>>IT'S A REAL HONOR TO BE ABLE
TO INTRODUCE DR. RON GERMAIN TO
THIS AUDIENCE.
NOW, OF COURSE IT'S RIDICULOUS,
THE IDEA THAT I WOULD INTRODUCE
RON TO THIS AUDIENCE.
THERE'S NO NEED TO DO THAT.
BUT IT IS ALWAYS EXHILARATING TO
GO BACK AND TO LOOK AT HOW
SOMEBODY LIKE RON GOT TO
SOMEPLACE LIKE HERE.
AND JUST TO RECAP A COUPLE OF
VERY BRIEF THINGS, RON GOT HIS
BACHELOR'S AND HIS MASTERS AT
BROWN IN FOUR YEARS.
IT DID TAKE HIM SIX YEARS TO GET
HIS M.D. AND PH.D. AT HARVARD,
AND THEN HE WENT FROM THAT TO BE
ON THE PATHOLOGY FACULTY AT
HARVARD FOR A COUPLE OF YEARS,
BEFORE COMING HERE AND THE GOOD
NEWS IS, NOT LEAVING.
AS YOU ALL KNOW, RON IS REALLY
QUITE A VISIONARY SCIENTIST, AND
HAS ALWAYS BEEN SO.
FROM HIS EARLY WORK ON T-CELL
BIOLOGY TO HIS CURRENT WORK
REALLY IN, I THINK -- I LOOK AT
IT AS MORE LYMPHOID BIOLOGY
GENERALLY.
AND ON THE ONE HAND, RON HAS
BEEN PUTTING TOGETHER, AS I'M
SURE HE'LL SHOW YOU,
EXTRAORDINARY GLOBAL PICTURES OF
WHAT GOES ON.
AS THEY SAY, THOUGH, THE DEVIL
IS IN THE DETAILS, AND RON HAS
NEITHER SHIED FROM THE DETAILS
NOR FAILED TO MEET THE DEVIL AND
STARE HIM DOWN, AND THE WORK
THAT HE HAS DONE, THE CLARITY
WHICH HE HAS BROUGHT TO IT, HAS
REALLY CHANGED THE WAY THAT WE
LOOK AT THESE THINGS.
THE OTHER THING THAT IS
REALLY -- I'VE RESPECTED
ENORMOUSLY ABOUT RON IS, I'VE
HAD THE EXPERIENCE OF HIM COMING
IN AS AN OUTSIDE INTERLOCULAR
FOR RESEARCH GROUPS.
COMING IN WITH TOPICS WITH WHICH
HE HAD NO PRIOR EXPERIENCE BUT
WAS ABLE, JUST BY DENT OF
THINKING HARD AND REASONING
CLEARLY TO HELP BRING TO THOSE
OF US THAT WERE MORE BEKNIGHTED,
SOME GUIDANCE AND SOME BETTER
PATH FORWARD.
SO IT'S REALLY A GREAT PRIVILEGE
TO INTRODUCE SOMEBODY WHO WAS
THE FOUNDER OF THE CENTER FOR
HUMAN IMMUNOLOGY AND WHO HAS
BEEN REALLY A MAJOR DRIVER FOR
IMMUNOLOGY IN THE UNITED STATES,
IN THE WORLD AND HERE ON THE
CAMPUS.
SO THANK YOU VERY MUCH FOR
LETTING ME TAKE THE TIME TO
INTRODUCE RON, AND TO BRING HIM
TO YOU.
>>THANK YOU, STEVE.
THIS IS THE PAUL LECTURE, IN
MEMORY OF WILLIAM E PAUL, AND
BILL, AND THAT'S WHAT I'LL USE
FROM NOW ON, WAS A REALLY
TOWERING FIGURE IN IMMUNOLOGY.
I'M JUST GOING TO SHOW YOU, HE
DID HAVE YOUNGER DAYS AND VERY
HAPPY DAYS, AND HE RECALLED IN A
FRONT PIECE THAT HE WROTE CALLED
"ENDLESS FASCINATION" HOW HE
BECAME AN IMMUNOLOGIST, WHICH IS
TO READ A SMALL VOLUME OF ESSAYS
BY MICHAEL HEIDELBERGER WHO WAS
STILL DOING RESEARCH IN HIS LAB
AT OVER 100 YEARS OLD, SO
SOMETHING ALL OF US COULD ASPIRE
TO.
HE ACTUALLY WAS DOING
ENDOCRINOLOGY HERE AT NIH FROM
'62 TO '64, BUT HE WENT AND
BECAME AN IMMUNOLOGIST BY
TRAINING WITH BARUJ AND NYU AND
MOVED WITH BARUJ TO NIH AROUND
1968, AND WHEN BARUJ LEFT IN
1970, BILL BECAME THE CHIEF OF
THE LABORATORY OF IMMUNOLOGY, A
POSITION HE HELD FOR 45 YEARS.
I RAN OUT OF SPACE ON MY SLIDE
DESCRIBING BILL'S CONTRIBUTIONS,
AND I'M NOT GOING TO READ THIS
TO YOU AND JUST POINT OUT THAT
HE DID VERY EARLY WORK ABOUT THE
DIFFERENCE BETWEEN
POLYSACCHARIDE AND PROTEIN
ANTIGENS THAT IN A WAY IS THE
PRECURSOR FOR CONJUGATE
VACCINES, DISCOVERED A AT IL4, E
DRY POLARIZATION OF T-CELLS BUT
DID MUCH, MUCH, MUCH MORE.
HE ALSO DID A LOT OF SPECTACULAR
TRAINING, AND PEOPLE WHO PASSED
THROUGH HIS LAB AND UNDER HIS
TUTELAGE INCLUDE RON SCHWARTZ,
LAURIE GLIMSHIRE, MELISSA BROWN,
MARK DAVIS, AND THEN OUR OWN
JEFF ZHU HERE AND OUR PREVIOUS
OWN JOSH MILNER.
WE ACTUALLY SHARED THE SAME
MENTOR.
I DID MY MD PH.D. AND MY PH.D.
PART WITH BARUJ, WHO YOU SEE IN
THE CENTER WITH BILL AND I
CELEBRATING ONE OF BARUJ'S
BIRTHDAYS.
THIS IS OBVIOUSLY A STAGED
PHOTOGRAPH.
WE DID NOT TYPICALLY ALWAYS WEAR
OUR WHITE COATS AND TIES IN THE
LAB.
ALTHOUGH BILL DID FAIRLY OFTEN.
BUT WE DID READ SEQUENCING GELS
TOGETHER IN THE DAYS WHEN THAT'S
HOW YOU DID SEQUENCING.
AND WHY IS THIS TITLED LOOKING
AT FUNDAMENTAL BIOLOGY.
AND MANY THINGS HE DID BEYOND
THE LABORATORY, MAKING IT THE
MOST CITED PUBLICATION IN
BIOMEDICAL SCIENCE, NOT JUST
IMMUNOLOGY, IS THAT HE PUBLISHED
MULTIPLE EDITIONS OF THE BOOK
"FUNDAMENTAL IMMUNOLOGY.
" THAT'S WHERE I REALLY WANT TO
START MY TALK TODAY, THINKING
ABOUT THE FUNDAMENTALS OF
IMMUNOLOGY.
WE THINK ABOUT THE IMMUNE SYSTEM
AS A PROTECTIVE SYSTEM THAT
PROTECTS US AGAINST THE
DANGEROUS EXTERNAL WORLD AND
RECENTLY WE THINK ABOUT IT A LOT
IN HAVING INFLUENCE IN PRO
TETING US AGAINST A DANGEROUS
INTERNAL WORLD.
THESE ARE VERY DIVERSE
PREDICTABLE DISEASE ENTITIES
THAT ARRIVE OR ARISE AT
DIFFERENT TIMES AND DIFFERENT
SITES IN THE BODY.
HOW DO YOU SURVEY EVERYTHING AND
KNOW AND ARE ABLE TO RECOGNIZE
WHAT'S GOING ON.
AND TO MEET THIS WHAT, WHEN AND
WHERE CHALLENGE, THE MY UNSYSTEM
USES SOLUBLE INFECT ORS,
ANTIBODIES COMPLEMENT
COMPONENTS, PENTRAXINS AND SO
ON.
MOBILE ELEMENTS AND THOSE I WILL
TALK ABOUT THAT CAN MOVE
DIRECTLY INTO THE RELEVANT
AFFECTED TARGET SITE, AND
ORGANIZE TISSUES.
AND I'LL TALK A LOT ABOUT THIS
TODAY, THAT ARE NECESSARY IN
ORDER -- ESPECIALLY FOR THE
ADAPTIVE IMMUNE SYSTEM TO DO ITS
JOB, GIVEN THAT IT ORIGINATES
ITS RESPONSE FROM VERY RARE
CELLS IN A VERY LARGE VOLUME OF
THE BODY.
SO WE HAVE CELLS FORMED IN THE
BONE MARROW, THESE ARE MYELOID
AND LYMPHOID CELLS.
THE MORE MATURE MYELOID CELLS
ARE READY TO GO WHEN THEY EXIT
AND THEY CAN FIND INFLAMED
ENDOTHELIUM, THEY CAN ADHERE,
AND EXIT TO MEDIATE EFFECTOR
FUNCTION.
SO PRECURSOR CELLS CAN ENTER THE
THYMUS AND GO THROUGH A WHOLE
DEVELOPMENTAL AND SELECTIVE
PROCESS TO GIVE RISE TO T-CELLS
THAT WIND UP BACK IN SECONDARY
LYMPHOID TISSUES, FOR EXAMPLE,
WITHIN THE LYMPH NODE.
WITHIN THE LYMPH NODE, THERE'S A
SEGREGATION BETWEEN THESE
T-CELLS AND ANOTHER FORM OF
LYMPHOCYTE, THE B LYMPHOCYTE IN
THESE FOLLICLES.
THEY EVENTUALLY HAVE TO GET
TOGETHER.
IMMUNOLOGISTS HAVE NEVER FIGURED
OUT WHY THEY NEED TO BE
SEPARATED BEFORE THEY GET
TOGETHER.
SOMETHING TO THINK ABOUT.
BUT WITHIN THE T-ZONE, THE
T-CELLS ARE SEARCHING FOR THEIR
COGNATE LIGAND AND THAT'S
TYPICALLY IN THE FORM OF A
PEPTIDE MAJOR HISTOCOMPATIBILITY
COMPLEX LIGAND ON THE SURFACE OF
A DENDRITIC CELL.
IF THERE'S A PRODUCTIVE
INTERACTION WHERE THAT LIGAND
MATCHES THE SPES FIS OF A CLONAL
RECEPTOR ON THE T-CELLS, THEY
GET ACTIVATED, THEY CAN
PROLIFERATE, DIFFERENTIATE, AND
LIKELY INNATE CELLS FIND
INFLAMED ENDOTHELIUM AND ENTER
INTO THE TISSUE TO MEDIATE
EFFECTOR FUNCTION.
AND IF WE THINK ABOUT WHAT I
JUST DESCRIBED AFTER ESPECIALLY
THE CELLS LEAVE THE BONE MARROW,
EVERYTHING THEY'RE DOING WHEN
THEY'RE NOT IN THE BLOOD PER SE
IS IN A TISSUE.
THEY'RE EITHER BECOMING
ACTIVATED IN THE TISSUE OR
THEY'RE MEDIATING EFFECT FOR
FUNCTION IN THE TISSUE, AND SO
WE CANNOT UNDERSTAND HOW THE
IMMUNE SYSTEM DOES ITS JOB IF WE
JUST LOOK AT ISOLATED CELLS AND
MOLECULES.
WE WILL NEVER UNDERSTAND EXACTLY
HOW THE SYSTEM WORKS, AND IN
ORDER TO LOOK AT WHAT'S GOING ON
IN A TISSUE WE NEED TO IMAGE,
BOTH DYNAMIC AND HIGH CONTENT
INFORMATION.
SO I'M GOING TO START WITH WHERE
WE AND OTHERS IN THE FIELD
STARTED ABOUT 20 YEARS AGO WITH
THE DYNAMIC IMAGING.
THIS IS MULTIPHOTON VITAL
MICROSCOPY.
IT'S TYPICALLY LIMITED TO FIVE
OR FEWER PARAMETERS AND THAT'S A
MAJOR LIMITATION AS WE'LL GET
INTO LATER.
IT'S POSSIBLE TO DO SIGNALING
MEASUREMENTS BUT IT'S TYPICALLY
PRETTY MUCH CALCIUM OR THE
ANALOG OF NFAT NUCLEAR
TRANSLOCATION.
YOU CAN LOOK FOR A FEW SECONDS
TO HOURS.
THERE ARE WINDOW PREPARATIONS
WHERE YOU CAN LOOK OVER LONGER
TIME PERIODS BUT THEN YOU'RE NOT
FOLLOWING THE SAME CELL OVER ALL
THAT TIME, YOU'RE LOOKING AT
ITERATIVE POPULATIONS OF CELLS
DURING THE TIMING AND IT'S NOT
REALLY APPLICABLE TO HUMAN
STUDY.
THERE'S NO INNERVATION, THERE'S
NO LYMPH FLOW, THERE'S NO
VASCULAR INVOLVEMENT, THERE ARE
NO CELLS COMING IN AND OUT IN
THE NORMAL WAY, AND SO THIS IS A
VERY LIMITED APPLICATION.
SO THE WAY YOU DO THIS IS
SURGICALLY EXPOS THE TISSUE OF
INTEREST.
YOU ONLY GET A FREE LUNCH IN THE
EAR SKIN WHERE YOU DON'T HAVE TO
DO SURGERY.
OF COURSE SURGERY IS DISRUPTIVE
AND PRO INFLAMMATORY SO YOU
SHOULD ASK WHEN YOU READ PAPERS
USING THIS METHOD, HOW DID
PEOPLE CONTROL FOR THE
BACKGROUND INFLAMMATION THAT
THEY CAUSE BY DOING THE SURGERY,
BUT ASSUMING THEY DID THAT WELL
AND PROPERLY, YOU TAKE A LASER
AND SCAN IT IN THE XY DIMENSION,
GET A PLANE OF IMAGING
INFORMATION, FOCUS UP OR DOWN,
GET A STACK OF INFORMATION, AND
THEN COLLECT ALL OF THAT OVER
TIME, SO FOUR-DIMENSIONAL
DATASETS THAT YOU PLAY BACK THIS
WAY, TWO TO 300 TIMES REAL
SPEED, AND COMPRESSED IN TWO
DIMENSIONS.
BUT WE DO HAVE THE
THREE-DIMENSIONAL INFORMATION.
HERE ARE T LYMPHOCYTES MOVING IN
THE T-ZONE OF A LYMPH NODE AND
IT LOOKS LIKE THEY'RE FREE TO
MOVE AROUND LIKE A GO FISH IN A
LITTLE BOWL YOU BOUGHT AT THE
STATE FAIR BUT THAT'S NOT TRUE.
THEY'RE IN THIS ENVIRONMENT.
THE BLACK IS NOT EMPTY SPACE.
THE BLACK IS JUST WHAT WE DON'T
MAKE VISUAL BY PUTTING
FLUORESCENCE INTO OUR IMAGES.
SO YOU SHOULD NEVER BELIEVE WHAT
AN IMAGER TELLS YOU BASED ON
WHAT HE OR SHE DOES NOT SEE, AND
YOU SHOULD ALWAYS WORRY ABOUT
WHAT'S IN THE BLACK.
BECAUSE IT IS INFLUENCING THE
BEHAVIOR VISUALIZING AND IT'S
NOT TO BE IGNORED.
SO I THINK EVIDE -- IF YOU BREEH
THEM, YOU GET RAPID INNATE
RESPONSE AND MORE SLOWLY -- THIS
IS WORK DONE BY A SPECTACULAR
COLLEAGUE WHO NOW HAS HIS OWN
LAB, AND WHAT STEFFAN WANTED TO
DO WAS TO OBSERVE MYELOID CELL
BEHAVIOR AND TO DO THAT, HE
CHOSE TO DO IT IN A NEW
PREPARATION LOOKING IN THE
PERITONEAL WALL.
THE REASON FOR THAT WAS WE KNOW
THERE IS THIS MONOCYTE
POPULATION TO PERITONEAL
MACROPHAGES BUT THERE IS ALSO
THESE EMBRYOLOGICALLY DERIVED
FIXED TISSUE MACROPHAGES IN THE
SEROSAL LINING IN THE PERITONEAL
CAVITY AND THEN IT'S EASY TO
RECRUIT NEUTROPHILS WITH A
CHALLENGE.
SO WE CAN LOOK AT ALL THESE
DIFFERENT MYELOID POPULATIONS AT
ONE TIME.
WHEN HE DID THAT, HE TOOK
ADVANTAGE OF A MODEL THAT TIM
LALLERMAN HAD DEVELOPED IN THE
LAB EARLIER WHERE WE DID STERILE
INJURY WITH LASER DAMAGE IN THE
DERMIS.
WHAT HE DID IS DO THE SAME TYPE
OF DAMAGE IN THE PERITONEAL
WALL.
WHEN HE DID THAT, WHAT HE SAW
WAS THE SAME TYPE OF NEUTROPHIL
SWARMING THAT YOU SEE HERE.
YOU SEE THE CORPSES IN WHITE
HERE ARE DESTROYED BUT THIS IS
THE PROBLEM.
HERE IS THE COLLAGEN THAT'S PART
OF THE TISSUE MATRIX, AND YOU
SEE THAT NEUTROPHIL SWARMING
DAMAGES THIS MATRIX.
THAT RAISES AN INTERESTING
QUESTION.
CELLS DIE IN TISSUES ALL THE
TIME.
IF THE NEUTROPHILS RESPOND TO
CELL DEATH BY SWARMING IN THE
WAY THAT I JUST SHOWED YOU, AND
THEN THEY CAUSE THIS COLLATERAL
DAMAGE, HOW WOULD YOU EVER
MAINTAIN TISSUE PHYSIOLOGY?
BECAUSE YOU'D HAVE THIS CONSTANT
INFLAMMATORY PROCESS BE THAT WAS
DISRUPTING THE TISSUE STRUCTURE
AND THAT DID NOT MAKE ANY SENSE,
SO WE NEEDED TO UNDERSTAND WHAT
WAS PREVENTING THIS, OR WE TREE
SEUM WAS PREVENTING THIS.
WHAT STEFFAN FIGURED OUT, WE
ALWAYS KILLED MASSES OF CELLS
WITH OUR LASER AND HE CHANGED
THIS AND KILLED A SINGLE
FIBROBLAST IN THE PERITONEAL
WALL.
THAT'S WHAT YOU SEE IN ORANGE IN
THE MIDDLE OF THIS IMAGE HERE.
AND THEN HE OBSERVED WHAT HAPPEN
WITH GENETICALLY LABELED MYELOID
CELLS.
HERE WE'RE LOOK AT THOSE
EMBRYOLOGICALLY DERIVED
MACROPHAGES.
WHAT HE SAW IS THEY RAPIDLY
SENSE THAT CELL DEATH, AND THEN
THEY DON'T MOVE THE CELL BODIES
BUT THEY SEND OUT PROCESSES THAT
ENCOMPASS THE CORPS.
SOMETHING WE CALL CLOAKING.
AND THAT TURNED OUT TO BE THE
KEY FEATURE.
SO NORMALLY IF WE DAMAGE
MULTIPLE CELLS, WE CAN SEE THIS
KIND OF TRACKING OF THE
NEUTROPHILS.
THEY SENSE IT, AND THEN THEY ALL
TURN AND RUN IN A VERY
DIRECTIONAL WAY TO CREATE THE
SWARMS THAT I SHOWED YOU A
MOMENT AGO.
BUT IF WE HAVE THE SINGLE CELL
DEATH, NEUTROPHILS DO SHOW UP IN
THE PREPARATION, BUT THEY DON'T
SHOW THAT HIGHLY DIRECTIONAL AND
RAPID MIGRATION, AND THE REASON
THEY DON'T IS THEY CAN'T SEE THE
CORPS.
AND WHAT TIM LAMBERMAN HAD
PREVIOUSLY SHOWN IS THAT THE
SWARMING IS INITIATED BY A LEAD
NEUTROPHIL SENSING AND ACTUALLY
TOUCHING THE DEAD MATERIAL AND
BEGINNING TO PRODUCE LBT4 WHICH
THEN INTRODUCES OTHER LIVING
NEUTROPHILS CREATING AN ENORMOUS
GRADIENT FOR THESE CELLS TO
FOLLOW TO GENERATE THE SWARMING
RESPONSE.
IT TURNS OUT THAT THIS
PROTECTIVE MECHANISM IS
CRITICAL.
IF WE CHRONICALLY DEPLETE THESE
FIXED TISSUE MACROPHAGES AND
LOOK AT A TISSUE UNDERGOING
CHRONIC STRESS, THE DIAG THE DI,
YOU'RE BREATHING ALL THE TIME
AND STRESSING THE MUSCLE FIBERS.
NOW WHAT WE SEE IS WE GET THESE
NECROTIC MUSCLE FIBERS WHEN
THOUGH MACROPHAGES ARE NOT THERE
TO CLOAK AND THESE ARE
ASSOCIATED WITH VERY INTENSE
KNEW CROWPHILIC INFILTRATION.
SO WHAT STEFFAN'S WORK SHOWED IS
THAT THE RESIDENT MACROPHAGES
PREVENT INNATE IMMUNE DAMAGE IN
RESPONSE TO INTERMITTENT CELL
DEATH ORE DAMAGE AND IT PERMITS
HEALING BY LOCAL REPLACEMENT.
THE NEXT NEIGHBORING FIBROBLAST
DIVIDES, FILLS IN THE GAP,
THERE'S NO RESOLUTION, POST
INFLAMMATION TYPE RESOLUTION AND
THERE'S NO SCARRING, SO IT'S
VERY BENIGN FOR THE TISSUE.
IF WE DO VERY CAREFUL WORK OVER
TIME AND IN AGING ANIMALS, WE
SEE THESE RATHER PROTECTIVE
MACROPHAGES BEGIN TO ACQUIRE
MORE INFLAMMATORY PROPERTIES,
AND SO WE THINK THAT THIS
PROCESS MAY BE SUBVERTED AND
CONTRIBUTE TO WHAT'S CALLED
INFLAMAGING AND IT MAY ALSO BE
THAT CHRONIC STIMULATION OF
THINGS LIKE MYELOID IN THE BRAIN
CAUSE MICROGLIAL CELLS TO MOVE
FROM THIS MORE PROTECTIVE STATE
TO A MORE INFLAMMATORY STATE
THAT CONTRIBUTES TO DISEASES
LIKE ALZHEIMER'S.
SO THIS KIND OF LIGHT IMAGING
HAS BEEN VERY VALUABLE LETTING
US SEE THINGS YOU COULD NOT
POSSIBLY SEE WITH EXTRACTED
CELLS IN IN VITRO CONDITIONS OR
BY SINGLE CELL ANALYSIS, AND
WE'VE USED TO LOOK AT DYNAMICS
IN LYMPH NODES TO DISCOVER
T-CELLS MOVE ON FIBROBLASTIC
RETICULAR CELL LOADS TO FIND
DENDRITIC CELLS WITHIN THE
T-CELL ZONE, TO LOOK AT DYNAMICS
IN GERMINAL CENTERS THAT ARE
MAKING ANTIBODIES TO LOOK AT
DENDRITIC CELLS REACHING INTO
THE GUT LUMEN TO SAMPLE THE
MICRO BIOTA, WORK THAT JACKSON
EGAN DID LOOKING AT FUNCTION IN
THE KIDNEY, AND ALSO WORK THAT
HE'S DONE WITH MYCOBACTERIAL
GRANULOMAS AT BOTH THE MYELOID
CELL AND LYMPHOCYTE LEVEL, TO
LOOK IN THE LUNG AS PARAS AD HAS
DONE AND INFLAMMATION RESPONSE
TO INFLUENZA OR TUBERCULOSIS
INFECTIONS, TO LOOK AT THESE
RAPID SWARMING RESPONSES IN
NEUTROPHILS IN THE EAR BUT ALSO
T-CELLS RESPONDING TO
HYPERSENSITIVITY INDUCERS, AND
AS ISHI DID IN THE LAB, TO LOOK
IN THE BONE MARROW AT
OSTEOCLASTOGENESIS.
IT'S BEEN APPLIED IN MANY
DIFFERENT TISSUES.
I WANT TO GIVE YOU A SAMPLE OF
THE WAY WE'VE USED IT OVER THE
YEARS BUT MANY LABORATORIES HAVE
USED THIS IN THEIR OWN WAYS.
THE PROBLEM IS THAT HERE WE SEE
IN BLUE A BILOBAR MOUSE LYMPH
NODE AND WE ONLY IMAGED THIS
SMALL VOLUME.
SO WE HAD A LOT IN THE BLACK.
I TOLD YOU YOU NEED TO WORRY
ABOUT WHAT'S IN THE BLACK, AND
SO I ENCOURAGED THE PEOPLE IN
THE LAB TO DO SOME HISTOLOGY TO
SECTION THE LYMPH NODES TO SEE
WHAT WE WEREN'T SEEING IN OUR
TWO PHOTON IMAGING.
THE PROBLEM WAS, WHEN WE WENT TO
DO THIS, WE REALIZED THAT THE
METHODS FOR DOING STAINING WERE
TWO OR THREE COLOR.
TYPICALLY YOU DID A RED AND A
GREEN STAIN, MAYBE YOU LOOKED
FOR YELLOW TO SEE IF THEY WERE
COINCIDENT, BUT MOST OF THE
PEOPLE IN AN IMMUNOLOGY LAB BY
THIS TIME WERE DOING FLOW
CYTOMETRY EXPERIMENTS BETWEEN
9 AND 15 PARAMETERS, BECAUSE IT
TOOK TWO TO FOUR PARAMETERS TO
IDENTIFY ANY GIVEN SUBSET OF
CELLS, SO TWO COLORS OBVIOUSLY
DIDN'T CUT IT.
AND SO WE AT T DECIDED WE NEEDEO
MOVE TO MULTIPLEX STATIC IMAGING
AND HERE WE'RE NOT GOING TO GET
THOSE REALTIME DYNAMICS BUT NOW
THE PARAMETERS INSTEAD BEING
LIMITED TO FIVE IS EASILY 30 TO
40 AND WE CAN PUSH IT ABOVE 80.
WE CAN LOOK AT SIGNALING AND
POST TRANSLATIONAL
MODIFICATIONS, WE CAN LOOK AT
PROTEINS AND RNA, AND OBVIOUSLY
WE CAN LOOK AT HUMAN MATERIAL.
WE STARTED WITH HISTOCYTOMETRY
MORE THAN A DECADE AGO, MIKE HAS
HIS OWN LAB AT THE UNIVERSITY OF
WASHINGTON NOW FOR A NUMBER OF
YEARS.
AND THE WAY THIS WORKED IS WE'VE
STAINED IN MULTIPLE -- WITH
MULTIPLE FLOOR CHROME
CONGREGATED ANTIBODIES, WE MIGHT
DECONVOLUTE TO IMPROVE THE
QUALITY OF THE IMAGES A LITTLE
BIT, WE COMPENSATE FOR ANY
CHANNEL SPILLOVER LIKE YOU WOULD
DO IN FLOW, USE SOFTWARE TOOLS
TO SEGMENT OBJECTS WITH THEIR
ASSOCIATED FLUORESCENCE, BUT
THOSE DATA ARE EQUIVALENT TO
CELLULAR OBJECTS WITH
FLUORESCENCE, SO WE COULD THEN
PUT THAT INFORMATION INTO FLOW
CYTOMETRY SOFTWARE AND GET
QUANTITATIVE DATA WHERE WE'RE
GAINING A MULTIPLEX EXPRESSION
OF MARKER SETS, BUT SINCE WE
KEPT THE XYZ COORDINATES OF
ANYTHING WE MIGHT GAIN, GATE, WD
IDENTIFY WHERE THOSE CELLS
RESIDED IN THE ORIGINAL TISSUE.
THIS METHOD WORKS NOT ONLY WITH
ANTI -- SO CALLED MARKER
ANTIBODY, MEANING MOSTLY CELL
SURFACE PROTEINS BUT ALSO
ANTIPHOSPHOPROTEIN ANTIBODIES
AND SOME ANTICYTOKINE ANTIBODIES
AND IT CAN RESOLVE THE
DIFFERENCE BETWEEN LOCALIZATION
OF TRANSCRIPTION FACTORS IN THE
CYTOPLASM VERSUS NUCLEUS SO THEN
IT MEANS WE CAN LOOK AT CELL
STATE, CELL FUNCTION AND THE
PHENOTYPE ALL IN THE CONTEXT OF
SPATIAL POSITIONING IN THE
TISSUE.
THIS IS INFORMATION YOU CANNOT
GET EVEN FROM MODERN SPATIAL
TRANSCRIPTOMIC METHODS THAT ONLY
LOOK AT THE RNA, THE SIGNALING
COMPONENTS, FOR EXAMPLE, ARE
INVISIBLE UNDER THOSE
CONDITIONS.
AND SO I'M JUST GOING TO
SUMMARIZE IN SOME WORDS HERE
COMBINING SOME OF THE EARLY
2 PHOTON IMAGE WITH WHAT FLOW
CYTOMETRY HAS TAUGHT US AND
THERE ARE TWO VERY IMPORTANT
LESSONS.
ONE IS THERE'S A VERY POLARIZED
DISTRIBUTION AT BARRIER SITES
THAT ENABLES THE SYSTEM TO SORT
OF HEAD OFF THE INNOVATION OF
THE INFECTIOUS AGENT BEFORE IT
GETS INTO THE SYSTEMIC
CIRCULATION AND THAT'S
CRITICALLY IMPORTANT.
THE OTHER IS FOR THE ADAPTIVE
IMMUNE SYSTEM, THESE CELLS ARE
NOT JUST GENERALLY CO-LOCALIZED
IN A TISSUE AND THEN RANDOMLY
DISPERSED OR MOVING AROUND
LOOKING FOR EACH OTHER.
BUT THEY ACTUALLY ARE VERY, VERY
PRECISELY ORGANIZED TO
FACILITATE THE RARE INTERACTIONS
YOU NEED TO DRIVE T OR B CELL
IMMUNITY.
AND SO I'M GOING TO TELL YOU ONE
STORY THAT GOES WITH THE FIRST
OF THOSE TWO MAJOR CONCLUSIONS,
WHICH IS VERY RECENT WORK THAT
WAS DONE IN THE LAB BY ANITA
GOLA WHO'S NOW WORKING WITH
ELAINE FUCHS AT ROCKEFELLER AND
THIS HAS TO DO WITH THE LIVER.
THE LIVER HAS BEEN UNUSUAL IN
THAT IT HAS TWO CIRCULATIONS.
IT HAS THE PORTAL CIRCULATION,
BRINGING ESSENTIALLY NUTRIENTS
FROM THE GUT INTO THE LIVER, AND
THEN IT'S GOT THE NORMAL
CIRCULATION, THERE'S A HEPATIC
ARTERY AND THEN BOTH OF THESE
INPUTS THEN FLOW OUT IN THE
HEPATIC VEIN.
IF WE SCALE IN, WE CAN SEE THAT
A LIVER LOBULE IS SET UP WHERE
THERE'S CALLED A PORTAL TRIAD
THAT HAS THE PORTAL VEIN, THE
ARTERY, AND A BILE DUCT, AND THE
FLOW IS TOWARD THE CENTRAL VEIN
THROUGH SINUSOIDS THAT ALIGNED
BY A NUMBER OF LYMPHOID
COMPARTMENTS MOSTLY NK AND NK
T-CELLS, AND THESE MYELOID CELLS
THAT ARE MEMBERS OF THE
MACROPHAGE LINEAGE, AND THEN YOU
HAVE THE ENDOTHELIAL CELLS
FORMING THE EDGES OF THE
SINUSOIDS.
AND IT'S WELL-KNOWN THAT THE
LIVER IS METABOLICALLY ZONE
ATED.
THERE ARE DIFFERENT THINGS THAT
HAPPEN TOWARD THE PER PERIPORTAL
REGION.
THEY SAID THAT'S NOT TRUE OF THE
IMMUNE SYSTEM.
BASICALLY THE T-CELLS WANDER IN
A RANDOM WAY SURVEYING WHAT'S
NEAR GOING ON IN THE SINUSOIDS
AND THE DISPLAY OF THOSE OTHER
CELLS I SHOWED YOU, THEY'RE JUST
DISTRIBUTED ALONG THE SINUSOIDAL
WALLS.
SO GOING BACK TO THE 2 PHOTON
IMAGING, THE BEHAVIOR OF NK
T-CELLS IN THE LIVER, WHICH WAS
ORIGINALLY DESCRIBED AS RANDOM
BEHAVIOR, BUT IF YOU LOOK AT
THESE TRACKS, UR SEE THEY'RE
CONCENTRATED HERE NEAR THE E CAD
ENRICHED STAINING AREAS, AND
THESE CONSTITUTE THE PERIPORTAL
REGION SO THERE'S NOT RANDOM
MOTION.
THE CELLS MOVE OUT AWAY FROM THE
PERIPORTAL REGION BUT ALSO RUN
BACK ALMOST LIKE A YOYO AND
SPEND MOST OF THEIR TIME IN THE
PERIPORTAL REGION.
IF WITH YOU THEN LOOK AT THE
KEUPFER CELLS, WHEN I ENHANCE
THE STAINING YOU SEE ALMOST ALL
THESE RED CELLS ARE NOT IN THE
CENTRAL REGION, WHICH IS WHERE
THE CENTRAL FEIGN IS.
THERVEIN S THERE'S AVERY STRICTS
DISTRIBUTION.
I'LL JUST SKIP OVER SOME OF THIS
MORE QUANTITATIVE DATA.
WELL, IS THAT, IN ESSENCE,
INBORN IN THE SAME WAY THE
METABOLIC ZONATION IS?
AND THE ANSWER IS NO.
IF WE LOOK AT A NEWBORN, WE
DON'T SEE THAT ZONATION AT ALL
FOR THE KUPFFER CELLS, AND THEN
AS THE ANIMAL AGES, WE SEE A
MAJOR TRANSITION AT AROUND DAY
20.
WELL, DAY 20 TURNS OUT TO BE
WHEN YOU WEAN THE MICE AND THEY
MARKETLY CHANGE THEIR
MICROBIOTA.
SO WE ASKED DOES IT MATTER, IF
YOU LOOK IN A GERM-FREE MOUSE,
NOW YOU DON'T SEE THE
POLARIZATION.
IF YOU LOOK IN GERM-FREE ANIMALS
THAT HAVE BEEN CO-HOUSED, THEIR
KUPFFER CELLS POLARIZE IN THEIR
DISTRIBUTION, AND IF YOU TAKE AN
SPF MOUSE AND TREAT IT WITH
ANTIBIOTICS, THEY LOSE THE
POLARIZATION.
SO ALL OF THIS TOLD US IT'S THE
MICROBIOTA AND SIGNALS FROM THE
MICROBIOTA THAT MATTER, THIS IS
NOT SOMETHING BUILT INTO THE
SYSTEM ON THE LOGICALLY.
SYSTEM ON TOTO LOGICALLY.
IF WE GET RID OF A MAJOR PROTEIN
THROUGH TOE-LIKE RECEPTORS AS
WELL AS FOR IL-1 -- THEN WE LOSE
THIS ASYMMETRY AND WE COULD SHOW
THAT KNOCKING OUT THINGS LIKE
TLR4 HAD PARTIAL EFFECT SO WE'RE
QUITE CONFIDENT THAT THIS IS
WHAT'S CALLED PAMP SIGNALING.
THAT S RESPONSE TO THINGS LIKE
ENDO TOXIN THAT ARE RELEASED
FROM THE GUT MICROFLORA AND
HIGHLY ENRICHED IN THE PORTAL
CIRCULATION BEING CARRIED TO THE
LIVER AND ENTERING IN THAT
PORTAL TRIAD REGION, THAT'S THE
HIGHEST CONCENTRATION, AND THEN
FLOWING OUT.
WHAT WE THEN SHOWED IS THEY
DON'T TALK DIRECTLY TO THE CUP
FER CELLS, THEY INSTRUCT THE
CELLS NOT FOR HOW MUCH CHEMOKINE
THEY MAKE THAT GUIDE THE KUPFFER
CELLS BUT HOW THEY RECONSTRUCT
THE EXTRACELLULAR MATRIX OF
AMINO GLYCANS TO WHICH THE
CHEMOKINES BIND.
SO THE GRADIENT CONTROL IS BASED
ON EXTRACELLULAR MATRIX
REMODELING THAT'S RESPONSIVE TO
THESE SIGNALS.
WHY IS THE SYSTEM POLARIZED THIS
WAY?
IN GREEN ARE THE KUPFFER CELLS.
IN WHITE ARE GOING TO BE
BACTERIA THAT ARE PUT INTO THE
VASCULAR TREE.
WHAT YOU SEE IS WHETHER THOSE
BACTERIA HIT THE LIVER, THEY
STICK DIRECTLY TO THE KUFFER
CELLS.
WE CREATED A MATHEMATICAL MODEL
THAT SAYS IF WE MAKE THEM ALL
EQUALLY STICKY TO BACTERIA
BECAUSE THAT'S SOMETHING WE
CAN'T EASILY CONTROL IF THEY DO
VARY IN THEIR STICKINESS ACROSS
THE SIGN SOY DAL DISTRIBUTION,
WHAT HAPPENS IF WE HAVE THEM
POLAR OIZED VERSUS
NON-POLARIZED?
THE ANSWER IS WHEN THEY'RE
HIGHLY POLARIZED, THE BACTERIA
ARE CAPTURED VERY CLOSE TO THE
PERIPORTAL REGION AND VERY FEW
REACH THE CENTRAL VEIN, MEANING
GOING INTO THE GENERAL
CIRCULATION.
BUT IF WE MAKE THEM UNIFORMLY
DISTRIBUTED, NOW LOTS OF THE
BACTERIA MAKE TO THE CENTRAL
VEIN AND GO INTO THE
CIRCULATION.
THIS WAS A MODEL, BUT ANITA DID
A HEROIC EXPERIMENT OF INJECTING
BACTERIA DIRECTLY INTO THE
PORTAL VEIN AND WHEN YOU DO
THAT, YOU GET EXACTLY THAT
RESULT.
IN ANIMALS WITH THE POLARIZED
KUPFFER CELLS, MOST OF THEM ARE
TRAPPED VERY CLOSE TO THE
PERIPORTAL REGION, VERY FEW
REACH THE SPLEEN OR THE BLOOD
HERE IN BLACK, BUT IF YOU USE A
GERM-FREE ANIMAL WITH A
NON-POLARIZED KUPFFER
DISTRIBUTION, NOW YOU SEE -- OR
PRESENT IN THE BLOOD.
SO THAT GIVES US A MODEL FOR
WHAT'S GOING ON HERE WHERE THIS
POLARIZATION IS MEANT TO ENABLE
THE SYSTEM TO MOST EFFICIENTLY
CAPTURE ANYTHING THAT'S ESCAPING
THE MUCUS AND EPITHELIAL
BARRIERS IN THE GUT, GETTING
INTO THE PORTAL CIRCULATION AND
POTENTIALLY GOING SYSTEMIC.
YOU NEED THAT POLARIZATION OF
THE IMMUNE SYSTEM TO DO THAT
EFFICIENTLY.
ALTHOUGH IT'S NOT ABSOLUTE, WHEN
YOU THINK ABOUT THESE BACTERIA
CHRONICALLY COMING OUT OF THE
GUT, IF YOU WEREN'T DOING THIS
ALL OF THE TIME, YOU'D BE AT
GREAT RISK FOR SYSTEMIC SEPTIC
EVENTS.
I THINK I'VE LOST MY CONTROL
HERE.
SO I'LL JUST SAY THAT THAT
FRACTAL BIOLOGY AS I CALL IT
ALSO APPLIES TO LYMPH NODE
STRUCTURE AND FUNCTION.
WE DON'T HAVE LYMPHOCYTES
EVERYWHERE, I MEAN, THEY'RE IN
THE BLOOD BUT THEY'RE NOT IN ALL
THE TISSUES.
THEY CONCENTRATE IN A LYMPH NODE
OR SPLEEN.
WITHIN, SAY, A LYMPH NODE, THEY
SEPARATE INTO THOSE T AND B
AREAS.
IN THE T AREAS YOU HAVE THIS
FIBROBLASTIC RETICULAR NETWORK.
THE T-CELLS WALK ON THAT NETWORK
AND THE REASON THEY DO THAT IS
IT REDUCES THEIR SEARCH VOLUME
LOOKING FOR DENDRITIC CELLS ON
EXACTLY THAT SAME NETWORK.
WITHIN THE CONTEXT OF THIS
NETWORK CONCENTRATION, IF YOU
GET AN ENGAGEMENT OF ONE T-CELL
WITH A DENDRITIC CELL, IT MAKES
CHEMOKINES THAT THEN BIAS THE
WALK ON THE FIBER NETWORK TO
DIRECT THOSE CELLS TO THE
INTERESTING DENDRITIC CELL, AND
THEN ANTONIO IN THE LAB SHOWED
THERE'S A POLARIZATION MATCH
BETWEEN DENDRITIC CELL SUBSETS
AND T-CELL SUBSET, THEY'RE NOT
RANDOMLY DISPERSED WITHIN THE
T-CELL ZONE, AND THAT
POLARIZATION IS CRITICAL TO THE
RIGHT TIMING FOR ACTIVATING THE
DIFFERENT SUBSETS OF
LYMPHOCYTES, AND IF YOU DESTROY
THAT POLARITY WITH NO OTHER
CHANGE IN THE LYMPH NODE, NOW
CD4 CELLS DON'T COOPERATE
PROPERLY WITH CD8 CELLS TO MAKE
GOOD MEMORY IN RESPONSE TO
VACCINES, FOR EXAMPLE.
SO IT'S THIS ORGANIZATIONAL
LEVEL IS ABSOLUTELY CRITICAL TO
ADAPTIVE IMMUNITY, AND IT BOTH
STRUCTURAL ORGANIZATION AND
CHEMICAL ORGANIZATION JUST AS I
SHOWED YOU IN THE LIVER.
SO WE'VE NOW APPLIED THIS TO
CANCER BIOLOGY.
VIVIAN MALTES IN THE LAB HAS
WORKED TOGETHER WITH CAITLYN
BERN INITIALLY AND NOW AT HER
OWN LAB IN OREGON IN A MODEL IN
WHICH IT'S A GERM -- GENETICALLY
ENGINEERED MODEL OF PANCREATIC
DUCTAL ADENOCARCINOMA, AND WHAT
KATE DID WAS TO CLONE INDIVIDUAL
CELLS FROM THOSE PRIMARY TUMORS,
AND IT TURNED OUT THAT THEY
DISTRIBUTED INTO TWO GROUPS,
WHAT WE WOULD TYPICALLY THESE
DAYS CALL COLD OR HOT TUMORS IN
TERMS OF THE T-CELL INFILTRATION
AND THE BIAS TOWARD SUPPRESSIVE
MACROPHAGES.
SO WHAT KATE AND VIVIAN DID WAS
TO STUDY ANIMALS THAT ARE
BEARING A HOT TUMOR WITH A
TREATMENT THAT COMBINES
CHECKPOINT THERAPY WITH AN
AGONIST CD4 ANTIBODY, IN CURRENT
CLINICAL TRIALS AND HIGHLY
EFFECTIVE IN THIS ANIMAL MODEL.
HERE THEY WERE LOOKING AND
VIVIAN IN PARTICULAR WAS LOOKING
VERY SHORTLY AFTER THE
COMBINATION THAT INVOLVES THE
ANTIHD1 ANTICTLA4 -- WHAT'S
STRIKING WHEN THIS IS DONE,
LOOKING HERE AT A DOT VERSION
RATHER THAN THE FINE GRAIN
PRIMARY ANALYSIS OF THE IMAGES
FOR T-CELLS IS THAT THERE IS A
MAJOR LOSS OF FOX P3 POSITIVE
T-CELLS.
SO WHAT ARE CALLED REGULATORY
T-CELLS.
AND THE FEW THAT REMAIN, AND YOU
SEE HOW MUCH THIS REDUCTION IS
HERE, ARE IN THE PERIPHERY.
AND THAT'S GOING TO BE IMPORTANT
WHEN WE DRAW SOME CONCLUSIONS A
BIT LATER ON, AND THIS JUST
QUANTIFIES THAT CHANGE IN
DISTRIBUTION.
NOW, IT SEEMED LIKE THAT WAS
ASSOCIATED UNIQUELY WITH THE
ANTICD40 AND THIS EXPERIMENT
SHOWS THAT THAT'S TRUE IF YOU
USE A CD40 KNOCKOUT ANIMAL, THIS
TREATMENT DOES NOT DEPLETE THE
REGULATORY T-CELLS, BUT THAT WAS
VERY SURPRISING BECAUSE CD40 IS
NOT EXPRESSED BY REGULATORY
T-CELLS SO WHY WOULD THIS
ANTIBODY GIVE YOU THIS
PHENOTYPE?
WELL, CD40 IS EXPRESSED BY
DENDRITIC CELLS, AND IF YOU GET
RID OF A PARTICULAR SUBSET OF
DENDRITIC CELLS IN A
TRANSCRIPTION FACTOR KNOCKOUT,
NOW THIS TREATMENT DOES NOT
CAUSE THE LOSS OF THE T REGS.
SO THE QUESTION IS WHAT'S
HAPPENING TO THE T REGS, AND WHY
ARE DENDRITIC CELLS INVOLVED IN
LOSING FOX P3 CELLS.
IT LED US TO WONDER WHETHER
THESE CELLS WERE LOSING FOX
P3 EXPRESSION RATHER THAN BEING
ELIMINATED AS T-CELLS ENTIRELY.
TO LOOK AT THAT, WE'VE TURNED TO
LINEAGE TRACKING, AND YOU DO
THAT A AN ANIMAL IN WHICH YOU
CAN GIVE TAMOXIFEN TO DO
INDUCIBLE TURN YON OF THE ROSA
TOMATO LOCUS SO YOU GET THE RED
FLUORESCENT PROTEIN EXPRESSED IN
CELLS THAT ARE EXPRESSING CRE
UNDER THE CONTROL OF FOX P3, SO
THE AUTHENTIC T REGS ARE GOING
TO SHOW AS THIS TRACKING WHEN WE
DO THAT.
THEN YOU GO THROUGH THE REGULAR
PROCEDURE, AND THE SURPRISING
RESULT, THE VERY STRIKING RESULT
IS AS YOU SEE HERE, WE GET THE
ELIMINATION OF MOST OF THE T
REGS AND THE RESIDUAL ONES BEING
ON THE PERIPHERY OF THE TUMOR AS
WE SAW BEFORE, BUT NOW WE GET A
VERY LARGE POPULATION OF
SO-CALLED XT REGS, CELLS THAT
HAVE THE LINEAGE PARKER BUT NO
LONGER HAVE FOX P3, THESE ARE
CD4 POSITIVE CD3 POSITIVE
T-CELLS AND THEY ALSO POPULATE
THE PERIPHERY.
AND THIS JUST QUANTIFIES THOSE
CHANGES.
AND IT WAS VERIFIED ALSO BY FLOW
CYTOMETRY.
SO HOW DOES THAT WORK?
WELL, THE DENDRITIC CELLS THAT
RELY ON BAT OF 3 ARE A MAJOR
IL-12 PRODUCER AND ANTICD40
INDUCES THE FORM OF IL12.
SO KATE AND VIVIAN LOOKED AT
IL-12 AND WHETHER THE
CYTOKINE -- AS SHOWN HERE, IF
YOU TREAT WITH EITHER ANTI-12 OR
ANTIINTERFERON GAMMA, YOU NO
LONGER LOSE THE MAJORITY OF THE
CONVENTIONAL FOX P3 T REGS AND
YOU DON'T MAKE NEARLY AS MANY OF
THESE EX--TREGS.
THERE'S THE CONVERSION AND LOSS
OF FOX P3 IS DEPENDENT ON
INTERFERON 12 AND INTERFERON
GAMMA.
INDUCE CELLS TO BECOME TH1s,
SO IS THIS HAPPENING AS THEY
LOSE FOX P3, SO HERE VIVIAN
TURNED TO ANOTHER TECHNOLOGY FOR
LOOKING AT TARGETS IN THE TISSUE
HERE, IT'S RNA FISH, AND I'LL
JUST SHOW YOU THE BLOWN-UP PART
WHICH SHOWS YOU THAT THESE
EX--TREGS ARE ACTIVELY MAKING
ANTIINTERFERON GAMMA MESSAGE,
THIS IS WITHOUT ANY
RE-STIMULATION.
SO WHAT'S HAPPENING HERE IS THAT
THE ANTICD40 ACTION PROBABLY
TOGETHER WITH SOME CONVENTIONAL
CD4 CELLS GIVES YOU A
COMBINATION OF IL-12 AND
INTERFERON GAMMA PRODUCTION
WHICH KOWS KAWS THE LOSS OF FOX
P3 EXPRESSION IN THE
CONVENTIONAL TREGS.
I DIDN'T SHOW YOU THIS BUT THEY
ALSO GAINED NT -- EXPRESSION AND
THEY ALSO MAKE -- IS CONSIDERED
A CANONICAL OUTCOME OF DOING
CHECKPOINT THERAPY, BUT IN THIS
MODEL, THE CHECKPOINT THERAPY
ALONE DOESN'T DO THAT, EVEN IN
THESE HOT TUMORS.
YOU NEED THE ANTI-CD40 AS WELL
TO GET THIS AT A HIGH LEVEL.
IN ADDITION, THE MOVEMENT OF ANY
RESIDUAL TREGS TO THE PERIPHERY
REMOVES THEM FROM PROXIMITY TO
THE RESPONDING CONVENTIONAL
CELLS, WHICH THEN LIMITS THEIR
ABILITY TO SUPPRESS THE
RESPONSE.
SO OVERALL, YOU GET A VERY
POTENT ANTITUMOR EFFECT BECAUSE
YOU'RE AT MULTIPLE LEVELS
REMOVING THE SUPPRESSION WITHIN
THE SYSTEM, BUT NOT ONLY THAT,
YOU'RE ACTUALLY ADDING TO THE
EFFECTOR FUNCTION THAT'S
ATTACKING THE TUMOR.
NOW, THE HISTOCYTOMETRY HAS BEEN
OBVIOUSLY VERY VALUABLE IN MANY
DIFFERENT WAYS, BUT IT'S NOT
ADEQUATE.
THERE ARE MANY MORE IMMUNE
CELLS, SUBSETS OR TYPES THAN
WE'RE ABLE TO LOOK AT IN A
NUMBER OF COLORS THAT I SHOWED
YOU, THE NUMBER OF PARAMETERS.
THERE ARE ALL THESE EPITHELIAL
AND STROMAL CELLS YOU WANT TO
LOOK AT IN THE TISSUE AT THE
SAME TIME, THERE ARE ACELLULAR
STRUCTURAL COMPONENTS AND IF
WE'RE INTERESTED IN TUMOR
BIOLOGY, THERE ARE OTHER THINGS
GOING ON WITH THE TUMOR CELLS.
AND SO ANDREA RADKE DID A
SPECTACULAR JOB IN THE LAB OF
SETTING UP A NEW ITERATIVE
STAINING METHOD WHICH IS CALLED
IBEX.
YOU STAIN IN SOMEWHERE BETWEEN
FOUR AND THESE DAYS MAYBE EVEN
BACK UP TO ABOUT 8 OR 9 COLORS.
YOU IMAGE, YOU BLEACH AND THEN
YOU ITERATE THIS PROCESS.
THEN SHE WORKED TO CREATE WAYS
OF REGISTERING ALL THESE
ITERATIVE IMAGING CYCLES BECAUSE
THERE ARE SMALL DIFFERENCES AS
YOU GO THROUGH THIS PROCESS SO
WE GET PIXEL LEVEL RESOLUTION
HERE AND THEN WE CAN GO INTO
OTHER SOFTWARE I'LL TALK ABOUT
FOR DOING ANALYSIS.
SO HERE'S AN EXAMPLE OF A HUMAN
LYMPH NODE STAINED WITH
82 DIFFERENT ANTIBODIES OF WHICH
60-SOME-ODD ACTUALLY REACT WITH
MOLECULES IN THAT ENVIRONMENT.
AND THEN THAT REALLY RAISES A
QUESTION, YOU COULD LOOK AT
COMBINATIONS OF TWO OR THREE
MARKERS AND SORT OF PERCEIVE
DIFFERENT PATTERNS, BUT HOW
WOULD YOU LOOK AT 62 DIFFERENT
PARAMETERS AT ANY GIVEN TIME AND
MAKE -- GAINING SOME
UNDERSTANDING OF WHAT'S
HAPPENING IN THIS TISSUE
ENVIRONMENT.
SO THIS IS A CHALLENGE TAKEN ON
AND THEN WITH HELP FROM SPENCER
GRANT IN THE LAB, AND WHAT
THEY'VE DEVELOPED IS A SOFTWARE
TOOL CALLED RAPID.
IT'S BASED ON MACHINE LEARNING.
AND IT BASICALLY DOES A COMPLEX
ANALYSIS TO SORT OF CLUSTER THE
DIFFERENT FLUORESCENT PIXELS YOU
GET IN THESE IMAGES INTO THINGS
THAT MORE REALISTICALLY
REPRESENT WHAT YOU WOULD SEE IF
YOU WERE DOING MULTIPLEX FLOW
CYTOMETRY.
NOW THE PIXEL APPROACH IS
IMPORTANT BECAUSE ALL
SEGMENTATION ALGORITHMS FAIL AT
A CERTAIN LEVEL WITH VERY
IRREGULAR CELLS AND THEY'RE NOT
VERY GOOD AT EXTRACELLULAR
ACELLULAR MATRIX COMPONENTS.
THE PIXELS DON'T CARE ABOUT
THOSE, AND THEY'RE MUCH MORE
USEFUL, BUT BUILT INTO THIS
SOFTWARE IS ALSO THE ABILITY
WHEN IT'S USEFUL TO DO
SEGMENTATION AND WE'VE COMPARED
THE SEGMENTATION ALGORITHM HERE
TO OTHER MODELS, AND IT'S AS
GOOD OR BETTER UNDER ALL THE
CONDITIONS THAT WE'VE TESTED.
SO AS AN EXAMPLE HERE, WITH
ANDREA'S HUMAN LYMPH NODE DATA,
HERE'S THE IMAGING PICTURE.
IT GETS TRANSLATED INTO THE
PSEUDOCOLOR IN RAPID, AND WHAT
YOU SEE HERE IS A GERMINAL
CENTER, AND THE PSEUDOCOLORS ARE
NOT SEGMENTED CELLS BUT THEY ARE
REPRESENTATIVE OF A CLUSTER OF
PIXELS THAT THE SOFTWARE HAS
DECIDED ARE UNIQUE AND DIFFERENT
FROM WHAT IS ADJACENT TO THEM.
AND THAT WINDS UP LOOKING LIKE
INDIVIDUAL CELLS.
IF WE GO AND ASK WHAT IT'S
SHOWING US, IT'S USING A
COMBINATION OF FOUR DIFFERENT
MARKERS OF NAIVE B CELLS THAT
LIVE OUTSIDE THE GERMINAL
CENTER, SIX DIFFERENT MARKERS TO
LOOK AT GERMINAL CENTER B CELLS
AND YOU CAN EVEN SEE IN THE
PIXEL IMAGE THE SHAPE DIFFERENCE
BETWEEN THE LIGHT AND THE DARK
ZONE B CELLS TO LOOK AT WHICH OF
THOSE B CELLS ARE PROLIFERATING,
TO LOOK AT TFHs AND CELL LINE.
WE DON'T ALL WANT TO WRITE
PYTHON CODE, AND SO NISHAN AND
SPENCER HAVE TURNED THIS INTO A
GRAFGRAPHIC USER INTERRER FACE.
CAN YOU LOAD IN YOUR IMAGE AND
DO SOME MANIPULATIONS OF IT.
YOU CAN DO EITHER OBJECT OR
PIXEL-BASED SEGMENTATION AND
CLUSTERING ANALYSIS, SO YOU WIND
UP WITH INFORMATION LOOKING LIKE
THIS ON YOUR SCREEN, AND YOU
ALSO GET THESE TABLES THAT
QUANTIFY WHAT THE CLUSTER AND
PIXEL CALLS ARE IN THE SOFTWARE,
SO HERE, FOR EXAMPLE, THE ONLY
STRONG SIGNAL WE SEE IS CD31,
THAT'S REPRESENTING ENDOTHELIAL
CELLS.
HERE WE HAVE A COMBINATION OF
HIGH CD3 AND CD8.
THOSE ARE CD8 T-CELLS BUT IF YOU
LOOK AT 68 PARAMETERS, MAYBE
THEY'RE CD44 HIGH SO THEY'RE NOT
NAIVE, FOR EXAMPLE, VERSUS THE
ONES THAT ARE CD62L POSITIVE AND
SO ON.
AND THEN YOU CAN DO A LIMITED
AMOUNT OF SPATIAL ANALYSIS, BUT
AS I TOLD YOU FOR
HISTOCYTOMETRY, THIS IS THE
INPUT FOR REALLY BEGINNING TO
LOOK AT WHAT'S HAPPENING IN
SPACE.
AND I SAY THAT FOR A REASON, AND
THERE ARE ALL THESE DIFFERENT
POSSIBLE PATTERNS ACROSS TISSUE
SCALES, THE ZONATION I ALREADY
TALKED ABOUT, THE BROADCAST
BEING OF SIGNALS BASED ON
CYTOKINES OR PHYSICAL CELLULAR
ENTANGLEMENTS OF TWO OR MORE
CELL TYPES.
SO ED HAS DONE A SPECTACULAR JOB
OF APPROACHING THIS AS A
MATHEMATICAL PROBLEM CREATING
ANOTHER SOFTWARE TOOL CALLED
SPACE THAT ENABLES YOU TO LOOK
AB INITIO AT WHAT'S HAPPENING WP
THE TISSUE IN ALL THESE
DIFFERENT PARAMETERS.
SO FIRST STEP IS TO GO INTO ARE
PID ANRAPID AND DO THIS CLUSTER,
AND THEN YOU DO THIS CENSUS OF
LOCAL NEIGHBORHOODS.
IT IS NOT A NEIGHBORHOOD
ALGORITHM OF THE TYPE YOU MAY
HAVE READ ABOUT IN THE
LITERATURE.
IT JUST USES THIS TO GATHER
INFORMATION ACROSS THE ENTIRE
TISSUE SPACE.
THEN HE GOES INTO A MATHEMATICAL
APPROACH CALLED GENERALIZED
MUTUAL INFORMATION, THAT
DETERMINES WHETHER THERE IS
STATISTICALLY RELEVANT ASYMMETRY
IN THE DISTRIBUTION OF VARIOUS
COMPONENTS IN THE TISSUE.
AND FROM THAT, YOU CAN LEARN
RELATIONSHIPS.
I'M NOT GOING TO WALK YOU
THROUGH ALL THIS.
THIS IS STILL BEING FINALIZED
BUT I'LL GIVE YOU AN EXAMPLE OF
HOW POWERFUL IT IS.
SO HERE WE'RE LOOKING AT A
42PLEX MOUSE LYMPH NODE THAT HAD
BEEN NOT ANALYZED PREVIOUSLY FOR
THIS PARTICULARLY IMAGE, BUT HAD
BEEN STUDIED PREVIOUSLY IN THE
LAB, AND WHAT HE WAS ABLE TO
SHOW AS I MENTIONED PREVIOUSLY
IS THAT THE CD4 AND CD8 T-CELLS
ARE BIASED IN THEIR DISTRIBUTION
IN THE T-CELL ZONE, AND THAT
THAT BIAS MATCHES THE DIFFERENT
DISTRIBUTIONS OF CDC1s AND
2s TO THOSE DIFFERENT T-CELL
POPULATIONS.
WHAT SPACE REVEALS OVER HERE ON
THE RIGHT-HAND SIDE IS THAT OF
COURSE B CELLS ARE SEPARATED
FROM THE TWO TYPES OF T-CELLS.
YOU SEE THAT IN THIS REDUCED
DIMENSIONALITY PLOT.
BUT FURTHER, THAT THERE IS A
DIFFERENCE BETWEEN WHERE THE CD4
T-CELLS AND THE CD8 T-CELLS ARE,
AND IF WE DO THAT IN A GRAPHIC
FORM, YOU SEE THAT THE
CD-4 CELLS ARE MORE PERIPHERAL
THAN THE CD-8s, EXACTLY WHAT
ANTONIO FOUND AFTER YEERLS OF
CAREFUL STUDY BY HISTOCYTOMETRY
BUT SOMETHING THAT COMES OUT
AUTOMATICALLY FROM APPLYING
SPACE.
THOSE USED WITH CODEX AND OTHER
MULTIPLEX IMAGING TOOLS WILL
NEVER REVEAL THESE TYPES OF
RELATIONSHIPS BECAUSE THEY'RE
BASED ON -- AND THE CELLS THAT
ARE TOUCHING IT, WHEREAS THIS
CAN LOOK ACROSS ALL THESE
SCALES.
THAT'S VERY NICE, BUT I TOLD YOU
EARLIER ON WE HAD A PROBLEM
LOOKING AT ANY GIVEN SECTION
THROUGH A TISSUE ALSO UNDER
SAMPLES, SO THEY GOT TOGETHER
AND SAID LET'S LOOK AT A VOLUME
RATHER THAN A SINGLE SECTION.
AND THEY DEVELOPED A NEW WAY OF
CLEARING THE TISSUE.
YOU SEE THE WAY THEY'VE MADE THE
TISSUE TRANSPARENT.
I WANT TO POINT OUT THAT BEFORE
WE DID THIS, THERE WERE MANY
METHODS ALREADY PUBLISHED FOR
CREATING CLEARED TISSUE.
THEY INCLUDED CLARITY, DISCO,
BABB. THE REASON WE INVENTED OR
REINVENTED A NEW APPROACH IS
NONE OF THOSE ENABLED YOU TO DO
HIGH SENSITIVITY DIRECT
MULTIPLEX STAINING.
YOU CAN TRACK FLUORESCENT
PROTEINS VERY WELL IF THEY'RE
VERY BRIGHT, YOU CAN DO A
LIMITED AMOUNT OF STAINING WITH
AN INDIRECT REAGENT FOR
AMPLIFICATION, BUT IF YOU WANT
TO DO MULTIPLEX, YOU NEEDED A
DIFFERENT METHOD.
AND THAT'S WHAT THEY DEVELOPED.
SOME HERE'SO HERE'S AN EXAMPLE H
NODE COMPLETELY INTACT, NO
SECTIONING, YOU SEE BEAUTIFUL
COLORS, THICK VESSELS, THE
AGVs, YOU SEE THE FULL VOLUME
OF THOSE PERIPHERAL B CELL
FOLLICLES, BUT OF COURSE WE CAN
LOOK IN ANY GIVEN SECTION AND
TREAT IT LIKE HISTOCYTOMETRY.
SO WE GET ALL OF THIS
INFORMATION IN 3D IN A WAY THAT
AN INDIVIDUAL SECTION WOULD FAIL
TO GIVE US THINGS LIKE THE
RAMIFICATIONS OF THOSE VESSELS.
SO HARRY, WHO NOW HAS HIS OWN
LAB AT MIT DID A SPECTACULAR JOB
COMBINING HISTOCYTOMETRY AND HIS
3D MULTIPLEX IMAGING TO STUDY
T-CELLS IN TISSUES.
LU HAD SHOWN PREVIOUSLY USING
ANTIPHOSPHOSTAT STAINING THAT
CONTRARY TO SOME EXPECTATIONS,
REGULATORY T-CELLS DIDN'T KEEP
AUTO REACTIVE T-CELLS FROM
BECOMING ACTIVATE.
THAT'S NOT HOW THEY REALLY
PREVENTED AUTOIMMUNITY.
YOU COULD SEE THAT THERE WAS THE
PRODUCTION OF IL-2, SO FULL
ACTIVATION OF CYTOKINE
PRODUCTION FROM WHAT WE COULD
SHOW BASED ON GERM-FREE MOUSE
ANALYSES WERE SELF-ROW ACTIVE
T-CELLS, BUT THEY WERE
SURROUNDED BY THESE CLUSTERS OF
WHAT JADHU IDENTIFIED AS
REGULATORY T-CELLS.
SO WE KNEW THAT THESE LITTLE
CLUSTERS SEEM TO HAVE A
RELEVANCE TO CONTROLLING
RESPONSES, BUT WE DIDN'T KNOW
HOW THEY DID IT.
AND THAT'S THE QUESTION HARRY
TOOK ON.
SO THE FIRST THING THEY DID IS
TO RE-ANALYZE THINGS, IT TURNS
OUT LOOKING FOR IL2 IS RATHER
DIFFICULT, HE FOUND PD-1
EXPRESSION IN THE STEADY
STATE -- WE SAME THE SAME IN
GERM-FREE MICE, AND THESE ARE
ALL P STAT5 NEGATIVE AS SHOWN
BEFORE.
HE WORKED ON SPATIAL STATISTICAL
METHODS THIS ALLOWED HIM TO LOOK
RELATIVE TO THE SURROUNDING
POPULATIONS OF REGULATORY CELLS
AND WHEN HE DID THAT, ARE WHAT
HE FOUND IS THAT THERE IS A
CLEAR CLUSTERING OF REGULATORY
CELLS AROUND UNIQUELY THE PD-1
POSITIVE ACTIVATED AUTO REACTIVE
CELLS AS OPPOSED TO ALL THE
OTHER CD4 CELLS, THE PD-1
NEGATIVE CELLS IN THE
POPULATION.
I WANT TO POINT OUT HERE
SOMETHING BECAUSE I JUST CAME
BACK FROM A MEETING WHERE THIS
WASN'T CLEAR TO EVERYBODY DOING
SPATIAL ANALYSES, YOU FEED TO GO
AND DONEED TO GODO RANDOM SAMPLE
CELLS IN THE VOLUME TO KNOW
WHETHER SOMETHING THAT LOOKS
LIKE AN INTERESTING ASSOCIATION
IS REALLY JUST RANDOM OR IS
REAL.
BECAUSE HE DID MULTIPLEX
IMAGING, HE COULD ACTUALLY BEGIN
TO CHARACTERIZE THE STATE OF
THOSE T REGS, SOMETHING THAT
WOULD BE DIFFICULT TO DO IF HE
WOULD HAVE TAKEN EVERYTHING
APARTMENT AND DONE SINGLE CELL
AND IS BEYOND THE RESOLUTION OF
THE CURRENT TRANSCRIPTOMIC
METHODS.
WHAT HE FOUND WAS THAT THE CELLS
CLUSTERING NEAR THESE AUTO
REACTIVE CELLS WERE HIGHLY
SUPPRESSIVE EFFECTOR T REGS, AND
EXPERIMENTS ARE NOW SHOWING YOU,
HE SHOWED THIS IS
IL-2 DEPENDENT, CONSISTENT TO
WHAT I SHOWED YOU.
T REGS ARE OPERATING IN A
NEGATIVE FEEDBACK MODE, THEY
WERE RESPONDING TO THE RESPONSE
OF THE AUTO REACTIVE CELLS, THEY
WERE SENSING THAT THOSE CELLS
WOULD BECOME ACTIVATED, THEY
WERE MAKING IL-2, AND THE T REGS
NEEDED TO DO SOMETHING, AND WHAT
THEY NEEDED TO DO WAS TO BECOME
SUPPRESSIVE, AND THAT'S WHAT WAS
HAPPENING HERE.
I SAID THEY WERE HIGH 67
POSITIVE AND AGAIN, THAT WAS
HIGHLY ENRICHED IN THE T REGS
VERY CLOSE TO THE RESPONDING
CELLS, SO THE QUESTION WAS
WHETHER THE DENSITY OF THESE T
REGS WAS A CHEMOKINE ATTRACTION
OR WHETHER IT COULD HAVE BEEN
PROLIFERATION LOCALLY IN
RESPONSE TO IL2, TO LOOK AT THAT
WE USED WHAT ARE CALLED CONFETTI
MICE THAT HAVE A WAY OF GIVING
YOU A KIND OF CLONAL PICTURE OF
CELLS BASED ON COLOR EXPRESSION,
AND YOU CAN SEE THESE SMALL
CLUSTERS OF LIKE-COLORED CELLS
WITHIN THESE T REG CLUSTERS AND
IF YOU LOOK AT THAT
STATISTICALLY, IT'S CLEAR THAT
THIS IS NON-RANDOM.
SO WHAT WAS HAPPENING IS THAT
THERE WAS PROLIFERATION GOING
ON, AND SO THERE'S A FEE FORWARD
AMPLIFICATION.
THE SYSTEM IS OPERATING IN A
NEGATIVE FEEDBACK ROAD
RESPONDING TO THE IL-2 AND WHEN
IT DOES THAT, IT FEEDS FORWARD
IN AN INTERESTING WAY BY
PROLIFERATING THESE HIGHLY
ACTIVATED REGULATORY CELLS.
THAT STILL DIDN'T ANSWER THE
QUESTION OF HOW THIS CONTROLS
AUTOIMMUNITY AND FOR THAT, WE
NEEDED THE CLEARING AND THE C3D
APPROACH.
THE REASON FOR THAT IS WE KNOW
IF YOU DISTURB RATIOS OR NUMBERS
OF CELLS WITHIN AN ANIMAL OR
HOST YOU IMBALANCE THE T REG AND
AUTO REACTIVE COMPONENTS SO WE
NEEDED TO LOOK AT PHYSIOLOGICAL
NUMBERS OF CELLS TO REALLY
ADDRESS THIS QUESTION.
WELL, IF WE TRY TO DO THAT WITH
A THIN SECTION, WE MIGHT SEE ONE
IF WE'RE LUCKY CELL IN A
SECTION.
IF WE LOOKED AT AN ENTIRE CLEAR
LYMPH NODE, THEN EVEN 10 OR 15
CELLS BEGIN TO BE STATISTICALLY
MEANINGFUL.
SO THAT'S WHAT HARRY DID.
HE WORKED OUT A WAY OF
TRANSFERRING INCREDIBLY SMALL
NUMBERS, SO IT WAS AT
PHYSIOLOGICAL FREQUENCIES FOR A
FEW CELLS OF AN ENTIRE LYMPH
NODE, AND WHEN HE DID THAT, HE
COULD SEE IN THIS LYMPH NODE, IN
THE RED CLOUD ARE ALL THE
ENDOGENOUS REGULATORY T-CELLS,
THESE FEW SPOTS ARE THE AUTO
REACTIVE TRANSGENIC CELLS OR
CONTROL CELLS, AND HE COULD
QUANTIFY WHAT HAPPENED OVER
TIME.
AND THERE, THERE WAS A VERY BIG
SURPRISE.
FOR THE FIRST THREE DAYS, THOSE
AUTO REACTIVE CELLS PROLIFERATED
EVEN IN THE PRESENCE OF A FULL
COHORT OF REGULATORY CELLS WHERE
WE KNEW THESE ANIMALS WOULD NOT
GET SICK.
BUT THEN AFTER A FEW MORE DAYS,
THEY ESSENTIALLY ALL DIED OFF.
SO THE WAY THE REGULATORY CELLS
WORK IS NOT TO PREVENT THE
ACTIVATION OF SELF-REACTIVE
CELLS, NOT EVEN TO PREVENT THEM
FROM PROLIFERATING, BUT TO CAUSE
THEM TO DIE WHEN THEY DO THAT.
SO THE WAY THIS ALL WORKS OUT IS
THAT WE BELIEVE THAT POSITIVE
SELECTION IN THE THYMUS GIVES US
THE CONVENTIONAL CELLS AND IS
SOMEWHAT LOWER SELF LIGAND --
THAN THE REGULATORY CELLS WHEN
THEY MEET UP WITH THEIR
DENDRITIC CELL YOU GET A LITTLE
BIT OF CD5 EXPRESSION ON THE
CONVENTIONAL CELLS AND A LITTLE
BIT OF IL-2.
THE T REGS WHEN THEY SEE THEIR
LIGAND PUT UP MORE CD25 AND THEY
HAVE PREFERENTIAL CAPACITY TO
CAPTURE IL-2, IN ESSENCE TO
STEAL IT AND WAY AND NOT LET IT
OCCUR AMONG THE COP VENGSAL
CELLS.
AT THE SAME TIME, THOSE K REGS
CAPTURING THE IL-2 PROLIFERATE
TO CREATE THIS VERY SUPPRESSIVE
ZONE TO MAKE SURE THE
CONVENTIONAL CELLS DON'T, QUOTE,
ESCAPE AND SEE IL-2.
WHY DOES THAT MATTER?"
L-2 IS USUALLY THOUGHT ABOUT AS
DRIVING T-SELL PROLIFERATION BUT
IT DOESN'T DO THAT.
IT ENABLES THEM TO SURVIVE.
THE CONVENTIONAL CELLS GO
INTO -- DUE TO TCR SIGNALING,
THEY MAKE IL-2 WHICH THEY WOULD
NORMALLY USE TO SURVIVE THAT
PROLIFERATIVE BURST, BUT THE T
REGS ARE STEALING ALL THAT
IL-2 SO THE PROLIFERATING
CONVENTIONAL CELLS DON'T SEE IT,
AND THEY DIE.
PRUNING THEM FROM THE REPERTOIRE
IN A CONTINUOUS BASIS AND
PREVENTING AUTOIMMUNITY.
SO WITH PARK AND SANG, WE
CREATED A MATH MATT CAT MODEL,
AND THAT MODEL SHOWS THAT SMALL
CHANGES OF LESS THAN TWOFOLD
WILL DISREGULATE THE SYSTEM I
JUST DESCRIBED.
SO FOR EXAMPLE, 50% LEVELS OF
CTLA4, A VERY ABUNDANT MOLECULE
ON REGULATORY CELLS WAS PREDICT
TODAY ALLOW ESCAPE FROM THIS
REGULATION AND FOR THE
CONVENTIONAL CELLS TO SEE THEIR
OWN IL-2 AND THAT'S WHAT WE SAW
EXPERIMENTALLY, AND THAT MATCHES
THE PHENOTYPE OF HETEROZYGOUS
HUMANS, WHERE
CTLA4 HETEROZYGOSITY OR THE
EQUIVALENT IN A MUTATION IN A
PROTEIN CONTROLLING
CTLA4 EXPRESSION GIVES YOU
AUTOIMMUNITY.
SO THIS BEGINS TO GIVE US A CLUE
ABOUT HOW GWAS HITS THINGS THAT
ARE EQTLs IN THE ORDER OF
TWOFOLD DIFFERENCES CAN BEGIN TO
FAVOR AN AUTOIMMUNE STATE.
AND SO VERY RECENTLY, WE'VE BEEN
COLLABORATING WITH ALEX MARSEN,
WHO HAS IDENTIFIED ENHANCERS
THAT OPERATE UNIQUELY TO CONTROL
CD25 EXPRESSION, THE
IL2 RECEPTOR ALPHA CHAIN
EXPRESSION IN REGULATORY CELLS
OR IN CONVENTIONAL CELLS.
AND WHEN YOU DO THAT, WHAT HE
SEES IS THAT IF YOU MODIFY THE
ENHANCER THAT WORKS IN THE
REGULATORY CELLS, THEY HAVE A
LOWER STEADY STATE LEVEL OF CD25
WI25BUT THAT GOES UP TO THE NORL
LEVEL WITHIN SIX HOURS OF THEM
TRIGGERS THEIR TCR.
SO VERY SUBTLE DIFFERENCE.
AND LIKEWISE FOR THE
CONVENTIONAL CELLS, THEY TAKE A
LITTLE BIT LONGER TO UPREGULATE
THEIR CD25, BUT THEY REACH THE
FULL LEVEL.
WELL, IN THE CASE OF THE
ENHANCER THAT WORKS IN THE
CONVENTIONAL CELLS, NOW IN THE
NOD MODEL OF AUTOIMMUNE
DIABETES, IF YOU DISADVANTAGE
THESE CONVENTIONAL CELLS FOR
THEIR CD25 EXPRESSION RELATIVE
TO THE NORMAL STATE, NONE OF THE
ANIMALS BECOME DIABETIC, AND
THAT'S TRUE EVEN IF YOU TREAT
THEM WITH ANTI-PD-1, THAT
NORMALLY GIVES YOU THIS
INCREDIBLY RAPID AND FULLY
PENETRANT DISEASE STATE.
AND CONVERSELY, IF YOU
DISENCUMBER THE T REG SLIGHTLY,
NOW DISEASE HAPPENS MUCH MORE
RAPIDLY AND IN A MORE PENETRANT
MANNER.
SO THESE VERY SMALL CHANGES THAT
I SHOWED YOU FROM THE STAINING
TRANSLATE INTO MAJOR DIFFERENCES
IN THE BIOLOGY AND IT SHOWS YOU
THAT THE T REG SYSTEM IS AT ONE
LEVEL ROBUST, BUT IT'S ALSO VERY
FRAGILE, WHICH IS EXACTLY WHAT
THE MATHEMATICAL MODELING SHOWED
US.
WITH A FEW PARAMETERS THAT HAVE
TO STAY IN A VERY NARROW RANGE
FOR THE SYSTEM TO WORK.
THIS BEGINS TO GET AT HOW
GENETIC VARIATION ACTUALLY
CAUSES HUMAN DISEASE.
SO I WANT TO END BY SAYING THAT
WE ALWAYS WANT MORE, AND MORE IS
TO GO FROM IBEX MULTIPLEX AND
C3D VOLUME AND PUT THEM TOGETHER
AND IT TAKES A TEAM, A TAKES A
VILLAGE TO DO THIS.
SO HIROSHI, ANDREA, COLIN, J.L.
AND -- PRETTY CLOSE TO HAVING
THIS RESOLVED.
THE WAY WE DO THIS IS A
COMBINATION OF IBEX WITH
ITERATIVE CYCLES THAT INVOLVE
THE CLEARING WITH THE C3D
METHOD, AND HERE IS ALUM IMAGE
THIS WAY IN A COUPLE OF HUNDRED
MICRONS BY HIROSHI IN 20
PARAMETERS.
AND YOU REALLY BEGIN TO SEE THAT
THIS GIVES YOU ALL SORTS OF
INFORMATION YOU DON'T GET WHEN
YOU'RE DOING LOAMPLEX OR VOLUME,
AND I'LL LET THIS PLAY A LITTLE
BIT MORE AND JUST POINT OUT THAT
THIS IS ESPECIALLY VALUABLE NOT
ONLY FOR VESSELS THAT I SHOWED
YOU IN THE LYMPH NODE IMAGE
BEFORE, BUT FOR EXAMINER OFS
THAT ARE VERY HARD TO SEE IN
THIN SECTIONS.
HERE'S ANOTHER 20 PLEX IN THE
GUT, AND THESE WILL CHANGE WITH
FOUR OR FIVE CLOFER FIVE COLORSH
CYCLE.
THIS SHOWS YOU WHAT YOU CAN GET
BY DOING THIS.
I'LL END BY SAYING WE REALLY
THINK THE WAY TO UNDERSTAND
IMMUNITY AS I STARTED OUT SAYING
IN THE VERY BEGINNING IS HIGH
ANALYSIS OF TISSUE ARCHITECTURE
IN CELL STATE.
WE CAN DO THIS BY 2P IMAGE, BY
HISTOCYTOMETRY, WE CAN IMPROVE
THAT WITH IBEX, WE CAN DO
HISTOCYTOMETRY IN 3D BUT THEN WE
CAN COMBINE ALL OF THAT TO C3D
IBEX.
WE HAVE SOFTWARE TOOLS RAPID AND
SPACE IN TICK THE INFORMATION WE
GET, QUANTIFYING IT AND PUTTING
IT IN CONTEXT.
I HAVEN'T HAD A CHANCE TO SHOW
YOU BUT WE'RE BEGINNING TO
COMBINE THIS WITH RNA PROBES SO
WE CAN COMBINE IT WITH THAT
APPROACH AND USE THE STAINING
PART FOR THOSE THINGS THAT ARE
UNIQUELY APPROACHABLE THAT WAY
LIKE THE PHOSPHOPROTEINS NUCLEAR
TRANSLOCATION AND SO FORTH.
LET ME END BY SAYING THAT I
DON'T HAVE THE TRADITIONAL SLIDE
WITH A THOUSAND NAMES ON IT.
I TRY TO TELL YOU WHO'S BEEN MY
COLLEAGUE AND COLLABORATOR
THROUGHOUT IN ALL THE WORK WE
DONE BUT I WANT TO THANK
EVERYBODY WHO'S BEEN IN THE LAB
BECAUSE THERE HAVE BEEN ALL
DIFFERENT LEVELS OF
CONTRIBUTIONS BESIDES THE PEOPLE
I'VE NAMED.
WE OBVIOUSLY HAVE HAD A LOT OF
COLLABORATIONS AND I'VE POINTED
THOSE OUT ALONG THE WAY AND
WE'VE REALLY BEEN GENEROUSLY
SUPPORTED BY THE INTRAMURAL
PROGRAM OF NIAID.
WE'VE ALSO HAD SUPPORT OVER THE
LAST FEW YEARS FOR WHAT WE CALL
THE CENTER FOR ADVANCED TISSUE
IMAWJING FROM THE NCI, SOME
BENCH TO BED SUPPORT AND --
THANKS VERY MUCH.
[APPLAUSE]
>>I LOVE THE MODEL FOR THE
T REGS.
I WAS WONDERING IF -- PRESUMABLY
THAT MODEL WORKS BEST IN THE
LYMPH NODE.
AND WHAT ABOUT IN THE PERIPHERAL
TISSUES?
HOW DO YOU THINK THE T REGS
WOULD WORK IN THE SAME WAY IN
THE PERIPHERAL TISSUES OR WOULD
THERE BE SOME OTHER MECHANISM?
>>SO I ALWAYS LEAVE SOMETHING
FOR THE FELLOWS THAT LOAF THE
LAB TO DO.
AND ACTUALLY HARRY IS MAKING A
MAJOR EFFORT LOOKING IN THE
TISSUES NOW USING THESE SAME
APPROACHES.
SO I DON'T KNOW WHAT HE'S FOUND,
HE'S JUST STARTED THE LAB.
HE'S LOOKING AT IT.
THERE IS REASON TO THINK IN SOME
CASES THAT IT STILL COULD BE ILT
THERAPY, FOR EXAMPLE, WORKS BY
EXPANDING CELLS FROM THESE
STEM-LIKE MEMORY CELLS.
MAYBE THAT'S DEPENDENT ON 15 AND
NOT JUST TWO AND THEN THE RULES
WOULD BE SOMEWHAT DIFFERENT SO I
DON'T WANT TO PUSH FOR THIS.
BUT GIVEN THAT THERE IS
PROLIFERATION UNDER THOSE
CONDITIONS, THEY AGAIN COULD
OPERATE IN PART BY STEALING.
THEY OBVIOUSLY -- I DIDN'T GO
INTO ALL OF IT.
THE MODEL INCLUDES THE
CTLA4 EFFECT BECAUSE IT'S NOT
JUST STEALING BUT LIMITING HOW
IL2 IS MADE BECAUSE YOU'RE
INTERFERING WITH CROW
STIMULATION.
THAT'S GOING TO APPLY IN THE
PERIPHERY AS WELL OBVIOUSLY, AND
WE KNOW YOU NEED TO
CO-STIMULATION FOR MANY OF THE
CYTOKINES.
THAT WOULD BE PRODUCED.
SO I THINK PARTS OF THE MODEL
WILL APPLY BUT I DON'T THINK ILL
IN THE PERIPHERY.
>>THANKS.
>>HI.
VERY IMPRESSIVE IMAGING.
I HAVE TWO QUESTIONS.
THE FIRST ONE IS REGARDING THE
ARCHITECTURE OF THE LIVER.
HOW COMPLEX DOES THE MICROBIOME
NEED TO BE FOR THE KUPFFER CELLS
TO BE POLARIZED?
THE SECOND QUESTION WOULD BE
REGARDING THE EX-T REGS, DO THEY
REMAIN SUPPRESSIVE?
>>FOR THE FIRST QUESTION, WE
CAN GET AT LEAST A PARTIAL
EFFECT BY USING LPS WHEN THEY'RE
NOT POLARIZED, AND WE CAN LOSE A
LOT OF THE POLARIZATION REMOVING
ONLY TLR4.
SO I DON'T THINK THE MICROBIOME
HAS TO BE ENORMOUSLY COMPLEX,
BUT IT CLEARLY THERE ARE
MULTIPLE DIFFERENT INPUTS
BECAUSE WE HAVE A DIFFERENCE
BETWEEN -- WE DIDN'T DO TRIF,
AND JUST THE TLR4.
BUT TLR2 KNOCKOUT ALSO HAD AN
EFFECT.
SO COMPLEXITY MATTERS BUT IT NOT
ESSENTIAL.
AS FAR AS THE EX-T REGS ARE
CONCERNED, WE HAVEN'T TRACED
THIS OUT OVER LONG PERIODS OF
TIME.
SO WE KNOW IN THAT ACUTE PERIOD
THEY CONVERT AND SPONTANEOUSLY
MAKE INTERFERON GAMMA IN VIVO.
LET ME CORRECT THAT STATEMENT.
I DIDN'T SLOW TO YOU BUT WE HAVE
A WAY OF VALIDATING WHETHER A
CELL IS ENGAGING TCR IN THE
TISSUES NOW, WITH THE HIGHEST
FRACTION OF CELLS SHOWING THAT
SIGNALING STATE RL THE XT REGS.
BUT WHETHER THEY PETER OUT, DIE,
I CAN'T TELL YOU YET.
>>THANK YOU.
>>LET ME SQUEEZE IN ONE FROM
HOME HERE.
OUR FRIEND HOWARD YOUNG ASKS,
HOW DO YOU DEAL WITH THE DATA
STORAGE?
IT MUST BE ENORMOUS.
>>WELL, OUR INSTITUTE IS VERY
HAPPY TO SUPPORT LARGE AMOUNTS
OF STORAGE SO WE JUST KEEP
DUMPING IT INTO THE H DRIVE.
THAT AND A LOT OF HARD DRIVES
HANGING AROUND.
>>HEY, RON, THAT WAS BEAUTIFUL.
SO I KNOW IN HARRY'S WORK, YOU
WERE LOOKING AT STEADY STATE TO
LOOK AT AUTO REACTIVITY, BUT IF
YOU START TO DO THAT IN THE
CONTEXT OF AN INFECTION WHERE
JUST SORT OF RELATED TO THE
QUESTION OF IS IL-2 RESCUING
LOWER AFFINITY CELLS AND HOW DO
T REGS FIT IN THERE?
>>SO WE DID THAT NOT IN AN
INFECTION YET.
WE'RE ALSO LOOKING AT THAT.
BUT THE WAY WE DID IT WAS TO USE
CYTOCHROME TRANSGENIC CELLS AND
ALTERED LIGANDS.
SO IF YOU USE A FULL AGONIST,
THEY WIND UP P STAD 5 POSITIVE
AND YOU BASICALLY SATURATE THE T
REGS.
SO ONCE YOU HIT SATURATION, THEY
CAN'T CONTROL ANYMORE AND THOSE
CELLS RESPOND.
BUT IF YOU USE A PARTIAL
AGONIST, THEN IT LOOKS LIKE THE
SELF-REACTIVE CELLS.
THEY DON'T BECOME P STAD
5 MOSSTIVE AND THAT'S ALSO WHY
THEY CAN'T EXPAND TO A
REASONABLE NUMBER.
102S.
>>YEAH, AND WHAT IF -- DO YOU
HAVE DIFFERENT TCRs THAT HAVE
DIFFERENT AFFINITY?
SO CAN YOU LOOK AT THAT?
>>WE DIDN'T DO THAT EXPERIMENT
WITH TCRs WITH DIFFERENT
AFFINITIES, WE DON'T HAVE A
SERIES WITH THE SAME LIGAND.
J.L. IS ACTUALLY GOING TO BUILD
THAT FOR A DIFFERENT REASON AND
WE COULD GO BACK AND CHECK THAT,
BUT -- PUBLISHED YEARS AGO THAT
WHEN YOU ELIMINATE T REGS, THEN
A POPULATION OF LOW AFFINITY
CD8s EMERGE AND THE RESPONSE
YOU DIDN'T SEE IF THE T REGS
WERE THERE, SO WE'D ONLY BE
REPEATING PUBLISHED DATA.
>>ONE OTHER QUESTION.
WHEN YOU WERE LOOKING AT THE --
IN ED'S WORK WHEN YOU WERE
LOOKING AT THE LOCALIZATION OF
THE T-CELLS VERSUS THE B CELLS,
WITHIN THAT B CELL PEAK, THERE'S
A SMALL PEAK OF CD8s.
DO YOU KNOW, ARE THOSE THE CXCR-
>>I'D HAVE TO ASK ED FOR THAT
PARTICULAR POINT, BUT I ALSO --
ONE OF THE THINGS YOU HAVE TO
KEEP IN MIND IS SOMETHING THAT
WE DID A NUMBER OF YEARS AGO.
THE B CELLS COME OUT ON HEVs,
AND THE HEVs ARE AT THE
PERIPHERY OF THE T-ZONE, BUT
THEY'RE IN THE T-ZONE, THEY'RE
NOT IN THE B CELL FOLLICLES.
SO ALL THE B CELLS, AND THIS IS
SOMETHING HE HAD IN HIS FIRST
PAPER ABOUT INTERACTIONS
DIRECTLY OUT OF THE -- PT B
CELLS HAVE TO RUN ACROSS A FIELD
OF T-CELLS TO GET INTO THE
FOLLICLE.
AND DEPENDING ON EXACTLY WHERE
YOU CUT THE LYMPH NODE AND HOW
YOU DO THAT, EVEN THOUGH WE SAY
CD4s ARE MORE PERIPHERAL, YOU
CAN HIT REGIONS WHERE YOU'RE --
THE WAY TO GET AROUND THEM, WE
DIDN'T DO THIS TREATMENT HERE,
IS TO TREAT THEM WITH ANTI62L.
SO NOW YOU ALLOW THE B CELLS TO
CLEAR THE T-ZONE, YOU GET A
BEAUTIFULLY CRISP BORDER AT
EXACTLY WHERE THE FRC NETWORK
ESSENTIALLY DISAPPEARS WHEN IT
TRYING TO GO INTO THE FOLLICLE,
AND THAT WOULD GIVE US A CLEANER
ANSWER.
>>THANKS SO MUCH.
>>ANOTHER ONE FROM HOME HERE.
I DON'T KNOW IF YOU ANSWERED
THIS, I'M SORRY.
HAVE YOU HAD ANY LUCK WITH
CDIBEX?
>>YES, IN TUMOR, IT DEPENDS ON
THE TUMOR.
WE'RE LOOKING AT THE FACT THAT
TUMORS HAVE A DIFFERENT LEVEL OF
NECROSIS AND AUTO FLUORESCENCE,
AND WE'RE WORKING TO GET AROUND
THAT.
WE'RE ALSO WORKING WITH FFPE
MATERIAL SINCE WE THINK THIS IS
ESPECIALLY USEFUL FOR HUMANS, SO
PRELIMINARILY, WE CAN GET AROUND
THAT, BUT YOU HAVE TO DO SOME
THINGS BEYOND WHAT I'VE SHOWN
YOU.
TO DO THAT.
AND WHAT WAS THE OTHER PART OF
THE QUESTION?
>>[INAUDIBLE]
>>YES.
>>THANKS, RON, FOR THIS GREAT
TALK.
BACK TO THE T REG MODEL.
OF THIS HANDFUL OF PARAMETERS
THAT YOU SAID HAVE TO BE VERY
TIGHTLY REGULATED WITHIN
TWOFOLD, HAVE ANY OF THEM BEEN
SHOWN TO ACTUALLY -- THAT THE
CHANGE CAUSES THE AUTOIMMUNE
DISEASE IN PATIENTS OR --
>>SO THE ENHANCERS THAT ALEX
MARSON HAS MANIPULATED ARE LOCI
OF SINGLE NUCLEOTIDE
POLYMORPHISMS THAT MATTER FOR
DISEASE IN HUMAN.
>>OKAY.
SO THEY'RE FROM -- ALL FROM GWAS
AND --
>>WELL, THE MOUSE IS NOT
IDENTICAL TO THE HUMAN, BUT
THEY'RE IN RELATIVE POSITIONS
WHERE THERE ARE HITS IN THE GWAS
STUDIES.
THERE'S A VERY HIGH-DENSITY OF
GWAS HITS IN CD25.
SO THAT'S WELL-KNOWN.
>>OKAY.
THANKS.
>>WE HAVE EIGHT QUESTIONS, BUT
WE'RE PAST THE HOUR AT THIS
POINT SO LET ME JUST ASK ONE
MORE.
>>I CAN TAKE EIGHT QUESTIONS.
>>OKAY, WE'LL SEE WHAT WE CAN
DO.
DO YOU THINK THE SHIELDING
MICROPHAGES, QUOTE-UNQUOTE, YOU
TALKED ABOUT REGARDING TISSUE
DAMAGE COULD BE INVOLVED IN
IMMUNOSUPPRESSION AT TUMOR
SITES?
>>YES, THEY COULD BE.
THESE CELLS ARE, IF YOU WANT TO
USE A VERNACULAR, M2-LIKE, AND,
THEREFORE, ANTI-INFLAMMATORY AND
YOU WANT INFLAMMATORY RESPONSES
TO BE ANTITUMOR.
HOWEVER, THESE AS I SAID ARE
EMBRYO LOGICALLY DERIVED CELLS,
AND MOST OF THE MACROPHAGES IN A
TUMOR HAVE BEEN SHOWN TO COME
PRIMARILY FROM MONOCYTES THAT
THEN ENTER, AND THEN POLARIZE IN
THIS DIRECTION.
SO WHETHER IT A SIMILAR PROCESS
BUT NOT WITH THESE MORE RESIDENT
CELLS, I CAN'T TELL YOU.
>>HAVE YOU BEEN ABLE TO
OPTICALLY CLEAR AND USE CE 3D
IMAGING IN TUMOR TISSUE, AND
HAVE YOU OBSERVED TERTIARY
LYMPHOD STRUCTURES.
>>IN MOUSE TISSUES, THAT'S
ONGOING, BUT IT'S BEEN DONE.
AGAIN, WE'RE TRYING TO FIGURE
OUT THE RULES FOR WHY SOME
TISSUES DON'T WORK SO WELL
VERSUS OTHERS.
IT SEEMS TO BE -- WE JUST HAD A
BIG MEETING YESTERDAY ABOUT
THIS, RELATED TO PERHAPS THE
LEVEL OF NECROTIC TISSUE WHICH
CAUSES SOME UNSPECIFIC STAINING,
AND THE BACKGROUND LEVEL OF AUTO
FLUORESCENCE, SO THOSE ARE SOME
CONFOUNDERS.
MOST MOUSE TUMORS DON'T SHOW
TERTIARY LYMPHOID STRUCTURES.
THEY'RE PREDOMINANT AND DOMINANT
AND VISIBLE IN MOSTLY HUMAN, AND
WE'RE JUST STARTING TO DO HUMAN
SAMPLES USING THIS METHOD.
>>OKAY.
NOT SURE IF THIS WAS ADDRESSED.
IN YOUR TUMOR STUDIES WITH
ANTICD40 AND SUBSEQUENT
CONVERSION OF T REGS TO
CONVENTIONAL CELLS, DO YOU HAVE
ANY INSIGHTS ON WHETHER THE
CELLS DOING THE CONVERSION ARE
NATURALLY THYMIC DERIVED T REGS
OR MORE PERIPHERAL DERIVED?
>>SO THE BEST WAY I CAN ANSWER
THAT QUESTION IS, 60 TO 70% OF
THE -- WHAT WE CALL T REGS IN
THE TUMOR VOLUME ARE HELIOS
POSITIVE.
WHEN WE START BEFORE TREATMENT.
AND THAT HELIOS GOES AWAY IN THE
EX-T REGS AND ACTUALLY FROM A
SUBSTANTIAL FRACTION OF THE
RESIDUAL SO-CALLED T REGS WHICH
WE THINK ARE IN THE PROCESS
PROBABLY OF CONVERTING, THEY
JUST HAVEN'T FIP PIS FINISHED LL
THEIR FOX P3.
SO THAT WOULD SUGGEST THAT AT
LEAST A LARGE FRACTION OF THEM
WOULD BE THYMICLY DERIVED
CONVENTIONAL T REGS BUT WE CAN'T
RULE OUT THERE ARE PT REGS IN
THE POPULATIONS AS WELL.
>>ALMOST DONE, THE LIGHTNING
ROUND HERE.
WE ALL BENEFIT FROM YOU AND YOUR
PROGRAM'S REMARKABLE CAREER.
THANK YOU FOR THE WONDERFUL
TALK.
IN A GIVEN AUTOIMMUNE DISEASE
AFFECTED TISSUE, DO YOU SEE A
RANGE OF CT REG-EX-T REG BALANCE
AND IN ORGANS, IS THERE SOME
SPATIAL ARRANGEMENT OF THESE
DIVERSE STATE?
EG ALONG THE TBUT OR WITHIN THEE
PANCREAS?
>>IT'S A VERY INTERESTING
QUESTION.
WE DON'T HAVE A GOOD METHOD FOR
ADDRESSING THAT IN THE HUMAN
SAMPLES AT THE PRESENT BECAUSE
WE CAN'T DO THE LINEAGE TRACING
TO IDENTIFY AN EX-T REG.
WHAT WE'RE TRYING TO DO IS
EITHER MINE EXISTING DATASETS
FROM SINGLE CELL RNA SEQ OR
CREATE SUCH DATASET THAT WOULD
ALLOW US TO IDENTIFY A FEW
MARKER PROTEINS THAT WE COULD
ADDRESS EITHER BY STAINING OR BY
FISH OR COMPARABLE
TRANSCRIPTOMIC METHODS TO
DISTINGUISH WHAT WAS AN EX-T REG
ASSUMING THAT THIS CONVERSION
DOESN'T CHANGE ALL THE
TRANSCRIPTIONAL PROFILE OF THE
CELL.
WHEN WE HAVE THAT INFORMATION,
THEN WE COULD DO THE TYPES OF
EXPERIMENT THAT ARE BEING ASKED
TO SEE WHAT THOSE BALANCES AND
THE DISTRIBUTIONS ARE IN VARIOUS
TISSUES.
WE COULD THEORETICALLY DO THAT
EXPERIMENT IN MICE WITH THE
LINEAGE SYSTEM, BUT WE HAVEN'T
DONE IT.
>>OKAY.
I WONDER IF IT IS POSSIBLE TO
CAPTURE CELL LOOR/TISSUE
DYNAMICS INCLUDING CELL
DIVISION/APOPTOSIS MOVING AND
INTERACTING IN 3D AS WELL, AND,
HAVE YOU LOOKED AT T-CELL
POPULATION IN THE CONTEXT OF
SOLID TUMORS AND THEIR LYMPH
NODES?
>>WE'RE DOING A LOT OF WORK ON
THE TUMORS RIGHT NOW.
TUMORS ARE ACTUALLY HARD TO
IMAGE BY 2P BECAUSE FROM THE
TIME THEY'RE AS LARGE AS YOU'D
LIKE THEM TO BE TO BEGIN
PERTURBING THEM OR DOING
TREATMENT, THEY HAVE A LOT OF
LIMITATIONS THAT MAKE TWO PHOTON
IMAGING DIFFICULT.
THERE HAVE BEEN SOME GOOD
STUDIES FROM MAX CROMELL AND A
FEW OTHER LABS BUT FEW ARE IN
NORMAL TISSUE SO THAT'S A
PROBLEM.
TO ANSWER THE FIRST QUESTION,
ALL OF THE IMAGING IS DONE IN --
LIMITED BY THE SPEED OF THE
CELLS MORE THAN THE OPTICAL
PROPERTIES.
IF A T-CELL MOVES MORE THAN ONE
CELL DIE AM TER BETWEEN EACH
CYCLE OF IMAGES YOU COLLECT, YOU
CAN'T TRACE THE TRACK FOR THE
PATH, SO THAT LIMIT YOU TO ALL
THE DEPTH OF ABOUT 100 MICRONS
BUT WITHIN THAT VOLUME, YES, YOU
CAN SEE INTERACTIONS, YOU CAN
SEE CELLS DIVIDING AND WE
PUBLISHED ON THAT, YOU CAN SEE
CELLS UNDERGOING POP PTOSIS
PERFECTLY WELL.
>>WHAT DRIVES THE SAMPLING OF
THE LUMEN IN T-CELLS IN THE GUT?
ARE THERE MICROBIOME-GENERATED
CHEMO ATTRACT ANTIS THAT ARE
KNOWN TO DO THIS?
>>THE ANSWER IS YES IN PART.
THE STUDYS THAT HAVE BEEN DONE
ON THIS KIND OF DENDRITIC CELL
SAMPLING SUGGEST THAT THERE ARE
SIGNALS TO THE EPITHELIAL CELLS
THAT THEN MAKE CHEMO -- IT MAY
ALSO CHANGE THEIR ADHESIVE
PROPERTY FOR THE CELLS TO EXPAND
THEIR PROCESSES AND DO THE
SAMPLING BUT THE DETAILS ARE NOT
AS WELL-KNOWN AS ONE WOULD LIKE,
SO IT'S A GOOD QUESTION.
>>IT WAS FROM NINA SCHOR.
VERY INTERESTING TALK.
T REGS DRAIN IL2 TO ELIMINATE
AUTO REACTIVE T-CELLS.
WOULD YOU SUGGEST
ANTIIL-2 THERAPY TO TREAT
AUTOIMMUNE DISEASE?
>>UNFORTUNATELY NO BECAUSE THAT
ALSO STARVES THE T REGS BECAUSE
IF YOU KILL THEM YOU CHANGE THE
BALANCE AGAIN, IN PRINCIPLE IN
FAVOR OF THE AUTO REACTIVE
CELLS.
SO IF ANYTHING, WHAT PEOPLE ARE
TRYING TO DO IS TO GIVE A LITTLE
BIT OF IL-2 IN A FORM THAT
FAVORS THE T-REGS BECAUSE OF
THEIR HIGHER CD T -- FROM REACTG
WITH CONVENTIONAL CELLS SO THAT
YOU GET T REG DOMINANCE OVER THE
CONVENTIONAL CELLS RATHER THAN
TRYING TO KILL THE CONVENTIONAL
CELLS BY STARVING THEM.
>>LAST ONE, IT'S FROM ED
SCHROM, IT'S THE LONGEST.
YOU MENTIONED THE T-CELLS FOLLOW
A RETICULAR NETWORK TO REDUCE
SEARCH TIMES WHEN ENCOUNTERING
DENDRITIC CELLS IN THE LYMPH
NODE.
BUT FOLLOWING THESE PREDEFINED
NETWORKS MIGHT ACTUALLY IMPEDE A
CELL'S ABILITY TO FOLLOW THE
CYTOKINE GRADIENT AS WHEN A
REGULATORY T-CELL MIGHT PREFER
TO MAKE A, QUOTE, BEELINE FOR
THE IL-2 PRODUCING CONVENTIONAL
T-CELLS AS IN HARRE'S WORK.
ARE T REGS LESS TIED TO THE
RETICULAR NETWORK?
AND/OR CAN T-CELLS ADAPTIVELY
MODULATE WHEN THEY ADHERE TO
VERSUS DISENGAGE FROM THE
RETICULAR NETWORK?
>>SO WE HAVE NOT SEEN T-CELLS
MOVE OFF OF THE NETWORK WITHIN
THE LYMPH NODE EXCEPT MAYBE WHEN
THEY'RE EXITING THROUGH THE
LYMPHATICS.
BUT IN TERMS OF THE BEELINE,
WHAT WE HAVE SHOWN BY DIRECT
IMAGING IS THAT WHEN A SOURCE
PRODUCES A CHEMOKINE AND WE DID
CAREFUL WORK WITH THE DENDRITIC
CELL BUT IT COULD BE THE SAME
CELL MAKING IL2 IF IT'S
PRODUCING A CUOMO KINE BY ITSELF
OR THROUGH ITS INTERACTION WITH
A DENDRITIC CELL THAT TYPICALLY
DOES THIS, THAT BIASES THE WALK
IN THE FRST NETWORK AND ALTHOUGH
THERE ARE SOME VERTICES YOU HAVE
TO GO THROUGH, THE ANSWER IS
IT'S A RELATIVELY VERY FAST PATH
AND YOU CAN GET ABOUT AN EIGHT
FOLD IMPROVEMENT IN FINDING THE
RELEVANT CELL COMPARED TO AN
EQUIDISTANT RELEVANT CELL.
SO I'M NOT SURE IT IS A
LIMITATION OF THE SYSTEM TO DO
THAT.
AND ED CAN FOLLOW UP OBVIOUSLY
IN THE LAB SINCE WE TALK ALL THE
TIME.
>>VERY GOOD.
THANK YOU.
ALL THAT'S REMAINING, LET'S
THANK THE SPEAKER.
[APPLAUSE]