>> GOOD AFTERNOON, EVERYONE. THANK YOU FOR COMING ON A THURSDAY FOR THE WEDNESDAY AFTERNOON LECTURE. THERE'S A GOOD REASON FOR THAT, WHICH YOU PROBABLY NOTICED YESTERDAY AND HAD YOUR LIFE DISRUPTED, NAMELY OUR LATEST SNOWSTORM, IF YOU WERE IN BOSTON WAS NOT A BIG DEAL BUT HERE IN D.C. IT WAS PARALYZING. PLEASED OUR GUEST SPEAKER WHO HAS BEEN HERE FOR A DAY AND A HALF, PROFESSOR ALAIN FISCHER, WAS ABLE TO MEET WITH MANY PEOPLE YESTERDAY BECAUSE WE'RE SUCH A HARDY GROUP OF PEOPLE, HE'S HAD INTERESTING CONVERSATIONS WITH INVESTIGATORS AND TRAINEES. NOW WE HAVE A CHANCE TO HEAR SOME THOUGHTS ABOUT PEDIATRIC IMMUNE DISEASES, AND ALL GENETICS IS PART OF HIS TITLE WITH A QUESTION MARK, THAT'S INTRIGUING TO SEE WHERE THAT MIGHT TAKE US. PROFESSOR FISCHER GOT HIS MD/PHD AT UNIVERSITY OF PARIS, POSTDOC UNIVERSITY COLLEGE OF LONDON, REMAINED IN VARIOUS ROLES OVER THE COURSE OF THE PAST 37 YEARS, WHERE HE IS NOW DIRECTOR AND PROFESSOR IN THE CHAIR OF MEDICINE EXPERIMENTAL AT THE COLLEGE DE FRANCE, ONCE HELD BY CLAUDE BERNARD, ALSO DIRECTOR OF IMAGINE AT THE UNIVERSITY PARIS DESCARTESE AWARDED MANY HONORS INCLUDING MEMBERSHIP IN THE FRENCH SCIENCE ACADEMY, FRENCH ACADEMY OF MEDICINE, SANTA FE PASTEUR PRIZE, KOCH PRIZE FROM BERLIN, JAPAN PRIZE, A BIG DEAL, A FOREIGN MEMBER OF THE U.S. NATIONAL ACADEMY OF MEDICINE. HIS RESEARCH HAS FOCUSED OVER ALL OF THIS TIME SPECIFICALLY ON PEDIATRIC IMMUNE DISEASES, HE TOLD ME HE'S NOT GOING TO SPEAK ABOUT GENE THERAPY FOR IMMUNE DISORDERS TODAY BUT HE IS WELL KNOWN TO MANY OF US AS PERHAPS THE LEAD INVESTIGATOR PUSHING FORWARD SUCCESSFUL APPLICATIONS, PARTICULARLY TO SEVERE COMBINED IMMUNODEFICIENCY, A REPORT IN THE YEAR 2000 OF EX VIVO THERAPEUTIC THAT I THINK GAVE US MANY HOPES AT A TIME WHERE GENE THERAPY WAS STRUGGLING QUITE A BIT, THIS COULD IN FACT BE SOMETHING THAT COULD BE APPLIED MORE BROADLY. MANY UPS AND DOWNS SINCE THEN BUT IT'S FAIR TO SAY THE FIELD OF GENE THERAPY IS RISING AT AN INTERESTING AND IMPORTANT JUNCTURE AND PROFESSOR FISCHER'S WORK HAD A LOT TO DO WITH THAT, EVEN THOUGH HE WILL TELL YOU TODAY THAT HE'S TALKING ABOUT OTHER THINGS AND INTERESTED IN OTHER THINGS. I'M ANXIOUS TO SEE HOW THAT PLAYS OUT. PLEASE IF YOU WOULD, JOIN ME IN WELCOMING PROFESSOR ALAIN FISCHER. [APPLAUSE] >> WELL, THANK YOU FOR THE VERY KIND INTRODUCTION AND FOR THE INVITATION, DELIGHTED TO SPEND TWO DAYS AT THE NIH TALKING TO OLD FRIENDS, NEW FRIENDS, GREAT SCIENTISTS, ALSO I FEEL YOU MANAGED TO HAVE KIND OF SNOWSTORM FOR ME YESTERDAY. VERY SPECIAL AND UNIQUE EVENT. I CHANGED THE TITLE, FROM DAY TO DAY MY MIND IS A BIT DIFFERENT. STILL THE SAME STORY. IS TH STUDY OF MONOGENIC DISEASE, MORE COMPLEX COMMON AND COMPLEX DISEASES, I'M NOT SURE I WILL ADDRESS FULLY THE QUESTION BUT WILL TRY TO DO IT A LITTLE BIT THROUGH THE DESCRIPTION OF A FEW EXAMPLES OF MONOGENIC NEONATAL DISEASES, TO GIVE YOU A PICTURE FOR THOSE WHO ARE NOT SPECIALISTS IN THE FIELD, THIS IS THE TIME OF THE DISCOVERY OF GENETIC DEFECTS WITH ALMOST ALL THE WAY THROUGH MENDELIAN INHERITTANTS. IN THE 1980s THERE WERE ZERO IDENTIFIED, BUT NOW WE'RE UP TO MORE THAN 350, OBVIOUSLY DIFFERENT PATTERNS OF INHERITANCE. WE ESTIMATE BASED ON REGISTRIES AND DIFFERENT STUDIES THAT APPROXIMATELY ONE CHILD IN 2,000 LIVE BIRTHS IS AFFECTED, WILL BE AFFECTED PHENOTYPICALLY SPEAKING, SOONER OR LATER, BY A GENETIC DEFECT OF THE IMMUNE SYSTEM. AS I SAID, MOST WITH MENDELIAN INHERITANCE, NOT ALL. PHENOTYPIC CONSEQUENCES ARE DIVERSE, VULNERABILITY TO INFECTION, BROAD OR NARROW, AUTOIMMUNITY, I WILL DISCUSS THAT, ALLERGY, INFLAMMATION, CANCER. THESE GENETIC DEFECTS ARE CAUSING PROBLEMS IN ALL ARMS OF THE IMMUNE SYSTEM, NOT EQUALLY BUT THAT'S ANOTHER STORY, AND OF COURSE OVER TIME THERE ARE STUDIES PROVIDED US WITH IMPORTANT INFORMATIONS OF THE ROLE OF KEY MOLECULES IN THE EFFECTOR FUNCTIONING OF THE IMMUNE SYSTEM, SO IN DEFINING PATHWAYS, FUNCTIONS, THE HUMAN SYSTEM, AND WITH IN VIVO ANALYSIS, OF COURSE THE STRENGTH OF HUMAN ANALYSIS. BASED ON THIS, I'D LIKE TO START TO DISCUSS TWO IMMUNODEFICIENCIES, RELATED TO T-CELL ACTIVATION, MORE SPECIFICALLY TO INDUCTION OF T CELL PROLIFERATION, THE FIRST OF MY THREE VIGNETTES TRYING TO ILLUSTRATE WHAT THE STUDY OF MONOGENIC DISEASES MAY TEACH US AND HOW THEY MAY HELP US TO UNDERSTAND MORE COMPLEX DISEASES. HERE YOU HAVE A PICTURE, A SIMPLIFIED PICTURE OF THE DIFFERENT MOLECULES AND PATHWAYS THAT ARE INVOLVED DOWNSTREAM OF THE T-CELL RECEPTOR, AND ARE LEADING TO MANY GENE ACTIVATION PATHWAYS, AS YOU KNOW, AND EVERY SINGLE MOLECULE THAT IS IN BROWN OVAL IS A MOLECULE THAT HAS BEEN FOUND TO BE DEFICIENT IN HUMANS WITH MUTATIONS OF GIVEN GENE CAUSING DEFECT. I WON'T REVIEW BUT WOULD LIKE TO FOCUS ON MOLECULE POINTED BY THE RED ARROW ON THE BOTTOM OF THE SLIDE, AND YOU WILL SEE IN A SECOND I WILL ALSO DISCUSS THE MOLECULE GFP 1, JUST UPSTREAM. WHAT IS CTPS1? IT'S A MOLECULE WHICH IS KEY HERE AS YOU CAN SEE IN RED, THAT IS TRANSFORMING UTP INTO CTP, A KEY STEP IN THE PATHWAY, IS THE WAY TO GENERATE CTP, USEFUL FOR MEMBRANE SYNTHESIS AS WELL AS OF COURSE RNA SYNTHESIS, AND TO LEAD TO THE SYNTHESIS OF DEOXY CTP, THIS IS REQUIRED FOR DNA SYNTHESIS, ALSO CELL PROLIFERATION. THERE'S A SECOND GENE ENCODING CTPS2, THEY ARE VERY DIFFERENT IN THE WAY THEY FUNCTION. A FEW YEARS AGO, WE FOUND PATIENTS, TODAY 19 OF THEM, WHO HAD SEVERE IMMUNODEFICIENCY WITH EBV, INTERESTING ENOUGH GIVEN THE CONTEXT OF DEFICIENCY THE ENZYME IS KEY. THE CHILDREN ARE GOING WELL, REPLACEMENT OF SKIN CELLS, EPITHELIAL CELLS IN THE GUT IS WORKING NORMALLY DESPITE THE ABSENCE OF KEY ENZYME. PHENOTYPE, BUT WHAT IS KEY IN THIS DISEASE DIVIDING IN VITRO WHEN STIMULATED THROUGH THE ANTIGEN RECEPTORS. ALL OF THE PATIENTS IDENTIFIED SO FAR ORIGINATED FROM THOSE IN ENGLAND, THEY ALL CARRIED THE SAME HOMOZYGOUS MUTATION IN THE GENE ENCODING, SO CTPS1, AND THEY HAVE HERE A MUTATION IN EXCESS OF INTERIM NUMBER 18, THAT PREVENTS THE GLOBAL PRESENCE OF THE LAST EXOME OF THE MOLECULE. THIS IS THE MUTATION HERE, WHAT IS THE CONSEQUENCE OF THE MUTATIONS? LOOKING AT THE CTPS1, CTPS2, LET'S LOOK AT CONTROL ACTIVATOR T CELLS, THE LEVEL OF EXPRESSION OF THIS ENZYME IN NON-ACTIVATED CELLS IS LOW, AND IT GOES UP QUITE SIGNIFICANTLY WITHIN THE FIRST TWO, THREE DAYS FOLLOWING T-CELL ACTIVATION, THROUGH THE T-CELL RECEPTOR, CTPS2 THERE IS SOME VARIATION BUT NOT THAT CHARACTERISTIC. YOU CAN SEE WITH PATIENT CELLS THERE'S A LITTLE BIT OF CTPS1 PRESENT, WITH REDUCED MOLECULE, CORRESPONDING TO LACK OF C-TERMINAL PART OF THE PROTEIN. BY THE WAY THIS MUTATED PROTEIN IS PRESENT IN THE MOTHER, PROBABLY ABNORMAL MOLECULES DESTABILIZED AS DIMER, TETRIMER WAS NORMAL FORM OF THE MOLECULE. TWO MESSAGES, ONE, THE ENZYME EXPRESSION IS HIGHLY ACTIVATED THROUGH ACTIVATION OF T CELLS EARLY ON. AND THE MUTATION RESULTS IN LOSS OF EXPRESSION THAT IS HYPERMORPHIC, RESIDUAL PROTEIN HAS NORMAL PRESERVED ACTIVITY, OVERALL IT'S A HYPERMORPHIC MUTATION. SEQUENCES ARE IMPAIRED PROLIFERATION, AS YOU CAN SEE ON THE LEFT-HAND SIDE, PROLIFERATION IN RED CIRCLES OF PATIENT CELLS. I'M SORRY. IT'S HERE, REDUCED COMPARD TO CONTROLS. DILUTION OF TRACER, YOU SEE THE DIFFERENCE. THE DIFFERENCE CAN BE COMPLEMENTED BY INTRODUCTION OF THE NORMAL COPY OF THE GENE, ALSO THE PROLIFERATIVE DEFECT CAN BE COMPENSATED BY CYTIDINE, ADDITION OF THE PRODUCT OF THIS ENZYME WHICH MAKES TOGETHER THE STORY VALID. IN TERMS OF ACTIVITY OF THE ENZYME, CAN YOU SEE HERE AGAIN ON THE LEFT-HAND SIDE PANEL THAT IT IS REDUCED IN RESTING T CELLS, NOT THAT MUCH REDUCED BUT STRIKINGLY NOW IF ONE LOOKS AT ACTIVATOR T CELLS OR EBV CELL LINES, YOU CAN SEE THE LEGEND IS NOT THE SAME SO THERE IS MUCH MORE ACTIVITY HERE, THE DISCREPANCY IN TERMS OF ACTIVITY BETWEEN CONTROLS AND PATIENT CELLS IS MUCH HIGHER. SO THE PROBLEM LOOKS TO BE QUITE IMPORTANT IN ACTIVATED CELLS AND IT IS THE SAME WITH EBV CELLS. IF ONE LOOKS AT THE PHENOTYPE, I'M NOT GOING TO, THERE ARE LITTLE VARIATION BETWEEN CONTROLS AND PATIENTS, WHATEVER YOU LOOK AT T CELLS AND K CELLS, B CELLS, MONOCYTES, DENDRITIC CELLS, CD4, CD8 AND B CELLS. ALSO THE DEFECT HAS NO DETECTSABLE IMPACT ON CELL DIFFERENTIATION AND HOMEOSTASIS. THERE ARE NO DIFFERENCES WITH ONE EXCEPTION, WE DO NOT UNDERSTAND TODAY, THAT IS PATIENT CELLS ARE LIST ABLE TO MAKE IL-2, THIS IS NOT THE CAUSE OF THE DEFECT IN T CELL PROLIFERATION SINCE ADDITION OF IL-2 DOES NOT RESTORE CELL PROLIFERATION. OKAY. SO HERE FOR ME THE KEY MESSAGE IS HERE, CTPS EXPRESSION, YOU SEE CTPS1 BECOMES DETECTABLE BETWEEN 24 TO 96 HOURS FOLLOWING T-CELL ACTIVATION, A LITTLE BIT OF VARIATION, NOT MUCH VARIATION FOR CTPS2. IN TERMS OF RNA EXPRESSION, THIS IS REGULATION OF TRANSCRIPTION, YOU CAN SEE THE LEVEL OF EXPRESSION OF THE MESSENGER RNA IN ACTIVATED T CELLS AFTER A FEW HOURS IS MUCH, MUCH, MUCH HIGHER, WITH THE EXCEPTION OF ACTIVATED B CELLS THAT ARE NOT SHOWN HERE ON THIS GRAPH. OVERALL WE MAY CONSIDER THE FOLLOWING PICTURE, BASED ON THESE ACTIVATION OF TRANSCRIPTION, YOU CAN SEE IT'S VERY QUICK, THE MAXIMUM DETECTION OF THE MESSENGER RNA IS ONE HOUR, 30 MINUTES, FOLLOWING T-CELL ACTIVATION. AND THIS IS THE ENZYMATIC ACTIVITY IN TERMS OF PROTEIN. THE CONCEPT IS THAT UPON T-CELL RECEPTOR OR B CELL RECEPTOR SIGNALING THERE IS MASSIVE AND RAPID INDUCTION OF CTPS1 ENABLING CELLS TO SYNTHESIZE MASSIVE CONCENTRATION OF CTP, REQUIRED FOR METABOLIC ADAPTATION, AND DNA SYNTHESIS, ENHANCED PROLIFERATION. WE THINK CTPS1 IS MAJOR CHECKPOINT IN REGULATION OF T CELL DIVISION FOLLOWING ACTIVATION. TODAY WE KNOW A LITTLE BIT MORE. WE KNOW THAT INHIBITORS, I WILL SAY MORE IN A SECOND, BLOCK CTPS1 EXPRESSION, (INDISCERNIBLE) MAY BIND TO ITS PROMOTER. INTERESTINGLY ENOUGH CTPS1 IS ACTIVE AS TETRIMER, PERHAPS FILAMENTS FORMED AND CAN BE DETECTED AND DELIVERED, OF WHICH CTP IS FORMED. THERE ARE SOME SETTINGS WHERE FILAMENTS MAY BE NOT ACTIVE, SOMETHING THAT IS NOT YET WELL UNDERSTOD. WE THOUGHT THAT CTPS1 MAY BE AN INTERESTING TARGET TO TRY TO BLOCK FOR IMMUNOSUPPRESSIVE -- IMMUNOSUPPRESSION OF, SAY, AUTOIMMUNE DISEASE OR GRAFT-VERSUS-HOST DISEASE BECAUSE OF ITS HIGHLY INDUCIBLE EXPRESSION IN T AND B CELLS. TODAY THERE'S BEEN A SMALL BIOTECH CREATED THAT IS SCREENING FOR INHIBITORS OF CTPS1, WE'LL SEE WHETHER THIS IS GOING TO DEVELOP INTO SOMETHING. ABOUT THIS PATHWAY, I'D LIKE TO FOCUS ON THIS MOLECULE, EXTANT FACTOR REGULATING RAS, AND ERK MAP KINASE PATH WAY AND WE FROM THE NEXT. THIS IS A FAMILY WITH TWO CHILDREN AFFECTED BY SEVERE EBV INFECTION ASSOCIATED WITH HODGKIN LYMPHOMA. YOU HAVE THE PICTURE HERE OF EBV LOAD IN THE BLOOD OF ONE. œYOU SEE A HIGH PEAK, EACH TIME THE CHILD WAS TREATED WITH ANTIBODY, EBV LOAD DROPPED BUT CAME BACK EACH TIME HE WAS TREATED BEFORE BEING TRANSPLANTED. OBVIOUSLY, THE CHILDREN, INCLUDING THE SISTER WHO DIED FROM LYMPHOMA, WE'RE NOT ABLE TO COPE WITH EBV INFECTION. EXOME SEQUENCING, HOMOZYGOUS MUTATION, INSERTION OF TWO NUCLEOTIDES EXACTLY HERE. C-TERMINAL PART OF THE PROTEIN IN TERMS OF PROTEIN, NOT GENES, OF COURSE. AND THAT'S THE CONSEQUENCES OF THE CAPACITY AFFECTING MESSENGER RNA, NOT SHOWN HERE, THE PROTEIN IS VIRTUALLY NOT DETECTED AS YOU CAN SEE HERE. SO IT'S LACK OF EXPRESSION OF MOLECULE. AS A CONSEQUENCE, BASED ON THE PATHWAY WHAT WE KNOW, MAP KINASE PATHWAY, IF ONE LOOKS AT ACTIVATION OF THIS PATHWAY THROUGH PHOSPHORYLATION OF ERK PATIENT CELLS COMPARED TO CONTROL THERE IS A MUCH REDUCED PHOSPHORYLATION OF ERK, DESPITE NORMAL PRESENCE OF PROTEIN, WHEREAS THE PROTEINS LIKE PLC GAMMA ONE NORMALLY PHOSPHORYLATED, INDEED THIS PATHWAY IS DEFECTIVE AND THE CONSEQUENCE FOR PATIENTS T CELLS, FORGOT TO MENTION, BASICALLY EXPRESSED IN T CELLS AND ALSO IN NK CELLS. SO THERE IS IMPAIRED PROLIFERATION OF T CELLS WHEN ACTIVATED THROUGH ANTI-CD3, THIS IS THE DILUTION OF TRACER, COMPLEMENTED BY ADDITION OF NORMAL COPY OF THE GENE. AS A CONSEQUENCE WE GUESS OF THIS DEFECTIVE PROLIFERATION, I'M NOT TALKING HERE ABOUT PHENOTYPE WHICH IS OF THE CELLS THAT IS VIRTUALLY ESSENTIALLY NORMAL, THE CONSEQUENCE IS THAT IF ONE LOOKS FOR THE PRESENCE OF EBV-SPECIFIC T CELLS BY USING PENTAMERS HARDLY DETECTED ANY, COMPARED TO CONTROL, 7% BINDING TO MIXTURE OF DIFFERENT PEPTIDES, BIND TO HLA11, THE CONTROL AS WELL AS THE PATIENT BEING HLA 11, VIRTUALLY NO EBV-SPECIFIC T CELLS. IN ADDITION TO DEFECT IN T-CELL ACTIVATION THERE'S ALSO DEFECT IN CAPACITY OF T CELLS TO PROLIFERATE THROUGH THE INTERACTION BETWEEN CD27 EXPRESSED BY ACTIVATED T CELLS, BINDING TO CD70 EXPRESSED BY EBV AFFECTED ACTIVATED B CELTS. ACTIVATED -- B CELLS. PATIENT CELLS THAT PROLIFERATE, HIGHLY DEFECTIVE, SO NOT ONLY IS RAS GFP REQUIRED FOR T-CELL RECEPTOR ACTIVATION OF PROLIFERATION BUT ALSO IT'S A PATHWAY THAT IS MAYBE CRITICAL FOR CONTROL OF INFECTION THROUGH THE -- ITS IMPORTANCE IN INTRODUCING PROLIFERATION WITH THE CO-ACTIVATOR. WE THINK THIS IS A PATHWAY CRITICAL FOR CONTROL OF INFECTION. THE PHENOTYPE IS PHENOCOPY OF THE DEFICIENCIES OF EAT CD27 OR CD70 REPORTED, WE REPORTD CD70 DEFICIENCY PHENOTYPE. SO FINALLY, THERE IS A LINK WITH CTPS1, BECAUSE IF ONE INDUCES DOWNREGULATION OF EXPRESSION IN CONTROL T CELLS WITH FLUORESCENT MARK YOU ARE YOU SEE DECREASE IN PROLIFERATION OF CELLS. MORE IMPORTANTLY, EXCUSE ME, MORE IMPORTANTLY THE CONTEXT OF DEFICIENCY, THE ACTIVATION OF EXPRESSION OF CTPS1 IS MUCH REDUCED, AND THIS CAN BE REPRODUCED BY INACTIVATING EXPRESSION IN T CELLS. SO THERE IS A LINK BETWEEN FUNCTIONAL LINK, SO WHICH FURTHER INDICATES THAT CTPS1 TRANSCRIPTION REGULATION IS UNDER NOT ONLY, BUT UNDER THE RAS MAP KINASE PATHWAY, PERHAPS ALL TOGETHER, MAYBE ALSO IMPORTANT FOR CD27 CO-STIMULATION OF T CELLS REQUIRED FOR CONTROL OF EBV. I'M SORRY TO GO TOO QUICKLY. HERE I THINK WE HAVE A PATHWAY THAT MAY BE AMENABLE FOR MODULATION BY IMMUNOSUPPRESSION WHEN REQUIRED, GIVEN KEY IMPORTANCE IN T-CELL ACTIVATION, PROLIFERATION AND CONTROL OF VIRAL INFECTION SO PERHAPS ALSO ACTIVATED TO CELLS. NOW I'D LIKE TO SWITCH TO MY SECOND VIGNETTE, AUTOIMMUNITY. AMONG MANY GENETIC DISORDERS CAUSING SUSCEPTIBILITY TO INFECTION, 150 CAUSING AUTOIMMUNE OR INFLAMMATION. IF YOU LOOK AT HOW FREQUENT ARE AUTOIMMUNE MANIFESTATIONS IN PATIENTS, GENETIC DISORDER AS HIGH AS 25%, IMPORTANT INFORMATION ABOUT PATHOPHYSIOLOGY OF AUTOIMMUNITY AND MAYBE A BIT FURTHER AS I WILL TRY TO TELL YOU NOW. SO THERE ARE MULTIPLE WAYS BY WHICH IMMUNE SYSTEM CONTROL ACTIVITY, I TOOK THIS SLIDE PUBLISHED MORE THAN TEN YEARS AGO, SO HE DIFFERENTED PHENOTYPICALLY SPEAKING AT LEAST 24 DIFFERET CHECKPOINTS OF THE CONTROL OF B OR T CELL AUTOIMMUNITY, BUT UP TO NOW NOT ALL OF THEM, FAR FROM THAT, HAVE BEEN MOLECULARLY DEFINED. SO PERHAPS SOME OF THE MONOGENIC DEFECTS CHARACTERIZED AND ASSOCIATED WITH AUTOIMMUNITY MAY CONTRIBUTE TO PATHOPHYSIOLOGY OF AUTOIMMUNITY. IF WE LOOK AT PHENOTYPE, AUTOIMMUNE PHENOTYPE OF SOME OF THESE DISEASES, THE MOST WELL KNOWN LIKE CTL 4 DEFICIENCY, GAIN OF FUNCTION, IPEX SYNDROME, FOXP3, SO ON, YOU CAN SEE FOR EVERY SINGLE GENETIC DISEASE, THE PHENOTYPE DIFFERS. FOR INSTANCE, HERE THE DEFICIENCY, AUTOIMMUNITY THAT IS NOT FOUND IN THE OTHER DISEASES, AND CONVERSELY. SO CLEARLY, THERE ARE DIFFERENT GENOTYPES LEADING TO DIFFERENT PHENOTYPES, RELATED TO DIFFERENT DISEASES WHERE WE DON'T KNOW HOW IT GOES EXACTLY. WHAT WE KNOW UP TO NOW THAT THERE ARE AT LEAST SIX DISEASES CHARACTERIZED TO BE CAUSING AUTOIMMUNITY IN CONTEXT OF MONOGENIC DISEASES, NOT GOING TO REVIEW IN DETAIL. DEFECTIVE REGULATORY T CELLS, I'M TALKING ABOUT FOXP3 REGULATORY T CELLS, ELIMINATION OF LYMPHOCYTES, B CELLS AND T CELLS REACTIVE TO CELLS IN THE PERIPHERY, THAT IS THE ROLE OF THE LIGAND INTERACTION. INHIBITION OF LYMPHOCYTE ACTIVATION AND OF COURSE CTLA-4 IS DOING SUCH A JOB. MANY REGULATING, CEASE ACTIVATION OF CELLS. B CELLS RELATED TO AID MOLECULE AND DIFFERENT FUNCTIONS IS WHAT WE KNOW. WE MAY GO A BIT FURTHER. BY TRYING A DIFFERENT APPROACH, JUST LET ME EXPLAIN TO YOU. IF WE LOOK AT RELATIVE RISK TO DEVELOP AUTOIMMUNE DISEASE, FOR PATIENTS COMPARED TO CONTROL POPULATION OF THE SAME AGE, YOU CAN SEE THAT RELATIVE RISK IS MUCH HIGHER FOR MANY OF THESE AUTOIMMUNE DISEASES. THIS IS ESPECIALLY IMPRESSIVE FOR AUTOIMMUNE CYTOPENIA, FOR CHILDREN THE RISK OF DEVELOPING SUCH AN ANEMIA IS 830 TIMES HIGHER IN PATIENTS WHO HAVE KNOWN GENETIC DEFECT COMPARED TO GENERAL POPULATION. IT STANDS ALSO FOR IBD AND OTHER DISEASES. WE SAW THE RISK IS SO HIGH, SO PERHAPS IF WE CONSIDER NOW THE GENERAL POPULATION OF CHILDREN TO THINK OF, COULD IT BE POSSIBLE THIS CHILD HAS GENETIC DISORDER, AUTOIMMUNITY, THIS WAS NOT THE WAY TO THINK ABOUT IT UP TO NOW. SO TO TRY TO ADDRESS THESE QUESTIONS, ANALYZING TWO COHORTS OF PATIENTS, THE FIRST PATIENTS WITH EVANS SYNDROME, 18 PATIENTS ANALYZED, YOU HAVE THE NAMES OF THE PEOPLE INVOLVED. AND THEN COHORT OF PATIENTS WITH LUPUS, THIS IS EARLY ONSET LUPUS, BELOW THE AGE OF 10. SO 95 PATIENTS. THEY WERE ANALYZED BY EXOME SEQUENCING. WHAT WAS FOUND AS FOLLOWS, EVANS SYNDROME, REMIND YOU IT'S A COMBINATION OF AUTOIMMUNE AND HEMOLYTIC ANEMIA, THROMBO CYTOPENIA, 80 PATIENTS ANALYZED SO FAR, MUTATIONS FOUND, VARIANTS IN 2/3 OF THEM. FOR SOME MUTATIONS WERE FOUND IN GENES ALREADY DESCRIBED TO BE MUTATED IN THE CONTEXT OF AUTOIMMUNITY AND PRIMARY IMMUNODEFICIENCIES, KIND OF CONFIRMATION. COMES ANOTHER 20 PATIENTS WHO HAVE MUTATIONS OR VARIANTS LIKELY THAT HAVE TO BE CAUTIOUS, YOU SEE THE CRITERIA USED TO CONSIDER HEPATOGENICITY ON THE BOTTOM OF THE SLIDES. WE FOUND VARIANTS IN 16 GENES. YOU WILL SEE IN SOME OCCASIONS, THAT MAY ACCOUNT FOR PREDISPOSITION TO AUTOIMMUNITY. MORE INTO DETAILS, SOME MUTATIONS AFFECT GENES TO BE INVOLVED IN THE FUNCTION OF REGULATORY T CELLS, THERE'S LIKELY A LOSS OF FUNCTION, HETEROZYGOUS LOSS OF FUNCTION IN ONE PATIENT. THEN THERE ARE MANY GAIN OF FUNCTION MUTATIONS, AND IN READY HAVE OUTLINED THE GENES FOR WHICH SEVERAL PATIENTS WERE FOUND TO BE MUTATED WHICH INCREASES THE CHANCE THAT INDEED THAT THESE MUTATIONS ARE PATHOGENIC. THIS INCLUDES AS YOU CAN READ JAK-1, WHICH MAKES SENSE, TNF RECEPTOR TYPE 2, VARIANTS FOUND IN GENES AFFECTING APOPTOSIS, SO THIS MAY FURTHER -- MAY GIVE US MORE INSIGHT INTO SOME ASPECTS OF MOLECULES IMPORTANT FOR THE CONTROL OF AUTOIMMUNITY BUT MEDICALLY SPEAKING MAY BE INTERESTING TOO BECAUSE WE THINK DIVERSITY OF POTENTIAL DISEASES TARGETED THERAPIES NOW CAN BE FORESEEN FOR 36%, SIGNIFICANT AND HELPFUL. THOSE PATIENTS WITH IMMUNE CELL LIKELY PATHOGENIC VARIANTS HAVE BEEN IDENTIFIED, TEND TO BE MORE SEVERE, IF YOU COMPARE THE TWO, 52 PATIENTS WITH VARIANTS DETECTED, THIS IS THOSE WITHOUT. SO THEY HAVE MORE FREQUENT AUTOMANIFESTATION, A TENDENCY TO HAVE MORE SEVERE DISEASE, A FEW WHO DIED AFTER SOME TIME, 20 TO 30 YEARS, LOOKS TO BE MORE SEVERE. IN LUPUS, UNFOLDED PROTEIN RESPONSE, SO MAYBE THIS MAY LEAD US TO SOME FURTHER INSIGHT INTO UNDERSTANDING ONE OF THE MECHANISMS THAT MAY CAUSE LUPUS, JUST TO ILLUSTRATE THIS POINT QUICKLY THIS IS THE FAMILY WHERE ACTUALLY THERE ARE FOUR PEOPLE, TWO DAUGHTERS HERE, AS WELL AS THE MOTHER, AND AN AUNT, WHO HAVE HETEROZYGOUS MUTATION, REQUIRING ENZYME 1 ALPHA THAT IS IMPORTANT FOR UPR RESPONSE. YOU HAVE THE CLINICAL MANIFESTATION, ILF ALPHA WITH PERK AND THERE MAY BE A RELATIONSHIP SOMEWHERE. HOW, I DON'T KNOW, BETWEEN THE VULNERABILITY TO DEVELOP LUPUS DISEASE, WITH THE UNFOLDED PROTEIN RESPONSE. HOW STILL WE DON'T KNOW. AS A CONCLUSION ABOUT GENETICS OF PROTEIN IN CHILDREN, I THINK DATA ARE STILL PRELIMINARY SUGGEST WE SHOULD STUDY OTHER COHORTS WITH THE SAME DISEASES AS OTHER AUTOIMMUNE DISEASES THAT LIKELY THESE FINDINGS MAY HAVE PROGNOSTIC FACTORS AND MAY HAVE IMPLICATIONS, MAY OFFER THE OPTION OF TARGETED THERAPY, FOR INSTANCE JAK-1, JAK-2 INHIBITORS FOR PATIENTS WHO HAVE GAIN OF FUNCTION MUTATION IN JAK-1 OR JAK-2 AS EXAMPLE. WE SHOULD SYSTEMATICALLY TEST ALL CHILDREN WITH AUTOIMMUNE DISEASE TO FIND OUT WHETHER THEY HAVE IMPORTANT GENETIC RISK FACTORS. HOPEFULLY THIS MAY IN THE FUTURE HELP US TO BETTER UNDERSTAND AUTOIMMUNE DISEASES LIKE PERHAPS THIS IS SPECULATIVE, THE POINT OF THE UNFOLDED PROTEIN RESPONSE. TO FINISH WITH, I'D LIKE TO PRESENT ANOTHER ASPECT OF OUR STUDY, NOW RELATES TO LYMPHOMAS. WITH THE IDEA THAT DEFINING GENOTYPE IN THAT CASE THAT YOU WILL SEE GERMLINE GENOTYPE, PHENOTYPE MAY HELP US TO UNDERSTAND PATHOPHYSIOLOGY, PERHAPS SOME INSIGHT FOR MORE COMMON DISEASES. THE DISEASE HERE THAT I'M GOING TO DISCUSS IS THE SO-CALLED SUBCUTANEOUS T CELL LYMPHOMA, RARE FORM OF T CELL LYMPHOMA, THAT VERY FREQUENTLY ASSOCIATED WITH -- RELATED TO T-CELL ACTIVATION. WE BECAME INTERESTED TOGETHER WITH OUR COLLEAGUE IN A COHORT OF PATIENTS WITH THE DISEASE, 27 OF THEM, BECAUSE FOR SOME THEY WERE CASES IN FAMILIES, IN THIS FAMILY THAT'S ONE, AND ALSO THIS ONE. SO THE QUESTION COULD IT BE THAT THIS IS GENETICALLY DETERMINED. INTERESTINGLY ENOUGH THE MAJORITY, NOT ALL, BUT THE MAJORITY OF PATIENTS ARE ORIGINATING FROM EAST ASIA, MORE FROM POLYNESIA. YOU MAY READ THIS FAMILY IS FROM POLYNESIA, THIS IS NEW ZEALAND, AND SO ON, MAYBE THERE'S A CONCENTRATION OF GEOGRAPHICAL FACTOR OF GENETIC ORIGIN. YOU CAN SEE THE LESION ON THE SKIN. PET SCAN, DETECT LESIONS HERE ALONG THIS. THE LESIONS ARE AS FOLLOWS. SO THIS IS T CELL, MONOCLONAL T CELL POPULATION, THAT IS EXPRESSING, THAT IS EXPRESSING CG 8 ACTIVATED, YOU CAN SEE THESE CELLS ARE LOCATED SURROUNDING ADIPOSEYTES, PHAGOCYTOSEIS, THE SAME IS PRESENT IN THE BONE MARROW, IN THE FAT OF THE BONE MARROW ONE CAN DETECT T CELLS AROUND ADIPOCYTES, EXPRESSING THE PERIPHERY. EXOME SEQUENCING, ALL OF THESE PATIENTS HAVE BLA MUTATION IN GENE WHICH IS ENCODING THE MOLECULE, FOUND IN 16 OUT OF THE 25 PATIENTS, AND ALL MULTIPLEX FAMILIES. TWO MUTATIONS FOUND, 82C IN THE DOMAIN SHOWN HERE OF THE MOLECULE, I WILL COME BACK TO THAT, OR THE OTHER MUTATION, AND MUTATION WAS DETECTED IN PATIENTS ORIGINATING FROM EAST ASIA AND POLYNESIA, THE OTHER IN PATIENTS FROM CAUCASIAN ORIGIN. THIS IS RECESSIVE DISEASE, GERMLINE MUTATIONS. A LOSS OF FUNCTION MUTATION, ACTUALLY THESE MUTATIONS ARE PERTURBING ORGANIZATION OF THE IG VARIABLE-LIKE DOMAIN OF THE MOLECULE I WILL SHOW YOU IN A SECOND. WHAT IS TEAM 3? IT STANDS FOR T CELL OF CELL IMMUNOGLOBULIN 3, A FAMILY OF MOLECULES, EXPRESSED AT MEMBRANE OF T CELLS, NK CELLS, DENDRITIC CELLS, MACROPHAGES, HAS GALATINE, NEGATIVE REGULATOR OF C CELL ACTIVATION, SHOWN HERE PD-1, PD-L1 INTERACTION. BINDING MAY DOWNREGULATE ACTIVATION OF T CELLS AS WELL AS CELLS OF THE IMMUNE SYSTEM, HIGHLY EXPRESSED BY SUPPOSEDLY EXHAUSTED T CELLS, USED IN CONJUNCTION WITH ANTI-PD-1 ANTIBODIES TO TREAT DIFFERENT FORMS OF CANCER. BACK TO THE MUTATIONS, WHAT ABOUT EXPRESSION? IF ONE LOOKS IN THE TUMORS, ONE DOESN'T DETECT SURFACE TEAM 3, DETECTED SHOWN HERE IN THE INSET, THE SURFACE OF T CELLS, IN STPCL WITHOUT MUTATIONS, THE BEST CONTROL WE CAN THINK OF. THAT'S NOT AT THE MEMBRANE, THIS IS SHOWN ALSO HERE ON BLOOD CELLS FROM SUCH PATIENTS. SO, MORE SPECIFICALLY, THERE IS EXPRESSION OF THE MUTATED FORMS OF THE MOLECULE IN THE CELL LINE. THIS IS NOT GOING THROUGH THE MEMBRANE AS WILDTYPE FORM DOES, IT IS AGGREGATED AROUND THE GOLGI, IF ONE LOOKS AT WHETHER THE PROTEIN IS WELL FOLDED OR NOT, BY SIZE EXCLUSION, ELUTION OF CHROMATOGRAPHY, COMPARING WILDTYPE FORM IN GREEN, RED ONES WITH OTHER MUTANT ONES, FOLDED MONOMER ONLY FORMED WITH WILDTYPE FORM. YOU CAN DETECT THEM HERE AND HERE, COMPARED TO THE CONTROL. THE PROTEIN IS MISFOLDED AND CAN'T GO TO THE MEMBRANE. SO TIM3 IS BINDING DIFFERENT LIGANDS, ANTI-INFLAMMATORY FUNCTION. IF YOU LOOK AT MONOCYTES FROM THE BLOOD, THIS IS ON THE BOTTOM HERE, THEY HAVE INCREASED (INDISCERNIBLE) UPON LPS STIMULATION, LOOKING AT PRODUCTION OF IL-1, IT'S ALSO INCREASED. THERE IS INDEED EXCESSIVE INFLAMMATION LIKELY ACCOUNTING FOR HLH PHENOTYPE. WE'LL SKIP THAT TO GO TO THE CONCLUSION. TIM-3 GERMLINE LOSS OF FUNCTION MUTATION, FOUND IN 60% OF CASES OF PATIENTS, IT MAY BE REGULATORY PROCESS WITH INCAPACITY OF CONTROLLING INFLAMMATION BUT STILL THERE IS MONOCLONAL T CELL POPULATION IN THE SCHEME, IT'S NOT CLEAR WHAT IT IS ABOUT, SO PERHAPS THESE PATIENTS SHOULD BE TREATED BY (INDISCERNIBLE) RATHER THAN CHEMOTHERAPY, INDEED THE CASE USING JAK-1, 2, INHIBITORS, SPECTACULAR AND GOES INTO THE SAME CONCEPT. PHYSIOLOGIC BASIS WHAT ARE REGULATORY FUNCTIONS OF THE TIM-3 MOLECULE, SPECIFICALLY IN ADIPOSE TISSUES GIVEN THE PHENOTYPE OBSERVED IN THESE PATIENTS? THIS IS A QUESTION FOR WHICH I HAVE UNFORTUNATELY HAVE NO ANSWER TODAY BUT I THINK THIS IS QUITE SPECTACULAR FINDING, THAT NEEDS TO BE FURTHER ADDRESSED. SO AS TIM-3 IS TARGET FOR CANCER IMMUNOTHERAPY, PERHAPS SHOULD BE A WORD OF CAUTION THERE MIGHT BE A RISK ASSOCIATED WITH THIS THERAPY TO REPRODUCE THE PHENOTYPE OF THE PATIENTS WHO DON'T EXPRESS TIM-3 NORMALLY. SO TO FINALLY CONCLUDE, I HOPE THROUGH THREE EXAMPLES I MAY HAVE CONVINCED YOU PEDIATRIC IMMUNOLOGY PATHOLOGIC DISEASE MORE LIKELY TO BE GENETICALLY DETERMINED THAN IN ADULTS, COULD BE MONOGENIC, OLIGO GENETIC DISEASES, STUDIES CONTRIBUTE TO UNDERSTANDING AND PERHAPS MEDICAL DECISION AS I SHOWED YOU, AND THERE MAY BE SOME INFORMATIONS THAT COULD BE DIRECT OR INDIRECT LIKE POTENTIALLY IMMUNOSUPPRESSION, THAT CAN BE DERIVED FROM THESE STUDIES THAT COULD BE APPLIED FOR MORE COMPLEX DISEASES. TO FINISH I'D LIKE TO MENTION A FEW PEOPLE WHO HAVE BEEN IMPORTANT AND MORE THAN IMPORTANT CRITICALLY IN THE STUDIES, SO SYLVAN AND SOME OTHER PEOPLE HERE HAVE BEEN INVOLVED IN THE DESCRIPTION OF THE CTPS1 DEFICIENCIES, AND THAT WAS A VERY FRUITFUL COLLABORATION FROM THIS GROUP, AND OTHERS HAVE BEEN INVOLVED IN THE STUDY OF THE AUTOIMMUNITY IN CHILDREN AND WITH THE HELP OF OTHER PEOPLE. THANK YOU VERY MUCH FOR YOUR ATTENTION. [APPLAUSE] >> THANK YOU, ALAIN, FOR THE WONDERFUL TALK. I WOULD INVITE WHOEVER WANTS TO ASK QUESTIONS TO COME TO THE MICROPHONE BECAUSE IT IS BEING VIDEOCAST. JOHN. >> TWO QUESTIONS, IF I MIGHT. WHEN YOU'RE TALKING ABOUT CTPS1 YOU USE THE WORD CHECKPOINT, I WAS WONDERING WITH CHECKPOINT BLOCK AID INHIBITORS DO YOU SEE CHANGES, REDUCED IN EXHAUSTED CELLS, INDUCED WHEN YOU RELIEVE THE EXHAUSTION? >> I'M NOT SURE I CAN TELL THE CELLS BECOME EXHAUSTED IF THEY ARE -- IF THEY DON'T EXPRESS CTPS1, IF IT WAS BLOCKED BY INHIBITOR BUT I CAN TELL YOU THE CELLS DON'T PROLIFERATE MUCH. THEY SURVIVE, MOST OF THEM SURVIVE. THERE IS VERY LITTLE DEATH. AND IF YOU ADD BACK, YOU RECOVER CAPACITY OF PROLIFERATING. IN TERMS TO BE MORE SPECIFIC ABOUT EXHAUSTED PHENOTYPES, THIS IS SOMETHING WE HAVE TO LOOK IN MORE DETAIL. >> I WAS ALSO WONDERING YOU PRESENTED PATIENTS WITH LUPUS AND PATIENTS WITH AUTOIMMUNE HEMOLYTIC ANEMIA. LUPUS PATIENTS HAVE THAT, WAS SURPRISED YOU DIDN'T SEE OVERLAP OF MUTATIONS AND I JUST -- WERE YOU SURPRISED THAT YOU DIDN'T SEE SOME OF THE SIMILAR MUTATIONS? >> I DON'T KNOW, I HAVE TO SAY. YOU HAVE TO KEEP IN MIND, THEY ARE NOT THAT BIG. 150 PATIENTS WITH VARIANT FOR AUTOIMMUNITY, AND 40, SO -- AND YOU SEE THE DIVERSITY OF VARIANTS WE HAVE FOUND. IT'S NOT A COMMON VARIANT, A BIG SURPRISE, BECAUSE IF THAT HAD BEEN THE CASE, SO I'M A BIT SURPRISED BUT NOT SO, IT COULD BE AT THE END, EVEN IF LUPUS MAY BE MANIFESTED BY AUTOIMMUNE HEMOLYTIC ANEMIA, THE DISEASE MAY BE AT LEAST IN PART DIFFERENT. >> GOOD AFTERNOON. BEAUTIFUL SEMINAR, DR. FISCHER. I'M WONDERING, YOU TALK ABOUT THEM IN TWO DIFFERENT BUCKETS, THE SEVERE INFECTIONS, LYMPHOMA, ALLERGY. I'M WONDERING IF YOU THINK THERE IS SOME SORT OF HOST MICROBE INTERACTION AND WHETHER YOU CAN -- IN THESE RARE PATIENTS, DEEP PHENOTYPING, WHETHER YOU MIGHT SEE SOME CORRELATIONS BETWEEN INFECTIONS AND PHENOTYPES EXPRESSED, PERHAPS EVEN DECADES LATER? >> I THINK THIS IS AN EXCELLENT QUESTION. I'M NOT SURE I WILL BE ABLE, UNFORTUNATELY, TO ANSWER IT TODAY BUT OBVIOUSLY THIS IS SOMETHING WE COLLECTIVELY HAVE TO DO FOR THE FUTURE TO CONSIDER THE MICROBIOTA OF THESE PATIENTS, THE GUT, THE MOUTH, SO ON, TO AT LEAST START WITH, TO CORRELATE THE FINDINGS WITH THEIR PHENOTYPE COULD WELL BE IT IS MODULATING FACING IN DIFFERENT WAYS. SO I'M VERY MUCH WITH YOU IN THE WAY OF THINKING BUT UNFORTUNATELY HAVE NOTHING PRESENT TODAY. >> THANK YOU, BEAUTIFUL TALK. GIVEN THE PAUCITY OF TREATMENTS FOR LUPUS, FROM YOUR MUTATIONS, DO YOU HAVE ANY INSIGHT INTO NOVEL APPROACHES THAT MIGHT -- DO YOU THINK IT'S NAMELY FOR THE PEDIATRIC POPULATION? >> WE HAVE TO LOOK, OF COURSE, ONCE WE LOOK BETTER, THE VARIANTS, VALIDATE AS MANY AS WE CAN, NOT AS EASY AS IMAGING, NEXT STEPS TO GO WITH SEGREGATION IN FAMILIES, AND THEN TO LOOK WHETHER FROM TIME TO TIME SIMILAR VARIANTS MAY BE FOUND IN OTHERS, BEFORE THINKING ABOUT THEIR PARTICULAR -- PARTICULAR INTERVENTION. FOR THE LUPUS DOESN'T LOOK TO ME AT THE MOMENT THAT OBVIOUS -- AUTOIMMUNITY SYNDROME, OBVIOUS SITUATIONS WHERE NOW WE CAN START TO PROPOSE ALTERNATIVE THERAPIES. FOR LUPUS I THINK WE HAVE MUCH LESS IN THE DATA, I'M NOT THAT CLEAR TO GO ALONG THIS LINE, ALTHOUGH I HOPE AT THE END THIS IS WHERE WE GO TOWARD SUCH A DIRECTION. >> HI. THANK YOU FOR A GREAT TALK. YOU MAKE A GOOD CASE FOR DOING SEQUENCING FOR PEDIATRIC POPULATIONS BUT I WAS WONDERING WHETHER YOU EXPLORED THE POSSIBILITY OF SOMATIC MUTATIONS IN ADULT POPULATIONS WITH THE SAME DISEASE. >> YEAH, THIS IS ANOTHER VERY GOOD QUESTION. I'M VERY SENSITIVE TO THIS HYPOTHESIS THAT WE SHARE FOR THE GROUP, WHO HAS WRITTEN BEAUTIFUL ABOUT IT, BUT THE POSSIBILITY SOMATIC MUTATIONS ARE KEY DRIVERS IN AUTOIMMUNITY, STILL THESE CAN BE DEMONSTRATED. THEY ARE A FEW EXAMPLES LIKE TOGETHER WE SHOWED IN THE PAST SOMATIC MUTATIONS IN THE GENE ENCODING MAY LEAD TO (INDISCERNIBLE), A BEAUTIFUL EXAMPLE, FIVE OR SIX UP TO NOW, I DO MENTION IN EVANS SYNDROME TWO PATIENTS WITH SOMATIC CLEAR HOUSE MUTATIONS, ANOTHER EXAMPLE. BUT THE DIFFICULTY THERE IS IN THE CHANCE TO FIND RELEVANT SOMATIC MUTATION IN THE GENERAL LYMPHOCYTE POPULATION I THINK IT'S VERY LOW. WHEN ASKED TO LOOK IN THE AUTOIMMUNE CAUSATIVE (INDISCERNIBLE) OR B CELLS, IT'S FEASIBLE BUT NOT THAT EASY, BUT I THINK THESE THINGS ARE GOING TO BE DONE IN MANY PLACES. >> THANK YOU. >> ALAIN, THANK YOU VERY MUCH. FANTASTIC TALK. SO I HAVE A QUESTION REGARDING YOUR CDS MUTATIONS AND WORK WITH FREDERICK, IF YOU HAVE DATA TO SHARE AND DO YOU THINK ACTING BY INTERACTING THROUGH OTHER MOLECULES. >> TALKING ABOUT THE (INDISCERNIBLE) IN THE CONTEXT OF EVAN SYNDROME, WE KNOW THIS PATIENT HAS T REGs, THAT'S ALL WE KNOW. >> OKAY. >> FOR NOW. >> GERMLINE MUTATION, YOU FIND FROM WHAT OOCYTE, SPERM, FERTILIZED ZYGOTE, YOU FIND GERMLINE MUTATION. >> INCLUDING MUTATION IN THE CONTEXT OF T CELL LYMPHOMA. >> YEAH, YOU FIND FROM WHAT MATERIAL, FROM THE OOCYTES, SPERM, OR FERTILIZED EMBRYO? BLOOD? >> YEAH, IN THE BLOOD. >> MUTATIONS WERE FOUND IN THE TUMOR AS WELL AS IN THE BLOOD. IN ALL CELLS. A FEW OCCASIONS IN OTHER CELLS TOO, IN THE FIBROBLAST CELLS. >> ALAIN, IF WE MAY ASK YOU ONE QUESTION MYSELF, ONE INTERESTING ASPECT WAS WHEN YOU SHOW EVANS SYNDROME PATIENT POPULATION, LOTS OF HETEROZYGOUS MUTATIONS, THE SAME FOR CTLA-4, AND WE SEE INCOMPLETE PENETRANCE. SO ARE YOU SAYING THE SAME PATTERN OR ARE THEY MOSTLY DE NOVO MUTATIONS? WHAT IS THE IMPACT OF INCOMPLETE PENETRANCE? HOW DO YOU EXPLAIN? >> THANKS FOR THIS SIMPLE QUESTION. [LAUGHTER] WE DON'T HAVE YET THE DATA SEGREGATION OF THESE MUTATIONS IN ALL OF THE FAMILIES, FOR THE CASES WHERE WE HAVE IT, IT'S MOSTLY DE NOVO BUT THERE ARE A FEW CASES WHERE PARENTS, BUT THERE ARE SOME OF THE PARENTS WHO HAVE HAVE DISEASE, WE STILL HAVE TO CONSIDER, CTLA-4 IS A BEAUTIFUL EXAMPLE, SO IS IT ENVIRONMENTALLY DETERMINED, IS IT MODIFIED GENE, WE HAVE TO CHECK AND SEE. IF YOU HAVE SOME IDEA, PLEASE. >> OKAY. >> ANY FINAL QUESTIONS? IF NOT, IF ANYBODY WANTS TO ASK PROFESSOR FISCHER ANY QUESTIONS, THERE WILL BE A RECEPTION IN THE LIBRARY. FEEL FREE TO JOIN US AND YOU CAN ASK QUESTIONS. THANK YOU AGAIN. [APPLAUSE]