT OF THE UNIVERSE AND
MEASURE ALL THE ANGLES.
SO I'M NOT SAYING THAT IMMUNOLOY
IS LIKE ASTROLOGY AND PHYSICS --
BUT IT'S KIND OF THE IDEA.
WE WANT TO MOVE TO WHERE TRYINGO
FIND CLUSTERS, WE ARE GOING TO Y
TO TAKE THIS COMPLEX AND FUNCTIL
DATA, MIGHT BE LOOKING AT T-CELL
DIFFERENTIATION OR AND YOU WANTO
PUT IN MACHINE LEARNING TOOLS.
WE ARE TRYING TO LEARN DIFFERENT
TYPE OF PEPTIDE DERIVED FROM --E
ARE USING -- VERY LARGE DATASET
TRYING TO SQUEEZE INTO SMALL SPE
TO LEARN DIFFERENT CLASSES -- HE
ON THE LEFT I'M SHOWING YOU WHAT
THE TWO DIMENSIONAL SPACE IN THE
MIDDLE LOOKS LIKE.
SO VERY HIGH DIMENSIONAL SPACE
WHERE WE HAVE A LOT OF CYTOKINE,
DEPENDING ON THE TYPE OF TUMOR,D
THE TYPE OF ONCOGENES, IT'S
DIFFICULT TO UNDERSTAND THE RHYE
OR REASON OF THE DIFFERENT
ACTIVATION.
VERY SIMPLE DATA MIX WHERE AGAIN
DEPENDING ON THE QUALITY OF
ONCOGENES, WE HAVE WILL DIFFERET
REGIONS OF SPACE DIFFERENTLY
ORGANIZED.
I'M GOING TO TRY TO CONVINCE YOO
USE TOOLS TO LEARN SOMETHING AND
THEN SHOW YOU SOME REAL NEAT
APPLICATION WE CAN REUSE TO LEAN
NEW STUFF.
SO VALIDATION IS ON THE RIGHT.
WE CAN QUANTIFY THIS MODEL -- YU
CAN THINK OF IT AS A MEASURE OF
BIOPHYSICS AND AGAIN THIS IS DOE
OBJECTIVELY BY LOOKING WITH MACE
LEARNING.
WHEN YOU COMPARE AND MEASURE BY
CHARACTERIZING THE PEPTIDE ON TE
PIECE, YOU SEE VERY GOOD
CORRELATION -- DIFFERENT QUANTIY
AND THE TUMORS CAUSE DIFFERENT
ORGANISMS.
YOU CAN SWITCH TO A HUMAN AND TE
SAME MODEL APPLIES.
SO SIMPLY BY CHARACTERIZING THE
VERY HIGH DIMENSIONAL SPACE AND
DYNAMICS OF THE CYTOKINE
PRODUCTION, WE CAN ABLE TO RELEN
THE MODEL OF THE WAY T-CELLS LOK
AT THE WORLD THE WAY THEY RECOGE
DIFFERENT SEQUENCE OF PEPTIDE AD
RELEARNING IN A WAY -- MODEL OF-
AGAIN THE BEAUTY OF THE COLOR IE
CAN GO BACK AND REALLY UNDERSTAD
WHAT THE CODE IS DOING.
SO PRACTICALLY WHAT IT DOES, AGN
TAKE THIS VERY HIGH DIMENSIONAL
SPACE, PUT IT INTO 2D SPACE, FAY
SIMPLE AND NOW WE CAN THINK OF O
PCAs IN A WAY AND VERY
COMPLICATED.
THESE TWO VARIABLES TELL US TWO
DIFFERENT MODES OF T-CELL
ACTIVATION.
WHEN WE HAVE A FUNCTION OF -- ON
THE X AXIS, WE HAVE A WAY THE
T-CELLS LOOK AT THE WORLD.
WHEN YOU WRAP UP, YOU SIMPLY WRP
UP THE POINT OF THE VARIABLE --
JUST ACTIVATING T-CELLS.
THE RED VARIABLE NUMBER 2 IS
INTERESTING.
WHAT YOU SEE AS YOU RAMP UP, YOU
FIRST GET COMPONENT AND THEN GOS
UP AND GETS ACTIVITY AS WELL.
THIS IS SOMETHING WE KNOW VERY L
FROM OVER THE YEARS, THE WAY
T-CELLS LOOK AT THE WORLD -- ONS
POSITIVE TRYING TO DRIVE ACTIVAN
AND ONE IS NEGATIVE TRYING TO BK
THE ACTIVATION AND MAKING SURE T
YOU DON'T GET -- SO WE CAN USE T
TO RECLASSIFY ALL THE CELLS WE E
IN THE LAB IN THE SAMPLES AND WE
ARE REALLY MEASURING THE
QUANTITATIVE MANNER AND POSITIVE
SIGNAL AND NEGATIVE SIGNAL.
SO, AGAIN VERY ABSTRACT BUT YOUT
A LOT OF -- ONE YEAR BEFORE COVD
SHOWED UP AND TOLD US YOU SHOULO
AND SPEAK -- LEARN ABOUT T-CELL
ACTIVATION AND SEE IF YOU CAN ME
SOMETHING MORE RELEVANT.
THIS IS WHAT WE DID.
WE SPOKE WITH IN THE POSTDOC INE
LAB AND THIS MODEL WHERE WE TAKE
THE ATOMS IN THE PCR AND DO WE
REALLY NEED THESE VERY COMPLICAD
MACHINERY?
NO WOOS DON'T LIKE ALL THE DETA.
WE WOULD LIKE TO SIMPLIFY THING.
HERE WE BUILT A MODEL IN THE LA.
WE HAD THIS DATA CHAIN IMITATED.
SO, WE LEARN TO WORK WITH HIM AD
WHAT WE DID IS TAKE THE CELLS AD
PUT IN ROBOT AND WE CAN AGAIN LK
AT THE WILDTYPE, WE CAN COMPOSEE
WAY THEY ARE ACTIVATING IN THE H
WAY AND WE LOOK AT THE MUTANT
T-CELLS WHICH DON'T CONTAIN
PHOSPHORYLATION, SIGNAL BETTER.
THE FIRST MODE ON THE X AXIS
DIFFERENT PEPTIDES.
AND YOU SEE NEW WAVE THOSE
RESPONSE -- YELLOW MODE WHICH IS
ACTIVATED AND MORE INTERESTING D
YOU SEE THE RED MODE WHICH IS
CORRESPONDING TO THE PATHWAY.
SO SUDDENLY INSTEAD OF JUST BEIG
ABSTRACT, YOU SEE THE COMPLETION
TWO MODES AND MAPS INTO SOME VEY
EXPLICIT BIOCHEMICAL PATHWAYS.
SO IN THIS MUTANT, WE ARE MISSIG
FEEDBACK AND WE SHOULD -- VERY
DEFECTIVE IN ACTIVATION.
-- IF YOU WANT SOMETHING
FUNCTIONAL, WE SCALE DIFFERENT -
NORMAL REGIME YOU HAVE DIFFERENT
TYPES OF T-CELLS VERY GOOD.
WILDTYPE ON THE LEFT.
AND WHEN YOU MOVE TO -- YOU HAVE
ONLY TWO CLASSES OF ONCOGENE.
EITHER NO ACTIVATION OR YOU DO.
WHEN WE LOOK AT IT THIS WAS
SOMETHING -- PRETTY CHANCE MODEE
ARE MISSING NEGATIVE FEEDBACK AD
ABLE TO INTERPRET A LOT OF
MEASUREMENTS BEING DONE.
SO ONE OF THE CONSEQUENCE OF THE
NEGATIVE FEEDBACK WE EXPECT -- O
THIS PART IS WHAT MAPS INTO --
KNOWN AS ANTAGONIST.
WHEN WE GO WE CAN CHECK IN THE
AFFECTED MUTANT, NOT ONLY MISSIG
FEEDBACK BUT NOT ABLE TO ANTAGOE
RESPONSE.
SO WE COULD DO AN ASSAY WHERE YU
MIX -- NORMAL T-CELL YOU GET NIY
DEFECT -- KEY CREASE CYTOKINE
PRODUCTION.
WHEN YOU MOVE TO MUTANT, YOU GET
INCREASE AND THAT'S WHAT -- JUST
WANT TO GIVE YOU A SENSE OF WHAE
ARE DOING IN TERMS OF TRYING TO
IMMUNOLOGY -- WE CAN GET FROM TE
ROBOT IN CYTOKINE -- CHARACTERIE
HIGH DIMENSIONAL SPACE.
ROBUST MANNER AND PRODUCTIVE ANT
WORKS WITH CORE SAMPLES AND WORS
IN COLLABORATION WHERE WE CAN GT
VERY QUICKLY MOVE AND DO THIS
MEASUREMENT AND WE CAN REALLY UE
THAT RICHNESS OF DATASET TO
COMPRESS DATA IN UNIQUE MANNER,
TRYING TO LINE IT UP AGAINST
FUNCTION AND REALLY RE-LEARN T-L
ACTIVATION AND USE THIS TO REALY
DESIGN SOME BETTER -- NO CLAPPI.
WE HAVE A SECOND PART.
>> THE TITLE OF MY TALK HAS
CHANGED.
PART OF IT IS WE HAD CHANGES INE
PROGRAM AND THEN BECAUSE OF LOTF
DIFFERENT ISSUES, I DECIDED I WD
BE THE LAST SPEAKER.
IT MADE SENSE TO SORT OF GIVE YU
THE SECOND PART OF THIS.
AND I WILL TRY TO CONVINCE YOU
ALSO -- SOME OF YOU MAY BE HARDR
TO CONVINCE THAN OTHERS BUT I WL
TRY TO CONVINCE YOU THAT EVEN
SOMETHING LIKE THINKING ABOUT
CHIMERIC ANTIGEN RECEPTORS DOES
HAVE A PLACE IN A SYMPOSIUM ON
BASIC IMMUNOLOGY.
WITH THAT AS MY CHALLENGE I WILL
MOVE FORWARD WITH THAT.
SO, WE ARE GOING TO COME BACK TO
TWO CHIMERIC ANTIGEN RECEPTORS D
THE VAST MAJORITY ARE AWARE THEY
ARE MADE UP OF BINDING SEGMENT O
TUMOR ANTIGEN AND THEN YOU HAVE
SIGNALING THAT GOES ON AND THAT
SIGNALING IS GOING TO LEAD TO TE
DEATH OF YOUR CELL IT'S REALLY
IMPORTANT TO SAY HERE IN THIS
MEETING, FRONTIERS AND BASIC
IMMUNOLOGY, ALL OF THIS CAN BE E
ONLY BECAUSE OF YEARS AND YEARSF
WORK ON TRYING TO UNDERSTAND HOW
THE T-CELL RECEPTOR WORKS.
WHEN I THINK ABOUT THE FOUR
PEOPLE -- I'M USING THE MICROPHE
HERE TOO.
THE FOUR PEOPLE WHO I THINK OF S
HAVING GIVEN AT LEAST ME AND THE
COMMUNITY A HUGE AMOUNT OF
INFORMATION ON TCR SIGNALING.
THERE IS ONE PERSON WHO ISN'T H,
ART WISE.
SO JUST PUTTING THAT TOGETHER, E
QUESTION BECOMES BASED ON WHAT -
IS MY -- DOES THE THING WORK?
THE POINTER?
SO ONE OF THE THINGS THAT HAPPES
THERE -- SORRY.
ONE OF THE THINGS THAT HAPPENS N
TERMS OF THINKING ABOUT WHAT WAS
JUST SAID, IS THERE -- WHAT IS E
INTERACTION BETWEEN THE SIGNALIG
THAT IS HAPPENING FROM THE CAR,
WHICH IS WHAT YOU'RE DOING WHEN
YOU'RE PUTTING INTO THE CELL AND
THE SIGNALING HAPPENING FROM THE
T-CELL RECEPTOR.
A LOT OF WORK WHERE PEOPLE HAVE
TAKEN THE T-CELL RECEPTOR OUT.
AND ALSO A FAIR AMOUNT OF DATA W
THAT SAYS THAT THE ENDOGENOUS
T-CELL RECEPTOR HAS IMPORTANCE R
THE CAR AND THE CAR IS THE
ASSOCIATE THE WITH SPECIFIC
ENDOGENOUS TCRs.
SO COMING BACK TO WHAT YOU HEARD
ABOUT FROM GREG, WE CAN TAKE THE
MODELS THAT THEY HAVE DEVELOPEDN
THEIR LAB AND WE CAN NOW LOOK AT
THIS AND THIS IS A MOUSE MODEL.
AND WE ARE LOOKING AT WHAT WE HD
ABOUT BEFORE IN TERMS OF THE LAT
SPACE AND SEEING WHAT HAPPENS WN
WE TAKE DIFFERENT ANTIGENS AGAIT
THE HUMAN TCR.
SO OT1 AND ALMOST NO RESPONSIVES
AND THEN GETTING TO HIGHER AND
HIGHER LEVELS OF ANTIGEN AND YOU
SEE MORE AND MORE RESPONSES.
WHAT HAPPENS WHEN YOU PUT IN A ?
SO YOU CAN SEE HERE THE LEVEL OF
RESPONSIVENESS THAT WE HAD WITH
CD19 CAR IS ACTUALLY MUCH LOWER
THAN WHAT WE HAD WHEN WE HAVE OR
BEST T-CELL RECEPTOR WITH A STRG
ANTIGEN.
SO A CD19 CAR WHICH IS REALLY TE
SORT OF ROLLS-ROYCE WHEN WE THIK
ABOUT IN TERMS OF CAR IS NOT
SIGNALING AS WELL AS OUR TCR
ANTIGENS.
BUT EACH MORE INTERESTING WHAT
HAPPENS IS, WE DON'T -- THE LOWT
LEVEL OF ANTIGEN HERE IS A LEVEF
RESPONSIVENESS LOWER THAN WHAT
YOU'RE SEEING WITH CD19 CAR.
SO THE QUESTION THEN BECOMES HOW
DOES THIS ALL WORK?
IS THERE AN INTERACTION AND WHAS
THE INTERACTION WHEN WE'RE LOOKG
AT A CD19 CAR AND AN OT1TCR?
WE DID THAT TO MODEL THESE THINS
FIRST.
WON'T TELL YOU A HUGE AMOUNT OF
DATA.
ALL THIS DATA IS UNPUBLISHED.
WE DECIDED TO PRESENT IT HERE
BECAUSE WE ARE IN THE PROCESS OF
TRYING TO THINK WHERE TO SEND IT
OUT.
SO WE WOULD BE HAPPY TO HAVE YOR
COMMENTS.
IT ALSO MEANS WE TAKE A SLIGHT K
BY SHARING TO A LARGE ROOM OF
PEOPLE.
BUT SINCE YOU'RE ALL OUR BEST
FRIENDS, WE KNOW THAT'S NOT AN
ISSUE.
WHAT WOULD EXPECT IS IF YOU HAVA
TCR SIGNAL WITH NOTHING AT ALL S
IS WRONG AND I APOLOGIZE.
THIS IS THE WRONG THING.
THIS IS WHAT WE ANTICIPATE THE E
RESULT WOULD BE.
IF YOU HAVE A CAR BY ITSELF, IT
WOULD GIVE YOU SOME RESPONSE.
THE MICE WOULD INDIVIDUALLY DIE.
THAT IF YOU HAD A CAR WITH A STG
TCR THAT WOULD GIVE YOU A REALLY
GOOD RESPONSE.
AND THEN IF YOU HAD A CAR WITH A
WEAK TCR IT WOULD BE SOMEWHERE N
THE MIDDLE.
BUT THAT'S NOT THE CASE.
THIS IS THE WRONG VERSION.
T-CELLS ARE BAD AT MATH AND THE
EXPERIMENTAL RESULT IS BASICALLY
THAT IN FACT THIS IS THE REAL
RESULTS WHICH IS IF YOU HAVE A K
TCR, THOSE MICE DIE MUCH EARLIE.
NOT ONLY DO THEY DIE MUCH EARLI,
BUT WHEN YOU LOOK AT CYTOKINES Y
ARE ALSO IN A CONTEXT WHERE THEY
ARE MUCH LOWER THAN WHAT YOU HAE
WITH A CAR ALONE.
YOU'RE HAVING AN EFFECT THAT IS
ANTAGONISTIC.
I THOUGHT ABOUT THIS, I LISTENEO
THE TALKS THIS MORNING FROM BOTH
LARRY AND JULIAN THINKING ABOUT
WHAT WE THINK ABOUT IN TERMS OFC
GAMMA ACTIVATION AND YOU CAN SEE
THAT IF WE HAVE A CD19 LOW CAR,D
WE LOOK AT T ARE CR ANTIGEN
STRENGTHS WITH JUST THE CAR ALO,
YOU HAVE A CERTAIN RESPONSE.
IF YOU THEN LOOK AND SEE WHAT
HAPPENS WHEN YOU HAVE LOW LEVELF
TCR ANTIGENS IT'S GONE DOWN AND
IT'S WHEN YOU HAVE VERY HIGH LES
OF ANTIGEN THAT IT COMES BACK U.
LOOKING AT MULTIPLE EXPERIMENTSU
CAN SEE THAT YOU CAN MODEL THISD
THIS IS WHAT YOU SEE WHEN YOU ML
IT.
SO, WE CAN MODEL THIS AND THIS S
THE KIND OF THING THAT YOU HEARD
ABOUT BEFORE.
WE CAN THEN MODEL FIT IT.
WE CAN MODEL FIT IT WITH ICANS
WHERE WE HAVE THREE ICHAN AND TN
WE CAN USE A MATHEMATICAL MODELD
WILL WHEN WE USE THIS MATHEMATIL
EQUATION YOU CAN SEE WHEN YOU HE
A WEAK TCR ANTIGEN, MOST OF THE
FEEDBACK YOU'RE GETTING IS
NEGATIVE.
SO YOU'RE CREATING AN ANTAGONISC
FEEDBACK.
SO THE QUESTION BECAME, CAN WE E
THIS IN THE CONTEXT WHICH WOULDE
THERAPEUTIC?
BECAUSE ONE OF THE ISSUES WITH S
IS THAT THE CAR IS NOT ABLE TO E
THE DIFFERENCE BETWEEN AN ANTIGN
THAT IS IT ON A HEALTHY CELL ANN
ANTIGEN THAT IS ON A TUMOR CELL.
IT'S GOING TO KILL BOTH OF THOS.
WHAT IS USUALLY DIFFERENT AND WH
WE ARE NOT TARGETING WITH A CARN
GENERAL, THE FACT THAT YOU'RE GG
TO HAVE PROTEINS MUTATED BECAUSE
YOU HAVE MANNER MORE PEWITATIONN
A TUMOR CELL THAN YOU HAVE A
HEALTHY CELL.
SO IN THE CONTEXT OF CD19 CARS,
WHEN YOU LOOK AT THIS EFFECT, WH
IS WHAT WE CALL AN EFFECT OF ON
TARGET BUT OFF TUMOR TOXICITY,
THAT'S NOT CAUSING HUGE AMOUNTSF
PROBLEMS BECAUSE YOU'RE KILLINGF
YOUR CD19 POSITIVE LEUKEMIA ANDE
FACT THAT YOU'RE KILLING OFF THE
POSITIVE B CELLS IS SOMETHING TT
YOU CAN DEAL WITH.
YOU CAN GIVE IMMUNOGLOBULIN.
BUT IT'S NOT SOMETHING THAT IS
GOING TO KILL YOUR PATIENT.
AT LEAST IN GENERAL.
AND THEN WE KNOW THE LONGER YOU
HAVE B-CELL APLASIA THAT'S A GOD
SIGN IN TERMS OF HAVING A CAR TT
WILL BE FUNCTIONAL.
IN THE CONTEXT OF SOLID TUMORS,E
OF THE PROBLEMS IS THAT A LOT OF
THE TARGETS WE HAVE IN SOLID TUS
HAVE TARGETS IN TISSUES THAT ARE
CRUCIAL.
SO FOR EXAMPLE, WHILE WE WANT TO
KILL OFF A HER2 POSITIVE CARCINA
YOU DON'T WANT TO KILL OFF LUNG
CELLS THAT ARE HER2 POSITIVE
BECAUSE THAT WOULD CAUSE
RESPIRATORY FAILURE AND DEATH.
THE QUESTION OF THIS YIN AND YAG
AND HOW TO DEAL WITH IT AND THE
TOXICITIES IS A HUGE PROBLEM.
AND IT'S DEFINITELY A LARGE PARF
THE REASON WHY IT'S BEEN SO MUCH
SIMPLER -- ONLY PART OF THE ISSE
BUT WHY IT'S BEEN SIMPLER TO HAE
SUCCESS IN THE TREATMENT OF LIQD
TUMORS AS COMPARED TO SOLID.
SO WHAT IS THE COMMUNITY?
THE COMMUNITY HAS DONE ALL SORTF
DIFFERENT THINGS AND DESIGNS TRG
TO SAY THAT YOU NEED TO -- IN OR
TO HAVE A CAR TO WORK FOR EXAMPE
AGAINST A SOLID TUMOR, YOU WOULD
HAVE TWO DIFFERENT TARGETS AND Y
BOTH HAVE TO BE THERE IN ORDER O
GET A RESPONSE.
OR THAT ONE HAS TO BE THERE ANDE
OTHER HAS TO BE GONE.
AND THAT YOU CAN PUT IT IN AND U
HAVE TO HAVE TWO TARGETS IN ORDR
TO GET SIGNALING AND THAT'S CALD
A LINKER.
SO THERE HAS BEEN A HUGE AMOUNTF
WORK ON THIS.
A LOT OF THESE PAPERS COME OUT N
SCIENCE, CELL, NATURE, BECAUSE
THERE IS A HUGE REQUEST TO SEE W
THIS WORKS.
BUT THERE ARE SOME SIGNIFICANT
LIMITATIONS EVEN WITH WHAT HAS N
DONE WITH WHAT WE CALL THE LOGIC
GATES.
AND THE PROBLEM IS WITH A CAR,
YOU'RE LOOKING AT SURFACE ANTIG.
BUT A HUGE AMOUNT OF THE PROTEIS
IN THE CELL IS NON SURFACED, ITS
INTRACELLULAR.
OVER 85% OF THE PROTEINS IN OUR
CELL ARE INSIDE THE CELL.
AND YOU WOULDN'T BE GETTING THAT
WITH A CAR.
SO YOU'RE ELIMINATING 85% OF THE
POSSIBLE TARGETS THAT YOU COULD
EVEN HAVE.
AND THEN THERE ARE OTHER ISSUES
THAT WE HAVE IN TERMS OF TISSUES
RESTRICTIONS, ANTIGENS, LOGISTIL
CHALLENGES.
SO THE QUESTION WE ASKED IS, CAE
CREATE A SITUATION WHERE IN THE
CONTEXT OF A STRONG ANTIGEN THAS
BEING SEEN BY THE TCR, WE WOULD
ENHANCE THE ACTIVATION.
WHEREAS WITH A WEAK ANTIGEN THEE
COULD USE THIS AS A WAY OF
RESTRAINING AND NOT HAVING ON
TARGET OFF TUMOR TOXICITY.
SO THE QUESTION IS, WILL THAT
HAPPEN?
IN ORDER TO DO THIS, WE, AND ITS
REALLY -- SITTING NEXT TO THE DR
AND RESPONSIBLE FOR A HUGE AMOUT
OF THIS WORK TOGETHER WITH FRANS
AND GEORGE TO DESIGN AND PUT INO
THAT CONSTRUCT A TCR AGAINST HH,
WHICH IS MUTATED IN SEVERAL TYPS
OF DIFFERENT KINDS OF TUMORS.
AND TO DO THAT WITH A REPORTER.
AND THEN THE QUESTION BECOMES, N
WE USE THIS NOW TO CHANGE WHAT S
OCCURRING IN THE CONTEXT OF TISE
CELLS AND TUMOR CELLS?
SO THE FIRST QUESTION IS, WE STT
TO LOOK AT THE RESPONSIVENESS OF
THIS TO TISSUE CELL ANTIGEN ANDE
ARE ALWAYS COMPARING WITH WHAT E
ARE SEEING FOR HER2 CAR.
SO WITH SELF ANTIGEN, OUR HHAT R
TWO CAR AGAINST TISSUE IS HAVING
LOWER LEVELS OF INTERFERON GAMMA
AND OTHERS AND AGAINST THE TUMOR
WHERE WE HAVE THE NEO-ANTIGEN OF
THE HHAT WE HAVE INCREASES IN SE
OF THESE INCLUDING INTERFERON GA
AND IL-2.
SO IT IS EXCITING TO SEE THAT WE
HAVE THIS YIN YANG WE CAN PLAY H
IN TERMS OF THE SELF ANTIGEN,
NEO-ANTIGEN CONTEXT.
SO WHAT HAPPENS WHEN WE START TO
LOOK AT IT?
SO HERE WE ARE LOOKING AT WHAT
HAPPENS IF WE HAVE THE TCR ALON,
THE HER2 CAR ALONE OR THE HER2 R
WITH AUTO TCR AGAINST HHAT.
SO IF YOU'RE NOT EXPRESSING HHA,
YOU WOULD EXPECT YOUR HER2 CAR D
HHAT WOULD BE THE SAME AND THEY
WOULD BLOCK PROLIFERATION IN THE
SAME WAY.
AND THAT'S THE CASE.
AND ALSO THE HHAT, TCR IS NOT DG
ANYTHING.
WHAT HAPPENS IF YOU HAVE THE TUR
TISSUE?
NOW IN THE TUMOR TISSUE WE HAVEE
NEO-ANTIGEN.
SO WITH THAT, YOU CAN SEE THAT E
TCR ITSELF IS BLOCKING
PROLIFERATION COMPARED TO THE
CONTROL.
AND IN THIS CASE, BOTH AGAIN THE
HER2 CAR AND THE MIXED CAR IS WH
THE TCR KILLING AT THE SAME RAT.
SO WHAT HAPPENS WHEN WE HAVE
TISSUE?
BECAUSE THIS IS THE REAL QUESTI.
NOW WE DON'T HAVE A NEW ANTIGENE
HAVE A SELF ANTIGEN.
AND THIS SHOULD BE WEAK.
DO WE SEE A DIFFERENCE?
THE ANSWER IS, YES.
YOU CAN SEE HERE THAT THE HER2 R
ALONE IS KILLING A HUGE NUMBER F
THE CELLS AND I DON'T KNOW WHY
MY -- I CAN'T GET THIS TO WORK.
THAT IS THE DIFFERENCE AND DEPTF
THE HEALTHY TISSUE.
WHEN WE HAVE THE MIXED CAR WITHE
TCR, WE HAVE MUCH LEGS DEATH OFE
HEALTHY TISSUE AS COMPARED TO TE
HER2 CAR.
SO THEN -- WE DECIDED TO GIVE TS
A NAME AND SEE IF IT WOULD WORK
IN-VIVO SITUATION.
SO THIS IS CALLED AN AUTO
MUSEMERGENCY BREAKING SYSTEM.
ABS.
THE IDEA IS YOU'RE SEEING A
DIFFERENCE WHEN YOU HAVE SELF AD
THAT WHEN IT DETECTIVES SELF ITS
AUTONOMOUS BREAKING THE CAR
ACTIVITY.
SO NOW TO BE ABLE TO LOOK AT TH,
WE'RE USING A MODEL WHERE WE'RE
USING THE TUMOR WHICH HAS THE
NEO-ANTIGEN WITH HHAT AND HER2 D
HEALTHY TISSUE AND THE HEALTHY
TISSUE IS BIOLUMINESCENT.
AND THAT IS EXPRESSING WILDTYPE
HHAT AND ALSO EXPRESSING HER2.
SO THE QUESTION IS, DOES THIS WK
IN-VIVO?
BECAUSE OF LOTS -- THERE ARE LOS
OF THINGS THAT GO INTO THIS.
THERE IS A HUMAN SYSTEM IN MSG
MOUSE.
YOU HAVE TO WORK THROUGH THE
DIFFERENT LEVELS OF THE ANTIGENS
YOU HAVE AND THESE ARE EXPERIMES
THAT TOOK OVER EIGHT MONTHS TO T
WORKING.
WHAT YOU SEE HERE IS THAT IF WE
LOOK AT THE TUMOR, AND NOW WE'RE
LOOKING AT THE TUMOR GROWTH, UNL
YOU CAN SEE THAT BOTH THE HER2 R
AND OUR AEBS CAR ARE DOING A REY
GOOD JOB OF CONTROLLING TUMOR
GROWTH.
SO WHAT HAPPENS NOW IN TERMS OFE
HEALTHY TISSUE?
I WOULDN'T BE SHOWING YOU THIS F
IT DIDN'T WORK.
IN MOCK CELLS YOUR HEALTHY LUNG
TISSUE CONTINUES TO BE PRESENT D
YOUR HHAT IS PRESENTS.
BUT YOUR HER2 CAR HAS QUICKLY
WITHIN THREE DAYS KILLED OFF ALL
YOUR LUNG TISSUE IN THIS MOUSE.
NOW THIS IS TRANSPLANTED IN THE
MICE BUT THE MICE ARE STILL ALI.
BUT WHAT CAN YOU SEE HERE IS WHE
THE AEBS CAR IS HAVING SOME KILG
OF THE HEALTHY TISSUE, YOU HAVEY
MORE HERE THAN WHAT YOU HAVE INE
HER2 CAR.
SO HOPEFULLY IN LESS THAN 15
MINUTES I HAD I CONVINCED YOU WE
HAVE ANTAGONISM ENFORCED BREAKIG
SYSTEM WITH A CART T-CELL WE CAN
USE AS AN ACCELERATOR WHEN WE HE
A NEO-ANTIGEN AND A BREAK WHEN E
HAVE A TISSUE ANTIGEN AND THAT E
CAN THEREBY PROTECT.
SO THIS IS A REALLY NEW CONCEPTN
TERMS OF THINKING ABOUT HOW WE N
CONTROL THE RESPONSIVENESS WE WD
HAVE IN TERMS OF IMMUNOTHERAPY.
AND SO THIS IS A SLIDE THAT I
REALLY LIKE.
WHEN WE THINK ABOUT WHAT HAPPEND
IN 2002 AND LOTS OF REALLY
WONDERFUL PEOPLE WHO TRIED TO ME
THIS HAPPEN.
SOMEONE WHO I WORKED WITH IN ISL
IS REALLY RESPONSIBLE FOR A HUGE
AMOUNT OF THE WORK AND A SINGLE
CHIMERIC TCR BETA THING.
AND NOW WE'RE AT THE POINT OF
HAVING NEXT GENERATION CARS WHEE
WE WENT FROM DRIVING OUR CELLS O
BEING ABLE TO HAVE AUTONOMOUS
DRIVING AND I CERTAINLY DON'T TK
WE ARE QUITE THERE YET BUT
HOPEFULLY THIS IS SOMETHING THAS
IT A NEW WAY OF THINKING ABOUT
THIS.
AND SO BASICALLY THE IDEA IS THT
USING THE MATHEMATICAL MODELS WE
DEVELOPED, WE SHOWN YOU AND
CONVINCED YOU THAT CAR TCR DOEST
EXIST AND WE CAN PREDICT WHAT IS
GOING TO HAPPEN IN DIFFERENT
PERTURBATIONS -- I DIDN'T HAVE E
TO SHOW YOU THAT -- THIS ANTAGOM
SIGNIFICANTLY IMPACTS TUMOR
PROGRESSION IN BOTH LIQUID AND
SOLID TUMORS AND REALLY MOST
IMPORTANTLY, BECAUSE THIS IS WHE
THE FIELD HAD A LOT OF PROBLEMS.
THIS ANTAGONISM CONFERS COMPETIE
SURVIVAL ADVANTAGE FOR TUMORS
DURING WEAK TCR LYINGANCE AND
HETEROGENOUS TUMOR POPULATIONS.
AND THAT WE CAN USE THIS TO REDE
OFF TARGET TOXICITY AGAINST HEAY
TISSUE.
AND AS WE SAID BEFORE, THIS IS
REALLY A HUGE AMOUNT OF WORK WIH
INCREDIBLE AMOUNT OF COMPITATORY
BETWEEN THREE GROUPS WORKING ON
MODELING SIDE AND WORKING ON THE
MATHEMATICAL SIDE AND WORKING ON
THE BIOLOGICAL SIDE AND WORKING
WITH OUR CLINICAL COLLEAGUES AS
WELL.
WE WERE INCREDIBLY FORTUNATE TO
HAVE FLEX FUNDING FROM THE NCI O
REALLY BE ABLE TO CARRY THIS OUS
A SYNERGY GRANT AND SOMETHING TT
NONE OF US HAD BEEN WORKING ON
BEFORE.
AND SO I JUST WANTED TO SAY THAT
THERE A HUGE NUMBER OF PEOPLE FM
ALL THESE LABS WHO HAVE BEEN
INVOLVED IN ALL OF THIS.
AND I DIDN'T TALK ABOUT LOTS OF
STUFF GOING ON IN OUR LAB,
METABOLIC WORK.
BUT I'M FORTUNATE TO HAVE MOVED
HERE TO THE NIH TO HAVE STARTEDN
INCREDIBLE GROUP AND I'M GRATEFL
TO EVERYONE.
AND WE ALL STARTED WHERE WE CAME
FROM AND VALERIE IS HERE SO I
WANTED TO GIVE A SHOUT OUT.
I SPENT 20 PLUS YEARS IN FRANCED
EVERYTHING I DID, I LEARNED WITH
THEM.
SO I REALLY OWE THEM A HUGE DEBF
GRATITUDE.
SO THANK YOU.
[ APPLAUSE ]
>> SO WE HAVE TIME FOR QUESTIONO
BOTH GREG AND NAOMI.
>> SO THIS IS REALLY FASCINATING
WORK.
MY QUESTION IS, FOR SOME TUMORS.
>> [ INDISCERNIBLE ]
>> I THINK FOR THE TUMORS THAT
MAYBE HAD NEW ANTIGEN, BUT AT TE
SAME TIME STILL EXPRESS THE
WILDTYPE ANTIGEN, HETEROZYGOUS
MUTATION.
WHAT IS GOING TO HAPPEN?
>> SO GREG HAS A SLIDE BUT I THK
THE BOTTOM LINE WOULD BE THAT TE
HIGH TCR SIGNALING WOULD BE
DOMINANT OVER THE LOW TCR
SIGNALING.
WHAT IS REALLY INTERESTING AND
STRIKING WHEN YOU LOOK AT THE DA
AND I'M SURE DAREIO CAN TELL YOU
THIS AS WELL.
YOU SELECT FOR TCRs.
YOU'RE GETTING TCRs AND WHAT IS
INTERESTING FOR EXAMPLE, IF YOU
LOOK AT PATIENTS WHO GOT CARS AD
THEN THEY GET ANTI-PD-1 AND THEN
LOOK AT THE TCs AVAILABLE
AFTERWARDS, YOU DON'T HAVE THE
SAME.
YOU'RE SELECTING FOR STRONG TCR
ANTIGENS RATHER THAN FOR
ANTAGONISTIC SIGNAL.
THOSE WILL BE THE NEXT QUESTIONS
AND TO REALLY BE ABLE TO MODEL S
WITH THE PEOPLE HERE AND SAY, HW
DO WE LOOK AT TCR LIGAND STRENGH
AND THINK ABOUT THIS IN TERMS OA
CAR THERAPY WILL BE SOMETHING TT
IS INCREDIBLY EXCITING.
>> BASED ON THE MODEL WE WANT TE
ABLE TO PREDICT FOR EVERYTHING
AGO - HOW FAR YOU ARE FROM THE L
WILL TELL YOU WHEREVER YOU ARE N
ANTAGONIST.
SO THE BIG PROBLEM IN CURRENT
MODELS -- THEY ASSUME BETTER
BIOPHYSICS THE MORE YOU ACTIVAT.
THERE IS NEGATIVE VAL I A YOU HE
TO PASS -- [ INAUDIBLE ]
MODELING IS REALLY CRITICAL.
>> MY QUESTION IS FOR GREG.
IN YOUR -- THAT MODEL YOU HAD IN
LATENT SPACE OUTPUT PARAMETERS,T
YOU'RE TRAINING THEM WITH CYTOKE
SIGNALS YOU'RE MEASURING.
SO IF YOU EXTRACT THE FEATURE
WEIGHTS OF THOSE NEURONS COULD U
LEARN SOMETHING ABOUT THE
IMPORTANCE OF WHAT THESE CYTOKIS
ARE?
>> THAT'S THE BEAUTY OF THESE
SIMPLE ENCODERS.
YOU CAN GO BACK AND ANALYZE EVEY
NEURON.
SO WE HAVE A BUNCH OF WEIGHT.
IF WE -- THAT'S WHAT YOU WOULD E
MONEY ON BECAUSE YOU HAVE A BUNH
OF THE WEIGHTS, AS YOU KNOW 0.5
INTERFERON GAMMA -- NONLINEAR
MANNER.
AND YOU CAN -- DISCOVER NEW STUF
WHICH IS IF YOUMENT TO MEASURE
ANTAGONISM VERY EARL WELLY ON.
IF YOU WANT TO MEASURE
RESPONSIVENESS WHAT YOU EXPECT,T
AGAIN WE CAN REALLY LOOK AT THIS
WEIGHT AND GO BACK TO -- IF YOU
TAKE AND YOU BUILD THESE CYTOKIS
YOU WILL BE ABLE TO --
IMMUNORESPONSE TO MORE ACTIVATIN
OR ANTAGONISM.
>> SO IN YOUR MODELS THEN, THOSE
ARE THE HIGHEST WEIGHTS?
>> RIGHT.
SO FIRST STEPS WE WERE USING MOY
12 CYTOKINE PANEL.
NOW WE ARE 45.
YOU WOULD BE AMAZED STUFF WHICHS
NOT EXPRESSED.
CD8 MAKING IL17.
YOU WOULD NOT EXPECT THAT.
IT CAN BE VERY SIGNIFICANT IN TS
OF INFLAMMATION AND WE CAN DECIE
AND PRIORITIZE THINGS BASED ON E
WEIGHTS.
>> I THINK THIS IS PROBABLY FOR
BOTH OF YOU.
BECAUSE IT REALLY HAS TO DO WITH
THE WAY THESE MODELS WORK.
THEY ARE STILL VERY TCH-BASED AD
WE SPENT A LOT OF TIME IN THE RT
OF THE LAST TWO DAYS TALKING ABT
THE DIFFERENCES IN THE CELL STAE
OF THE DIFFERENT T-CELL
POPULATIONS.
AND I'M ASSUMING THAT THEY ARE
GOING TOEL REALTER THE OPERATIOF
THE MODEL AND THE WEIGHTS OF THE
DIFFERENT SIGNAL FRANCE DEDUCTIN
PARAMETERS AND THE FEEDBACKS GIE
YOU THE NEGATIVE EFFECTS OF THE
LATENT SPACE TOO.
SO I GUESS -- OR A THIRD LATENT
SPACE WHICH MIGHT COME IN AS WE.
THAT THE IS SOMETHING THAT AS A
CAR-T CELL DESIGNER, YOU MIGHT E
ABLE TO PLAY WITH.
A LOT OF PEOPLE ARE TRYING TO PY
WITH IT.
SO I GUESS I WANTED TO JUST ASK
YOU, HOW YOU ARE THINKING ABOUT
THAT AND IS THERE WORK UNDERWAYO
SEE HOW ROBUST THESE ONE CELL SE
BUILT MODELS ARE AS YOU MOVE TO
OTHER KINDS OF T-CELLS, VIRTUAL
MEMORY, CD8 CELLS, NAIVE CD8 CE,
EXHAUSTIVE CD8 CELLS.
>> IT'S SOMETHING WE THOUGHT A T
ABOUT.
I WOULD SAY THAT WE WERE PLEASAY
SURPRISED -- WE OBVIOUSLY STARTD
THESE EXPERIMENTS IN A MODEL OF
MICE WHERE ALL OF OUR T-CELLS WE
NAIVE.
WE DIDN'T HAVE TO PLAY WITH THE
TCR.
WE DIDN'T HAVE TO DO ANY
TRANSDUCTIONS AND THEN WE WENT O
HUMAN CELLS AND WE SAW SOMETHING
THAT WAS VERY SIMILAR.
SO I THINK IN TERMS OF MODELINGD
I THINK THAT ONE OF THE REASONSY
WE WERE ABLE TO -- SEEING A VERY
SIMILAR DIFFERENCE, AT LEAST ASE
STATE-OF-THE-ART IS TODAY FOR
THINGS LIKE CAR-T CELLS OR ADOPE
T-CELL THERAPIES, EVERYTHING IS
BEING ACTIVATED AND IT'S BEING
ACTIVATED IN A WAY THAT IS HUGEY
POWERFUL.
SO IF YOU WERE TAKING YOUR T-CEL
ALONE AND HAD YOUR CAR AND TCR N
IT, YOU WOULD HAVE HUGE DIFFERES
DEPENDING ON YOUR SUB SET.
WE ARE TAKING A TCR BEING
STIMULATED WITH CD3, CD28 AND I2
AND THEN SEEING WHAT IS HAPPENI.
BECAUSE OF THE FACT THAT WE HAVE
THAT UPSTREAM, WE ARE ACTUALLY
NEGATING THE DIFFERENCES BETWEEN
THE DIFFERENT SUBSETS.
THERE ARE GOING TO BE DIFFERENCS
AT SOME POINT.
ONE OF THE THINGS WE ARE SURPRID
AT IN THE LAB IS -- I THINK WE L
ASSUME WHEN WE LOOK AT T-CELLS M
PATIENTS WHO HAVE THE GO EN TONF
CHEMOTHERAPIES, THOSE WOULD BE E
T-CELLS THAT WOULD HAVE THE LEAT
RESPONSIVENESS WHEN WE LOOK AT
ANTI-TUMOR RESPONSES WHEN PUTTIG
IT IN TO SEE IT.
IN FACT, IT'S NOT THE PATIENT
T-CELLS THAT ARE THE WORST.
IT'S EVERY SINGLE DONOR IS
DIFFERENT.
PEOPLE WHO TELL YOU THEY DO A
EXPERIMENT WITH HUMAN CELLS 5 TS
AND WILL USING THE SAME DONOR
THAT'S CRAP.
BECAUSE -- SORRY.
THERE IS SO MANY TIMES WHEN PEOE
SAY THEY USE THE SAME BATCH OF
T-CELLS.
THEY ARE COMPLETELY DIFFERENT.
A HUGE DIFFERENCE IN ALL OF OUR
MAPS IS REALLY BASED ON DONORS.
SO THERE ARE HUGE DIFFERENCES TT
ARE DONOR BASED.
AND THAT'S SOMETHING WE NEED TO
SPEND MORE AND MORE TIME ON.
>> WHEN YOU BUILD THE MODEL, ITS
THE BEAUTY OF COLLABORATION.
SO MANY DIFFERENT TYPES OF DONO,
SO MANY DIFFERENT TYPES OF TCR M
NEGATIVE MOUSE TO ALL THE WAY
EXHAUSTIVE AND EXPANDED CELLS, U
CAN STILL LEARN A MODEL WHICH IS
UNIVERSAL.
SO ALL CYTOKINES IF YOU LOOK AT
GAMMA AND IL-2, IT GOES FROM LIE
ONE TO 10 NANOMOLAR.
BECAUSE YOU CAN NORMALIZE IT WIH
ALL THE OTHER DIMENSIONS AND STL
LEARN THE WAY THE TCR IS WIRED,E
WAY THE CAR IS WIRED.
THAT'S WHY WE ARE GOING TO GO FM
MOUSE TO HUMANS IN A UNIVERSAL .
>> WE DON'T UNDERSTAND ENOUGH
BECAUSE WITH GREG, WHAT WE HAVE
DONE AND AGAIN THIS IS UNPUBLIS,
BUT CRYSTAL AND RALEIGH SHAW ARE
BOTH RUNNING TRIALS OF THE SAME
CD22 CAR.
ONE IN STANFORD AND ONE NCI.
THE PATIENTS ARE DIFFERENT.
LOTS OF DIFFERENCES IN PEDIATRIC
POPULATION VERSUS ADULT.
WE COULD BLINDLY WITH GREG IN
TESTING THE SAMPLES, WE KNEW WHO
WAS TREATED AT STANFORD AND WHOS
TREATED AT THE NIH BECAUSE THE
MANUFACTURING WAS DIFFERENT.
NOW HOW THAT MANUFACTURING IS WL
PLAY INTO SOMETHING LIKE DAREIOS
CLINICAL TRIALS LOOKING AT LAG D
HOW THOSE CELLS ARE BEING USED
DIFFERENTLY IS SOMETHING THAT WL
BE FASCINATING.
BUT I COULD SHOW YOU THE DATA AD
YOU WOULD KNOW EXACTLY WHICH
PATIENT WAS WHERE.
>> THIS IS, I BELIEVE, A QUESTIN
MORE FOR GREG.
YOU REDUCED THE RESPONSES DOWN O
SIX TYPES OF ANTIGENS AND WAS TT
JUST FOR THE OT1 SYSTEM OR
GENERALIZABLE?
IF YOU EXTENDED TO STUDIES, MORE
QUALITIES AND COULD YOU REDUCE
THOSE CATEGORIES TO IMMUNOSPEAK,
MEANING IS THERE ANTAGONIST TYPI
AND 2 WHICH MIGHT HAVE DIFFERENT
CYTOKINES OR SOMETHING THAT I CD
THEN PUT INTO CLUSTERS?
--
[ MULTIPLE SPEAKERS ]
>> THAT'S EXACTLY WHAT WE ARE
DOING.
WE WANT TO DISCOVER MORE.
SO WE DO MORE SINGLE CELLS SO IS
NOT SIX CLASSES.
WE ARE MORE AT LIKE 12.
AND BEING ABLE TO ASSIGN WHICH
CLASS WILL BE THE CHALLENGE.
SO WHEN PEOPLE DO SINGLE CELL
RNA-SEQ AND -- THEY SEE EVERY CE
SEEMS TO BE ABLE TO GO INTO A OE
POT WHICH IS VERY DIFFERENT AND
BEING ABLE TO MAP THESE THINGS S
REALLY GOING TO BE THE CHALLENGE
BUT I THINK IT'S NOT JUST
ANTAGONIST.
WE ARE REALLY GOING TO DISPARATE
THINGS.
IF YOU WANT TO, YOU CAN HAVE A T
OF CLASSES AND AGAIN FOR THE
CLINICAL, I THINK IT WILL BE VEY
IMPORTANT BECAUSE IT CAUSES YOUO
REALLY PARSE RESPONSIBILITY IN A
BETTER WAY.
>> [ OFF MICROPHONE ]
>> WE DON'T WANT LAKES.
WE THINK IT'S A CONTINUOUS
CONTINUUM.
IF YOU GIVE ME A PCR, I CAN PUTT
IN PRECISE MANNER SO YOU CAN DEE
IT AS A CLASS.
WHEN YOU START TO DO A SINGLE
MUTANT AND THEN YOU CAN REALLY E
ABLE TO WORK ON IT.
>> YOU CAN EVEN MAYBE CLASS 1,
CLASS 2 INDEPENDENT LIGAND.
>> THAT WAS BEAUTIFUL.
IN YOUR MODELS YOU'RE REALLY
FOCUSING ON SIGNALING TCR
SIGNALING.
AND GOING BACK TO OLD WORK OF
YOURS, GREG, JUST WONDERING IS
THERE A FEEDBACK TO THE CYTOKINE
SIGNALING ITSELF?
SO THAT THERE IS --
[ MULTIPLE SPEAKERS ]
>> RIGHT NOW WE ARE MOTHERING
RIGOROUS ENGINEERING.
TAKE A CYTOKINE AND MAP BACK THE
TCR.
IT'S CONSISTENT.
IF YOU LOOK AT THE MODEL, IF WEE
ABLE TO MEASURE THINGS IN 10
MINUTES, WE COULD REALLY PREDICT
EXACTLY WHAT IS GOING TO HAPPENN
22 HOURS.
THERE IS A LOT OF REINFORCEMENT
FROM THE CYTOKINE BEING PRODUCE,
FEEDBACK ON TO THE TCR TO ENABLE
YOU TO BUILD THIS.
>> IN YOUR OLD DATA, ONE WOULD
ARGUE THAT WEAK TCR SIGNALING WL
DESENSITIZE YOU TO CYTOKINE
SIGNALS --
[ MULTIPLE SPEAKERS ]
>> THE FACT THAT WEAK TCR IN OTR
WORDS YOU TO SWITCH OFF -- QUICY
AND RAMP UP -- NOT GIVE YOU AS H
CONSUMPTION BECAUSE YOU DON'T HE
A LOT OF C25T WILL GIVE YOU A
DIFFERENT -- COMPARED TO STRONG
WHICH PRODUCES MORE CYTOKINE AND
CONSUMES MUST FASTER.
WE ARE BLIND TO THESE DIFFERENCS
BUT THAT'S WHAT THE MODEL LEARN.
SO I'M NOT ABLE TO TELL YOU EXAY
WHICH KINASE IS DOING IT, BUT WE
CAN SEE IT FROM THE DATA.
TO BUILD THE DIFFERENT -- WE CAN
USE --
>> BUT AMAZINGLY, EVERY CENTER S
DOING THEIR OWN DIFFERENT CYTOKS
WHEN THEY ARE MAKING T-CELLS FOR
ADOPTIVE TRANSFER.
WHAT WE ARE CONVINCED OF, WHAT L
TAKE A LONG TIME TO FIGURE OUT,W
ARE YOU CHANGING THE TCR CAR
BALANCE IF YOU'RE MAKING YOUR CS
WITH IL7 OR IL15 VERSUS MAKING M
WITH IL-2.
AND YOU'RE GOING TO BE SELECTING
OUT DIFFERENCES IN TERMS OF THA.
I DON'T THINK IT'S A QUESTION OF
DIVERSITY, PER SE BUT YOU PROBAY
WILL BE SEEING DIFFERENCES IN TS
OF WHAT YOU'RE SEEING FOR ANT JN
INTERACTIONS.
AND ESPECIALLY FOR LIQUID TUMORS
WHERE THE CAR TARGET IS NOT
PRESENT, THAT'S GOING TO BE EASR
TO DEAL WITH BUT WITH SOLID TUMS
WHERE YOU WILL HAVE THE INTERACN
T WILL BE MORE INTERESTING.
I DON'T THINK WE ARE OUT OF A JB
ANY TIME SOON.
>> [ OFF MICROPHONE ]
>> THANK YOU.
[ APPLAUSE ]
>> YOU MADE IT TO THE END.
WE MADE IT TO THE END.
WE REALLY JUST WANTED TO THANK .
JEN WHERE ARE YOU?
YOU WANT TO SAY SOMETHING?
COME SAY SOMETHING.
IT'S REALLY BEEN A HUGE PLEASUR.
THIS IS THE FIRST TIME WE'VE HAD
THE BASIC IMMUNOLOGY MEETING SIE
COVID.
IT'S SUCH A PLEASURE TO SEE PEOE
WHO ARE HERE IN PERSON.
WE REALLY WANTED TO COME AND SAY
THAT PLEASE DO TELL YOUR FRIEND,
YOUR NEIGHBORS, YOUR PEN PALS
ACROSS THE WORLD, THESE ARE FREE
MEETINGS.
WE REALLY WANT TO BE ABLE TO REH
PEOPLE WHO CAN'T GO TO MEETINGSD
MONTH CAN BENEFIT FROM TWO DAYS.
SO SEND IT TO THEM.
AND THEN THE NEXT MEETING WILL A
CELEBRATION OF 50 YEARS OF STEVE
ROSENBERG AT THE NCI.
IT WILL ALSO BE A FREE MEETING.
IT WILL ALSO BE BEFORE OCTOBER .
THERE IS A REASON WHY THAT HAPPS
FOR THOSE OF YOU WHO CAN GUESS.
AGAIN, IT'S REALLY A HUGE PLEASE
AND I'LL LET JIM FINISH.
>> MY LAST WORD IS, THANK YOU
EVERYONE FOR YOUR QUESTIONS.
THANK YOU FOR YOUR PARTICIPATIO.
THIS IS ALL COMING FROM YOU GUYS
AND I HAVE TO SAY ABOUT 1000
REGISTRANTS, SOME JUST CAME IN.
WE HAD OVER THE TIME, CONTINUOUS
TWO50 PEOPLE ALWAYS ATTENDING ON
LINE.
SO THIS WAS IN MY VIEW, A HUGE
SUCCESS.
WE BEAT COVID.
WE BEAT -- FOR ALL THE SPEAKERSO
CAME, I JUST HAVE TO SAY THANK U
AND THEN, NAOMI GAVE EVERYONE A
CREDIT EXCEPT FOR HERSELF, SO I
WANT TO SAY THANK YOU, NAOMI FOR
LEADING THIS ONE.
[ APPLAUSE ]
AND THE NEXT MEETING FOR THIS BC
IMMUNOLOGY WILL BE IN THREE YEAS
AND I'M VERY SURE THAT I'M NOT
GOING TOL CHAIR BUT SOMEBODY ELE
WILL.
THANK YOU AND HAVE A GOOD NIGHT.
>> AND GIVE YOURSELF A HAND.
THANK YOU.