THIS, OF COURSE, IS THE MOST FAMOUS BRIDGE IN THE WESTERN WORLD, BROOKLYN BRIDGE, 1883 WE ARE THE PEOPLE ON THE CATWALK OF PEOPLE INTERESTED IN MEDICINE, DISEASE, PATIENTS AND SO FORTH AND ON THE OTHER HAND, THOSE WHO ARE PURSUING THE FUNDAMENTAL QUESTIONS OF BIOLOGY AND ENGINEERING WHICH HAVE PROFOUND IMPLICATIONS IN CLINICAL MEDICINE. BUT THE PROBLEM IS WE DON'T NORMALLY TALK TO ONE ANOTHER SO LIKE THESE TWO CHAPS ON THE BRIDGE BETWEEN BROOKLYN AND NEW YORK, THIS COURSE IS DESIGNED FOR COMMUNICATION AND WE URGE YOU TO COMMUNICATE WITH THE SPEAKERS, ALL THE INFORMATION, ALL THE POWERPOINTS, EVERYTHING FROM OF COURSE THE PAST 19 YEARS ON THE NIH WEBSITE SO YOU CAN SEE WHAT WE HAVE BEEN UP TO. TODAY WE'RE GOING TO HAVE AN EXCITING DISCUSSION ON AUTOIMMUNITY AND FORGIVE THE POETIC REFERENCE BUT I FIND IT ONE OF THE MOST INTERESTING FRONTIERS AND WE FIRST HAVE TO LOOK BACKWARD TO GO FORWARD AND IT WAS REALLY PAUL ERLICH IN THE 19TH CENTURY THAT CAME UP WITH THE DISORDER MEANING THINGS GOING ON IN THE HUMAN BODY THAT CAN CAUSE DISEASE AND NOT FROM STUFF ON THE OUTSIDE BUT THE FAILURE OF THE THE IMMUNOLOGIC STRUCTURE THAT PROTECTS US. AND IN THIS CONCEPT THAT HAS BEEN AROUND A LONG TIME, AND BY THE WAY, IT RECEIVED A GREAT ADDITION AND ALSO IN THE 19TH CENTURY WITH THE DISCOVERY OF THE MICROBIOME, METCHNIKOFF PROPOSED IT WAS THE MICROBIOME OF THE SOURCE THAT CAUSED THE ORGANISM TO ACT IN A MATTER THAT OVERCAME THE IMIMMUNOLOGIC SYSTEM AND AS WE KNOW NOW, THE IMMUNE SYSTEM CAN ATTACK ITSELF WHICH IS WHAT PROVIDES THE MOST FUNCTION OF THE AUTOIMMUNE DISEASE. SO IT WAS DISCOVERED THERE WAS A PORTAL ANDOGEN AND BODY THAT PRODUCED THE DISEASE AND THAT PRODUCED THE DISCUSSION OF SO-CALLED AUTO ANTIGENS CAUSING THE RESPONSE AND WE WILL HEAR ABOUT ALL OF THE RESPONSES TODAY BUT WHETHER THEY ARE PATHOGIC IS A HUGE QUESTION WHICH HAS NOT BEEN TRULY RESOLVED AND WHETHER THE DIAGNOSTIC HAS ADVANCED A LITTLE BIT OR PROGNOSTIC FOR WHETHER THEY ARE RELATED IN A PATHOGENETIC WAY TO THE DISEASE IS ONE OF THE CHALLENGES. SO IT SEEMS TO ME AND I AM SURE THERE ARE MANY MORE OF THEM BUT THE KEY ISSUE OF AUTOIMMUNITY WE WILL HEAR ABOUT TODAY IS WHETHER THE RESPONSES REPRESENT SOME MODIFICATION OF A NORMAL RESPONSE OR DO THEY REALLY ILLICIT A COMPLETELY FUNCTIONAL AND DIFFERENT PATHWAY AND CAST OF CHARACTERS? NOW, AUTOIMMUNE, THERE'S SOME RELATIONSHIP FOR TOLERANCE SO WHAT IS THAT RELATIONSHIP? AND FUNDAMENTALLY, OF COURSE, WHAT IS THE CELLULAR AND MOLECULAR BASIS OF AUTOIMMUNITY? SO TODAY'S LECTURE FEATURES TWO CHARACTERS WHO WILL REPRESENT THE CHARACTERS IN OUR BROOKLYN BRIDGE ON THIS HUGE PROBLEM. SO THE FIRST SPEAKER IS Dr. JAMES KATZ WHO WENT TO MEDICAL SCHOOL AT CASE WESTERN RESERVE, DID AN INTERNCY AND WAS A FIGURES FOR A NUMBER OF YEARS BEFORE COMING TO NIAMS. RECENTLY HE WAS APPOINTED AS THE DIRECTOR OF THE NIAMS FELLOWSHIP AND TRAINING BRANCH. Dr. KATZ IS MORE THAN AN ACCOMPLISHED CLINICIAN. HE IS A SUPERB EDUCATOR AND FROM HIS PUBLICATIONS AND INTERESTS, HAS EXTREME KNOWLEDGE OF WHERE THINGS STAND WITH RESPECT TO PARTICULARLY THE CLINICAL DIAGNOSTIC AND THERAPEUTIC ASPECTS OF AUTOIMMUNE DISEASE AND PARTICULARLY ARTHRITIS. AND THEN OUR SECOND SPEAKER IS RON GERMA A IN WHO IS CHIEF OF IMMUNE SYSTEM BIOLOGY AND CHIEF OF THE LEUKOCYTE BIOLOGY SECTION. HE IS AT MD, PHD GRADUATE FROM HARVARD WHO TRAINED THERE, WAS IN THE DEPARTMENT OF PATHOLOGY BEFORE HE RECRUITED TO COME HERE IN 1987 WHERE HE RAPIDLY BECAME THE DIRECTOR OF THE LABORATORY OF SYSTEMS BIOLOGY WHICH IS SUBSEQUENTLY BEEN CHANGED. RON IS WIDELY KNOWN INTERNATIONALLY FOR HIS EXTRAORDINARY WORK IN LOOKING AT THE IMMUNE MECHANISMS FROM THE STANDPOINT OF THE ACTUAL MOLECULAR DYNAMICS USING A VARIETY OF TECHNIQUES OF INDIGEN NATURE GIVEN THE DYNAMICS OF THE CELLULAR AND MOLECULAR LEVEL OF WHAT GOES ON WHEN THEY REALITY IN VIVO. HE HAS BEEN ELECTED TO THE NATIONAL ACADEMY OF MEDICINE, INSTITUTE OF MEDICINE AND A DISTINGUISHED INVESTIGATORS IN THE FIELD OF AUTOIMMUNEOLOGY. SO GREAT YOU ARE WITH US AND Dr. KATZ, LET'S BEGIN. >> THANK YOU SO MUCH. I AM REALLY QUITE FLATTERED TO COME HERE AND I ALSO SEE A LOT OF NIAMS FRIENDS. THANKS FOR COMING FOR YOUR SHOW OF SUPPORT AND ALSO YOU KNOW I AM HUMBLED TO BE WITH Dr. GERMAIN AND I CONSIDER MYSELF THE ADVANCE ACT OR THE COVER ACT BEFORE YOU GET THE REALLY GOOD STUFF. I AM AN EMPLOYEE OF THE FEDERAL GOVERNMENT. I HAVE NO DISCLOSURES THAT ARE PERTINENT TO THIS PARTICULAR TALK AND MY AGENDA HERE IS TO DO SOMETHING A LITTLE BIT DIFFERENT. YOU KNOW, I LOVE RHEUMATOLOGY, RIGHT? I EVEN MADE UP A JOKE ABOUT RHEUMATOLOGY THAT I WILL SHARE WITH YOU. HOW MANY RHEUMATOLOGISTS DOES IT TAKE TO SCREW IN A LIGHT BULB? AT LEAST TWO AS IT IS ALWAYS A JOINT EFFORT? [LAUGHTER] >> I AM NOT JUST A SPEAKER, I AM A DOCTOR AND I SEE ARTHRITIS WHICH IS AN BE A -- ABERRATION IN THE IMMUNE SYSTEM AND SO MANY GET ARTHRITIS, AND HOPEFULLY BY THE END OF MY SHORT TALK YOU WILL KIND OF UNDERSTAND I AM MERELY A SYSTEMIC INFLAMMATION. BUT I WANT TO TRY TO GIVE YOU A SENSE OF THE LIVED EXPERIENCE OF AUTOIMMUNE DISEASE, TO MAKE IT REAL, TO BRING IT HOME BECAUSE FROM THERE, I HOPE TO INSHIRE YOU TO THINK ABOUT THE IMMUNE SYSTEM AND WHAT THE IMMUNE SYSTEM HAS TO DO WITH THE LIVED EXPERIENCE OF AUTO IMMUNITY AND THEN TO REALLY INSPIRE YOU TO FOLLOW THE FOOTSTEPS OF Dr. GERMAIN AND GO INTO THE LAB AND STUDY IT MORE, BECAUSE THAT IS WHAT WE NEED. SO HOW DO I DO THIS? HOW DO I BRING IT TO LIFE? I DO IT BY STARTING WITH A GAME. I AM GOING TO PLAY A GAME WITH ALL OF YOU AND THAT MEANS I NEED SOME VOLUNTEERS. I NEED THREE VOLUNTEERS. I NEED SOMEONE WHO WILL BE MY JUDGE AND TWO CONTESTANTS YOU SAY GO, YOU HAVE TO DO THE ARTHRITIC BUTTONING. >> ON YOUR MARK, GET SET, GO. [LAUGHTER] >> ONE BUTTON OVER HERE. YAY! TWO BUTTONS. OH, ONE BUTTON. [LAUGHTER] >> AND IF WE HAVE A WINNER, YOU CAN CALL IT. >> WE HAVE A WINNER. >> WILL THE WINNER PLEASE COME TO THE STAND AND EXPLAIN WHAT YOU JUST DID? >> WELL, I CHEATED. [LAUGHTER] >> I USED A DEVICE CALLED A BUTTON HOOK WHICH IS USED PRIMARILY FOR PEOPLE WITH ARTHRITIS WHO HAVE DIFFICULTY PUTTING ON THINGS LIKE THIS. >> HOW DOES IT WORK? CAN EVERYBODY SEE HOW IT WORKS? YOU CAN TAKE YOUR GLOVES OFF. >> SURE, THE WAY IT WORKS IS YOU PUT THE DEVICE THROUGH THE LOOP FIRST AND THEN YOU GO AHEAD AND GRAB YOUR BUTTON AND IT HAS A SPACE FOR YOU TO GRAB IT AND PULL IT ON THROUGH AND IT MAKES IT QUITE EASY AND YOU JUST HAVE TO TWIST IT OFF SO IT MAKES IT QUITE EASY TO PUT ON BUTTONS. >> WE'LL PASS IT AROUND SO YOU CAN SEE WHAT IT LOOKS LIKE BUT CAN YOU TELL EVERYBODY WHAT IT FEELS LIKE TO HAVE ARTHRITIS? >> OH, MY GOD, IT IS VERY HUMBLING. I DIDN'T REALIZE LIKE DEXTERALLY, IT WAS SO DIFFICULT AND THEN WHILE I WAS TRYING, THERE WAS AN EMOTIONAL COMPONENT LIKE WOW, I CAN'T DO THIS. SO THERE WAS A FEELING COMPONENT AND THEN AN EMOTIONAL COMPONENT THAT I CAN'T DO THIS BASIC FUNCTION. >> YOU WERE FRUSTRATED. >> YES. >> THANK YOU SO MUCH. CAN WE HAVE A HAND WITH OUR ARTHRITIS PATIENTS? YOU WERE UPSET WE HAD A CHEATER? >> OKAY, LET'S TALK MEDICINE BUT BEFORE WE DO THAT, I AM TALKING, OUR ARTHRITIS PATIENTS COME TO ME AND SAY I KNOW WHAT IT FEELS LIKE TO GET OLD. THEY SAY I AM NORMALLY A STRONG PERSON AND I CAN'T EVEN OPEN A JAR, TURN A KEY IN THE DOOR, BUTTON BUTTONS. THEY LEAVE THE JAR TOPS OFF IN THE REFRIGERATOR SO THEY DON'T HAVE TO DEAL WITH IT. AND WE DON'T HAVE A LANGUAGE FOR PAIN. WHEN JOINTS HURT, WE SAY IT HURTS BUT PEOPLE WILL COME UP WITH ALL KINDS OF VIVID WAYS OF WHO SAID TO ME I FEEL LIKE I AM WALKING ON BROKEN GLASS. IT IS KIND OF THREATENING TO OUR PERSONAL SENSE OF IMMORTALITY TO SEE OURSELVES DETERIORATE WITH JOINT DAMAGE, IS A THREAT TO OUR PERFECT IDENTITY. AND IT IS A BEAUTIFUL PARALLEL METAPHOR BECAUSE AUTOIMMUNITY IS A THREAT TO OURSELF AS A WELL. WHEN YOUR OWN IMMUNE SYSTEM RECOGNIZES YOU AS DANGEROUS, AS AUTOXICUS, WHO IS GOING ON WITH AND YOU WHO YOU ARE? SO I AM AN EXISTENTIAL RHEUMATOLOGIST. NOW, WHO CAN TELL ME WHO PAINTED THIS? ARE THERE ANY HISTORIANS IN THE AUDIENCE? ANYBODY RECOGNIZE THIS? THANK YOU, RENOIR. HOW DID YOU KNOW THAT? [ OFF MIC ] >> YOU CAN TELL. PIERRE AUGUST RENOIR. AND WHY WOULD I WANT TO TALK ABOUT RENOIR? WELL, PARTLY BECAUSE HE WAS AN IMPRESSIONIST, PART OF THE WHOLE IMPRESSIONIST MOVEMENT. HE WAS VERY FAMOUS BECAUSE HE WAS AMONG THE VERY FIRST OF THE IMPRESSIONISTS AND THE LATER ON DECIDED HE DIDN'T LIKE IMPRESSIONISM AND TOTALLY CHANGED HIS STYLE. BUT HE HAD RHEUMATOID ARTHRITIS. BACK THEN, IT WAS CALLED RHEUMATISM. AND NOW WE SEPARATE OUT ALL THE DIFFERENT KINDS OF INFLAMMATORY ARTHRITIS THAT AUTOIMMUNITY CAN CAUSE AND WE NO LONGER LUMP IT IN THE TERM OF RHEUMATISM BUT WE TALK ABOUT SYSTEM ELK LUPUS AND MYECITIS AND RHEUMATOID ARTHRITIS. HERE IS A PICTURE OF HIM WHEN HE IS OLDER AND THE ARTHRITIS HAS DONE SOME DAMAGE TO HIS HANDS. BUT THIS IS ONE OF THE PAINTINGS THAT HE DREW AND ONE TIME-OUT HIGHLIGHT. I BELIEVE IF YOU WANT TO GO SEE IT, YOU HAVE TO GO TO LOS ANGELES AND IT IS A PICTURE, IF I AM NOT MISTAKEN, OF HIS SON WHO WOULD GO HUNTING BUT IT HAS BEEN TAUGHT BY HISTORIANS THAT IT WAS AN ATTEMPT TO FIGHT HIS ARTHRITIS AND DISABILITY BY PORTRAYING STRENGTH, BOLDNESS, UPRIGHTNESS AND THAT IT WAS, FOR HIM, A WAY OF SAYING I AM NOT GIVING IN TO RHEUMATOID ARTHRITIS. BUT HOW BIG IS 68 INCHES? HOW TALL IS THAT? 172 CENTIMETERS, SO THAT IS ABOUT FIVE FEET, RIGHT? AND THIS IS RENOIR. HE WAS CONFINED TO A WHEELCHAIR. NOT ONLY THAT, HE HAD FROZEN SHOULDER. HE COULD NOT REACH OVERHEAD. HIS HANDS WERE DEFORMED AND HIS WIFE WOULD TAPE OR WRAP THE PAINTBRUSH IN HIS HAND. IT WAS PUT BETWEEN TWO FINGERS, IT WAS WRAPPED TO HIS HAND AND THAT IS HOW HE WOULD PAINT. BUT I AM STILL LEFT ASKING YOU, HOW COULD HE REACH, AT FIVE FOOT, THE TOP OF HIS PAINTING? SO HE WAS ALSO AN INVENTOR. HE GOT A BICYCLE CHAIN AND GEARS AND DEVELOPED A CRANK SO THAT HE COULD CRANK THE PICTURE UP OR DOWN SO HE COULD PAINT AT ONE LEVEL AND DIDN'T HAVE TO LIFT HIS FROZEN SHOULDER. THERE ARE NO PICTURES OF THAT DEVICE BUT IT HAS BEEN WRITTEN ABOUT EXTENSIVELY AND THEN IN THE BIBLIOGRAPHY, THERE ARE A LOT OF PAPERS YOU CAN READ ABOUT IT. SO LET'S KEEP IT SIMPLE. RHEUMATOID ARTHRITIS, NOT WHAT IT WAS CALLED BUT I AM GOING TO TALK ABOUT IT AS A PROTOTYPE OF THE CONDITION THAT WAS PARTICULARLY DEVASTATING TO RENOIR. WE DON'T KNOW WHAT CAUSES IT BUT I WILL ELIMINATE SOME OF WHAT IT IS ALL ABOUT AND WHAT WE DO KNOW. WE KNOW VERY LITTLE. WE KNOW IT IS VARIABLE IN ITS PRESENTATION. WE KNOW IT IS CHRONIC AND PROGRESSIVE IF YOU DON'T INTERVENE, YOU WILL END UP WITH DAMAGE TO JOINTS AND DAMAGE MEANS DEFORMITIES. AND HERE YOU CAN SEE SOME EXAMPLES OF WHAT IS CALLED THE DEVIATIONS, SOMETIMES CALLED THE WINDSWEPT HAND. YOU CAN ALSO SEE THE ENLARGED KNUCKLES, THE PROGRESSMENT AND WASTING OF THE MUSCLES OF THE HAND AND THAT IS NOW EVIDENCE HOW DISABLING IT IS. YOU FELT IT WHEN YOU CAME DOWN HERE AND YOU TRIED TO BUTTON BUTTONS WITH GLOVES. AND R ARE -- RENOIR WAS TYPICALLY 45 TO 50 WHEN IT STARTED AND IT IS MORE PREVALENT IN WOMEN THAN MEN BUT THERE ARE SOME POPULATIONS THAT ARE ENRICHED WITH RHEUMATOID ARTHRITIS. THERE ARE SOME POTENT INFLUENCES, INCLUDING THIS GENETIC FINDING BECAUSE IT ALSO RELATES TO OTHER AUTOIMMUNE DISEASES AND NOT JUST RHEUMATOID ARTHRITIS. IT IS NOT ONLY A MARKER FOR RA BUT A MARKER FOR RA SEVERITY. AND ALTHOUGH YOU CAN'T SEE IT VERY WELL, THIS IS A STILL PICTURE OF RENOIR FROM A MOVIE THAT YOU CAN GO ON YOUTUBE AND SEE AND HE IS SMOKIN IN THIS VIDEO THAT HAS BEEN TAKEN OF HIM. I BELIEVE IT IS HARD TO SEE BUT THE CIGARETTE IS RIGHT THERE IN HIS HAND AND WE KNOW THAT IS A STRONG RISK FACTOR AMONG OTHER ENVIRONMENTAL CONSIDERATIONS TO DEVELOPING RHEUMATOID ARTHRITIS. SO HOW DO I DIAGNOSED RHEUMATOID ARTHRITIS IN THE CHIN? THE RULE OF S'S. SEM METRIC SYNOVITIS MEANING INFLAMMATION OF THE JOINTS, A LONGSTANDING DURATION GOING ON WEEKS OR MONTHS. A VIRAL ARTHRITIS WILL PRESENT LIKE RHEUMATOID ARTHRITIS BUT GO AWAY ON ITS OWN. RHEUMATOID ARTHRITIS PATIENTS ARE MAKING BODIES THAT ARE PRESENT IN THE BLOOD, THE MORE COMMON ONE, CCP ANTIBODY. AND THEN FOR YEARS WE HAVE KNOWN ABOUT THE SED RATE. YOU DRAW BLOOD, SIT IT ON THE COUNTER FOR AN HOUR AND SEE HOW FAST THE RED CELLS SETTLE AS A MARKER OF INFLAMMATION. SO IT IS AN INFLAMMATORY DISEASE WE CAN MEASURE IN BLOOD WITH THE SED RATE AND AN AUTOIMMUNE DISEASE WE CAN MEASURE WITH AUTO ANTI-BODIES AND ONE YOU CAN SEE HERE IN PICTURES. THIS IS AN EARLIER PICTURE OF RENOIR AND YOU CAN SEE ENLARGED KNUCKLES, HIS THUMB ENLARGED FROM THE SWELLING. THIS I BELIEVE IS HIS WALKING CANE AND THIS IS WHEN HIS RHEUMATOID ARTHRITIS WAS ACTUALLY EARLY IN THE INFLAMMATORY PROCESS. BUT LIKE MOCITIS, SCLERODERMA, RHEUMATOID ARTHRITIS IS A MULTISYSTEM DISEASE. IT DOES NOT JUST AFFECT JOINTS. YOU CAN GET SKIN THINGS, THIS IS A HUMANNODULE, YOU CAN GET SEROSYVITIS WHICH IS THE INFLAMED LINING AND YOU CAN SEE THAT IN A CAT SCAN. IT CAUSES FEVERS. IT CAN CAUSE HEART PROBLEMS. IT LOVES TO CAUSE EYE INFLAMMATION. THIS IS TRUE AUTO IMMUNITY. A CLOSE-UP OF WHAT A SWOLLEN JOINT LOOKS LIKE IS HERE. WE USE THE TERM HERE BECAUSE WHEN YOU PUSH ON THE JOINTS, IT IS SQUISHY AND SPONGEY-FEELING. IT IS A MIX SURE -- MIXTURE OF FLUID AND INFLAMED TISSUE ON THE JOINT AND I WILL POINT OUT TO YOU THE YELL WILL HE NAILS STRONGLY SUGGEST WHAT ABOUT THIS PATIENT? SMOKING HISTORY. IF YOU WANT ANOTHER VIVID IMAGING OF INFLAMMATION, EVEN THOUGH IT IS AN OLD FASHIONED BONE SCAN, AND YOU SAW THOSE FINGERS SWOLLEN IN THE JOINTS, YOU DO A BONE SCAN AND IT IS IN MULTIPLE JOINTS AND IMAGINE WAKING UP WITH THIS INFLAMMATION OF JOINTS AND FEELING STIFF AND ACHY AND SORE. AND MY PATIENTS ARE NOTORIOUSLY GRUMPY WHEN THEY HAVE THIS ARTHRITIS. BUT THIS IS WHAT I AM FIGHTING AND THIS IS OVER A TEN-YEAR PERIOD. THIS IS THE BEGINNING OF ARTHRITIS WITH SOFT TISSUE SWAYING, HERE IS THINNING OUT OF THE BONE AT THE MARGIN OF THE JOINT AND HERE, IT LOOKS LIKE A RAT TOOK A BITE OUT OF THAT JOINT. THIS IS CALLED AN ARRASIAN AND THIS DAMAGE IS NOT SOMETHING WE CAN REPAIR WITH PILLS OR SHOTS OR MEDICINES, LEAVING US TO WANT TO TREAT IT HERE TO PREVENT THAT. YOU WILL ALSO SEE THAT THE JOINT SPACE IS COMPROMISED OVER HERE AND YOU BEGIN TO REALLY UNDERSTAND THAT THE SHEER BURDEN OF MULTIPLE JOINT DAMAGE CAN LEAD TO THE DEFORMITIES THAT YOU SAW IN THE PICTURES OF RENOIR. THIS IS DAMAGE TO EACH OF THESE JOINTS -- THE WRISTS, THE THUMB, THESE JOINTS HERE AND THEY LEAD TO HANDS THAT LOOK LIKE THAT. NOW, YOU CAN STILL HAVE A GRIP BUT YOU WON'T HAVE AS STRONG A GRIP. AND I DO WANT TO POINT OUT THAT THESE ARE THE NODULES, NOT THE SWELLING OF THE JOINT. THERE ARE TWO DIFFERENT ISSUES GOING ON HERE. HERE IS THE FAMOUS BOOTINIERE'S DEFORMITY FAMED FOR THE PEOPLE WHO WOULD SEW ALL THE TIME. AND THIS IS THE HYPER EXTENSION AT THE DIP AND THESE ARE CALLED NONREDUCABLE DEFORMTIES. THAT MEANS THAT THEY HAVE DAMAGED THE JOINTS SO MUCH YOU CANNOT REALIGN THE JOINTS BACK TO NORMAL. SO WHAT IS GOING ON? LET'S TALK ABOUT THE TISSUE LEVEL. IF YOU TAKE A SCOPE AND YOU LOOK INSIDE A RHEUMATOID ARTHRITIS JOINT, WELL, A NORMAL ONE IS GOING TO LOOK LIKE THIS. EARLY IN RHEUMATOID ARTHRITIS, YOU ARE STARTING TO SEE THE INCREASE IN RED BLOOD SUPPLY, A LITTLE EXTRA TISSUE AND THEN LATER IN THE DISEASE, YOU GET THESE FROG-LIKE EXTENSIONS OF THE SYNOVIAL TISSUE, THE LINING TISSUE OF THE JOINT THAT IS PROLIFERATING LIKE A TUMOR. THAT IS WHAT I DO, I FIGHT A BENIGN TUMOR CALLED RHEUMATOID ARTHRITIS. AND EVENTUALLY YOU HAVE EATEN AWAY AT THE CARTILAGE SO YOU HAVE EXPOSED BONE AND DEBRIS FROM ALL THE CARTILAGE THAT IS CHEWED UP. IF YOU WANT TO ZOOM IN A LITTLE MORE WITH THE MICROSCOPE, ON THIS SIDE IS THE NORMAL LINING OF THE TISSUE ON THE JOINT AND HERE IS THE VERY INTENSELY INFLAMED LINING OF A RHEUMATOID JOINT, EXACTLY WHERE YOU ARE GOING TO SEE THESE GERMINAL ARRANGEMENTS THAT WE'RE GOING TO TALK ABOUT AND Dr. GERMAIN WILL POINT OUT WHERE THE T-CELLS AND IMMUNE CELLS AND ALL THE CELLS ARE MEETING UP TO PRODUCE THEIR NASTY PLAN AND AUTO ANTIBODIES AND JOINT DAMAGE. SO THIS IS A NORMAL BONE INTERFACE WITH CARTILAGE AND THIS IS THE LINING AND IT IS THIS THING CALLED PANNUS THAT I HAVE BEEN SHOWING YOU WHERE YOU WILL FIND T-CELLS, MICROPHAGES BUT YOU WILL ALSO SEE FIBROBLASTS AND INCREASEDEPITHELIAL CELLS. SO PANNUS IS A VAST TISSUE, YOU HAVE SCAR TISSUE AND STIMULUS TO BRING IN NEW BLOOD FLOW, NEW BLOOD VESSELS TO SUPPLY THAT VERY ENLARGED TISSUE. BUT THE STORY, THE IMMUNOLOGY STORY OF RHEUMATOID ARTHRITIS IS COMPLEX AND I AM VERY LOATHE TO SAY IT STARTS HERE AND ENDS HERE BUT THE STORY IS VERY FAMILIAR ACROSS ALL MY AUTOIMMUNE DISEASES. MANY OF THEM WILL HAVE SOME STIMULUS OR ABERRATION OF THE AILING IMMUNE SYSTEM EARLY ON. MANY WILL HAVE EVIDENCE OF IMMUNE AUTOBODIES WITH THAT OCCURRING IN THE JOINT, THE PEP TIDES CERTAINLY MARKERS AND PROBABLY PLAY A ROLE IN THE AUTOIMMUNE PROCESS AND YOU HAVE THE TRADITIONAL ANTIGEN PRESENTATION THAT WILL PERPETUATE THE DISEASE. SO WE HAVE INCITEING EVENTS AND THE ANTIGEN AND IF YOU WANT TO COMBAT RHEUMATOID ARTHRITIS, YOU HAVE TO WORK ON BOTH TO MAKE AN IMPACT. T-CELLS DO PLAY AN IMPORTANT ROLE IN RHEUMATOID ARTHRITIS. WE KNOW THEY CONSTITUTE A LARGE NUMBER OF CELLS IN THE SYNOVIUM AND I WANT TO POINT OUT THAT THIS IS KIND OF IRONIC BECAUSE -- IRONIC BECAUSE INSIDE THE SYNOVIAL FLUID, THE MAJOR CELL TYPE IS ACTUALLY NEUTROPHILS SO WE HAVE A LOT TO LEARN ABOUT RHEUMATOID ARTHRITIS. I AM RUNNING LOW ON TIME BUT I WANTED TO POINT OUT THIS ENVIRONMENT IS ALSO A PRO OSTEOCLAST ENVIRONMENT, AN ENVIRONMENT THAT EATS AWAY AT BONE. YOU SAW THIS WHEN YOU SAW THE EROSION. I AM POINTING OUT THAT RENOIR BROKE HIS ARM TWICE, THE SAME ARM HE WAS PAINTING WITH AND PEOPLE LIKED TO POINT OUT HOW RESILIENT HE IS BUT THAT IS NOT A SURPRISE. OSTEOBLASTS ARE TURNED OFF IN RHEUMATOID DISEASES AND AS YO -- AND OSTEO CLASTS ARE PRESENT. ALFALFA WAS ORIGINALLY CALLED WHAT WHEN IT WAS DISCOVERED? THEY FOUND IT IN HIGH LEVELS AND IT WAS A WASTING PROTEIN AND INDEED WE TREAT RHEUMATOID ARTHRITIS WITH THE ANTI-CACHEXUM. I CAN'T DO IT BY MYSELF, SOMETIMES I HAVE TO HAVE THE HELP OF OTHER SURGEONS, PHYSICIAN THERAPISTS, A PHARMACIST IN THE CLINIC AND I SPEND A LOT OF TIME EDUCATING MY PATIENTS ABOUT THE MEANING WHAT HAVE THEY ARE GOING THROUGH AND THE OPTIONS FOR MANAGING IT. SO EARLY DIAGNOSIS AND AGGRESSIVE MANAGEMENT IS WHAT WE'RE REALLY TALKING ABOUT. THE STATE-OF-THE-ART SMALL MOLECULES IN METHOTREJ -- METHOTREXATE, A ONCE A WEEK MEDICATION VERSUS ONCE A DAY AND REQUIRES FREQUENT EVALUATION BY THE PHYSICIANS TO MAKE SURE IT IS BEING USED SAFELY. SO BIOLOGICAL AGENTS ARE QUICKLY TAKING OVER THE SCENE AND IN SOME WAYS, SAFER THAN METTHOTREXATE BUT MORE TARGETED IN SUPPRESSING KEY ASPECTS OF THE IMMUNE SYSTEM. I ALREADY MENTIONED TO YOU ANTI--TNF AND IT IS NICE BECAUSE IT DOES WHAT YOU WANT IT TO DO, SUPPRESSED IL-6 AND THESE ARE THE MAIN AGENTS THAT PEOPLE ARE USING THESE DAYS. BUT WE HAVE OTHER WAYS OF INTERFERING WITH THE IMMUNE SYSTEM. WE CAN INTERFERE WITH THE CTA-4 SYSTEM, INTERFERE WITH B-CELLS, INTERFERE WITH THE INFLAMMOSOME AND INTERFERE WITH INTERLUKIN-6. SO THAT IS THE DEFINITION OF THE DISEASE, I HOPE I HAVE GIVEN YOU SOME IDEA AND THE SYMPATHY FOR THOSE WITH THE DISEASE AND AS WE SET THE STAGE FOR THE T-CELL THAT RELATES TO AUTOIMMUNE DISEASE. [APPLAUSE] >> THANK YOU VERY MUCH. WAIT A MINUTE, WE HAVE SOME TIME. PLEASE, IF YOU HAVE QUESTIONS FOR DOCTOR KATZ, EITHER WAVE YOUR HAND OR GO TO THE MICROPHONE. >> HI -- OH, SORRY, WERE YOU GOING FIRST? I WILL LET YOU GO. >> GO AHEAD. >> SO THIS IS NOT GOING TO SOUND AS EXTREME BUT IN CASES WHERE IT DEVELOPS TO THE POINT WHERE THE HANDS ESSENTIALLY ARE NO LONGER FUNCTIONAL, IS ELECTIVE AMPUTATION EVER PRESENTED AS A POTENTIAL OPTION FOR& INTERVENTION IN REGARDS TO AT LEAST RECENTLY ASSUMING THE PATIENT'S ELIGIBLE, THERE ARE NOT PROSTHETICS THAT WOULD PROBABLY ALLOW AT LEAST A RELIABLE GRIP ENOUGH THAT PERFORMANCE ON BASIC ACTIVITIESIVITY OF DAILY LIVING WOULD BE BETTER THAN THEY WERE GIVEN THEIR STATE. SO IS THAT EVER A TOPIC OF DISCUSSION? >> NOT IN MY CLINIC. I WOULD SCARE OFF A LOT OF PATIENTS -- >> YEAH, I KNOW BUT -- >> BUT YOUR POINT IS WELL TAKEN. PROSTHETICS, PROSTHESIS AND BIOMECHANICAL ASSISTIVE DEVICES ARE A HOT TOPIC FOR MANY OF OUR PATIENTS. YOU SEE MANY PEOPLE WALKING WITH AN EXOSKELETON AND WE ARE NOT THAT FAR WITH THE SMALL DIGITS OF THE HAND. THEY ARE REALLY HARD TO ADDRESS BOTH WITH SURGERY AND SPLINTING IS NOTORIOUSLY A BIG FAILURE. SURGERY TO FUSE A WRIST ON PURPOSE DOES WORK QUITE WELL. YOU LOSE FLEXIBILITY BUT YOU GAIN STRENGTH SO BEFORE WE TALK ABOUT ANYTHING AS EXOTIC AS YOU ARE MENTIONING, THERE IS A LOT THAT THE SURGEONS CAN DO ALTHOUGH I AM NOT A SURGEON, TO KEEP PATIENTS SOMEWHAT FUNCTIONAL. THEY MAY NOT HAVE THE PEST GOLF GRIP BUT WE CAN PRESERVE IT IN MANY CASES SURGICALLY. >> YOU USED RENOIR AS AN EXAMPLE BUT HE IS A MALE AND THIS IS NORTH -- NOTORIOUSLY A FEMALE DISEASE. I WOULD LIKE TO HEAR MORE ABOUT THAT AND DOES THIS PERSIST IN CHILDREN? >> THANK YOU SO MUCH. SO THE AUTOIMMUNE DISEASES AS A GENERAL RULE AFFECT WOMEN MORE COMMONLY THAN MEN. IN WEAKNESS, IT MIGHT BE NINE TIMES MORE FREQUENT IN WOMEN. TO MAKE MATTERS WORSE, THE AUTOIMMUNE DISEASES PARTICULARLY EFFECT WOMEN IN CHILD BEARING YEARS CAUSING GREAT STRESS BECAUSE THAT IMPACTS FAMILY DECISION-MAKING BOTH IN TERMS OF WHAT MEDICINES WE USE AND IN TERMS OF THEIR FUNCTION AND ABILITY TO CARE FOR THEMSELVES. IT IS A HOT TOPIC AND I DON'T EVEN WANT TO TOUCH THAT WITH A 10-FOOT POLE BECAUSE THE ENDOCRINE SYSTEM DOES PROFOUNDLY IMPACT THE IMMUNE SYSTEM BUT I CAN'T TEASE IT OUT ENOUGH TO MAKE GROUP SENSE FOR WHY THAT WOULD BE. >> ANY OTHER QUESTIONS THAT YOU HAVE? OKAY, WELL, THERE WILL BE TIME AFTER Dr. GERMAIN'S TALK SO WE CAN ASK YOU SOME MORE. THANK YOU VERY MUCH, Dr. KATZ. [APPLAUSE] >> IT IS ACTUALLY VERY CHALLENGING TO FOLLOW Dr. KATZ' TALK. HE HEADS A TRAINING PROGRAM BUT HIS EDUCATIONAL SKILLS ARE NOT APPROPRIATELY RECOGNIZED UNTIL YOU ACTUALLY HEAR HIM GIVE A TALK LIKE THAT. IT IS REALLY -- AND NOW FOR SOMETHING COMPLETELY DIFFERENT AND I HAVE A DIFFERENT APPROACH TO WHAT I AM GOING TO TELL YOU WITH SOME DETAILS. I WENT TO QUITE A FEW OF THIS YEAR'S LECTURES AND I WAS SURPRISED THAT IT WASN'T EDUCATING ALONE AND THINGS WERE A BIT MORE HIGH-END. SO I AM GOING TO ASSUME THAT SOME OF YOU WILL ACTUALLY UNDERSTAND REAL DATA, I WILL EXPLAIN TERMS AS WE GO ALONG AND SEE WHERE WE END UP. SO DISCLOSURES, I AM A FEDERAL EMPLOYEE AND OBJECTIVES, SO YOU CAN UNDERSTAND WHAT THE IMMUNE SYSTEM IS DOING THROUGH OPTIC IMAGING, TRYING TO UNDERSTAND THE ROLE OF SPATIAL ORGANIZATION WHICH YOU ONLY CAN DO IF YOU WORK AT SOMETHING AND NOT TEAR IT APART LIKE PEOPLE DO WITH GENOMIC ANALYSES AND WITH RESPECT TO OUR TALK ABOUT RELEVANT -- REGULATORY T-CELLS, BECAUSE OF THE T-CELL LEVEL, WHAT I MAKE CLEAR EARLY IN THE TALK IS THE NOTION THAT YOU LEARN WHAT THE SELF IS ACCURATELY IS CLEARLY NOT THE CASE AND YOU HAVE TO BE PREVENTED FROM DOING BAD THINGS AND IF YOU ARE NOT PREVENTED FROM DOING THE BAD THINGS, THEN THINGS GO TO H IN A HANDBASKET. SO LET'S TALK ABOUT THIS. THERE ARE ALL KINDS OF DANGEROUS FUNGI, PARASITES, BACTERIA, CAN COME IN AT ANY TIME AND ENTER THE BODY. THERE IS ALSO THE INTERNAL WORLD, CELLS TRANSFORMING IN TISSUES ALL THE TIME THAT YOU WOULD LIKE TO SUPPRESS AND THE ROLE OF THE IMMUNE SYSTEM IN PREVENTING AND NOT JUST TREATING CANS WHICH YOU HEAR ABOUT IN THE NEWS ALL THE TIME IS NOW ESTABLISHED. THIS IS A MAJOR ACTIVITY OF THE IMMUNE SYSTEM SO DEALING WITH MODIFIED SEPARATE IN AN APPROPRIATE WAY ALL THE TIME. AND IT POSES A CHALLENGE FOR THE IMMUNE SYSTEM. THERE ARE ALL THESE DIVERSE TARGETS IT HAS TO DEAL WITH, A VERY DIFFERENT COMPOSITION IN NATURE AND HAS TO DO WITH MANY DIFFERENT PLACES THROUGHOUT THE BODY AND THE QUESTION IS HOW DOES THE IMMUNE SYSTEM SOLVE THAT PROBLEM OF MANY DIFFERENT TARGETS AT MANY DIFFERENT TIMES IN MANY DIFFERENT PLACES AND THE ANSWER IS IT COMBINES AN INTERESTING SET OF STRATEGIES. IT HAS MOBILE CELLS THAT ARE ABLE TO SCAN THROUGHOUT THE ORGANISM SO THAT COVERS TO SOME EXTENT THE ISSUE OF WHERE THINGS MIGHT HAPPEN. WE'LL TALK ABOUT THE ISSUE OF SPECIFICITY AND PAYING ATTENTION TO THE DETAILS OF WHAT HAPPENS, BUT THERE'S A PROBLEM. IF YOU HAVE TO HAVE ALL THE DIFFERENT CELL TYPES EVERYWHERE IN THE BODY, THEN EVERY CELL WOULD BE AN IMMUNE CELL AND YOU WOULD HAVE NO HEART OR LIVER OR ANYTHING ELSE. SO THEY HAVE TO COMPROMISE AND WHEN YOU DISTRIBUTE THESE CELLS AND THEY ARE NOT ALLOWED TO TALK TO EACH OTHER, THAT IS A PROBLEM BECAUSE THEY ARE NOT IN THE RIGHT PLACE TO DO THINGS. SO THE OTHER PART OF THE EQUATION IS NOT JUST THE CELLS PRIMARILY FORMED IN THE BLOOD WHETHER LYMPHOCYTES OR PWHRAOL CELLS BUT WE HAVE FIXED TISSUES. WE HAVE BONE MARROW WHICH IS THE GENERATOR OF MOST OF THE CELLS AND AS THEY REACH MATURITY, WE HAVE THE CLASSIC LIMP CLASSIC LYMPH NODE S AND SO THERE ARE THESE INTERESTING THINGS TO THINK ABOUT, HOW DO THE CELLS MOVE THROUGH AND BETWEEN THOSE TISSUES, HOW DO THEY ADJUST THE RATES THROUGH WHICH THEY MOVE SO THEY ARE THERE LONG ENOUGH BUT NOT TOO LONG TO LOOK AT SOMETHING. SO THEY KNOW THAT SO THE ADAPTIVE IMMUNE SYSTEM, IMMUNE TO USERS BUT NOT NECESSARILY INFLUENZA, THAT IS SPECIFIC TO THE LYMPHOCYTE AND THERE ARE TWO TYPES AND I WILL FOCUS ON T-CELLS TODAY. THEY EXIST AS DO B-CELLS AS CLONAL ENTITIES. A SPECIAL PROTEIN ON THE SURFACE OR RECEPTOR HAS UNIQUE SPECIFICITY. IT LOOKS AT MEASLES, IT LOOKS AT MUMPS BUT NOT EBOLA OR INFLUENZA SO THAT IS INHERENT IN THE STRUCTURE I WILL REFER TO AS THE T-CELERY SEPTEMBER TORE OR T T-CELL RECEPTOR. AND THIS GOES BACK TO THE GENETIC PREDISPOSITION OF THE DISEASE THAT Dr. KATZ TALKED ABOUT, THE DR MOLECULE HE TALKED ABOUT, WHEN IT IS A PARTICULAR TYPE, GIVES YOU A RISK FOR RHEUMATOID ARTHRITIS, THOSE ARE THE PROTEINS THAT ARE FINDING LITTLE PIECES, PEPTIDES THAT COME FROM THE INFLUENZA, THE GLUTEN FROM THE NUCLEAR PROTEIN THAT COME FROM BACTERIA SO YOU MAKE THIS LITTLE FLAG FOR RECOGNITION BY THE CLONAL RECEPTOR, THE TCR, ON THAT T-CELL SO WHEN THE ENGAGEMENT TAKES PLACE, IT GETS SIGNALS, IT ACTIVATES, IT DIFFERENTIATES INTO WHAT WE CALL INFECTOR CELLS AND THEN GOES OUT OF THE CIRCULATION, LEAVES THE SECONDARY LYMPHOID TISSUE LIKE THIS NODE AND GET TO OUT WHERE THE INFECTION IS AND NOT NECESSARILY HERE IN THE LYMPH NODE. AND HERE IS WHERE THE PROBLEM IS. IF YOU WANT TO GIVE EACH OF THOSE CELLS A CLONAL RECEPTOR, YOU HAVE TWO WAYS TO THINK ABOUT THAT. YOUR RNA HAS MILLIONS OF THOSE RECEPTORS ALL ENCODED AND SOMEHOW TO PICK ONE AND NOT ANOTHER TO BE EXPRESSED IN A GIVEN CELL. BUT WHAT NATURE HAS DECIDED TO DO IS CREATE THOSE de NOVO FROM DISPERSE GENETIC ELEMENTS FROM EACH CELL AND WHAT THAT SAYS YOU CAN INHERIT A SET OF RECEPTORS BUT THOSE CELLS DON'T KNOW WHAT THEY MAKE UP. SO IF YOU ARE MAKING THEM ESSENTIALLY AT RANDOM, COMBINING DIFFERENT PIECES OF THESE MOLECULES IN THE GENOME, YOU ALSO CAN MAYBE LOOK AT THE ALBUMEN IN THE SIR YUM AS A PEP TIED MOLECULE AND THEY COULD REACT TO IT. SO THIS IS THE REARRANGEMENT PROCESS. THE SYSTEM TRIES TO DEAL WITH THAT PROBLEM AT TWO LEVELS. ONE IS CALLED CENTRAL THYMUS. SOME CELLS NEED TO GO TO THE THYMUS TO BE MATURE. THAT IS WHEN THEY ACQUIRE THIS STRONG ACTIVE TO SELF. SO THE THYMUS HAS DEVELOPED A WAY IN WHICH IT RUNS A FILTER THAT HAS THESE MOLECULES WITH ALL CELLS HAVE PRESENTED SO YOU CAN ELIMINATE THOSE. IF YOU SEE THAT WHEN THEY ARE IMMATURE, THEY DIE SO YOU TAKE THEM OUT OF THE REPERTOIRE AND FILTER THEM OUT. THEN SOMETHING ELSE THAT TOOK A LONG TIME BEFORE PEOPLE FIGURED THIS OUT, IT ACTIVATES ALMOST ALL THE GENES IN YOUR PERIPHERAL TISSUES LIKE YOUR LIVER OR YOUR EYE OR YOUR KIDNEY EVEN THOUGH YOU DON'T NEED THEM FOR ANY STRUCTURAL REASON TO PUT PEPTIDES IN THE MOLECULE TO TRY TO ADDRESS TISSUE RECEPTIVE CELLS. BUT THAT IS NOT AN EFFICIENT PROCESS. IT IS USEFUL BUT YOU CAN'T PUT ANENORMOUS AMOUNT OF THOSE IN AND MAKE THE WHOLE SYSTEM WORK SO THEY ARE CELLS THAT SNEAK OUT AND GET IN THE PERIPHERY. SO YOU KNOW THERE ARE CELLS YOU HAVE TO DEAL WITH WHEN THE IMMATURE T-CELLS ARE INCAPABLE OF RESPONSE. SO IN THIS CASE CELLS THAT WERE GENERATED CALLED REGULATORY CELLS, THE THINGS THAT CAN'T BECOME RECEPTORS FOR EPIGENETIC REASONS, BECOME A COMPONENT OF THE IMMUNE SYSTEM AND KEEP THINGS IN CHECK AND THIS IS WHERE YOU GET INTO A PROBLEM. YOU HAVE THESE RECEPTORS THAT CAN RECOGNIZE THE MOLECULES AND CELLS GET ACTIVATED BUT THESE CAN'T BE SUPER-STRONG BECAUSE THEY PREVENT YOUR RESPONSE TO MUMPS AND MEASLES AND RUBELLA AND E. COLI AND EVERYTHING ELSE SO THEY HAVE TO BE TITRATED IN SOME WAY AS TO INVOGUE THE IMMUNE RESPONSE. SO WHAT IS LEFT, THEY ARE A LITTLE WEAKER AND YOU CAN TITRATE TO THAT LEVEL OF STIMULATION AND HOPE THAT THE MEASLES AND MUMPS AND RUBELLA RESPONSES ARE WITHIN REGULATION BUT IF IT IS NOT APPROPRIATE, YOU CAN PREDICT WHAT WILL HAPPEN AND NOW THESE CELLS WILL BEGIN TO RESPOND. SO WE HAVE ALL THESE AUTOIMMUNE DISORDERS, HERE IS YOUR ARTHRITIS AND I HAVE SHOWN YOU A LOT OF CARTOONS, BEGIN YOU BACKGROUND INFORMATION, BUT HOW DO YOU ACTUALLY LOOK IN TISSUES AT WHAT IS REALLY HAPPENING? WHAT CELLS ARE HAPPENING TO OTHER CELLS, WHAT ARE THE SIGNALS AND STIMULI THAT CAUSE OTHER THINGS TO WORK? IF I TELL YOU T-CELLS HAVE TO BE NEAR OTHER T-CELLS TO DO THEIR JOB, TO TURN THEM OFF, THEN THEY MUST GET CLOSE ENOUGH TO ONE ANOTHER IN ORDER TO DO THAT AND YET I TELL YOU THESE CELLS ARE VERY CLONAL SO IS EVERYBODY TALKING TO EVERYBODY? AND IF THERE ARE ONLY A FEW CELLS TOGETHER, HOW DO THEY END UP IN THE RIGHT PLACE IN ALL OF YOUR DISSEMINATED IMMUNE SYSTEM? SO WE CAN DRAW THESE KINDS OF CARTOONS OF THE T-CELLS AND ACTIVATING THEM BUT IT DOESN'T TELL YOU ANYTHING THAT HAPPENS IN THE TISSUE. SO HERE IS THAT HISTOLOGY SECTION OF THE LYMPH NODE, THERE IS A T-CELL HERE AND WE ARE TALKING ENORMOUS DISTANCES. YOUR FRIEND CALLS UP, SAYS I WANT YOU TO PICK ME UP AT THE AIRPORT. SO THEN YOU HAVE THREE TO CHOOSE FROM AND I WILL TELL YOU WHAT TIME THE FLIGHT IS GETTING IN BUT THEY EXPECT YOU TO BE AT THE DOOR TO PICK THEM UP. AND WHAT ARE THE CHANCES FOR THAT HAPPENING? THE ANSWER IS ZERO AND THAT IS ESSENTIALLY THE SAME FOR THESE LYMPHOCYTES. THEY HAVE TO FIND EACH OTHER WHERE YOU ARE TALKING 5-MICRONS, EVEN HUMAN CELLS ARE A CENTIMETER SO YOU ARE ORDERS OF MAGNITUDE, A BILLION CELLS AND THERE ARE THREE OF THEM THAT HAVE TO FIND EACH OTHER IN THAT SPACE IN A REASONABLE TIME. SO HOW DO YOU TRANSLATE THESE CARTOONS INTO THAT ISSUE SHALY RESTRAINED DYNAMIC VIEWS OF WHAT IS HAPPENING? AND SO OBVIOUSLY WE HAVE THE HOSTAGE BUT WE HAVE A LOT OF CHALLENGES. LOOKING AT INDIVIDUAL CELLS, WE HAVE PRETTY GOOD RESOLUTION. BUT WE HAVE THINGS MOVING AROUND SO WE NEED TEMPORAL RESOLUTION. WE DO THAT IMAGING AND IF WE DO IT LIVE, WE HAVE MESSED UP THE PHYSIOLOGY AND THAT WILL NOT BE VERY HELPFUL. WE WANT TO LOOK AT ONE THING AT A TIME SO YOU CAN MULTIPLEX DETECTION, LOOK AT ONE THING AT A TIME TO BE INTERESTING, THEN YOU HAVE A LOT OF DATA AND HAVE TO FIGURE OUT WHAT TO DO WITH IT AND THEN YOU HAVE TO SHOW IT TO PEOPLE. SO THERE ARE A LOT OF CHALLENGES AND WAYS TO DO I AM MAINLYING BUT NONE OF THEM GIVES US THE SPECIFICITY WE ARE LOOKING FOR SO YOU REALLY HAVE TO GO TO MICROSCOPY AND SEVERAL YEARS AGO, THEY SAID MULTIPROTON WILL DO THAT. SO IF SOMEONE IS SAYING THEY ARE LOOKING AT NORMAL TISSUES BUT DIDN'T TELL YOU WHERE THEY CAME FROM, IT IS JUST PAPER IN A JOURNAL. IF YOU TAKE IT AND SCAN IT X-Y IN AN I AM MAINLYING PLANE, THEN TAKE ANOTHER X-Y IMAGE AND YOU DO THAT THROUGH A WHOLE STACK SO YOU HAVE THREE DIMENSIONAL DATA AND THEN YOU HAVE REPEAT STACK AND SO THOSE IT OF YOU WHO HAD THOSE LITTLE FLIP BOOKS WHEN YOU WERE YOUNG, THAT IS WHAT WE'RE DOING. SO NOW WE LOOK AT T-CELLS IN THE LYMPH NODE, MOVING TWO TO THREE TIMES THE REAL SPEED AT WHICH THEY ARE MOVING AND EVERYTHING IS COMPRESSED. WELL, THAT IS VERY NICE BUT THESE ARE MISLEADING. SO YOU SEE THESE CELLS AND YOU SAY OH, THEY CAN DO WHATEVER THEY WOULD LIKE TO DO. THEY ARE ACTUALLY IN THIS SPACE. AND SO AGAIN, YOU READ A PAPER, DON'T EVER BELIEVE WHAT AN I AM MAINLYR -- AN IMAGER TELLS YOU WITHOUT TELLING YOU WHAT HE DOESN'T SEE. SO HERE ARE THE B-CELL AREAS, THE T-CELL ZONE AND THERE ARE STRUCTURAL ELEMENTS. IT IS NOT JUST A LITTLE BAG OF LYMPHOCYTES SITTING IN THERE. IF THEY HAVE TO MOVE AROUND AND FIND EACH OTHER, THEY WILL EITHER BUMP INTO THESE THINGS OR TAKE ADVANTAGE OF THEM AND THE QUESTION IS WHAT IS THE RIGHT ANSWER? SO WE CAN MAKE THESE FIBROBLAST CELLS VISUAL, THEY COME OUT THROUGH THESE LITTLE HOLES OF THE LYMPH NODE AND WE SEE IN IMAGING THEY ARE ACTUALLY WALKING ON THE FIBROBLASTIC CELL. SO THEY ARE NOT MOVING OR MUG LING AND CURLING AROUND EACH OTHER, THEY ARE MOVING ON THESE ROADWAYS. SO ISN'T THAT INTERESTING? IT TURNS OUT DENDRITIC CELLS THAT ARE TRYING TO TALK TO THEM LIVE ON THE SAME FIBERS SO NOW YOU HAVE THESE ROADWAYS AND THE ONLY THING T-CELLS CAN DO IS BUMP INTO DENDRITIC CELLS ALL THE TIME AND THAT MAKES SENSE IF YOU ARE TRYING TO GET THEM TOGETHER. BUT IF THEY ARE WALKING AROUND, SAY THIS IS THE DENDRITIC CELL WITH ANTIGEN ON IT BUT THIS CELL DOESN'T. WELL, DO YOU WANT TO CHOOSE THIS ONE HALF THE TIME AND THIS ONE HALF THE TIME WHEN THEY ARE THE SAME DISTANCE APART? THE ANSWER IS NO. IF THIS DENDRITIC CELL INTERACTS WITH THE T-CELL, THAT COMBINATION PUTS DOWN A HANSEL AND GET TALE TRACK OF BLOOD CELLS THAT THE RECEPTOR IS ABLE TO SEE AND THE CELL GOES OVER HERE EIGHT TO TEN TIMES MORE OFTEN THAN OVER HERE. SO IT IS NOT RANDOM, IT IS DIRECTED AND THAT MAKES IT MORE EFFICIENT. SO IF WE BLOCK THESE CHEMOTINES, THE WHOLE SYSTEM FAILS. SO NOW WITHIN THE FUNCTION OF BIOLOGY STRUCTURE, YOU HAVE ALL THESE CIRCULATING CELLS BUT YOU HAVE TO ACCUMULATE THEM INTO A PARTICULAR PLACE TOGETHER WITH THE CELLS TRYING TO PRESENT TO THEM THE RELEVANT INFORMATION, THE DENDRITIC CELLS. THE T-CELLS ARE IN A SUBREGION AND IN THOSE ARE THE DENDRITIC CELLS ON THOSE ROADWAYS WHERE THEY MOVE AND SO YOU HAVE THE CONCENTRATED REACTANTS WITHIN THOSE THREE ZONES, THEN USE THESE TO DIRECT THE MOTION ON THE FIBERS AND IN VERY RECENT WORK FROM THE LAB, IT TURNS OUT THE DIFFERENT FLAVORS OF DENDRITIC CELLS LIVE IN DIFFERENT REGIONS OF THE LYMPH NOTE AND THE RECEPTORS LIVE IN THIS STEADY STATE IN ASSOCIATION WITH THE DENDRITIC CELLS THAT MATTER. SO AGAIN, LOCAL CONCENTRATION WILL INCREASE THE EFFICIENCY OF THIS SYSTEM. SO INSTEAD OF THINKING CELLS WANDER AIMLESSLY AROUND WHICH WAS THE FIRST OF THE TWO MOVIES, IT IS A VERY ORGANIZED SYSTEM BECAUSE YOU HAVE VERY RARE CELLS TRYING TO FIND EACH OTHER IN BIG VOLUMES AND UNLESS YOU ORGANIZE IT THIS WAY, IT JUST WON'T HAPPEN. NOW THAT IS HOW YOU START THE RESPONSE. THE T-CELLS GET ACTIVATED, NOW THE RECEPTOR CELLS AND WHAT DO THEY DO? IF THEY WERE JUST HANGING OUT IN THE -- LYMPH NODE, IT WOULD BE NO PROBLEM BUT IT IS NOT AS SIMPLE AS THAT. WE CAN HAVE LOTS OF CELLS WHOSE T-CELL RECEPTOR THAT WE KNOW. WE CAN THEN ACTIVATE BY TWO ANTIGENS, THE GREEN IS T-CELLS AND THEN SOME OF THE OTHER T-CELLS IN THAT HOST. WE CAN USE THE OTHER ANTIGEN TO CREATE INFLAMMATION IN THE EAR WITH THE ANIMAL GETTING DELAYED HYPER SENSITIVITY REACTION. THAT ATTRACTS THE PREACTIVATED CELLS, THE GREEN CELL INTO THE TISSUE BUT THERE IS NONE OF THEIR ANTIGEN THERE AND WE DO THAT FOR A PARTICULAR REASON. IF WE WANT TO LOOK LIVE AT WHAT IS HAPPENING, WE NEED TO COORDINATE WHEN THINGS OCCUR. IF WE JUST LET THE CELLS COME IN RANDOM, WE WOULDN'T KNOW IF THEY CAME IN TWO DAYS AGO, AN HOUR AGO OR TWO WEEKS AGO. NOW WE CAN PUT IN THE RELEVANT ANTIGEN AND VERY RAPIDLY AND IN AN ACUTE MANNER SAY HOW DO THEY BEHAVE AND THAT TURNS OUT TO BE INCREDIBLY INTERESTING. SO THIS IS 14 HOURS OF IMAGING IN A LIVE ANIMAL AND THESE ARE THE T-CELLS, THEY ARE RUNNING AROUND, WE PUT IN THE ANTIGEN AND THEY ALL STOP. THEY ARE NOT MOVING AROUND ANYMORE. NOW WE WATCH FOR HOURS AND YOU SEE SOME OF THE OTHER CELLS COME INTO THE FIELD, THE ONES THAT STOPPED BEGIN TO MOVE AGAIN, LET THIS RUN A LITTLE LONGER AND SOME OF THEM STOP AGAIN. SO WHEN A T-CELL IS WAITING FOR A T-CELL ECE -- RECEPTOR, IT STOPS MOVING. NOW THINK ABOUT WHY THEY START MOVING AGAIN AND HOW DOES THAT RELATE TO THEIR FUNCTION? THAT TURNS OUT TO BE VERY INTERESTING. THEY ARE NOT MAKING ANY AFFECTIVE CYTOKINES WHEN THEY ARE MOVING AROUND, WE INJECT THE CELLS AT THE SITE, THEY START MOVING. THEY ONLY STOP WHEN THEY ARE ON A PRESENTING CELL SEEING THE LIGAND. AND THAT ONLY MAKES SENSE, THEY ARE LOCATING THE PATHOGEN IN THE TISSUE. YOU DON'T WANT TO RANDOMIZE DISTRIBUTE TOXIC INFORMATION TO A CELL THAT DOESN'T WANT IT. SO WHY? ONE POSSIBILITY, IT JUST WENT AWAY, MAYBE THE CELL TURNED IT AWAY FASTER THAN THOUGHT, MAYBE THE T-CELL ATE IT UP. BUT THE OTHER POSSIBILITY IS THAT THE T-CELLS ARE NOT PAYING ATTENTION TO THE ANTIGEN. IT IS STILL THERE, THEY ARE NOT PAYING ATTENTION TO IT ANYMORE. SO HOW CAN WE TELL THESE APART? I WILL SHOW YOU THAT IN A MINUTE. SO EVERYTHING YOU SEE HERE LOOKS REAL COMPLICATED BUT IT IS WHAT I HAVE ALREADY EXPLAINED. WE GO THROUGH THIS CYCLE OF STOPPING AND STARTING AGAIN BY PUTTING ANTIGEN IN. THEN WE MAKE MORE CELLS IN A SPECIAL COLOR THAT DON'T HAVE ANY SPECIFICITY FOR THE ANTIGEN IN THE HERE AND PUT THEM IN. IF YOU READ ABOUT THESE, YOU ALWAYS NEED TO HAVE CONTROLS IN THE SAME IMAGING FIELD TO KNOW IT IS THE SAME STATE OF INFLAMMATION, THE SAME ARTIFACT FOR WHAT EVER DO YOU GO FOR THE TREATMENT OF THAT ANIMAL. SO HERE THEY ARE MOVING AS THEY SHOULD AND THEY ARE, 6-MICRONS PER MINUTE. THESE ARE THE CELLS WE STOPPED THAT ARE ALMOST BACK TO FULL SPEED THAT SEEM TO BE IGNORING ANTIGEN AND HERE ARE THE CELLS WE PUT IN, YOU CAN SEE THEY ARE STOPPED. SO THERE IS PLENTY OF ANTIGEN THERE BUT THESE CELLS ARE IGNORING IT WHICH MEANS THEY ARE BEING ACTIVELY INHIBITED BY SOMETHING ELSE IN THE SYSTEM AND THAT IS A LITTLE HARD TO BELIEVE BECAUSE THEN THESE CELLS SHOULD ALSO BE INHIBITED. OR AND THIS IS THE REAL QUESTION, EVERY ANTIGEN HAS SOME REGULATION, SOMETHING THAT TURNS OFF THE PROCESS AND BRINGS IT BACK TO THE HOMEOSTATIC STATE. SO THIS IS A VERY FAMOUS HOLIDAY COOL FOR IMMUNOTHERAPY AND I WILL COME BACK TO THAT IN A SECOND AND THAT TENDS TO TURN THE T-CELL BACK DOWN AGAIN AFTER IT HAS BEEN ACTIVATED AND THE TREATMENT YOU READ ABOUT IN THE NEWSPAPER AND THE NEWSPAPERS NOW ARE TARGETING THESE REGULATORS AND TAKING THE BRAKES BACK OFF TO ACTIVATE THE CELL. SO WHAT WE WANT TO LOOK AT IS WHETHER THE CELLS WE WERE LOOKING AT THAT WERE MOVING AND STOPPED AND THEN STARTED MOVING AGAIN WERE BLUNT TO THE ANTIGEN THAT WAS PRESENT AND SURE ENOUGH, IT GOES UP AT THAT TWO-HOUR PEAK, STARTING TO TURN EVERYTHING OFF, THIS ONE DOES NOT SO WE CAN TEST WHETHER THIS MATTERS BY BLOCKING THE LIGAND FOR PD1 AND WHEN WE DO THAT, NOW THEY PERSIST AND STAY STOPPED ON THE ANTIGEN PRESENTING CELLS. SO WHAT IS CLEAR IS THE CELLS GO GO THROUGH THIS CYCLE AND YOU ARE LISTENING TO ME CAREFULLY, YOU WOULD SAY THIS DOESN'T MAKE ANY SENSE. IF THE ANTIGEN IS THERE, THE T-CELLS MUST KNOW IT IS THERE, WHY DOES IT TURN OFF AND THAT DOESN'T MAKE SENSE FOR THE IMMUNE SYSTEM. SO I WILL MAKE IT HARDER. WE DIDN'T GIVE IT A LIVING ANTIGEN THAT WOULD MAKE A PATHOGEN. THESE T-CELLS ARE SENSING THERE THAT IS AT LEAST STASIS IF NOT DECLINE IN THE ANTIGEN LEVEL WHICH MEANS IT IS NOT GROWING AND CONTRIBUTING MORE ANTIGEN, IT IS AT A STEADY STATE. SO THESE CELLS TUNE TO THIS LEVEL OF ANTIGEN TURNED OFF. THE NEW ONES WE PUT IN WHO HAVEN'T GONE THROUGH THIS EXPERIENCE, DON'T HAVE THE REGULATORS TURNED ON ARE ABLE TO SEE THIS AND THIS IS VERY WELL-KNOWN. IF YOU TRY TO TREAT A TOMB OF AN ADOPTIVE TRANSFER WITH A WHOLE BUNCH OF LYMPHOCYTES, YOU ARE HARD-PRESSED TO HAVE AN EFFECT. IF YOU HAVE WHAT ARE CALLED STEM-LIKE CELLS THAT KEEP GENERATING NEW EFFECTORS BUT THEN ENTER THE TISSUE LIKE OUR SECONDARY TRANSFER CELLS, THEY CAN COME IN AND THAT REDUCED LEVEL OF ANTIGEN IS STILL STIMULATORY FOR THEM, THAT CAN DRIVE DOWN THE TUMOR CELLS SO WE ARE ON THIS RATCHET, A CONTINUOUS RAT CHET, NOT A REAL RATCHET, SOME TURN OFF, OTHERS KEEP WORKING AND THAT IS A MAJOR FUNCTION OF THE IMMUNE SYSTEM WITH MINIMAL TISSUE DAMAGE. SO THESE ACTIVATED CELLS, AS YOU HAVE HEARD BEFORE, CAN BE VERY DAMAGING SO THEY TITRATE AND THE SYSTEM IS TRYING TO SAY AM I WINNING OR LOSING. IF I AM WINNING, I AM NOT GOING TO KEEP DOING THIS STUFF BECAUSE IT WILL JUST CAUSE MORE DAMAGE BUT YOU HAVE TO HAVE THE RIGHT SOURCE OF CELLS OR YOU LOSE. NOW, THERE IS A PROBLEM. WHAT I JUST TOLD YOU ABOUT HAD NOTHING TO DO WITH TUMORS. THIS MOLECULE COMES UP ALL THE TIME. IT REGULATES THE SYSTEM. AND IF YOU BLOCK IT, YOU TURN OFF THIS NEGATIVE REGULATION. SO WHAT I JUST SAID IS YOU GET EXCESS EFFECTOR FUNCTION. THAT IS WHERE YOU ALLOW CELLS TO CAUSE TISSUE DAMAGE. TISSUE DAMAGE IS AUTOIMMUNITY IF YOU ARE LOOKING AT THE WRONG THING. SO AS LONG AS YOU CAN GET TO THE EFFECTOR STATE AND WE HAVE TO FIGURE OUT HOW TO GET THERE, THEN YOU TURN THIS MOLECULE OFF AND THEN YOU WILL HAVE A PROBLEM, ALL RIGHT? SO THIS IS THE BALANCE THAT IS HAPPENING WITH CHECKPOINT THERAPY IN TUMORS BECAUSE IT POTIENTIATES AUTOIMMUNE FACTORS. SO THAT IS THE LIVE IMAGING AND WE CAN DO IT ON NODES AND WATCH THE STRANDS AND MAKING ANTIBODIES AND LOOK AT THE GUT FLORA AND WHAT HAPPENS IN THE KIDNEY AND WHAT HAPPENS IN GRANULAR AND VARIOUS TISSUES, A LITTLE MORE DIFFICULT, WE CAN LOOK IN THE LUNG. WE CAN LOOK IN THE EAR SKIN, LIKE THE NEUTROPHILS COMING IN TO FIGHT AN INFECTION, YOU CAN LOOK AT THE CELLS I HAVE BEEN QUANTIFYING, LOOK AT THE MARROW AND OTHER PLACES WE CAN IMAGE AND IT IS ALL GREAT BUT THE MOUSE LYMPH NODE, THAT IS THE VOLUME WE IMAGE SO IT IS BLACK EVERYWHERE WE LOOK SO PEOPLE SAID WELL, WE WILL HAVE TO FIX THAT. SO WE STARTED DOING HISTOLOGY AND REALIZED THAT IT MAKES ONE OR TWO OR THREE COLORS AND EVERYBODY WAS GOING DOWN THE HALLWAY AND DOING FOUR, FIVE, MANY COLORS OF IMAGERY WHICH WAS A REAL DISCONNECT. SO MY LAB DECIDED TO FIGURE OUT HOW TO FIX THAT, DOING HISTOCYTOMETRY, WE FIX THE IMAGE A LITTLE BIT BY MATH MATHEMATICAL SOFTWARE TOOLS, COMPENSATE FOR THE DIFFERENT COLORS WE USE BECAUSE THEY SPILL OVER ONE ANOTHER WHEN WE'RE TRYING TO DETECT THEM AND THEN YOU USE SOFTWARE TO FIND THE DATA SETS AND THAT IS INTERESTING BECAUSE WE CAN COLLECT ALL THE CLASSIC INFORMATION ASSOCIATED WITH THAT CELLULAR OBJECT AND CELL DATA WITH FLUORESCENCE IS FLOWCYTOMETRY DATA SO NOW YOU CAN PUT THIS IN ANY SOFTWARE BASED ON THE PARAMETERS YOU WANT TO USE. SO A MOLECULE CALLED CD3, CD4, YOU LOOK AT THAT COMBINATION AND YOU CAN LOOK FOR THOSE T-CELLS IN YOUR DATA SET. BUT WE HAVE THE X, Y AND Z COORDINATES SO WE CAN GAUGE AND SAY WHERE IS IT IN THE TISSUE. WITH THE COMPLEX CYTOMETRY, YOU NEVER KNOW THAT INFORMATION. WE GET THE INFORMATION. SO IF WE PHENOTYPE THE CELLS, WE CAN LOOK AT THE SIGNALING, WE CAN LOOK AT THEIR FUNCTION IN TERMS OF CYTOKINES, GROWTH FACTORS, WHATEVER THEY ARE MAKING AND LOOK AT A SPECIAL HIGH RESOLUTION IMAGE IN THE TISSUE. SO TALKING ABOUT T-CELLS, THEY SORT OF PREVENT CELLS YOU DON'T WANT TO BE MAKING DAMAGING RESPONSES FROM MAKING THEM AND THE WAY PEOPLE THOUGHT ABOUT THIS A FEW YEARS AGO IS THEY PREVENT THE ACTIVATION OF THESE CELLS THAT ESCAPE FROM THE CLIMATE. WELL, THEN DOING EXPERIMENTS USING THESE STANDING APPROACHES, YOU WANT TO BEGIN TO LOOK AT SIGNALING THROUGH PMOSPHO-STAT LOCATION AND SO WE STARTED STUDYING THOSE AND TURNS OUT OF A OF THOSE CELLS ARE REGULATORY T-CELLS AND THAT MEANS THEY ARE GETTING A SIGNAL THROUGH THE RECEPTIVE, BLOCKING THIS, THIS IS ALL IL-2, KNOWN AS T-CELL GROWTH FACTOR. SO WHO IS MAKING THAT? AND LET ME JUST BACK UP A STEP. NOW, YOU COULD SAY WOW, THESE ARE WHAT ARE CALLED SPFS, JUST SITTING AROUND, THEY ARE EXPOSED TO AMBIENT PATHOGEN, HAVE THIS BACTERIA AND KNOWN FOR THE STEADY STATE RESPONSE THAT IS GOING ON SO THERE ARE SOME CELLS GETTING ACTIVATED, THEY MAKE THE T-REGS GET BRIGHT. EXCEPT THIS IS WHAT YOU SEE IN GERM-FREE MAPS. SAME NUMBERS OF CLUSTERS, SAME NUMBER OF T-CELLS, THERE IS NO EVIDENCE THIS COMES FROM COMMENCERS OR ANTI-ANTIGEN WHICH MEANS THERE IS SOMETHING MAKING IL2 ALL THE TIME EVEN IN THE ABSENCE OF A MICROBIOME. SO THOSE CELLS ARE CONVENTIONAL T-CELLS AND LOOKING AT SELF-ANTIGENS. SO THEY CAN PREVENT SOME CELLS FROM ACTIVATING, WE CAN'T PROVE THE NEGATIVE BUT IT DOESN'T PREVENT THESE CELLS. WE CAN SEE THIS CELL MAKING IT AN IL2 IN THE MIDDLE OF THE P5 CLUSTER AND THAT MEANS THAT IT MUST DO SOMETHING ELSE AND I WILL NOT BE ABLE TO TELL YOU THE WHOLE STORY TODAY BUT THESE CELLS GET ACTIVATED, MAKE IL2 BUT TURNS OUT THEY HAVE THE IL2 RECEPTOR CELL AT A HIGH LEVEL. AND THIS HAS BEEN PROVEN BY A FELLOW AT NCI, THEY CAPTURE ALL OF THIS THAT THE CELL IS MAKING AND THIS CELL NEVER SEES ITS OWN IL2. IT ALSO IS UP PRESSES ACTIVITY AND SOME DATA SUGGESTS IT MAY CAUSE THEM TO PRO LIVE RATE AND PUT MORE T-REGS AROUND THIS LOCATION AND THAT INHIBITS THE FUNCTION OF THOSE EFFECTOR CELLS. SO YOU DON'T STOP THEM FROM BEING ACTIVATED BUT YOU STOP THEM FROM BEING DANGEROUS REGULATORS. NOW, THIS DOESN'T EXPLAIN ALL OF THIS BUT PEOPLE WORKING ON THIS FOR DECADES CERTAINLY DID NOT KNOW THERE ARE TINY CLUSTERS WHERE THIS SELF-EXPRESSION IS IMPORTANT. IF IT SEES ITS OWN IL-2, IT IS DANGEROUS SO A TISSUE ARGUMENT. THE PROBLEM IS I ONLY SHOWED YOU A COUPLE OF PARAMETERS. I DIDN'T SHOW YOU 10 OR 15 AND IN FACT IF YOU MADE THIS CELL WHEN IS SITTING THERE, MAYBE THE BOTTOM LINE 30 ONES OR SO IN THE LYMPH MODE, IT TAKES ABOUT 40 MARKERS TO LOOK AT THAT. SO HOW DO WE GO THERE? EVELYN, YOU CAN RECOGNIZE IT. THEY BASICALLY HAVE A STAINING METHOD SO YOU CAN GO THROUGH ANY COMBINATION OF THREE, FOUR, FIVE, SIX, EIGHT COLORS AT A TIME TO GET TO THE 40. SO THIS IS REALLY SPECTACULAR AND YOU DON'T NEED TO TAKE ANYTHING AWAY FROM THE INDIVIDUAL CHANNELS I AM GOING TO SHOW YOU BUT BASICALLY I AM JUST WALKING YOU THROUGH THE STRAINING IN ONE LYMPH NODE SECTION FOR 45 DIFFERENT PARAMETERS. OKAY, NOW Dr. KATZ HAD A CHALLENGE FOR YOU BEFORE. SO I AM GOING TO CHALLENGE YOU. HOW MANY OF YOU ARE WILLING TO TELL ME WHAT THE DISTRIBUTION IS FOR ANY OF THOSE MARKERS AT RANDOM? HOW ABOUT ANY PAIR? ANY TRIPLET? SO THIS BECOMES A PROBLEM. YOU ARE TRYING TO LOOK AT HOW CELLS ARE RELATED TO ONE ANOTHER, YOU ARE LOOKING AT MULTIPLE MARKERS ON THE CELLS, HOW DO YOU PROCESS THE DATA? THE PICTURES ARE VERY NICE AND YOU CAN GAIN INFORMATION FROM IT BUT THE TOTAL TKWHROEBL POOL OF INFORMATION IS MUCH GREATER AND IF YOU SUPER IMPOSE IT, THIS IS WHAT IT LOOKS LIKE. SO I HAVE DONE SOMETHING INCREDIBLY COOL, TAKING 45 CHANNELS IN THAT PARTICULAR CASE AND COMPRESSES THEM DOWN MATHEMATICALLY TO 3 CHANNELS. THAT IS HOW YOU CREATE THE IMAGE ON YOUR TV SCREEN, YOU TAKE RED, BLUE AND GREEN AND MIX THE COLORS. SO NOW HE HAS TAKEN ALL THE PARAMETERS INCLUDING THE 45, PUT THEM IN THE THREE CHANNELS AND COLORED THEM SO THAT PICTURE NOW BECOMES THIS PICTURE AND WE ARE VERY GOOD AS HUMANS TO PICK OUT PATTERNS VISUALLY AND IF YOU LOOK AT THAT, YOU WILL BEGIN TO SEE PATTERNS THAT YOU DIDN'T SEE BEFORE. SO HERE IS THE B-CELL FOLLICLE, HAS A DARK AND A LIGHT CELL AND DENDRITIC CELLS SITTING THERE AND I CAN TELL YOU THEY ARE THERE BUT HE DIDN'T SEGMENT THE INDIVIDUAL CELLS WHICH IS VERY TIME-CONSUMING AND DIFFICULT TO DO. AND THIS IS WHAT IT LOOKS LIKE BLOWN UP. YOU ACTUALLY SEE THE CELLS EVEN THOUGH WE DIDN'T BLOW THEM UP AND GAIN ON THEM. SO WE'RE GREEDY ABOUT THE IMAGING BECAUSE WE NEED TO SEE WHAT IS IN THE TISSUE MORE HOLISTICALLY THAN WHAT I HAVE ALREADY TOLD YOU. SOTHEY GOT TOGETHER AND LOOKED AT THE VOLUME AND THERE ARE MANY METHODS OUT THERE BUT THEY DON'T DO WHAT THIS METHOD DOES WHICH IS TO PRESERVE FORENSIC MOLECULES, THESE ARE IN MICE AND YOU CAN SEE THEM AT ANY GIVEN TIME WITH THE COLORS. SO THAT EARLIER MOVIE I SHOWED YOU WHERE THE LYMPHOCYTES ARE COMING IN, THESE ARE THE PERIPHERAL B-CELL FOLLICLES BUT OF COURSE WE CAN LOOK IN THE COMPUTER AT THESE SLIDES SO IF WE DID MORE PARAMETERS THAN THE FIVE, WE COULD IDENTIFY EVERY ONE OF THE MILLIONS OF CELLS IN THIS ONE CUBE IN POSITION TO THE OBJECT AND EVERYONE ELSE SO WE'RE NOW USING THAT TO STUDY AUTOIMMUNEOLOGY. SO NOW WE HAVE A LYMPH NODE, ALL OF THESE ARE REGULATORY CELLS AND HERE WE HAVE GREEN AND PURPLE. GREEN ARE THE IRRELEVANT CELLS THAT DON'T LOOK AT AN AUTO ANTIGEN AND THE PURPLE ONES THAT DO AT A FREQUENCY THAT YOU CAN'T GET IN ANY OTHER ANIMAL. EVEN TEN CELLS GIVE US QUANTIFIABLE DATA AND YOU CAN SEE THESE AUTOIMMUNE CELLS ARE GETTING FAT BECAUSE THEY ARE REACTING AND BLASTING WHEREAS THE OTHER CELLS ARE SMALLER BUT WE CAN USE SPATIAL STATISTICS TO DETERMINE WHETHER THEY ARE ACTIVATED IN A MORE COHERENT COHORT OF REGULATORY CELLS THAN A RANDOM CELL AND THAT IS TRUE. SO WE LOOKED AT THREE KINDS OF DATA SETS IN 3D, HERE LOOKING AT THE INTESTINE BUT THAT IS ONLY A COUPLE OF COLORS AT A TIME SO HOW DO WE IT TRAIT THAT? BECAUSE SOAKING ANTI-BODIES IN AND OUT OF VOLUME WOULD TAKE FOREVER. SO WE'RE COMBINING A TRICK DONE BY GARY'S LAB IN STANFORD AND TAKE THE NUCLEOTIDES AND PUT THEM ON THE TISSUE AND THEN COMBINE THEM WITH FLUORESCENT LEVELS, THREE, FIVE, EIGHT AT A TIME, THIS ERR VERY SMALL, YOU IMAGE AND THEN USE THE ASSOCIATED BUFFER TO COME IN WITH A WHOLE SET OF OLIGODES. THIS IS A COUPLE OF MIGHT BE CHRONICS THICK, A FAKE SELL YOU CAN DO IN A SHORT PERIOD OF TIME DOING THIS APPROACH. SO I WILL END BY SAYING THIS PUSH FOR DOING SINGLE-CELL TRANSCRIPT GENETICS. WHEN YOU DON'T HAVE A GOOD ANTI-BODY TO IT, HOW DO YOU DEFINE THE DATA WITH THE SUBPOPULATION OF CELLS? SO YOU DO IT WITH FISH. HERE IS 3-7B8 D COMBINATION OF STAINING AND FISH. AND SO WHEN THEY WORKED OUT HOW TO DO THAT AND WE CARE ABOUT THE MICROBIOME. THESE ARE SINGLE FILL -- FILAMENT BACTERIUM THAT EXIST ON THE EDGE OF THE GUT. SO IT COULD BE AFFECTED TISSUE, WE CAN DO THE CYTOMETRY, GO INTO 3D, ADD THE FISH, SO WE CAN GET AN ENORMOUS AMOUNT OF INFORMATION ABOUT WHAT IS HAPPENING IN THE TISSUE SETTING TO TRY TO UNDERSTAND SOME OF THE QUESTIONS THAT Dr. KATZ RAISED BEFORE. THANKS VERY MUCH. >> YOU HAVE TWO DYES, HOW DO YOU EXUDE OVERLAP? IT'S COMBINATION OF THINGS, WE USE SINGLE COLOR CONTROLS AND SUBTRACT THE KNOWN OR SPILLOVER, DEPENDING ON THE FILTER SET WE ARE USING. RATHER THAN DO 12 AT ONE TIME, YOU MAY ONLY DO 3 OR 4 OF THE 12 IN ONE SCAN AND PICK WHICH ONES YOU ARE DOING SO YOU CUT LESS OF THE FLOUREXINS OFF WHEN YOU DO THAT GIVING YOU GREATER PRECISION AND CONTROL TO MAKE SURE WE'RE NOT MISDETECTING BUT WE HAVE TO BE VERY CAREFUL IF WE'RE LOOKING AT A VERY BRIGHT AND ABUNDANT FLUOROCHROME AND NOT ANOTHER SO WE BRIGHT THE PANEL TO AVOID THIS SPILL OVER AND IT SOLVES THE PROBLEM. >> IS THERE A ROLE OF THE TYPE OF STUDIES, LIKE WHAT JENNIFER SCHWARZ HAS DONE WITH THE LASER SHIELD TECHNOLOGY WHERE INDIVIDUAL COMPONENTS, MAYBE UP TO 15 OR 16 ARE DYNAMICALLY LABELED IN A LIVING CELL? NOW, THAT IS A VIRUS OR A -- >> WHAT I SAID ABOUT THE -- ABOUT THE IMAGING CELL, IF YOU TRY TO GO IN AND LOOK AT AN INDIVIDUAL CELL, YOU WILL ONLY SEE THAT CELL. IS IT REPRESENTATIVE OF ALL THE OTHER CELLS IN THE TISSUE OR THE LYMPH NODE THAT IT BEHAVIORS THAT WAY? YOU CAN GET THAT INFORMATION FROM INSIDE THE CELLS. THE NUCLEOTRANSCRIPTION FACTORS, SOME ARE ACTIVATED AS OPPOSED TO STEADY STATE AND CAN TELL THEY ARE SIGNALE SO THERE ARE MANY THINGS I TALKED ABOUT THAT GET AT THE STATES OF THE CELLS BUT WE ARE NOT LOOKING AT THEM AND WHY. BECAUSE THE CELLS ARE MOVE UP AND DOWN WITH THE STUDIES HE REFERENCED WITH JENNIFER, Z AND X AND Y, YOU CORRECT FOR THAT IN VERY FANCY CASES BUT FOR MOST TIMES, THAT IS A PROBLEM. SO UNDERSTANDING WHETHER A MOLECULE IS MOVING, YOU HAVE TO COMPENSATE FOR THE CHANGE IN INTENSITY THAT COMES FROM BEING IN A DIFFERENT Z-LEVEL AND THAT HAS TURNED OUT TO BE EXTREMELY DIFFICULT TO DO. THERE ARE ONLY ONE OR TWO VERY GOOD PAPERS IN THE LITERATURE USING THIS DYNAMIC IMAGING FOR LOOKING INSIDE THE PRO TOEPLASMM AND NUCLEUS. >> I WOULD REMIND YOU THAT AT TOMORROW'S LECTURE, ERIC WILL BE TALKING ABOUT HIS WORK WITH THAT. >> THIS IS AMAZING BUT APPLYING IT TO THE ISSUE OF AUTOIMMUNITY IS THE TOPIC HERE AND HAVE YOU WORKED ON LOOKING AT -- I THINK WE SAW THE SEQUENTIAL ARRANGEMENT OF HOW THAT DEVELOPS BUT CAN YOU DO THIS TO SHOW THE LESIONS THAT MOVE -- >> ONE OF MY FELLOW ASSOCIATES, EARLY ON, AFTER HE WENT BACK TO INDIANA, I WENT OVER AND WE WORKED TOGETHER SO WE COULD WORK ON HOW TO IMAGE THE JOINTS OF MICE USING THE TWO-KEY APPROACH WHICH THEY HAVE DONE. SO IT IS APPLICABLE IN AN ANIMAL MODEL AND THAT IS ONE THING. I TALKED ABOUT AN EARLIER VERSION OF THIS TYPE OF HISTOCYTOMETRY AT THE TARGETED THERAPEUTICS, THE RHEUMATOLOGY MEETING THAT IS SMALLER AND SOME THAT WILL EUROPEANS RUN AND I WAS OFFERED 300 BIOPSY SAMPLES FROM PATIENTS TO LOOK AT WHICH I DIDN'T ACCEPT BECAUSE I DON'T HAVE ANYONE TO LOOK AT 300 SAMPLES BUT YES, AS YOU ARE SUGGESTING, WE CAN LOOK AT THESE SAMPLES FOR BACKGROUND AND SEE WHAT THE APPLICATIONS ARE FOR THESE METHODS AT LEAST FOR STATIC WORK. OBVIOUSLY DINE -- DYNAMIC WORK IS DIFFERENT AND I AM NOT SURE HOW RELEVANT IT IS FOR SOME OF THE QUESTIONS BUT YES, WE HAVE DONE THIS PROCEDURE IN LIVER, INTESTINAL SAMPLES, LYMPH NODE SAMPLES. IT SEEMS TO WORK IN DIFFERENT TYPES OF TISSUES. WE HAVE DONE MOUSE BRAIN, KIDNEY AND OTHER TISSUES SO PRETTY BROADLY APPLICABLE. YOU JUST NEED PEOPLE DEDICATED TO DO THE WORK. IT DOESN'T HAPPEN INSTANTANEOUSLY WHEN YOU DON'T KNOW WHAT YOU ARE LOOKING AT. >> SO SEEMS LIKE THE T-CELLS PLAY A BIG ROLE IN WHAT CAUSES AUTOIMMUNE DISEASE AND IF YOU COULD REMOVE THE T-CELLS, WOULD THAT STOP IT OR WHAT PERCENTAGE OF -- >> THERE IS T-CELL INDEPENDENT AND ANTIBODY AND THEY COULD BE ACTIVATED THROUGH THE RECEPTOR TRIGGERS AND OTHER SIGNALS THAT ALLOW THEM TO RESPOND IN THE ABSENCE OF VERY SPECIFIC T-CELLS. BUT IF YOU LOOK AT MANY, AND I DON'T KNOW IF YOU REMEMBER THE DISEASES ON THE LIST YOU SAW BEFORE, THE HIGHEST ASSOCIATION WITH GENE GENOTYPE MAPS TO GENES WE KNOW CONTROL ANTIGEN PRESENTATION AND THEREFORE THE BEST UNDERSTANDING IS EVEN THROUGH PROJECT DEVELOPMENT EFFECTS OR PERIPHERAL PRESENTATION, T-CELLS ARE CONVENTIONAL T-CELLS THAT IT USE THESE MOLECULES OR TRIGGER THEM TO PLAY A CONTROL AND THAT IS LIKELY TO BE THE CASE SO I THINK THAT IS YOUR REASONABLE STATEMENT. THE PD1 PART, AS I SAID, IT DOES NOT TAKE A NAIVE CELL AND MAKE IT ACTIVATED BUT IT WILL TAKE A CELL THAT IS ACTIVATED AND ALLOW IT TO ESCAPE REGULATION. SO IT STILL HAS TO BE WHAT I WAS POINTING OUT TO YOU, THAT ALL OF THE MECHANISMS WE KNOW DON'T PREVENT ORAL CELLS FROM UNDERGOING ACTIVATION, AND THEY NEED TO BE ARRESTED AT LATER PHASES AND IF THEY EVER ESCAPE IN THE TISSUE, THERE IS A ROLE FOR PD1 BEING INHIBITED BY IL1 ON THE STROMAL CELL OR PARENCHYMAL CELLS AND THEN YOU HAVE A PROBLEM. SO ONE OF THE ISSUES WITH THE THERAPY, ARE YOU GETTING TO KNOW ABOUT IMMUNITY OR ARE YOU TAKING PEOPLE WHO HAVE IMMUNE CONDITIONS AND ACTUALLY RELEASING THOSE CELLS TO CAUSE MORE PROBLEMS. SO ONE OF THEM WORKED WITH ANTI-BODIES AND THAT MAY BE THE PRO NOVO PART OF THE DISEASE BUT YOU ARE ACHIEVING THE DISEASE PROCESS AND PUTTING IT IN SOMEONE AND TRYING TO DESTABILIZE IT. >> THERE ARE SOME PARTICULARLY INTESTINAL AUTOIMMUNE DISEASES, COLITIS WHERE CHANGING THE MICROPIAL POPULATION, SO-CALLED FECAL TRANSPLANTS, HAVE ACTUALLY BEEN THERAPEUTIC PEN -- BENEFICIALLY. >> IF IT DEVIATES THE RESPONSE TO NONDAMAGING RESPONSES AND THEN YOU MOVE THEM OVER TO A DAMAGING RESPONSE TYPE, THEY ARE NOT REALLY ATTACKING THE TISSUE CELLS, THEY ARE CAUSING CHRONIC DISRUPTION THAT DISRUPTS THE GUT FUNCTION AND THAT IS WHAT YOU SEE AS THE COLITIS AND CYTOKINE CAN CAUSE GROWTH EFFECTS ON THE WALL OF THINGS YOU LOOK AT IN SAMPLES. SO IT MAY BE THE RESPONSE TO THE MICROBIAL PEPTIDES AND NOT NECESSARILY THE CELLS THEMSELVES IN THOSE CASES >> FROM A CLINICAL PERSPECTIVE AND INVESTIGATIVE STUDIES, HAVE PEOPLE INCRIMINATED THE MICROBIOME IN ANY ASPECT OF RHEUMATOID ARTHRITIS? >> OKAY, SO YES. IF YOU TAKE MICE AND RAISE THEM IN A GERM-FREE ENVIRONMENT, YOU WILL HAVE A DIFFERENT MANIFESTATION OF THEIR AUTOIMMUNE DISEASE. IT IS TRUE. BUT WHY? THAT IS TOUGH. DON'T HAVE A GOOD ANSWER. >> WELL, WE WANT TO THANK YOU BOTH FOR A REALLY EXCITING TALK. [APPLAUSE]