MY NAME IS YASMINE DOCKET AND I'M A SENIOR INVESTIGATOR AT NIAID, AND IT'S MY TRUE PLEASURE TO WELCOME YOU TO TD COVID-19 SPECIAL INTEREST GROUP LECTURE SERIES. SO WE'RE VERY HONORED TODAY TO HAVE JOHN WHERRY, AS OUR SPEAKER HE'S THE DISTINGUISHED PROFESSOR, CHAIR OF THE DEPARTMENT SCHOOL OF MEDICINE AND DIRECTOR OF UBAN INSTITUTE TRAINING. VERY FEW WORDS FOR INTRODUCTION, HE DOES NOT NEED MUCH INTRODUCTION TO THE FIELD OF IMMUNOLOGY, HE RECEIVED HIS Ph.D. AT THOMAS JEFFERSON UNIVERSITY AND FOLLOWED BY A FELLOWSHIP AT NYU UNIVERSITY ANDA LEADER IN THE FIELD OF AND [INDISCERNIBLE]. DURING THAT TIME JOHN MADE SEMINOLE CONTRIBUTIONS ON UNDERSTANDING IMAGERY [INDISCERNIBLE] AND THE FILAMENT OF T-CELL EXHAUSTION, IT WAS A LINE OF RESEARCH THAT BECAME THE [INDISCERNIBLE] PROGRAM. TOGETHER WITH [INDISCERNIBLE] AT THE NIH, JOHN'S WORK CONTRIBUTED TO THE FUNDAMENTAL ROLE OF PD1 IN RESTRAINING T-CELLS IN CHRONIC INFECTION, HE WAS ALSO THE FIRST TO DEMONSTRATE TARGETING RECEPTORS AND CAN PUT TOGETHER EFFICACY, SOMETHING OF COURSE THAT HAD FUNDAMENTAL IMPLICATION AND [INDISCERNIBLE] FOR CANCER PATIENTS. FOR HAD REMARKABLE ACHIEVEMENT, JOHN HAS RECEIVED NUMEROUS AWARDS AND DISTINCTIONS. HIS ABILITY TO CONDUCT INDEPTH STUDIES USING ANIMAL MODELS AND AT THE SAME TIME PUSHING AND BRIDGING FUNDAMENTED AMILLIO RESEARCH AND TRAINING, IT CAN BE EXEMPLIFIED TODAY IN THE CONTEXT OF THE PANDEMIC CRISIS WHERE BASED ON THE UNIQUE STRUCTURE HE DEVELOPED IN ORDER TO EXPLORE THE HUMAN SYSTEM, USING A SYSTEM BIOLOGY OPROACH, HE DID REMARKABLE WORK AND REFERRING [INDISCERNIBLE] COVID-19 PATIENTS AND UNCOVERED--NIH COVID-19 SPECIAL INTEREST GROUP SERIES. SO I WOULD LIKE TO FIRST REMIND A COUPLE OF ITEMS, FIRST WE OFFER CMI CREDIT FOR THIS LECTURE ANDHAVE ANY QUESTIONS-- >> THANKS YASMINE, AND THANKS FOR HAVING ME AND IT'S GREAT TO BE HERE AND SEE OLD FRIENDS, AT LEAST VIRTUALLY, HOPEFULLY WE CAN DO HAD THIS IN PERSON AT SOME POINT IN THE NOT TOO DISTANT FUTURE. I WILL TELL YOU ABOUT THE WORK WE'VE BEEN DOING AT PENN, WITH IMMUNEOLOGICAL BACKGROUND WITH PATIENTS WITH HOSPITALIZED COVID-19 DISEASE, TO GO THROUGH A BIT OF PUBLISHED WORK, I WILL TALK ABOUT IT TO YOU THE DIRECTIONS WE ARE HEADING IN WITH THAT PUBLISHED WORK AND I WILL END WITH A SHORT VIGNETTE ON WORK WE'RE GETTING READY TO SUBMIT, THAT I THINK HAS IMPLICATIONS MORE BROADLY TO WHAT WE'RE SEEING COVID-19 SEES, OBVIOUSLY THERE'S NOTHING ELSE GOING ON AND I KNOW ALL OF YOU CAN'T REALLY COMMENT, BUT I'M AWARE THAT THERE ARE DISTRACTIONS AND AT THIS TIME OF DAY, THERE ARE PIECES OF NEWS COMING OUT EVEN AS WE SPEAK, NEWS CONFERENCE IN NEVADA AT THE MOMENT AND THINGS HAPPENING IN GEORGIA, AND PENNSYLVANIA DURING THIS HOUR, SO HOPEFULLY, I CAN KEEP YOUR ATTENTION AND MAYBE DISTRACT YOU A BIT FROM THE ONGOING NEWS. SO WHEN WREE THINK ABOUT WHAT WE KNOW FOR COVID-19 IMMUNOLOGY, THE FIELD IS MOVING VERY, VERY QUICKLY, WE'RE SEEING NEW INFORMATION AND JUST VERY GENERAL POINTS HERE, EACH OF WHICH COULD WARRANT THEIR OWN OWN HOUR-LONG DISCUSSION, WE KNOW IN COVID-19 PATIENTS, ESPECIALLY HOSPITALIZED PATIENTS THAT WE HAVE ELEVATED INFLAMMA TORRESPONSES, AT LEAST IN MANY PATIENTS BUT THE RESPONSE TO ANTIINFLAMMATORY MEDICATIONS HAS BEEN MIXED SO THIS IS PROBABLY BEST ILLUSTRATED BY THE RESPONSE TO BLOCKING IL6, OR USING TOSUMUSLIBMAB, IN THE PATIENTS THERE'S A SUBSET THAT BENEFITS BUT THE VAST MAJORITY DON'T GET BETTER WHEN YOU BLOCK THE IL6 PATHWAY. DEXAMETHA SEWN HAS RECEIVED ATTENTION IN THE PAST FEW MONTHS HAS SOME BENEFIT IN SOME PATIENTS BUT ACTUALLY ANOTHER GROUP OF PATIENTS SEEMS TO IN FACT BE HARMED BY SUPPRESSING THE IMMUNE SYSTEM WITH THE STEROIDS LIKE DEXAMETHA SONE. SO IT'S NOT A SIMPLE EQUATION, WE KNOW THERE'S TREMENDOUS T-CELL ACTIVATION AND I WILL SHOW YOU EVIDENCE FROM OUR OWN WORK BUT GOOD EVIDENCE FOR THIS LITERATURE AND THERE'S ALSO EVIDENCE FOR PERHAPS T-CELL HYPOFUNCTION OR POOR FUNCTIONALITY PERHAPS EXHAUSTION AND WE CAN TALK ABOUT THAT AS WE GO THROUGH SOME OF THE DATA. WE DON'T REALLY--YOU CAN STILL UNDERSTAND THE TEMPORAL KINETICS AND SOME OF THAT RELATES TO IDENTIFYING PATES AT THE TIME OF INFECTION OR RECONSTRUCTING PREVIOUS INFECTIOUS HISTORY EVENTS, BUT THE TEMPORAL KINETICS REMAIN PUZZLING AND AS WE GO THROUGH SOME OF THE DATA PERHAPPINGS IT'S PUZZLING BECAUSE ALL PATIENTS DON'T FOLLOW THE SAME TRAJECTORY. WE KNOW THIS TREMENDOUS LYMPHPENIA IN THE SICKET PATIENTS WE DON'T UNDERSTAND HOW THIS RELATES TO T-CELL ACTIVATION OR IMMUNE CHANGES. WE KNOW THERE ARE VERY ROBUST PLASMA BLAST RESPONSES TO THIS INFECTION, WE CAN SEE THESE CELLS IN THE BLOOD DURING THE ACUTE PHASE OF INFECTION, WE ALSO KNOW THAT HOSPITALIZED PATIENTS MAKE ANTIBODY INCLUDING MOST HOSPITALIZED PATIENTS MAKING NEUTRALIZING ANTIBODIES BUT THESE PATIENTS ARE STILL SICK DESPITE THE FACT THERE'S NEUTRALIZING ANTIBODY ON ONBOARD. CONVALESCENT PLASMA THERAPY OR MONOCLONAL THERAPY SEEM TO HAVE SOME BENEFIT BUT IT'S NOT CLEAR IF THEY'RE ADDING MORE BEANT BODY ON TOP OF ANTIBODY WILL BE BENEFICIAL IN CASES. ONE OF KEY QUESTIONS THAT'S STILL IN NOVEMBER WAS DRIVING OUR INITIAL STUDIES BACK IN MARCH AND APRIL WAS WHAT WAS THE TYPICAL IMMUNE RESPONSE TO SARS-COV-2 AND COVID-19 PATIENTS LOOK LIKE. THIS IS DRIVEN BY THE GENERAL CONCEPT OF WHAT HAPPENS DURING A VIRAL INFECTION ESPECIALLY A RESPIRATORY VIRAL INFECTION WHERE IF YOU FAIL FOCUSED ON MOUNT AN APPROPRIATE OR ROBUST ENOUGH IMMUNE RESPONSE, YOU CAN COULD GET VIRUS INDUCED TISSUE DAMAGE, VIRAL DISSEMINATION AND DYSFUNCTION IMMUNE CELLS OR TISSUES, LEAVING TO DISEASE. AND SO THIS CAN OCCUR BECAUSE OF A FAILURE TO MOUNT AN IMMUNE RESPONSE OR IMMUNE O SUPPRESSION OR VIRAL EVASION, I WILL HAVE GOOD EVIDENCE FOR SARS-COV-2, AT LEAST FROM TYPE 1 INTERFERON RESPONSE AND THAT SORT OF FAILURE OR DELAY OR INACTIVITY OF THE IMMUNE RESPONSE COMMUNITY TO SEVERE PATHOLOGY, HOWEVER ON THE OTHER SIDE OF THE EQUATION, IF YOU RESPOND TOO VIGOROUSLY, ESPECIALLY TO RESPIRATORY INFECTIONS WE'RE PRESERVING AIRWAY FUNCTIONS SO IMPORTANT, CAN YOU HAVE PATHOLOGY BECAUSE YOU HAVE TOO STRONG OF AN IMMUNE RESPONSE AND IT'S LIKELY THEN IN TYPICAL INFLUENZA INFECTION, BOTH OF THESE OCCUR, PERHAPS AT DIFFERENT PHASES OF THE DISEASE AND IN COVID-19 PATIENTS, IT'S VERY LIKELY THAT AT LEAST SOME PATIENTS EXPERIENCE THIS SORT OF HYPER INFLAMMATION THAT THERE'S, YOU KNOW PATHOLOGY ON 1 END, PATHOLOGY ON THE OTHER END AND SORT OF A SWEET SPOT AND BELL CURVE IN THE MITSD BUT LIKELY THERE ARE MORE DIMENSIONS TO THIS ACCESS FOR DIFFERENT KINDS OF PATHOLOGY THAT WE'LL TALK ABOUT. SO THE KEY QUESTION IS, WE DON'T REALLY KNOW WHERE WE ARE ON THIS CURVE, LET'S SAY, AND WE CERTAINLY DON'T KNOW WHAT ALL THE PLAYERS ARE AND HOW TO MEASURE THEM. SO STARTING BACK IN LATE MARCH AND EARLY APRIL AT PENN WE SET UP A TRANSLATIONAL IMMUNOLOGY PROFILING PIPELINE, THIS INCLUDED THE TRANSLATIONAL SIDE OF ACQUIRING SAMPLES FROM OUR HOSPITALIZED PATIENTS AND WE WORKED WITH CRIT CRITICAL CARE WITH PENN, AND A LARGE TEAM OF CRITICAL CARE PHYSICIANS IN NOT ONLY AMAZING IN HAVING STUDIES TO STUDY SEPS SIS AND NANDS AT THE TIME THE PANDEMIC STRUCK BUT ALSO SAMPLES FROM THESE VERY DIFFICULT TO TREAT PATIENTS AND THIS GOES WITHOUT SAYING ABOUT YOU OUR ABILITY TO MANAGE THESE HOSPITALIZED AND I CU PATIENTS HAS IMPROVED DRAMATICALLY OVER THE LAST 9 OR 10 MONTHS AND IT'S REALLY THE SKILL OF THESE CRITICAL CARE PHYSICIANS THAT'S LED THE WAY IN BEING ABLE TO SORT OF JUST MANAGE SOME OF THESE MOST SICK PATIENTS THROUGH THE CRITICAL DISEASE AND NOW SEEING MUCH BETTER OUTCOMES BUT AT THE TIME WE WERE DOING THIS, THIS STILL REMAIN AID VERY, VERY DICEY SITUATION CLINICALLY. WE WERE ABLE TO OBTAIN SASMYS FOR WELL OVER 250 HOSPITALIZED PATIENTS, MANY CASES GET REPEATED BLOOD DRAWS, WE BUILT A NEW COVID PROCESSING UNIT TO HANDLE ALL THESE SAMPLES TO PROCESS BLOOD AND OTHER SAMPLES AND THEN FED THE BPMW SAMPLES INTO A DEEP IMMUNE PROFILE PIPELINE THAT WE BUILT TO DO CANCER IMMUNOTHERAPY AND THIS INCLUDED HIDIMENSIONAL SIGNIFY TOMETRY WHICH I WILL SHOW YOU A LOT OF DATA FROM--ALSO SERIES POINTSOLOGY, CYTOKINE ANALYSIS, RNASEQ AND OTHER TYPES OF MEASUREMENTS OF THE IMMUNE RESPONSE. THIS WAS ALL BUILT IN CLOSE LABERATION WITH MIKE BETTS AND WAS DRIVEN BY 2 POST DOCKERS IN MIKE'S LAB AND ARE 7 PEOPLE IN MY LAB, POST DOCS, GRADUATE STUDENTS AND CLINICAL FELLOWS WHO LED THESE STUDIES. I MENTION THESE NAMESOT SLIDE BECAUSE THEY ENDED UP AS CO-FIRST AUTHORSOT 2 PAPERS THAT CAME OUT AND REALLY WAS EQUAL CONTRIBUTION FROM EVERYONE, AND OUR COHORT ACTUALLY ENDED UP BEING THESE WERE DESIGNATED 14 DAYS SINCE LAST SYMPTOM AND LAST PART OF THE TALK, SOME OF OUR RECENT WORK ON PEDIATRIC PATIENTS ABOUT HALF OF WHOM ARE THESE MIS-C KIDS OR MULTIINFLAMMATORY SYNDROME IN CHILDREN PATIENTS. SO THIS INITIAL COHORT, I WILL USE THIS COLOR COATING THROUGHOUT, COVID PATIENTS IN RED, RECOVERED DONORS IN BLUE AND HEALTHY IN GREEN. WE HAD 149 COVID PATIENTS, 125 OF WHOM WE HAD SAMPLES FOR HIGH DIMENSIONAL FLOW SIGNIFY TEMETRY, ABOUT 35 RECOVERED DONORS AND SIBLGHT HEALTH CONTROLS WHO WE ANALYZE THROUGHOUT WHAT I WILL SHOW YOU. WE MATCH THESE AS WELL AS I COULD DEMON GRAPHICALLY AT THIS TIME. IN THE MIDDLE OF THE PANDEMIC IT WAS NOT THAT TRIVIAL TO RECRUIT AND GET SAMPLES FROM HEALTHY CONTROLS AND RECOVERED DONORS SO WE WE'RE A LITTLE BIT AGE SKEWED AND THAT MAY REFLECT THE DISEASE PATHOGENESIS WHERE OUR RECOVERED PATIENTS THAT ARE NOT HOSPITALIZED ARE A BIT YOUNGER. OVERALL IN OUR COVID PATIENTS WE ARE 67% AFRICAN AMERICAN WITH A WIDE DISTRIBUTION OF OTHER RACIAL CONTRIBUTION AS WELL AS SOCIOECONOMIC CONTRIBUTIONS, SO REALLY QUITE A DISCIPLINARY SEARS COHORT COMPARED TO MANY OF THE OTHER COHORTS IN THE LITERATURE. AND 1 OF THE REALLY NICE THINGS ABOUT THIS COHORT IS WE HAVE VERY, VERY DEEP CLINICAL DATA SO NOT ONLY DID WE ACQUIRE A LOT OF IMMUNEOLOGICAL DATA BUT WE ACQUIRED AT LEAST 50 DISCREET FEATURES OF CLINICAL DAT HA WERE WELLAN OITATED AND COULD BE USED TO HELP MAKE SENSE OF WHAT WAS HAPPENING IMMUNEOLOGICALLY SO I'M SHOWING A FEW OF THESE HERE, BENCHMARKING PARTIALITYS COME IN AND HAVE HIGH FERIA TIN, DIMER, AND CRP IS ELEVATED AND YOU CAN SEE WHITE BLOOD CELL COUNTS ARE MARE--MANY ARE IN THE NORMAL RANGE BUT MOST PATIENTS ARE LYMPHPENIC AND HAVE SLIGHTLY ELVITTED MONOCYTES AND ALL PATIENTS, I'LL SHOW YOU ON THE NEXT SLIDE HAVE ELEVATED NUTRIFILLS IN THIS COHORT. YOU CAN THEN TAKE THESE CLINICAL FEATURES AND START TO ASK ABOUT THEIR INTERACTIONS OR AT LEAST CO CORRELATION, THE BUOYANT OF THIS BEING NOT TO POINT OUT ANY INDIVIDUAL FEATURES BUT JUST TO ILLUSTRATE THAT ACTUALLY WE CAN START TO SEE, KINDS OF CLINICAL DATA ON AGGREGATING AND THAT IS JUST SORT OF A FORESHADOWING OF WHAT WE CAN DO WITH THIS DATA IF YOU MAKE 1 OF THEAXIS IN THE ACTUAL DATA RATHER THAN CLINICAL DATA. SO WE WILL COME BACK AND USE THIS DATAs WE GO THROUGH. THIS IS WHAT WE INITIALLY DID, THIS IS FROM MIKE BET'S LABS AND BETTY IN MIKE'S LAB LOOKING AT THE OVERALL DISTRIBUTION OF MAJOR LEUCOCYTE POPULATIONS IN PERIPHERAL BLOOD AND WE CAN SEE THAT THE SEVERE COVID PATIENTS AND HOSPITALIZED COVID PATIENTS HAVE A REDISTRIBUTION OF SORT OF THE NORMAL LANDSCAPE OF MYELOID CELLS, LYMPHOCYTES IN THE BLOOD AND WE JUST MAKE A COUPLE VERY, VERY SIMPLE POINTS. NUTRIFILLS ARE ELEVATED IN JUST ABOUT ALL PATIENTS, WE SEE SOME OTHER PROTURBATIONS BUT THIS IS REALLY THE BIGGEST WHEN YOU TAKE A VERY CRUDE OR 10,000-FOOT VIEW LANDSCAPE PICTURE OF WHAT'S HAPPENING. WE SEE A LOSS OF MAIT CELLS, CD8 61 T-CELLS AND THAT'S CONFIRMED BY OTHER GROUPS USING OTHER MARKERS FROM THESE CELLS AND YOU SLEEP APNEA AND OBESITYY A PRETTY DRAMATIC CHANGE IN THE NK POPULATIONS INCLUDING BOTH ON NK CELLS AND MYELOID CELLS, DOWN REGULATION OF CD16 AND LOSS OF CD57 IN THE CASE OF THE NK-CELLS. SO NUTRIFILLS WERE INCREASED, ACENOFILLS WERE INCREASED AND DEBD RIDDIC CELLS,--DENDRITIC CELLS, PDCS AND K-CELLS ARE DOWN, YOU SEE A DRAMATIC LOSS OF INNATE CELLS AND OTHER GROUP VS SEEN THAT AND ILCs ARE ALSO REDUCED IN THESE PATIENTS. I WILL NOT GO INTO A LOT MORE DETAIL IN THE NONLYMPHOCYTE POPULATIONS HERE. SOME OF THAT IS IN THE PAPER FROM MIKE'S LAB. WHAT I WILL TALK ABOUT ARE THE LYMPHOCYTE PROTURBATIONS WE SEE IN THE PATIENTS, ON THE B-CELL AND T-CELL CHARACTERISTICS SO ILLUSTRATING HERE, WE CAN AGAIN ON CD4 AND 8 T-CELLS AND THE GATES ON THE LAST SLIDE AND WE GET IN ON B-CELLS AND WE SEE A PROPORTIONAL INCREASE AND THAT REFLECTS T-CELL, B-CELL FREQUENCIES AND NUMBERS ARE ACTUALLY PRETTY WELL PRESERVED AND EVENOT T-CELL SIDE, THE LYMPHPENIA IS DRAMATICALLY BIS ONED AND TAKING THE CD4 AND CD8 RATIO WHY THIS IS INCREASED REFLECTING A PREFERENTIAL LOSS OF THE CELLS AND YOU CAN ACTUALLY CONVERT THIS INTO ABSOLUTE COUNTS LOOKING AT THE CELLS OF PER MICROLITER. THIS IS KIND OF UNUSUAL WHEN YOU SEE LIESM O PENIA AND OTHER INFECTIONS IT ATTENDS TO INFECT ALL LYMPHOCYTES MORE OR LESS EQUALLY, CAN YOU SEE OCCASIONALLY EXAMPLES OF CD8 DRIVEN LYMPHPENIA AND 1 PLACE IT'S NOTE INDEED THE LITERATURE IS ACTUALLY EBOLA INFECTION OF PRIMATES WHERE THERE TENDS TO BE A CD8 DRIVEN LYMPHPENIA IN THAT SETTING. WE WANTED TO USE THE SURROGATES AT THIS EARLY TIME IN THE PANDEMIC WE DIDN'T HAVE A LOT OF ANTIGEN SPECIFIC TOOLS, WE WANTED TO USE SURROGATES TO START TO GET A PICTURE OF WHAT THE I HAVE RUTS SPECIFIC T-CELL RESPONSE MIGHT LOOK LIKE,--OR EXPRESSION OF KI67 AND THIS IS EXAMPLES FROM JOE MILLER'S TABLE MORE THAN 10 YEARS AGO LOOKINGA THE LIVE ATTENUATE VAC NATION, SO BASICALLY VIRAL INFECTION IN HUMANS WHERE YOU SEE A ROBUST IN CELLS THAT CO EXPRESS CD8 AND 38 AT PEAK OF THE VIRAL REPLICATION AND THEN AGAIN WITH THE ANTIVIRAL T-CELL RESPONSE, PT ON DAY 12-15, YOU GET A SIMILAR PICTURE IF YOU LOOK AT CD4 T-CELLS AND INNUCLEOTIDESSENSA INFECTION, IN THE MICHAELS GROUP AND YOU CAN SEE KIEXPEVEN 38, OR IMMUNE KI67 AND AGAIN IN THIS CASE, WHICH IS MORE LIKE A RECALL RESPONSE, YOU SEE A PEAK OF THIS ANTIVIRAL T-CELL RESPONSE AROUND DAY 7. IN THE CASE OF THE VACCINIA AND YELLOW FEVER STUDIES THAT WERE FIRST DONE HERE BY JOE MILLER, CAN YOU LOOK IN THESE POPULATIONS AND FIND YOUR TETRAMERPOSITIVE CELLS OR TRUE KNOWN VIRUS SPECIFIC T-CELLS FOLLOWING INTO THESE QUADRANTS, THESE ACTIVATION MARKERS. SO THE QUESTION IS, WHAT HAPPENS IN THESE COVID-19 PATIENTS, AND JUST USING HLA-DRAND CD38 ACROSS THE TOP HERE, CAN YOU SEE IN OUR HEALTHY CONTROLS OR RECOVERED DONORS, A RELATIVELY LOW LEVEL OF BASE LEVEL BACKGROUND T-CELL ACTIVATION. IN OUR COVID-19 PATIENTS HOWEVER, CAN YOU SEE VERY ROBUST RESPONSES, ACTUALLY CAN BE BY HLADR AND CD38 CAN BE MORE THAN 30% OF YOUR CIRCULATING CD8 T-CELLS ACTUALLY HAVE THIS ACTIVATION FEE CONSISTING WITH ANTIVIRAL T-CELL RESPONSES, YOU SEE A VERY SIMILAR PICTURE IF YOU SEE KI67 WHERE AGAIN MORE THAN 30% OF YOUR CIRCULATING CD8 T-CELLS, ACTUALLY HAVE THIS ROBUST ACTIVATION CONSIST WENT ANTIVIRAL T-CELL RESPONSES. WELL YOU KNOW NOTICE IN BOTH CASES, YOU HAVE EXAMPLES OF PATIENTS THAT HAVE NO STATISTICAL OR NO NUMERICAL INCREASE IN ANTIVIRAL LIKE T-CELL ACTIVATION COMPARED TO THE CONTROLS. AND THROUGHOUT A NUMBER OF THE GRAPHS, I'M GOING STHOA YOU, THERE'S THIS GREEN LINE, THAT GREEN LINE IN THESE GRAPHS REFLECTS THE 90th PERCENTILE OF OUR HEALTHY CONTROL INDICATING THAT SOME OF THE COVID-19 PATIENTS SEEM TO FAIL TO MOWBT A RESPONSE CONSISTING WITH THE OTHER COVID-19 PATIENTS. CAN YOU LOOK AT THIS LANDSCAPE IN A LITTLE MORE DETAIL AND SO WHAT WE HAD HERE WAS A 27 COLOR FLOW CYTOMETRY PANEL TO GET A LOT OF RESOLUTION, IN THE ACTIVATION AND DIFFERENTIATION STATUS OF THE T-CELLS AND JUST PROJECTING THIS, IN HIGH DIMENSIONAL SPACE, CAN YOU SEE THAT THE OVERALL LANDSCAPE OF NONNAIVE CD8 T-CELLS IN DONORS AND THERE MAY BE SOME DONORS BEING RELATIVELY EARLY AFTER LAST SYMPTOM BUT IF YOU LOOK AT OUR COVID-19 PATIENTS YOU SEE A MASSIVE REORGANIZATION OF THE LANDSCAPE OF PREVIOUSLY ACTIVATED OR CURRENTLY ACTIVATED T-CELLS AND SO LOOKING AT THAT IN A LITTLE MORE DETAIL BECAUSE WE ARE KIND OF T-CELL PEEKS AND WE LIKE DOING THIS, YOU CAN ACTUALLY FIND SOME BENCHMARKS IN THE SPACE, THE VALLEY DOWN THE MIDDLE HERE AND THIS IS DOWN HERE IN SORT OF THE BOTTOM MIDDLE, YOU CAN LOOK FOR OTHER MARKERS OF NAIVE CELLS OR EASKTOR CELLS OR IN THIS CASE, FAS, WHICH WILL LIGHT UP NAIVE CELLS ON THE LEFT BUT ALSO ACTIVATED KAS ACTIVATED CELLS AND YOU CAN LOOK FOR MORE EFFECTOR LIKE CELLS SO MARKETED BY T-BET OR CX3 CR 1, AND THIS IS INTERESTINGOT CD8 SIDE BECAUSE WHILE NORMAL SUBJECT VS PRETTY GOOD T-BET POSITIVE OR CRCX POSITIVE CELLS IN THE BLOOD, YOU SEE THAT THAT AREA OF THE HIGH DIMENSIONAL SPACE AND LARGELY MISSING IN THE COVID-19 PATIENTS, AND IT'S LOOKING INDIVIDUAL PATIENTS A LITTLE BIT LATER, IT'S NOT MISSING IN ALL PATIENTS AND CERTAINLY SOMETHING DIFFERENT IN THE AGGREGATE DATA I'M SHOWING YOU HERE. YOU CAN USE FLOWS TO IDENTIFY CLUSTERS AND ASK WHAT THEIR IDENTITIES ARE WITH THEIR TYPIC MARKERS AND PROJECT THAT AND SO, THIS CLUSTER DOWN HERE NUMBER 14 CONTAINS ALL OF THESE ACTIVATED PROLIFERATING CELLS THAT I WAS SHOWING YOU ON THE LAST SLIDE, HAVE YOU OTHER CLUSTERS INCLUDING THIS CXC3 CR1 CLUSTER OVER HERE, CLUSTER 11 WHICH IS DOWN HERE AND THE 1 THAT'S MISSING FROM THE COVID-19 PATIENTS AND SO ON. SO CAN YOU DO THIS AND ASK WHICH CLUSTERS ARE INCREASED OR DECREASE INDEED YOUR COVID-19 PATIENTS AND FIND THIS CLUSTER 14 THESE HIGHLY ACTIVATED PROLIFERATING KI67 PD1 POSITIVE CD8 T-CELLS ARE ENRICHED IN THE COVID PATIENTS WHERE THE T-BET CLUSTER IS MISSING OR DEFICIENT MOST ALTHOUGH NOT ALL THROUGH ASHING 19 PATIENTS. AND I'LL POINT OUT THERE'S EVIDENCE IN THE LITERATURE OR SOME SUGGEST IN THE LITERATURE THAT T-CELLS IN COVID-19 PATIENTS ARE EXHAUSTED. INDEED THERE ARE HIGH EXPRESS AND WILL COME BACK TO THIS A BIT TOWARDS THE END. YOU CAN DO THE SAME THING ON THE CD4 SIDE, I WILL NOT GO INTO THIS IN AS MUCH DETAIL BUT YOU SEE DRAMATIC INCREASES IN ACTIVATION AND PROLIFERATION IN MOST COVID-19 PATIENTS, YOU SEE PD1 IS INCREASED, YOU SEE HELPER CELLS ARE INCREASED BY FREQUENCY AND IN CONTRAST, YOU ACTUALLY SEE AN INCREASE IN THIS CLUSTER DOWN HERE ON THE LOWER RIGHT. YOU CAN SEE A LITTLE BIT OF IT HERE IN THE HEALTHY CONTROLS BUT THAT AREA IS MUCH DARKER IN THE COVID-19 PATIENTS, ALTHOUGH THERE ARE SUBTLYS, HERE, YOU CAN IDENTIFY THEM BY MARKERS AND GET THE ANSWER THAT YES ON CD4 SIDE, YOU SEE THE ROBUST ACTIVATION OR PROLIFERATION BY KI67, CD38 AND CLUSTER 14, BUT NOW THIS CLUSTER 15 THAT WAS RELATIVELY TEFICIENTOT CD8 SIDE, YOU SEE INCREASED ON THE CD4 SIDE. SO YOU CAN THEN USE THIS INFORMATION TO GO BACK AND SAY, OKAY, THAT'S GREAT, HAVE YOU T-CELL ACTIVATION, HOW DOES THAT RELATE TO THE WAY THAT PATIENT'S PRESENT CLINICALLY. SO YOU CAN START TO TAKE THESE DIFFERENT SUBPOPULATIONS AND CD4S, AND ASK HOW THEIR FREQUENCY OR THEIR ABUNDANCE CORRELATES WITH VARIOUS CLINICAL FEATURES, AND SO LET ME WALK YOU THROUGH THIS FOR 1 OR 2 EXAMPLES. SO IF YOU HAVE A LOT OF ACTIVATED CD4 T-CELLS BY HLADR, CD38 OR KI67, THAT CORRELATES VERY STRONGLY WITH SOMETHING CALLED THE APACHE SCORE WHICH IS A MEASURE OF THE MULTIORGAN DYSFUNCTION OR HIGH LEVELS OF FERIA TIN, HIGH PATROL CALCA TONEIN AND A LOT OF NUTRIFILLS. WHAT'S INTERESTING IS YOU SEE THAT THESE 2 ACTIVATION FEATURES NEGATIVELY CORRELATE WITH THE NUMBER OF LYMPHOCYTES IN THAT THERE'S SOME RELATIONSHIP BETWEEN LYMPHPENIA IN T-CELL ACTIVATION, AND YOU CAN GO ON DOWN THROUGH THIS LIST, AND I'M SHOWING YOU THE STARS INDICATE THE P-VILLE RAUE AND THE BOX OROUND THESE SQUARES AND INDICATES WHETHER THIS IS SIGNIFICANCE BY .05. WHEN WE USE THAT CONVENTION THROUGHOUT THE TALK, SO THE DARKER RED IT IS THE MORE POSITIVELY IT CORRELATES, THE DARKER BLUE, THE MORE NEGATIVELY IT DOESN'T CORRELATE. AND JUST TO TALK THROUGH THIS, THE THESE CORRELATE WITH THE CALTONEIN AND JUST A BIT OF A LYMPHPENIA EFFECT HERE. T-CELL ACTIVATION CORRELATES WITH MARKERS AND DEC SEVERITY INCLUDING THE APACHE SCORE, KI67 NEGATIVELY CORRELATES WITH LYMPHOCYTE COUNT, LYMPHPENIA DRIVEN T-CELL ACTIVATION, OR HISM O PENIA PLAYING A ROLE IN OVERALL ACTIVATION, THERE ARE INTERESTING THINGS HERE THAT EVEN WITH 150 PATIENTS WE FEEL ARE A BIT UNDERPOWERED TO DETECT ROBUSTLY, BUT THERE ARE SOME CONNECTION TO ABO BLOOD TYPE AS WELL AS AGE, BMI AND POTENTIAL CO-INFECTIONS AND AT THIS TIME BEFORE WE WERE TREATING EVERYBODY WITH DEXAMETHA ZONE WHETHER PATIENTS GOT STEROIDS OR NOT. AND 1 OF THE LESSONS FROM POINTING OUT THIS LAST BULLET POINT HERE IS THAT EACH AT 150 PATIENTS, THERE'S SUCH HETEROGENEITY BOTH INUNEOLOGICALLY AND THE WAY THE PATIENTS PRESENT CLINICALLY THAT 150 PATIENTS REMAINS UNDER POWER TO SEE MINGS THAT MAY EXIST IN THE DATA. WE ALSO HAD PROFOUND B-CELL RESPONSES IN THESE PATIENTS AND IF YOU JUST LOOK AGAIN, VERY SIMPLY, LOOKING AT PLASMA BLASTS IN THE BLOOD AND SOME OF THESE HAVE INCREDIBLE LIE PLASMA PLAOF THE RESPONSES AND THE IPAD MAY BE ABOVE BACKGROUND, QUARTER PERCENT OF PLASMA BLASTS YOU SLEEP APNEA AND OBESITYY IN THIS 1 EXAMPLE, OF A COVID PATIENT. WE'RE TIP LALLY DOWN AROUND A QUARTER OF%, RATHER THAN 3-QUARTERS OF A% HERE, BUT THIS LEVEL OF PLASMA BLASTS 2030, 40% IS REMARKABLE. I THINK OUR RECORD FOR A MAISHT THAT DIDN'TEND UP IN THIS COHORT WAS 80% OF THE CIRCULATING B-CELLS FOR PLASMA BLASTS. TO PUT THIS IN PERSPECTIVE TO SORT OF REGRAPHING DATA FROM [INDISCERNIBLE]. IN ACUTE INFLUENZA INFECTION, YOU MIGHT HAVE 10-15% OF YOUR CIRCULATING B-CELLS ARE PLASMA BLASTS AND ACUTE EBOLA, NOW YOU GET TO THE LEVEL YOU'RE SEEING HERE IN THESE ACUTE SARS-COV-2 INFECTED PATIENTS, THE ONLY 2 SETTINGS WE'VE NOTICED IN THE LITERATURE IS ACUTE EBOLA OR ACUTE DENGUE FEVER, I WILL NOT SHOW THE DATA, MANY OF THESE ARE EXPANDED, WE DON'T YET HAPPEN ABOUT THEIR PESSPISSITY ENTIRELY AND IT'S INTERESTING THAT ACTUALLY THE PLASMA BLASTS FREQUENCY IN THE BLOOD DOES NOT CORRELATE WITH THE LEVEL OF OF ANTISCARS COV-2 RECEPTOR BINDING IGG, THAT'S 1 SPECIFICITY OUT OF MANY SO MAYBE IT'S ASKING A LOT, AND PLASMA BLASTS FREQUENCY HOWEVER DOES NOT CORRELATE WITH ACTIVATED TFH IN THE BLOOD WHERE INSTEAD OF--EXTRA FOLLICULAR B-CELL RESPONSES PLAYING A ROLE HERE OR YOU WOULDN'T EXPECT THEM TO CORRELATE. HOWEVER YOUR ACTIVATED TFH, WITH ANTIBODY RESPONSES WITH IGM OR IGG, AND THE RECEPTOR BINDING DOMAIN AND PROTEIN, IT DOES APPEAR THAT AT LEAST SOME RESPONSES ARE COORDINATED. ALL RIGHT, SO 1 OF THE THINGS WE WANT TO UNDERSTAND, WAS HOW THESE RESPONSES WERE CHANGING OVERTIME IN OUR HOSPITALIZED PATIENTS. AND IF YOU JUST LOOK AT THE LEVEL OF OF ALL PATIENTS, AS HIGH PROJUKS OF THE TYPE, CD8 T-CELLS OR B-CELLS, YOU DON'T REALLY HAVE TO LOOK TOO CLOSELY, NONAPOPTOTIC THE A WHOLE LOT SEEMS TO CHANGE WHEN YOU AGGREGATE ALL PATIENTS AND ALTHOUGH YOU CAN GET A P-VALUE DOWN HERE FOR SOME OF THESE PAIRED COMPARISONS ABOUT CHANGES IN KI67 OR HLADR OVER TIME AND SO ON, WHAT THIS MORE TOLD US LOOKINGA THE DATA ACROSS THE BOTTOM WAS THAT THERE ARE GROUPS PATIENTS THAT WERE STAYING THE SAME, GROUPS OF PARENTS GOING UP AND GROUPS OF PATIENTS GOING DOWN. SOPHISTICATEDY WE DECIDED TO ACTUALLY ASK WHETHER THERE WERE DIFFERENT TEMPORAL GROUPS IN THESE PATIENTS THAT ARE COMING TO THE HOSPITAL. PERHAPS BECAUSE WE WERE CATCHING THEM AT DIFFERENT TIMES AFTER INITIAL INFECTION OR PERHAPS BECAUSE THERE WERE DIFFERENT TRAJECTORIES OF THE IMMUNE RESPONSE. SO TAKING THAT AS A THE HYPOTHESIS, WE JUST SEPARATED PATIENTS INTO GROUPS WHERE YOU SAW INCREASES IN CERTAIN IMMUNE FEATAURS OVERTHE 1 WEEK WHERE WE HAD MULTIPLE SAMPLES, WHERE THOSE FEATURES STAYED THE SAME OVER TIME OR THOSE FEATURES DECLINED AND SO WALKING THROUGH THIS AS AN EXAMPLE JUST FOR PLASMA BLAST WHERE THE FREQUENCY OF PLASMA BLASTS INCREASES BETWEEN DAY 0 AND 7 AND YOU CAN SEE THAT ILLUSTRATED IN THE FLOW CYTOMETRY REAR, THIS MIDDLE GROUP IS QUITE INTERESTING EPECIALLY FOR THE PLASMA BLASTS AND SO PLACENTAs MA BLASTS FOLLOWING INFECTIONS TEND TO HAVE A VERY NARROW FEAT, AND YOU CAN DETECT THEM IN THE BLOOD FOR A COUPLE DAYS AND THEN THEY DISAPPEAR. IN CONTRAST, IN THE COVID-19 PATIENTS, A LOT OF PATIENTS HAVE VERY STABLE PLASMA BLASTS FREQUENCY IN THE BLOOD FOR QUITE A LONG TIME. I'M SHOWING YOU OVER THE COURSE OF 1 WEEK, WE HAVE PATIENTS THAT HAVE STABLE HIGH LEVELS OF PLASMA BLASTS FOR WEEKS IN THE PERIPHERAL BLOOD. WE ALSO HAVE OTHER PATIENTS WHERE WE MIGHT--CONSIDER THESE EVEN MORE TYPICAL RESPONSE TO ACUTE VIRAL INFECTION AND WE SEE A HIGH PEAK AT PRESENTATION AND THEN THEY DECLINE OVER THE COURSE OF STUDY FOR 1 WEEK IN THE HOSPITAL. AND YOU CAN DO THIS FOR ALL THE DIFFERENT FEATURES OF THE IMMUNE RESPONSE AND THEN ASK, HOW DOES CHANGE IN THE IMMUNE RESPONSE CORRELATE WITH CLINICAL SYMPTOMS, SO I WILL USE ANOTHER 1 OF THESE SORT OF MATRIX PRESENT ANTICIPATIONS AND LET ME WALK YOU THROUGH 1 SET OF IMMUNEOLOGICAL FEATURES COMPARED TO THE CLINICAL FEATURES AND THEN I WILL OPEN UP AND SHOW YOU THE OTHERS. O IF YOU LOOK AT KI67 NCD8 T-CELLS AND SEGBREAKTHROUGH GRIT PATIENTS INTO THOSE WHERE KI67 INCREASED OVER 1 WEEK OR REMAINED STABLE, WHERE NO PATIENTS HAD 67 DECREASE, AND THEN ASK IF KEYE 67 IS INCREASING WHAT DOES THAT CORRELATE WITH CLINICALLY. WELL IT CORRELATES WITH A LOW LEVEL OF VIRUS SPECIFIC ANTIBODY AT THE TIME OF CLINICAL PRESENTATION AND WITH AN INCREASE IN ANTIBODY FROM DAY 0 TO DAY 7 OF HOSPITALIZATION, SO I THINK WHAT THIS IS TELLING US IS THAT THE PATIENTS HAVE AN INCREASE IN KI67 AND CD8S OVER TIME WERE CATCHING THEM AT THE BEGINNING OR THE UPSWING OF THE IMMUNE RESPONSE. IN CONTRAST, PATIENTS WHO HAVE STABLE KI67 OVER TIME THAT POSITIVELY CORRELATES WITH HAVING ANTIBODY AT DAY 0, SO THEY'VE ALREADY MOUNTED ANTIBODY RESPONSE AND IT NEGATIVELY CORRELATES WITH ANTIBODY RESPONSE INCREASING OVER TIME, OKAY? THESE PATIENTS HAVE MADE THEIR ANTIBODY AND THEY'RE AT THE POINT OF HAVING STABLE ANTIBODY. YOU CAN DO THIS FOR A BUNCH OF OTHER FEATURES OF THE IMINE SYSTEM, CAN YOU USE T-CELL ACTIVATION, RATHER THAN T-CELL PROLIFERATION, AND GIVEN THIS IS A BROADER DEFINITION, PROBABLY IS DETECTABLE FOR A LONGER TOWARD PERIOD OF TIME. YOU SEE SLIGHTLY SUBTLEY DIFFERENT RELATIONSHIPS WITH THE FEATURES INCLUDING PRESENCE OF ANTIBODY PRESENTATION, INCREASING ANTIBODY OVERTIME BUT THE SAME GENERAL IDEA HOLDS THAT PATIENTS WHO HAVE KNOWA INCREASE IN T-CELL ACTIVATION, OVER TIME SEEMED TO HAVE AN INCREASING ANTIBODY RESPONSE AND NOT MUCH EABT BODY AT THE TIME THEY COME TO THE HOSPITAL. WHEREAS PATIENTS THAT HAVE T-CELL ACTIVATION, SEEM TO ALREADY HAVE ANTIBODY AT THE TIME THEY COME TO THE HOSPITAL AND DON'T SEE A FURTHER INCREASE IN ANTIBODY OVER TIME, MOST OF THE TIME. THEY'RE ALSO RELATIONSHIPS THE WAY THE PATIENTS WERE TREATED, WHETHER THEY'RE GIVEN NONTERROIDAL ANTIINFLAMMA TORES, AND YOU CAN LOOK ACROSS IMINE RESPONSES AND I WILL NOT GO INTO EACH OF THESE AND I WILL POINT OUT AND I COME BACK AND SUMMARIZE SOME OF THIS AS WE GO FORWARD THAT YOU CAN SEE DIFFERENT RELATIONSHIPS TO ACTIVATED TFH OVER TIME OR PLASMA BLAST CHANGES OVER TIME. SO OVERALL, HAVING HIGH ANTIBODY ON DAY 0 NEGATIVELY CORRELATES CAN T-CELL ACTIVATION OVER TIME, SUGGESTING THEY MOUNTED AN IMMUNE RESPONSE BUT THE ANTIBODY RESPONSE IS INCREASING, YOU SEEM TO HAVE OTHER FOUGHTURES OF THE IMMUNE SYSTEM ALSO INCREASING IN A COORDINATED FASHION. --IMMUNE RESPONSENESS CONNECTED TO OVERALL DISEASE SEVERE MITRAL SERIES POINTSITY. WE USE THE NIH DISEASE SEVERITY, BUT THE POINT IS THE DARKER RED THE MORE SEVERE YOUR DISEASE IS IN PRESENTATION, THE MORE SEVERE YOUR DISEASE IS, THE MORE LYMPHPENIA THAT EXISTS BUT YOU CAN SEE A DUBTLE TREND THAT IT'S NOR SEVERE ON THE CD8 SIDE THAN THE CD4 SIDE, CAN YOU SEE TRENDS FOR INCREASING PROLIFERATION OR ACTIVATION AND MORE SEVERE DISEASES, AND THAT MAY DIFFER A LITTLE BIT DEPENDING ON WHETHER YOU'RE LOOKING AT CD8 OR CD4S OR PLASMA BLAST. CAN YOU START TO PUT THESE THINGS TOGETHER AND HIGH DIMENSIONAL SPACE AGAIN, SO WE BUILT, BASICALLY A WAY TO ELECTRIC AT THE RELATIONSHIP BETWEEN DIFFERENT FEATURES OF THE IMMUNE SYSTEMS, SORT OF MANUELLY SELECTED FEATURES OF THE IMMUNE SYSTEM AND THE CHANGES IN DISEASE SEVERITY OVER TIME YOU CAN START TO SEE FEATURES OF THE IMMUNE SYSTEM BEGIN TO CO AGGREGATE WITH EACH OTHER AS IT RELATES TO DIFFERENT CLINICAL FEATURES ACROSS THE TOP AND AGAIN HERE, I'M SHOWING YOU THE COVID PATIENTS IN RED AND THE HEALTHY CONTROLS AND RECOVER DONORRED IN BLUE. BUT WHAT YOU MAY BE ABLE TO DISCERN IS MAYBE 3 OR 4 DIFFERENT GROUPS OF COVID PATIENTS THAT SEEM TO SEGGREGATE, MAYBE THIS GROUP 2, HAS A HIGHER DEGREE OF MORTALITY OR SEVERE DISEASE AND THAT SEEMS TO ACTUALLY ALIGN WITH ESPECIALENTIALLY CD4 T-CELL ACTIVATION WHERE THIS GROUP 3 SEEMS TO ACTUALLY HAVE LESS CD4 T-CELL ACTIVATION AND PERHAPS MORE MORTALITY. CAN YOU DO THIS FOR B-CELLS AND GET DIFFERENT PATTERNS, MAYBE THEOR THERE ARE 3 GROUPS, BUT WHAT WAS VERY CLEAR FROM THIS IS THAT WE HAD MANUELLY SELECTED THESE NEW FEATURES AND BY DOING SO, WE SORT OF IMPRINTED OUR OWN BIAS IN WHICH PART OF THE IMMUNE SYSTEM MIGHT BE RELEVANT. SO IT WAS CLEAR AS WE STARTED LOOKING AT THE DIFFERENT PATTERNS THAT WE NEEDED A MORE UNBIASED APPROACH TO DO THIS TO INTEGRATE ALL IMMUNE FEATURES WITH THE CLINICAL FEATURES. AND SO NOT TO BE A LITTLE BIT PIDANTIC, BUT WE CHOSE TO USE SOMETHING CALLED UNIFORM MANIFOLD APPROXIMATION AND PROJECTION WHICH IS AN APPROACH TO REDUCE TO 2 DIMENSIONAL APPROACH, AND I KNOW MANY OF YOU ARE FAMILIAR WITH THIS BUT TO SET THE STAGE THIS, IS A NONLINEAR WAY OF REDUCING DIMENSIONALITY AND GIVING YOU A LOW DIMENSIONALITY OF THE LOAD STRUCTURE AND THE DID THEA SPACE, SO HERE WE HAVE 27 INDIVIDUAL MARKERS IN THE FLOW CYTOMETRY PANEL, WE HAVE A VERY, VERY LARGE NUMBER OF INDIVIDUAL AND UNIQUE FEATURES. THIS APPROACH IS SIMILAR TO [INDISCERNIBLE], BUT IT PRESERVES THE LOCAL STRUCTURE WHICH IS IMPORTANT GIVEN THE WAY THE FLOW CYTOMETRY PANELS WORK AND THEN YOU HAVE MORE TYPES AND SUBTLE DIFFERENCES BASED ON ACTIVATION MARKERS AND THAT SORT OF THING. BUT WITH THIS DATA IT'S MUCH, MUCH FASTER TO RUN UMAP COMPARED TO TSNE, SO OUR FLOW CYTOMETRY GAVE US 200 DISCREET FEATURINGS OF THE IMMUNE SYSTEM TO BUILD THE UMAP, BUT THEN WE HAD 500 INDIVIDUAL FEATURES WE COULD PROJECT INTO THAT SPACE AS WELL AS 50 CLINICAL PARAMETERS THAT WE COULD USE IN THIS PROJECTION. SO THIS IS WHAT IT LOOKS LIKE WHEN YOU RESOLVE ALL OF THAT DOWN TO 2 DIMENSIONAL SPACE BASED ON IMINE FEATURES COLLECTED BY FLOW CYTOMETRY, THE HEALTHY CONTROLS AND RECOVERED DONORS ARE OVER HERE ON THE LEFT AND YOU CAN SEE THEY SEGGREGATE AWAY FROM THE COVID-19 PATIENTS FOR THE MOST PART, THERE ARE A FEW STRAGGLERS HERE, YOU CAN ALSO SEE IF YOU LOOK AT THIS THAT BASICALLY COMPONENT 1 IN THE HORIZONTAL DIRECTION SEEMS TO BE CAPTURING SOME OF THE DISEASE SEVERITY, SO AGAIN A DARKER RED IS MORE SEVERE. OW PATIENT WHO IS ARE DECEASED ARE IN TRIANGLES, SO YOU CAN GET SOME SENSE OF THIS AND IN FACT, IF YOU PLOT DISEASE SEVERITY DIRECTLY, YOU YOU ACTUALLY CAN SEE THAT THIS COMPONENT 1, WHILE IT'S BUILT WITH IMMUNEOLOGICAL FEATURES ALONE, CAPTURES ACTUALLY DISEASE SEVERITY VERY, VERY NICELY. SO YOU CAN ALSO THEN TAKE ADVANTAGE OF THIS AND PROJECT SOME OF THE INDIVIDUAL IMMUNE FEATURES AND SO IF YOU TAKE CD4 T-CELL ACTIVATION, BY HLADR AND CD38, THAT'S CONTRIBUTING TO THIS SORT OF DIAGONAL, VERTICAL DIAGONAL DIRECTION HERE WHERE MANY PATIENT SAMPLES IN THE UPPER RIGHT HAVE A HIGH DEGREE OF C4 T-CELL ACTIVATION, CAN YOU SEE THERE'S MORE DARK RED AND MORE MORTALITY UP HERE. CAN YOU SEE, IF YOU REMEMBER, THE CD8 T-CELLS ARE MISSING FROM MANY COVID-19 PATIENTS AND THEY TRACK IF HAD A DIRECTION THAT'S COMPLETELY ORTHOGONAL TO DISEASE SEVERITY SO THEY'RE TRACKING ALMOST UNIFORMLY, WHERE THE PLASMA BLASTS ARE TRACKING IN THE HORIZONT AT DIRECTION AND CONTRIBUTING TO COMPONENT 1. SO YOU CAN THEN USE THIS INSTEAD OF TAKING A WHOLE BUNCH OF INDIVIDUAL FEATURES, CAN YOU THEN ASK HOW THESE UMAP COMPONENTS 1 AND 2 RELATE TO SOME OF THOSE IMMUNE FEATURES. INCLUDING THE CLUSTER 14 WHICH IS THE HLADR ACTIVATED T-CELLS AS WELL AS T-BET POSITIVE ACTIVATED T-CELLS HERE WHERE ON THE CD8 SIDE THAT, COMPONENT CORRELATES NEGATIVELY WITH EFFECTOR LIKE OR T-BET POSITIVE EFFECTOR LIKE CELLS, WHEREAS COMPONENT 2 CORRELATES POSITIVELY. YOU HAVE OTHER THINGS HERE INCLUDING ON COMPONENT 1, A NEAR ABSENCE OR VERY, VERY STRONG NEGATIVE CORRELATION OF TFH AS COMPONENTS 2 ANDLY AND THE B-CELLS YOU SEE A PLASMA BLAST CORRELATION WITH CLUSTER 5 WITH COMPOPENT 1 WITH A NEGATIVE ASSOCIATION WITH SEVERAL OTHER ACTIVATED B-CELL FRACTIONS THERE, CAN YOU THEN ALSO RELATE THIS TO CLINICAL DISEASE FEATURES WHERE WHAT IT TURNS OUT IS COMPONENT 1 ACTUALLY CORRELATES VERY, VERY STRONGLY WITH THINGS LAKE APACHE SCORE AND THE NIERK H DISEASE AND SEVERITY INDEX AND OTHER MARKERS OF DISEASE SEVERITY, SOME OF WHICH REACH FDR POINT OF 5, AND SOME OF WHICH DON'T. IN CONTRAST COMPONENT 2 DOESN'T CORRELATE WITH DISEASE SEVERITY AS MUCH BUT HAS NEGATIVE RELATIONSHIPS WITH A FEW OTHER THINGS IN MINOR WAITS BUT IS NOT CORRELATED AS STRONG WITH CLINICAL FEATURES. SO WE HAVE 2 IMMUNE O TYPES. IMMUNE O TYPE 1 IS CHARACTERIZED BY HIGHLY ACTIVATED CD4 AND CD8 T-CELLS AND A--BITS SENSE REALLY OF TYPICAL CIRCULATING TFH AND VERY, VERY ROBUST PLASMA BLAST. IMMUNE O TYPE 2 AS EFFECTOR LIKE CD4 T-CELLS AND HAS MEMORY AND PROLIFERATING B-CELLS THAT DOES NOT ASSOCIATED WITH DISEASE SEVERITY. SO WHAT WAS INTERESTING ABOUT ALL THIS, IS, YOU COULD LOOK AT THE WAY THESE FEATURES CHANGED OVER TIME NOW IN THIS HIGH DIMENSIONAL SPACE AND IF YOU LOOK THERE ARE PATIENTS THAT ACTUALLY CHANGE VERY DRAMATICALLY, THAT IS THEIR LOCATION, IN THIS VIEW MAP SPACE, MOVES A LOT, AND THERE ARE OTHER PATIENTS WHOSE LOCATIONS, MOVES BARELY AT ALL OVER 1 WEEK IN THE HOSPITAL. YOU CAN THEN ASK ABOUT HOW THAT TRAJECTORY INFLUENCES OR IS LINKED TO THEIR CLINICAL DISEASE FEATURES, SO IF YOU HAVE COMPONENT 1 THAT'S CHANGING DRAMATIC LOAMACYY BETWEEN DAY 0 AND DAY 7, THAT IS A COMPONENT 1, IS GOING UP OVER TIME, THIS IS NEGATIVELY CORRELATING WITH CO INFECTION, WITH YOU'RE NUTRIFILLS BUT IT'S POSITIVELY CORRELATING WITH ANTIBODIES INCREASING OVER TIME SO THIS IS EVIDENCE OF A MOUNTING IMMUNE RESPONSE. IN THIS IS GOING DOWN OVER TIME, THESE PATIENTS ARE QUITE SICK AND IT SUPPORTS DISEASE SEVERITY. GOING UP AND/OR DOWN OVER 2,OR CORRELATES WITH DISEASE SEVERITY IF YOU LOOK THEA THE MAGNITUDE OF CHANGE OVER TIME, A BIGGER CHANGE OR TIME IS POSITIVELY CORRELATING WITH CHANGES IN YOUR ANTIBODIES MOSTLY, SO YOU'RE TELLING US ROUGHLY SPEAKING WHETHER WE'RE ON THE UPSWING OF THE IMMUNE RESPONSE, HAVE ALREADY PEAKED AND ARE STABLE AND WHETHER THAT RELATES TO DISEASE SEVERITY. SO COMPONENT 1 CHANGING, CORRELATES WITH METHOD 1 CHANGING, WITH THE APACHE SCORE, COAGULATION AND INFECTION, COMPONENT 2 CHANGING SEEMS TO BE SOMETHING SOMEHOW LINKED TO BLOOD TYPE PERHAPS CRP LEVELS, THE AMOUNT OF CHANGE SEEMS TO CORRELATE WITH ANTIBODY CHANGE OVER TIME. THERE'S 1 THING THAT WAS BOTHERING US STILL IN THIS DATA SET AND STILL YOU DON'T UNDERSTAND EVERYTHING EVEN WHO I JUST TOLD YOU, 1 THEY THINK THAT WAS BOTHERING US IS THE GREEN LINE AND SO THIS GREEN LINE, IT'S NOT LIKE A BLUE WALL BUT IT IS AN IMPORTANT LINE. IT TOLD US THERE'S A SUBSET OF PATIENTS MAYBE 15-20% OF THESE PATIENTS THAT ACTUALLY HAD NO DETECTABLE IMMUNE ACTIVATION AT LEAST IN THEIR CELLULAR COMPARTMENT OF THEIR IMMUNE SYSTEM. WE WANTED TO UNDERSTAND THESE PATIENTS A LITTLE BIT BETTER, AND IT TURNS OUT THAT WHEN YOU PROJECT INTO THIS UMAP SPACE, WHERE THESE PATIENTS ARE, THEY FALL SORT OF IN THIS COMPONENT 2 LOW, COMPONENT 1 ESSENTIALLY 0 RANGE. THEY ACTUALLY NEGATIVELY CORRELATE WITH BOTH IMMUNE O TYPE 1 AND 2, OR COMPONENTS 1 AND 2 AND NEGATIVELY CORRELATE WITH DISEASE SEVERITY. I SAY THAT BUT IF YOU LOOK CAREFULLY AT THE TRIANGLES WE HAVE 1 PATIENT WHO SUCCUMBED HERE WITH WHAT WE'RE CALLING IMMUNE O TYPE 3. YOU ALSO HAVE PATIENT WHO IS SUCCUMBED UP TO THE TOP HERE THAT HAVE LOW COMPONENT 1,IMEUNE O TYPE 1 BUT ARE GREAT EXAMPLES OF UMPIRESUNE O TYPE 2, AND MOST OF OUR MORTALITY IS OUT HERE ALONG THIS RIM, WHERE WE HAVE VERY STRONG IMMUNE O TYPE 1 SIGNATURE THAT IS HYPER INFLAMMATORY SIGNATURE. SO, THAT'S WHERE WE STAND WITH THIS, TRYING TO UNDERSTAND THINGS, I WILL SHOW YOU 3 WAYS WE'RE TAKING THIS NEXT, ALL OF WHICH ARE VERY, VERY EARLY AND PRELIMINARY BUT START TO ANSWER, I THINK FOR US SOME OF THE KEY OPEN QUESTIONS ABOUT THESE ADULT COVID-19 DAT A.--REALLY CLEAR FROM ONGOING EVOLVING LITERATURE THAT WE NEED TO IDENTIFY TRUE KNOWN VIRUS SPECIFIC T-CELLS, SO LIKE MANY OTHERS IN THE FIELD, WE ARE USING THE ACTIVATION WHOSE MARKER ASSAY, THAT SHANE [INDISCERNIBLE] AND SHANE [INDISCERNIBLE] STUDIED FOR THE SARS-COV2, AND PEPTIDE MEGAPOOLS AND YOU TAKE PBMCs AND YOU STIMULATE WITH MEGAPOOLS FOR 24 HOURS AND THEN YOU LOOK AT THE UPREGULATION OF MARKERS LIKE CD69, CD200 AND OTHERS WHERE YOU CAN NONAPOPTOTIC GATE ON CELLS THAT RESPONDED IN AN ANTIGENERATED SPECIFIC WAY TO THE STIMULATION. THERE'S BEEN SOME THOUGHT SOME OF THIS COULD RESULT FROM BI STANDER ACTIVATION FROM CYTOKINES BUT CAN YOU DO THESE ASSAYS WHERE YOU ALSO INCLUDE CYTOKINE INCRETION AND BLOCK MOST OF THAT ACTIVITY. AND THEN YOU CAN VALIDATE THESE THINGS WITH OTHER METHODS. ONE OF THE THINGS WE HAVE TO DO IS UNDERSTAND WHAT THIS LOOKS LIKE FOR RECOVERED DONORS, AND THE MAGNITUDE QUALITY AND STABILITY OF THESE MEMORY T-CELLS AND UNDERSTAND THE TFH AND ACTIVATED CD8 RESPONSES DURING ACUTE INFECTION FROM THESE TYPES IS REALLY 1 OF THE KEYS SOPHISTICATEDY WE WANT TO UNDERSTAND NOW THAT YOU SORT OF EMERGING ACCEPTANCE OF AN IDEA WHERE THERE'S PATIENT WHO IS HAVE LONG-TERM INFECTION WHICH IS IS A HEAVY POST COVID COMPLICATIONS IN PATIENT WHO IS ARE SUSCEPTIBLE TO REINFECTION AND THAT'S WHERE WE NEED ANTIGEN SPECIFIC ASSAYS THAT WILL COMPLEMENT WITH ANTIGEN SPECIFIC SERIES POINTSOLOGY AS WELL. AGAIN LIKE EVERYBODY ELSE, WE ARE DOING A LOT OF RIN,A SEQ ON THESE PATIENTS AND IN OUR FIRST SET OF DATA ON THIS THAT I'M JUST SHOWING YOU A FEW EXAMPLES FROM, WE ACTUALLY RAN ABOUT 16 SUBJECTS, WE RAN 12 SUBJECTS, COVID-19 4 HEALTHY CONTROLS AND BUT WE SELECTED 3 FOR EACH IMMUNE O TYPE AND INDEED THEY FALL IN DIFFERENT PLACES IN THE HIGH DIMENSIONAL SINGLE CELL RNA SEQ SPACE. THIS WAS 5 PRIME WITH 10 X SO WE CAN GET TCRs AND PC Rs ACTUALLY AND LOOK AT HOW THOSE CLONAL EXPANSIONS MIGHT FALL IN DIFFERENT REGIONS OF T-CELL ACTIVATION FOR EXAMPLE FROM THE DIFFERENT TYPES. THE OTHER THING THAT'S POSSIBLE TO DO IS USE ALL THE PUBLICLY AVAILABLE DATA SO WE FILLED A LARGE AGGREGATED SINGLE CELL RNA SEQ DATA SET FROM 2 INDIVIDUALS SAMPLES THAT HAVE BEEN PUBLISHED AND FROM THAT DATA YOU CAN ALSO DERIVE QUITE A BIT OF INFORMATION. SO HOPEFULLY WE WILL GET THOSE DATA VERY SOON. AND THE LAST THING I WANT TO MENTION HERE BEFORE ENDING WITH A FEW SLIDES ON CHILDREN. IS THAT WE'RE VERY INTERESTED IN THE COAGULATION COMPLICATIONS AND THINK OF THIS AS AN ORTHOGONAL WAY THESE PATIENTS ARE SICK. AND WE BECOME INTERESTED IN WHAT THE PLATELETS ARE DOING THESE IN PATES, THEY CAN BE HIGHLY IMMUNEOLOGICALLY ACTIVE AND ARE PLAYING A ROLE IN THE PATHOLOGY INCLUDING THROMBOSIS AND MICROTHROMBI THAT WE'RE SEEING HERE IN 1 PUBLICATION IN GENE CIRCULATION IN AUGUST. SO WHAT WE'VE DONE IS BUILD AN ASSAY WHERE WE LOOK AT PLATELET ACTIVATION EXVIVO AND THE WAY YOU DO THIS IS YOU TAKE A PEPTIDE FROM AND ACTIVATES THE THROMBIN RECEPTOR ON PLATELETS, YOU STIMULATE THE PLATELETS AND LOOK AT UPREGULATION OF MARKERS AND LIKE CD62 P AND ALLOW ITS TO ROLE HERE AND YOU CAN STIMULATE BRIEFLY INVITRO, YOU CAN LOOK AT BASAL ACTIVATION SO EXPRESSION CD62 B BUT THEN TAKE THE PLATELETS AND ACTIVATE THEM WITH THE PEPTIDE AND LOOK AT DEGREE OF UPREGULATION, AND IT'S CALLED THE TRAP ASIGNIFY. WHEN YOU DO THAT, YOU CAN SEE THAT THE BASAL LEVEL OF PLATELET ACTIVATION IS INCREASED IN THESE COVID-19 IN-PATIENTS SORT OF CONTROLS, SO IT'S A BIT OF LINGERING EFFECT IN THE CONVALESCE ENSEL OR RECOVERED DONORS, YOU DO THE TRAP ASSAY, THAT IS YOU ACTIVATE THE PLATELETS EXVIVO AND YOU SEE THAT THINGS ARE PRETTY CLOSE, ALTHOUGH THE HEALTHY CONTROLS ALSO SEEM TO DO A BIT BETTER BUT IF YOU TAKE THE RATIO TO THE BASE THAT'S ACTIVATED, IT'S CLEAR THE PLATELETS COMING OUT OF THE COVID PATIENTS TO DO A LITTLE BIT WORSE IN BEING ABLE TO ACTIVATE THEMSELVES. TURNS OUT, THAT THERE'S SOME RELATIONSHIP BETWEEN THIS ACTIVATION STATE OF THE PLATELETS AND CO AGZULATION COMPLICATION OR CARDIOVASCULAR RISK, IT'S NOT THAT ROBUST YET BUT IT'S A SMALL NUMBER OF PATIENTS. IT ALSO TURNS OUT THAT PLATELETS ARE A TOTAL PAIN TO WORK WITH, APOLOGIES TO ANYBODY WHO, WOS WITH PLATELETS AND SHAKING THE TUBE A BIT AS IT COMES FROM THE CLINIC TO THE LAB COULD RESULT IN THIS KIND OF PLATELET ACTIVATION, SO WE DECIDED DO TOR DO BECAUSE OF THE LARGE VARIANCE IN THESE STUDIES WAS JUST USE SERUM OR PLASMA FROM THE COVID-19 PATIENTS AND PUT THAT ON TO HEALTHY PLATELETS FROM DIFFERENT CONTROL INDIVIDUALS AND IN DO THAT YOU CAN GET GOOD ACTIVATION OF PLATELETS AND YOU CAN ASK ABOUT WHAT ARE THE FEATURES ON THE PLATELETS THAT ARE IMPORTANT IN ALLOWING SERUM FROM SICK PATIENTS TO ACTIVATE THEM. SO CAN YOU BLOCK F-C-RECEPTORS AND BLOCK IL6 BLOCK COMPLEMENT AND SO ON, CAN YOU BLOCK ALL OF THEM AND ACTUALLY DRAMATICALLY BUT THE CLEAR EXAMPLE FROM THESE DATA SO FAR IS THAT ACTIVATION OF PLATELETS--ACTIVATION OF HEALTHY PLATELETS WITH THE PLASMA FROM COVID-19 PATIENTS SEEMS TO BE DEPENDENT ON ANTIBODY AND COMPLEMENT. SO WE CAN BLOCK COMPLEMENT C3A OR C5A, EITHER INDIVIDUALLY OR TOGETHER WITH DEPLETING IMMUNE O GLOBUE LYNN FROM THE PLASMA AND WHEN YOU BLOCK ALL 3, THAT'S GET RID OF ANTIBODY AND BLOCK COMPLEMENT RECEPTORS BASICALLY, CAN YOU SEE YOU CAN RETURN PLATEMENT ACTIVATION BACK TO BASE LINE. ONE WOULD BE THE BASE LINE HERE AND WHAT THIS SUPG JEST SYSTEM THAT SOME DEGREE OF ANTIBODY, PERHAPS IMMUNE COMPLEXES OR COMPLEMENT EITHER FREE COMPLEMENT OR COMPLEMENT DEPOSITED BY THE COMPLEXES IN THE PLASMA COVID-19 PATIENTS IS PLAYING A ROLE IN ACTIVATING PLATELETS, PERHAPS CONTRIBUTING TO THE MICROTHROMBI, WE SEE, AND YOU WILL ALSO NIGH THERE'S A GOOD DEGREE OF HETEROGENEITY SUGGESTING THAT SOME DEGREE OF HETEROGENEITY IS 1 REASON WHY WE'RE SEEING DIFFERENCES IN THE WAY PATIENTS HAVE THIS CARDIOVASCULAR COMPLICATIONS. ALL RIGHT, SO I KNOW THERE'S A LOT GOING ON AND SUMMARIZE THAT THATTA WHERE WE STAND WITH ADULT PATIENTS. I WANT TAKE A MINUTE TO SHOW YOU 3 OR 4 MORE SLIDES ON WHAT WE'RE DOING IN CHILDREN, AND I THINK THIS IS INFORMATIVE AND RELATES BACK TO WHAT WE CAN LEARN FROM THE DISCREET PATHOLOGIES OF COVID-19 INFECTION, AND NIGHT INFORM ALL PATIENTS. SO WE WANTED TO STUDY WHAT WAS HAPPENING IN KIDS AND IN PARTICULAR, UNDERSTAND THESE MIS-C KIDS THAT HAVE THIS REALLY UNIQUE OR DISCREET CLINICAL PRESENTATION PHENOTYPE THAT LOOKS A LOT LIKE KAWASAKIS DISEASE, HALF OF THEM HAVE THE MIS-C, THEY LOOK LIKE,A DULTS, THE COUNTS ARE LOW, T-CELLS ARE ACTIVATED SO I'M SHOWING HERE ACCIDENT THE RED AND THE GRAY ARE VERY, VERY SIMILAR DATA TO WHAT I SHOWED YOU ON THE FITTER PART FROM,A DULTS, WE COULD NOT RECRUIT HEALTHY CHILDREN DURING THE PANDEMIC SO WE HAVE OUR HEALTHYA, DULTS FOR COMPARISON, OUR NONMIS-C CHILDREN IN THE LIGHT BLEW AND CIRCLES AND THE OUR MIS-C CHILDREN IN THE GREEN, YOU CAN SEE THERE'S LYMPHPENIA IN THE MIS-C KIDS, PERHAPS MORE PROFOUND THAN THE NONMIS-C KIDS AND THIS IS TRUE FOR OF NONNAIVE T-CELL COULD YOU WANTS IN KIDS. THESE KIDS HAVE GREATER T-CELL ACTIVATION, ADULT LIKE T-CELLAC VACATION COMPARED TO OTHER COVID-19 CHILDREN, EVEN THOSE WITH ARDS AND YOU CAN SEE THAT HERE WITH A LEVEL OF CD8 T-CELL ACTIVATION AND THIS RIVALS OUR SICKEST ADULT PATIENTS IN THE DARK RED THERE. CAN YOU DO THIS A NUMBER OF DIFFERENT WAYS AND 1 OF THE INTERESTING THINGS ABOUT MIS-C KIDS IS THEY PRESENT A RANGE OF 3-4 WEEKS AFTER THE SUSPECTED INFECTIOUS EVENT OCCURRED SO THIS MIGHT BE AN INDICATOR OF LONG IMMUNE RESPONSE, THE KID VS MUCH HIGHER TD1 THAN OTHER COVID KIDS AND IN FACT, THAT'S TRUEOT CD4 SIDE, THE CD8 SIDE AND IF YOU START USING MARKERS THAT DON'T ACTUALLY LOOK MORE INDICATIVE, OF T-CELL EXHAUSTION THAT IS CO EXPRESSION OF OTHER INHIBITORY RECEPTORS, THE MIS-C KIDS ARE AT LEAST AS HIGH AS THE VERY SICKEST ADULTS WHERE WE SEE VERY LITTLE OF THIS IN THE YOUNG KIDS. BUT THE 1 THING I WANT TO TELL YOU ABOUT AND I WILL WRAP UP, IS IDENTIFICATION OF THE POPULATION OF CXCRX CD1 T-CELLS THAT ARE HIGHLY ACTIVATED IN THE MIS-C KIDS. THIS IS INTERESTING BECAUSE THIS ACTS AS CXCR1, AND CXC3 CL1 IS INVOLVED IN A LOT OF THINGS AND 1 OF WHICH IS THE ABILITY OF CD8 T-CELLS TO POETIC TROLL THE VASCULATURE, THESE CELLS ARE MORE EFFECTOR LIKE, AND CYTOTOXIC AND BASED ON GENETICS 32'S A CLEAR ROLE IN THE PATHWAY, AND SUGGESTING PERHAPS THESE CELLS ARE POETIC TROLLING BLOOD VESSELS THEY MIGHT HAVE A ROLE IN THE INTERESTING PATHOGENESIS AND BEYOND. THE NUMBER OF THESE CELLS ARE FREQUENCY OF THESE CELLS IS NOT DIFFERENT IN ANY OF THE PATIENT GROUPS WE LOOKED AT, BUT WHEN YOU LOOK AT THE ACTIVATION STATUS, OF CXCR31 POSITIVE CD8 T-CELLS THEY'RE DRAMATICALLY MORE ACTIVATED, THERE'S MORE ACTIVATION IN THE CXCR31 NEGATIVE CELLS AS I SHOWEDOT LAST SLIDE BUT LOOKING AT THE RIGHT, THE ACTIVATION STATUS OF THESE CXCR31 CELLS IS DRAMATICALLY HIGHER IN THE COVID KIDS IN THE MIS-C KIDS COMPARED TO THE NONMIS-C COVID KIDS. SO DOES THAT HAVE ANY RELATION TO WHETHER THEY'RE EXPERIENCING CASK LARK OR CARDIOVASCULAR COMPLICATIONS AND INDEED THE VAST MAJORITY OF--BUT HOW THAT IMPACTS THINGS HERE WE'RE NOT TOO CLEAR. SO TAKING A LEAVE FROM THAT, IF YOU LOOK AT ADULTS, IF THEY HAVE ACTIVATION OF THIS CONTROLLING DEPARTMENT OF CD8 T-CELLS THEY'RE MUCH MORE LIKELY TO HAVE VASCULAR COMPLICATIONS INCLUDING [INDISCERNIBLE]. SO THEY WILL SKIP OVER THE LAST PART OF THIS IN THE INTEREST OF TIME AND JUST SUMMARIZE THAT WE HAVE TREMENDOUS DEGREE OF IMMUNE HETEROGENEITY IN COVID-19 WITH ADULTS AND KIDS, THERE'S A LOT OF LYMPHPENIA THAT MAY DRIVE THE T-CELL ACTIVATION AND PLASMA BLAST ACTIVATION THAT WE SEE IN ACUTE EBOLA AND DENGUE DISEASE, IN SOME CASES THE ACTIVATION OF THE T-CELL OR BCELL RESPONSE IS REMARKABLY STABLE OVER TIME AND THAT MAY CORRELATE WITH DISEASE SEVERITY. WE IDENTIFIED 3 ENUMERATORUNE O TYPES THAT WERE CALLED TO,A DULTS AND DIFFERENT CLINICAL AND IMMUNEOLOGICAL PRESENTATIONS AND IDENTIFIED AS POTENTIAL VASCULAR PATROLLING ACTIVATION PHENOMENON THAT WE IDENTIFY INDEED THE MIS-C KIDS THAT MAY HAVE RELEVANCE TO THE NONMIS-C AS WELL. SO WE'RE USING THIS TO TRY TO IDENTIFY LINKS BETWEEN THE CLINICAL TREATMENTS AND CLINICAL FEATURES AND SPRVEG IMMUNE CELL POPULATIONS AND THE IMMUNE ACTIVATION. LET ME JUST WRAP UP AND THANK EVERYBODY IN THE LAB, BECAUSE EVERYBODY IN THE GROUP REALLY CONTRIBUTED TO THIS, BUT THERE ARE REALLY 7 PEOPLE ON MY END THAT CONTRIBUTED TO THE ADULTS THAT WERE CO-FIRST AUTHORS INCLUDING DIVIJ, JOSEPHINE, AMY AND THE LIKE, WE SET UP A TESTING CENTER, AROUND CAMPUS, WE COULD NONAPOPTOTIC THE HAVE DONE THIS WITHOUT THIS GROUP TIRELESSLY PROCESSING BLOOD SAMPLES DAY IN AND DAY IDENTITY DURING THE PANDEMIC. THE LAST PEDIATRIC WORK I SHOWED YOU IS ACTUALLY WORK DONE BY LAURA VELLA, IN COLLABORATION WITH THIS WHOLE WORK AGAIN. WE HAD TREMENDOUS CLINICAL COLLABORATION WITH [INDISCERNIBLE] WITH MIKE BETTS GROUP AND MANY ON THE PEDIATRIC SIDE AND WITH A ROBUST CLINICAL ACCORD NEAGZ TEAM HERE. SO I WILL STOP HERE, HOPEFULLY LEFT AT LEAST A FEW MINUTES IF ARE QUESTION FIST THERE ARE ANY. SO THANKS A LOT. >> THANK YOU JOHN, THAT WAS INCREDIBLE. I REALLY GREAT TALK. THIS IS PAM. SCHWARTZ BERG AND I WILL FORWARD SOME QUESTIONS TO YOU. SO THE FIRST QUESTION FROM KRISHNA [INDISCERNIBLE], VERY INTERESTING TALK, THEY HAVE 2 QUESTIONS, ARE THE IMMUNE O TYPES COVID-19 SPECIFIC? SO DO YOU SEE THIS IN SEVERE FLU FOR EXAMPLE? OR OTHER COMPARISONS WITH OTHER TYPES, I THINK YOU MENTIONED DENGUE AND OTHER THINGS. >> YEAH, GREAT QUESTION. THANKS. WE HAVE NO EVIDENCE WAY WAY OR THE OTHER. BUT OUR BIASES THAT YES, THOSE IMMUNE O TYPES WILL EXIST OTHER PLACES, ALL OF US HAVE OUR OWN KIND OF IMMUNE FINGERPRINT THAT WILL BE SHAPED NOT ONLY BY GENETICS BUT BY OUR IMMUNEOLOGICAL EXPERIENCE, WHAT WE THINK IS THAT THE SARS-COV2 IS SUCH A MASSIVE LANDSCAPE ON THE ENUMERATORUNE SYSTEM, SO IT WILL BE EASIER TO SEE THEM. WE'RE NOW LOOKING TO TEST THAT WITH OTHER PROTURBATIONS OF THE IMMUNE SYSTEM, OTHER NATURAL LIKE OTHER INFECTIONS AND SOME INSTATE REGULATORIGATED SUCH AS YEARLY VACCINATION. >> AND SO, THEN ALSO THERE WAS A REQUEST ABOUT DO THE ACTIVATED CD4 CELLS ASSOCIATE WITH THE DISEASE SEVERITY AND IS THAT COVID 2 SPECIFIC, SARS-COV2 SPECIFIC OR THROUGH BI STANDER ACTIVATION OF SELF-REACTIVE CD4 CELLS THAT ARE CAUSING'MUNE O PATHOLOGY. >> YEAH, REALLY IMPORTANT QUESTION, I CAN'T ANSWER THE SECOND PART OF THAT SO WE DON'T HAVE ENOUGH DATA YET ON SPECIFICITY OF THOSE CELLS, AND SO IT COULD EASILY BE CROSS REACTIVE, AUTOREACTIVE, SPECIFIC FOR SOME OTHER PATHOGEN, SOME OF THESE PATIENTS ARE CO INFECT WIDE BACTERIAL PATHOGENS AS WELL, BUT TO COMMENTOT FIRST PART OF YOUR--COMMENT ON THE FIRST PART OF YOUR QUESTION OR STATEMENT, THESE ACTIVATED CELLS SEEM TO BE STRONGER CORRELATES WITH THE SEVERE SCEEZ, AT LEAST IN THE PATIENT WHO IS HAVE PROBABLY THE IMMUNE O PATHOLOGY SIDE OF THE EQUATION, CD4, CDs SEEM TO BE STRONGER CORRELATES IN THAT SETTING. >> GREAT. AND SOMEBODY SAID OBVIOUSLY NEUTRALIZING ANTIBODIES ARE CONSIDERED THE GOLD STANDARD FOR PROTECTION BUT THAT IS THERE BEEN ANY INVESTIGATIONS INTO NONNEUTRALLIZING ANTIBODIES AND PARTICULARLY GIVEN THE DISRUPTIONS AND NK CELLS IS IT POSSIBLE THAT ADCC COULD PLAN AN IMPORTANT ROLE? >> YEAH, GREAT QUESTION. YOU MUST BE THE 1 PERSON WHO READ OUR OLD PLUS PATHOGENS PAPER ON NONNEUTRALLIZING ANTIBODIES AND PROTECTION FROM INFLUENZA INFECTION,--FROM OTHERS SUGGESTING THE ACTIVATION OF THE ANTIBODY MAY PLAY AN IMPORTANT ROLE. THIS ACUTE INFECTED PATIENTS THE INTERESTING THING IS THEY HAVE NEUTRALIZING ANTIBODIES BUT IT DOESN'T SEEM TO BE HELPING, AT LEAST SOME OF THEM RESOLVED IN THE DISEASE, SO I SUSPECT 1 OF THE INTERESTING THINGS WE'RE SEEING IS THAT FOR PREVENTION OF COURSE NEUTRALIZING ANTIBODY IT WILL BE THE GOLD STANDARD, ONCE YOU'RE INFEBTED, I SUSPECT THAT OTHER FEATURE OF THE ANTIBODY WILL BECOME INCREASINGLY IMPORTANT AND THEY MAY HAVE TO DO WITH OW THE VIRUS SPREADS THROUGHOUT THE BODY SO I WOULD NOT RULE OUT A ROLE FOR NONNEUTRALLIZING ANTIBODIES RIGHT HERE. GREAT QUESTION BUT WE DON'T KNOW. >> YEAH, A LOT OF INTERESTING THINGS COMING OUT WITH THESE ANTIBODY RESPONSES, I THINK. ANOTHER--EVERYONE'S TARTING OUT SAYING HOW INTERESTING THE TALK IS, 1 PERSON WANTED TO KNOW ABOUT THE--WHETHER YOU TOOK INTO ACCOUNT THIS TIME FROM SYMPTOM ONSET, AND WEIRD THAT THE IMMUNE RESPONSE IS REALLY STARTING FROM? >> YEAH, IT'S A REALLY GOOD QUESTION, THERE'S A BIT OF DATA IN THE PAPER THAT I GROSSED OVER, IESM NOT PERSONALLY SURHOW CONFIDENT WE CAN BE IN IT. BUT SELF-REPORTED ONSET OF SYMPTOMS WE'RE PROBABLY GETTING THE FIRST SAMPLE OF 7-10 DAYS OF LIKELY INFECTION, BUT YOU KNOW PEOPLE REPORT THIS DIFFERENTLY, SO IT DID NOT SEEM TO EXPLAIN THIS SORT OF TRAJECTORY DYNAMICS THAT WE SAW. WE ACTUALLY THINK THE IMMUNE RESPONSE WE CAN CHARACTERIZE MAY BE BETTER AT GIVING US A PSEUDOTIME ESTIMATE OF WHETHER WE'RE IN THE EARLY PHASE AND UPSPRING OF THE IMMUNE RESPONSE VERSE US SORT OF THE PLATEAU, THEN DOES PATIENT REPORTED ONSET OF SYMPTOMS, ALL SORTS OF REASONS FOR THAT, IT'S A GREAT QUESTION, MAYBE WITH BETTER QUESTIONNAIRES WE CAN DO BET OR THAT. >> ONE QUESTION FROM JOHN SANG ABOUT WHAT YOU THOUGHT ABOUT DISTINGUISHING CD8 T-CELL ACTIVATION VERSUS EXHAUSTION, ESPECIALLY IN THE ACUTE PHASE OR EARLY PHASE. >> YEAH, WE--SO WE LIKE CD38, CD39 AND PD1 CO EXPRESSION AND WE'VE DONE GENOMICS ON THAT, THAT SMALL SUBPOPULATION THAT'S EACH PRESCRIBINGENT IN HEALTHY CONTROLS, DOES HAVE ALL THE FEATURES INCLUDING EPIDENETTIC FEATURES OF EXHAUSTIVE T-CELLS IN SETTING WHERE YOU HAVE HIGHLY ACTIVATED T-CELLS WE HAVE TO BE CARE EMPLOY. SO THE 1 REASON I MENTION IT IN THE PEDIATRIC SETTINGS BECAUSE WE SUSPECT THOSE KIDS HAVE HAD AN ONGOING IMMUNE RESPONSE FOR AT LEAST 3 WEEKS, MAYBE LONGER. WE DON'T SEE EVEN IN HUMANS A REALLY CLEAR SWITCH TO THE MOLECULAR PROGRAM OF EXHAUSTION, FOR 2-3 WEEKS. SO IN THESE ACUTE PATIENTS, MOST OF WHOM ARE PROBABLY IN THE LATTER PART OF THE FIRST WEEK, MAYBE SECOND WEEK OF INFECTION, I THINK THE DATA WE ARE PREMATURE ON SAY ANYTHING ABOUT EXHAUSTION. NOW WE HAVE THESE LONG-TERM PATIENTS, SOME OF WHOM ARE I HAVE REAMIC FOR SEVERAL WEEKS, THAT'S A SETTING WHERE I THINK LOOKING AT THE LITERATURE THERE ARE A FEW EXAMPLES NOW, SNEAKING OUT WHERE WE HAVE TO THINK BENEFIT EXHAUSTION IN THESE SETTING, SO WE HAVE TO BE CAUTIOUS, BECAUSE OF COURSE A MOLECULAR PROGRAM WILL BE DIFFERENT. HOW WE MANAGE THE DISEASE THAT MAY BE CAUSED BY OR ASSOCIATE WIDE EXHAUSTION VERSUS EFFECTOR CELLS WILL ALSO BE DIFFERENT SO WE'RE BEING CIRCUMSPECT ABOUT THIS AT THIS POINT UNTIL WE HAVE MORE DATA. >> I THINK 1 LAST QUESTION BECAUSE I KNOW YOU ALSO HAVE TO GO, 1 PERSON WOULD LIKE TO KNOW WHAT PLASMA BASED BIOMARKERS WOULD YOU SUGGEST TO EVALUATE AND SAMPLES FROM CLINICAL STUDIES WHERE NO IMMUNE CELLS ARE AVAILABLE? >> YEAH, REALLY GOOD QUESTION. SO NO 1 CLINICAL PLASMA BASED MARKER IS GOOD ENOUGH TO GIVE US THIS DIVERSITY. FERRET AND D-DIMER ALL GIVE USEFUL INFORMATION, CRP, ACTUALLY CAN YOU START TO USE AGGREGATES OF THAT. SO IF YOU GET DENSE ENOUGH CLINICAL DATA WE ARE STARTING TO SEE EMERGENCE OF DIFFERENT CLUSTERS THAT RELATE TO THESE DIFFERENT IMMUNE O TYPES. IT'S NOT PERFECT YET. WE'RE SEE WORKING ON IT SO I CAN'T GIVE SPECIFICS BECAUSE I DON'T KNOW THEM YET. BUT I WOULD SAY, PROBABLY COMBINATIONS OF CLINICAL MARKERS THAT GIVE YOU SLIGHTLY DIFFERENT PICTURES SO FERRET AND D-DIMER AND A FEW OTHERS WILL GIVE YOU 1 TYPE OF CLINICAL MANIFESTATION. I WOULD LOOK FOR CLINICAL MEASURES THAT GIVE YOU SOMETHING DIFFERENT FROM THAT, AND THEN CAN YOU START TO SEE A LITTLE BIT OF RESOLUTION, THAT'S WHAT WE'RE DOING RIGHT NOW. IT'S A REALLY, REALLY KEY QUESTION GOING FORWARD FOR THE FIELD. >> I WILL LEAVE WITH 1 COMMENT IS A QUESTION BUT IT'S A COMMENT FOR GOING FORWARD THERE, WANTED TO KNOW WHETHER YOU HAD STARTED TO CORRELATE ANY OF THESE RESPONSES TO THE STRAIN OR DIFFERENCES IN THE VIRUS ITSELF. >> IT'S A COMMENT BECAUSE THE ANSWER'S NO, IT'S A REALLY IMPORTANT COMMENT. >> OKAY, WITH THAT, I THINK WE WILL HAVE TO CLOSE BECAUSE DR. WHERRY HAS TO GO TO ANOTHER MEETING. JOHN IF YOU COULD STAY ON THE LINE FOR ONE MINUTE FOR MICHAEL GOTTESMAN THANK YOU