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>>IT'S A REAL HONOR TO BE
ABLE

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TO INTRODUCE DR. RON GERMAIN TO

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THIS AUDIENCE.

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NOW, OF COURSE IT'S RIDICULOUS,

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THE IDEA THAT I WOULD INTRODUCE

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RON TO THIS AUDIENCE.

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THERE'S NO NEED TO DO THAT.

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BUT IT IS ALWAYS EXHILARATING TO

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GO BACK AND TO LOOK AT HOW

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SOMEBODY LIKE RON GOT TO

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SOMEPLACE LIKE HERE.

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AND JUST TO RECAP A COUPLE OF

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VERY BRIEF THINGS, RON GOT HIS

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BACHELOR'S AND HIS MASTERS AT

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BROWN IN FOUR YEARS.

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IT DID TAKE HIM SIX YEARS TO GET

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HIS M.D. AND PH.D. AT HARVARD,

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AND THEN HE WENT FROM THAT TO BE

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ON THE PATHOLOGY FACULTY AT

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HARVARD FOR A COUPLE OF YEARS,

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BEFORE COMING HERE AND THE GOOD

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NEWS IS, NOT LEAVING.

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AS YOU ALL KNOW, RON IS REALLY

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QUITE A VISIONARY SCIENTIST, AND

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HAS ALWAYS BEEN SO.

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FROM HIS EARLY WORK ON T-CELL

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BIOLOGY TO HIS CURRENT WORK

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REALLY IN, I THINK -- I LOOK AT

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IT AS MORE LYMPHOID BIOLOGY

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GENERALLY.

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AND ON THE ONE HAND, RON HAS

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BEEN PUTTING TOGETHER, AS I'M

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SURE HE'LL SHOW YOU,

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EXTRAORDINARY GLOBAL PICTURES OF

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WHAT GOES ON.

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AS THEY SAY, THOUGH, THE DEVIL

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IS IN THE DETAILS, AND RON HAS

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NEITHER SHIED FROM THE DETAILS

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NOR FAILED TO MEET THE DEVIL AND

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STARE HIM DOWN, AND THE WORK

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THAT HE HAS DONE, THE CLARITY

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WHICH HE HAS BROUGHT TO IT, HAS

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REALLY CHANGED THE WAY THAT WE

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LOOK AT THESE THINGS.

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THE OTHER THING THAT IS

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REALLY -- I'VE RESPECTED

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ENORMOUSLY ABOUT RON IS, I'VE

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HAD THE EXPERIENCE OF HIM COMING

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IN AS AN OUTSIDE INTERLOCULAR

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FOR RESEARCH GROUPS.

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COMING IN WITH TOPICS WITH WHICH

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HE HAD NO PRIOR EXPERIENCE BUT

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WAS ABLE, JUST BY DENT OF

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THINKING HARD AND REASONING

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CLEARLY TO HELP BRING TO THOSE

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OF US THAT WERE MORE BEKNIGHTED,

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SOME GUIDANCE AND SOME BETTER

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PATH FORWARD.

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SO IT'S REALLY A GREAT PRIVILEGE

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TO INTRODUCE SOMEBODY WHO WAS

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THE FOUNDER OF THE CENTER FOR

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HUMAN IMMUNOLOGY AND WHO HAS

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BEEN REALLY A MAJOR DRIVER FOR

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IMMUNOLOGY IN THE UNITED STATES,

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IN THE WORLD AND HERE ON THE

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CAMPUS.

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SO THANK YOU VERY MUCH FOR

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LETTING ME TAKE THE TIME TO

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INTRODUCE RON, AND TO BRING HIM

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TO YOU.

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>>THANK YOU, STEVE.

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THIS IS THE PAUL LECTURE, IN

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MEMORY OF WILLIAM E PAUL, AND

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BILL, AND THAT'S WHAT I'LL USE

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FROM NOW ON, WAS A REALLY

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TOWERING FIGURE IN IMMUNOLOGY.

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I'M JUST GOING TO SHOW YOU, HE

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DID HAVE  YOUNGER DAYS AND VERY

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HAPPY DAYS, AND HE RECALLED IN A

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FRONT PIECE THAT HE WROTE CALLED

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"ENDLESS FASCINATION" HOW HE

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BECAME AN IMMUNOLOGIST, WHICH IS

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TO READ A SMALL VOLUME OF ESSAYS

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BY MICHAEL HEIDELBERGER WHO WAS

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STILL DOING RESEARCH IN HIS LAB

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AT OVER 100 YEARS OLD, SO

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SOMETHING ALL OF US COULD ASPIRE

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TO.

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HE ACTUALLY WAS DOING

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ENDOCRINOLOGY HERE AT NIH FROM

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'62 TO '64, BUT HE WENT AND

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BECAME AN IMMUNOLOGIST BY

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TRAINING WITH BARUJ AND NYU AND

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MOVED WITH BARUJ TO NIH AROUND

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1968, AND WHEN BARUJ LEFT IN

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1970, BILL BECAME THE CHIEF OF

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THE LABORATORY OF IMMUNOLOGY, A

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POSITION HE HELD FOR 45 YEARS.

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I RAN OUT OF SPACE ON MY SLIDE

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DESCRIBING BILL'S CONTRIBUTIONS,

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AND I'M NOT GOING TO READ THIS

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TO YOU AND JUST POINT OUT THAT

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HE DID VERY EARLY WORK ABOUT THE

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DIFFERENCE BETWEEN

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POLYSACCHARIDE AND PROTEIN

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ANTIGENS THAT IN A WAY IS THE

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PRECURSOR FOR CONJUGATE

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VACCINES, DISCOVERED A AT IL4, E

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DRY POLARIZATION OF T-CELLS BUT

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DID MUCH, MUCH, MUCH MORE.

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HE ALSO DID A LOT OF SPECTACULAR

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TRAINING, AND PEOPLE WHO PASSED

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THROUGH HIS LAB AND UNDER HIS

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TUTELAGE INCLUDE RON SCHWARTZ,

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LAURIE GLIMSHIRE, MELISSA BROWN,

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MARK DAVIS, AND THEN OUR OWN

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JEFF ZHU HERE AND OUR PREVIOUS

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OWN JOSH MILNER.

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WE ACTUALLY SHARED THE SAME

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MENTOR.

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I DID MY MD PH.D. AND MY PH.D.

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PART WITH BARUJ, WHO YOU SEE IN

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THE CENTER WITH BILL AND I

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CELEBRATING ONE OF BARUJ'S

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BIRTHDAYS.

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THIS IS OBVIOUSLY A STAGED

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PHOTOGRAPH.

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WE DID NOT TYPICALLY ALWAYS WEAR

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OUR WHITE COATS AND TIES IN THE

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LAB.

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ALTHOUGH BILL DID FAIRLY OFTEN.

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BUT WE DID READ SEQUENCING GELS

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TOGETHER IN THE DAYS WHEN THAT'S

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HOW YOU DID SEQUENCING.

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AND WHY IS THIS TITLED LOOKING

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AT FUNDAMENTAL BIOLOGY.

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AND MANY THINGS HE DID BEYOND

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THE LABORATORY, MAKING IT THE

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MOST CITED PUBLICATION IN

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BIOMEDICAL SCIENCE, NOT JUST

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IMMUNOLOGY, IS THAT HE PUBLISHED

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MULTIPLE EDITIONS OF THE BOOK

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"FUNDAMENTAL IMMUNOLOGY.

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" THAT'S WHERE I REALLY WANT TO

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START MY TALK TODAY, THINKING

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ABOUT THE FUNDAMENTALS OF

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IMMUNOLOGY.

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WE THINK ABOUT THE IMMUNE SYSTEM

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AS A PROTECTIVE SYSTEM THAT

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PROTECTS US AGAINST THE

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DANGEROUS EXTERNAL WORLD AND

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RECENTLY WE THINK ABOUT IT A LOT

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IN HAVING INFLUENCE IN PRO

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TETING US AGAINST A DANGEROUS

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INTERNAL WORLD.

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THESE ARE VERY DIVERSE

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PREDICTABLE DISEASE ENTITIES

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THAT ARRIVE OR ARISE AT

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DIFFERENT TIMES AND DIFFERENT

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SITES IN THE BODY.

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HOW DO YOU SURVEY EVERYTHING AND

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KNOW AND ARE ABLE TO RECOGNIZE

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WHAT'S GOING ON.

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AND TO MEET THIS WHAT, WHEN AND

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WHERE CHALLENGE, THE MY UNSYSTEM

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USES SOLUBLE INFECT ORS,

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ANTIBODIES COMPLEMENT

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COMPONENTS, PENTRAXINS AND SO

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ON.

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MOBILE ELEMENTS AND THOSE I WILL

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TALK ABOUT THAT CAN MOVE

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DIRECTLY INTO THE RELEVANT

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AFFECTED TARGET SITE, AND

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ORGANIZE TISSUES.

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AND I'LL TALK A LOT ABOUT THIS

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TODAY, THAT ARE NECESSARY IN

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ORDER -- ESPECIALLY FOR THE

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ADAPTIVE IMMUNE SYSTEM TO DO ITS

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JOB, GIVEN THAT IT ORIGINATES

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ITS RESPONSE FROM VERY RARE

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CELLS IN A VERY LARGE VOLUME OF

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THE BODY.

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SO WE HAVE CELLS FORMED IN THE

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BONE MARROW, THESE ARE MYELOID

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AND LYMPHOID CELLS.

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THE MORE MATURE MYELOID CELLS

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ARE READY TO GO WHEN THEY EXIT

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AND THEY CAN FIND INFLAMED

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ENDOTHELIUM, THEY CAN ADHERE,

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AND EXIT TO MEDIATE EFFECTOR

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FUNCTION.

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SO PRECURSOR CELLS CAN ENTER THE

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THYMUS AND GO THROUGH A WHOLE

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DEVELOPMENTAL AND SELECTIVE

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PROCESS TO GIVE RISE TO T-CELLS

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THAT WIND UP BACK IN SECONDARY

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LYMPHOID TISSUES, FOR EXAMPLE,

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WITHIN THE LYMPH NODE.

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WITHIN THE LYMPH NODE, THERE'S A

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SEGREGATION BETWEEN THESE

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T-CELLS AND ANOTHER FORM OF

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LYMPHOCYTE, THE B LYMPHOCYTE IN

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THESE FOLLICLES.

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THEY EVENTUALLY HAVE TO GET

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TOGETHER.

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IMMUNOLOGISTS HAVE NEVER FIGURED

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OUT WHY THEY NEED TO BE

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SEPARATED BEFORE THEY GET

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TOGETHER.

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SOMETHING TO THINK ABOUT.

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BUT WITHIN THE T-ZONE, THE

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T-CELLS ARE SEARCHING FOR THEIR

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COGNATE LIGAND AND THAT'S

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TYPICALLY IN THE FORM OF A

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PEPTIDE MAJOR HISTOCOMPATIBILITY

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COMPLEX LIGAND ON THE SURFACE OF

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A DENDRITIC CELL.

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IF THERE'S A PRODUCTIVE

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INTERACTION WHERE THAT LIGAND

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MATCHES THE SPES FIS OF A CLONAL

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RECEPTOR ON THE T-CELLS, THEY

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GET ACTIVATED, THEY CAN

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PROLIFERATE, DIFFERENTIATE, AND

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LIKELY INNATE CELLS FIND

225
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INFLAMED ENDOTHELIUM AND ENTER

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INTO THE TISSUE TO MEDIATE

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EFFECTOR FUNCTION.

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AND IF WE THINK ABOUT WHAT I

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JUST DESCRIBED AFTER ESPECIALLY

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THE CELLS LEAVE THE BONE MARROW,

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EVERYTHING THEY'RE DOING WHEN

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THEY'RE NOT IN THE BLOOD PER SE

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IS IN A TISSUE.

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THEY'RE EITHER BECOMING

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ACTIVATED IN THE TISSUE OR

236
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THEY'RE MEDIATING EFFECT FOR

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FUNCTION IN THE TISSUE, AND SO

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WE CANNOT UNDERSTAND HOW THE

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IMMUNE SYSTEM DOES ITS JOB IF WE

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JUST LOOK AT ISOLATED CELLS AND

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MOLECULES.

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WE WILL NEVER UNDERSTAND EXACTLY

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HOW THE SYSTEM WORKS, AND IN

244
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ORDER TO LOOK AT WHAT'S GOING ON

245
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IN A TISSUE WE NEED TO IMAGE,

246
00:08:50,040 --> 00:08:51,720
BOTH DYNAMIC AND HIGH CONTENT

247
00:08:51,720 --> 00:08:52,280
INFORMATION.

248
00:08:52,280 --> 00:08:53,680
SO I'M GOING TO START WITH WHERE

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00:08:53,680 --> 00:08:54,800
WE AND OTHERS IN THE FIELD

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00:08:54,800 --> 00:08:56,280
STARTED ABOUT 20 YEARS AGO WITH

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00:08:56,280 --> 00:08:57,920
THE DYNAMIC IMAGING.

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00:08:57,920 --> 00:09:02,440
THIS IS MULTIPHOTON VITAL

253
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MICROSCOPY.

254
00:09:02,960 --> 00:09:06,120
IT'S TYPICALLY LIMITED TO FIVE

255
00:09:06,120 --> 00:09:08,440
OR FEWER PARAMETERS AND THAT'S A

256
00:09:08,440 --> 00:09:09,280
MAJOR LIMITATION AS WE'LL GET

257
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INTO LATER.

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IT'S POSSIBLE TO DO SIGNALING

259
00:09:11,800 --> 00:09:12,800
MEASUREMENTS BUT IT'S TYPICALLY

260
00:09:12,800 --> 00:09:16,880
PRETTY MUCH CALCIUM OR THE

261
00:09:16,880 --> 00:09:18,400
ANALOG OF NFAT NUCLEAR

262
00:09:18,400 --> 00:09:18,760
TRANSLOCATION.

263
00:09:18,760 --> 00:09:20,400
YOU CAN LOOK FOR A FEW SECONDS

264
00:09:20,400 --> 00:09:20,920
TO HOURS.

265
00:09:20,920 --> 00:09:22,200
THERE ARE WINDOW PREPARATIONS

266
00:09:22,200 --> 00:09:24,000
WHERE YOU CAN LOOK OVER LONGER

267
00:09:24,000 --> 00:09:26,360
TIME PERIODS BUT THEN YOU'RE NOT

268
00:09:26,360 --> 00:09:29,520
FOLLOWING THE SAME CELL OVER ALL

269
00:09:29,520 --> 00:09:32,200
THAT TIME, YOU'RE LOOKING AT

270
00:09:32,200 --> 00:09:33,120
ITERATIVE POPULATIONS OF CELLS

271
00:09:33,120 --> 00:09:34,560
DURING THE TIMING AND IT'S NOT

272
00:09:34,560 --> 00:09:36,440
REALLY APPLICABLE TO HUMAN

273
00:09:36,440 --> 00:09:36,760
STUDY.

274
00:09:36,760 --> 00:09:38,080
THERE'S NO INNERVATION, THERE'S

275
00:09:38,080 --> 00:09:39,480
NO LYMPH FLOW, THERE'S NO

276
00:09:39,480 --> 00:09:40,880
VASCULAR INVOLVEMENT, THERE ARE

277
00:09:40,880 --> 00:09:42,320
NO CELLS COMING IN AND OUT IN

278
00:09:42,320 --> 00:09:44,400
THE NORMAL WAY, AND SO THIS IS A

279
00:09:44,400 --> 00:09:46,600
VERY LIMITED APPLICATION.

280
00:09:46,600 --> 00:09:48,040
SO THE WAY YOU DO THIS IS

281
00:09:48,040 --> 00:09:50,960
SURGICALLY EXPOS THE TISSUE OF

282
00:09:50,960 --> 00:09:51,640
INTEREST.

283
00:09:51,640 --> 00:09:53,280
YOU ONLY GET A FREE LUNCH IN THE

284
00:09:53,280 --> 00:09:54,320
EAR SKIN WHERE YOU DON'T HAVE TO

285
00:09:54,320 --> 00:09:55,000
DO SURGERY.

286
00:09:55,000 --> 00:09:56,800
OF COURSE SURGERY IS DISRUPTIVE

287
00:09:56,800 --> 00:09:58,600
AND PRO INFLAMMATORY SO YOU

288
00:09:58,600 --> 00:09:59,720
SHOULD ASK WHEN YOU READ PAPERS

289
00:09:59,720 --> 00:10:01,680
USING THIS METHOD, HOW DID

290
00:10:01,680 --> 00:10:03,400
PEOPLE CONTROL FOR THE

291
00:10:03,400 --> 00:10:04,240
BACKGROUND INFLAMMATION THAT

292
00:10:04,240 --> 00:10:08,000
THEY CAUSE BY DOING THE SURGERY,

293
00:10:08,000 --> 00:10:09,040
BUT ASSUMING THEY DID THAT WELL

294
00:10:09,040 --> 00:10:10,680
AND PROPERLY, YOU TAKE A LASER

295
00:10:10,680 --> 00:10:13,920
AND SCAN IT IN THE XY DIMENSION,

296
00:10:13,920 --> 00:10:16,080
GET A PLANE OF IMAGING

297
00:10:16,080 --> 00:10:16,920
INFORMATION, FOCUS UP OR DOWN,

298
00:10:16,920 --> 00:10:19,400
GET A STACK OF INFORMATION, AND

299
00:10:19,400 --> 00:10:20,600
THEN COLLECT ALL OF THAT OVER

300
00:10:20,600 --> 00:10:22,320
TIME, SO FOUR-DIMENSIONAL

301
00:10:22,320 --> 00:10:24,120
DATASETS THAT YOU PLAY BACK THIS

302
00:10:24,120 --> 00:10:26,640
WAY, TWO TO 300 TIMES REAL

303
00:10:26,640 --> 00:10:28,480
SPEED, AND COMPRESSED IN TWO

304
00:10:28,480 --> 00:10:28,800
DIMENSIONS.

305
00:10:28,800 --> 00:10:30,960
BUT WE DO HAVE THE

306
00:10:30,960 --> 00:10:32,000
THREE-DIMENSIONAL INFORMATION.

307
00:10:32,000 --> 00:10:34,080
HERE ARE T LYMPHOCYTES MOVING IN

308
00:10:34,080 --> 00:10:35,800
THE T-ZONE OF A LYMPH NODE AND

309
00:10:35,800 --> 00:10:37,040
IT LOOKS LIKE THEY'RE FREE TO

310
00:10:37,040 --> 00:10:38,440
MOVE AROUND LIKE A GO FISH IN A

311
00:10:38,440 --> 00:10:39,640
LITTLE BOWL YOU BOUGHT AT THE

312
00:10:39,640 --> 00:10:41,400
STATE FAIR BUT THAT'S NOT TRUE.

313
00:10:41,400 --> 00:10:43,240
THEY'RE IN THIS ENVIRONMENT.

314
00:10:43,240 --> 00:10:45,000
THE BLACK IS NOT EMPTY SPACE.

315
00:10:45,000 --> 00:10:47,960
THE BLACK IS JUST WHAT WE DON'T

316
00:10:47,960 --> 00:10:51,040
MAKE VISUAL BY PUTTING

317
00:10:51,040 --> 00:10:52,120
FLUORESCENCE INTO OUR IMAGES.

318
00:10:52,120 --> 00:10:53,640
SO YOU SHOULD NEVER BELIEVE WHAT

319
00:10:53,640 --> 00:10:55,080
AN IMAGER TELLS YOU BASED ON

320
00:10:55,080 --> 00:10:57,160
WHAT HE OR SHE DOES NOT SEE, AND

321
00:10:57,160 --> 00:10:58,680
YOU SHOULD ALWAYS WORRY ABOUT

322
00:10:58,680 --> 00:11:01,320
WHAT'S IN THE BLACK.

323
00:11:01,320 --> 00:11:03,080
BECAUSE IT IS INFLUENCING THE

324
00:11:03,080 --> 00:11:04,320
BEHAVIOR VISUALIZING AND IT'S

325
00:11:04,320 --> 00:11:05,440
NOT TO BE IGNORED.

326
00:11:05,440 --> 00:11:14,080
SO I THINK EVIDE -- IF YOU BREEH

327
00:11:14,080 --> 00:11:17,160
THEM, YOU GET RAPID INNATE

328
00:11:17,160 --> 00:11:21,440
RESPONSE AND MORE SLOWLY -- THIS

329
00:11:21,440 --> 00:11:24,600
IS WORK DONE BY A SPECTACULAR

330
00:11:24,600 --> 00:11:27,000
COLLEAGUE WHO NOW HAS HIS OWN

331
00:11:27,000 --> 00:11:28,880
LAB, AND WHAT STEFFAN WANTED TO

332
00:11:28,880 --> 00:11:31,280
DO WAS TO OBSERVE MYELOID CELL

333
00:11:31,280 --> 00:11:32,720
BEHAVIOR AND TO DO THAT, HE

334
00:11:32,720 --> 00:11:34,360
CHOSE TO DO IT IN A NEW

335
00:11:34,360 --> 00:11:35,640
PREPARATION LOOKING IN THE

336
00:11:35,640 --> 00:11:36,560
PERITONEAL WALL.

337
00:11:36,560 --> 00:11:38,760
THE REASON FOR THAT WAS WE KNOW

338
00:11:38,760 --> 00:11:41,680
THERE IS THIS MONOCYTE

339
00:11:41,680 --> 00:11:43,240
POPULATION TO PERITONEAL

340
00:11:43,240 --> 00:11:44,960
MACROPHAGES BUT THERE IS ALSO

341
00:11:44,960 --> 00:11:46,480
THESE EMBRYOLOGICALLY DERIVED

342
00:11:46,480 --> 00:11:48,880
FIXED TISSUE MACROPHAGES IN THE

343
00:11:48,880 --> 00:11:50,880
SEROSAL LINING IN THE PERITONEAL

344
00:11:50,880 --> 00:11:52,600
CAVITY AND THEN IT'S EASY TO

345
00:11:52,600 --> 00:11:53,800
RECRUIT NEUTROPHILS WITH A

346
00:11:53,800 --> 00:11:54,080
CHALLENGE.

347
00:11:54,080 --> 00:11:55,560
SO WE CAN LOOK AT ALL THESE

348
00:11:55,560 --> 00:11:56,800
DIFFERENT MYELOID POPULATIONS AT

349
00:11:56,800 --> 00:11:57,200
ONE TIME.

350
00:11:57,200 --> 00:11:58,600
WHEN HE DID THAT, HE TOOK

351
00:11:58,600 --> 00:12:00,720
ADVANTAGE OF A MODEL THAT TIM

352
00:12:00,720 --> 00:12:02,560
LALLERMAN HAD DEVELOPED IN THE

353
00:12:02,560 --> 00:12:04,160
LAB EARLIER WHERE WE DID STERILE

354
00:12:04,160 --> 00:12:05,960
INJURY WITH LASER DAMAGE IN THE

355
00:12:05,960 --> 00:12:06,360
DERMIS.

356
00:12:06,360 --> 00:12:08,800
WHAT HE DID IS DO THE SAME TYPE

357
00:12:08,800 --> 00:12:09,760
OF DAMAGE IN THE PERITONEAL

358
00:12:09,760 --> 00:12:10,040
WALL.

359
00:12:10,040 --> 00:12:11,840
WHEN HE DID THAT, WHAT HE SAW

360
00:12:11,840 --> 00:12:14,600
WAS THE SAME TYPE OF NEUTROPHIL

361
00:12:14,600 --> 00:12:19,880
SWARMING THAT YOU SEE HERE.

362
00:12:19,880 --> 00:12:21,040
YOU SEE THE CORPSES IN WHITE

363
00:12:21,040 --> 00:12:26,240
HERE ARE DESTROYED BUT THIS IS

364
00:12:26,240 --> 00:12:26,680
THE PROBLEM.

365
00:12:26,680 --> 00:12:28,520
HERE IS THE COLLAGEN THAT'S PART

366
00:12:28,520 --> 00:12:31,760
OF THE TISSUE MATRIX, AND YOU

367
00:12:31,760 --> 00:12:35,360
SEE THAT NEUTROPHIL SWARMING

368
00:12:35,360 --> 00:12:36,240
DAMAGES THIS MATRIX.

369
00:12:36,240 --> 00:12:38,000
THAT RAISES AN INTERESTING

370
00:12:38,000 --> 00:12:38,640
QUESTION.

371
00:12:38,640 --> 00:12:40,080
CELLS DIE IN TISSUES ALL THE

372
00:12:40,080 --> 00:12:40,280
TIME.

373
00:12:40,280 --> 00:12:41,720
IF THE NEUTROPHILS RESPOND TO

374
00:12:41,720 --> 00:12:42,560
CELL DEATH BY SWARMING IN THE

375
00:12:42,560 --> 00:12:44,880
WAY THAT I JUST SHOWED YOU, AND

376
00:12:44,880 --> 00:12:46,840
THEN THEY CAUSE THIS COLLATERAL

377
00:12:46,840 --> 00:12:48,280
DAMAGE, HOW WOULD YOU EVER

378
00:12:48,280 --> 00:12:49,480
MAINTAIN TISSUE PHYSIOLOGY?

379
00:12:49,480 --> 00:12:52,240
BECAUSE YOU'D HAVE THIS CONSTANT

380
00:12:52,240 --> 00:12:54,400
INFLAMMATORY PROCESS BE THAT WAS

381
00:12:54,400 --> 00:12:56,240
DISRUPTING THE TISSUE STRUCTURE

382
00:12:56,240 --> 00:12:57,600
AND THAT DID NOT MAKE ANY SENSE,

383
00:12:57,600 --> 00:12:58,920
SO WE NEEDED TO UNDERSTAND WHAT

384
00:12:58,920 --> 00:13:03,720
WAS PREVENTING THIS, OR WE TREE

385
00:13:03,720 --> 00:13:06,240
SEUM WAS PREVENTING THIS.

386
00:13:06,240 --> 00:13:07,960
WHAT STEFFAN FIGURED OUT, WE

387
00:13:07,960 --> 00:13:08,840
ALWAYS KILLED MASSES OF CELLS

388
00:13:08,840 --> 00:13:10,280
WITH OUR LASER AND HE CHANGED

389
00:13:10,280 --> 00:13:12,560
THIS AND KILLED A SINGLE

390
00:13:12,560 --> 00:13:13,360
FIBROBLAST IN THE PERITONEAL

391
00:13:13,360 --> 00:13:13,560
WALL.

392
00:13:13,560 --> 00:13:14,800
THAT'S WHAT YOU SEE IN ORANGE IN

393
00:13:14,800 --> 00:13:15,960
THE MIDDLE OF THIS IMAGE HERE.

394
00:13:15,960 --> 00:13:19,640
AND THEN HE OBSERVED WHAT HAPPEN

395
00:13:19,640 --> 00:13:21,000
WITH GENETICALLY LABELED MYELOID

396
00:13:21,000 --> 00:13:21,640
CELLS.

397
00:13:21,640 --> 00:13:23,720
HERE WE'RE LOOK AT THOSE

398
00:13:23,720 --> 00:13:25,440
EMBRYOLOGICALLY DERIVED

399
00:13:25,440 --> 00:13:25,840
MACROPHAGES.

400
00:13:25,840 --> 00:13:26,800
WHAT HE SAW IS THEY RAPIDLY

401
00:13:26,800 --> 00:13:29,200
SENSE THAT CELL DEATH, AND THEN

402
00:13:29,200 --> 00:13:30,480
THEY DON'T MOVE THE CELL BODIES

403
00:13:30,480 --> 00:13:33,560
BUT THEY SEND OUT PROCESSES THAT

404
00:13:33,560 --> 00:13:35,000
ENCOMPASS THE CORPS.

405
00:13:35,000 --> 00:13:37,160
SOMETHING WE CALL CLOAKING.

406
00:13:37,160 --> 00:13:38,800
AND THAT TURNED OUT TO BE THE

407
00:13:38,800 --> 00:13:42,560
KEY FEATURE.

408
00:13:42,560 --> 00:13:44,120
SO NORMALLY IF WE DAMAGE

409
00:13:44,120 --> 00:13:45,080
MULTIPLE CELLS, WE CAN SEE THIS

410
00:13:45,080 --> 00:13:46,720
KIND OF TRACKING OF THE

411
00:13:46,720 --> 00:13:47,040
NEUTROPHILS.

412
00:13:47,040 --> 00:13:49,160
THEY SENSE IT, AND THEN THEY ALL

413
00:13:49,160 --> 00:13:51,320
TURN AND RUN IN A VERY

414
00:13:51,320 --> 00:13:52,400
DIRECTIONAL WAY TO CREATE THE

415
00:13:52,400 --> 00:13:54,800
SWARMS THAT I SHOWED YOU A

416
00:13:54,800 --> 00:13:55,400
MOMENT AGO.

417
00:13:55,400 --> 00:13:58,000
BUT IF WE HAVE THE SINGLE CELL

418
00:13:58,000 --> 00:13:59,840
DEATH, NEUTROPHILS DO SHOW UP IN

419
00:13:59,840 --> 00:14:01,240
THE PREPARATION, BUT THEY DON'T

420
00:14:01,240 --> 00:14:04,760
SHOW THAT HIGHLY DIRECTIONAL AND

421
00:14:04,760 --> 00:14:07,480
RAPID MIGRATION, AND THE REASON

422
00:14:07,480 --> 00:14:09,360
THEY DON'T IS THEY CAN'T SEE THE

423
00:14:09,360 --> 00:14:09,800
CORPS.

424
00:14:09,800 --> 00:14:12,520
AND WHAT TIM LAMBERMAN HAD

425
00:14:12,520 --> 00:14:13,400
PREVIOUSLY SHOWN IS THAT THE

426
00:14:13,400 --> 00:14:16,360
SWARMING IS INITIATED BY A LEAD

427
00:14:16,360 --> 00:14:18,000
NEUTROPHIL SENSING AND ACTUALLY

428
00:14:18,000 --> 00:14:21,600
TOUCHING THE DEAD MATERIAL AND

429
00:14:21,600 --> 00:14:25,120
BEGINNING TO PRODUCE LBT4 WHICH

430
00:14:25,120 --> 00:14:27,840
THEN INTRODUCES OTHER LIVING

431
00:14:27,840 --> 00:14:29,400
NEUTROPHILS CREATING AN ENORMOUS

432
00:14:29,400 --> 00:14:30,360
GRADIENT FOR THESE CELLS TO

433
00:14:30,360 --> 00:14:31,840
FOLLOW TO GENERATE THE SWARMING

434
00:14:31,840 --> 00:14:33,440
RESPONSE.

435
00:14:33,440 --> 00:14:35,480
IT TURNS OUT THAT THIS

436
00:14:35,480 --> 00:14:36,920
PROTECTIVE MECHANISM IS

437
00:14:36,920 --> 00:14:37,240
CRITICAL.

438
00:14:37,240 --> 00:14:39,880
IF WE CHRONICALLY DEPLETE THESE

439
00:14:39,880 --> 00:14:41,960
FIXED TISSUE MACROPHAGES AND

440
00:14:41,960 --> 00:14:43,720
LOOK AT A TISSUE UNDERGOING

441
00:14:43,720 --> 00:14:47,920
CHRONIC STRESS, THE DIAG THE DI,

442
00:14:47,920 --> 00:14:49,080
YOU'RE BREATHING ALL THE TIME

443
00:14:49,080 --> 00:14:50,600
AND STRESSING THE MUSCLE FIBERS.

444
00:14:50,600 --> 00:14:53,000
NOW WHAT WE SEE IS WE GET THESE

445
00:14:53,000 --> 00:14:55,080
NECROTIC MUSCLE FIBERS WHEN

446
00:14:55,080 --> 00:14:56,720
THOUGH MACROPHAGES ARE NOT THERE

447
00:14:56,720 --> 00:14:57,720
TO CLOAK AND THESE ARE

448
00:14:57,720 --> 00:14:58,920
ASSOCIATED WITH VERY INTENSE

449
00:14:58,920 --> 00:15:00,880
KNEW CROWPHILIC INFILTRATION.

450
00:15:00,880 --> 00:15:02,720
SO WHAT STEFFAN'S WORK SHOWED IS

451
00:15:02,720 --> 00:15:04,920
THAT THE RESIDENT MACROPHAGES

452
00:15:04,920 --> 00:15:07,520
PREVENT INNATE IMMUNE DAMAGE IN

453
00:15:07,520 --> 00:15:09,200
RESPONSE TO INTERMITTENT CELL

454
00:15:09,200 --> 00:15:11,280
DEATH ORE DAMAGE AND IT PERMITS

455
00:15:11,280 --> 00:15:12,800
HEALING BY LOCAL REPLACEMENT.

456
00:15:12,800 --> 00:15:15,280
THE NEXT NEIGHBORING FIBROBLAST

457
00:15:15,280 --> 00:15:16,720
DIVIDES, FILLS IN THE GAP,

458
00:15:16,720 --> 00:15:21,280
THERE'S NO RESOLUTION, POST

459
00:15:21,280 --> 00:15:22,520
INFLAMMATION TYPE RESOLUTION AND

460
00:15:22,520 --> 00:15:23,600
THERE'S NO SCARRING, SO IT'S

461
00:15:23,600 --> 00:15:24,840
VERY BENIGN FOR THE TISSUE.

462
00:15:24,840 --> 00:15:27,760
IF WE DO VERY CAREFUL WORK OVER

463
00:15:27,760 --> 00:15:30,160
TIME AND IN AGING ANIMALS, WE

464
00:15:30,160 --> 00:15:31,720
SEE THESE RATHER PROTECTIVE

465
00:15:31,720 --> 00:15:32,920
MACROPHAGES BEGIN TO ACQUIRE

466
00:15:32,920 --> 00:15:34,400
MORE INFLAMMATORY PROPERTIES,

467
00:15:34,400 --> 00:15:35,800
AND SO WE THINK THAT THIS

468
00:15:35,800 --> 00:15:37,720
PROCESS MAY BE SUBVERTED AND

469
00:15:37,720 --> 00:15:41,560
CONTRIBUTE TO WHAT'S CALLED

470
00:15:41,560 --> 00:15:43,640
INFLAMAGING AND IT MAY ALSO BE

471
00:15:43,640 --> 00:15:45,080
THAT CHRONIC STIMULATION OF

472
00:15:45,080 --> 00:15:47,240
THINGS LIKE MYELOID IN THE BRAIN

473
00:15:47,240 --> 00:15:48,320
CAUSE MICROGLIAL CELLS TO MOVE

474
00:15:48,320 --> 00:15:49,960
FROM THIS MORE PROTECTIVE STATE

475
00:15:49,960 --> 00:15:53,520
TO A MORE INFLAMMATORY STATE

476
00:15:53,520 --> 00:15:55,560
THAT CONTRIBUTES TO DISEASES

477
00:15:55,560 --> 00:15:56,000
LIKE ALZHEIMER'S.

478
00:15:56,000 --> 00:15:58,200
SO THIS KIND OF LIGHT IMAGING

479
00:15:58,200 --> 00:15:59,520
HAS BEEN VERY VALUABLE LETTING

480
00:15:59,520 --> 00:16:00,520
US SEE THINGS YOU COULD NOT

481
00:16:00,520 --> 00:16:01,560
POSSIBLY SEE WITH EXTRACTED

482
00:16:01,560 --> 00:16:03,440
CELLS IN IN VITRO CONDITIONS OR

483
00:16:03,440 --> 00:16:05,400
BY SINGLE CELL ANALYSIS, AND

484
00:16:05,400 --> 00:16:08,600
WE'VE USED TO LOOK AT DYNAMICS

485
00:16:08,600 --> 00:16:11,520
IN LYMPH NODES TO DISCOVER

486
00:16:11,520 --> 00:16:15,120
T-CELLS MOVE ON FIBROBLASTIC

487
00:16:15,120 --> 00:16:16,800
RETICULAR CELL LOADS TO FIND

488
00:16:16,800 --> 00:16:18,120
DENDRITIC CELLS WITHIN THE

489
00:16:18,120 --> 00:16:19,960
T-CELL ZONE, TO LOOK AT DYNAMICS

490
00:16:19,960 --> 00:16:21,600
IN GERMINAL CENTERS THAT ARE

491
00:16:21,600 --> 00:16:24,440
MAKING ANTIBODIES TO LOOK AT

492
00:16:24,440 --> 00:16:25,880
DENDRITIC CELLS REACHING INTO

493
00:16:25,880 --> 00:16:29,480
THE GUT LUMEN TO SAMPLE THE

494
00:16:29,480 --> 00:16:31,200
MICRO BIOTA, WORK THAT JACKSON

495
00:16:31,200 --> 00:16:32,480
EGAN DID LOOKING AT FUNCTION IN

496
00:16:32,480 --> 00:16:34,960
THE KIDNEY, AND ALSO WORK THAT

497
00:16:34,960 --> 00:16:38,440
HE'S DONE WITH MYCOBACTERIAL

498
00:16:38,440 --> 00:16:39,520
GRANULOMAS AT BOTH THE MYELOID

499
00:16:39,520 --> 00:16:41,720
CELL AND LYMPHOCYTE LEVEL, TO

500
00:16:41,720 --> 00:16:45,560
LOOK IN THE LUNG AS PARAS AD HAS

501
00:16:45,560 --> 00:16:47,040
DONE AND INFLAMMATION RESPONSE

502
00:16:47,040 --> 00:16:49,360
TO INFLUENZA OR TUBERCULOSIS

503
00:16:49,360 --> 00:16:51,480
INFECTIONS, TO LOOK AT THESE

504
00:16:51,480 --> 00:16:55,720
RAPID SWARMING RESPONSES IN

505
00:16:55,720 --> 00:16:57,120
NEUTROPHILS IN THE EAR BUT ALSO

506
00:16:57,120 --> 00:17:00,880
T-CELLS RESPONDING TO

507
00:17:00,880 --> 00:17:02,520
HYPERSENSITIVITY INDUCERS, AND

508
00:17:02,520 --> 00:17:04,920
AS ISHI DID IN THE LAB, TO LOOK

509
00:17:04,920 --> 00:17:08,840
IN THE BONE MARROW AT

510
00:17:08,840 --> 00:17:09,640
OSTEOCLASTOGENESIS.

511
00:17:09,640 --> 00:17:10,480
IT'S BEEN APPLIED IN MANY

512
00:17:10,480 --> 00:17:11,280
DIFFERENT TISSUES.

513
00:17:11,280 --> 00:17:15,120
I WANT TO GIVE YOU A SAMPLE OF

514
00:17:15,120 --> 00:17:16,080
THE WAY WE'VE USED IT OVER THE

515
00:17:16,080 --> 00:17:17,400
YEARS BUT MANY LABORATORIES HAVE

516
00:17:17,400 --> 00:17:18,720
USED THIS IN THEIR OWN WAYS.

517
00:17:18,720 --> 00:17:22,760
THE PROBLEM IS THAT HERE WE SEE

518
00:17:22,760 --> 00:17:27,560
IN BLUE A BILOBAR MOUSE LYMPH

519
00:17:27,560 --> 00:17:29,640
NODE AND WE ONLY IMAGED THIS

520
00:17:29,640 --> 00:17:30,080
SMALL VOLUME.

521
00:17:30,080 --> 00:17:32,280
SO WE HAD A LOT IN THE BLACK.

522
00:17:32,280 --> 00:17:33,360
I TOLD YOU YOU NEED TO WORRY

523
00:17:33,360 --> 00:17:34,680
ABOUT WHAT'S IN THE BLACK, AND

524
00:17:34,680 --> 00:17:37,920
SO I ENCOURAGED THE PEOPLE IN

525
00:17:37,920 --> 00:17:39,600
THE LAB TO DO SOME HISTOLOGY TO

526
00:17:39,600 --> 00:17:41,120
SECTION THE LYMPH NODES TO SEE

527
00:17:41,120 --> 00:17:43,720
WHAT WE WEREN'T SEEING IN OUR

528
00:17:43,720 --> 00:17:44,600
TWO PHOTON IMAGING.

529
00:17:44,600 --> 00:17:45,600
THE PROBLEM WAS, WHEN WE WENT TO

530
00:17:45,600 --> 00:17:47,360
DO THIS, WE REALIZED THAT THE

531
00:17:47,360 --> 00:17:49,320
METHODS FOR DOING STAINING WERE

532
00:17:49,320 --> 00:17:50,640
TWO OR THREE COLOR.

533
00:17:50,640 --> 00:17:51,840
TYPICALLY YOU DID A RED AND A

534
00:17:51,840 --> 00:17:53,440
GREEN STAIN, MAYBE YOU LOOKED

535
00:17:53,440 --> 00:17:55,360
FOR YELLOW TO SEE IF THEY WERE

536
00:17:55,360 --> 00:17:56,040
COINCIDENT, BUT MOST OF THE

537
00:17:56,040 --> 00:17:57,680
PEOPLE IN AN IMMUNOLOGY LAB BY

538
00:17:57,680 --> 00:17:59,720
THIS TIME WERE DOING FLOW

539
00:17:59,720 --> 00:18:01,120
CYTOMETRY EXPERIMENTS BETWEEN

540
00:18:01,120 --> 00:18:02,680
9 AND 15 PARAMETERS, BECAUSE IT

541
00:18:02,680 --> 00:18:04,520
TOOK TWO TO FOUR PARAMETERS TO

542
00:18:04,520 --> 00:18:06,720
IDENTIFY ANY GIVEN SUBSET OF

543
00:18:06,720 --> 00:18:08,240
CELLS, SO TWO COLORS OBVIOUSLY

544
00:18:08,240 --> 00:18:10,440
DIDN'T CUT IT.

545
00:18:10,440 --> 00:18:14,800
AND SO WE AT T DECIDED WE NEEDEO

546
00:18:14,800 --> 00:18:16,960
MOVE TO MULTIPLEX STATIC IMAGING

547
00:18:16,960 --> 00:18:19,320
AND HERE WE'RE NOT GOING TO GET

548
00:18:19,320 --> 00:18:20,480
THOSE REALTIME DYNAMICS BUT NOW

549
00:18:20,480 --> 00:18:23,240
THE PARAMETERS INSTEAD BEING

550
00:18:23,240 --> 00:18:24,600
LIMITED TO FIVE IS EASILY 30 TO

551
00:18:24,600 --> 00:18:27,840
40 AND WE CAN PUSH IT ABOVE 80.

552
00:18:27,840 --> 00:18:30,480
WE CAN LOOK AT SIGNALING AND

553
00:18:30,480 --> 00:18:31,440
POST TRANSLATIONAL

554
00:18:31,440 --> 00:18:34,280
MODIFICATIONS, WE CAN LOOK AT

555
00:18:34,280 --> 00:18:35,520
PROTEINS AND RNA, AND OBVIOUSLY

556
00:18:35,520 --> 00:18:42,520
WE CAN LOOK AT HUMAN MATERIAL.

557
00:18:42,520 --> 00:18:44,520
WE STARTED WITH HISTOCYTOMETRY

558
00:18:44,520 --> 00:18:47,200
MORE THAN A DECADE AGO, MIKE HAS

559
00:18:47,200 --> 00:18:48,280
HIS OWN LAB AT THE UNIVERSITY OF

560
00:18:48,280 --> 00:18:49,600
WASHINGTON NOW FOR A NUMBER OF

561
00:18:49,600 --> 00:18:50,480
YEARS.

562
00:18:50,480 --> 00:18:53,000
AND THE WAY THIS WORKED IS WE'VE

563
00:18:53,000 --> 00:18:58,160
STAINED IN MULTIPLE -- WITH

564
00:18:58,160 --> 00:19:01,200
MULTIPLE FLOOR CHROME

565
00:19:01,200 --> 00:19:02,920
CONGREGATED ANTIBODIES, WE MIGHT

566
00:19:02,920 --> 00:19:03,760
DECONVOLUTE TO IMPROVE THE

567
00:19:03,760 --> 00:19:05,240
QUALITY OF THE IMAGES A LITTLE

568
00:19:05,240 --> 00:19:06,680
BIT, WE COMPENSATE FOR ANY

569
00:19:06,680 --> 00:19:07,680
CHANNEL SPILLOVER LIKE YOU WOULD

570
00:19:07,680 --> 00:19:09,280
DO IN FLOW, USE SOFTWARE TOOLS

571
00:19:09,280 --> 00:19:11,800
TO SEGMENT OBJECTS WITH THEIR

572
00:19:11,800 --> 00:19:13,960
ASSOCIATED FLUORESCENCE, BUT

573
00:19:13,960 --> 00:19:18,680
THOSE DATA ARE EQUIVALENT TO

574
00:19:18,680 --> 00:19:19,800
CELLULAR OBJECTS WITH

575
00:19:19,800 --> 00:19:20,960
FLUORESCENCE, SO WE COULD THEN

576
00:19:20,960 --> 00:19:22,960
PUT THAT INFORMATION INTO FLOW

577
00:19:22,960 --> 00:19:24,720
CYTOMETRY SOFTWARE AND GET

578
00:19:24,720 --> 00:19:27,320
QUANTITATIVE DATA WHERE WE'RE

579
00:19:27,320 --> 00:19:28,880
GAINING A MULTIPLEX EXPRESSION

580
00:19:28,880 --> 00:19:30,400
OF MARKER SETS, BUT SINCE WE

581
00:19:30,400 --> 00:19:33,040
KEPT THE XYZ COORDINATES OF

582
00:19:33,040 --> 00:19:37,120
ANYTHING WE MIGHT GAIN, GATE, WD

583
00:19:37,120 --> 00:19:38,160
IDENTIFY WHERE THOSE CELLS

584
00:19:38,160 --> 00:19:39,680
RESIDED IN THE ORIGINAL TISSUE.

585
00:19:39,680 --> 00:19:41,880
THIS METHOD WORKS NOT ONLY WITH

586
00:19:41,880 --> 00:19:43,520
ANTI -- SO CALLED MARKER

587
00:19:43,520 --> 00:19:45,160
ANTIBODY, MEANING MOSTLY CELL

588
00:19:45,160 --> 00:19:47,560
SURFACE PROTEINS BUT ALSO

589
00:19:47,560 --> 00:19:49,320
ANTIPHOSPHOPROTEIN ANTIBODIES

590
00:19:49,320 --> 00:19:51,520
AND SOME ANTICYTOKINE ANTIBODIES

591
00:19:51,520 --> 00:19:52,920
AND IT CAN RESOLVE THE

592
00:19:52,920 --> 00:19:53,920
DIFFERENCE BETWEEN LOCALIZATION

593
00:19:53,920 --> 00:19:56,640
OF TRANSCRIPTION FACTORS IN THE

594
00:19:56,640 --> 00:20:00,480
CYTOPLASM VERSUS NUCLEUS SO THEN

595
00:20:00,480 --> 00:20:02,960
IT MEANS WE CAN LOOK AT CELL

596
00:20:02,960 --> 00:20:04,200
STATE, CELL FUNCTION AND THE

597
00:20:04,200 --> 00:20:05,920
PHENOTYPE ALL IN THE CONTEXT OF

598
00:20:05,920 --> 00:20:06,720
SPATIAL POSITIONING IN THE

599
00:20:06,720 --> 00:20:07,320
TISSUE.

600
00:20:07,320 --> 00:20:08,440
THIS IS INFORMATION YOU CANNOT

601
00:20:08,440 --> 00:20:10,960
GET EVEN FROM MODERN SPATIAL

602
00:20:10,960 --> 00:20:12,160
TRANSCRIPTOMIC METHODS THAT ONLY

603
00:20:12,160 --> 00:20:14,320
LOOK AT THE RNA, THE SIGNALING

604
00:20:14,320 --> 00:20:15,680
COMPONENTS, FOR EXAMPLE, ARE

605
00:20:15,680 --> 00:20:16,360
INVISIBLE UNDER THOSE

606
00:20:16,360 --> 00:20:17,760
CONDITIONS.

607
00:20:17,760 --> 00:20:19,320
AND SO I'M JUST GOING TO

608
00:20:19,320 --> 00:20:22,120
SUMMARIZE IN SOME WORDS HERE

609
00:20:22,120 --> 00:20:24,960
COMBINING SOME OF THE EARLY

610
00:20:24,960 --> 00:20:27,440
2 PHOTON IMAGE WITH WHAT FLOW

611
00:20:27,440 --> 00:20:29,240
CYTOMETRY HAS TAUGHT US AND

612
00:20:29,240 --> 00:20:30,440
THERE ARE TWO VERY IMPORTANT

613
00:20:30,440 --> 00:20:30,680
LESSONS.

614
00:20:30,680 --> 00:20:34,320
ONE IS THERE'S A VERY POLARIZED

615
00:20:34,320 --> 00:20:35,920
DISTRIBUTION AT BARRIER SITES

616
00:20:35,920 --> 00:20:37,440
THAT ENABLES THE SYSTEM TO SORT

617
00:20:37,440 --> 00:20:38,440
OF HEAD OFF THE INNOVATION OF

618
00:20:38,440 --> 00:20:42,040
THE INFECTIOUS AGENT BEFORE IT

619
00:20:42,040 --> 00:20:43,720
GETS INTO THE SYSTEMIC

620
00:20:43,720 --> 00:20:44,680
CIRCULATION AND THAT'S

621
00:20:44,680 --> 00:20:45,200
CRITICALLY IMPORTANT.

622
00:20:45,200 --> 00:20:46,720
THE OTHER IS FOR THE ADAPTIVE

623
00:20:46,720 --> 00:20:48,360
IMMUNE SYSTEM, THESE CELLS ARE

624
00:20:48,360 --> 00:20:50,680
NOT JUST GENERALLY CO-LOCALIZED

625
00:20:50,680 --> 00:20:52,720
IN A TISSUE AND THEN RANDOMLY

626
00:20:52,720 --> 00:20:53,720
DISPERSED OR MOVING AROUND

627
00:20:53,720 --> 00:20:54,840
LOOKING FOR EACH OTHER.

628
00:20:54,840 --> 00:20:56,920
BUT THEY ACTUALLY ARE VERY, VERY

629
00:20:56,920 --> 00:20:58,880
PRECISELY ORGANIZED TO

630
00:20:58,880 --> 00:21:00,960
FACILITATE THE RARE INTERACTIONS

631
00:21:00,960 --> 00:21:02,800
YOU NEED TO DRIVE T OR B CELL

632
00:21:02,800 --> 00:21:03,240
IMMUNITY.

633
00:21:03,240 --> 00:21:04,800
AND SO I'M GOING TO TELL YOU ONE

634
00:21:04,800 --> 00:21:07,600
STORY THAT GOES WITH THE FIRST

635
00:21:07,600 --> 00:21:08,600
OF THOSE TWO MAJOR CONCLUSIONS,

636
00:21:08,600 --> 00:21:09,920
WHICH IS VERY RECENT WORK THAT

637
00:21:09,920 --> 00:21:12,640
WAS DONE IN THE LAB BY ANITA

638
00:21:12,640 --> 00:21:15,640
GOLA WHO'S NOW WORKING WITH

639
00:21:15,640 --> 00:21:17,360
ELAINE FUCHS AT ROCKEFELLER AND

640
00:21:17,360 --> 00:21:18,680
THIS HAS TO DO WITH THE LIVER.

641
00:21:18,680 --> 00:21:19,880
THE LIVER HAS BEEN UNUSUAL IN

642
00:21:19,880 --> 00:21:21,360
THAT IT HAS TWO CIRCULATIONS.

643
00:21:21,360 --> 00:21:24,040
IT HAS THE PORTAL CIRCULATION,

644
00:21:24,040 --> 00:21:25,080
BRINGING ESSENTIALLY NUTRIENTS

645
00:21:25,080 --> 00:21:26,880
FROM THE GUT INTO THE LIVER, AND

646
00:21:26,880 --> 00:21:28,520
THEN IT'S GOT THE NORMAL

647
00:21:28,520 --> 00:21:29,840
CIRCULATION, THERE'S A HEPATIC

648
00:21:29,840 --> 00:21:32,240
ARTERY AND THEN BOTH OF THESE

649
00:21:32,240 --> 00:21:33,760
INPUTS THEN FLOW OUT IN THE

650
00:21:33,760 --> 00:21:35,960
HEPATIC VEIN.

651
00:21:35,960 --> 00:21:39,600
IF WE SCALE IN, WE CAN SEE THAT

652
00:21:39,600 --> 00:21:41,400
A LIVER LOBULE IS SET UP WHERE

653
00:21:41,400 --> 00:21:42,960
THERE'S CALLED A PORTAL TRIAD

654
00:21:42,960 --> 00:21:45,360
THAT HAS THE PORTAL VEIN, THE

655
00:21:45,360 --> 00:21:47,840
ARTERY, AND A BILE DUCT, AND THE

656
00:21:47,840 --> 00:21:50,640
FLOW IS TOWARD THE CENTRAL VEIN

657
00:21:50,640 --> 00:21:51,920
THROUGH SINUSOIDS THAT ALIGNED

658
00:21:51,920 --> 00:21:54,240
BY A NUMBER OF LYMPHOID

659
00:21:54,240 --> 00:21:56,400
COMPARTMENTS MOSTLY NK AND NK

660
00:21:56,400 --> 00:21:59,120
T-CELLS, AND THESE MYELOID CELLS

661
00:21:59,120 --> 00:22:01,440
THAT ARE MEMBERS OF THE

662
00:22:01,440 --> 00:22:02,720
MACROPHAGE LINEAGE, AND THEN YOU

663
00:22:02,720 --> 00:22:04,840
HAVE THE ENDOTHELIAL CELLS

664
00:22:04,840 --> 00:22:07,120
FORMING THE EDGES OF THE

665
00:22:07,120 --> 00:22:08,120
SINUSOIDS.

666
00:22:08,120 --> 00:22:10,160
AND IT'S WELL-KNOWN THAT THE

667
00:22:10,160 --> 00:22:12,920
LIVER IS METABOLICALLY ZONE

668
00:22:12,920 --> 00:22:13,360
ATED.

669
00:22:13,360 --> 00:22:14,800
THERE ARE DIFFERENT THINGS THAT

670
00:22:14,800 --> 00:22:21,960
HAPPEN TOWARD THE PER PERIPORTAL

671
00:22:21,960 --> 00:22:23,760
REGION.

672
00:22:23,760 --> 00:22:25,160
THEY SAID THAT'S NOT TRUE OF THE

673
00:22:25,160 --> 00:22:25,920
IMMUNE SYSTEM.

674
00:22:25,920 --> 00:22:28,720
BASICALLY THE T-CELLS WANDER IN

675
00:22:28,720 --> 00:22:33,080
A RANDOM WAY SURVEYING WHAT'S

676
00:22:33,080 --> 00:22:35,240
NEAR GOING ON IN THE SINUSOIDS

677
00:22:35,240 --> 00:22:36,320
AND THE DISPLAY OF THOSE OTHER

678
00:22:36,320 --> 00:22:39,080
CELLS I SHOWED YOU, THEY'RE JUST

679
00:22:39,080 --> 00:22:41,680
DISTRIBUTED ALONG THE SINUSOIDAL

680
00:22:41,680 --> 00:22:42,000
WALLS.

681
00:22:42,000 --> 00:22:44,760
SO GOING BACK TO THE 2 PHOTON

682
00:22:44,760 --> 00:22:46,960
IMAGING, THE BEHAVIOR OF NK

683
00:22:46,960 --> 00:22:48,680
T-CELLS IN THE LIVER, WHICH WAS

684
00:22:48,680 --> 00:22:51,760
ORIGINALLY DESCRIBED AS RANDOM

685
00:22:51,760 --> 00:22:52,720
BEHAVIOR, BUT IF YOU LOOK AT

686
00:22:52,720 --> 00:22:54,600
THESE TRACKS, UR SEE THEY'RE

687
00:22:54,600 --> 00:22:57,000
CONCENTRATED HERE NEAR THE E CAD

688
00:22:57,000 --> 00:22:58,760
ENRICHED STAINING AREAS, AND

689
00:22:58,760 --> 00:23:01,800
THESE CONSTITUTE THE PERIPORTAL

690
00:23:01,800 --> 00:23:03,120
REGION SO THERE'S NOT RANDOM

691
00:23:03,120 --> 00:23:03,840
MOTION.

692
00:23:03,840 --> 00:23:06,720
THE CELLS MOVE OUT AWAY FROM THE

693
00:23:06,720 --> 00:23:08,360
PERIPORTAL REGION BUT ALSO RUN

694
00:23:08,360 --> 00:23:10,800
BACK ALMOST LIKE A YOYO AND

695
00:23:10,800 --> 00:23:14,280
SPEND MOST OF THEIR TIME IN THE

696
00:23:14,280 --> 00:23:14,840
PERIPORTAL REGION.

697
00:23:14,840 --> 00:23:18,560
IF WITH YOU THEN LOOK AT THE

698
00:23:18,560 --> 00:23:21,600
KEUPFER CELLS, WHEN I ENHANCE

699
00:23:21,600 --> 00:23:27,280
THE STAINING YOU SEE ALMOST ALL

700
00:23:27,280 --> 00:23:29,720
THESE RED CELLS ARE NOT IN THE

701
00:23:29,720 --> 00:23:31,760
CENTRAL REGION, WHICH IS WHERE

702
00:23:31,760 --> 00:23:33,320
THE CENTRAL FEIGN IS.

703
00:23:33,320 --> 00:23:37,960
THERVEIN S THERE'S AVERY STRICTS

704
00:23:37,960 --> 00:23:38,560
DISTRIBUTION.

705
00:23:38,560 --> 00:23:39,680
I'LL JUST SKIP OVER SOME OF THIS

706
00:23:39,680 --> 00:23:42,080
MORE QUANTITATIVE DATA.

707
00:23:42,080 --> 00:23:45,040
WELL, IS THAT, IN ESSENCE,

708
00:23:45,040 --> 00:23:47,440
INBORN IN THE SAME WAY THE

709
00:23:47,440 --> 00:23:48,280
METABOLIC ZONATION IS?

710
00:23:48,280 --> 00:23:49,160
AND THE ANSWER IS NO.

711
00:23:49,160 --> 00:23:51,920
IF WE LOOK AT A NEWBORN, WE

712
00:23:51,920 --> 00:23:53,120
DON'T SEE THAT ZONATION AT ALL

713
00:23:53,120 --> 00:23:55,840
FOR THE KUPFFER CELLS, AND THEN

714
00:23:55,840 --> 00:23:59,160
AS THE ANIMAL AGES, WE SEE A

715
00:23:59,160 --> 00:24:00,880
MAJOR TRANSITION AT AROUND DAY

716
00:24:00,880 --> 00:24:01,040
20.

717
00:24:01,040 --> 00:24:03,080
WELL, DAY 20 TURNS OUT TO BE

718
00:24:03,080 --> 00:24:05,080
WHEN YOU WEAN THE MICE AND THEY

719
00:24:05,080 --> 00:24:08,760
MARKETLY CHANGE THEIR

720
00:24:08,760 --> 00:24:09,640
MICROBIOTA.

721
00:24:09,640 --> 00:24:12,240
SO WE ASKED DOES IT MATTER, IF

722
00:24:12,240 --> 00:24:13,920
YOU LOOK IN A GERM-FREE MOUSE,

723
00:24:13,920 --> 00:24:15,000
NOW YOU DON'T SEE THE

724
00:24:15,000 --> 00:24:15,320
POLARIZATION.

725
00:24:15,320 --> 00:24:18,520
IF YOU LOOK IN GERM-FREE ANIMALS

726
00:24:18,520 --> 00:24:23,120
THAT HAVE BEEN CO-HOUSED, THEIR

727
00:24:23,120 --> 00:24:24,520
KUPFFER CELLS POLARIZE IN THEIR

728
00:24:24,520 --> 00:24:26,920
DISTRIBUTION, AND IF YOU TAKE AN

729
00:24:26,920 --> 00:24:28,360
SPF MOUSE AND TREAT IT WITH

730
00:24:28,360 --> 00:24:31,200
ANTIBIOTICS, THEY LOSE THE

731
00:24:31,200 --> 00:24:31,520
POLARIZATION.

732
00:24:31,520 --> 00:24:34,920
SO ALL OF THIS TOLD US IT'S THE

733
00:24:34,920 --> 00:24:37,280
MICROBIOTA AND SIGNALS FROM THE

734
00:24:37,280 --> 00:24:38,320
MICROBIOTA THAT MATTER, THIS IS

735
00:24:38,320 --> 00:24:40,080
NOT SOMETHING BUILT INTO THE

736
00:24:40,080 --> 00:24:44,000
SYSTEM ON THE LOGICALLY.

737
00:24:44,000 --> 00:24:47,520
SYSTEM ON TOTO LOGICALLY.

738
00:24:47,520 --> 00:24:53,280
IF WE GET RID OF A MAJOR PROTEIN

739
00:24:53,280 --> 00:24:55,040
THROUGH TOE-LIKE RECEPTORS AS

740
00:24:55,040 --> 00:24:58,640
WELL AS FOR IL-1 -- THEN WE LOSE

741
00:24:58,640 --> 00:24:59,960
THIS ASYMMETRY AND WE COULD SHOW

742
00:24:59,960 --> 00:25:03,560
THAT KNOCKING OUT THINGS LIKE

743
00:25:03,560 --> 00:25:05,000
TLR4 HAD PARTIAL EFFECT SO WE'RE

744
00:25:05,000 --> 00:25:06,240
QUITE CONFIDENT THAT THIS IS

745
00:25:06,240 --> 00:25:08,360
WHAT'S CALLED PAMP SIGNALING.

746
00:25:08,360 --> 00:25:10,640
THAT S RESPONSE TO THINGS LIKE

747
00:25:10,640 --> 00:25:11,920
ENDO TOXIN THAT ARE RELEASED

748
00:25:11,920 --> 00:25:14,480
FROM THE GUT MICROFLORA AND

749
00:25:14,480 --> 00:25:16,040
HIGHLY ENRICHED IN THE PORTAL

750
00:25:16,040 --> 00:25:16,800
CIRCULATION BEING CARRIED TO THE

751
00:25:16,800 --> 00:25:18,640
LIVER AND ENTERING IN THAT

752
00:25:18,640 --> 00:25:20,520
PORTAL TRIAD REGION, THAT'S THE

753
00:25:20,520 --> 00:25:22,160
HIGHEST CONCENTRATION, AND THEN

754
00:25:22,160 --> 00:25:26,200
FLOWING OUT.

755
00:25:26,200 --> 00:25:27,760
WHAT WE THEN SHOWED IS THEY

756
00:25:27,760 --> 00:25:30,360
DON'T TALK DIRECTLY TO THE CUP

757
00:25:30,360 --> 00:25:32,200
FER CELLS, THEY INSTRUCT THE

758
00:25:32,200 --> 00:25:34,800
CELLS NOT FOR HOW MUCH CHEMOKINE

759
00:25:34,800 --> 00:25:37,480
THEY MAKE THAT GUIDE THE KUPFFER

760
00:25:37,480 --> 00:25:39,560
CELLS BUT HOW THEY RECONSTRUCT

761
00:25:39,560 --> 00:25:42,160
THE EXTRACELLULAR MATRIX OF

762
00:25:42,160 --> 00:25:43,680
AMINO GLYCANS TO WHICH THE

763
00:25:43,680 --> 00:25:44,680
CHEMOKINES BIND.

764
00:25:44,680 --> 00:25:47,720
SO THE GRADIENT CONTROL IS BASED

765
00:25:47,720 --> 00:25:49,080
ON EXTRACELLULAR MATRIX

766
00:25:49,080 --> 00:25:54,120
REMODELING THAT'S RESPONSIVE TO

767
00:25:54,120 --> 00:25:57,600
THESE SIGNALS.

768
00:25:57,600 --> 00:26:01,040
WHY IS THE SYSTEM POLARIZED THIS

769
00:26:01,040 --> 00:26:01,560
WAY?

770
00:26:01,560 --> 00:26:05,320
IN GREEN ARE THE KUPFFER CELLS.

771
00:26:05,320 --> 00:26:06,600
IN WHITE ARE GOING TO BE

772
00:26:06,600 --> 00:26:10,160
BACTERIA THAT ARE PUT INTO THE

773
00:26:10,160 --> 00:26:13,480
VASCULAR TREE.

774
00:26:13,480 --> 00:26:15,040
WHAT YOU SEE IS WHETHER THOSE

775
00:26:15,040 --> 00:26:17,200
BACTERIA HIT THE LIVER, THEY

776
00:26:17,200 --> 00:26:20,640
STICK DIRECTLY TO THE KUFFER

777
00:26:20,640 --> 00:26:21,320
CELLS.

778
00:26:21,320 --> 00:26:23,000
WE CREATED A MATHEMATICAL MODEL

779
00:26:23,000 --> 00:26:25,520
THAT SAYS IF WE MAKE THEM ALL

780
00:26:25,520 --> 00:26:26,920
EQUALLY STICKY TO BACTERIA

781
00:26:26,920 --> 00:26:27,720
BECAUSE THAT'S SOMETHING WE

782
00:26:27,720 --> 00:26:29,800
CAN'T EASILY CONTROL IF THEY DO

783
00:26:29,800 --> 00:26:32,320
VARY IN THEIR STICKINESS ACROSS

784
00:26:32,320 --> 00:26:33,640
THE SIGN SOY DAL DISTRIBUTION,

785
00:26:33,640 --> 00:26:34,960
WHAT HAPPENS IF WE HAVE THEM

786
00:26:34,960 --> 00:26:38,000
POLAR OIZED VERSUS

787
00:26:38,000 --> 00:26:38,360
NON-POLARIZED?

788
00:26:38,360 --> 00:26:39,760
THE ANSWER IS WHEN THEY'RE

789
00:26:39,760 --> 00:26:41,800
HIGHLY POLARIZED, THE BACTERIA

790
00:26:41,800 --> 00:26:44,440
ARE CAPTURED VERY CLOSE TO THE

791
00:26:44,440 --> 00:26:45,440
PERIPORTAL REGION AND VERY FEW

792
00:26:45,440 --> 00:26:47,200
REACH THE CENTRAL VEIN, MEANING

793
00:26:47,200 --> 00:26:48,640
GOING INTO THE GENERAL

794
00:26:48,640 --> 00:26:48,960
CIRCULATION.

795
00:26:48,960 --> 00:26:53,520
BUT IF WE MAKE THEM UNIFORMLY

796
00:26:53,520 --> 00:26:55,080
DISTRIBUTED, NOW LOTS OF THE

797
00:26:55,080 --> 00:26:56,400
BACTERIA MAKE TO THE CENTRAL

798
00:26:56,400 --> 00:26:57,800
VEIN AND GO INTO THE

799
00:26:57,800 --> 00:26:58,120
CIRCULATION.

800
00:26:58,120 --> 00:27:02,720
THIS WAS A MODEL, BUT ANITA DID

801
00:27:02,720 --> 00:27:04,800
A HEROIC EXPERIMENT OF INJECTING

802
00:27:04,800 --> 00:27:05,440
BACTERIA DIRECTLY INTO THE

803
00:27:05,440 --> 00:27:06,520
PORTAL VEIN AND WHEN YOU DO

804
00:27:06,520 --> 00:27:07,920
THAT, YOU GET EXACTLY THAT

805
00:27:07,920 --> 00:27:08,160
RESULT.

806
00:27:08,160 --> 00:27:11,520
IN ANIMALS WITH THE POLARIZED

807
00:27:11,520 --> 00:27:13,320
KUPFFER CELLS, MOST OF THEM ARE

808
00:27:13,320 --> 00:27:15,440
TRAPPED VERY CLOSE TO THE

809
00:27:15,440 --> 00:27:17,000
PERIPORTAL REGION, VERY FEW

810
00:27:17,000 --> 00:27:18,080
REACH THE SPLEEN OR THE BLOOD

811
00:27:18,080 --> 00:27:23,440
HERE IN BLACK, BUT IF YOU USE A

812
00:27:23,440 --> 00:27:25,440
GERM-FREE ANIMAL WITH A

813
00:27:25,440 --> 00:27:27,920
NON-POLARIZED KUPFFER

814
00:27:27,920 --> 00:27:31,440
DISTRIBUTION, NOW YOU SEE -- OR

815
00:27:31,440 --> 00:27:32,000
PRESENT IN THE BLOOD.

816
00:27:32,000 --> 00:27:33,320
SO THAT GIVES US A MODEL FOR

817
00:27:33,320 --> 00:27:35,560
WHAT'S GOING ON HERE WHERE THIS

818
00:27:35,560 --> 00:27:38,200
POLARIZATION IS MEANT TO ENABLE

819
00:27:38,200 --> 00:27:40,520
THE SYSTEM TO MOST EFFICIENTLY

820
00:27:40,520 --> 00:27:43,160
CAPTURE ANYTHING THAT'S ESCAPING

821
00:27:43,160 --> 00:27:45,000
THE MUCUS AND EPITHELIAL

822
00:27:45,000 --> 00:27:46,000
BARRIERS IN THE GUT, GETTING

823
00:27:46,000 --> 00:27:51,800
INTO THE PORTAL CIRCULATION AND

824
00:27:51,800 --> 00:27:53,120
POTENTIALLY GOING SYSTEMIC.

825
00:27:53,120 --> 00:27:54,640
YOU NEED THAT POLARIZATION OF

826
00:27:54,640 --> 00:27:56,840
THE IMMUNE SYSTEM TO DO THAT

827
00:27:56,840 --> 00:27:57,360
EFFICIENTLY.

828
00:27:57,360 --> 00:27:58,800
ALTHOUGH IT'S NOT ABSOLUTE, WHEN

829
00:27:58,800 --> 00:28:00,640
YOU THINK ABOUT THESE BACTERIA

830
00:28:00,640 --> 00:28:02,400
CHRONICALLY COMING OUT OF THE

831
00:28:02,400 --> 00:28:03,600
GUT, IF YOU WEREN'T DOING THIS

832
00:28:03,600 --> 00:28:05,040
ALL OF THE TIME, YOU'D BE AT

833
00:28:05,040 --> 00:28:07,520
GREAT RISK FOR SYSTEMIC SEPTIC

834
00:28:07,520 --> 00:28:08,520
EVENTS.

835
00:28:08,520 --> 00:28:13,680
I THINK I'VE LOST MY CONTROL

836
00:28:13,680 --> 00:28:14,200
HERE.

837
00:28:14,200 --> 00:28:17,160
SO I'LL JUST SAY THAT THAT

838
00:28:17,160 --> 00:28:17,800
FRACTAL BIOLOGY AS I CALL IT

839
00:28:17,800 --> 00:28:19,600
ALSO APPLIES TO LYMPH NODE

840
00:28:19,600 --> 00:28:20,120
STRUCTURE AND FUNCTION.

841
00:28:20,120 --> 00:28:21,760
WE DON'T HAVE LYMPHOCYTES

842
00:28:21,760 --> 00:28:22,680
EVERYWHERE, I MEAN, THEY'RE IN

843
00:28:22,680 --> 00:28:23,720
THE BLOOD BUT THEY'RE NOT IN ALL

844
00:28:23,720 --> 00:28:25,480
THE TISSUES.

845
00:28:25,480 --> 00:28:27,880
THEY CONCENTRATE IN A LYMPH NODE

846
00:28:27,880 --> 00:28:28,760
OR SPLEEN.

847
00:28:28,760 --> 00:28:30,280
WITHIN, SAY, A LYMPH NODE, THEY

848
00:28:30,280 --> 00:28:33,280
SEPARATE INTO THOSE T AND B

849
00:28:33,280 --> 00:28:33,480
AREAS.

850
00:28:33,480 --> 00:28:35,960
IN THE T AREAS YOU HAVE THIS

851
00:28:35,960 --> 00:28:37,160
FIBROBLASTIC RETICULAR NETWORK.

852
00:28:37,160 --> 00:28:38,520
THE T-CELLS WALK ON THAT NETWORK

853
00:28:38,520 --> 00:28:39,920
AND THE REASON THEY DO THAT IS

854
00:28:39,920 --> 00:28:41,680
IT REDUCES THEIR SEARCH VOLUME

855
00:28:41,680 --> 00:28:43,920
LOOKING FOR DENDRITIC CELLS ON

856
00:28:43,920 --> 00:28:48,000
EXACTLY THAT SAME NETWORK.

857
00:28:48,000 --> 00:28:49,560
WITHIN THE CONTEXT OF THIS

858
00:28:49,560 --> 00:28:51,200
NETWORK CONCENTRATION, IF YOU

859
00:28:51,200 --> 00:28:53,480
GET AN ENGAGEMENT OF ONE T-CELL

860
00:28:53,480 --> 00:28:55,640
WITH A DENDRITIC CELL, IT MAKES

861
00:28:55,640 --> 00:28:57,840
CHEMOKINES THAT THEN BIAS THE

862
00:28:57,840 --> 00:28:59,480
WALK ON THE FIBER NETWORK TO

863
00:28:59,480 --> 00:29:00,960
DIRECT THOSE CELLS TO THE

864
00:29:00,960 --> 00:29:05,400
INTERESTING DENDRITIC CELL, AND

865
00:29:05,400 --> 00:29:06,400
THEN ANTONIO IN THE LAB SHOWED

866
00:29:06,400 --> 00:29:08,680
THERE'S A POLARIZATION MATCH

867
00:29:08,680 --> 00:29:10,000
BETWEEN DENDRITIC CELL SUBSETS

868
00:29:10,000 --> 00:29:12,520
AND T-CELL SUBSET, THEY'RE NOT

869
00:29:12,520 --> 00:29:13,720
RANDOMLY DISPERSED WITHIN THE

870
00:29:13,720 --> 00:29:15,680
T-CELL ZONE, AND THAT

871
00:29:15,680 --> 00:29:17,880
POLARIZATION IS CRITICAL TO THE

872
00:29:17,880 --> 00:29:20,280
RIGHT TIMING FOR ACTIVATING THE

873
00:29:20,280 --> 00:29:21,560
DIFFERENT SUBSETS OF

874
00:29:21,560 --> 00:29:23,120
LYMPHOCYTES, AND IF YOU DESTROY

875
00:29:23,120 --> 00:29:24,200
THAT POLARITY WITH NO OTHER

876
00:29:24,200 --> 00:29:27,160
CHANGE IN THE LYMPH NODE, NOW

877
00:29:27,160 --> 00:29:27,720
CD4 CELLS DON'T COOPERATE

878
00:29:27,720 --> 00:29:29,760
PROPERLY WITH CD8 CELLS TO MAKE

879
00:29:29,760 --> 00:29:32,080
GOOD MEMORY IN RESPONSE TO

880
00:29:32,080 --> 00:29:34,040
VACCINES, FOR EXAMPLE.

881
00:29:34,040 --> 00:29:36,280
SO IT'S THIS ORGANIZATIONAL

882
00:29:36,280 --> 00:29:38,000
LEVEL IS ABSOLUTELY CRITICAL TO

883
00:29:38,000 --> 00:29:40,080
ADAPTIVE IMMUNITY, AND IT BOTH

884
00:29:40,080 --> 00:29:42,160
STRUCTURAL ORGANIZATION AND

885
00:29:42,160 --> 00:29:43,880
CHEMICAL ORGANIZATION JUST AS I

886
00:29:43,880 --> 00:29:49,920
SHOWED YOU IN THE LIVER.

887
00:29:49,920 --> 00:29:52,160
SO WE'VE NOW APPLIED THIS TO

888
00:29:52,160 --> 00:29:54,200
CANCER BIOLOGY.

889
00:29:54,200 --> 00:29:56,520
VIVIAN MALTES IN THE LAB HAS

890
00:29:56,520 --> 00:29:58,480
WORKED TOGETHER WITH CAITLYN

891
00:29:58,480 --> 00:30:00,360
BERN INITIALLY AND NOW AT HER

892
00:30:00,360 --> 00:30:02,200
OWN LAB IN OREGON IN A MODEL IN

893
00:30:02,200 --> 00:30:05,040
WHICH IT'S A GERM -- GENETICALLY

894
00:30:05,040 --> 00:30:07,920
ENGINEERED MODEL OF PANCREATIC

895
00:30:07,920 --> 00:30:09,440
DUCTAL ADENOCARCINOMA, AND WHAT

896
00:30:09,440 --> 00:30:13,760
KATE DID WAS TO CLONE INDIVIDUAL

897
00:30:13,760 --> 00:30:15,920
CELLS FROM THOSE PRIMARY TUMORS,

898
00:30:15,920 --> 00:30:17,800
AND IT TURNED OUT THAT THEY

899
00:30:17,800 --> 00:30:18,880
DISTRIBUTED INTO TWO GROUPS,

900
00:30:18,880 --> 00:30:20,720
WHAT WE WOULD TYPICALLY THESE

901
00:30:20,720 --> 00:30:23,240
DAYS CALL COLD OR HOT TUMORS IN

902
00:30:23,240 --> 00:30:27,400
TERMS OF THE T-CELL INFILTRATION

903
00:30:27,400 --> 00:30:29,040
AND THE BIAS TOWARD SUPPRESSIVE

904
00:30:29,040 --> 00:30:29,520
MACROPHAGES.

905
00:30:29,520 --> 00:30:31,680
SO WHAT KATE AND VIVIAN DID WAS

906
00:30:31,680 --> 00:30:34,400
TO STUDY ANIMALS THAT ARE

907
00:30:34,400 --> 00:30:38,440
BEARING A HOT TUMOR WITH A

908
00:30:38,440 --> 00:30:40,200
TREATMENT THAT COMBINES

909
00:30:40,200 --> 00:30:43,280
CHECKPOINT THERAPY WITH AN

910
00:30:43,280 --> 00:30:46,480
AGONIST CD4 ANTIBODY, IN CURRENT

911
00:30:46,480 --> 00:30:47,280
CLINICAL TRIALS AND HIGHLY

912
00:30:47,280 --> 00:30:51,640
EFFECTIVE IN THIS ANIMAL MODEL.

913
00:30:51,640 --> 00:30:52,920
HERE THEY WERE LOOKING AND

914
00:30:52,920 --> 00:30:54,240
VIVIAN IN PARTICULAR WAS LOOKING

915
00:30:54,240 --> 00:30:55,560
VERY SHORTLY AFTER THE

916
00:30:55,560 --> 00:31:03,760
COMBINATION THAT INVOLVES THE

917
00:31:03,760 --> 00:31:04,480
ANTIHD1 ANTICTLA4 -- WHAT'S

918
00:31:04,480 --> 00:31:08,080
STRIKING WHEN THIS IS DONE,

919
00:31:08,080 --> 00:31:09,600
LOOKING HERE AT A DOT VERSION

920
00:31:09,600 --> 00:31:11,800
RATHER THAN THE FINE GRAIN

921
00:31:11,800 --> 00:31:14,080
PRIMARY ANALYSIS OF THE IMAGES

922
00:31:14,080 --> 00:31:18,480
FOR T-CELLS IS THAT THERE IS A

923
00:31:18,480 --> 00:31:21,080
MAJOR LOSS OF FOX P3 POSITIVE

924
00:31:21,080 --> 00:31:21,440
T-CELLS.

925
00:31:21,440 --> 00:31:23,480
SO WHAT ARE CALLED REGULATORY

926
00:31:23,480 --> 00:31:23,680
T-CELLS.

927
00:31:23,680 --> 00:31:26,760
AND THE FEW THAT REMAIN, AND YOU

928
00:31:26,760 --> 00:31:28,960
SEE HOW MUCH THIS REDUCTION IS

929
00:31:28,960 --> 00:31:30,280
HERE, ARE IN THE PERIPHERY.

930
00:31:30,280 --> 00:31:32,680
AND THAT'S GOING TO BE IMPORTANT

931
00:31:32,680 --> 00:31:34,640
WHEN WE DRAW SOME CONCLUSIONS A

932
00:31:34,640 --> 00:31:36,600
BIT LATER ON, AND THIS JUST

933
00:31:36,600 --> 00:31:40,440
QUANTIFIES THAT CHANGE IN

934
00:31:40,440 --> 00:31:41,240
DISTRIBUTION.

935
00:31:41,240 --> 00:31:43,600
NOW, IT SEEMED LIKE THAT WAS

936
00:31:43,600 --> 00:31:45,680
ASSOCIATED UNIQUELY WITH THE

937
00:31:45,680 --> 00:31:46,800
ANTICD40 AND THIS EXPERIMENT

938
00:31:46,800 --> 00:31:48,800
SHOWS THAT THAT'S TRUE IF YOU

939
00:31:48,800 --> 00:31:50,840
USE A CD40 KNOCKOUT ANIMAL, THIS

940
00:31:50,840 --> 00:31:53,880
TREATMENT DOES NOT DEPLETE THE

941
00:31:53,880 --> 00:31:57,080
REGULATORY T-CELLS, BUT THAT WAS

942
00:31:57,080 --> 00:31:58,640
VERY SURPRISING BECAUSE CD40 IS

943
00:31:58,640 --> 00:32:00,360
NOT EXPRESSED BY REGULATORY

944
00:32:00,360 --> 00:32:01,560
T-CELLS SO WHY WOULD THIS

945
00:32:01,560 --> 00:32:02,520
ANTIBODY GIVE YOU THIS

946
00:32:02,520 --> 00:32:03,320
PHENOTYPE?

947
00:32:03,320 --> 00:32:05,760
WELL, CD40 IS EXPRESSED BY

948
00:32:05,760 --> 00:32:06,840
DENDRITIC CELLS, AND IF YOU GET

949
00:32:06,840 --> 00:32:09,680
RID OF A PARTICULAR SUBSET OF

950
00:32:09,680 --> 00:32:11,200
DENDRITIC CELLS IN A

951
00:32:11,200 --> 00:32:13,280
TRANSCRIPTION FACTOR KNOCKOUT,

952
00:32:13,280 --> 00:32:14,240
NOW THIS TREATMENT DOES NOT

953
00:32:14,240 --> 00:32:19,840
CAUSE THE LOSS OF THE T REGS.

954
00:32:19,840 --> 00:32:21,040
SO THE QUESTION IS WHAT'S

955
00:32:21,040 --> 00:32:23,440
HAPPENING TO THE T REGS, AND WHY

956
00:32:23,440 --> 00:32:29,560
ARE DENDRITIC CELLS INVOLVED IN

957
00:32:29,560 --> 00:32:38,880
LOSING FOX P3 CELLS.

958
00:32:38,880 --> 00:32:40,520
IT LED US TO WONDER WHETHER

959
00:32:40,520 --> 00:32:43,160
THESE CELLS WERE LOSING FOX

960
00:32:43,160 --> 00:32:44,480
P3 EXPRESSION RATHER THAN BEING

961
00:32:44,480 --> 00:32:46,640
ELIMINATED AS T-CELLS ENTIRELY.

962
00:32:46,640 --> 00:32:48,640
TO LOOK AT THAT, WE'VE TURNED TO

963
00:32:48,640 --> 00:32:50,960
LINEAGE TRACKING, AND YOU DO

964
00:32:50,960 --> 00:32:53,600
THAT A AN ANIMAL IN WHICH YOU

965
00:32:53,600 --> 00:32:56,440
CAN GIVE TAMOXIFEN TO DO

966
00:32:56,440 --> 00:32:58,680
INDUCIBLE TURN YON OF THE ROSA

967
00:32:58,680 --> 00:33:01,120
TOMATO LOCUS SO YOU GET THE RED

968
00:33:01,120 --> 00:33:02,240
FLUORESCENT PROTEIN EXPRESSED IN

969
00:33:02,240 --> 00:33:06,040
CELLS THAT ARE EXPRESSING CRE

970
00:33:06,040 --> 00:33:08,360
UNDER THE CONTROL OF FOX P3, SO

971
00:33:08,360 --> 00:33:10,520
THE AUTHENTIC T REGS ARE GOING

972
00:33:10,520 --> 00:33:11,720
TO SHOW AS THIS TRACKING WHEN WE

973
00:33:11,720 --> 00:33:12,160
DO THAT.

974
00:33:12,160 --> 00:33:13,840
THEN YOU GO THROUGH THE REGULAR

975
00:33:13,840 --> 00:33:15,480
PROCEDURE, AND THE SURPRISING

976
00:33:15,480 --> 00:33:17,000
RESULT, THE VERY STRIKING RESULT

977
00:33:17,000 --> 00:33:19,000
IS AS YOU SEE HERE, WE GET THE

978
00:33:19,000 --> 00:33:20,520
ELIMINATION OF MOST OF THE T

979
00:33:20,520 --> 00:33:22,160
REGS AND THE RESIDUAL ONES BEING

980
00:33:22,160 --> 00:33:24,360
ON THE PERIPHERY OF THE TUMOR AS

981
00:33:24,360 --> 00:33:29,040
WE SAW BEFORE, BUT NOW WE GET A

982
00:33:29,040 --> 00:33:31,240
VERY LARGE POPULATION OF

983
00:33:31,240 --> 00:33:32,800
SO-CALLED XT REGS, CELLS THAT

984
00:33:32,800 --> 00:33:34,560
HAVE THE LINEAGE PARKER BUT NO

985
00:33:34,560 --> 00:33:39,280
LONGER HAVE FOX P3, THESE ARE

986
00:33:39,280 --> 00:33:40,480
CD4 POSITIVE CD3 POSITIVE

987
00:33:40,480 --> 00:33:41,880
T-CELLS AND THEY ALSO POPULATE

988
00:33:41,880 --> 00:33:42,440
THE PERIPHERY.

989
00:33:42,440 --> 00:33:45,800
AND THIS JUST QUANTIFIES THOSE

990
00:33:45,800 --> 00:33:46,600
CHANGES.

991
00:33:46,600 --> 00:33:49,640
AND IT WAS VERIFIED ALSO BY FLOW

992
00:33:49,640 --> 00:33:49,920
CYTOMETRY.

993
00:33:49,920 --> 00:33:52,600
SO HOW DOES THAT WORK?

994
00:33:52,600 --> 00:33:57,200
WELL, THE DENDRITIC CELLS THAT

995
00:33:57,200 --> 00:34:02,120
RELY ON BAT OF 3 ARE A MAJOR

996
00:34:02,120 --> 00:34:05,480
IL-12 PRODUCER AND ANTICD40

997
00:34:05,480 --> 00:34:07,440
INDUCES THE FORM OF IL12.

998
00:34:07,440 --> 00:34:10,920
SO KATE AND VIVIAN LOOKED AT

999
00:34:10,920 --> 00:34:13,200
IL-12 AND WHETHER THE

1000
00:34:13,200 --> 00:34:14,040
CYTOKINE -- AS SHOWN HERE, IF

1001
00:34:14,040 --> 00:34:16,920
YOU TREAT WITH EITHER ANTI-12 OR

1002
00:34:16,920 --> 00:34:18,280
ANTIINTERFERON GAMMA, YOU NO

1003
00:34:18,280 --> 00:34:20,680
LONGER LOSE THE MAJORITY OF THE

1004
00:34:20,680 --> 00:34:23,360
CONVENTIONAL FOX P3 T REGS AND

1005
00:34:23,360 --> 00:34:30,080
YOU DON'T MAKE NEARLY AS MANY OF

1006
00:34:30,080 --> 00:34:32,280
THESE EX--TREGS.

1007
00:34:32,280 --> 00:34:34,040
THERE'S THE CONVERSION AND LOSS

1008
00:34:34,040 --> 00:34:38,440
OF FOX P3 IS DEPENDENT ON

1009
00:34:38,440 --> 00:34:39,760
INTERFERON 12 AND INTERFERON

1010
00:34:39,760 --> 00:34:42,000
GAMMA.

1011
00:34:42,000 --> 00:34:43,880
INDUCE CELLS TO BECOME TH1s,

1012
00:34:43,880 --> 00:34:45,440
SO IS THIS HAPPENING AS THEY

1013
00:34:45,440 --> 00:34:47,400
LOSE FOX P3, SO HERE VIVIAN

1014
00:34:47,400 --> 00:34:49,720
TURNED TO ANOTHER TECHNOLOGY FOR

1015
00:34:49,720 --> 00:34:51,760
LOOKING AT TARGETS IN THE TISSUE

1016
00:34:51,760 --> 00:34:54,160
HERE, IT'S RNA FISH, AND I'LL

1017
00:34:54,160 --> 00:34:55,760
JUST SHOW YOU THE BLOWN-UP PART

1018
00:34:55,760 --> 00:35:00,000
WHICH SHOWS YOU THAT THESE

1019
00:35:00,000 --> 00:35:03,600
EX--TREGS ARE ACTIVELY MAKING

1020
00:35:03,600 --> 00:35:05,200
ANTIINTERFERON GAMMA MESSAGE,

1021
00:35:05,200 --> 00:35:06,800
THIS IS WITHOUT ANY

1022
00:35:06,800 --> 00:35:10,840
RE-STIMULATION.

1023
00:35:10,840 --> 00:35:14,040
SO WHAT'S HAPPENING HERE IS THAT

1024
00:35:14,040 --> 00:35:16,120
THE ANTICD40 ACTION PROBABLY

1025
00:35:16,120 --> 00:35:18,920
TOGETHER WITH SOME CONVENTIONAL

1026
00:35:18,920 --> 00:35:19,920
CD4 CELLS GIVES YOU A

1027
00:35:19,920 --> 00:35:21,800
COMBINATION OF IL-12 AND

1028
00:35:21,800 --> 00:35:23,000
INTERFERON GAMMA PRODUCTION

1029
00:35:23,000 --> 00:35:25,400
WHICH KOWS KAWS THE LOSS OF FOX

1030
00:35:25,400 --> 00:35:26,960
P3 EXPRESSION IN THE

1031
00:35:26,960 --> 00:35:27,840
CONVENTIONAL TREGS.

1032
00:35:27,840 --> 00:35:29,240
I DIDN'T SHOW YOU THIS BUT THEY

1033
00:35:29,240 --> 00:35:34,520
ALSO GAINED NT -- EXPRESSION AND

1034
00:35:34,520 --> 00:35:38,240
THEY ALSO MAKE -- IS CONSIDERED

1035
00:35:38,240 --> 00:35:41,120
A CANONICAL OUTCOME OF DOING

1036
00:35:41,120 --> 00:35:42,400
CHECKPOINT THERAPY, BUT IN THIS

1037
00:35:42,400 --> 00:35:43,920
MODEL, THE CHECKPOINT THERAPY

1038
00:35:43,920 --> 00:35:45,280
ALONE DOESN'T DO THAT, EVEN IN

1039
00:35:45,280 --> 00:35:46,280
THESE HOT TUMORS.

1040
00:35:46,280 --> 00:35:49,200
YOU NEED THE ANTI-CD40 AS WELL

1041
00:35:49,200 --> 00:35:51,920
TO GET THIS AT A HIGH LEVEL.

1042
00:35:51,920 --> 00:35:54,480
IN ADDITION, THE MOVEMENT OF ANY

1043
00:35:54,480 --> 00:35:56,120
RESIDUAL TREGS TO THE PERIPHERY

1044
00:35:56,120 --> 00:35:58,520
REMOVES THEM FROM PROXIMITY TO

1045
00:35:58,520 --> 00:36:00,160
THE RESPONDING CONVENTIONAL

1046
00:36:00,160 --> 00:36:02,160
CELLS, WHICH THEN LIMITS THEIR

1047
00:36:02,160 --> 00:36:04,880
ABILITY TO SUPPRESS THE

1048
00:36:04,880 --> 00:36:05,400
RESPONSE.

1049
00:36:05,400 --> 00:36:07,720
SO OVERALL, YOU GET A VERY

1050
00:36:07,720 --> 00:36:08,480
POTENT ANTITUMOR EFFECT BECAUSE

1051
00:36:08,480 --> 00:36:10,000
YOU'RE AT MULTIPLE LEVELS

1052
00:36:10,000 --> 00:36:12,440
REMOVING THE SUPPRESSION WITHIN

1053
00:36:12,440 --> 00:36:13,440
THE SYSTEM, BUT NOT ONLY THAT,

1054
00:36:13,440 --> 00:36:15,520
YOU'RE ACTUALLY ADDING TO THE

1055
00:36:15,520 --> 00:36:16,400
EFFECTOR FUNCTION THAT'S

1056
00:36:16,400 --> 00:36:20,440
ATTACKING THE TUMOR.

1057
00:36:20,440 --> 00:36:22,880
NOW, THE HISTOCYTOMETRY HAS BEEN

1058
00:36:22,880 --> 00:36:24,600
OBVIOUSLY VERY VALUABLE IN MANY

1059
00:36:24,600 --> 00:36:26,720
DIFFERENT WAYS, BUT IT'S NOT

1060
00:36:26,720 --> 00:36:27,800
ADEQUATE.

1061
00:36:27,800 --> 00:36:30,080
THERE ARE MANY MORE IMMUNE

1062
00:36:30,080 --> 00:36:32,520
CELLS, SUBSETS OR TYPES THAN

1063
00:36:32,520 --> 00:36:34,360
WE'RE ABLE TO LOOK AT IN A

1064
00:36:34,360 --> 00:36:35,680
NUMBER OF COLORS THAT I SHOWED

1065
00:36:35,680 --> 00:36:37,000
YOU, THE NUMBER OF PARAMETERS.

1066
00:36:37,000 --> 00:36:39,280
THERE ARE ALL THESE EPITHELIAL

1067
00:36:39,280 --> 00:36:40,760
AND STROMAL CELLS YOU WANT TO

1068
00:36:40,760 --> 00:36:42,280
LOOK AT IN THE TISSUE AT THE

1069
00:36:42,280 --> 00:36:43,800
SAME TIME, THERE ARE ACELLULAR

1070
00:36:43,800 --> 00:36:44,800
STRUCTURAL COMPONENTS AND IF

1071
00:36:44,800 --> 00:36:46,120
WE'RE INTERESTED IN TUMOR

1072
00:36:46,120 --> 00:36:47,200
BIOLOGY, THERE ARE OTHER THINGS

1073
00:36:47,200 --> 00:36:48,400
GOING ON WITH THE TUMOR CELLS.

1074
00:36:48,400 --> 00:36:50,840
AND SO ANDREA RADKE DID A

1075
00:36:50,840 --> 00:36:52,120
SPECTACULAR JOB IN THE LAB OF

1076
00:36:52,120 --> 00:36:53,800
SETTING UP A NEW ITERATIVE

1077
00:36:53,800 --> 00:36:56,000
STAINING METHOD WHICH IS CALLED

1078
00:36:56,000 --> 00:36:56,880
IBEX.

1079
00:36:56,880 --> 00:36:59,920
YOU STAIN IN SOMEWHERE BETWEEN

1080
00:36:59,920 --> 00:37:01,000
FOUR AND THESE DAYS MAYBE EVEN

1081
00:37:01,000 --> 00:37:05,200
BACK UP TO ABOUT 8 OR 9 COLORS.

1082
00:37:05,200 --> 00:37:07,520
YOU IMAGE, YOU BLEACH AND THEN

1083
00:37:07,520 --> 00:37:08,560
YOU ITERATE THIS PROCESS.

1084
00:37:08,560 --> 00:37:14,760
THEN SHE WORKED TO CREATE WAYS

1085
00:37:14,760 --> 00:37:16,800
OF REGISTERING ALL THESE

1086
00:37:16,800 --> 00:37:18,160
ITERATIVE IMAGING CYCLES BECAUSE

1087
00:37:18,160 --> 00:37:21,760
THERE ARE SMALL DIFFERENCES AS

1088
00:37:21,760 --> 00:37:23,040
YOU GO THROUGH THIS PROCESS SO

1089
00:37:23,040 --> 00:37:24,840
WE GET PIXEL LEVEL RESOLUTION

1090
00:37:24,840 --> 00:37:26,480
HERE AND THEN WE CAN GO INTO

1091
00:37:26,480 --> 00:37:27,680
OTHER SOFTWARE I'LL TALK ABOUT

1092
00:37:27,680 --> 00:37:29,080
FOR DOING ANALYSIS.

1093
00:37:29,080 --> 00:37:31,280
SO HERE'S AN EXAMPLE OF A HUMAN

1094
00:37:31,280 --> 00:37:33,720
LYMPH NODE STAINED WITH

1095
00:37:33,720 --> 00:37:36,480
82 DIFFERENT ANTIBODIES OF WHICH

1096
00:37:36,480 --> 00:37:37,960
60-SOME-ODD ACTUALLY REACT WITH

1097
00:37:37,960 --> 00:37:38,760
MOLECULES IN THAT ENVIRONMENT.

1098
00:37:38,760 --> 00:37:40,320
AND THEN THAT REALLY RAISES A

1099
00:37:40,320 --> 00:37:42,240
QUESTION, YOU COULD LOOK AT

1100
00:37:42,240 --> 00:37:43,920
COMBINATIONS OF TWO OR THREE

1101
00:37:43,920 --> 00:37:45,120
MARKERS AND SORT OF PERCEIVE

1102
00:37:45,120 --> 00:37:46,320
DIFFERENT PATTERNS, BUT HOW

1103
00:37:46,320 --> 00:37:49,080
WOULD YOU LOOK AT 62 DIFFERENT

1104
00:37:49,080 --> 00:37:52,400
PARAMETERS AT ANY GIVEN TIME AND

1105
00:37:52,400 --> 00:37:53,360
MAKE -- GAINING SOME

1106
00:37:53,360 --> 00:37:54,360
UNDERSTANDING OF WHAT'S

1107
00:37:54,360 --> 00:37:55,120
HAPPENING IN THIS TISSUE

1108
00:37:55,120 --> 00:37:55,640
ENVIRONMENT.

1109
00:37:55,640 --> 00:37:59,280
SO THIS IS A CHALLENGE TAKEN ON

1110
00:37:59,280 --> 00:38:00,840
AND THEN WITH HELP FROM SPENCER

1111
00:38:00,840 --> 00:38:02,680
GRANT IN THE LAB, AND WHAT

1112
00:38:02,680 --> 00:38:04,120
THEY'VE DEVELOPED IS A SOFTWARE

1113
00:38:04,120 --> 00:38:05,520
TOOL CALLED RAPID.

1114
00:38:05,520 --> 00:38:09,200
IT'S BASED ON MACHINE LEARNING.

1115
00:38:09,200 --> 00:38:12,800
AND IT BASICALLY DOES A COMPLEX

1116
00:38:12,800 --> 00:38:16,080
ANALYSIS TO SORT OF CLUSTER THE

1117
00:38:16,080 --> 00:38:17,320
DIFFERENT FLUORESCENT PIXELS YOU

1118
00:38:17,320 --> 00:38:20,400
GET IN THESE IMAGES INTO THINGS

1119
00:38:20,400 --> 00:38:23,200
THAT MORE REALISTICALLY

1120
00:38:23,200 --> 00:38:24,320
REPRESENT WHAT YOU WOULD SEE IF

1121
00:38:24,320 --> 00:38:27,040
YOU WERE DOING MULTIPLEX FLOW

1122
00:38:27,040 --> 00:38:27,400
CYTOMETRY.

1123
00:38:27,400 --> 00:38:29,800
NOW THE PIXEL APPROACH IS

1124
00:38:29,800 --> 00:38:32,000
IMPORTANT BECAUSE ALL

1125
00:38:32,000 --> 00:38:33,080
SEGMENTATION ALGORITHMS FAIL AT

1126
00:38:33,080 --> 00:38:34,840
A CERTAIN LEVEL WITH VERY

1127
00:38:34,840 --> 00:38:35,880
IRREGULAR CELLS AND THEY'RE NOT

1128
00:38:35,880 --> 00:38:37,800
VERY GOOD AT EXTRACELLULAR

1129
00:38:37,800 --> 00:38:38,800
ACELLULAR MATRIX COMPONENTS.

1130
00:38:38,800 --> 00:38:40,520
THE PIXELS DON'T CARE ABOUT

1131
00:38:40,520 --> 00:38:43,040
THOSE, AND THEY'RE MUCH MORE

1132
00:38:43,040 --> 00:38:44,360
USEFUL, BUT BUILT INTO THIS

1133
00:38:44,360 --> 00:38:45,480
SOFTWARE IS ALSO THE ABILITY

1134
00:38:45,480 --> 00:38:47,880
WHEN IT'S USEFUL TO DO

1135
00:38:47,880 --> 00:38:49,840
SEGMENTATION AND WE'VE COMPARED

1136
00:38:49,840 --> 00:38:51,520
THE SEGMENTATION ALGORITHM HERE

1137
00:38:51,520 --> 00:38:53,240
TO OTHER MODELS, AND IT'S AS

1138
00:38:53,240 --> 00:38:55,000
GOOD OR BETTER UNDER ALL THE

1139
00:38:55,000 --> 00:38:56,400
CONDITIONS THAT WE'VE TESTED.

1140
00:38:56,400 --> 00:39:00,920
SO AS AN EXAMPLE HERE, WITH

1141
00:39:00,920 --> 00:39:02,240
ANDREA'S HUMAN LYMPH NODE DATA,

1142
00:39:02,240 --> 00:39:03,880
HERE'S THE IMAGING PICTURE.

1143
00:39:03,880 --> 00:39:06,080
IT GETS TRANSLATED INTO THE

1144
00:39:06,080 --> 00:39:07,720
PSEUDOCOLOR IN RAPID, AND WHAT

1145
00:39:07,720 --> 00:39:09,920
YOU SEE HERE IS A GERMINAL

1146
00:39:09,920 --> 00:39:12,120
CENTER, AND THE PSEUDOCOLORS ARE

1147
00:39:12,120 --> 00:39:15,520
NOT SEGMENTED CELLS BUT THEY ARE

1148
00:39:15,520 --> 00:39:17,280
REPRESENTATIVE OF A CLUSTER OF

1149
00:39:17,280 --> 00:39:18,920
PIXELS THAT THE SOFTWARE HAS

1150
00:39:18,920 --> 00:39:20,440
DECIDED ARE UNIQUE AND DIFFERENT

1151
00:39:20,440 --> 00:39:22,560
FROM WHAT IS ADJACENT TO THEM.

1152
00:39:22,560 --> 00:39:24,360
AND THAT WINDS UP LOOKING LIKE

1153
00:39:24,360 --> 00:39:27,720
INDIVIDUAL CELLS.

1154
00:39:27,720 --> 00:39:29,800
IF WE GO AND ASK WHAT IT'S

1155
00:39:29,800 --> 00:39:33,640
SHOWING US, IT'S USING A

1156
00:39:33,640 --> 00:39:34,960
COMBINATION OF FOUR DIFFERENT

1157
00:39:34,960 --> 00:39:36,640
MARKERS OF NAIVE B CELLS THAT

1158
00:39:36,640 --> 00:39:38,480
LIVE OUTSIDE THE GERMINAL

1159
00:39:38,480 --> 00:39:39,320
CENTER, SIX DIFFERENT MARKERS TO

1160
00:39:39,320 --> 00:39:41,000
LOOK AT GERMINAL CENTER B CELLS

1161
00:39:41,000 --> 00:39:42,960
AND YOU CAN EVEN SEE IN THE

1162
00:39:42,960 --> 00:39:44,240
PIXEL IMAGE THE SHAPE DIFFERENCE

1163
00:39:44,240 --> 00:39:45,520
BETWEEN THE LIGHT AND THE DARK

1164
00:39:45,520 --> 00:39:47,680
ZONE B CELLS TO LOOK AT WHICH OF

1165
00:39:47,680 --> 00:39:49,600
THOSE B CELLS ARE PROLIFERATING,

1166
00:39:49,600 --> 00:39:55,920
TO LOOK AT TFHs AND CELL LINE.

1167
00:39:55,920 --> 00:39:57,720
WE DON'T ALL WANT TO WRITE

1168
00:39:57,720 --> 00:40:02,520
PYTHON CODE, AND SO NISHAN AND

1169
00:40:02,520 --> 00:40:05,760
SPENCER HAVE TURNED THIS INTO A

1170
00:40:05,760 --> 00:40:09,800
GRAFGRAPHIC USER INTERRER FACE.

1171
00:40:09,800 --> 00:40:12,440
CAN YOU LOAD IN YOUR IMAGE AND

1172
00:40:12,440 --> 00:40:14,920
DO SOME MANIPULATIONS OF IT.

1173
00:40:14,920 --> 00:40:17,120
YOU CAN DO EITHER OBJECT OR

1174
00:40:17,120 --> 00:40:18,560
PIXEL-BASED SEGMENTATION AND

1175
00:40:18,560 --> 00:40:20,520
CLUSTERING ANALYSIS, SO YOU WIND

1176
00:40:20,520 --> 00:40:21,640
UP WITH INFORMATION LOOKING LIKE

1177
00:40:21,640 --> 00:40:22,760
THIS ON YOUR SCREEN, AND YOU

1178
00:40:22,760 --> 00:40:25,600
ALSO GET THESE TABLES THAT

1179
00:40:25,600 --> 00:40:29,000
QUANTIFY WHAT THE CLUSTER AND

1180
00:40:29,000 --> 00:40:30,560
PIXEL CALLS ARE IN THE SOFTWARE,

1181
00:40:30,560 --> 00:40:33,600
SO HERE, FOR EXAMPLE, THE ONLY

1182
00:40:33,600 --> 00:40:35,920
STRONG SIGNAL WE SEE IS CD31,

1183
00:40:35,920 --> 00:40:38,320
THAT'S REPRESENTING ENDOTHELIAL

1184
00:40:38,320 --> 00:40:38,720
CELLS.

1185
00:40:38,720 --> 00:40:41,320
HERE WE HAVE A COMBINATION OF

1186
00:40:41,320 --> 00:40:42,840
HIGH CD3 AND CD8.

1187
00:40:42,840 --> 00:40:46,160
THOSE ARE CD8 T-CELLS BUT IF YOU

1188
00:40:46,160 --> 00:40:48,040
LOOK AT 68 PARAMETERS, MAYBE

1189
00:40:48,040 --> 00:40:50,240
THEY'RE CD44 HIGH SO THEY'RE NOT

1190
00:40:50,240 --> 00:40:51,240
NAIVE, FOR EXAMPLE, VERSUS THE

1191
00:40:51,240 --> 00:40:53,960
ONES THAT ARE CD62L POSITIVE AND

1192
00:40:53,960 --> 00:40:54,600
SO ON.

1193
00:40:54,600 --> 00:40:56,160
AND THEN YOU CAN DO A LIMITED

1194
00:40:56,160 --> 00:40:58,080
AMOUNT OF SPATIAL ANALYSIS, BUT

1195
00:40:58,080 --> 00:41:00,120
AS I TOLD YOU FOR

1196
00:41:00,120 --> 00:41:01,040
HISTOCYTOMETRY, THIS IS THE

1197
00:41:01,040 --> 00:41:02,640
INPUT FOR REALLY BEGINNING TO

1198
00:41:02,640 --> 00:41:04,960
LOOK AT WHAT'S HAPPENING IN

1199
00:41:04,960 --> 00:41:08,800
SPACE.

1200
00:41:08,800 --> 00:41:10,520
AND I SAY THAT FOR A REASON, AND

1201
00:41:10,520 --> 00:41:11,640
THERE ARE ALL THESE DIFFERENT

1202
00:41:11,640 --> 00:41:14,720
POSSIBLE PATTERNS ACROSS TISSUE

1203
00:41:14,720 --> 00:41:15,680
SCALES, THE ZONATION I ALREADY

1204
00:41:15,680 --> 00:41:17,600
TALKED ABOUT, THE BROADCAST

1205
00:41:17,600 --> 00:41:19,680
BEING OF SIGNALS BASED ON

1206
00:41:19,680 --> 00:41:22,080
CYTOKINES OR PHYSICAL CELLULAR

1207
00:41:22,080 --> 00:41:23,200
ENTANGLEMENTS OF TWO OR MORE

1208
00:41:23,200 --> 00:41:25,080
CELL TYPES.

1209
00:41:25,080 --> 00:41:26,800
SO ED HAS DONE A SPECTACULAR JOB

1210
00:41:26,800 --> 00:41:29,000
OF APPROACHING THIS AS A

1211
00:41:29,000 --> 00:41:30,320
MATHEMATICAL PROBLEM CREATING

1212
00:41:30,320 --> 00:41:33,400
ANOTHER SOFTWARE TOOL CALLED

1213
00:41:33,400 --> 00:41:38,640
SPACE THAT ENABLES YOU TO LOOK

1214
00:41:38,640 --> 00:41:42,200
AB INITIO AT WHAT'S HAPPENING WP

1215
00:41:42,200 --> 00:41:44,160
THE TISSUE IN ALL THESE

1216
00:41:44,160 --> 00:41:45,120
DIFFERENT PARAMETERS.

1217
00:41:45,120 --> 00:41:46,480
SO FIRST STEP IS TO GO INTO ARE

1218
00:41:46,480 --> 00:41:51,240
PID ANRAPID AND DO THIS CLUSTER,

1219
00:41:51,240 --> 00:41:55,280
AND THEN YOU DO THIS CENSUS OF

1220
00:41:55,280 --> 00:41:55,960
LOCAL NEIGHBORHOODS.

1221
00:41:55,960 --> 00:41:57,760
IT IS NOT A NEIGHBORHOOD

1222
00:41:57,760 --> 00:41:59,320
ALGORITHM OF THE TYPE YOU MAY

1223
00:41:59,320 --> 00:42:00,120
HAVE READ ABOUT IN THE

1224
00:42:00,120 --> 00:42:00,440
LITERATURE.

1225
00:42:00,440 --> 00:42:02,320
IT JUST USES THIS TO GATHER

1226
00:42:02,320 --> 00:42:03,600
INFORMATION ACROSS THE ENTIRE

1227
00:42:03,600 --> 00:42:04,080
TISSUE SPACE.

1228
00:42:04,080 --> 00:42:05,840
THEN HE GOES INTO A MATHEMATICAL

1229
00:42:05,840 --> 00:42:07,560
APPROACH CALLED GENERALIZED

1230
00:42:07,560 --> 00:42:08,880
MUTUAL INFORMATION, THAT

1231
00:42:08,880 --> 00:42:13,200
DETERMINES WHETHER THERE IS

1232
00:42:13,200 --> 00:42:16,960
STATISTICALLY RELEVANT ASYMMETRY

1233
00:42:16,960 --> 00:42:19,880
IN THE DISTRIBUTION OF VARIOUS

1234
00:42:19,880 --> 00:42:21,440
COMPONENTS IN THE TISSUE.

1235
00:42:21,440 --> 00:42:22,960
AND FROM THAT, YOU CAN LEARN

1236
00:42:22,960 --> 00:42:23,280
RELATIONSHIPS.

1237
00:42:23,280 --> 00:42:24,680
I'M NOT GOING TO WALK YOU

1238
00:42:24,680 --> 00:42:25,880
THROUGH ALL THIS.

1239
00:42:25,880 --> 00:42:27,360
THIS IS STILL BEING FINALIZED

1240
00:42:27,360 --> 00:42:30,080
BUT I'LL GIVE YOU AN EXAMPLE OF

1241
00:42:30,080 --> 00:42:30,640
HOW POWERFUL IT IS.

1242
00:42:30,640 --> 00:42:34,560
SO HERE WE'RE LOOKING AT A

1243
00:42:34,560 --> 00:42:36,560
42PLEX MOUSE LYMPH NODE THAT HAD

1244
00:42:36,560 --> 00:42:38,640
BEEN NOT ANALYZED PREVIOUSLY FOR

1245
00:42:38,640 --> 00:42:40,160
THIS PARTICULARLY IMAGE, BUT HAD

1246
00:42:40,160 --> 00:42:43,800
BEEN STUDIED PREVIOUSLY IN THE

1247
00:42:43,800 --> 00:42:45,240
LAB, AND WHAT HE WAS ABLE TO

1248
00:42:45,240 --> 00:42:47,840
SHOW AS I MENTIONED PREVIOUSLY

1249
00:42:47,840 --> 00:42:51,280
IS THAT THE CD4 AND CD8 T-CELLS

1250
00:42:51,280 --> 00:42:53,720
ARE BIASED IN THEIR DISTRIBUTION

1251
00:42:53,720 --> 00:42:55,880
IN THE T-CELL ZONE, AND THAT

1252
00:42:55,880 --> 00:43:00,080
THAT BIAS MATCHES THE DIFFERENT

1253
00:43:00,080 --> 00:43:06,320
DISTRIBUTIONS OF CDC1s AND

1254
00:43:06,320 --> 00:43:08,400
2s TO THOSE DIFFERENT T-CELL

1255
00:43:08,400 --> 00:43:08,720
POPULATIONS.

1256
00:43:08,720 --> 00:43:10,160
WHAT SPACE REVEALS OVER HERE ON

1257
00:43:10,160 --> 00:43:11,000
THE RIGHT-HAND SIDE IS THAT OF

1258
00:43:11,000 --> 00:43:12,520
COURSE B CELLS ARE SEPARATED

1259
00:43:12,520 --> 00:43:14,200
FROM THE TWO TYPES OF T-CELLS.

1260
00:43:14,200 --> 00:43:16,760
YOU SEE THAT IN THIS REDUCED

1261
00:43:16,760 --> 00:43:17,400
DIMENSIONALITY PLOT.

1262
00:43:17,400 --> 00:43:19,800
BUT FURTHER, THAT THERE IS A

1263
00:43:19,800 --> 00:43:23,240
DIFFERENCE BETWEEN WHERE THE CD4

1264
00:43:23,240 --> 00:43:25,640
T-CELLS AND THE CD8 T-CELLS ARE,

1265
00:43:25,640 --> 00:43:28,080
AND IF WE DO THAT IN A GRAPHIC

1266
00:43:28,080 --> 00:43:29,960
FORM, YOU SEE THAT THE

1267
00:43:29,960 --> 00:43:31,160
CD-4 CELLS ARE MORE PERIPHERAL

1268
00:43:31,160 --> 00:43:35,120
THAN THE CD-8s, EXACTLY WHAT

1269
00:43:35,120 --> 00:43:37,240
ANTONIO FOUND AFTER YEERLS OF

1270
00:43:37,240 --> 00:43:40,440
CAREFUL STUDY BY HISTOCYTOMETRY

1271
00:43:40,440 --> 00:43:43,480
BUT SOMETHING THAT COMES OUT

1272
00:43:43,480 --> 00:43:45,360
AUTOMATICALLY FROM APPLYING

1273
00:43:45,360 --> 00:43:46,000
SPACE.

1274
00:43:46,000 --> 00:43:49,160
THOSE USED WITH CODEX AND OTHER

1275
00:43:49,160 --> 00:43:50,600
MULTIPLEX IMAGING TOOLS WILL

1276
00:43:50,600 --> 00:43:53,480
NEVER REVEAL THESE TYPES OF

1277
00:43:53,480 --> 00:43:55,560
RELATIONSHIPS BECAUSE THEY'RE

1278
00:43:55,560 --> 00:43:56,760
BASED ON -- AND THE CELLS THAT

1279
00:43:56,760 --> 00:43:58,520
ARE TOUCHING IT, WHEREAS THIS

1280
00:43:58,520 --> 00:43:59,760
CAN LOOK ACROSS ALL THESE

1281
00:43:59,760 --> 00:44:04,880
SCALES.

1282
00:44:04,880 --> 00:44:07,400
THAT'S VERY NICE, BUT I TOLD YOU

1283
00:44:07,400 --> 00:44:11,880
EARLIER ON WE HAD A PROBLEM

1284
00:44:11,880 --> 00:44:13,320
LOOKING AT ANY GIVEN SECTION

1285
00:44:13,320 --> 00:44:19,040
THROUGH A TISSUE ALSO UNDER

1286
00:44:19,040 --> 00:44:20,160
SAMPLES, SO THEY GOT TOGETHER

1287
00:44:20,160 --> 00:44:22,120
AND SAID LET'S LOOK AT A VOLUME

1288
00:44:22,120 --> 00:44:23,560
RATHER THAN A SINGLE SECTION.

1289
00:44:23,560 --> 00:44:27,160
AND THEY DEVELOPED A NEW WAY OF

1290
00:44:27,160 --> 00:44:28,320
CLEARING THE TISSUE.

1291
00:44:28,320 --> 00:44:29,720
YOU SEE THE WAY THEY'VE MADE THE

1292
00:44:29,720 --> 00:44:30,320
TISSUE TRANSPARENT.

1293
00:44:30,320 --> 00:44:31,680
I WANT TO POINT OUT THAT BEFORE

1294
00:44:31,680 --> 00:44:33,360
WE DID THIS, THERE WERE MANY

1295
00:44:33,360 --> 00:44:37,640
METHODS ALREADY PUBLISHED FOR

1296
00:44:37,640 --> 00:44:38,480
CREATING CLEARED TISSUE.

1297
00:44:38,480 --> 00:44:45,520
THEY INCLUDED CLARITY, DISCO,

1298
00:44:45,520 --> 00:44:47,280
BABB.  THE REASON WE INVENTED OR

1299
00:44:47,280 --> 00:44:48,360
REINVENTED A NEW APPROACH IS

1300
00:44:48,360 --> 00:44:50,560
NONE OF THOSE ENABLED YOU TO DO

1301
00:44:50,560 --> 00:44:55,440
HIGH SENSITIVITY DIRECT

1302
00:44:55,440 --> 00:44:55,960
MULTIPLEX STAINING.

1303
00:44:55,960 --> 00:44:58,520
YOU CAN TRACK FLUORESCENT

1304
00:44:58,520 --> 00:44:59,320
PROTEINS VERY WELL IF THEY'RE

1305
00:44:59,320 --> 00:45:00,400
VERY BRIGHT, YOU CAN DO A

1306
00:45:00,400 --> 00:45:01,680
LIMITED AMOUNT OF STAINING WITH

1307
00:45:01,680 --> 00:45:03,800
AN INDIRECT REAGENT FOR

1308
00:45:03,800 --> 00:45:04,440
AMPLIFICATION, BUT IF YOU WANT

1309
00:45:04,440 --> 00:45:07,240
TO DO MULTIPLEX, YOU NEEDED A

1310
00:45:07,240 --> 00:45:08,720
DIFFERENT METHOD.

1311
00:45:08,720 --> 00:45:11,920
AND THAT'S WHAT THEY DEVELOPED.

1312
00:45:11,920 --> 00:45:17,000
SOME HERE'SO HERE'S AN EXAMPLE H

1313
00:45:17,000 --> 00:45:19,720
NODE COMPLETELY INTACT, NO

1314
00:45:19,720 --> 00:45:21,600
SECTIONING, YOU SEE BEAUTIFUL

1315
00:45:21,600 --> 00:45:23,520
COLORS, THICK VESSELS, THE

1316
00:45:23,520 --> 00:45:26,240
AGVs, YOU SEE THE FULL VOLUME

1317
00:45:26,240 --> 00:45:28,520
OF THOSE PERIPHERAL B CELL

1318
00:45:28,520 --> 00:45:29,160
FOLLICLES, BUT OF COURSE WE CAN

1319
00:45:29,160 --> 00:45:30,640
LOOK IN ANY GIVEN SECTION AND

1320
00:45:30,640 --> 00:45:32,840
TREAT IT LIKE HISTOCYTOMETRY.

1321
00:45:32,840 --> 00:45:37,120
SO WE GET ALL OF THIS

1322
00:45:37,120 --> 00:45:38,600
INFORMATION IN 3D IN A WAY THAT

1323
00:45:38,600 --> 00:45:39,680
AN INDIVIDUAL SECTION WOULD FAIL

1324
00:45:39,680 --> 00:45:44,680
TO GIVE US THINGS LIKE THE

1325
00:45:44,680 --> 00:45:46,000
RAMIFICATIONS OF THOSE VESSELS.

1326
00:45:46,000 --> 00:45:48,200
SO HARRY, WHO NOW HAS HIS OWN

1327
00:45:48,200 --> 00:45:52,840
LAB AT MIT DID A SPECTACULAR JOB

1328
00:45:52,840 --> 00:45:55,920
COMBINING HISTOCYTOMETRY AND HIS

1329
00:45:55,920 --> 00:45:58,320
3D MULTIPLEX IMAGING TO STUDY

1330
00:45:58,320 --> 00:46:01,960
T-CELLS IN TISSUES.

1331
00:46:01,960 --> 00:46:06,360
LU HAD SHOWN PREVIOUSLY USING

1332
00:46:06,360 --> 00:46:07,440
ANTIPHOSPHOSTAT STAINING THAT

1333
00:46:07,440 --> 00:46:09,640
CONTRARY TO SOME EXPECTATIONS,

1334
00:46:09,640 --> 00:46:11,640
REGULATORY T-CELLS DIDN'T KEEP

1335
00:46:11,640 --> 00:46:13,440
AUTO REACTIVE T-CELLS FROM

1336
00:46:13,440 --> 00:46:13,960
BECOMING ACTIVATE.

1337
00:46:13,960 --> 00:46:16,640
THAT'S NOT HOW THEY REALLY

1338
00:46:16,640 --> 00:46:17,240
PREVENTED AUTOIMMUNITY.

1339
00:46:17,240 --> 00:46:18,640
YOU COULD SEE THAT THERE WAS THE

1340
00:46:18,640 --> 00:46:21,960
PRODUCTION OF IL-2, SO FULL

1341
00:46:21,960 --> 00:46:23,600
ACTIVATION OF CYTOKINE

1342
00:46:23,600 --> 00:46:24,640
PRODUCTION FROM WHAT WE COULD

1343
00:46:24,640 --> 00:46:28,360
SHOW BASED ON GERM-FREE MOUSE

1344
00:46:28,360 --> 00:46:30,920
ANALYSES WERE SELF-ROW ACTIVE

1345
00:46:30,920 --> 00:46:32,240
T-CELLS, BUT THEY WERE

1346
00:46:32,240 --> 00:46:36,520
SURROUNDED BY THESE CLUSTERS OF

1347
00:46:36,520 --> 00:46:38,960
WHAT JADHU IDENTIFIED AS

1348
00:46:38,960 --> 00:46:39,640
REGULATORY T-CELLS.

1349
00:46:39,640 --> 00:46:41,800
SO WE KNEW THAT THESE LITTLE

1350
00:46:41,800 --> 00:46:43,320
CLUSTERS SEEM TO HAVE A

1351
00:46:43,320 --> 00:46:44,800
RELEVANCE TO CONTROLLING

1352
00:46:44,800 --> 00:46:45,840
RESPONSES, BUT WE DIDN'T KNOW

1353
00:46:45,840 --> 00:46:47,280
HOW THEY DID IT.

1354
00:46:47,280 --> 00:46:48,720
AND THAT'S THE QUESTION HARRY

1355
00:46:48,720 --> 00:46:49,160
TOOK ON.

1356
00:46:49,160 --> 00:46:52,800
SO THE FIRST THING THEY DID IS

1357
00:46:52,800 --> 00:46:54,560
TO RE-ANALYZE THINGS, IT TURNS

1358
00:46:54,560 --> 00:46:58,440
OUT LOOKING FOR IL2 IS RATHER

1359
00:46:58,440 --> 00:47:00,920
DIFFICULT, HE FOUND PD-1

1360
00:47:00,920 --> 00:47:04,120
EXPRESSION IN THE STEADY

1361
00:47:04,120 --> 00:47:08,080
STATE -- WE SAME THE SAME IN

1362
00:47:08,080 --> 00:47:09,520
GERM-FREE MICE, AND THESE ARE

1363
00:47:09,520 --> 00:47:12,400
ALL P STAT5 NEGATIVE AS SHOWN

1364
00:47:12,400 --> 00:47:14,120
BEFORE.

1365
00:47:14,120 --> 00:47:18,080
HE WORKED ON SPATIAL STATISTICAL

1366
00:47:18,080 --> 00:47:23,560
METHODS THIS ALLOWED HIM TO LOOK

1367
00:47:23,560 --> 00:47:24,840
RELATIVE TO THE SURROUNDING

1368
00:47:24,840 --> 00:47:25,960
POPULATIONS OF REGULATORY CELLS

1369
00:47:25,960 --> 00:47:27,080
AND WHEN HE DID THAT, ARE WHAT

1370
00:47:27,080 --> 00:47:29,600
HE FOUND IS THAT THERE IS A

1371
00:47:29,600 --> 00:47:33,200
CLEAR CLUSTERING OF REGULATORY

1372
00:47:33,200 --> 00:47:35,320
CELLS AROUND UNIQUELY THE PD-1

1373
00:47:35,320 --> 00:47:37,400
POSITIVE ACTIVATED AUTO REACTIVE

1374
00:47:37,400 --> 00:47:41,360
CELLS AS OPPOSED TO ALL THE

1375
00:47:41,360 --> 00:47:42,960
OTHER CD4 CELLS, THE PD-1

1376
00:47:42,960 --> 00:47:43,560
NEGATIVE CELLS IN THE

1377
00:47:43,560 --> 00:47:43,960
POPULATION.

1378
00:47:43,960 --> 00:47:45,000
I WANT TO POINT OUT HERE

1379
00:47:45,000 --> 00:47:46,000
SOMETHING BECAUSE I JUST CAME

1380
00:47:46,000 --> 00:47:47,800
BACK FROM A MEETING WHERE THIS

1381
00:47:47,800 --> 00:47:50,320
WASN'T CLEAR TO EVERYBODY DOING

1382
00:47:50,320 --> 00:47:51,840
SPATIAL ANALYSES, YOU FEED TO GO

1383
00:47:51,840 --> 00:47:59,320
AND DONEED TO GODO RANDOM SAMPLE

1384
00:47:59,320 --> 00:48:00,720
CELLS IN THE VOLUME TO KNOW

1385
00:48:00,720 --> 00:48:01,400
WHETHER SOMETHING THAT LOOKS

1386
00:48:01,400 --> 00:48:02,480
LIKE AN INTERESTING ASSOCIATION

1387
00:48:02,480 --> 00:48:04,840
IS REALLY JUST RANDOM OR IS

1388
00:48:04,840 --> 00:48:07,920
REAL.

1389
00:48:07,920 --> 00:48:09,680
BECAUSE HE DID MULTIPLEX

1390
00:48:09,680 --> 00:48:12,640
IMAGING, HE COULD ACTUALLY BEGIN

1391
00:48:12,640 --> 00:48:13,600
TO CHARACTERIZE THE STATE OF

1392
00:48:13,600 --> 00:48:15,280
THOSE T REGS, SOMETHING THAT

1393
00:48:15,280 --> 00:48:16,600
WOULD BE DIFFICULT TO DO IF HE

1394
00:48:16,600 --> 00:48:18,000
WOULD HAVE TAKEN EVERYTHING

1395
00:48:18,000 --> 00:48:19,680
APARTMENT AND DONE SINGLE CELL

1396
00:48:19,680 --> 00:48:21,680
AND IS BEYOND THE RESOLUTION OF

1397
00:48:21,680 --> 00:48:23,600
THE CURRENT TRANSCRIPTOMIC

1398
00:48:23,600 --> 00:48:24,400
METHODS.

1399
00:48:24,400 --> 00:48:26,400
WHAT HE FOUND WAS THAT THE CELLS

1400
00:48:26,400 --> 00:48:28,840
CLUSTERING NEAR THESE AUTO

1401
00:48:28,840 --> 00:48:35,200
REACTIVE CELLS WERE HIGHLY

1402
00:48:35,200 --> 00:48:38,040
SUPPRESSIVE EFFECTOR T REGS, AND

1403
00:48:38,040 --> 00:48:40,040
EXPERIMENTS ARE NOW SHOWING YOU,

1404
00:48:40,040 --> 00:48:46,280
HE SHOWED THIS IS

1405
00:48:46,280 --> 00:48:46,920
IL-2 DEPENDENT, CONSISTENT TO

1406
00:48:46,920 --> 00:48:48,360
WHAT I SHOWED YOU.

1407
00:48:48,360 --> 00:48:50,240
T REGS ARE OPERATING IN A

1408
00:48:50,240 --> 00:48:51,920
NEGATIVE FEEDBACK MODE, THEY

1409
00:48:51,920 --> 00:48:53,680
WERE RESPONDING TO THE RESPONSE

1410
00:48:53,680 --> 00:48:55,360
OF THE AUTO REACTIVE CELLS, THEY

1411
00:48:55,360 --> 00:48:57,000
WERE SENSING THAT THOSE CELLS

1412
00:48:57,000 --> 00:48:58,160
WOULD BECOME ACTIVATED, THEY

1413
00:48:58,160 --> 00:49:01,840
WERE MAKING IL-2, AND THE T REGS

1414
00:49:01,840 --> 00:49:03,040
NEEDED TO DO SOMETHING, AND WHAT

1415
00:49:03,040 --> 00:49:06,040
THEY NEEDED TO DO WAS TO BECOME

1416
00:49:06,040 --> 00:49:07,680
SUPPRESSIVE, AND THAT'S WHAT WAS

1417
00:49:07,680 --> 00:49:08,320
HAPPENING HERE.

1418
00:49:08,320 --> 00:49:09,760
I SAID THEY WERE HIGH 67

1419
00:49:09,760 --> 00:49:10,880
POSITIVE AND AGAIN, THAT WAS

1420
00:49:10,880 --> 00:49:12,960
HIGHLY ENRICHED IN THE T REGS

1421
00:49:12,960 --> 00:49:14,400
VERY CLOSE TO THE RESPONDING

1422
00:49:14,400 --> 00:49:15,800
CELLS, SO THE QUESTION WAS

1423
00:49:15,800 --> 00:49:19,720
WHETHER THE DENSITY OF THESE T

1424
00:49:19,720 --> 00:49:22,240
REGS WAS A CHEMOKINE ATTRACTION

1425
00:49:22,240 --> 00:49:24,320
OR WHETHER IT COULD HAVE BEEN

1426
00:49:24,320 --> 00:49:25,160
PROLIFERATION LOCALLY IN

1427
00:49:25,160 --> 00:49:28,400
RESPONSE TO IL2, TO LOOK AT THAT

1428
00:49:28,400 --> 00:49:30,160
WE USED WHAT ARE CALLED CONFETTI

1429
00:49:30,160 --> 00:49:31,280
MICE THAT HAVE A WAY OF GIVING

1430
00:49:31,280 --> 00:49:33,760
YOU A KIND OF CLONAL PICTURE OF

1431
00:49:33,760 --> 00:49:35,760
CELLS BASED ON COLOR EXPRESSION,

1432
00:49:35,760 --> 00:49:38,880
AND YOU CAN SEE THESE SMALL

1433
00:49:38,880 --> 00:49:42,520
CLUSTERS OF LIKE-COLORED CELLS

1434
00:49:42,520 --> 00:49:43,840
WITHIN THESE T REG CLUSTERS AND

1435
00:49:43,840 --> 00:49:45,640
IF YOU LOOK AT THAT

1436
00:49:45,640 --> 00:49:47,040
STATISTICALLY, IT'S CLEAR THAT

1437
00:49:47,040 --> 00:49:48,920
THIS IS NON-RANDOM.

1438
00:49:48,920 --> 00:49:50,200
SO WHAT WAS HAPPENING IS THAT

1439
00:49:50,200 --> 00:49:51,720
THERE WAS PROLIFERATION GOING

1440
00:49:51,720 --> 00:49:54,760
ON, AND SO THERE'S A FEE FORWARD

1441
00:49:54,760 --> 00:49:55,280
AMPLIFICATION.

1442
00:49:55,280 --> 00:49:56,920
THE SYSTEM IS OPERATING IN A

1443
00:49:56,920 --> 00:49:57,960
NEGATIVE FEEDBACK ROAD

1444
00:49:57,960 --> 00:49:59,800
RESPONDING TO THE IL-2 AND WHEN

1445
00:49:59,800 --> 00:50:01,240
IT DOES THAT, IT FEEDS FORWARD

1446
00:50:01,240 --> 00:50:03,800
IN AN INTERESTING WAY BY

1447
00:50:03,800 --> 00:50:05,320
PROLIFERATING THESE HIGHLY

1448
00:50:05,320 --> 00:50:09,240
ACTIVATED REGULATORY CELLS.

1449
00:50:09,240 --> 00:50:10,240
THAT STILL DIDN'T ANSWER THE

1450
00:50:10,240 --> 00:50:11,920
QUESTION OF HOW THIS CONTROLS

1451
00:50:11,920 --> 00:50:13,120
AUTOIMMUNITY AND FOR THAT, WE

1452
00:50:13,120 --> 00:50:17,640
NEEDED THE CLEARING AND THE C3D

1453
00:50:17,640 --> 00:50:18,480
APPROACH.

1454
00:50:18,480 --> 00:50:19,720
THE REASON FOR THAT IS WE KNOW

1455
00:50:19,720 --> 00:50:21,720
IF YOU DISTURB RATIOS OR NUMBERS

1456
00:50:21,720 --> 00:50:23,520
OF CELLS WITHIN AN ANIMAL OR

1457
00:50:23,520 --> 00:50:26,280
HOST YOU IMBALANCE THE T REG AND

1458
00:50:26,280 --> 00:50:28,000
AUTO REACTIVE COMPONENTS SO WE

1459
00:50:28,000 --> 00:50:29,720
NEEDED TO LOOK AT PHYSIOLOGICAL

1460
00:50:29,720 --> 00:50:32,880
NUMBERS OF CELLS TO REALLY

1461
00:50:32,880 --> 00:50:34,840
ADDRESS THIS QUESTION.

1462
00:50:34,840 --> 00:50:36,240
WELL, IF WE TRY TO DO THAT WITH

1463
00:50:36,240 --> 00:50:38,560
A THIN SECTION, WE MIGHT SEE ONE

1464
00:50:38,560 --> 00:50:43,000
IF WE'RE LUCKY CELL IN A

1465
00:50:43,000 --> 00:50:43,440
SECTION.

1466
00:50:43,440 --> 00:50:46,640
IF WE LOOKED AT AN ENTIRE CLEAR

1467
00:50:46,640 --> 00:50:48,640
LYMPH NODE, THEN EVEN 10 OR 15

1468
00:50:48,640 --> 00:50:50,600
CELLS BEGIN TO BE STATISTICALLY

1469
00:50:50,600 --> 00:50:51,120
MEANINGFUL.

1470
00:50:51,120 --> 00:50:52,280
SO THAT'S WHAT HARRY DID.

1471
00:50:52,280 --> 00:50:54,040
HE WORKED OUT A WAY OF

1472
00:50:54,040 --> 00:50:58,120
TRANSFERRING INCREDIBLY SMALL

1473
00:50:58,120 --> 00:51:00,360
NUMBERS, SO IT WAS AT

1474
00:51:00,360 --> 00:51:01,640
PHYSIOLOGICAL FREQUENCIES FOR A

1475
00:51:01,640 --> 00:51:03,520
FEW CELLS OF AN ENTIRE LYMPH

1476
00:51:03,520 --> 00:51:04,880
NODE, AND WHEN HE DID THAT, HE

1477
00:51:04,880 --> 00:51:07,920
COULD SEE IN THIS LYMPH NODE, IN

1478
00:51:07,920 --> 00:51:09,880
THE RED CLOUD ARE ALL THE

1479
00:51:09,880 --> 00:51:11,760
ENDOGENOUS REGULATORY T-CELLS,

1480
00:51:11,760 --> 00:51:15,320
THESE FEW SPOTS ARE THE AUTO

1481
00:51:15,320 --> 00:51:16,640
REACTIVE TRANSGENIC CELLS OR

1482
00:51:16,640 --> 00:51:17,920
CONTROL CELLS, AND HE COULD

1483
00:51:17,920 --> 00:51:19,840
QUANTIFY WHAT HAPPENED OVER

1484
00:51:19,840 --> 00:51:20,160
TIME.

1485
00:51:20,160 --> 00:51:22,560
AND THERE, THERE WAS A VERY BIG

1486
00:51:22,560 --> 00:51:23,880
SURPRISE.

1487
00:51:23,880 --> 00:51:26,000
FOR THE FIRST THREE DAYS, THOSE

1488
00:51:26,000 --> 00:51:27,880
AUTO REACTIVE CELLS PROLIFERATED

1489
00:51:27,880 --> 00:51:30,520
EVEN IN THE PRESENCE OF A FULL

1490
00:51:30,520 --> 00:51:31,920
COHORT OF REGULATORY CELLS WHERE

1491
00:51:31,920 --> 00:51:33,520
WE KNEW THESE ANIMALS WOULD NOT

1492
00:51:33,520 --> 00:51:34,280
GET SICK.

1493
00:51:34,280 --> 00:51:36,760
BUT THEN AFTER A FEW MORE DAYS,

1494
00:51:36,760 --> 00:51:39,600
THEY ESSENTIALLY ALL DIED OFF.

1495
00:51:39,600 --> 00:51:41,440
SO THE WAY THE REGULATORY CELLS

1496
00:51:41,440 --> 00:51:43,080
WORK IS NOT TO PREVENT THE

1497
00:51:43,080 --> 00:51:44,600
ACTIVATION OF SELF-REACTIVE

1498
00:51:44,600 --> 00:51:46,960
CELLS, NOT EVEN TO PREVENT THEM

1499
00:51:46,960 --> 00:51:48,240
FROM PROLIFERATING, BUT TO CAUSE

1500
00:51:48,240 --> 00:51:51,440
THEM TO DIE WHEN THEY DO THAT.

1501
00:51:51,440 --> 00:51:52,760
SO THE WAY THIS ALL WORKS OUT IS

1502
00:51:52,760 --> 00:51:54,800
THAT WE BELIEVE THAT POSITIVE

1503
00:51:54,800 --> 00:51:56,080
SELECTION IN THE THYMUS GIVES US

1504
00:51:56,080 --> 00:51:58,160
THE CONVENTIONAL CELLS AND IS

1505
00:51:58,160 --> 00:52:00,840
SOMEWHAT LOWER SELF LIGAND --

1506
00:52:00,840 --> 00:52:02,160
THAN THE REGULATORY CELLS WHEN

1507
00:52:02,160 --> 00:52:05,200
THEY MEET UP WITH THEIR

1508
00:52:05,200 --> 00:52:07,240
DENDRITIC CELL YOU GET A LITTLE

1509
00:52:07,240 --> 00:52:09,680
BIT OF CD5 EXPRESSION ON THE

1510
00:52:09,680 --> 00:52:10,440
CONVENTIONAL CELLS AND A LITTLE

1511
00:52:10,440 --> 00:52:11,960
BIT OF IL-2.

1512
00:52:11,960 --> 00:52:14,120
THE T REGS WHEN THEY SEE THEIR

1513
00:52:14,120 --> 00:52:16,600
LIGAND PUT UP MORE CD25 AND THEY

1514
00:52:16,600 --> 00:52:18,240
HAVE PREFERENTIAL CAPACITY TO

1515
00:52:18,240 --> 00:52:21,240
CAPTURE IL-2, IN ESSENCE TO

1516
00:52:21,240 --> 00:52:23,800
STEAL IT AND WAY AND NOT LET IT

1517
00:52:23,800 --> 00:52:25,320
OCCUR AMONG THE COP VENGSAL

1518
00:52:25,320 --> 00:52:25,600
CELLS.

1519
00:52:25,600 --> 00:52:27,680
AT THE SAME TIME, THOSE K REGS

1520
00:52:27,680 --> 00:52:29,520
CAPTURING THE IL-2 PROLIFERATE

1521
00:52:29,520 --> 00:52:31,640
TO CREATE THIS VERY SUPPRESSIVE

1522
00:52:31,640 --> 00:52:32,760
ZONE TO MAKE SURE THE

1523
00:52:32,760 --> 00:52:35,400
CONVENTIONAL CELLS DON'T, QUOTE,

1524
00:52:35,400 --> 00:52:39,520
ESCAPE AND SEE IL-2.

1525
00:52:39,520 --> 00:52:42,160
WHY DOES THAT MATTER?"

1526
00:52:42,160 --> 00:52:43,960
L-2 IS USUALLY THOUGHT ABOUT AS

1527
00:52:43,960 --> 00:52:47,320
DRIVING T-SELL PROLIFERATION BUT

1528
00:52:47,320 --> 00:52:48,520
IT DOESN'T DO THAT.

1529
00:52:48,520 --> 00:52:50,480
IT ENABLES THEM TO SURVIVE.

1530
00:52:50,480 --> 00:52:54,600
THE CONVENTIONAL CELLS GO

1531
00:52:54,600 --> 00:52:56,880
INTO -- DUE TO TCR SIGNALING,

1532
00:52:56,880 --> 00:52:59,280
THEY MAKE IL-2 WHICH THEY WOULD

1533
00:52:59,280 --> 00:53:01,280
NORMALLY USE TO SURVIVE THAT

1534
00:53:01,280 --> 00:53:02,480
PROLIFERATIVE BURST, BUT THE T

1535
00:53:02,480 --> 00:53:05,360
REGS ARE STEALING ALL THAT

1536
00:53:05,360 --> 00:53:06,320
IL-2 SO THE PROLIFERATING

1537
00:53:06,320 --> 00:53:08,560
CONVENTIONAL CELLS DON'T SEE IT,

1538
00:53:08,560 --> 00:53:11,000
AND THEY DIE.

1539
00:53:11,000 --> 00:53:13,520
PRUNING THEM FROM THE REPERTOIRE

1540
00:53:13,520 --> 00:53:15,640
IN A CONTINUOUS BASIS AND

1541
00:53:15,640 --> 00:53:17,280
PREVENTING AUTOIMMUNITY.

1542
00:53:17,280 --> 00:53:19,040
SO WITH PARK AND SANG, WE

1543
00:53:19,040 --> 00:53:20,520
CREATED A MATH MATT CAT MODEL,

1544
00:53:20,520 --> 00:53:22,920
AND THAT MODEL SHOWS THAT SMALL

1545
00:53:22,920 --> 00:53:25,000
CHANGES OF LESS THAN TWOFOLD

1546
00:53:25,000 --> 00:53:26,600
WILL DISREGULATE THE SYSTEM I

1547
00:53:26,600 --> 00:53:27,600
JUST DESCRIBED.

1548
00:53:27,600 --> 00:53:34,360
SO FOR EXAMPLE, 50% LEVELS OF

1549
00:53:34,360 --> 00:53:37,280
CTLA4, A VERY ABUNDANT MOLECULE

1550
00:53:37,280 --> 00:53:41,320
ON REGULATORY CELLS WAS PREDICT

1551
00:53:41,320 --> 00:53:42,520
TODAY ALLOW ESCAPE FROM THIS

1552
00:53:42,520 --> 00:53:44,320
REGULATION AND FOR THE

1553
00:53:44,320 --> 00:53:45,960
CONVENTIONAL CELLS TO SEE THEIR

1554
00:53:45,960 --> 00:53:48,360
OWN IL-2 AND THAT'S WHAT WE SAW

1555
00:53:48,360 --> 00:53:49,600
EXPERIMENTALLY, AND THAT MATCHES

1556
00:53:49,600 --> 00:53:52,480
THE PHENOTYPE OF HETEROZYGOUS

1557
00:53:52,480 --> 00:53:55,440
HUMANS, WHERE

1558
00:53:55,440 --> 00:53:56,400
CTLA4 HETEROZYGOSITY OR THE

1559
00:53:56,400 --> 00:53:58,360
EQUIVALENT IN A MUTATION IN A

1560
00:53:58,360 --> 00:54:01,760
PROTEIN CONTROLLING

1561
00:54:01,760 --> 00:54:02,800
CTLA4 EXPRESSION GIVES YOU

1562
00:54:02,800 --> 00:54:03,880
AUTOIMMUNITY.

1563
00:54:03,880 --> 00:54:07,080
SO THIS BEGINS TO GIVE US A CLUE

1564
00:54:07,080 --> 00:54:09,960
ABOUT HOW GWAS HITS THINGS THAT

1565
00:54:09,960 --> 00:54:11,280
ARE EQTLs IN THE ORDER OF

1566
00:54:11,280 --> 00:54:13,680
TWOFOLD DIFFERENCES CAN BEGIN TO

1567
00:54:13,680 --> 00:54:16,160
FAVOR AN AUTOIMMUNE STATE.

1568
00:54:16,160 --> 00:54:18,320
AND SO VERY RECENTLY, WE'VE BEEN

1569
00:54:18,320 --> 00:54:21,960
COLLABORATING WITH ALEX MARSEN,

1570
00:54:21,960 --> 00:54:23,760
WHO HAS IDENTIFIED ENHANCERS

1571
00:54:23,760 --> 00:54:27,040
THAT OPERATE UNIQUELY TO CONTROL

1572
00:54:27,040 --> 00:54:29,560
CD25 EXPRESSION, THE

1573
00:54:29,560 --> 00:54:30,400
IL2 RECEPTOR ALPHA CHAIN

1574
00:54:30,400 --> 00:54:31,600
EXPRESSION IN REGULATORY CELLS

1575
00:54:31,600 --> 00:54:34,000
OR IN CONVENTIONAL CELLS.

1576
00:54:34,000 --> 00:54:36,680
AND WHEN YOU DO THAT, WHAT HE

1577
00:54:36,680 --> 00:54:39,960
SEES IS THAT IF YOU MODIFY THE

1578
00:54:39,960 --> 00:54:41,840
ENHANCER THAT WORKS IN THE

1579
00:54:41,840 --> 00:54:44,600
REGULATORY CELLS, THEY HAVE A

1580
00:54:44,600 --> 00:54:46,240
LOWER STEADY STATE LEVEL OF CD25

1581
00:54:46,240 --> 00:54:49,520
WI25BUT THAT GOES UP TO THE NORL

1582
00:54:49,520 --> 00:54:51,240
LEVEL WITHIN SIX HOURS OF THEM

1583
00:54:51,240 --> 00:54:52,640
TRIGGERS THEIR TCR.

1584
00:54:52,640 --> 00:54:53,560
SO VERY SUBTLE DIFFERENCE.

1585
00:54:53,560 --> 00:54:55,680
AND LIKEWISE FOR THE

1586
00:54:55,680 --> 00:54:56,760
CONVENTIONAL CELLS, THEY TAKE A

1587
00:54:56,760 --> 00:54:58,400
LITTLE BIT LONGER TO UPREGULATE

1588
00:54:58,400 --> 00:54:59,960
THEIR CD25, BUT THEY REACH THE

1589
00:54:59,960 --> 00:55:03,320
FULL LEVEL.

1590
00:55:03,320 --> 00:55:06,200
WELL, IN THE CASE OF THE

1591
00:55:06,200 --> 00:55:07,560
ENHANCER THAT WORKS IN THE

1592
00:55:07,560 --> 00:55:13,960
CONVENTIONAL CELLS, NOW IN THE

1593
00:55:13,960 --> 00:55:15,280
NOD MODEL OF AUTOIMMUNE

1594
00:55:15,280 --> 00:55:16,400
DIABETES, IF YOU DISADVANTAGE

1595
00:55:16,400 --> 00:55:17,880
THESE CONVENTIONAL CELLS FOR

1596
00:55:17,880 --> 00:55:19,320
THEIR CD25 EXPRESSION RELATIVE

1597
00:55:19,320 --> 00:55:24,000
TO THE NORMAL STATE, NONE OF THE

1598
00:55:24,000 --> 00:55:25,200
ANIMALS BECOME DIABETIC, AND

1599
00:55:25,200 --> 00:55:26,360
THAT'S TRUE EVEN IF YOU TREAT

1600
00:55:26,360 --> 00:55:28,560
THEM WITH ANTI-PD-1, THAT

1601
00:55:28,560 --> 00:55:30,520
NORMALLY GIVES YOU THIS

1602
00:55:30,520 --> 00:55:32,200
INCREDIBLY RAPID AND FULLY

1603
00:55:32,200 --> 00:55:35,400
PENETRANT DISEASE STATE.

1604
00:55:35,400 --> 00:55:38,560
AND CONVERSELY, IF YOU

1605
00:55:38,560 --> 00:55:42,400
DISENCUMBER THE T REG SLIGHTLY,

1606
00:55:42,400 --> 00:55:44,520
NOW DISEASE HAPPENS MUCH MORE

1607
00:55:44,520 --> 00:55:46,160
RAPIDLY AND IN A MORE PENETRANT

1608
00:55:46,160 --> 00:55:46,720
MANNER.

1609
00:55:46,720 --> 00:55:48,040
SO THESE VERY SMALL CHANGES THAT

1610
00:55:48,040 --> 00:55:49,920
I SHOWED YOU FROM THE STAINING

1611
00:55:49,920 --> 00:55:51,480
TRANSLATE INTO MAJOR DIFFERENCES

1612
00:55:51,480 --> 00:55:52,800
IN THE BIOLOGY AND IT SHOWS YOU

1613
00:55:52,800 --> 00:55:55,520
THAT THE T REG SYSTEM IS AT ONE

1614
00:55:55,520 --> 00:55:58,440
LEVEL ROBUST, BUT IT'S ALSO VERY

1615
00:55:58,440 --> 00:56:00,160
FRAGILE, WHICH IS EXACTLY WHAT

1616
00:56:00,160 --> 00:56:02,960
THE MATHEMATICAL MODELING SHOWED

1617
00:56:02,960 --> 00:56:04,160
US.

1618
00:56:04,160 --> 00:56:05,480
WITH A FEW PARAMETERS THAT HAVE

1619
00:56:05,480 --> 00:56:07,200
TO STAY IN A VERY NARROW RANGE

1620
00:56:07,200 --> 00:56:08,280
FOR THE SYSTEM TO WORK.

1621
00:56:08,280 --> 00:56:10,920
THIS BEGINS TO GET AT HOW

1622
00:56:10,920 --> 00:56:12,200
GENETIC VARIATION ACTUALLY

1623
00:56:12,200 --> 00:56:13,000
CAUSES HUMAN DISEASE.

1624
00:56:13,000 --> 00:56:15,240
SO I WANT TO END BY SAYING THAT

1625
00:56:15,240 --> 00:56:17,560
WE ALWAYS WANT MORE, AND MORE IS

1626
00:56:17,560 --> 00:56:21,880
TO GO FROM IBEX MULTIPLEX AND

1627
00:56:21,880 --> 00:56:23,800
C3D VOLUME AND PUT THEM TOGETHER

1628
00:56:23,800 --> 00:56:26,440
AND IT TAKES A TEAM, A TAKES A

1629
00:56:26,440 --> 00:56:27,720
VILLAGE TO DO THIS.

1630
00:56:27,720 --> 00:56:33,840
SO HIROSHI, ANDREA, COLIN, J.L.

1631
00:56:33,840 --> 00:56:34,720
AND -- PRETTY CLOSE TO HAVING

1632
00:56:34,720 --> 00:56:35,560
THIS RESOLVED.

1633
00:56:35,560 --> 00:56:36,760
THE WAY WE DO THIS IS A

1634
00:56:36,760 --> 00:56:38,240
COMBINATION OF IBEX WITH

1635
00:56:38,240 --> 00:56:40,000
ITERATIVE CYCLES THAT INVOLVE

1636
00:56:40,000 --> 00:56:43,200
THE CLEARING WITH THE C3D

1637
00:56:43,200 --> 00:56:47,000
METHOD, AND HERE IS ALUM IMAGE

1638
00:56:47,000 --> 00:56:48,920
THIS WAY IN A COUPLE OF HUNDRED

1639
00:56:48,920 --> 00:56:51,800
MICRONS BY HIROSHI IN 20

1640
00:56:51,800 --> 00:56:52,200
PARAMETERS.

1641
00:56:52,200 --> 00:56:54,160
AND YOU REALLY BEGIN TO SEE THAT

1642
00:56:54,160 --> 00:56:55,680
THIS GIVES YOU ALL SORTS OF

1643
00:56:55,680 --> 00:56:57,200
INFORMATION YOU DON'T GET WHEN

1644
00:56:57,200 --> 00:57:01,240
YOU'RE DOING LOAMPLEX OR VOLUME,

1645
00:57:01,240 --> 00:57:02,400
AND I'LL LET THIS PLAY A LITTLE

1646
00:57:02,400 --> 00:57:03,920
BIT MORE AND JUST POINT OUT THAT

1647
00:57:03,920 --> 00:57:05,640
THIS IS ESPECIALLY VALUABLE NOT

1648
00:57:05,640 --> 00:57:06,960
ONLY FOR VESSELS THAT I SHOWED

1649
00:57:06,960 --> 00:57:10,560
YOU IN THE LYMPH NODE IMAGE

1650
00:57:10,560 --> 00:57:12,160
BEFORE, BUT FOR EXAMINER OFS

1651
00:57:12,160 --> 00:57:16,000
THAT ARE VERY HARD TO SEE IN

1652
00:57:16,000 --> 00:57:19,840
THIN SECTIONS.

1653
00:57:19,840 --> 00:57:21,440
HERE'S ANOTHER 20 PLEX IN THE

1654
00:57:21,440 --> 00:57:23,080
GUT, AND THESE WILL CHANGE WITH

1655
00:57:23,080 --> 00:57:27,440
FOUR OR FIVE CLOFER FIVE COLORSH

1656
00:57:27,440 --> 00:57:28,080
CYCLE.

1657
00:57:28,080 --> 00:57:30,080
THIS SHOWS YOU WHAT YOU CAN GET

1658
00:57:30,080 --> 00:57:30,920
BY DOING THIS.

1659
00:57:30,920 --> 00:57:32,400
I'LL END BY SAYING WE REALLY

1660
00:57:32,400 --> 00:57:34,360
THINK THE WAY TO UNDERSTAND

1661
00:57:34,360 --> 00:57:36,160
IMMUNITY AS I STARTED OUT SAYING

1662
00:57:36,160 --> 00:57:38,400
IN THE VERY BEGINNING IS HIGH

1663
00:57:38,400 --> 00:57:40,800
ANALYSIS OF TISSUE ARCHITECTURE

1664
00:57:40,800 --> 00:57:41,440
IN CELL STATE.

1665
00:57:41,440 --> 00:57:45,880
WE CAN DO THIS BY 2P IMAGE, BY

1666
00:57:45,880 --> 00:57:46,680
HISTOCYTOMETRY, WE CAN IMPROVE

1667
00:57:46,680 --> 00:57:50,440
THAT WITH IBEX, WE CAN DO

1668
00:57:50,440 --> 00:57:51,760
HISTOCYTOMETRY IN 3D BUT THEN WE

1669
00:57:51,760 --> 00:57:55,760
CAN COMBINE ALL OF THAT TO C3D

1670
00:57:55,760 --> 00:57:56,440
IBEX.

1671
00:57:56,440 --> 00:57:59,400
WE HAVE SOFTWARE TOOLS RAPID AND

1672
00:57:59,400 --> 00:58:01,680
SPACE IN TICK THE INFORMATION WE

1673
00:58:01,680 --> 00:58:02,960
GET, QUANTIFYING IT AND PUTTING

1674
00:58:02,960 --> 00:58:03,600
IT IN CONTEXT.

1675
00:58:03,600 --> 00:58:04,920
I HAVEN'T HAD A CHANCE TO SHOW

1676
00:58:04,920 --> 00:58:06,920
YOU BUT WE'RE BEGINNING TO

1677
00:58:06,920 --> 00:58:08,800
COMBINE THIS WITH RNA PROBES SO

1678
00:58:08,800 --> 00:58:10,240
WE CAN COMBINE IT WITH THAT

1679
00:58:10,240 --> 00:58:11,920
APPROACH AND USE THE STAINING

1680
00:58:11,920 --> 00:58:14,360
PART FOR THOSE THINGS THAT ARE

1681
00:58:14,360 --> 00:58:16,360
UNIQUELY APPROACHABLE THAT WAY

1682
00:58:16,360 --> 00:58:17,960
LIKE THE PHOSPHOPROTEINS NUCLEAR

1683
00:58:17,960 --> 00:58:18,960
TRANSLOCATION AND SO FORTH.

1684
00:58:18,960 --> 00:58:21,000
LET ME END BY SAYING THAT I

1685
00:58:21,000 --> 00:58:22,400
DON'T HAVE THE TRADITIONAL SLIDE

1686
00:58:22,400 --> 00:58:23,200
WITH A THOUSAND NAMES ON IT.

1687
00:58:23,200 --> 00:58:25,120
I TRY TO TELL YOU WHO'S BEEN MY

1688
00:58:25,120 --> 00:58:27,840
COLLEAGUE AND COLLABORATOR

1689
00:58:27,840 --> 00:58:28,960
THROUGHOUT IN ALL THE WORK WE

1690
00:58:28,960 --> 00:58:29,960
DONE BUT I WANT TO THANK

1691
00:58:29,960 --> 00:58:31,240
EVERYBODY WHO'S BEEN IN THE LAB

1692
00:58:31,240 --> 00:58:32,200
BECAUSE THERE HAVE BEEN ALL

1693
00:58:32,200 --> 00:58:32,960
DIFFERENT LEVELS OF

1694
00:58:32,960 --> 00:58:33,960
CONTRIBUTIONS BESIDES THE PEOPLE

1695
00:58:33,960 --> 00:58:35,280
I'VE NAMED.

1696
00:58:35,280 --> 00:58:37,320
WE OBVIOUSLY HAVE HAD A LOT OF

1697
00:58:37,320 --> 00:58:39,280
COLLABORATIONS AND I'VE POINTED

1698
00:58:39,280 --> 00:58:40,280
THOSE OUT ALONG THE WAY AND

1699
00:58:40,280 --> 00:58:42,400
WE'VE REALLY BEEN GENEROUSLY

1700
00:58:42,400 --> 00:58:43,840
SUPPORTED BY THE INTRAMURAL

1701
00:58:43,840 --> 00:58:45,360
PROGRAM OF NIAID.

1702
00:58:45,360 --> 00:58:46,560
WE'VE ALSO HAD SUPPORT OVER THE

1703
00:58:46,560 --> 00:58:50,480
LAST FEW YEARS FOR WHAT WE CALL

1704
00:58:50,480 --> 00:58:53,880
THE CENTER FOR ADVANCED TISSUE

1705
00:58:53,880 --> 00:58:55,800
IMAWJING FROM THE NCI, SOME

1706
00:58:55,800 --> 00:59:00,440
BENCH TO BED SUPPORT AND --

1707
00:59:00,440 --> 00:59:00,840
THANKS VERY MUCH.

1708
00:59:00,840 --> 00:59:02,880
[APPLAUSE]

1709
00:59:02,880 --> 00:59:05,600
>>I LOVE THE MODEL FOR
THE 

1710
00:59:05,600 --> 00:59:09,040
T REGS.

1711
00:59:09,040 --> 00:59:13,000
I WAS WONDERING IF -- PRESUMABLY

1712
00:59:13,000 --> 00:59:14,520
THAT MODEL WORKS BEST IN THE

1713
00:59:14,520 --> 00:59:16,160
LYMPH NODE.

1714
00:59:16,160 --> 00:59:17,880
AND WHAT ABOUT IN THE PERIPHERAL

1715
00:59:17,880 --> 00:59:19,520
TISSUES?

1716
00:59:19,520 --> 00:59:23,040
HOW DO YOU THINK THE T REGS

1717
00:59:23,040 --> 00:59:24,280
WOULD WORK IN THE SAME WAY IN

1718
00:59:24,280 --> 00:59:26,240
THE PERIPHERAL TISSUES OR WOULD

1719
00:59:26,240 --> 00:59:27,560
THERE BE SOME OTHER MECHANISM?

1720
00:59:27,560 --> 00:59:29,320
>>SO I ALWAYS LEAVE
SOMETHING

1721
00:59:29,320 --> 00:59:30,840
FOR THE FELLOWS THAT LOAF THE

1722
00:59:30,840 --> 00:59:33,040
LAB TO DO.

1723
00:59:33,040 --> 00:59:34,920
AND ACTUALLY HARRY IS MAKING A

1724
00:59:34,920 --> 00:59:35,960
MAJOR EFFORT LOOKING IN THE

1725
00:59:35,960 --> 00:59:38,320
TISSUES NOW USING THESE SAME

1726
00:59:38,320 --> 00:59:38,680
APPROACHES.

1727
00:59:38,680 --> 00:59:40,520
SO I DON'T KNOW WHAT HE'S FOUND,

1728
00:59:40,520 --> 00:59:42,480
HE'S JUST STARTED THE LAB.

1729
00:59:42,480 --> 00:59:43,240
HE'S LOOKING AT IT.

1730
00:59:43,240 --> 00:59:44,760
THERE IS REASON TO THINK IN SOME

1731
00:59:44,760 --> 00:59:50,880
CASES THAT IT STILL COULD BE ILT

1732
00:59:50,880 --> 00:59:52,600
THERAPY, FOR EXAMPLE, WORKS BY

1733
00:59:52,600 --> 00:59:53,760
EXPANDING CELLS FROM THESE

1734
00:59:53,760 --> 00:59:56,600
STEM-LIKE MEMORY CELLS.

1735
00:59:56,600 --> 00:59:58,800
MAYBE THAT'S DEPENDENT ON 15 AND

1736
00:59:58,800 --> 01:00:00,080
NOT JUST TWO AND THEN THE RULES

1737
01:00:00,080 --> 01:00:01,280
WOULD BE SOMEWHAT DIFFERENT SO I

1738
01:00:01,280 --> 01:00:02,600
DON'T WANT TO PUSH FOR THIS.

1739
01:00:02,600 --> 01:00:05,080
BUT GIVEN THAT THERE IS

1740
01:00:05,080 --> 01:00:06,080
PROLIFERATION UNDER THOSE

1741
01:00:06,080 --> 01:00:08,040
CONDITIONS, THEY AGAIN COULD

1742
01:00:08,040 --> 01:00:09,760
OPERATE IN PART BY STEALING.

1743
01:00:09,760 --> 01:00:11,720
THEY OBVIOUSLY -- I DIDN'T GO

1744
01:00:11,720 --> 01:00:12,840
INTO ALL OF IT.

1745
01:00:12,840 --> 01:00:15,640
THE MODEL INCLUDES THE

1746
01:00:15,640 --> 01:00:16,560
CTLA4 EFFECT BECAUSE IT'S NOT

1747
01:00:16,560 --> 01:00:19,240
JUST STEALING BUT LIMITING HOW

1748
01:00:19,240 --> 01:00:20,960
IL2 IS MADE BECAUSE YOU'RE

1749
01:00:20,960 --> 01:00:21,840
INTERFERING WITH CROW

1750
01:00:21,840 --> 01:00:22,200
STIMULATION.

1751
01:00:22,200 --> 01:00:23,800
THAT'S GOING TO APPLY IN THE

1752
01:00:23,800 --> 01:00:25,440
PERIPHERY AS WELL OBVIOUSLY, AND

1753
01:00:25,440 --> 01:00:27,280
WE KNOW YOU NEED TO

1754
01:00:27,280 --> 01:00:28,480
CO-STIMULATION FOR MANY OF THE

1755
01:00:28,480 --> 01:00:29,480
CYTOKINES.

1756
01:00:29,480 --> 01:00:30,680
THAT WOULD BE PRODUCED.

1757
01:00:30,680 --> 01:00:34,040
SO I THINK PARTS OF THE MODEL

1758
01:00:34,040 --> 01:00:37,840
WILL APPLY BUT I DON'T THINK ILL

1759
01:00:37,840 --> 01:00:38,880
IN THE PERIPHERY.

1760
01:00:38,880 --> 01:00:43,840
>>THANKS.

1761
01:00:43,840 --> 01:00:44,800
>>HI.

1762
01:00:44,800 --> 01:00:46,040
VERY IMPRESSIVE IMAGING.

1763
01:00:46,040 --> 01:00:47,200
I HAVE TWO QUESTIONS.

1764
01:00:47,200 --> 01:00:50,120
THE FIRST ONE IS REGARDING THE

1765
01:00:50,120 --> 01:00:52,120
ARCHITECTURE OF THE LIVER.

1766
01:00:52,120 --> 01:00:53,720
HOW COMPLEX DOES THE MICROBIOME

1767
01:00:53,720 --> 01:00:57,200
NEED TO BE FOR THE KUPFFER CELLS

1768
01:00:57,200 --> 01:00:58,200
TO BE POLARIZED?

1769
01:00:58,200 --> 01:00:59,880
THE SECOND QUESTION WOULD BE

1770
01:00:59,880 --> 01:01:01,480
REGARDING THE EX-T REGS, DO THEY

1771
01:01:01,480 --> 01:01:02,920
REMAIN SUPPRESSIVE?

1772
01:01:02,920 --> 01:01:06,360
>>FOR THE FIRST QUESTION,
WE

1773
01:01:06,360 --> 01:01:10,720
CAN GET AT LEAST A PARTIAL

1774
01:01:10,720 --> 01:01:13,240
EFFECT BY USING LPS WHEN THEY'RE

1775
01:01:13,240 --> 01:01:15,960
NOT POLARIZED, AND WE CAN LOSE A

1776
01:01:15,960 --> 01:01:18,120
LOT OF THE POLARIZATION REMOVING

1777
01:01:18,120 --> 01:01:19,960
ONLY TLR4.

1778
01:01:19,960 --> 01:01:21,480
SO I DON'T THINK THE MICROBIOME

1779
01:01:21,480 --> 01:01:24,360
HAS TO BE ENORMOUSLY COMPLEX,

1780
01:01:24,360 --> 01:01:26,560
BUT IT CLEARLY THERE ARE

1781
01:01:26,560 --> 01:01:27,720
MULTIPLE DIFFERENT INPUTS

1782
01:01:27,720 --> 01:01:29,800
BECAUSE WE HAVE A DIFFERENCE

1783
01:01:29,800 --> 01:01:31,760
BETWEEN -- WE DIDN'T DO TRIF,

1784
01:01:31,760 --> 01:01:35,640
AND JUST THE TLR4.

1785
01:01:35,640 --> 01:01:38,920
BUT TLR2 KNOCKOUT ALSO HAD AN

1786
01:01:38,920 --> 01:01:40,080
EFFECT.

1787
01:01:40,080 --> 01:01:42,320
SO COMPLEXITY MATTERS BUT IT NOT

1788
01:01:42,320 --> 01:01:42,600
ESSENTIAL.

1789
01:01:42,600 --> 01:01:44,720
AS FAR AS THE EX-T REGS ARE

1790
01:01:44,720 --> 01:01:45,920
CONCERNED, WE HAVEN'T TRACED

1791
01:01:45,920 --> 01:01:49,280
THIS OUT OVER LONG PERIODS OF

1792
01:01:49,280 --> 01:01:49,720
TIME.

1793
01:01:49,720 --> 01:01:51,480
SO WE KNOW IN THAT ACUTE PERIOD

1794
01:01:51,480 --> 01:01:53,760
THEY CONVERT AND SPONTANEOUSLY

1795
01:01:53,760 --> 01:01:56,160
MAKE INTERFERON GAMMA IN VIVO.

1796
01:01:56,160 --> 01:01:57,480
LET ME CORRECT THAT STATEMENT.

1797
01:01:57,480 --> 01:01:59,600
I DIDN'T SLOW TO YOU BUT WE HAVE

1798
01:01:59,600 --> 01:02:01,480
A WAY OF VALIDATING WHETHER A

1799
01:02:01,480 --> 01:02:07,720
CELL IS ENGAGING TCR IN THE

1800
01:02:07,720 --> 01:02:09,080
TISSUES NOW, WITH THE HIGHEST

1801
01:02:09,080 --> 01:02:10,520
FRACTION OF CELLS SHOWING THAT

1802
01:02:10,520 --> 01:02:14,440
SIGNALING STATE RL THE XT REGS.

1803
01:02:14,440 --> 01:02:17,600
BUT WHETHER THEY PETER OUT, DIE,

1804
01:02:17,600 --> 01:02:18,360
I CAN'T TELL YOU YET.

1805
01:02:18,360 --> 01:02:19,920
>>THANK YOU.

1806
01:02:19,920 --> 01:02:21,800
>>LET ME SQUEEZE IN ONE
FROM

1807
01:02:21,800 --> 01:02:23,680
HOME HERE.

1808
01:02:23,680 --> 01:02:25,000
OUR FRIEND HOWARD YOUNG ASKS,

1809
01:02:25,000 --> 01:02:26,440
HOW DO YOU DEAL WITH THE DATA

1810
01:02:26,440 --> 01:02:27,080
STORAGE?

1811
01:02:27,080 --> 01:02:31,360
IT MUST BE ENORMOUS.

1812
01:02:31,360 --> 01:02:32,880
>>WELL, OUR INSTITUTE IS
VERY

1813
01:02:32,880 --> 01:02:34,600
HAPPY TO SUPPORT LARGE AMOUNTS

1814
01:02:34,600 --> 01:02:36,600
OF STORAGE SO WE JUST KEEP

1815
01:02:36,600 --> 01:02:38,400
DUMPING IT INTO THE H DRIVE.

1816
01:02:38,400 --> 01:02:40,040
THAT AND A LOT OF HARD DRIVES

1817
01:02:40,040 --> 01:02:43,160
HANGING AROUND.

1818
01:02:43,160 --> 01:02:45,840
>>HEY, RON, THAT WAS
BEAUTIFUL.

1819
01:02:45,840 --> 01:02:47,600
SO I KNOW IN HARRY'S WORK, YOU

1820
01:02:47,600 --> 01:02:49,680
WERE LOOKING AT STEADY STATE TO

1821
01:02:49,680 --> 01:02:51,800
LOOK AT AUTO REACTIVITY, BUT IF

1822
01:02:51,800 --> 01:02:53,680
YOU START TO DO THAT IN THE

1823
01:02:53,680 --> 01:02:55,320
CONTEXT OF AN INFECTION WHERE

1824
01:02:55,320 --> 01:02:56,920
JUST SORT OF RELATED TO THE

1825
01:02:56,920 --> 01:02:59,480
QUESTION OF IS IL-2 RESCUING

1826
01:02:59,480 --> 01:03:01,960
LOWER AFFINITY CELLS AND HOW DO

1827
01:03:01,960 --> 01:03:03,400
T REGS FIT IN THERE?

1828
01:03:03,400 --> 01:03:05,760
>>SO WE DID THAT NOT IN
AN

1829
01:03:05,760 --> 01:03:06,440
INFECTION YET.

1830
01:03:06,440 --> 01:03:08,320
WE'RE ALSO LOOKING AT THAT.

1831
01:03:08,320 --> 01:03:12,640
BUT THE WAY WE DID IT WAS TO USE

1832
01:03:12,640 --> 01:03:14,400
CYTOCHROME TRANSGENIC CELLS AND

1833
01:03:14,400 --> 01:03:15,280
ALTERED LIGANDS.

1834
01:03:15,280 --> 01:03:17,800
SO IF YOU USE A FULL AGONIST,

1835
01:03:17,800 --> 01:03:20,720
THEY WIND UP P STAD 5 POSITIVE

1836
01:03:20,720 --> 01:03:22,920
AND YOU BASICALLY SATURATE THE T

1837
01:03:22,920 --> 01:03:23,520
REGS.

1838
01:03:23,520 --> 01:03:25,080
SO ONCE YOU HIT SATURATION, THEY

1839
01:03:25,080 --> 01:03:27,360
CAN'T CONTROL ANYMORE AND THOSE

1840
01:03:27,360 --> 01:03:27,920
CELLS RESPOND.

1841
01:03:27,920 --> 01:03:29,560
BUT IF YOU USE A PARTIAL

1842
01:03:29,560 --> 01:03:31,600
AGONIST, THEN IT LOOKS LIKE THE

1843
01:03:31,600 --> 01:03:33,280
SELF-REACTIVE CELLS.

1844
01:03:33,280 --> 01:03:35,880
THEY DON'T BECOME P STAD

1845
01:03:35,880 --> 01:03:37,840
5 MOSSTIVE AND THAT'S ALSO WHY

1846
01:03:37,840 --> 01:03:39,240
THEY CAN'T EXPAND TO A

1847
01:03:39,240 --> 01:03:41,560
REASONABLE NUMBER.

1848
01:03:41,560 --> 01:03:41,760
102S.

1849
01:03:41,760 --> 01:03:43,840
>>YEAH, AND WHAT IF -- DO
YOU

1850
01:03:43,840 --> 01:03:46,000
HAVE DIFFERENT TCRs THAT HAVE

1851
01:03:46,000 --> 01:03:47,320
DIFFERENT AFFINITY?

1852
01:03:47,320 --> 01:03:48,960
SO CAN YOU LOOK AT THAT?

1853
01:03:48,960 --> 01:03:51,120
>>WE DIDN'T DO THAT
EXPERIMENT

1854
01:03:51,120 --> 01:03:53,200
WITH TCRs WITH DIFFERENT

1855
01:03:53,200 --> 01:03:53,960
AFFINITIES, WE DON'T HAVE A

1856
01:03:53,960 --> 01:03:55,920
SERIES WITH THE SAME LIGAND.

1857
01:03:55,920 --> 01:03:57,320
J.L. IS ACTUALLY GOING TO BUILD

1858
01:03:57,320 --> 01:03:58,960
THAT FOR A DIFFERENT REASON AND

1859
01:03:58,960 --> 01:04:01,680
WE COULD GO BACK AND CHECK THAT,

1860
01:04:01,680 --> 01:04:04,760
BUT -- PUBLISHED YEARS AGO THAT

1861
01:04:04,760 --> 01:04:06,400
WHEN YOU ELIMINATE T REGS, THEN

1862
01:04:06,400 --> 01:04:09,560
A POPULATION OF LOW AFFINITY

1863
01:04:09,560 --> 01:04:10,560
CD8s EMERGE AND THE RESPONSE

1864
01:04:10,560 --> 01:04:12,480
YOU DIDN'T SEE IF THE T REGS

1865
01:04:12,480 --> 01:04:14,000
WERE THERE, SO WE'D ONLY BE

1866
01:04:14,000 --> 01:04:14,880
REPEATING PUBLISHED DATA.

1867
01:04:14,880 --> 01:04:15,640
>>ONE OTHER QUESTION.

1868
01:04:15,640 --> 01:04:18,800
WHEN YOU WERE LOOKING AT THE --

1869
01:04:18,800 --> 01:04:20,200
IN ED'S WORK WHEN YOU WERE

1870
01:04:20,200 --> 01:04:21,160
LOOKING AT THE LOCALIZATION OF

1871
01:04:21,160 --> 01:04:23,240
THE T-CELLS VERSUS THE B CELLS,

1872
01:04:23,240 --> 01:04:25,800
WITHIN THAT B CELL PEAK, THERE'S

1873
01:04:25,800 --> 01:04:27,640
A SMALL PEAK OF CD8s.

1874
01:04:27,640 --> 01:04:34,880
DO YOU KNOW, ARE THOSE THE CXCR-

1875
01:04:34,880 --> 01:04:37,280
>>I'D HAVE TO ASK ED FOR
THAT

1876
01:04:37,280 --> 01:04:39,640
PARTICULAR POINT, BUT I ALSO --

1877
01:04:39,640 --> 01:04:40,520
ONE OF THE THINGS YOU HAVE TO

1878
01:04:40,520 --> 01:04:41,480
KEEP IN MIND IS SOMETHING THAT

1879
01:04:41,480 --> 01:04:44,200
WE DID A NUMBER OF YEARS AGO.

1880
01:04:44,200 --> 01:04:47,600
THE B CELLS COME OUT ON HEVs,

1881
01:04:47,600 --> 01:04:50,000
AND THE HEVs ARE AT THE

1882
01:04:50,000 --> 01:04:51,120
PERIPHERY OF THE T-ZONE, BUT

1883
01:04:51,120 --> 01:04:52,280
THEY'RE IN THE T-ZONE, THEY'RE

1884
01:04:52,280 --> 01:04:53,600
NOT IN THE B CELL FOLLICLES.

1885
01:04:53,600 --> 01:04:55,800
SO ALL THE B CELLS, AND THIS IS

1886
01:04:55,800 --> 01:04:58,160
SOMETHING HE HAD IN HIS FIRST

1887
01:04:58,160 --> 01:05:00,160
PAPER ABOUT INTERACTIONS

1888
01:05:00,160 --> 01:05:02,760
DIRECTLY OUT OF THE -- PT B

1889
01:05:02,760 --> 01:05:04,840
CELLS HAVE TO RUN ACROSS A FIELD

1890
01:05:04,840 --> 01:05:06,280
OF T-CELLS TO GET INTO THE

1891
01:05:06,280 --> 01:05:07,520
FOLLICLE.

1892
01:05:07,520 --> 01:05:08,440
AND DEPENDING ON EXACTLY WHERE

1893
01:05:08,440 --> 01:05:10,360
YOU CUT THE LYMPH NODE AND HOW

1894
01:05:10,360 --> 01:05:13,680
YOU DO THAT, EVEN THOUGH WE SAY

1895
01:05:13,680 --> 01:05:15,080
CD4s ARE MORE PERIPHERAL, YOU

1896
01:05:15,080 --> 01:05:19,800
CAN HIT REGIONS WHERE YOU'RE --

1897
01:05:19,800 --> 01:05:22,200
THE WAY TO GET AROUND THEM, WE

1898
01:05:22,200 --> 01:05:23,400
DIDN'T DO THIS TREATMENT HERE,

1899
01:05:23,400 --> 01:05:25,240
IS TO TREAT THEM WITH ANTI62L.

1900
01:05:25,240 --> 01:05:26,840
SO NOW YOU ALLOW THE B CELLS TO

1901
01:05:26,840 --> 01:05:29,600
CLEAR THE T-ZONE, YOU GET A

1902
01:05:29,600 --> 01:05:32,080
BEAUTIFULLY CRISP BORDER AT

1903
01:05:32,080 --> 01:05:35,600
EXACTLY WHERE THE FRC NETWORK

1904
01:05:35,600 --> 01:05:36,480
ESSENTIALLY DISAPPEARS WHEN IT

1905
01:05:36,480 --> 01:05:39,040
TRYING TO GO INTO THE FOLLICLE,

1906
01:05:39,040 --> 01:05:40,360
AND THAT WOULD GIVE US A CLEANER

1907
01:05:40,360 --> 01:05:40,880
ANSWER.

1908
01:05:40,880 --> 01:05:44,920
>>THANKS SO MUCH.

1909
01:05:44,920 --> 01:05:46,320
>>ANOTHER ONE FROM HOME
HERE.

1910
01:05:46,320 --> 01:05:47,800
I DON'T KNOW IF YOU ANSWERED

1911
01:05:47,800 --> 01:05:48,800
THIS, I'M SORRY.

1912
01:05:48,800 --> 01:05:55,400
HAVE YOU HAD ANY LUCK WITH

1913
01:05:55,400 --> 01:05:55,600
CDIBEX?

1914
01:05:55,600 --> 01:05:58,760
>>YES, IN TUMOR, IT
DEPENDS ON

1915
01:05:58,760 --> 01:05:59,840
THE TUMOR.

1916
01:05:59,840 --> 01:06:01,000
WE'RE LOOKING AT THE FACT THAT

1917
01:06:01,000 --> 01:06:02,800
TUMORS HAVE A DIFFERENT LEVEL OF

1918
01:06:02,800 --> 01:06:04,320
NECROSIS AND AUTO FLUORESCENCE,

1919
01:06:04,320 --> 01:06:05,920
AND WE'RE WORKING TO GET AROUND

1920
01:06:05,920 --> 01:06:06,400
THAT.

1921
01:06:06,400 --> 01:06:08,320
WE'RE ALSO WORKING WITH FFPE

1922
01:06:08,320 --> 01:06:09,680
MATERIAL SINCE WE THINK THIS IS

1923
01:06:09,680 --> 01:06:12,280
ESPECIALLY USEFUL FOR HUMANS, SO

1924
01:06:12,280 --> 01:06:13,360
PRELIMINARILY, WE CAN GET AROUND

1925
01:06:13,360 --> 01:06:15,200
THAT, BUT YOU HAVE TO DO SOME

1926
01:06:15,200 --> 01:06:18,040
THINGS BEYOND WHAT I'VE SHOWN

1927
01:06:18,040 --> 01:06:19,160
YOU.

1928
01:06:19,160 --> 01:06:20,320
 TO DO THAT.

1929
01:06:20,320 --> 01:06:21,160
AND WHAT WAS THE OTHER PART OF

1930
01:06:21,160 --> 01:06:25,960
THE QUESTION?

1931
01:06:25,960 --> 01:06:26,760
>>[INAUDIBLE]

1932
01:06:26,760 --> 01:06:27,080
>>YES.

1933
01:06:27,080 --> 01:06:28,840
>>THANKS, RON, FOR THIS
GREAT

1934
01:06:28,840 --> 01:06:29,720
TALK.

1935
01:06:29,720 --> 01:06:32,440
BACK TO THE T REG MODEL.

1936
01:06:32,440 --> 01:06:33,560
OF THIS HANDFUL OF PARAMETERS

1937
01:06:33,560 --> 01:06:35,040
THAT YOU SAID HAVE TO BE VERY

1938
01:06:35,040 --> 01:06:37,760
TIGHTLY REGULATED WITHIN

1939
01:06:37,760 --> 01:06:40,320
TWOFOLD, HAVE ANY OF THEM BEEN

1940
01:06:40,320 --> 01:06:43,920
SHOWN TO ACTUALLY -- THAT THE

1941
01:06:43,920 --> 01:06:47,080
CHANGE CAUSES THE AUTOIMMUNE

1942
01:06:47,080 --> 01:06:48,480
DISEASE IN PATIENTS OR --

1943
01:06:48,480 --> 01:06:52,880
>>SO THE ENHANCERS THAT
ALEX

1944
01:06:52,880 --> 01:06:55,600
MARSON HAS MANIPULATED ARE LOCI

1945
01:06:55,600 --> 01:06:57,800
OF SINGLE NUCLEOTIDE

1946
01:06:57,800 --> 01:06:58,680
POLYMORPHISMS THAT MATTER FOR

1947
01:06:58,680 --> 01:06:59,320
DISEASE IN HUMAN.

1948
01:06:59,320 --> 01:07:00,080
>>OKAY.

1949
01:07:00,080 --> 01:07:02,240
SO THEY'RE FROM -- ALL FROM GWAS

1950
01:07:02,240 --> 01:07:02,440
AND --

1951
01:07:02,440 --> 01:07:04,000
>>WELL, THE MOUSE IS NOT

1952
01:07:04,000 --> 01:07:06,200
IDENTICAL TO THE HUMAN, BUT

1953
01:07:06,200 --> 01:07:08,160
THEY'RE IN RELATIVE POSITIONS

1954
01:07:08,160 --> 01:07:10,840
WHERE THERE ARE HITS IN THE GWAS

1955
01:07:10,840 --> 01:07:11,320
STUDIES.

1956
01:07:11,320 --> 01:07:12,840
THERE'S A VERY HIGH-DENSITY OF

1957
01:07:12,840 --> 01:07:15,840
GWAS HITS IN CD25.

1958
01:07:15,840 --> 01:07:16,760
SO THAT'S WELL-KNOWN.

1959
01:07:16,760 --> 01:07:18,040
>>OKAY.

1960
01:07:18,040 --> 01:07:20,480
THANKS.

1961
01:07:20,480 --> 01:07:23,680
>>WE HAVE EIGHT
QUESTIONS, BUT

1962
01:07:23,680 --> 01:07:24,920
WE'RE PAST THE HOUR AT THIS

1963
01:07:24,920 --> 01:07:26,200
POINT SO LET ME JUST ASK ONE

1964
01:07:26,200 --> 01:07:29,760
MORE.

1965
01:07:29,760 --> 01:07:32,040
>>I CAN TAKE EIGHT
QUESTIONS.

1966
01:07:32,040 --> 01:07:33,760
>>OKAY, WE'LL SEE WHAT WE
CAN

1967
01:07:33,760 --> 01:07:34,040
DO.

1968
01:07:34,040 --> 01:07:36,320
DO YOU THINK THE SHIELDING

1969
01:07:36,320 --> 01:07:37,160
MICROPHAGES, QUOTE-UNQUOTE, YOU

1970
01:07:37,160 --> 01:07:38,520
TALKED ABOUT REGARDING TISSUE

1971
01:07:38,520 --> 01:07:41,440
DAMAGE COULD BE INVOLVED IN

1972
01:07:41,440 --> 01:07:42,440
IMMUNOSUPPRESSION AT TUMOR

1973
01:07:42,440 --> 01:07:43,440
SITES?

1974
01:07:43,440 --> 01:07:45,600
>>YES, THEY COULD BE.

1975
01:07:45,600 --> 01:07:48,120
THESE CELLS ARE, IF YOU WANT TO

1976
01:07:48,120 --> 01:07:52,880
USE A VERNACULAR, M2-LIKE, AND,

1977
01:07:52,880 --> 01:07:53,520
THEREFORE, ANTI-INFLAMMATORY AND

1978
01:07:53,520 --> 01:07:55,440
YOU WANT INFLAMMATORY RESPONSES

1979
01:07:55,440 --> 01:07:59,040
TO BE ANTITUMOR.

1980
01:07:59,040 --> 01:08:00,880
HOWEVER, THESE AS I SAID ARE

1981
01:08:00,880 --> 01:08:02,880
EMBRYO LOGICALLY DERIVED CELLS,

1982
01:08:02,880 --> 01:08:07,120
AND MOST OF THE MACROPHAGES IN A

1983
01:08:07,120 --> 01:08:08,320
TUMOR HAVE BEEN SHOWN TO COME

1984
01:08:08,320 --> 01:08:10,840
PRIMARILY FROM MONOCYTES THAT

1985
01:08:10,840 --> 01:08:12,920
THEN ENTER, AND THEN POLARIZE IN

1986
01:08:12,920 --> 01:08:15,520
THIS DIRECTION.

1987
01:08:15,520 --> 01:08:17,240
SO WHETHER IT A SIMILAR PROCESS

1988
01:08:17,240 --> 01:08:19,800
BUT NOT WITH THESE MORE RESIDENT

1989
01:08:19,800 --> 01:08:23,960
CELLS, I CAN'T TELL YOU.

1990
01:08:23,960 --> 01:08:25,760
>>HAVE YOU BEEN ABLE TO

1991
01:08:25,760 --> 01:08:30,600
OPTICALLY CLEAR AND USE CE 3D

1992
01:08:30,600 --> 01:08:32,440
IMAGING IN TUMOR TISSUE, AND

1993
01:08:32,440 --> 01:08:36,160
HAVE YOU OBSERVED TERTIARY

1994
01:08:36,160 --> 01:08:37,720
LYMPHOD STRUCTURES.

1995
01:08:37,720 --> 01:08:40,320
>>IN MOUSE TISSUES,
THAT'S

1996
01:08:40,320 --> 01:08:41,800
ONGOING, BUT IT'S BEEN DONE.

1997
01:08:41,800 --> 01:08:43,920
AGAIN, WE'RE TRYING TO FIGURE

1998
01:08:43,920 --> 01:08:45,640
OUT THE RULES FOR WHY SOME

1999
01:08:45,640 --> 01:08:47,000
TISSUES DON'T WORK SO WELL

2000
01:08:47,000 --> 01:08:47,720
VERSUS OTHERS.

2001
01:08:47,720 --> 01:08:50,440
IT SEEMS TO BE -- WE JUST HAD A

2002
01:08:50,440 --> 01:08:52,000
BIG MEETING YESTERDAY ABOUT

2003
01:08:52,000 --> 01:08:53,280
THIS, RELATED TO PERHAPS THE

2004
01:08:53,280 --> 01:08:55,800
LEVEL OF NECROTIC TISSUE WHICH

2005
01:08:55,800 --> 01:08:59,400
CAUSES SOME UNSPECIFIC STAINING,

2006
01:08:59,400 --> 01:09:01,880
AND THE BACKGROUND LEVEL OF AUTO

2007
01:09:01,880 --> 01:09:03,760
FLUORESCENCE, SO THOSE ARE SOME

2008
01:09:03,760 --> 01:09:04,400
CONFOUNDERS.

2009
01:09:04,400 --> 01:09:06,600
MOST MOUSE TUMORS DON'T SHOW

2010
01:09:06,600 --> 01:09:08,760
TERTIARY LYMPHOID STRUCTURES.

2011
01:09:08,760 --> 01:09:10,560
THEY'RE PREDOMINANT AND DOMINANT

2012
01:09:10,560 --> 01:09:12,160
AND VISIBLE IN MOSTLY HUMAN, AND

2013
01:09:12,160 --> 01:09:14,360
WE'RE JUST STARTING TO DO HUMAN

2014
01:09:14,360 --> 01:09:15,360
SAMPLES USING THIS METHOD.

2015
01:09:15,360 --> 01:09:16,120
>>OKAY.

2016
01:09:16,120 --> 01:09:17,640
NOT SURE IF THIS WAS ADDRESSED.

2017
01:09:17,640 --> 01:09:21,680
IN YOUR TUMOR STUDIES WITH

2018
01:09:21,680 --> 01:09:22,440
ANTICD40 AND SUBSEQUENT

2019
01:09:22,440 --> 01:09:25,640
CONVERSION OF T REGS TO

2020
01:09:25,640 --> 01:09:27,000
CONVENTIONAL CELLS, DO YOU HAVE

2021
01:09:27,000 --> 01:09:28,040
ANY INSIGHTS ON WHETHER THE

2022
01:09:28,040 --> 01:09:32,640
CELLS DOING THE CONVERSION ARE

2023
01:09:32,640 --> 01:09:34,480
NATURALLY THYMIC DERIVED T REGS

2024
01:09:34,480 --> 01:09:36,320
OR MORE PERIPHERAL DERIVED?

2025
01:09:36,320 --> 01:09:37,760
>>SO THE BEST WAY I CAN
ANSWER

2026
01:09:37,760 --> 01:09:42,360
THAT QUESTION IS, 60 TO 70% OF

2027
01:09:42,360 --> 01:09:43,920
THE -- WHAT WE CALL T REGS IN

2028
01:09:43,920 --> 01:09:47,760
THE TUMOR VOLUME ARE HELIOS

2029
01:09:47,760 --> 01:09:48,120
POSITIVE.

2030
01:09:48,120 --> 01:09:49,560
WHEN WE START BEFORE TREATMENT.

2031
01:09:49,560 --> 01:09:52,760
AND THAT HELIOS GOES AWAY IN THE

2032
01:09:52,760 --> 01:09:55,600
EX-T REGS AND ACTUALLY FROM A

2033
01:09:55,600 --> 01:09:56,560
SUBSTANTIAL FRACTION OF THE

2034
01:09:56,560 --> 01:09:57,880
RESIDUAL SO-CALLED T REGS WHICH

2035
01:09:57,880 --> 01:09:59,520
WE THINK ARE IN THE PROCESS

2036
01:09:59,520 --> 01:10:00,640
PROBABLY OF CONVERTING, THEY

2037
01:10:00,640 --> 01:10:04,760
JUST HAVEN'T FIP PIS FINISHED LL

2038
01:10:04,760 --> 01:10:06,000
THEIR FOX P3.

2039
01:10:06,000 --> 01:10:06,920
SO THAT WOULD SUGGEST THAT AT

2040
01:10:06,920 --> 01:10:08,160
LEAST A LARGE FRACTION OF THEM

2041
01:10:08,160 --> 01:10:11,320
WOULD BE THYMICLY DERIVED

2042
01:10:11,320 --> 01:10:12,720
CONVENTIONAL T REGS BUT WE CAN'T

2043
01:10:12,720 --> 01:10:14,440
RULE OUT THERE ARE PT REGS IN

2044
01:10:14,440 --> 01:10:16,560
THE POPULATIONS AS WELL.

2045
01:10:16,560 --> 01:10:17,960
>>ALMOST DONE, THE
LIGHTNING

2046
01:10:17,960 --> 01:10:18,960
ROUND HERE.

2047
01:10:18,960 --> 01:10:20,720
WE ALL BENEFIT FROM YOU AND YOUR

2048
01:10:20,720 --> 01:10:21,480
PROGRAM'S REMARKABLE CAREER.

2049
01:10:21,480 --> 01:10:23,200
THANK YOU FOR THE WONDERFUL

2050
01:10:23,200 --> 01:10:23,480
TALK.

2051
01:10:23,480 --> 01:10:26,440
IN A GIVEN AUTOIMMUNE DISEASE

2052
01:10:26,440 --> 01:10:28,200
AFFECTED TISSUE, DO YOU SEE A

2053
01:10:28,200 --> 01:10:33,080
RANGE OF CT REG-EX-T REG BALANCE

2054
01:10:33,080 --> 01:10:35,160
AND IN ORGANS, IS THERE SOME

2055
01:10:35,160 --> 01:10:36,920
SPATIAL ARRANGEMENT OF THESE

2056
01:10:36,920 --> 01:10:39,440
DIVERSE STATE?

2057
01:10:39,440 --> 01:10:44,840
EG ALONG THE TBUT OR WITHIN THEE

2058
01:10:44,840 --> 01:10:45,120
PANCREAS?

2059
01:10:45,120 --> 01:10:45,720
>>IT'S A VERY INTERESTING

2060
01:10:45,720 --> 01:10:46,080
QUESTION.

2061
01:10:46,080 --> 01:10:47,840
WE DON'T HAVE A GOOD METHOD FOR

2062
01:10:47,840 --> 01:10:49,680
ADDRESSING THAT IN THE HUMAN

2063
01:10:49,680 --> 01:10:50,960
SAMPLES AT THE PRESENT BECAUSE

2064
01:10:50,960 --> 01:10:54,240
WE CAN'T DO THE LINEAGE TRACING

2065
01:10:54,240 --> 01:10:56,640
TO IDENTIFY AN EX-T REG.

2066
01:10:56,640 --> 01:10:57,880
WHAT WE'RE TRYING TO DO IS

2067
01:10:57,880 --> 01:10:59,600
EITHER MINE EXISTING DATASETS

2068
01:10:59,600 --> 01:11:02,120
FROM SINGLE CELL RNA SEQ OR

2069
01:11:02,120 --> 01:11:03,640
CREATE SUCH DATASET THAT WOULD

2070
01:11:03,640 --> 01:11:06,480
ALLOW US TO IDENTIFY A FEW

2071
01:11:06,480 --> 01:11:07,800
MARKER PROTEINS THAT WE COULD

2072
01:11:07,800 --> 01:11:10,680
ADDRESS EITHER BY STAINING OR BY

2073
01:11:10,680 --> 01:11:12,920
FISH OR COMPARABLE

2074
01:11:12,920 --> 01:11:14,600
TRANSCRIPTOMIC METHODS TO

2075
01:11:14,600 --> 01:11:17,760
DISTINGUISH WHAT WAS AN EX-T REG

2076
01:11:17,760 --> 01:11:19,080
ASSUMING THAT THIS CONVERSION

2077
01:11:19,080 --> 01:11:21,240
DOESN'T CHANGE ALL THE

2078
01:11:21,240 --> 01:11:21,880
TRANSCRIPTIONAL PROFILE OF THE

2079
01:11:21,880 --> 01:11:22,240
CELL.

2080
01:11:22,240 --> 01:11:23,880
WHEN WE HAVE THAT INFORMATION,

2081
01:11:23,880 --> 01:11:25,400
THEN WE COULD DO THE TYPES OF

2082
01:11:25,400 --> 01:11:26,520
EXPERIMENT THAT ARE BEING ASKED

2083
01:11:26,520 --> 01:11:28,800
TO SEE WHAT THOSE BALANCES AND

2084
01:11:28,800 --> 01:11:30,560
THE DISTRIBUTIONS ARE IN VARIOUS

2085
01:11:30,560 --> 01:11:30,800
TISSUES.

2086
01:11:30,800 --> 01:11:32,320
WE COULD THEORETICALLY DO THAT

2087
01:11:32,320 --> 01:11:35,400
EXPERIMENT IN MICE WITH THE

2088
01:11:35,400 --> 01:11:38,120
LINEAGE SYSTEM, BUT WE HAVEN'T

2089
01:11:38,120 --> 01:11:38,360
DONE IT.

2090
01:11:38,360 --> 01:11:42,680
>>OKAY.

2091
01:11:42,680 --> 01:11:45,000
I WONDER IF IT IS POSSIBLE TO

2092
01:11:45,000 --> 01:11:46,880
CAPTURE CELL LOOR/TISSUE

2093
01:11:46,880 --> 01:11:51,480
DYNAMICS INCLUDING CELL

2094
01:11:51,480 --> 01:11:53,120
DIVISION/APOPTOSIS MOVING AND

2095
01:11:53,120 --> 01:11:55,600
INTERACTING IN 3D AS WELL, AND,

2096
01:11:55,600 --> 01:11:57,280
HAVE YOU LOOKED AT T-CELL

2097
01:11:57,280 --> 01:11:59,680
POPULATION IN THE CONTEXT OF

2098
01:11:59,680 --> 01:12:01,320
SOLID TUMORS AND THEIR LYMPH

2099
01:12:01,320 --> 01:12:02,840
NODES?

2100
01:12:02,840 --> 01:12:05,400
>>WE'RE DOING A LOT OF
WORK ON

2101
01:12:05,400 --> 01:12:06,880
THE TUMORS RIGHT NOW.

2102
01:12:06,880 --> 01:12:08,760
TUMORS ARE ACTUALLY HARD TO

2103
01:12:08,760 --> 01:12:11,720
IMAGE BY 2P BECAUSE FROM THE

2104
01:12:11,720 --> 01:12:13,680
TIME THEY'RE AS LARGE AS YOU'D

2105
01:12:13,680 --> 01:12:15,680
LIKE THEM TO BE TO BEGIN

2106
01:12:15,680 --> 01:12:17,320
PERTURBING THEM OR DOING

2107
01:12:17,320 --> 01:12:20,800
TREATMENT, THEY HAVE A LOT OF

2108
01:12:20,800 --> 01:12:22,760
LIMITATIONS THAT MAKE TWO PHOTON

2109
01:12:22,760 --> 01:12:23,440
IMAGING DIFFICULT.

2110
01:12:23,440 --> 01:12:24,880
THERE HAVE BEEN SOME GOOD

2111
01:12:24,880 --> 01:12:27,800
STUDIES FROM MAX CROMELL AND A

2112
01:12:27,800 --> 01:12:30,760
FEW OTHER LABS BUT FEW ARE IN

2113
01:12:30,760 --> 01:12:31,880
NORMAL TISSUE SO THAT'S A

2114
01:12:31,880 --> 01:12:32,200
PROBLEM.

2115
01:12:32,200 --> 01:12:34,400
TO ANSWER THE FIRST QUESTION,

2116
01:12:34,400 --> 01:12:39,440
ALL OF THE IMAGING IS DONE IN --

2117
01:12:39,440 --> 01:12:40,400
LIMITED BY THE SPEED OF THE

2118
01:12:40,400 --> 01:12:41,520
CELLS MORE THAN THE OPTICAL

2119
01:12:41,520 --> 01:12:42,000
PROPERTIES.

2120
01:12:42,000 --> 01:12:43,760
IF A T-CELL MOVES MORE THAN ONE

2121
01:12:43,760 --> 01:12:45,480
CELL DIE AM TER BETWEEN EACH

2122
01:12:45,480 --> 01:12:49,640
CYCLE OF IMAGES YOU COLLECT, YOU

2123
01:12:49,640 --> 01:12:51,160
CAN'T TRACE THE TRACK FOR THE

2124
01:12:51,160 --> 01:12:54,040
PATH, SO THAT LIMIT YOU TO ALL

2125
01:12:54,040 --> 01:12:56,160
THE DEPTH OF ABOUT 100 MICRONS

2126
01:12:56,160 --> 01:12:57,600
BUT WITHIN THAT VOLUME, YES, YOU

2127
01:12:57,600 --> 01:12:59,560
CAN SEE INTERACTIONS, YOU CAN

2128
01:12:59,560 --> 01:13:00,880
SEE CELLS DIVIDING AND WE

2129
01:13:00,880 --> 01:13:03,400
PUBLISHED ON THAT, YOU CAN SEE

2130
01:13:03,400 --> 01:13:06,160
CELLS UNDERGOING POP PTOSIS

2131
01:13:06,160 --> 01:13:06,560
PERFECTLY WELL.

2132
01:13:06,560 --> 01:13:08,760
>>WHAT DRIVES THE
SAMPLING OF

2133
01:13:08,760 --> 01:13:12,480
THE LUMEN IN T-CELLS IN THE GUT?

2134
01:13:12,480 --> 01:13:16,200
ARE THERE MICROBIOME-GENERATED

2135
01:13:16,200 --> 01:13:17,400
CHEMO ATTRACT ANTIS THAT ARE

2136
01:13:17,400 --> 01:13:20,640
KNOWN TO DO THIS?

2137
01:13:20,640 --> 01:13:23,440
>>THE ANSWER IS YES IN
PART.

2138
01:13:23,440 --> 01:13:24,640
THE STUDYS THAT HAVE BEEN DONE

2139
01:13:24,640 --> 01:13:27,680
ON THIS KIND OF DENDRITIC CELL

2140
01:13:27,680 --> 01:13:28,680
SAMPLING SUGGEST THAT THERE ARE

2141
01:13:28,680 --> 01:13:31,320
SIGNALS TO THE EPITHELIAL CELLS

2142
01:13:31,320 --> 01:13:36,440
THAT THEN MAKE CHEMO -- IT MAY

2143
01:13:36,440 --> 01:13:38,880
ALSO CHANGE THEIR ADHESIVE

2144
01:13:38,880 --> 01:13:40,800
PROPERTY FOR THE CELLS TO EXPAND

2145
01:13:40,800 --> 01:13:42,680
THEIR PROCESSES AND DO THE

2146
01:13:42,680 --> 01:13:44,120
SAMPLING BUT THE DETAILS ARE NOT

2147
01:13:44,120 --> 01:13:45,840
AS WELL-KNOWN AS ONE WOULD LIKE,

2148
01:13:45,840 --> 01:13:47,720
SO IT'S A GOOD QUESTION.

2149
01:13:47,720 --> 01:13:50,440
>>IT WAS FROM NINA SCHOR.

2150
01:13:50,440 --> 01:13:52,000
VERY INTERESTING TALK.

2151
01:13:52,000 --> 01:13:55,000
T REGS DRAIN IL2 TO ELIMINATE

2152
01:13:55,000 --> 01:13:56,360
AUTO REACTIVE T-CELLS.

2153
01:13:56,360 --> 01:13:59,000
WOULD YOU SUGGEST

2154
01:13:59,000 --> 01:14:01,080
ANTIIL-2 THERAPY TO TREAT

2155
01:14:01,080 --> 01:14:05,240
AUTOIMMUNE DISEASE?

2156
01:14:05,240 --> 01:14:07,400
>>UNFORTUNATELY NO
BECAUSE THAT

2157
01:14:07,400 --> 01:14:12,040
ALSO STARVES THE T REGS BECAUSE

2158
01:14:12,040 --> 01:14:13,640
IF YOU KILL THEM YOU CHANGE THE

2159
01:14:13,640 --> 01:14:15,160
BALANCE AGAIN, IN PRINCIPLE IN

2160
01:14:15,160 --> 01:14:16,320
FAVOR OF THE AUTO REACTIVE

2161
01:14:16,320 --> 01:14:16,600
CELLS.

2162
01:14:16,600 --> 01:14:18,240
SO IF ANYTHING, WHAT PEOPLE ARE

2163
01:14:18,240 --> 01:14:19,600
TRYING TO DO IS TO GIVE A LITTLE

2164
01:14:19,600 --> 01:14:23,520
BIT OF IL-2 IN A FORM THAT

2165
01:14:23,520 --> 01:14:25,240
FAVORS THE T-REGS BECAUSE OF

2166
01:14:25,240 --> 01:14:31,720
THEIR HIGHER CD T -- FROM REACTG

2167
01:14:31,720 --> 01:14:32,880
WITH CONVENTIONAL CELLS SO THAT

2168
01:14:32,880 --> 01:14:35,080
YOU GET T REG DOMINANCE OVER THE

2169
01:14:35,080 --> 01:14:36,200
CONVENTIONAL CELLS RATHER THAN

2170
01:14:36,200 --> 01:14:38,280
TRYING TO KILL THE CONVENTIONAL

2171
01:14:38,280 --> 01:14:42,640
CELLS BY STARVING THEM.

2172
01:14:42,640 --> 01:14:46,000
>>LAST ONE, IT'S FROM ED

2173
01:14:46,000 --> 01:14:47,440
SCHROM, IT'S THE LONGEST.

2174
01:14:47,440 --> 01:14:48,880
YOU MENTIONED THE T-CELLS FOLLOW

2175
01:14:48,880 --> 01:14:50,760
A RETICULAR NETWORK TO REDUCE

2176
01:14:50,760 --> 01:14:52,960
SEARCH TIMES WHEN ENCOUNTERING

2177
01:14:52,960 --> 01:14:55,800
DENDRITIC CELLS IN THE LYMPH

2178
01:14:55,800 --> 01:14:56,040
NODE.

2179
01:14:56,040 --> 01:14:58,200
BUT FOLLOWING THESE PREDEFINED

2180
01:14:58,200 --> 01:14:59,640
NETWORKS MIGHT ACTUALLY IMPEDE A

2181
01:14:59,640 --> 01:15:01,840
CELL'S ABILITY TO FOLLOW THE

2182
01:15:01,840 --> 01:15:03,120
CYTOKINE GRADIENT AS WHEN A

2183
01:15:03,120 --> 01:15:04,320
REGULATORY T-CELL MIGHT PREFER

2184
01:15:04,320 --> 01:15:07,880
TO MAKE A, QUOTE, BEELINE FOR

2185
01:15:07,880 --> 01:15:10,600
THE IL-2 PRODUCING CONVENTIONAL

2186
01:15:10,600 --> 01:15:13,320
T-CELLS AS IN HARRE'S WORK.

2187
01:15:13,320 --> 01:15:15,960
ARE T REGS LESS TIED TO THE

2188
01:15:15,960 --> 01:15:16,840
RETICULAR NETWORK?

2189
01:15:16,840 --> 01:15:20,440
AND/OR CAN T-CELLS ADAPTIVELY

2190
01:15:20,440 --> 01:15:23,840
MODULATE WHEN THEY ADHERE TO

2191
01:15:23,840 --> 01:15:26,360
VERSUS DISENGAGE FROM THE

2192
01:15:26,360 --> 01:15:29,880
RETICULAR NETWORK?

2193
01:15:29,880 --> 01:15:32,040
>>SO WE HAVE NOT SEEN
T-CELLS

2194
01:15:32,040 --> 01:15:34,120
MOVE OFF OF THE NETWORK WITHIN

2195
01:15:34,120 --> 01:15:35,680
THE LYMPH NODE EXCEPT MAYBE WHEN

2196
01:15:35,680 --> 01:15:37,240
THEY'RE EXITING THROUGH THE

2197
01:15:37,240 --> 01:15:37,880
LYMPHATICS.

2198
01:15:37,880 --> 01:15:39,080
BUT IN TERMS OF THE BEELINE,

2199
01:15:39,080 --> 01:15:44,520
WHAT WE HAVE SHOWN BY DIRECT

2200
01:15:44,520 --> 01:15:47,520
IMAGING IS THAT WHEN A SOURCE

2201
01:15:47,520 --> 01:15:49,360
PRODUCES A CHEMOKINE AND WE DID

2202
01:15:49,360 --> 01:15:51,120
CAREFUL WORK WITH THE DENDRITIC

2203
01:15:51,120 --> 01:15:52,120
CELL BUT IT COULD BE THE SAME

2204
01:15:52,120 --> 01:15:54,600
CELL MAKING IL2 IF IT'S

2205
01:15:54,600 --> 01:15:56,280
PRODUCING A CUOMO KINE BY ITSELF

2206
01:15:56,280 --> 01:15:58,680
OR THROUGH ITS INTERACTION WITH

2207
01:15:58,680 --> 01:15:59,600
A DENDRITIC CELL THAT TYPICALLY

2208
01:15:59,600 --> 01:16:01,680
DOES THIS, THAT BIASES THE WALK

2209
01:16:01,680 --> 01:16:04,440
IN THE FRST NETWORK AND ALTHOUGH

2210
01:16:04,440 --> 01:16:05,680
THERE ARE SOME VERTICES YOU HAVE

2211
01:16:05,680 --> 01:16:06,800
TO GO THROUGH, THE ANSWER IS

2212
01:16:06,800 --> 01:16:08,640
IT'S A RELATIVELY VERY FAST PATH

2213
01:16:08,640 --> 01:16:10,720
AND YOU CAN GET ABOUT AN EIGHT

2214
01:16:10,720 --> 01:16:12,680
FOLD IMPROVEMENT IN FINDING THE

2215
01:16:12,680 --> 01:16:17,320
RELEVANT CELL COMPARED TO AN

2216
01:16:17,320 --> 01:16:18,520
EQUIDISTANT RELEVANT CELL.

2217
01:16:18,520 --> 01:16:19,840
SO I'M NOT SURE IT IS A

2218
01:16:19,840 --> 01:16:22,560
LIMITATION OF THE SYSTEM TO DO

2219
01:16:22,560 --> 01:16:22,920
THAT.

2220
01:16:22,920 --> 01:16:24,160
AND ED CAN FOLLOW UP OBVIOUSLY

2221
01:16:24,160 --> 01:16:25,560
IN THE LAB SINCE WE TALK ALL THE

2222
01:16:25,560 --> 01:16:25,760
TIME.

2223
01:16:25,760 --> 01:16:26,400
>>VERY GOOD.

2224
01:16:26,400 --> 01:16:28,280
THANK YOU.

2225
01:16:28,280 --> 01:16:29,680
ALL THAT'S REMAINING, LET'S

2226
01:16:29,680 --> 01:16:30,280
THANK THE SPEAKER.

2227
01:16:30,280 --> 01:16:40,440
[APPLAUSE]