Evolution of Adaptive Immunity in Vertebrates


WELCOME TO THE FIRST OF THIS SEASON’S WALS LECTURES AND TODAY’S A SPECIAL OCCASION, IT IS THE WILLIAM E. PAUL LECTURE AND I WILL START WITH THIS IS THE PROFESSIONAL PICTURE OF BILL BUT I PREFER THAT PICTURE OF BILL BECAUSE I THINK ALL OF US THAT KNEW HIM WELL REMEMBER HIM MUCH MORE IN EBUULENT MODE THAN THE SOMBER PICTURE THAT PRECEDED THIS IS AND I BEING THE TITLE WAS CORRECT, HE WAS IMMUNOLOGYST AND HE EVEN RECOUNTED IN HIS BRIEF AUTOBIOGRAPHY ENDLESS FASCINATION HOW HE CAME TO CHOOSE PHYSIOLOGY AND SMALL ESAYS AND DECIDED ON A BROOKLYN SUBWAY HE WOULD BECOMA AN IMMUNOLOGYST AND HE THEN UNFORTUNATELY DEVELOPMENTAL PATH VERY BRIEFLY WHEN HE WAS AT NIH FROM 62-64 TO DO ENDOCRINOLOGY BUT THEN HE SAW THE LIGHT, JOINED THE NASARUS LAB IN NEW YORK AND MOVED TO THE NIH, AND FOLLOWING BARUK’S MOVE IN 1970 BILL GAME THE CHIEF OF IMONNOLOGY, A POST HE HELD UNTIL HE PASSED AWAY IN TWEBT 15 VENLT SO CAN YOU BACK CALCULATE HOW YOUNG HE WAS WHEN HE WAS APPOINTED AND HOW LONG HE HELD THAT POST AND HOW WONDERFUL THE LABORATORY WAS DIRKING THAT TIME PERIOD. NOW FOR THOSE WHO ARE NOT FULLY AWARE, HE MADE MANY DISC CONTRIBUTIONS, THE 1 THAT HE IS PERHAPS BEST RECOGNIZED FOR IS THE DISCOVERY OF BSF 1 WHICH IS WHAT YOU NOW KNOW AS INTERLUKEIN 4. HE REALLY DID AN ENORMOUS AMOUNT OF WORK ON UNRAVELING HOW IT CONTRIBUTED TO IGE RESPONSES, SIGNALING OCCURRED AND HOW IT CONTRIBUTED TO TH2 DEVELOPMENT, HE WAS IN LARGE MEASURE RESPONSIBLE FOR A CLASSIC PARADIGM IN IMMUNOLOGY NOW ABOUT THE POSITIVE CYTOKINE FEEDBACK REGULATION, POLARIZATION OF CD4 T-CELLS. HE WAS INVOLVED IN DESCRIBING ISOTYPE SWITCHING, THAT WAS MEDIATED BY PARTICULAR CYTOKINES. HE DISCOVERED THAT MASS CELLS AND BASAL-FILLS HA MAKE THESE TH2 CYTOKINES AND HE SPENT A LOT OF TIME DEALING WITH IF YOU WANT TO CALL THEM THEORETICAL TREATMENTS OF HOW SELF-TOLERANCE IS MAINTAINED IN WORK HE DID WITH [INDISCERNIBLE] GROSSMAN BUT THIS IS A VERY ABBREVIATED LIST THAT BILL CONTRIBUTED SCIENTIFICALLY AND I URGE THOSE OF YOU WHO ARE NOW FAMILIAR WITH THE REST OF THE LIST TO GO AND LOOK IT UP. NOW HE MADE MANY OTHER CONTRIBUTIONS OUTSIDE OF THE LABORATORY WITH DR. FAUCI AND HAROLD VARMIS, HE PLAYED AN IMPORTANT ROLE IN THE CREATION OF THE VACCINE RESEARCH CENTER, SOMETHING THAT OCCURRED WHEN HE WAS THE FIRST DIRECTOR OF OFFICE OF AIDS RESEARCH. HE WAS ON AN ENORMOUS MEMBER ADVISORY PANELS. I STILL HAVE A COPY OF HIS CV AND YOU HAVE TO WADE THROUGH MANY, MANY PAGES TO GET THROUGH ALL THE LISTS OF WAYS THAT HE HELPED OTHER PEOPLE IN SCIENCE, SELECTION OF HONORIFIC AWARDS, ON ACADEMIC BOARDS AND EVEN ON DISEASE FOCUS FUNDING ORGANIZATION SUCH AS THE LUPUS FOUNDATION ANDLY ALSO IS AN EDUCATOR. MANY OF US KNOW ABOUT HIM THROUGH HIS BOOK FUNDAMENTAL IMMUNOLOGY, MAYBE NOT EVERYBODY RECOGNIZES THE FACT THAT HE WAS THE INITIAL EDITOR AND THE EDITOR FOR NEARLY 30 YEARS AT THE ANNUAL REVIEW OF IMMUNOLOGY AND DURING HIS TENURE IT WAS THE MOST CITED PUBLICATION IN BIOMEDICINE, NOT IMMUNOLOGY, BIOMEDICINE WORLD WIDE. HE WAS A GREAT MENTOR. YOU CAN QUICKLY READ THIS LIST AND LOOK AT NAMES LIKE CHARLIE JANWAY, RON SCHWARTZ, LORI GLEMSURE, JUST PEOPLE WHO MIGHT KNOW FROM HERE BUT MANY RPGHTS OVER THE COURSE OF HIS CAREER AND I WROTE A FEW WORDS WHEN HE PASSED AWAY THAT I STILL THINK RING TRUE AND IS WORTH POINTING OUT BUT I WILL NOT READ ALL THIS AND JUST PARAPHRASE IT THAT YOU KNOW SCIENCE IS MADE UP OF MANY LITTLE PIECES THAT HAVE TO GET WOVEN TOGETHER INTO A FULL TAPESTRY. SOME PEOPLE CONTRIBUTE LITTLE THREADS, OTHER PEOPLE ARE REALLY THE WEAVERS OF THAT TAPESTRY AND BILL WAS A WEAVER AND THAT’S SOMETHING WE SHOULD ALL KEEP IN MIND AND THAT IS A GREAT SEGUE TO TODAY’S SPEAKER, MAX COOPER BECAUSE MAX HAS MANY OF THOSE SAME QUALITIES HE RECEIVED HIS M. D. FROM TULANE, HE THEN FOR REASONS THAT ARE UNCLEAR DECIDED TO BRAVE THE NORTHERN WILES AND WENT TO MICHIGAN BREFLY, THEN RETURNED TO THE SOUTH. HE THEN BECAME A LITTLE P A RAPPETTIC AND THEN WEND TO LONDON AND UCSF AND MINNESOTA WHICH HE ASSURES ME IS COLDER THAN MICHIGAN AND THERE HE WORKED WITH BOB GOOD AND BEGAN TO DO THE GROUND BREAKING WORK THAT LED TO THIS YEAR’S LASKER AWARD IN DISSECTING WAS A SINGLE SMALL POPULATION OF NONDESCRIPT CELLS CALLED LYMPHCITES INTO 2 MAJOR SUBSETS OF THE B & T-CELLS AND ASSUMED DIFFERENT ROLES IN THE ADAPTIST IMMUNE SYSTEM. HE SPENT 40 YEARS IN THE FACULTY OF UNIVERSITY OF ALABAMA BIRMINGHAM HAD WHERE HE DID ENORM AMOUNTS OF WORK DISCOVERING ISOTYPE SWITCHING AND WITH OWENS AND RAF, THE BONE MARROW ORIGIN OF MAMMALIAN B-CELLS HE DID A LOT OF TRANSLATIONAL WORK USING STUDIES ON THE OTOGEIN, Y, OF B-CELLS TO INFORM STUDIES OF CHILDHOOD LEUKEMIA AND FOR THE LAST 15 YEARS HE WORKED ON THE TYPIC HE WILL ADDRESS TODAY AND RATHER THAN GOING THROUGH THE LONGER DEKRIPGZ OF THAT, BECAUSE I AWLTZ WONDER WHAT INTRODUCERS GIVE UP WHAT THEY’RE GOING TO LECTURE BUT ABOUT IF YOU YOU DON’T KNOW ABOUT IT YOU WILL BE GLAD YOU ARE HERE TO HEAR ABOUT IT. HE’S WON MANY AWARDS BESIDES THE LASKER AWARD, ACADEMY OF SCIENCES, ACADEMY OF MEDICINE AND FRENCH ACADEMY OF CONSCIENCES AND I WILL RUN OUT OF BREATH READING THE REST OF THIS LIST, HE’S BEEN THE PRESIDENT OF MANY SOCIETIES INCLUDING AMERICAN ASSOCIATION OF IMMUNOLOGYSTS AND WON A NUMBER OF AWARDS FOR THE LAST YEAR’S JAPAN PRIZE AND I HAVE 1 OFFICIAL ANNOUNCEMENT I NEED TO MAKE THIS SEMINAR WAS SPONSORED BY THE IMMUNOLOGY INTEREST GROUP AND THE FAES IS SPONSORING A SMALL RECEPTION AFTERWARDS TO WHICH EVERYONE IS INVITED AND WITHOUT FURTHER ADOEUR SPEAKER DR. MAX COOPER.>>THANK YOU RON FOR THAT GRACIOUS INTRODUCTION AND I’M HONORED TO BE HERE TO GIVE THE WILLIAM PAUL LECTURE. ESPECIALLY HONORED THAT MARILYN, YOU WOULD ATTEND. THANK YOU SO MUCH. BILL WAS AN IMMUNOLOGYST’S IMMUNOLOGYST AND HE WAS A GREAT FRIEND TO SO MANY OF US. HE TALKED ALL OF US A LOT, HE CERTAINLY TAUGHT ME MANY THINGS. I NEVER PUBLISHED WITH HIM BUT WE DID DO ACTUALLY EXPERIMENTS TOGETHER AT 1 TIME. AND HE BROUGHT HIS STUDENT JOE DAVEY WHO IS AT THE TOP OF THAT LIST, AS RON JUST SHOWED TO–THEY KNEW HOW TO DO AUTORADIOGRAPHY AND WE WERE TRYING TO FIND OUT HOW FREQUENT WERE B-CELLS THAT WOULD BIND A PARTICULAR ANTIGEN. AND WE WERE RIGHT ON THE NOSE FOR 1 AND 10-TO-THE-FIFTH AT THAT POINT WE NEVER PUBLISHED THE DATA ACTUALLY AND I FORGET THE REASON WHY, BUT–[LAUGHTER] –BILL AND I ENJOYED A LOT OF FRIENDSHIP TOGETHER AND HE CERTAINLY WAS VERY SUPPORTIVE OF ME AND MY CAREER AND MANY DIFFERENT WAYS. I’M REALLY HONORED TO BE ABLE TO GIVE THIS LECTURE IN HIS HONOR. WELL, I’M GOING TO GO BACK AND TRY TO TRACE THE ORIGIN OF OUR ADAPTIVE IMMUNE SYSTEM, SO THE QUESTION IS HOW DID OUR ADAPTIVE IMMUNE SYSTEM ARISE AND WE ALL CAME FROM THE SEA, IT’S THOUGHT, BUT HOW IS IT THAT WE GOT THE DEVELOPMENT OF THE KIND OF ADAPTIVE IMMUNE SYSTEM THAT WE HAVE. I STARTED WITH QUESTION TO TRY AND SEEK THE ANSWER TO THIS QUESTION WITH JAN KLEIN WHO IS INTERESTED IN EVOLUTION, WAS A FAMOUS MHC CLASS MAJOR HISTOCOMPATIBILITY CLASS GENE SCIENTIST AND WE AGREED TO GO BACK AND START TOGETHER TO WORK ON TRYING TO SEEK THE ORIGIN OF OUR ADAPTIVE IMMUNE SYSTEM, WE KNEW AT THE TIME THAT ALL JAW VERTEBRATES ALL THE WAY BACKS TO SHARKS, SKINKS AND RAYS, HAD BASICALLY THE SAME KIND OF IMMUNE SYSTEM WE HAVE. THEY HAD A THYMUS WHERE A GENERATED T-CELLS, THEY GENERATED BCELLS IN THEIR BONE MARROW OR HEMATOPOIETIC TISSUES, THEY HAD MAJOR HISTOCOMBATTIBILITY GENES, CLASS 1, CLASS 2, THEY HAD RAG 1, RAG 2 RECOMBIN ACE ACTIVATING GENES 1 AND 2 THAT WERE INVOLVED IN THE ASSEMBLY OF T& B-CELL RECEPTORS AND THEY ALL HAD B-CELL RECEPTORS ALPHABETTA, GAMMA, DELTA,-TELERECEPTORS AS SHOWN BY MEN INVESTIGATORS SHOWING IN HOW THE EVOLUTION OF ADAPTIVE IMMUNITY IN JAW VERTEBRATES. BUT WE COULDN’T GO BACK AND LOOK AT EARLIER JAW VERTEBRATES BECAUSE WE KNEW THEM ONLY THROUGH THEIR FOSTER NURSED FOE REMAINS. THEY HAD ALL DIES OUT. IT WAS THOUGHT SOMEWHERE AROUND 360 OR MILLION YEARS AGO BUT THERE WERE JAWLESS VERTEBRATES, LAMP RAYS AND HAG FISH THAT WERE THOUGHT TO BE EVEN MORE ANCESTRAL OR BASAL THAN VERTEBRATES THAN JAW VERTEBRATES SO JAN KLEIN AND I SET OUT TO WORK TOGETHER TO STUDY THESE JAWLESS VERTEBRATES AND THE SPECIES THAT WE PICK WERE THOSE SEA LAMP RAYS, VETRIMYOSIN RENUS, THEY WERE 1 OF 40 SPECIES OF LAMP RAYS THAT LIVE WORLD WIDE IN DISTRIBUTION. ABOUT HALF OF THEM ARE PREDATORS LIKE THE SEA LAMPREGISTERED P–LAMPREY, AND THEY SPEBD A SHORT TIME AS PREDATORS WHEN THEY CAME INTO THE GREAT LAKES OF NORTH AMERICA, THEY WIPED OUT THE FISH. THEY HAVE A VERY RAS LIKE TUNK AND THEY HAVE A RING OF SHORP TEETH AND AT A LATCH ON TO THE SIDES OF THESE FISH PREY AND THEY DRIVE THEIR NUTRIENTS FROM THE BLOOD AND TISSUE FLUIDS AND 1 OF THESE LAMPREYS IT’S ESTIMATED CAN KILL ABOUT 40 KILOGRAMS OF FISH IN A YEAR. THEY REALLY DID WIPE OUT THE NATIVE FISH WHEN THEY MADE THEIR WAY FROM THE LANTIC SEA THROUGH CANAL CONNECTIONS INTO THE GREAT LAKES OF NORTH AMERICA. THEY MAKE AND THEN THE LIFE CYCLE STARTS OVER WITH FERTILIZED EGGS, THE LARVA A LIVE FOR 3 OR MORE YEARS IN THESE SMALL STREAMS THAT FLOW INTO THE OCEAN OR GREAT LAKES IN THIS CASE, BEFORE THEY UNDERGO METAMORPHOSEIS, GET LARGER RETRACT THE HOODS OF THEIR EYES SO THEY CAN SEE THEIR PREY AND THE LIFE CYCLE GOES THIS WAY. AFTER THE ADULTS MATE, THEY DIE. SO IT’S A COMPLICATED LIFESTYLE. IF YOU CUT 1 OF THOSE LARVA WHICH IS WHAT WE STUDY MOSTLY BECAUSE IT’S DIFFICULT TO GET AND KEEP THE ADULTS, YOU SEE SOMETHING LIKE THIS: THERE’S A ROUND THIS VERY SIMPLE INTESTINE, IS A TISSUE A HEMEAT O POTENT STATIC TISSUE CALLED THE LAMPREY SPLEEN AND JAN KLEIN AND HIS COLLEAGUES HAD FOUND A GENE, A TRANSCRIPTION F ACTOR–THEY SHOW HYBRIDIZATION OF A ROUND CELL AND RAISE THE QUESTION COULD THIS BE A LYMPHOCYTE AND JAN KLEIN AND I SET OUT TOGETHER. HE WAS FUNDED AT THAT TIME BY THE MAX PLANK INSTITUTE AND IN ADDITION TO NIH SUPPORT I HAD HOWARD HUGHES SUPPORT AND THAT ALLOWED US TO GO BACK ON THIS FISHING EXPEDITION. I THOUGHT THAT WE COULD ISOLATE THOSE CELLS THAT MIGHT BE LYMPHOCYTES BY THEIR LIGHT SCATTER CHARACTERISTICS AND JUST BY FORWARD AND SIDE SCATTER LIGHT REFLECTION. AND INDEED, THERE WAS A SMALL BUTTON OF CELLS THAT LOOK LIKE INTESTINAL EPITHELIAL LYMPHOCYTES PHYSICAL STRUCTURE OR LIGHT SCATTER CHARACTERISTICS WHEN WE SORTED THEM, THEY LOOK LIKE SMALL LYMPHOCYTES EVEN BY ELECTRON MICROSCOPY THEY LOOKED JUST LIKE OUR LYMPHOCYTES EXCEPT THEY WERE A LITTLE SMALLER, BUT THAT’S A VERY INDESCRIPT CELL PICTURE. ALL IT MEANS IS THAT MOST OF THE CELL IS NUCLEUS AND IN THE CYTOPLASM WHICH IS SORT OF SCANT THEY’RE NOT MANY ORGANLES THERE. SO WE MADE CDNA LIBRARIES TO TRY AND GET SEQUENCES OF GENES THAT THEY WERE EXPRESSING TO SEE IF THEY EXPRESS THE GENES THAT OUR LYMPHOCYTES EXPRESS. AND WHEN WE GOT SOME 2000 SEQUENCES WHICH WAS QUITE A LOT JUST IN THE EARLY PART OF THIS MILLENNIUM, WE–AS WE CONVERTED THE SEQUENCES INTO AMINO ACIDS AND ANNOTATED OUR CATCH, WE COULDN’T FIND ANY OF THESE CARDINAL ELEMENTS OF OUR ADAPTIVE IMMUNE SYSTEM, ALTHOUGH WE FOUND LOTS OF GENES BEING EXPRESSED THAT OUR LYMPHOCYTES EXPRESS FOR DIFFERENTIATION, FOR MIGRATION AND FOR OTHER ESSENTIAL FUNCTIONS. WE WENT BACK ACTUALLY ZETH P A NCER, WHO WAS A MOLECULAR AND MARINE BIOLOGIST JOINED THE LABORATORY AT THIS TIME AND ZEB AND I TRIED AGAIN WITH THE SAME RESULT TO SEE IF WE COULD CATCH CELLS IN THE ACT OF RESPONDING AND OTHERS HAD SHOWN INCLUDING COLLEAGUES BOB GOOD WHOM RON MENTIONED WHO WAS MY MENTOR HAD SHOWN MANY YEARS EARLIER THAT THESE CREATURES COULD RESPOND, THESE LAMPREY’S COULD MAKE DPLIEWTENS IN THEIR SERUM THAT WOULD CLUMP AN ANTIGEN LIKE A RED BLOOD CELL, A HUMAN O-POSITIVE RED BLOOD CELL IN A SPECIFIC FASHION, BUT NO 1 COULD FIND EVIDENCE FOR IMMUNE O GLOBUE LYNN OR WHAT THE BASIS OF THIS WAS AND THAT RESULTED HAD BEEN DISCREDITED BUT SWRAM SQUARELY FIT I DECIDED WE WOULD GO BACK AGAIN. THIS TIME WE WOULD STIMULATE THE LAMPREYULAR VEY AND THEN SEE–LARVA A AND THEN SEE IF WE COULD CATCH CELLS RESPONDING TO THE IMMUNIZATION IN THE ACT. SO TO SPEAK. AND SO, WE INJECTED LAMPREY LARVA WITH LIVE BACTERIA, E.COLI, RED BLOOD CELLS AND 2 PLANT MIGHT O GENS THAT SIMULATE JAW IN VERTEBRATES AND AFTER THAT WE SAW EXPANSION OF A POPULATION OF CELLS THAT BY LIGHT SCATTER WERE LARGER AND WHEN WE ISOLATED THOSE, THEY LOOKED EXACTLY LIKE LYMPHBLASTS THAT WE–THAT OUR LYMPHBLASTS HAD RESPOND TO SIMUE LANTS SUCH AS THESE. SO WE MADE A DIFFERENTIAL LIBRARY, CDNA LIBRARY FROM THIS POPULATION OF BLASTS AND THEN GOT SEQUENCING AND THEN STARTED ANNOTATING OUR CATCH AND WE RAN INTO NOT–STILL WE SAW NONE OF THE CARDINAL ELEMENTS OF OUR ADAPTIVE IMMUNE SYSTEM, BUT WE SAW LOTS OF LUCINE REPEATS WHICH WE DIDN’T THINK WAS SO INTERESTING BECAUSE LUCINE RICH REPEATS ARE USED BY EVERYTHING ON OUR PLANET, TOTAL LIGHT RECEPTORS ARE GOOD EXAMPLE OF LUCINE RICH REPEATS AND THAT INCLUDES TOLL LIKE RECEPTOR SEQUENCES IN THE END, WE HAD SOME STUDENTS FROM UNIVERSITY OF MICHIGAN LIMITED PARTNERSHIPPED BIOLOGY TECHNIQUES AND WE ASSIGNED HER TASK OF SEQUENCING THESE–THESE SEESTs EXPRESSED SEQUENCE TAGS AND SHE DID THIS AND IT SHOWED THE FACT THAT EACH 1 WAS DIFFERENT FROM THE OTHER. THEY HAD THE ENDS THAT WERE CONSTANT? SEQUENCE INVARIANT BUT IN THE MIDDLE THEY HAD DIFFERENT NUMBERS OF THESE LUCINE RICH REPEAT SEGMENTS AND THE 1S THEY SHARED WERE ALL DIFFERENT FROM THE OTHER AS YOU CAN TELL FROM THE SCORE CARD, THE COLOR CODING. AND FINALLY, THE LIGHT CAME ON FOR US THAT ALTHOUGH THIS WAS NOT WHAT WE STARTED OUT LOOKING FOR, PERHAPS THIS WAS A KIND OF VARIABILITY THAT COULD GIVE A RECOGNITION SYSTEM THAT WOULD ALLOW DISCRIMINATION BETWEEN DIFFERENT ANTIGENS. NOW THAT LONGEST SEQUENCE IS DEPICTED IN THE CARTOON HERE AND THE SEQUENCE HERE AND INTERESTING THINGS ABOUT IT, IS THAT THE PART THAT’S HIGHLY VARIABLE IN THE OTHERS ARE THAT EACH OF THESE HAD THE END TERMINAL AND C-TERMINAL RR-REGION WHICH ARE FAIRLY LENGTH SKPE BAKUGAN SHORTER LRR-1 AND THESE GREEN BLOCKS OF ALL OF 24 RESIDUES IN THEMSELVES PLUS THE CONNECTIVE PEPTIDE THAT WERE–WERE THE VARIABILITY WAS OCCURRING. STOP REGION WAS INTERESTING, TOO IN SOME WAYS, IT WAS RICH IN 3-ANINEs AND PROLEANS SUGGESTING MAYBE IT HAD FLEXIBILITY. IT HAD AT THE CAR BOXY TERMINAL REGION SEVERAL CYSTINS, 8 IN ALL AND I WILL COME BACK TO THOSE BUT THE INTERESTING OTHER SIGNATURE WE COULD SEE WAS THE SEQUENCE THAT SUGGESTED THAT THEY MIGHT BE CLIPPED AT THIS–AT THIS SITE AND EXPRESSED ON CELLS BY AGPI LINKAGE INSTEAD OF EXPRESSING ALL OF THE CAR BOXY TERMINAL SEQUENCE. NOW WE WONDERED JUST HOW VARIABLE THESE MIGHT BE AND THESE ARE–THIS IS A PHILO-GENETIC CARTOON THAT SHOWS HOW CLOSELY RELATED THE FIRST 112 VLR CLONES THAT WE SEQUENCED AND THESE CAME FROM 13 DIFFERENT LARVA AND ALSO WE FOUND LATER THAT THE SAME FROM ADULTS AND THE WAY TO LOOK AT THIS DIAGRAM IS THAT THESE 2 ARE THE CLOSEST TO EACH OTHER THAN ANY OF THE OTHERS AND THEY’RE 5, BACK UP, 10% DIFFERENT IN SEQUENCE FROM EACH OTHER AND SO ON AROUND THIS CLOCK. THERE ARE A COUPLE OF POINTS I WOULD LIKE TO MAKE FROM THIS. THERE WERE ONLY DUPLICATES OR–THE SAME SEQUENCES WERE SEEN 3 TIMES IN ALL OF THOSE CAME FROM THE STIMULATED LARVA THAT I JUST MENTIONED AND ALSO THIS ASTERISK MEANS THAT MEANS THAT SINGLE CELL POLYMER ACE CHAIN REACTIONS WAS DONE AND EACH OF THEM ONLY EXPRESSED 1 SEQUENCE IMPLYING THAT EACH OF THE CELLS THAT EXPRESSED THEM WERE MONOALLELIC IN THEIR EXPRESSION. SO WE WONDERED OF COURSE WHAT’S THE GENETIC BASIS FOR ALL THIS DIVERSITY. ARE THERE MANY GENES LIKE THE TOLL LIKE RECEPTORS IN SEA URCHINS OR PLANT RESISTANT GENES IN PLANTS THAT ARE INVOLVED IN DEFENSE THEY ENCODED BY EACH 1 BY A DIFFERENT GENE OR ARE THERE MULTIPLE IF THERE’S SOME OTHER BASIS. SO AT THAT TIME, WE FOUND BY CONTACTING CHRIS AMAMIA IN SEATTLE WHO HAD MADE A GENOMIC LIBRARY OF PIECES OF DNA FROM THE SPERM OF A SINGLE LAMPREY. AND WE LOOKED THROUGH HIS LIBRARY AND WE COULD ONLY FIND 1 BLR GENE AND IT WAS VERY INCOMPLETE. IT ENCODED FOR A SIGNAL PEPTIDE, THE EPPED TERMINAL REGION OF THE LRR AND THE C-TERMINAL REGION IN 2 PIECES BUT NONE OF THE OTHER BLOCKS, THE GREEN OR THE LIGHT BLUE THAT I SHOWED BEFORE, IN BETWEEN THESE PIECES OF THE INCOMPLETE GERM LINE GENE WERE LARGE STRETCHES OF ABOUT 6 KB EACH OF NONCODING INTERMEANING SEQUENCE. AND THEN THE STOP REGION, THE INVARIANT STOP REGION. WHEN WE COULD GET SEQUENCES IN THE FLANKING REGIONS AND THAT WAS NOT SO EASY BECAUSE THEY–THERE ARE LOTS OF REPEATS AND IT’S HARD TO GET CONTINUOUS SEQUENCES WHICH IS STILL A PROBLEM FOR LOOKING FOR THE GENOME OF THESE KRETTURES ALTHOUGH IT’S MUCH BETTER AT THIS POINT BUT WE FOUND FLANKING THIS INCOMPLETE GERM LINE GENE GENES THAT IF BROUGHT IN SOMEHOW AND STITCHED INTO THE GERM LINE GENE, AND DELETION OF THE NONCODING INTERVENING SEQUENCE COULD GIVE RISE TO THIS MATURE VLR GENE THAT ARE THOSE GENES THAT WE WERE ENCODING, FINDING IN OUR ANALYSIS. ORIGINAL OUR LYMPHOCYTE CHARACTERISTICS LIKE THE 1S I SHOWED EARLIER, AND RED BLOOD CELLS AND THEN DID USING THESE FORWARD AND REVERSE PRIMERS, WE COULD FIND THAT THE LARGE GERM LINE GENE PRODUCT WAS PRESENT IN BOTH TYPES OF CELLS BUT ONLY IN THE LYMPHOCYTE POPULATION WERE THESE SHORTER, SMALLER PRODUCTS AND WE SEQUENCED MANY OF THESE CLONES AND LIKE THE OTHERS THAT I MENTIONED ALL OF THEM WERE DIFFERENT IN SEQUENCE, 1 FROM THE OTHER. SO WE STARTED OUT WITH THIS MODEL OF OUR RESULT SO WE–BY THIS TIME WE HAD OVER 600 DIFFERENT–WE CALL THEM VARIABLE LYMPHOCYTE RECEPTOR GENES BECAUSE WE COULD SHOW IF WE EXPRESSED A CONSTRUCT WITH THE TAG IN MOUSE THYMOCYTES,
T-CELLS THAT PRODUCT TO THE SURFACE WE COULD FIND WITH OUR TAG AND ALSO WE COULD CLEAVE THEM FROM THE SURFACE BY FOSTER NURSED FOCUSED ON LINEUP ACE C, INDICATING THAT I HAD A GPI LINKAGE TO THE CELLS THAT THEY WERE EXPRESSED BY. SO WE CALL THEM VARIABLE LYMPHOCYTE RECEPTORS OR VLRs. WE SUBMITTED OVER 600 DIFFERENT SEQUENCES TO IGOR [INDISCERNIBLE] AND MAX [INDISCERNIBLE] AT NCBI AND THEY CALCULATED THESE COMPUTATIONAL BIOLOGISTS THAT THEY POTENTIAL REPERTOIRE FOR THESE–FOR LAMPREY LYMPHOCYTES EXPRESSING THESE VLRs WOULD BE GREATER THAN 10 TO THE 14th POWER. AND ACTUALLY IT TURNS OUT TO BE HIGHER THAN THAT BECAUSE THEY WERE CALCULATED ON THE BASIS OF EACH 1 OF THESE DONOR SEGMENTS BEING COMPLETE AND THEY’RE GIIVE IN SMALL FRAGMENTS. BUT THE–THAT’S EQUIVALENT TO THE POTENTIAL REPERTOIRE OF OUR B-CELLS IN THE LIGHT AND HEAVY CHAIN ASSEMBLIES ABOUT 10-TO THE 14th IN REPERTOIRE DIVERSITY. WE ASSUME THAT THEY WOULD PROBABLY BE SECRETED OR RELEASED IN SOME WAY AS MULTIMERS IN ORDER TO EXPLAIN THE GLUTENINS LIKE THE 1S THAT ARE GLIEWTINNATED O-POSITIVE HUMAN RED BLOOD CELLS IN AN APPARENT SPECIFIC WAY. WE SET OUT TO TEST THIS HYPOTHESIS BY IMMUNIZING LAMPREY LARVA AND HERE YOU GET AN IDEA OF THE SIZE, THEY ARE ABOUT THE SIZE OF A SMALL PENCIL, ABOUT 12-CENTIMETERS AND THIS IS AN INSULIN SYRINGE. YOU CAN SEE THE FINGERS OF THE IMMUNIZER AND INJECT THEM INTO THE–THEIR CAVITY WHICH IS EQUIVALENT TO OUR PEROTONEALL CAVITY. OUR ABDOMINAL CAVITY. YOU CAN ONLY INJECT A SMALL AMOUNT BECAUSE IT LIKES OUT IF YOU GIVE MORE AND WE IMMUNIZE THEM AND THEN LOOKED 2 WESTBOUNDS LATER, ALL OF THEM MADE GLUTINENs IN A FAIRLY LOW TITER OF 1 TO A HUNDRED ON AVERAGE BUT IF WE GAVE A BOOSTER IMMUNIZATION, WE GOT A 20 FOLD INCREASE IN THE LEVEL OF THESE GLUTENINs, WE’VE MADE MOUSE MONOCLONAL ANTIBODY THAT WAS SPECIFIC FOR THAT INVARIANT STALK REGION AND IF WE ABSORBED THIS SERUM, WITH THAT ANTIBODY, WE COULD REMOVE ALL OF THIS GLUTENIN ACTIVITY. SO IT WAS CLEAR THAT WHERE THESE GLUTENINs WERE THE ACTUAL PROTEINS VARIABLE RECEPTORS THAT WERE CAUSING THE GLUTENINNATION, AND IF WE USE THE EABT BODY WITH A GREEN FLUORESCENT TAG TO IDENTIFY CELLS THAT WE’RE EXPRESSING WE CALL THEM VLRB AND WITH A RED TAG THAT WAS INTORPERATED BY CELLS THAT WERE SENTHICIZING DNA, AND BLUE TAG JUST TO LIGHT UP ANY NUCLEATED CELL, YOU COULD SEE THAT IN THIS–THESE ARE CROSS SECTIONS OF GILL FOLDS THAT CUT CROSS WISE AND IN THESE SMALL VESSELS YOU CAN SEE THERE ARE LOTS OF THESE VLRB PRODUCING CELLS. THIS IS BEFORE AND AFTER IMMUNIZATION AND THESE ARE X-FORS, AND IT’S EITHER DIVIDING AND MAKING THESE GREEN OR INCREASED IN NUMBER AFTER IMMUNIZATION THE CELLS THOUGH THAT WERE ACTUALLY SECRETED THESE ARE THE SMALL OR LARGER 1S, THAT CAME FROM THE CELLS THAT HAD THESE LIGHT SCATTERED THEY EXPRESSED THEM ON THEIR SURFACE VLRB THAT MATT ALD ER AN MD-Ph.D. STUDENT COULD SHOW BY AN ELI AS SPOT ASSAY, THESE WERE THE 1S WE’RE SECRETING ANTIBODIES THESE ARE PLASMA BLASTS AND JUST LIKE OUR PLASMA BLASTS. AND IN THE CHANNELS THAT ARE TYPIFIED PLASMA CELLS, SO THEY HAD PLASMA CELLS THAT ARE IDENTICAL TO IN APPEARANCE TO OURS BUT WE WOULDN’T DETECT THEM BECAUSE AT THIS POINT, AT THE POINT OF PLASMA CELL DIFFERENTIATION, THEY WOULD HAVE LOST THEIR SURFACE VLR. BRANDT HERON A POST DOC IN THE LAB DID A WESTERN BLOT USING THIS STALK REGION ANTIBODY THAT SEES ALL OF THESE VLRBs REGARDLESS OF THEIR LUCINE RICH REPEAT VARIABILITY AND YOU CAN SEE THAT IN THE SERUM THERE ARE VERY LARGE PROTEINS MUCH LARGER THAN ANY OF OUR STANDARDS BUT IF WE REDUCED THE DISCIPLINARY SULFIDE BONDS, AND REMEMBER, THOSE CYSTINES AT THE CAR BOXY TERMINUS, THEN HE COULD REDUCE THEM TO MUCH SMALLER BLOTS. YOU CAN SEE THAT MAYBE ABOUT 10 OF THESE OR MORE WOULD BE IN THE POLYMERIC FORM OF THESE VLRBs, EACH HAVING 1 OF THESE. YOU CAN ALSO SEE THAT THERE ARE DIFFERENT IN SIZES AT BOTH TREATMENTS. AND AS YOU WOULD EXPECT GIIVE THE FACT THAT THEY CAN HAVE DIFFERENT NUMBERS OF THESE GREEN BLOCKS AND THEREFORE DIFFERENT LINKS SO THIS IS NOT SOMETHING THAT YOU COULD BEGIN TO DO BIOCHEMICAL ANALYSIS, SO, BRANDT HERON DESIGNED A WAY TO MAKE MONOCLONAL VLR-B ANTIBODIES. SO HE IMMUNIZED LAMPREYs AND PREPARED RNA AND CONSTRUCTIVE CDNA LIBRARIES THAT HE EXPRESSED 1 BY 1 IN HUMAN EMBRYONIC KIDNEY CELLS, AND THEN, PLATED THESE CELLS IN AND THE REASON FOR ACTIVITY AND HUMAN O-POSITIVE ERYTHROCYTE AND KEN RUES AT FLORIDA STATE UNIVERSITY TOOK THE SAMPLE AND DID THE NEGATIVE IMAGE WITH THIS KIND OF IMAGE HERE’S THE CARTOON THAT IPT GREATER TERPRETINAL LOCATIONSAGE THAT CAN DERIVE SMALL ANTIBODY. AND SO THERE ARE 5 PAIRS OF DISCIPLINARY MERS THAT ARE JOINED BY THESE DISCIPLINARY SULFIDE BONDS AT THE BASE OF THE STALK REGION. I MENTIONED THAT THERE WERE 8 OF THESE CYSTINES THAT COULD CREATE THESE DISCIPLINARY SULFIDE BONDS AT LEAST 4 OF THEM ARE REQUIRED FOR SECRETING IN THIS POLYMER, THIS MULTIMERFASHION. IF BRANDT TRUNCATED THE CONSTRUCTS TO NOT HAVE THE SEQUENCE FOR THESE CAR BOXY TAIL THEN THEY WERE RELEASED FROM THE VANS FECTED CELLS IN SINGULATE FORMS. SO, THIS STALK REGION IF YOU REMOVE THIS CAR BOXY TERMINAL THAT ACCOUNTS FOR THEIR BEING HELD TOGETHER AT THE BASE, IS A WAY THEY’RE EXPRESSED. WE STILL DON’T KNOW EXACTLY HOW THIS CONVERSION FROM GPI LINKAGE TO THE SECRETEATORY PLASMA CELLS AND THIS MODEL WITH 1 OF THE QUESTIONS WE HAD IN HERE AND B-CELLS BECAUSE THERE IS A SIMPLER WAY, PROTECTIVE PURPOSES AND TO HAVE PRESENTING CELLS CAN ONLY SEE ANTIGENS IN PIECES IN THE MAIN, AND SO, THIS WOULD BE AN EARLIER WAY OF ANTIBODIES AND AGAIN, THIS 2 OF THESE VLR GENES THAT WERE ALSO FRANKED BY MULTIPLE–FOUND A THIRD 1 IS IN THE HAGFISH AND LAMPREY’S AS WELL, ALL THESE GERM LINES ARE INCOMPLETE MAINLY HAVING BITS AND PIECES MISSING IN THE MIDDLE, VARIABLE PORTIONS OF THESE GENES BUT FLANKING THEM HUNDREDS OF THESE POTENTIAL DONORS THAT COULD BE BROUGHT IN BIT BY BIT AND THEN ASSEMBLES USING SHORT STRETCH OF SIMILARITY OR HOMOLOGY BETWEEN THE DONOR AND THE RECIPIENT TO BRING IN FIRST 1 LIKE THIS, IT HAS THE DARK BLUE WITH THE MATCHING SEQUENCE HELD IN PLACE REAL WELL, AND THIS GETS COPIED IN AND THEN TO GET FROM THERE 1 HAS TO HAVE 1 THAT HAS THESE GREEN SEQUENCES AND SO ON DOWN TO THE END AND DELETING IT THE SAME TIME THIS–THESE NONCODING INTERVENING SEQUENCES AND A GROUP FROM TOKYO [INDISCERNIBLE] AND HIS COLLEAGUES SHOWED THAT THIS COULD START ON EITHER END FROM THE 5 PRIME END AND GO THROUGH TO COMPLETION OR FROM THE 3 PRIME END AND GO BACK THE OTHER DIRECTION TO FASHION A COMPLETELY ASSEMBLED VLR-B GENE. BUT, IT ONLY HAPPENS ON 1 ALLELE AND ONLY STARTS AT 1 END OR THE OTHER AND NEVER BOTH AT THE SAME TIME. AND THEN USING MONOCLONAL ANTIBODIES THAT WOULD DISSCRIMINATE BETWEEN THESE INVARIANT SEQUENCES OF THE VLR A, B, C, TYPE CELLS THAT EXPRESS THOSE GENES WE MADE MONOCLONAL ANTIBODIES MASAHARANO, AND [INDISCERNIBLE] AND OTHERS IN THE LABORATORY THAT COULD DISCRIMINATE 3 POPULATIONS OF LYMPHOCYTES. AND OUR DATA SUGGESTS THAT THEY ARE–ALL DERIVED FROM A LYMPHOID PROGENITOR. AND IN A THYMUS EQUIVALENT AT THE TIPS OF THOSE GILL FILAMENTS THAT I SHOWED YOU ACROSS SECTIONS OF BEFORE THERE WERE SITES WHERE GENES ARE EXPRESSED THAT ARE NORMALLY REQUIRED OR HAVE BEEN SHOWN TO BE REQUIRED FOR T-CELL DIFFERENTIATION IN MICE AND ALSO IN SOME CASES BY GENETIC DEFECTS IN HUMANS AS WELL. TO GIVE RISE TO 2 PROTOTYPIC LINEAGES OF AND THEY ARE TRANSMEMBRANE AND NOT SECRETED AS VLRB, THE GENES ARE B-CELLS ARE LIKE AND AGAIN REGOSIN AND THE [INDISCERNIBLE] IN HIS OWN LAB DISCOVERED 2 GENES THAT ARE LIKE ACTIVATION INDUCED DEAMNAIS WHICH IS ESSENTIAL FOR A GENERATION WITH THE B-CELL CLASS SWITCHING AND BY AFFINITY MATURATION AND ALSO FOR GENE CONVERSION THAT BIRDS USED FOR GENERATING DIVERSITY OF B-CELLS. BUT IN THE THYMUS EQUIVALENT IS ANOTHER–THE SECOND OF THE CYTODINE DEAMNAISS THAT ARE LIKE A. I.D. IN US THAT’S EXPRESSED AND WE COULD SHOW THAT IT’S NORMALLY EXPRESSED IN THIS THYME O REGION. AND THEY ARE ASSEMBLED FIRST IN THE ORDER OF VLRC AND THEN VLR A. AND THE GAMMA DELTA T-CELLS AND COMPLETE EVIDENCE. AND B-CELLS BEFORE AND ABOUT WHICH DIFFERENTIATE INTO MULTIPLE DIFFERENT HELPER INHIBITORY, ALL DIFFERENT KINDS OF T-LINEAGE CELLS. WE HAVE DATA THAT’S SUGGESTED THAT THEY ARE EXPRESSING CYTOKINES IN THIS CASE LIKE INTERLUKEIN 17 THAT SEES THE RECEPTOR ON THE B-CELLS THAT’S MADE BY THE V LR-A CELLS THAT SUGGEST THEY HAVE CROPPED IMMUNE O ACTIONS THROUGH THE CYTOKINES AND CYTOKINE RECEPTORS THAT THEY EXPRESS. BUT REALLY WE KNOW NOT SO MUCH YET ABOUT EXACTLY WHAT THEY DO, SO HERE’S THE EXPRESSION PATTERNS THAT LED TO THAT CONCLUSION OR SOME OF THE DATA THAT LED TO THAT CONCLUSION. IF YOU ISOLATE VLR-A CELLS, VLR-C, CELLS THEY EXPRESS GENES THAT ARE INVOLVED IN T-CELL DIFFERENTIATION MUCH LIKE OUR ALPHABETTA AND GAMMA DELTA T-CELLS. WHEREAS THERE’S SOME GENES THAT ARE DIFFERENTIALLY EXPRESSED BY 1 OR THE OTHER. THESE EXPRESSED IN PARTICULAR BY A GAMMA CELTA LINEAGE OF T-CELLS IN MICE AND HUMANS AND THESE EXPRESSED MORE PREFERENTIALLY IN ALPHABETTA TYPE T-CELLS AND SOME THEY SHARE IN EXPRESSION. THE VLRBs EXPRESS JEERNS THAT ARE MUCH–THAT ARE LOOK ALIKES TO CELLS LIKE THE CDA2 THAT I MENTIONED AND OTHER GENES THAT WE KNOW TO BE ESSENTIAL IN B-CELL DIFFERENTIATION, HERE’S AN EXCEPTION, A CD-4 LIKE GENE, IT WOULD NORMALLY BE EXPRESSED BY ALPHABETTA T-CELLS IN US IMU IN THIS CASE, IN THE LAMPREY’S EXPRESSED IF B-LIKE CELLS. SO 3 DIFFERENT LINEAGES OF CELLS T-LIKE CELLS THAT DON’T SECRETE PRODUCTS THAT CAN RESPOND TO PROLIFERATION WHEN THEY SEE–WHEN THEY’RE IMMUNIZED WITH OTHER LAMPREY’S THAT ARE GENERATEDET RIKELY DIFFERENT FROM THEM BECAUSE THEY’RE OUT THERE THESE ARE THE 2 GENES THAT I MENTIONED, THEY AID-LIKE IS A MULTIAXON GENE, KREBS CYCLE, DA 1 IS A SIGNAL, AN INTRON-LESS GENE AND THIS IS THE 1 THAT’S EXPRESSED DURING T-CELL–THE VLR A& C GENE ASSEMBLY ON T-LIKE CELLS. THIS 1 HAS OTHER TRANSCRIPTS THAT ARE NOT SHOWN HERE BECAUSE PART OF THE GENE IS MISSING IN THIS INITIAL REPORT BUT WITH THE LONGER SEQUENCES HERE, THESE ALL HAVE DNA’S EXPRESSIVITY OR FUNCTION AND THEY’RE ALL MUTE O GENIC, THESE IN PARTICULAR ARE HIGHLY SO. SO, THIS IS THE CURRENT MODEL AND I’D LIKE TO SPEND THE REST OF THE TIME DISCUSSING MORE ABOUT THE ANTIBODY PRODUCTS THAT THESE DLRs PRODUCE. BUT FIRST WHAT DO WE CONCLUDE FROM THIS DATA? WELL, FIRST OF ALL WE CONCLUDE THAT THE GENETIC PROGRAM FOR THESE 2 PROTOTYPIC T-LIKE LINEAGES IN LAMPREY’S AND HAGFISH AND B-LIKE LINEAGE THAT ARE VERY SIMILAR TO THE GENETIC PROGRAM FOR OUR T& B CELLS, THOSE GENES MUST OF EXISTED IN A COMMON ANCESTOR, THE LAST TIME WE HAD A COMMON ANCESTOR, IS ESTIMATED TO BE A LITTLE OVER 500 MILLION YEARS AGO, SO, THIS PROGRAM WAS ALREADY PRESENT BY THEN, BEFORE THE CONVERGENT EVOLUTION OF DIFFERENT KINDS OF RECEPTORS THAT GENERATE DIVERSE REPERTOIRE OF ANTIGEN RECEPTORS IN JAWLESS AND JAWED VERTEBRATES. WHAT THEY WERE USING BEFORE THEN IS 1 OF THE MANY UNANSWERED QUESTIONS. THE–ALSO IT SUGGESTS THAT THE AID OR CYTODINE DEAMNAIS EQUIVALENT IN JAWLESS VERTEBRATES, THE GENE FOR IT WAS ALREADY PRESENT IN BOTH AN ANCESTOR OF JAWLESS AND JAWLESS VERTEBRATES. BUT THE AS I MENTIONED EARLIER, THE JAWLESS VERTEBRATES DON’T HAVE ANY OF THESE CARDINAL ELEMENTS THAT IS USED TO MAKE ANTIGEN RECEPTORS OR TMV LIKE CELLS INSTEAD THEY HAVE THESE VLR A, B, AND C GENES SO WE RECKON THIS ISA 2 DIFFERENT EVOLUTION TO THE PUZZLE OF TRYING TO CREATE A DIVERSE PREPPER TWOAR WHICH IS WILL ALLOW FOR MEMORY AND SPECIFICITY IN REACTIVITY OF THE TMB CELL LINEAGES. SO WHAT ABOUT THESE B-CELL RECEPTORS THAT ARE SECRETED, THESE LAPPREYANTIBODIES IF YOU WISH? THIS FIRST MONOCLONAL THAT BRANDT PRODUCED THAT WAS SPECIFIC FOR O-POSSIVE ERYTHROCYTES THAT’S AGAINST 3 SACCHARIDE COMBINATION, H-TRI SACCHARIDE AND YOUNG WU HAN IN IAN WILSON’S LABORATORY AT SCRIPTS WAS ABLE TO GET CRYSTALS AND SOLVE THE STRUCTURE OF THE THIS LIGAND OR ANTIGEN INTERACTION WITH THIS ANTIGEN BINDING SITE AND THIS CARTOON SHOWS ON A RIBBON DIAGRAM, SO IN THIS, A HYDROGEN BOND BETWEEN RESIDUE IN THIS LRRV2, THERE ARE 2 HYDROGEN BONDS IN THIS NLLRV, AND IN ADDITION THEY’RE IMPORTANT INTERACTIONS WITH THE TRANSCRIPT O FAN BETWEEN THIS H-TRISACCHARIDE ON A LOOP THAT’S HIGHLY ENCODED AND VARIABLE AND ENCODED IN THIS C-TERMINAL PORTION OF LRRTHAT’S HIGHLY VARIABLE. AND I WILL–IN A SPACE FILLING MODEL IT’S EASIER TO SEE, THE GRAY IS THE H-TRI SACCHARIDE AND AS YOU CAN SEE, IT’S SCORT OF LIKE THE–SORT OF LIKE THE THUMB THAT’S STICKING OUT HERE FROM THE BASE OF A HAND, CURVED PALM OF THE HAND AND WITH THIS 1 LOOP THAT ALSO IS BINDING FORMS OF POCKET FOR THIS VLR ANTIGEN. SO, I HAVEN’T MENTIONED BEFORE WHAT IS INDUCING THIS HIGHLY VARIABLE REGION IS I’D LIKE TO JUST SHOW YOU BECAUSE IT GIVES A LITTLE BIT OF A INSIGHT INTO HOW THIS HUGE VARIABILITY ACTUALLY CAN BE GENERATED OF GREATER THAN 10-14th OR 15th OR MORE. SO, HERE ARE-WE DON’T HAVE CONTINUOUS DNA SEQUENCE FOR THE WHOLE GENOME AND THIS IS ACTUALLY FOR JAPANESE LAMPREY AND I’VE BEEN TELLING YOU ABOUT WORK DONE MAINLY ON THE SEA LAMPREY FROM THE SEA. AND THE JAPANESE LAMPREY HAS 2 OF THESE EXTRA BITS IN THE MIDDLE OF THE GERM LINE GENE WHICH IS THIS CONFIGURATION. AND THESE ARE 2 LARGE CONTIGER DATABASES OR OF DNA SEQUENCE THAT HAVE BEEN PUT TOGETHER BY [INDISCERNIBLE] COMPUTATIONAL BIOLOGIST IN OUR GROUP AND YOU CAN SEE THAT THERE ARE MULTIPLE 1S OF THESE POTENTIAL DONORS IN THESE 1, 2, AND 3, SCAFFOLDS FROM A CONTINUOUS SEQUENCES. SO TO START AND IF YOU STARTED ON THIS END, YOU WOULD HAVE TO BRING IN 1 OF THESE OR SEQUENCES FROM 1 OF THESE PIECES THAT WOULD BE COMPLIMENTARY BY A SHORT STRETCH OF DNA SEQUENCE WITH THE GERM LINE SEQUENCE IN ORDER TO GET TO THE NEXT STAGE. AND IN ORDER TO MOVE IT FORWARD, THEY WOULD ALSO HAVE TO HAVE 1 OF THIS SEQUENCE FROM THIS BLUE WHICH COMES NEXT. THEN TO GET FROM THE BLUE TO THE JEANS, YOU WOULD HAVE TO HAVE THESE DONOR SEQUENCES THAT HAD AT LEAST SOME OF THE BLUE AND THE GREEN. OR THEY COULD HAVE MORE AS SHOWN HERE. IN THIS COFTIG, THERE ARE NONE THAT COULD TAKE IT TO THE NEXT STAGE AND NOTHING HERE EITHER FOR THESE GREEN BLOCKS, WHICH ARE MORE NUMEROUS, THERE ARE LOTS OF THESE POTENTIAL DONORS IN ALL OF THESE CONTIGER DATABASES EXCEPT FOR THE SHORTER 1 WHERE THERE ARE NONE, AND YOU CAN SEE EVIDENCE OF DUPLICATIONS TO SEE AND MECHANISM, AND GENERATING THESE DONOR SEQUENCES TO GO ALL THE WAY TO HERE 1 HAS TO HAVE SEQUENCE THAT HAS THIS A LITTLE BIT OF GREEN ALONG WITH THE CONNECTING PEPTIDE AND NOT A BIT OF THIS YELLOW ART OR C-TERMINAL REGION. AND YOU CAN SEE THAT THERE, YOU FIND THEM IN BOTH OF THESE, NOT IN THE SHORT STRETCH FOR THE LAST BIT TO CHANGE HERE YOU HAVE TO HAVE THESE GENES SEGMENTS AND THERE ARE A FEW OF THEM IN THIS 1 THAT IN THIS SHORTER 1, THERE’S MULTIPLE 1S OF THESE. AND THE JUNCTION OF THAT LAST BIT TO HERE IS WHAT CAUSES–EXCUSE ME–THIS HIGHLY VARIABLE REGION IN THIS CAR BOXY C-TERMINAL LRR THAT FORMS THIS LOOP. SO THESE WOULD FORM THE LOOP IN 85% OF THESE ALL HAVE CODING REGIONS FOR THIS HIGHLY VARIABLE LOOPS WHEREAS THESE ARE SHORTER AND THEY WOULD NOT–15% DON’T HAVE THE LOOP AT ALL BUT THEY STILL ARE MADE SO IF YOU LOOKED AS A COMPARISON OF THE MOUSE ANTIBODY, THE LIGHT CHAIN INHIBIT CHAIN VARIABLE REGIONS, WITH AN ANTIGEN AND HERE AGAIN THE–THIS IS A TRI SACCHARIDE CARBOHYDRATE ANTIGEN AND THIS IS THE LAMPREY ANTIBODY I SHOWED YOU EARLIER SO THERE ARE 3 LOOPS FOR THE LIGHT CHAIN AND 3 FOR THE HEAVY CHAIN THAT CAN HAVE WIGGLE CAPABILITY, CONFIRMATIONAL ADAPTABILITY SO THAT’S PROBABLY THE REASON THAT THESE REGIONS WHICH CONTACT THE ANTIGENS MAKE ANTIBODIES OF THE KIND THAT CONVENTIONAL ANTIBODIES THAT WE IN MICE AND RABBITS AND SO FORTH MAKE, THEY ARE HIGHLY PROMISCUOUS OR FOR EXAMPLE, CAN YOU DIAMOND CIRCLE ACHIEVER ANY MONOCLONE AT ANTIBODY AND IF YOU SCREEN FOR REACTIVITY, CAN YOU FIND OTHER ANTIGENS IT WILL BIND, MANY OF THEM HAVING NO DETECTABLE SEQUENCE IDENTITY. WHEREAS IN THESE LAMPREY ANTIBODIES THIS IS THE ONLY PART THAT HAS CONFIRMATIONAL ADAPTABILITY. AND THAT’S TD REASON THIS STRUCTURAL REASON WE THINK IS 1 OF THE REASONS THAT THE LAMPREY ANTIBODIES ARE SO HIGHLY SPECIFIC IN EVERY COMPARISON WE’VE MADE WITH THE MULTIPLE CLONES OR CLONED ANTIBODIES FOR MICE WITH THOSE FROM LAPPREWHY,’S, THEY’RE ALWAYS MORE SPECIFIC IN THERE REACTIVITY. HERE’S AN EXAMPLE OF THEIR REACTIVITY OF THIS MONOCLONAL THAT I SHOWED YOU EARLIER WITH GL YCANS OR SUGAR DETERMINANTS. THESE L AMPREY’S ARE PARTICULARLY GOOD TO MAKING CARBOHYDRATE ANTIGENS SO THR FEAS ENLINKED CARBOHYDRATES, 611 CONFIGURATIONS ARE LOADED ON TO THIS GLASS SLIDE FOR SCREENING PURPOSES, AND THE MONOCLONAL LAMPREY ANTIBODY THAT I’M SHOWING YOU REACTS WITH ONLY CERTAIN 1S OF THEM AND NONE AT ALL WITH THE OTHERS AND THE 1S THAT IT SEES ALL HAVE A FUCOS AS THE RESIDUE CARBOHYDRATE WITH A LINKAGE TO GALACTOSE, WHICH IS LINK INDEED 1-4 LINKAGE TO THE NEXT SUGAR DOWN THE LINE WHICH CAN VARY 1 FROM THE OTHER. NOW WE MADE AND SCREENED BY A MORE EFFECTIVE SCREENING MECHANISM USING YEAST SURFACE DISPLAY WHERE WE CAN SCREEN MILLIONS OF CLONESSA THE 1 TIME, 2 OTHER MONOCLONALS THAT REACT WITH THIS H-TRYSACCHARIDE AND BY CHANGING 1 OF WHICH IS MORE SPECIFIC THAN ANYTHING THAT’S EVER BEEN SEEN AGAINST THE O-POSITIVE BLOOD GROUP THE TRI SACCHARIDE AND BY LOOKING TO SEE THE DIFFERENCES IN THEM, THEY’RE ALMOST SIMILAR, THEY’RE ABOUT 80% SIMILAR IN SEQUENCE. BUT FIXING OUT RESIDUES THAT YOU CAN CHANGE BY STRUCTURE GUIDED MUTATION, YOU CAN AMPLIFY AND INCREASE THE SPECIFICITY SO THAT NO LONGER REACTS WITH THESE CARBOHYDRATES WHICH ARE VERY SIMILAR AS DOES THE PARENT 1. SO THEY ARE SMALL, THEY HAVE HIGH AFFINITYS, AND LOW AFFINITYS, SOME OF THEM. THEY CAN HAVE FUNCTIONAL EFFECTS AND THEY–THEY HAVE–THEY CAN RECOGNIZE PROTEIN ANTIGENS AS WELL AS NON–SO THIS–I WON’T GO INTO IN DETAIL BUT I JUST WANT TO MAKE 1 POINT, THE LITTLE VARIABLE REGION THAT’S ENCODED BY THIS LOOP THAT COMES OUT THAT I MENTIONED EARLIER IS NOT ALWAYS INVOLVED IN BINDING TO ANTIGENS. THESE ARE 1S THAT HAVE BEEN SEEN BEFORE, THIS IS THE 1 THAT I’VE BEEN TALKING ABOUT MOST BUT THESE EITHER DON’T HAVE THE LOOP FOR REASONS THAT I ALLUDED TO EARLIER OR THEY DON’T USE THE LOOP EVEN IF THEY HAVE IT IN BINDING TO THE ANTIGEN. SO, BECAUSE OF THESE ADVANTAGES ANDAISE ENGINEERING BECAUSE THERE’S ONLY 1 TYPE OF GENE, WE THOUGHT PERHAPS BECAUSE OF THE PHILOGENETIC DISTANCE AND LACK OF TOLERANCE CONSTRAINTS THAT WE HAVE, THAT THEY MIGHT BE ABLE TO SEE ANTIGENS THAT ARE NORMALLY IGNORED BY OUR CONVENTIONAL ANTIBODIES AND WE HAVE 2 EXAMPLES THAT I WOULD LIKE TO BRIEFLY SHOW IN CLOSING THAT ARE PROOF OF PRINCIPLES OF THIS PREDICTION. THIS IS AN ANTIBODY THAT [INDISCERNIBLE] MADE BY IMMUNIZING WITH MULTIPLE MYELOMA CELLS, THE MALIGNANT PLASMA CELLS AND HE SELECTED MONOCLONAL LAMPREY ANTIBODY THAT ONLY REACTS WITH PLASMA CELLS. BOTH HUMAN PLASMA CELLS AND NONHUMAN PRIMATES AS WELL, IT DOESN’T SEE ANY OF THESE EARLY STAGES OF B-LINEAGE DEVELOPMENT NOR OF OTHER CELL TYPES PRESENT IN SPLEEN, BLOOD, TISSUE, TONS ILLEGALSS, HERE’S ANOTHER 1 THAT GOETZPAYS EHRHARDT MADE AND THAT 1 TURNS OUT TO BE SPECIFIC FOR ACTUALLY A WELL KNOWN SURFACE ECTOENZYME CALLED CD38. AND THAT WAS SURPRISING BECAUSE CD38 IS A CELL SURFACE PROTEIN THAT IS EXPRESSED BY ALL BLOOD LINEAGES AND SUBPOPULATIONS OF T-CELLS WHICH WAS A GREAT SURPRISE TO US THAT WAS SEEING THIS PARTICULAR ANTIGEN, IT TURNS OUT A JAPANESE GROUP HAS SHOWN THAT THIS MAINLY ON THE SURFACE OF CELLS, HIGHER LEVELS IT FORMS A DIMER AND AT HIGHEST LEVELS IT FORMS A DIMER OF DIMERS AND A CARBOHYDRATE ADDITION AT THAT POINT PREVENTS FURTHER MULTIPERRIZATION OF THIS CD38 MOLECULE THIS MM3 RECOGNIZES THE DIMER OF DIMER FORMS AND IT’S–IT SEES A SITE THAT IS CLOSE TO OR MAYBE IT A SITE WHERE ECTOENZYME ACTIVITY BECAUSE NANAND GLYCO HIDE ROLLIZE ACTIVITY, USING A NONSUBSTRATE WILL BLOCK BY THIS ANTIBODY AND VICE VERSA THE ANTIBODY WILL BLOCK THE BINDING OR FUNCTIONAL ACTIVITY OF THIS FOSTER NURSED FOCUSED ON LIAISON FACE. FOSTER NURSED FOCUSED ON HIDE ROL ICE, EXCUSE ME THIS 1 IS ANOTHER 1 HE MADE BY IMMUNIZING AND USING PLASMA CELL LINES. AND IT SEES ONLY MEMORY B-CELLS IN PLASMA CELLS, AND NOT–IT SEES BOTH MEMORY B-CELLS THAT STILL EXPRESS IGD OR NOT BUT DOESN’T SEE THE OTHER TYPES OF CELLS HERE WE’RE LOOKING AT BLOOD AND TONS ILLEGALSS. –TONS ILLEGALSS. THIS 1 TURNS OUT MORE SURPRISINGLY TO SEE A MOLECULE, MHC CLASS 1 THAT’S PRESENT ON EVERY CELL IN OUR BODIES EXCEPT RED BLOOD CELLS THAT HAVE LOST THEIR NUCLEUS AND THIS CELL AND THIS ANTIBODY SEES A SPECIAL SULFATED TIARAS ARE SEEN ON THAT CLASS 1 MOLECULE THAT’S MADE BY MEMORY B-CELLS AND PERSISTS INTO THE PLASMA CELL STAGE. AND IF YOU DELETE 1 OF THE 2 ENZYMES THAT CAN CAN ADD THE SULFA TAIS INHIBITOR, THEN YOU LOSE THE REACTIVITY. SO, I WOULD LIKE TO CLOSE WITH BY SAYING THAT MANY PEOPLE HAVE PARTICIPATED IN THIS WORK, I MENTION MANY OF THEM DURING THE TALK. AND INVOLVE THE WORK OF MANY DIFFERENT COLLEAGUES AND COLLABORATORS. I DIDN’T MENTION ENOUGH THOMAS BOONE WITH WHOM WE’VE COLLABORATED TO DEFINE THE THYMUS EQUIVALENT IN LAMPREY’S AND MANY OTHER THINGS IN IN MODEL AND IN CLOSING I WOULD LIKE TO ACKNOWLEDGE THAT I’M A CONFOUNDER OF THE EARLY STAGE COMPANY NOVA, THEY HAVE A NOVEL ANTIBODIES THAT MAKES LAMPREYANTIBODIES ON POTENTIAL FOR BIOMEDICAL USES AND WITH THAT THANK YOU VERY MUCH FOR YOUR KIND ATTENTION. [ APPLAUSE ]>>THANK YOU MAX AND ALTHOUGH WE ARE RUNNING A FEW MINUTES LATE. I’M SURE MAX WOULD BE HAPPY TO ANSWER A COUPLE OF BURNING QUESTIONS SOPHISTICATEDY PLEASE USE THE MICROPHONE, OF COURSE, JAY?>>BEAUTIFUL SERIES OF STUDIES. SO, EVEN THOUGH THESE ANTI–THESE VLR ANTIBODIES SEE CARBOHYDRATES AND SOME SOME HYDRO FILIB RESIDUES IN THE SITES THE FACT THAT THE FRAMEWORK IS MADE OF LUCINE RICH REGIONS DOES THAT EFFECT THE RANGE OF SPECIFICITIES AT ALL PERHAPS FAVORING HYDROPHOBIC ANTIGENS OR ANTIGENS THAT HAVE HYDROPHOBIC REGIONS THAT CAN ALSO INTERACT WITH THESE FRAMEWORK LUCINE RICH REGIONS?>>I DON’T HAVE A GOOD ANSWER TO THAT QUESTION. WE HAVEN’T NOTICED ANY CONSTRAINTS IN THAT RESPECT BUT, I COULDN’T SAY THAT THERE ARE NONE.>>MAX, GREAT TALK, DO YOU KNOW ANYTHING ABOUT ANTIGEN PRESENTATION TO THESE VLR A-CELLS? DO THEY SEE NATIVE ANTIGEN SORE SOME FORM PROCESSED ANTIGEN OR NO MHC ON MOLECULES SO WHO?>>WE KNOW THAT THE VLR A AND C CELLS, ARE T-LIKE CELLS RESPOND WITH PROLIFERATION, PREFERENTIALLY TO ALOE STIMULATION. WHEREAS THE VLR-Bs RESPOND EQUALLY WELL TO ALOE OR ANY OTHER ANTIGEN PROTEIN OR CARBOHYDRATES. BUT WE DON’T KNOW WHAT THE ALOE DETERMINANTS ARE AND THAT’S A KEY QUESTION. AND THEREFORE, CAN’T ANSWER ANYTHING ABOUT ANTIGEN PRESENTATION AND SO FORTH.>>LAST QUESTION.>>IN THE ABSENCE OF IDENTIFIABLE RAG GENES WHAT ARE THE PROTEINS AND MECHANISMS THAT MEDIATE THE RECOMBINATION ACTIVITY FOR THE VLRs?>>I’M NOT SURE WHAT BUT THEY HAVE LOTS OF THE SAME CHARACTERISTICS AS THE AID ASSEMBLY OR DIVERSIFICATION OF THE GENE CONVERSION DIVERSIFICATION OF AVIAN ANTIBODIES. WE HAVE LOTS OF–WE DON’T KNOW THE BASIC BIOCHEMISTRY. I DIDN’T POINT OUT CLEARLY THE DONOR SEQUENCES DON’T CHANGE LOCATION. THEY ARE NEVER MOVED FROM THEIR ORIGINAL LOCATION, THEY MERELY SERVE AS TEMPLATES FOR COPYING IN THE SEQUENCES AND THYSELF SIGHTA DINE DEAMNAISS ARE CREDITED TO BE INVOLVED AS KEYS TO THAT FUNCTION BUT NOT–IT’S A KNCHT MECHANISM THAN REGULAR GENE CONVERSION, SEQUENCES SOMETHING THAT’S ALREADY THERE, THESE ACTUALLY BRING IN SEQUENCES FROM THE DONOR TEMPLATE USING IT AS A TEMPLATE. WE DO NOW HAVE THOMAS BOONE HAS UNPUBLISHED DATA AND SO DO WE THAT’S NOT QUITE AS FAR ADVANCED AS HIS THAT IF YOU DO KNOCK OUT USING CRSPR-CAS SYSTEM, CAN YOU PREVENT THOSE KNOCK OUTS LACK THE ABILITY TO EXPRESS A ASSEMBLED VLRB GENES SO THEY ARE ESSENTIAL LIKE AID, IN A GENE CONVERSION LIKE MECHANISM AND MOLECULAR CHARACTERISTICS OF WHICH ARE DETAILS OF WHICH WE STILL DON’T KNOW. >>THANK YOU.>>I THINK WE’RE GOING TO HAVE TO MOVE ON. SO LET’S THANK MAX AGAIN THERE WILL BE A SMALL RECEPTION IN THE
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