IT'S MY GREAT PLEASURE TO INTRODUCE TODAY'S IIG SPEAKER, DR. LYNN HEDRICK. LYNN RECEIVED HER PH.D. IN BIOCHEMISTRY FROM WAKE FOREST UNIVERSITY SCHOOL OF MEDICINE& WHERE SHE STUDIED LDL METABOLISM. AND I THINK YOU GOT YOUR FIRST EXPERIENCE WITH MYELOID CELLS DURING YOUR PH.D.? AS A POSTDOC, SHE JOINED LABORATORY OF DR. JAKE AT UCLA WHERE SHE PERFORMED RESEARCH ON HDL AND THE FACTS OF PROTEIN A2. AS A JUNIOR FACULTY MEMBER IN DIVISION OF CARDIOLOGY AT UCLA, SHE FOCUSED ON HOW INFLAMMATION EFFECTS ENDOTHELIAL FUNCTION. IN 2000, LYNNE JOINED THE DEPARTMENT OF MEDICINE AT THE UNIVERSITY VIRGINIA AND VERY CLOSE TO US QUICKLY RISING THROUGH THE RANKS IN 2009 SHE WAS AWARDED THE HARRISON CHAIR OF MOLECULAR PHYSIOLOGY AND BIOLOGICAL PHYSICS. HOWEVER, LYNNE HAD A CHANGE OF HEART AND DECIDED TO MOVE HER LAB TO THE WEST COAST TO LAHOYA INSTITUTE FOR ALLERGY AND IMMUNOLOGY WHERE SHE IS CURRENTLY PROFESSOR AT THE DIVISION OF INFORMATION BIOLOGY. NEVERTHELESS, HER WORK CONTINUES TO BE CENTERED AROUND THE CROSS TALK BETWEEN MILEID AND EPITHELIAL CELLS, LIPID METABOLISM AND LIPID MEDIATORS, AND THE GOAL TO BETTER DEFINE HOW FUNCTION OF IMMUNE CELLS CHANGES DURING PROGRESSION OF CARDIOVASCULAR DISEASES AND CANCER. LYNNE DISCOVERED THAT THE TRANSCRIPTIONAL FACTOR ON NA4A1, NEURO77, SO TRANSLATION QUICK, CONTROLS THE DIFFERENTIATION OF LY6 LOW MONOCYTES AND FURTHERMORE, THAT SHE IDENTIFIED HER GROUP IDENTIFIED THE SUPER ENHANCER IN THE SAME GENE THAT CONTROLS THE DIFFERENTIATION OF THESE CELLS BUT DOES NOT EFFECT THE FUNCTION OF MACROPHAGES. IMPORTANTLY, HER GROUP HAD LEVERAGED THEIR EXPERIENCE TO EXPLODE SPECIFIC MYELOID SUBSETS FOR IMMUNOTHERAPY OF CANCER AND I GUESS THAT IS THE TOPIC OF YOUR TALK TODAY. SO PLEASE WELCOME LYNNE WITH ME. [ APPLAUSE ] >> I'D LIKE TO THANK YOU FOR THE NICE INVITATION. THIS IS MY FIRST TIME SPEAKING AT NIH ALTHOUGH I HAVE SEEN SOME OLD FRIENDS, SOME OLD COLLEAGUES, I HAVE MET SOME NEW COLLABORATORS, AND I HAVE TO POINT OUT MY THREE NEWEST COLLABORATORS ROSIE, STEVEN AND DAVE. WE DECIDED TO WRITE AN INTRAMURAL EXTRAMURAL NIH GRANT ABOUT A YEAR AGO, YEAR AND A HALF AGO, AND FINGERS CROSSED, WE MAY HAVE IT, BUT ANYWAY, SO WE HOPE WE CAN DO THE STUDIES THAT WE REALLY WANT TO DO TOGETHER. YOU'RE GOING TO INVITE ME AGAIN. SO, ANYWAY, YOUR TRAINEES CAN SEE AS A BIOCHEMIST, I'M NOW AN IMMUNOLOGIST SO MY -- YOUR CAREER WILL TAKE MANY TURNS IN SCIENCE. BUT EMBRACE THEM BECAUSE IT IS DEFINITELY A LOT OF FUN. SO, I'M GOING TO TALK TO YOU TODAY ABOUT MONOCYTE HETEROGENEITY IN CANCER. I HAVE TWO ASPECTS OF MY LAB. ONE STUDIES CARDIOVASCULAR DISEASE AND ARTHROSCLEROSIS. I HAVE DONE THAT WORK FOR MANY, MANY YEARS AND IN THE PAST FEW YEARS WE HAVE TAKEN WHAT WE LEARNED IN ARTHEROSCLEROSEIS AND TRYING TO MOVE THAT TO CANCER. EVEN THOSE DISEASES ARE DIFFERENT, THEY HAVE A LOT OF SIMILARITIES AND THE THINGS YOU LEARN FROM ONE DISEASE ARE APPLICABLE TO ANOTHER. SO FOR THOSE OF YOU WHO DON'T THINK ABOUT MONOCYTES ALL THE TIME, THIS IS THE COOLEST THING, HONESTLY. I'LL JUST BRIEFLY GO OVER A SIMPLISTIC VIEW OF MONOCYTE DEVELOPMENT. THEY ARE MADE IN THE BONE MARROW FROM STEM CELL PROGENITORS. THE MDP AND CMOP. THE CMOP, COMMON MYELOID PROGENITOR GIVES RISE TO AT LEAST IN MICE ARE TWO WELL-DEFINED SUBSETS, THE LY6C HIGH AND LOW MONOCYTE. THESE THEN ARE EGRESS FROM THE BONE MARROW TO THE BLOOD. THE LOWS TONED HAVE FUNCTIONS IN THE VASCULATURE WHICH WE'LL TALK ABOUT. AND THEN THE HIGHS CAN MIGRATE INTO TISSUES. SO CAN THE LOWS BUT IT THE HIGHS ARE THE ONES WHO DIFFERENTIATE INTO MACROPHAGES. IT'S NOT CLEAR THAT THE LOWS WILL DIFFERENTIATE INTO A MACROPHAGE. SO THAT'S A FUNDAMENTAL DIFFERENCE BETWEEN THE TWO. IT'S NOT THAT THEY DON'T. IT'S JUST TO NO ONE CAN FIND THEM. WE THINK THERE IS ONE SPECIALIZED SUBSET IN THE THYMUS THAT DERIVES FROM THESE CELLS BUT IN GENERAL, WE THINK THAT MOST OF THE MONOCYTE-DERIVED MACROPHAGES IN THE TISSUE ARE DERIVED FROM THE LY6C HIGH SUBSET. SO THIS IS A VERY SIMPLISTIC VIEW OF MONOCYTES THAT WILL CHANGE TOWARD THE END OF MY TALK. BUT FOR THOSE OF YOU I JUST WANT TO SET THE STAGE AND GET PEOPLE ON THE SAME PAGE. THERE IS TWO WHAT WE CALL CLASSICAL AND NON-CLASSICAL SUBSETS N MICE WE DIFFERENTIATE THE TWO OR DISTINGUISH THE TWO BASED ON THE SURFACE ANTIGEN LY6C HIGH AND LOW. IN HUMAN, THERE ARE COUNTERPARTS THAT ARE FUNCTIONALLY SIMILAR AND WE DISTINGUISH THESE CURRENTLY BASED ON THE MARKER CD14 AND CD16. CD14 HIGH MONOCYTOSES ARE CLASSICAL. THERE IS A DOUBLE POSITIVE MONOCYTE CALLED AN INTERMEDIATE AND THEN THE NON-CLASSICAL MONOCYTE IS CD14dim. SO THE CLASSICAL MONOCYTE AS MANY OF YOU KNOW ARE THE MOST FAMOUS MONOCYTE SEMINOLE COUNTY SUBSET. THEY ARE THE MONOCYTE SUB SET THAT GETS RECRUITED RAPIDLY IN RESPONSE TO INFLAMMATION AND BEEN SHOWN BY JEFF POLLARD AND OTHERS TO PROMOTE TUMOR GROWTH. THE NON CLASSICAL MONOCYTE IS A DIFFERENT BEAST. IT IS VERY INVOLVED IN IMMUNE SURVEILLANCE, PATROLLING. AND THIS WAS IDENTIFIED, THIS PATROLLING QUALITY WHICH I'LL SHOW YOU WAS IDENTIFIED BY FREDRICK GRACE MAN SEVERAL YEARS AGO. SO THE QUESTION IS, WHAT IS THE ROLE OF THIS SUBSET IN CANCER IF THIS SUBSET PROMOTES TUMOR GROWTH? THAT IS WHAT OUR LAB SET OUT TO FIND OUT A FEW YEARS AGO. SO TO DO THIS, WE DO A LOT OF IMAGING ON LIVE ANIMALS AND WE USE TWO MODELS TO REALLY STUDY THE NON-CLASSICAL MONOCYTES, THOSE ARE OUR FAVORITE MONOCYTES WE USE REPORTER MICE MADE BY CHRIS. NR4A1 IS ALSO CALLED 77 UP AND IT PLACE A ROLE IN T-CELL SELECTION. SO THOSE WHO KNOW ABOUT T-CELLS ARE VERY FAMILIAR WITH THIS MOUSE MODEL. AND THEN SO THE NON CLASSICAL MONOCYTES ARE HIGH FOR EXPRESSION LIKE HIGHER LOGS FOLD THAN CLASSICAL AND THE NON-CLASSICAL ARE ALSO EXTREMELY HIGH FOR EXPRESSION OF THE RECEPTOR CX3CR1. SO USING THESE REPORTER MICE WE CAN SELECTIVELY FIND THE NON-CLASSICAL MONOCYTES IN-VIVO AND IMAGE THEM. SO, WE ROUTINELY USE BOTH OF THESE MODEL AND PRETTY MUCH SOMEWHAT INTERCHANGEABLY. SO THIS IS PATROLLING. SO THIS IS IN-VIVO IN A MOUSE EAR IN CASE YOU REALLY NEVER SEEN PATROLLING. S BASICALLY THE BLOOD IS GOING FROM THE TOP TO THE BOTTOM OF THE SCREEN. AND WHAT YOU DON'T SEE ARE CLASSICAL MONOCYTES AND EVERYTHING ELSE FLOWING THROUGH RAPIDLY. BUT WHAT YOU SEE HERE IN GREEN, AND THIS IS USING A MOUSE THAT IS SELECTIVELY LABELING THE NON CLASSICAL MONOCYTES.& YOU CAN SEE THE CELL CRAWLING ALONG THE VESSEL COMPLETELY ACROSS THE VESSEL KIND OF -- THIS DOESN'T SEEM TO BE DISTURBED AT ALL BY THE MASSIVE TSUNAMI FLOW GOING AGAINST IT. AND IT IS CRAWLING VERY SLOWLY REALLY SURVEYING THE VASCULATURE AND SO WE KNOW THAT FROM OUR WORK AND WORK OF NOW MANY IN THE FIELD AND PARTICULARLY FREDRICK, THAT THESE CELLS ARE INVOLVED IN& THE RECOGNITION AND REMOVAL OF PATHOGENS, FOR INSTANCE VIRUSES, AND DYING CELLS, AND WE BELIEVE THAT THEY ARE INVOLVED IN REPAIRING THE VASCULATURE AND WE ALSO BELIEVE THAT THEY ARE ANTI-INFLAMMATORY IN NATURE. AND SO REALLY THESE ARE THE CENTURY CELL OF THE VASCULATURE. THEY REALLY DO STAY IN CIRCULATION. WE THINK THEY HAVE CRITICAL FUNCTIONS IN THE VASCULATURE. AND SO WHERE THEY ARE KIND OF LIKE -UE CAN THINK OF THEM AS FIRST CELL RESPONSE FOR THE BODY. SO, IN TERMS OF CANCER, IT'S KNOWN BY POLLARD AND OTHERS THAT THESE CCR2 EXPRESSING MONOCYTES WHICH ARE IN MICE LY6C HIGH, THAT THESE MONOCYTES ACTUALLY ARE PRO-TUMORAL. THEY GET RECRUITED BY THE TUMOR SELLS AND DIFFERENTIATE INTO MACROPHAGES THAT PROMOTE TUMOR GROWTH AND THERE IS LOTS OF LITERATURE ON THIS PROCESS. AND SO CLASSICAL MONOCYTES ARE THOUGHT TO BE BAD FOR CANCER. PRO TUMORAL. THE NON CLASSICAL MONOCYTES, WE SHOWED THEY PLAY A ROLE IN PREVENTING TUMOR METASTASES. WHAT THEY DO IN TERMS OF PRIMARY TUMOR GROWTH, ARE NOT SURE YET SO OUR FOCUS HAS BEEN ON METASTASES. ONE OF THE MAIN QUESTIONS IS WHAT DO THEY DO? DO THEY PLAY A ROLE? IF SO WHAT? A LOT OF WHAT WE HAVE DONE IS KIND OF OUR WORKHORSE MODEL IS A VERY ARTIFICIAL MODEL OF LOOKING AT METASTASES WHERE WE TAKE VARIOUS TUMORS, B16, OR LLC, AND WE INJECT IV AND OF COURSE IT WILL GO TO THE LUNG. THERE IS A LOT OF MONOCYTES IN THE LUNG BUT THEN THAT ALLOWS US TO IMAGE THE INTERACTION OF THE MONOCYTE WITH THE TUMOR CELLS AS THEY ARE SEEDING VERY EARLY ON. AND SO A LOT OF OUR WORK HAS BEEN USING THIS MODEL ALTHOUGH WE HAVE USED OTHER MODELS, INCLUDING THE SPONTANEOUS PYMT MODEL. SO THIS IS LIVE IMAGE OF THE LUNG AND IN THIS CASE, THE TUMOR IS IN RED HERE AND THE NON CLASSICAL MONOCYTES SO YOU CAN SEE THESE ARE THE MONOCYTES. AND WHAT YOU SEE IF YOU WATCH THIS GUY UP HERE MAYBE, I THINK THAT HE IS GOING TO MAYBE AT SOME POINT TRY TO -- HE SENSES TE TUMOR AND GOES AROUND IT AND YOU CAN SEE THAT THEY ARE KIND OF INTERACTING WITH THE TUMOR QUITE READILY. AND YOU CAN SEE HERE WE CAN TRACK THESE OF COURSE USING SOFTWARE. SO THIS IS UNTREATED. EACH ONE OF THESE TRACKS IS A MONOCYTE IS MOVING AND YOU CAN SEE THAT BECAUSE OF PATROLLING, THEY ARE GOING -- WE GET HAIR PINS, LOOPS, DIFFERENT DIRECTIONS, DIFFERENT DISTANCES. AND THAT IS CLASSICAL -- THAT IS TYPICAL NON CLASSICAL MONOCYTE PATROLLING PATTERN. SO THIS IS WITHOUT TUMOR AND YOU CAN SEE EVEN FOUR HOURS AFTER GIVING A TUMOR, YOU CAN SEE THAT THE CELLS ARE STARTING TO REST AND SLOW DOWN AND BY 24 HOURS, ALL OF THESE MONOCYTES HAVE KIND OF ARRESTED AROUND THE TUMOR. WE CAN OBVIOUSLY CALCULATE THEIR SPEEDS AND YOU CAN SEE THAT AGAIN AT 24 HOURS, AND EVEN AT FOUR HOURS, THEY ARE SLOWING DOWN AROUND THE TUMOR. SO THEY SEE THESE TUMOR CELLS AND THEY ARE IMMEDIATELY GOING TO IT SO THEY ARE SENSING SOMETHING. AND THEY ARE GOING AND INTERACTING WITH THAT TUMOR. SO, TO STUDY HOW WE WANTED TO STUDY HOW THE NON CLASSICAL MONOCYTES MAY INTERACT WITH THE TUMOR CELLS SO WE DECIDED TO USE TWO MODELS THAT LACK NON CLASSICAL MONOCYTES. ONE OF THEM IS THE NR4A1 KNOCKOUT. THIS IS A GLOBAL MOUSE. GLOBAL KNOCKOUT MOUSE, AND THEN WE USED THE NR44A1 MOUSE THAT WE CROSS WITH THE CRE. SO THIS RANKS NON CLASSICAL MONOCYTES BUT OBVIOUSLY NR44A1 IS IMPORTANT FOR T-CELLS SO THIS MOUSE HAS T-CELL CHANGES AS WELL. AND IN THIS ANIMAL, IT IS FLOCKED SO MYELOID SPECIFIC BUT IT HITS BOTH THE CLASSICALS AND THE NON CASS CALLS AS WELL AS MACROPHAGES. BUT THIS MOUSE DOES LACK NON CLASSICAL MONOCYTES. SO AT THE TIME, WE STARTED THESE STUDIES, THESE WERE TO THE BEST MODELS WE HAD. AND AGAIN IN BOTH MODELS, THE NR4A1 WE DISCOVERED THE MASTER TRANSCRIPTIONAL REGULATOR FOR THE DEVELOPMENT OF THE NON CLASSICAL MONOCYTES. SO IF YOU DON'T HAVE NR4A1, YOU DO NOT HAVE NON CLASSICAL MONOCYTES IN EITHER MODEL. THESE WERE THE TOOLS WE STARTED WITH WHEN WE FIRST EMBARKED ON THESE STUDIES. SO BASICALLY WHAT WE FOUND IS IF YOU DON'T HAVE NON CLASSICAL MONOCYTES IN YOUR BODY, THAT YOU GET MORE LUNG CANCER METASTASES. AND HERE THIS IS JUST A BAR GRAPH OF FLOW CYTOMETRY TO SHOW THAT IN THIS CASE, WE ARE USING THE FLOCKED ANIMAL. YOU CAN SEE THAT THE CLASSICAL MONOCYTE COMPARTMENT IS NORMAL BUT THEY ARE MISSING ABOUT 50% OF MONOCYTES AND MISSING PRETTY MUCH ALL THEIR LY6C LOW MONOCYTES. THEY GET MORE TUMOR VERY EARLY ON. THIS IS A LUCIFERASE TUMOR AND YOU CAN SEE THE LUCIFERASE COUNTS HERE AND IF YOU WANT TO LOOK AT THE METASTASES, YOU CAN COUNT THEM AND YOU CAN SEE AGAIN IF YOU DON'T HAVE NON CLASSICAL MONOCYTES, YOU GET MORE TUMOR METASTASES SUGGESTING THESE CELLS ARE VERY IMPORTANT EARLY ON IN PREVENTING METASTASES. SO, WE DID -- THIS WAS A CRAZY EXPERIMENT. THIS TOOK OVER 250 MICE TO DO. SO WE HAD TO ACTUALLY ISOLATE NON CLASSICAL MONOCYTES AND CLASSICAL MONOCYTES FROM MICE AND THEN ACTUALLY ADD THEM BACK TO THE KNOCKOUT. AND SO I'LL TAKE YOU THROUGH THIS EXPERIMENT. SO BASICALLY, DAY ZERO WE INJECTED EITHER CLASSICAL OR NON CLASSICAL MONOCYTES. THESE ARE WILDTYPE MONOCYTES. AND THEN WE INJECTED TUMOR CELLS AND WE WAITED 7 DAYS AND IMAGED THE TUMOR. SO HERE JUST TO START, THIS IS THE LUCIFERASE TUMOR. YOU CAN SEE WILDTYPE NO TUMOR AND IN THE KNOCKOUT YOU HAVE AT 7 DAYS YOU HAVE A METASTASES. AND AGAIN, THESE DO NOT HAVE NON CLASSICAL MONOCYTES. SO IF YOU ADD IN CLASSICAL MONOCYTES, YOU GET EXACERBATED TUMOR GROWTH LIKE BEEN SHOWN BY POLLARD AND OTHERS. SO THIS IS CONFIRMED THAT IT IS MUCH GREATER THAN HERE. IT'S THE SAME TIME POINT. 7 DAYS. BUT IF YOU ADD IN THE NON CLASSICAL MONOCYTES, YOU SEE THAT YOU PREVENT TUMOR GROWTH. SO THIS WAS I THINK A KEY EXPERIMENT THAT SHOWED US THAT NON CLASSICAL MONOCYTES ARE EXTREMELY IMPORTANT FOR REGULATING TUMOR METASTASES BUT WE HAVE NO IDEA WHY. THAT THE POINT. SO I MENTIONED THAT THE OTHER MICE THAT WE HAVE USED, THE NR4A1 KNOCKOUT GLOBAL AND THE FLOCKS CFS1 MOUSE, HAD CAVEATS. BECAUSE NR4A1 IS EXPRESSED IN T-CELLS AND NON CLASSICAL AND CLASSICAL AND MACROPHAGES. SO EVEN THOUGH THAT RESCUE EXPERIMENT TRULY TOLD US THE NON CLASSICALS WERE IMPORTANT, IT DIDN'T REALLY RULE OUT MACROPHAGES IN THE CASE OF THE FLOCKED ANIMAL, CLASSICAL MONOCYTES IN THE CASE OF THE FLOCKED ANIMAL OR T-CELLS IN THE CASE OF THE GLOBAL KNOCKOUT. WE SET OUT TO TRY TO MAKE A MOUSE THAT ONLY LACKED NON CLASSICAL MONOCYTES. AND TO DO THAT AT THE SAME TIME WE WERE INTERESTED IN NR4A1 AND ITS REGULATION BECAUSE WE THOUGHT SINCE IT'S A TRANSCRIPTION FACTOR THAT REGULATES NON CLASSICAL MONOCYTE DEVELOPMENT, THE MORE WE KNOW ABOUT NR4A1 REGULATION, THE MORE WE THINK WE CAN TOGGLE NON CLASSICAL MONOCYTE LEVELS AND FUNCTION IN HUMANS, WHICH IS WHAT WE WANT TO DO. SO, WE SET OUT ON A SERIES OF STUDIES TO TRY TO UNDERSTAND NR4A1 REGULATION AND THIS PAPER WAS PUBLISHED LAST YEAR IN IMMUNITY. AND BASICALLY WHAT WE DID WAS WE DECIDED TO DO CHIP SEQ OF THE YR4A1 ENHANCER REGION AND WE DID THIS IN MONOCYTES AND SO WE LOOKED AT H3K27 ACETYLATION AND THE H3K FOR DIMETHYL TO TRACKS TO SEE, IDENTIFY ENHANCER REGIONS UPSTREAM OF NR4A1 AND THE LY6C LOWS IN RED AND THE LOWS THAT ARE IN BLUE. SO YOU HAVE ABOUT, IF YOU LOOK AT THESE TWO BARS, WHAT YOU SEE HERE IS IN THE LY6C LOWS YOU SEE ACTIVE ENHANCER REGION OF 20KB. SO THIS IS -- THIS REGION IS PRESENT BUT IT'S ACTIVE ONLY IN THE LY6C LOWS. SO THIS IS SUPER ENHANCER REGION. YOU CAN SEE THAT IT IS ACTIVE HERE IN THE LOWS. AND SO SUPER ENHANCERS ARE THOUGHT TO BE IMPORTANT REGULATORS OF LINEAGE COMMITMENT AND SO WE KNEW THAT SINCE NR4A1 WAS IMPORTANT FOR THIS MONOCYTE SUB SET LINEAGE, WE THOUGHT THIS REGION WOULD BE IMPORTANT FOR US TO INVESTIGATE. SO WHAT WE DID WAS WE WORKED IN COLLABORATION WITH CHRIS GLASS AT UCSD WHO IS A MASTER AT UNDERSTANDING EPIGENETIC REGULATION IN MACROPHAGES. SO WE WORKED WITH HIM AND IDENTIFIED 12 ENHANCER REGIONS. THESE ARE 1500 BASE PAIRS. AND WHAT WE DID WAS WE BASICALLY CLONED EACH OF THESE ENHANCERS. AND JUST SO YOU SEE HERE THE LY6C LOW, THIS IS THE ACTIVE ENHANCER REGION HERE AND THERE IS A LOT MORE IN THE LOWS THAN IN THE HIGHS AND VERY FEW IN THE PROGENITORS WHICH FITS WHAT WE KNOW ABOUT NR4A1 EXPRESSION IN PROGENITORS. SO WHAT WE DID WAS WE CLONED THESE 12 LITTLE ENHANCER REGIONS AND WE PUT THEM IN AN IN-VITRO SYSTEM UPSTREAM OF THE PROMOTER REGION OF NR4A1 AND WE BASICALLY TRANSFECTED THESE INTO A MACROPHAGE CELL LINE WHICH WAS ALL WE COULD DO AT THE TIME, AND WE WANTED TO SEE IF ANY OF THESE HAD ENHANCER ACTIVITY AND COULD DRIVE LUCIFERASE EXPRESSION BY ACTIVATING THE NR4A1 PROMOTER. IN DOING THAT, WE FOUND THREE REGIONS WHICH WE ARBITRARILY NAMED E2, EICS AND E9. AND YOU CAN SEE HERE THAT HERE IS THE DATA. IT'S ABOUT A TWO-FOLD INDUCTION. THIS IS A RAW CELL LINE SO IT'S NOT THAT GREAT BUT AT LEAST IT IS OVER BASELINE. SO WE SAID OKAY, WE ARE JUST GOING TO GO WITH IT. RIGHT? SO WHAT WE DID THEN IS TO MAKE AND USE CHRIS PER CAS9 TO MAKE KNOCKOUT MICE FOR EACH OF THESE ENHANCERS. SO WE MADE FOUR MICE SO WE MADE E2, E6 AND E9 AND THEN DELETED THIS ENTIRE REGION. AND SO EACH OF THESE IS A 1500 BASE PARODY LESION ONLY. AND IN DOING SO, WHAT WE FOUND WAS THAT THIS E2 REGION, WHICH IF YOU GO BACK HERE, IS REALLY LOCATED HERE, IS ACTUALLY CAUSES A ALMOST A COMPLETE DELETION OF THE NON CLASSICAL MONOCYTE LINEAGE. THE E6 NO DIFFERENCE IN THE E9 NO DIFFERENCE AND THIS IS WILDTYPE HERE. SO IF YOU'RE NOT FAMILIAR WITH THESE FLOW PLOTS, THIS IS LOW CLASS TWO LOW, NON CLASSICAL MONOCYTES HERE. SO WE DID ALMOST A COMPLETE LOSS. SO THIS SUGGESTS THAT THIS E21500 BASE PAIR REGION IS ESSENTIAL FOR NON CLASSICAL MONOCYTE DEVELOPMENT. AND SO A LOT OF WHAT WE HAVE DONE SUBSEQUENT TO THAT IS TO FIGURE OUT WHAT TRANSCRIPTION FACTORS CAN BIND IN THAT 1500 BASE PAIR REGION BECAUSE WE WANT TO KNOW HOW NR4A1 IS REGULATED. I'M NOT GOING TO SHOW THAT DATA BUT I'M HAPPY TO TALK TO YOU ABOUT THAT AFTER. SO WE HAVE SOME GOOD IDEAS OF HOW WE CAN REGULATE NR4A1 IF WE CAN'T DO IT DIRECTLY, HOW WE CAN DO IT THROUGH THESE OTHER TRANSCRIPTION FACTORS. BUT WHAT IS IMPORTANT ABOUT THIS MOUSE TWO-FOLD. ONE, WE ARE SELECTIVELY, LOST THE NON CLASSICAL MONOCYTES, WHICH IS GREAT, AND I CAN TELL YOU I'M NOT SHOWING DATA BUT THE T-CELL COMPARTMENT WE LOOKED AT AT LEAST AT HOMEOSTASIS, IS COMPLETELY NORMAL. SO THERE IS NOT A T-CELL DEFECT THAT WE SEE IN THE GLOBAL KNOCKOUT AT HOMEOSTASIS. THAT IS NICE. AND THEN IF WE LOOK AT THE MACROPHAGE RESPONSE, THE MACROPHAGES RESPONSES APPEAR TO BE NORMAL IN THIS MOUSE MODEL. THERE IS PROBABLY THE BEST EXAMPLE IS HERE. IF YOU GIVE MICE OR MACROPHAGES FROM THESE MICE LPS, THE NR4A1 KNOCKOUT AND THE FLOCKED ANIMAL HAVE AN EXACERBATED RESPONSE. THE MACROPHAGE VS. AN EXACERBATED RESPONSE AS DO THE CLASSICAL MONOCYTES COMPARED TO WILDTYPE. AND YOU CAN SEE THAT OUR E2 KNOCKOUT HAS A PRETTY NORMAL RESPONSE. THERE SEEMS TO BE NORMAL NR4A1 INDUCTION AND A NORMAL RESPONSE TO LPS AT LEAST BY THESE MACROPHAGES. THIS TOLD US THAT WE HAVE A NEW MOUSE MODEL THAT IS SELECTIVELY MISSING NON CLASSICAL MONOCYTES, HAVE A NORMAL T-CELL COMPARTMENT AND AS FAR AS WE CAN TELL, A NORMAL MACROPHAGE COMPARTMENT. SO THIS GIVES US A NEW MODEL TO SELECTIVELY TEST IN-VIVO THE FUNCTION OF NON CLASSICAL MONOCYTES IN DISEASE. SO WE HAVE USED THIS MOUSE NOW IN CANCER AND ARTHROSCLEROSIS. SO WHAT WE HAVE DONE IN CANCER IS WE JUST LOOKED AT METASTASES USING THIS MODEL. SO HERE ARE LUNG SECTIONS AND WILDTYPE AND THIS IS THE GLOBAL KNOCKOUT YOU CAN SEE THE METS AND HERE IS OUR E2 KNOCKOUT HERE. SO AND THAT IS QUANTIFIED HERE. SO YOU CAN SEE AGAIN, IN BOTH OF THESE MODELS, THAT DON'T HAVE NON CLASSICAL MONOCYTES AND THIS IS SPECIFIC, YOU SEE A VERY SIMILAR IMPACT OF INCREASED TUMOR METASTASES. SO I THINK THIS IS FURTHER REALLY WHEN YOU TAKE IT ALL TOGETHER, PRETTIED CONVINCING EVIDENCE THAT NON CLASSICAL MONOCYTES PLAY A CRITICAL FLOW METASTASES. SO, WHAT DO THEY DO? WE KNOW THEY TAKE AT LEAST TUMOR EXOSOMES, WE CALLED THEM DEBRIS OR PARTICLES. WE DON'T KNOW EXACTLY T IS OBVIOUSLY NOT WHOLE TUMOR CELLS. BUT PIECES, EXOSOMES OF -- THEY INGEST THEM. THEY PREFERENTIALLY INGEST THEM OVER THE CLASSICALS. THE CLASS CALLS CAN TAKE THEM UP BUT THE CLASSICALS MONOCYTES TAKE UP MUCH, MUCH FEWER AND IT IS A NON CLASSICAL MONOCYTE-DRIVEN EVENT. SO THEY INGEST THESE PARTICLES AND WE LOOKED THAT THE BY FLOW CYTOMETRY. THIS IS JUST -- IMAGES ARE PRETTY. THIS IS THE TUMOR IN RED, THE NON CLASSICAL MONOCYTES ARE IN GREEN AND THEY HAVE TAKEN UP THE TUMOR DEBRIS HERE INSIDE. AND THEN THIS IS MOUSE AND THIS IS HUMAN. SORRY THE COLORS ARE DIFFERENT BUT SO I DON'T WANT TO CONFUSE YOU BUT IN THIS CASE, WE CAN ONLY LABEL THE NON CLASSICAL CALLS CD16 IN BLUE. SO THE NON CLASSICALS ARE IN BLUE AND THE TUMOR IS IN GREEN. BUT YOU CAN SEE THAT THERE ARE TUMOR CELL DEBRIS OR MAYBE EXOSOMES INSIDE THESE MONOCYTES. SO WE KNOW THAT THEY CAN TAKE THESE PARTICLES UP. WE ALSO KNOW THAT THEY CAN TAKE THE PARTICLES TO THE LYMPH NODE, THE LUNG DRAINING LYMPH NODES AND PRESENT ANTIGEN TO CD4s AND CD8s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s FROM THEM AND WE BASICALLY DID MASS CYTOMETRY AND THEN A WHOLE BUNCH OF ANALYSIS AND THIS IS WHERE -- THIS TAKES YOU A FEW DAYS, A FEW WEEKS TO ACTUALLY OPTIMIZE BUT ONCE YOU GET YOUR PANELS, IT TAKES A FEW DAYS TO DO. IF YOU'RE REALLY GOOD, THIS WILL TALKED YOU MANY, MANY WEEKS TO ANALYZE. I THINK THE PROBLEM WITH THE FIELD NOW YOU IS GET SO MUCH DATA AND PEOPLE JUST, ESPECIALLY POSTDOCS AND STUDENTS, IT'S HARD TO SIT AT THE COMPUTER UNLESS YOU'RE A COMPUTATIONAL GUY, MATHEMATICIAN, I MEAN IT'S HARD FOR PEOPLE TO SIT THERE AND LOOK THAT THE DATA. YOU WANT TO GET TO THE BENCH AND DO YOUR NEXT EXPERIMENT BUT THE BEAUTY IS, THERE IS SO MUCH DATA HERE, IF YOU TAKE THE TIME TO ANALYZE IT CORRECTLY, YOU HAVE MANY, MANY PROJECTS AND MANY, MANY PAPERS AND CAREER, PERHAPS. THIS IS SOME OF THE MARKERS IN OUR PANEL. SO WHAT WE DID WAS WE HAVE A PANEL THAT ALLOWS US, WE FIGURED OUT BY MAKING MANY MISTAKES THAT YOU NEED DIVERSITY. SO WE KEEP T-CELLS IN AND B-CELLS AND STUFF. BUT WE HAVE HEAVILY BIASED OUR PANELS TOWARDS MYELOID CELLS, NEUTROPHILS, MONOCYTES, PROGENITORS, AND SO THESE ARE JUST SOME OF THE MARKERS IN THE PANEL. THIS IS JUST ONE OF OUR PANELS. WE HAVE SEVERAL DEPENDING ON ON WHAT WE ARE DOING. BUT THIS WHAT WE DID WAS WE RAN OVER 120 ANTIBODIES BY CONVENTIONAL FLOW TO SEE ARE THEY EXPRESSED ON ANY MYELOID CELL AT SCALL IF THEY WERE THEN WE ADDED IT TO THE PANEL. AND SO, OVER IN THE LAST COUPLE OF YEARS, I GUESS, WE HAVE MADE THESE REALLY NICE PANELS F ANYBODY WANTS TO KNOW OUR PANELS, I'M HAPPY TO SHARE THEM BECAUSE NOBODY SHOULD REALLY HAVE TO REDO THIS. IT'S A LOT OF WORK. BUT YOU CAN SEE THAT WE HAVE CELL LINEAGE, CELL DEATH, ADHESION, ANTIGEN PRESENTATION, ACTIVATION, MIGRATION, SCAVENGER RECEPTORS AND CHEMOKINE RECEPTORS. AND SO WE HAVE DONE A VISITING PLOT. WE HAVE ACTUALLY RUN PBMCs WITH A MYELOID-BASE PANEL BUT WE HAVE OTHER MARKERS ON IT AND SO WE WHAT I'M NOT SHOWING IS ANOTHER AREA WHERE WE HAVE FOUND OUR MONOCYTES AND THEN WE ACTUALLY ARE LOOKING CLOSER AT THEM. SO THIS IS A MONOCYTE. WE HAVE GOTTEN RID OF THE ALL OF B-CELLS AND T-CELLS BECAUSE WE ARE HONING IN ON OR INTERESTED IN THESE CELLS NOT THE OTHERS AT THE MOMENT. SO WHAT WE DID HERE IS -- IF YOU'RE NOT FAMILIAR WITH THESE, THESE PLOTS REDUCE THIS INTO 2D SO OUR EYES CAN UNDERSTAND IT BETTER. SO YOU HAVE -- IT BASICALLY PUTS SIMILAR CELL TYPES, CLUSTERS THEM -- THAT'S NOT THE RIGHT WORD BUT GROUPS THEM TOGETHER SO THAT THIS IS ONE BIG POPULATION OF CELLS THAT IS FAIRLY SIMILAR AND THIS CELL TYPE IS SIMILAR BUT IT MAY BE SLIGHTLY DIFFERENT. SO WHAT WE HAVE DONE HERE IS BASED ON THESE RAINBOW PLOTS, YOU CAN OVERLAY YOUR MARKERS SO JUST FOR FUN, I OVERLAID CD14 AND CD16 SO THOSE OF YOU WHO KNOW WILL KNOW THAT THESE ARE ALL CLASSICAL MONOCYTES LIKELY, AND THESE ARE MAYBE MOSTLY NON CLASSICAL MONOCYTES AND MAYBE THESE ARE SOMEWHERE IN BETWEEN. AND SO WE GET TONSE OF THESE BUT SO THIS IS JUST AN EXAMPLE OF A PLOT. AND WHAT WE HAVE DONE IS TAKE THIS BASIC PLOTS AND THEN RUN MULTIPLE ALGORITHMS. THIS IS A FLOW ALGORITHM BUT THERE IS PHENOGRAPH AND OTHERS THAT ALLOWS YOU TO CLUSTER AND THIS IS AGAIN MACHINE LEARNING AND IT CLUSTERS BASED ON 41 MARKERS AND TRIES TO TELL YOU WHICH GROUPS ARE MORE SIMILAR TO OTHERS. AND SO, IF YOU GO BACK TO THIS NOW, WE HAVE RUN CLUSTERING ON THIS AND USING PHENOGRAPH, WE FOUND EIGHT CLUSTERS AND SO THESE ARE ALL THE CLASSICALS THAT WE SEE AND YOU CAN SEE THAT THERE IS -- THEY ARE JUST RANDOMLY NUMBERED HERE. THESE ARE INTERMEDIATES AND THESE ARE THE THREE IN PURPLE ARE NON CLASSICALS. SO WE IDENTIFIED THESE THREE NON CLASSICALS AND THIS IS JUST A HEAT MAP OF ALL THE MARKERS WE RAN AND SO, AGAIN IF YOU LOOK FOR CD14, YOU SEE THAT THESE ARE THESE CLUSTERS HERE AND CD16 IS -- I DON'T WHEREIN IT IS. YOU CAN PROBABLY SEE IT. I CAN'T SEE IT. MAYBE IS THAT IT? THOSE ARE THE CLUSTERS HERE. AND THESE ARE INTERMEDIATE. SO THAT KIND OF TELLS UD WHAT YOU HAVE, RIGHT? AND SO THEN WITHIN EACH OF THESE IS HETEROGENEITY. SO IS THERE IS A TON OF DIVERSITY HERE. AND THIS AGAIN SHOWS US THE VARIABILITY SO IN THIS CASE THESE ARE HEALTHY HUMANS. WE DID 12 PATIENTS. YOU CAN SEE HERE IS THIS ALL THE MONOCYTES SO OBVIOUSLY IN HUMANS DIFFERENT THAN MICE, THERE IS A LOT MORE CLASSICALS THAN NON CLASSICALS THAN INTERMEDIATES BUT EACH BAR IS ONE INDIVIDUAL. AND YOU CAN SEE THAT IN HEALTH HUMANS THERE IS HUGE VARIABILITY IN THE NUMBER OF CLUSTERS OR THE AMOUNT OF EACH CLUSTER. AND WHAT WE DID IS BECAUSE WE ARE INTERESTED IN NON CLASSICAL, WE JUST BLUE THIS UP HERE. BUT YOU CAN SEE THAT EVERYBODY HAS I THINK PRETTY MUCH EVERY CLUSTER. IT IS JUST THAT THERE IS DIFFERENT AND SO FAR WE HAVE SEEN THAT. EVERYONE HAS PRETTY MUCH EVERY CLUSTER BUT THERE IS JUST DIFFERENT FREQUENCIES AND HOW THEN THESE ARE HEALTHY HUMANS AND CHANGES IN DISEASES IS OF COURSE ANOTHER STORY. AND SO THIS IS JUST TO SHOW YOU. THIS IS A DIFFUSION MAP F YOU'RE INTERESTED IN MONOCYTE CONVERSION, THAT WHOLE THEORY, THAT CLASSICALS BECOME NON CLASSICALS, THIS MAP SUGGESTS THIS MIGHT BE TRUE. THESE ARE THE FOUR CLASSICAL SUBSETS UP HERE AND HERE IS THE INTERMEDIATE AND HERE ARE THE THREE NON CLASSICAL SUBSETS. THIS IS A LITTLE DIFFERENT AND I'LL SHOW YOU WHY IN A MINUTE. THESE ARE JUST COMMON MARKERS THAT WE FOUND. DIFFUSION MAP IS SHOWING THESE ARE THE THREE CLUSTERS THAT WE FOUND. AND THEY ARE BASED ON THE EXPRESSION OF WIERD GLYCOPROTEIN THAT IS ON PSGL1 IN MONOCYTES CALLED SLAN. THERE ARE PUBLICATIONS PROBABLY MAY BE OF YOU HEARD IT. AND ACTUALLY SLAN IS IN HUMANS. WE CANNOT AT LEAST SEE IT IN MICE AND THERE AREN'T ANY GOOD TOOLS TO SEE IT. IF ANYBODY KNOWS OF ONE, PLEASE LET ME KNOW. AND ALSO CD9. SO THEY WERE THE WOL MARKERS THAT REALLY SEEMED TO PULL OUT OUR THREE NON CLASSICAL SUBSETS AND WE JUST PUT THE INTERMEDIATE ON HERE AS AN EXAMPLE. SO YOU CAN SEE THE INTERMEDIATE HAS HIGHER CCR2 THAN THE NON CLASSICALS AND ALSO HIGHER CD36 EXCEPT FOR THIS GUY. AND I MEAN THIS COULD -- YOU KNOW. BASICALLY ALL THIS INFORMATION HELPS US DEFINE THESE SUBSETS A LITTLE BIT BETTER. SO THE CD9 WE FOUND OUT BINDS PLATELETS. SO CD9 IS ON PLATELET AND THERE IS ONE CLASSICAL SUBSET AND ONE NON CLASSICAL MONOCYTE SUBSET AND THEY ARE BOTH CD9 POSITIVE AND THEY BOTH BIND PLATELETS. THESE ARE THE NON CLASSICAL MONOCYTES WITH THE CD9 PLATELETS ON IT AND THIS IS ENDOTHELIUM. SO, EVERYONE HAS -- ESPECIALLY IN THE ARTHROSCLEROSIS FIELD EVERYONE THOUGHT MONOCYTE PLATELET INTERACTIONS WERE PRO THROMBOTIC AND ATHGENIC AND EVERYONE ASSUMED THAT ALL MONOCYTES BIND PLATELETS BUT WE JUST SHOWED YOU THAT NOT OWL DO. ONLY TWO SUBSETS OUT OF SEVERAL. SO IT'S INTERESTING WHY DO THOSE BIND PLATELETS? WHY DO THOSE -- HOW ARE THOSE SUBSETS CHANGED IN DISEASE? SO IF YOU'RE INTERESTED IN THAT, YOU'RE MORE THAN WELCOME TO TALK TO ME ABOUT THAT. SO THEN WE DID RNA-SEQ ON THE SUBSETS AND THEN A CLASSICAL INTERMEDIATE. YOU CAN SEE ALL THE NON CLASSICALS PRETTY FIT TOGETHER PRETTY WELL AND THERE IS NOT THAT MANY GENE DIFFERENCES BETWEEN THE TWO OR AMONG THE THREE. AND IN THIS CASE, WE ARE FOCUSING NOW WITH NOT FOCUSING ON THE CD9 BUT WE JUST BROKE IT DOWN BY VAN POSITIVE AND SLAIN NEGATIVE. JONATHAN HAD A MASS CYTOMETRY PAPER WHERE HE ALSO FOUND THIS. IN THIS CASE WITH RNA-SEQ ONLY A FEW GENES ARE REALLY DIFFERENT. ONE IS CXCR6 REALLY IMPORTANT FOR MIGRATION. AND OF COURSE THE ENZYME THAT MAKES SLA! VERY HIGH. BUT A LOT OF microRNAs AND THINGS SO WE DON'T REALLY KNOW YET. SO WE ARE JUST TRYING TO FIGURE OUT WHAT MAKES A SLAN POSITIVE CELL DIFFERENT THAN A NEGATIVE ONE AND HOW ARE THEY IMPORTANT? I CAN TELL YOU ONE PIECE OF DATA QUICKLY. WE STARTED WORKING WITH DR. PATEL AT UCSD IN THE MOORES CANCER CENTER AND HE STUDIES -- DOES A LOT OF CLINICAL TRIALS WITH MELANOMA AND WE HAVE GOTTEN SOME PATIENTS AND DOING SIGH TO HAVE ON MONOCYTES AND T-CELLS AND THIS IS JUST AN EXAMPLE OF SOME OF THE PATIENTS. SO BASICALLY THEY ARE EITHER GIVEN ANTI-PD-1 OR ANTI-CTLA4 OR COMBO, AND AND THEY EITHER RESPOND TO THE THERAPY OR THEY DON'T. AND SO WE ARE INTERESTED IN WHY DO THEY RESPOND AND WHY DO THEY NOT RESPOND. AND WE HAVE JUST STARTED DOING THIS BUT WE HAVE ONE PIECE OF DATA THAT BASICALLY SAYS IF YOU RESPOND TO ANTI-PD-1, YOU HAVE VERY HIGH SLAN POSITIVE NON CLASSICAL MONOCYTES. THESE MONOCYTES ARE THOUGHT TO BE PRO-INFLAMMATORY. SO IT COULD BE -- IT MAKES SENSE THAT MAYBE THEY ARE BEING INDUCED BY THIS TO ACTUALLY BECOME MORE INFLAMMATORY AND ALLOW THESE PATIENTS TO RESPOND. SO THAT IS WHERE WE ARE IN TERMS OF THAT PROJECT. BUT WE ARE HOPING TO -- DOING MORE OF THAT AS WE ARE NOW MOVING FORWARD. SO IN SUMMARY, NON CLASSICAL MONOCYTES PLAY AN IMPORTANT ROLE IN THE VASCULATURE IN RECOGNIZING TUMOR CELLS AND ORCHESTRATING THEIR KILLING AND MAYBE KILLING THEM DIRECTLY, I DON'T KNOW. THERE IS SIGNIFICANT MONOCYTE DIVERSITY IN HUMANS. I THINK WE ARE JUST KIND OF DISCOVERING THAT AND WHAT ALL THAT MEANS BUT I THINK THAT IF WE CAN LINK THIS TO DISEASE AND TO THERAPY, THAT IS A GOOD THING, I THINK. AND I THINK REGULATION OF NON CLASSICAL MONOCYTE NUMBERS OR SUBSETS OR FUNCTION COULD AID IN CANCER THERAPY. AND WE ALSO SEE THE SAME THING WE SEE INCREASE IN SLAN POSITIVE NON CLASSICALS WITH SOME OF THE TREATMENTS FOR ARTHERO. SO I THINK THIS IS ALL A GOOD THING. BUT JUST BEGINNING. SO THE PEOPLE THAT ACTUALLY DOT WORK -- I WRITE THE GRANTS. THIS IS GRAHAM THOMAS WHO DID ENHANCER WORK. AND ANMENT OUK DID A LOT OF THE SIGH TO HAVE. MANY OF YOU KNOW THE FXR PROJECT AND LINDSAY, A NEW POSTDOC WHO HAS DONE SOME OF THE MELANOMA WORK WITH T-CELLS AND THE MONOCYTES. AND I'D LIKE TO THANK CHRIS GLASS AND SAN DIP AND STEFAN AND MAX AND RICHARD WHO GOT US STARTED IN ALL THIS. HE WAS A GREAT POSTDOC SEVERAL YEARS AGO WHO DID THE ORIGINAL MONOCYTE WORK IN TUMOR METASTASES AND I HAVE TO THANK ALL OF OUR FUNDING. SO THANK YOU. [ APPLAUSE ] >> -- [ OFF MICROPHONE ] VARY WHEN YOU LOOK AT DIFFERENT TUMOR MOD ELSE? >> WE ONLY LOOKED AT THE ONE. WE ARE DOING IT NOW. IT'S A GREAT QUESTION. >> ENHANCER OF NR4A1 AND ENHANCER AND -- FUNCTION AND USE CRISPR IDENTIFY ENHANCER 2 SO THAT CRITICAL FOR THE EXPRESSION OF NR4A1. SO I'M THINKING WHAT IS THE MECHANISM THAT ENHANCER NOT 11 THAT PLAYS A CRITICAL ROLE FOR GENE EXPRESSION? >> YOU KNOW, WE DON'T KNOW. WE KNOW WHAT BINDS TO THAT ENHANCER, KLF2 AND 4 AND KLF24 KNOCK-OUT MICE ACTUALLY HAVE SOME CHANGES IN THEIR CLASSICAL MONOCYTES. THE KLF2 MICE WE ARE COLLABORATING AND THEY DO NOT HAVE NON CLASSICAL MONOCYTES. >> THAT IS A QUESTION I'M THINKING THAT PROMOTOR SIGNATURE AT THIS ENHANCER SITE SO WHICH PROBABLY INDICATES THAT TRANSCRIPTION OF NON CODING RNA AT THE ENHANCER. >> IT COULD BE. WE TRIED TO LOOK THERE. I MEAN WE TRIED TO LOOK AT THAT BUT I'M NOT A RNA CODING PERSON BUT WE TRIED AND HAVEN'T SEEN ANYTHING BUT VERY WELL COULD BE. >> YOU MENTIONED UPTAKE OF EXOSOMES FROM TUMOR CELLS BY THE NON CLASSICAL MONOCYTES AND THEN LATER TALKED ABOUT CD9 EXPRESSION AND CD9 IS A MARKER OF EXOSOMES. SO I'M WONDERING WHETHER THERE IS SOME CONNECTION BETWEEN THOSE TWO OBSERVATIONS OR -- >> WE HAVEN'T LOOKED IN THE CONTEXT OF CANCER EITHER. SO THOSE WERE ALL HEALTHY PEOPLE, PRESUMABLY. WE KNOW THAT. THAT'S A GREAT QUESTION. WE THOUGHT ABOUT THAT AND WE DON'T HAVE AN ANSWER FOR YOU ON THAT. WE AGREE. YES. >> THANKS. >> HI, ROSIE. >> GREAT TALK, I ENJOYED IT. MY QUESTION FOR YOU IS ABOUT THE LOCATION OF THESE CELLS OVER TIME. ARE TRADITIONALLY NON CLASSICAL MONOCYTES IN THE VASCULATURE? AND DO YOU THINK THIS CONVERSION IF THEY COULD DO TO CLASSICAL IS WHEN THEY MOVE OUT OF THE VASCULATURE? OR IF YOU HAVE BEEN ABLE TO DO LONG-TERM STUDIES TO SEE IF THEY ARE LONG-LIVED? CAN YOU BE A NON CLASSICAL MONOCYTE YOUR WHOLE LIFE LIVING IN THE BLOODSTREAM? >> NON CLASSICAL MONOCYTES LIVE -- PEOPLE REPORTED 9-12 DAYS WHEREAS CLASSICAL MONOCYTES TEND TO TURN OVER MUCH MORE RAPIDLY. IN TERMS OF WHERE THIS CONVERSION OCCURS, IT IS ACTUALLY HARD -- SO IT'S ONE OF THE THINGS WE WERE TALKING ABOUT EARLIER. EVERYONE BELIEVES THAT CONVERSION OCCURS BUT NO ONE CAN PROVE IT. THERE HAS BEEN SOME BRDU LABELING AND STEFAN YOUNG HAS BEAUTIFUL WORK ABOUT THIS AND IT'S CONVINCING. BUT NOBODY HAS BEEN ABLE TO ACTUALLY TRACE IT. WE DON'T HAVE THE TOOLS TO TRACE IT. LIKE WE HAVE BEAUTIFUL FOX P3, YOU KNOW, THINGS FOR T-CELLS BUT FOR THIS MYELOID COMPARTMENT, WE ARE JUST ALMOST SCREWED. WE DON'T HAVE THE ABILITY TO SHOW THAT. SO WE DON'T KNOW WHETHER THAT WOULD HAPPEN. THE THOUGHT IS THAT YOU'RE RIGHT. IS THAT MAYBE WHEN THEY START TO EXTRAPOLATE MAYBE THEY CONVERT UBUT YOU KNOW -- WE DON'T KNOW. WE REALLY DON'T KNOW. WE DON'T KNOW WHERE IT HAPPENS? AND YOU ONLY LOOKED IN THE BLOOD IN TERMS OF THE HUMAN SAMPLES? YOU VIOLENT LOOKED IN OTHER COMPARTMENTS TO SEE IF THE PREAK CEASE ARE DIFFERENT? LIKE LYMPH NODE OR TUMOR TISSUE? >> NOT IN HUMANS NO. MAYBE WE CAN. >> YOU MENTIONED KLF4. ISN'T THAT LINKED TO THE CD DIFFERENTIATION? >> SO KLF4 IS. KLF2 SEEMS TO BE IMPORTANT FOR NON CLASSICAL MONOCYTES. >> OKAY. >> AND WE THINK IT IS THROUGH NR4A1. >> BC2 PHENOTYPE IS REALLY BIZARRE. I STILL NOT COMPLETELY UNDERSTAND IT. KEN MURPHY THEORY THAT THERE ARE DIFFERENT -- OKAY. NO MORE QUESTIONS?