>> I APOLOGIZE FOR THE DELAY. I WOULD LIKE TO WELCOME YOU ALL. BEFORE I INTRODUCE CHENG ZHU, I WOULD LIKE TO MAKE AN ANNOUNCEMENT THAT WE HAD A TECHNICAL GLITCH WITH THE MOVIES SO THE TALK WILL BE ON VIDEO CAST BUT NOT ON ADOBE CONNECT. I'M SORRY ABOUT THAT. ANYWAY -- >> [INDISCERNIBLE]. >> THERE ARE TWO KINDS OF WEB CAST, ONE THROUGH ADOBE CONNECT AND ONE THROUGH THE WEB CAST AND ONE OF THEM IS UNAVAILABLE TODAY. >> [LOW AUDIO]. >> YES. I WOULD LIKE THE WELCOME YOU ALL FORKER TODAY'S IIG SEMINAR. IT'S MY PLEASURE TO INTRODUCE CHENG ZHU WHO IS REGENTS PROFESSOR OF BIOMEDICAL ENGINEERING AT GEORGIA TECH FOR MECHANICAL ENGINEERING AND PHYSICS. CHENG ZHU TRAINED AS A [INDISCERNIBLE] PHYSICIST AND DID MATHEMATICAL MODELLING FOR HIS GRADUATE WORK AND POST DOC WORK AT COLUMBIA UNIVERSITY AS WELL AS AT UNIVERSITY OF CALIFORNIA, SAN DIEGO, AND HE'S DESCRIBES HIMSELF AS A SELF-START EXPERIMENTALIST. HE SET UP HIS OWN LAB. OVER THE PAST 20 YEARS HE HAS DEDICATED HIS LIFE TO STUDYING TWO DIMENSIONAL INTERACTIONS, ESSENTIALLY INTERACTION BETWEEN PROTEINS THAT ARE EXPRESSED ON TWO CELL SURFACES, AND HE'S BASICALLY ASKED THE SIMPLE QUESTION WHETHER TWO DIMENSIONAL INTERACTION KINETICS ARE ANY DIFFERENT FROM DI KI NE TICKS WE MEASURE IN SOLUTION TECHNIQUES SUCH AS [INDISCERNIBLE], AND HE HAS [INDISCERNIBLE] BIOPHYSICAL TOOLS BASED ON A VERY SIMPLE [INDISCERNIBLE] OF CALCULATING THE POWER OF ADHESION BETWEEN TWO SURFACES AS YOU BRING THEM TOGETHER AND TAKE THEM APART. HE'S USEDED THIS SYSTEM OVER THE YEARS AND DESCRIBE THE KINETICS FOR MOLECULES SUCH AS INTEGRINS, SELECTIN, Fc RECEPTORS AND THE FIRST ONE TO DESCRIBE A CATCH BOND FOR SELECTINS AND INTEGRINS WITH THEIR RESPECTED LIGANDS. IT'S INITIALLY A BOND THAT INCREASES BECAUSE OF THE FORCE ON THE MOLECULE IN LOCATIONS SUCH AS IN THE BLOOD WHERE YOU HAVE SHEER FORCES. TODAY HE'S GOING TO TELL US ABOUT HIS WORK WITH TCR-MHC RETRACTIONS AND [INDISCERNIBLE] CORE RETRACTIONS WHICH HE'S BEEN DOING IN THE PAST FIVE TO SEVEN YEARS AND HAS HAD A LONG-STANDING COLLABORATION WITH BRIAN EVAVOLD'S LAB AND HAS DISCOVERED SOME UNUSUAL CHARACTERISTICS ABOUT THESE THINGS AND HE'S GOING TO TELLS ABOUT HIS WORK. I'D LIKE TO WELCOME, CHENG ZHU. [APPLAUSE] >> THANK YOU, RAJAT VARMA, FOR THE KIND INTRODUCTION. I CAN'T TELL YOU HOW MUCH I FEEL HONORED TO BE ABLE TO STAND HERE AS A MECHANICAL ENGINEERING BY TRAINING, NOT EVEN THEORETICAL PHYSICS TO TALK ABOUT IMMUNE IMMUNENOLOGY TO THE BEST RESEARCHERS. IF I DO NOT SPEAK WELL, THAT'S PART OF THE REASON. I WANT TO TELL ME, UM, WHAT WE HAVE DONE FIRST DEVELOPING TWOS AT THE SURFACE OF LIVE CELLS ACROSS THE MEMBRANE BETWEEN TWO CELLS, THOSE TRANCE INTERACTIONS, AND THEN USE THOSE TO ANALYZE INTERACTIONS OF MOLECULES THAT ARE IMPORTANT TO IMMUNOLOGY AND T CELL RECEPTOR BEING ONE OF THEM AND WE HAVE ALSO DONE OTHER SYSTEMS. I GOT THIS IMAGE FROM MIKE DUSTIN WHO I COLLABORATE WITH MORE THAN A DECADE. HERE YOU SEE A T CELL AND AN ANTIGEN PRESENTING CELL AND THE MOLECULES THAT -- WHOOP, HOW COME'S NOT SHOWING? SO OF COURSE WE WOULD TALK ABOUT T CELL PRIMARILY THAT INTERACT WITH PEPTIDE COMPLEX AND [INDISCERNIBLE]. WE HAVE DONE WORK WITH L PAY ONE INTERACTING WITH [INDISCERNIBLE] ONE AND MAYBE I WILL SHOW ONE SLIDES OR ONE PANEL DATA THERE. WE HAVE PUBLISHED INTERACTIONS BETWEEN CD 50 A AND CD TWO, BUT I'M NOT GOING TO TALK ABOUT THAT TODAY, AND WE HAVE NOT DONE THIS INTERACTION YET BUT WE'RE IN THE PROCESS OF DOING THAT. SO RAJAT VARMA ELUDED TO THE DIFFERENCE BETWEEN WHAT I CALLED IT THREE DIMENSIONAL BINDING VERSES TWO DIMENSIONAL BINDING. THESE TERMS ARE LOOSELY DEFINED. THE IF YOU REALLY WANT TO LOOK AT RIGOROUS DEFINITION, THEY DON'T HOLD WATER. SO WITH THAT CAVEAT, LET'S LOOK AT WHAT I MEAN BY 3 D. YOU HAVE ONE MOL KUL MOLECULE IN SOLUTION LIKE A GROWTH FACTOR CYTOKIND. THE RECEPTOR IN THE CELL MEMBRANE TYPICALLY HAS A HUNDRED THOUSAND COPIES. THEN YOU GET NANO GRAM OR MICROGRAM, LOTS OF LOOSE MOLECULES THERE. YOU ALL USE DETERMINUS TICK KI NE I CANS TO DESCRIBE THE INTERACTION, AND THERE ARE [INDISCERNIBLE] METHODS THAT EXIST FOR MEASUREMENTS OF KINETICS AND AFFINITY. LIKE [INDISCERNIBLE] IN THIS CASE, IN THAT CASE IT'S THE [INDISCERNIBLE] PLASMIC RESIDENT TECHNIQUE, OKAY. NOW, THEN WE LOOK AT 2D BIDING WAS BETWEEN TWO CELLS ACROSS THE JUNCTIONAL SPACE OR BETWEEN A CELL AND EXTRA CELLAR MATRIX SUB STREAM WHEN BOTH MOLECULES ARE SERVICE BOUND OR SOME PEOPLE REFER THAT TO [INDISCERNIBLE] BIDING AND HOW CAN IT BE [INDISCERNIBLE] IT'S STILL SYSTEM? THE WORK I DESCRIBE TO YOU TODAY ARE SMALL SYSTEM, SO IT'S [INDISCERNIBLE] FRAMEWORK TO MEASURE IT, AND NOW THERE WERE TWO TYPE OF METHODS THAT ARE AVAILABLE. ONE BASED ON FLUORESCENTS AND THE OTHER BASED ON MECHANICAL DETECTIONS. SO I WANT TO KIND OF RUN THROUGH TWO SLIDES TO SUMMARIZE THEM. THE FIRST IS METHOD YOU CAN DO IT WITH LOSS SYSTEM. THE FIRST EK LIB RI YUM ANALYSIS WAS DONE BY MIKE DUSTIN NOW AT NCU. -- NYU. THIS IS NOT ME, IT'S SOMEBODY ELSE ALSO WITH THE SAME LAST NAME. THEY PUBLISHED TWO PAPERS, FIRST ONE IN 1996 AND THEY CALLED IT TWO DIMENSIONAL [INDISCERNIBLE] AND THEN THEY [INDISCERNIBLE]. NOW THE KINETIC ANALYSIS I CONTRIBUTED IN COLLABORATION WITH MIKE DUSTIN WITH TWO PAPERS PUBLISHED THREE E YEARS AGO IN BIO PHYSICAL JOURNAL AND THAT'S WHERE WE ANALYZE THE CD 2 CDNA INTERACTION. IT'S CALLED COUNTY AREA FRAP. THERE WAS ALSO A SINGLE-BOUND BASE SO [INDISCERNIBLE], AND MARK DAVIS' LAB AT STANFORD, JONATHAN HOOPER IS THE FIRST AUTHOR PUBLISHED IN SINGLE MOLECULE [INDISCERNIBLE]. HERE'S THE MECHANICAL METHOD AND SO FAR ALL OF THESE ARE SMALL-SYSTEM BASED. WE HAVE CONTRIBUTED ALL OF THEM AND STARTED OUT LONG TIME AGO [INDISCERNIBLE] OF SETTING MORE RECENTLY -- >> [LOW AUDIO]. >> >> CAN WE TAKE YOUR CELL PHONE? >> OH, MY CELL PHONE? >> I THINK IT'S INTERFERING WITH THE AUDIO. [LAUGHTER] >> DON'T DIAL ANY NUMBERS. [LAUGHTER] BETTER? >> SO FAR. >> OKAY. WHERE AM I? [LAUGHTER] WE'RE TALKING ABOUT KINETIC ANALYSIS. SO THESE ARE THE TWO ASSAYS THAT I'M GOING TO DESCRIBE THE TO YOU TODAY. UP WITH PUBLISHED IN 1998 AND THEN ANOTHER ONE WE PUBLISHED TEN YEARS AFTER THAT. WE'RE GOING TO APPLY THIS METHODS TO THE ANALYSIS OF THIS INTERACTION, A T CELL RECEPTOR INTERACTING WITH A PEPTIDE MHC AND ALSO HAVE THE CORECEPTOR OF CD 8. OF COURSE, TO THIS AUDIENCE, IT'S OBVIOUS WHY WE'RE INTERESTED IN STUDYING THAT BECAUSE THE FUNDAMENTAL HYPOTHESIS IS THAT THIS INTERACTION DIVIDING PARAMETERS OF THIS INTERACTION PLAY A DETERMINING ROLE OF T CELL PEPTIDES BECAUSE TCI IS THE ONLY MOLECULE ON THE T CELL THAT FUELS THE PEP SIDE. A LOFT PEOPLE LIKE TO USE THE WORD SEIZE THE PEPTIDE. I THINK WE SHOULD USE THE WORDS FEEL, IT'S MORE APPROPRIATE. THIS IS THE [INDISCERNIBLE] THAT GAVE US ALL THE TROUBLE IN THE ADOBE FLASH. OH. NOW IF IT DOESN'T PLAY, I REALLY GET PISSED. [LAUGHTER] SO I THINK WHAT HAPPENS IS I PUT IT INTO THE FOLDER IN ORDER TO COPY TO THE OTHER COMPUTER. SO I JUST HAVE TO PLAY IT HERE. SO IT'S NOT LINKED BUT IT PLAYS. OKAY, SO THIS IS THE ADHESION FREQUENCY ASSAY. I DIDN'T HAVE IT LOOP, BUT YOU GET -- YOU GET ON THIS CELL THIS SIDE A [INDISCERNIBLE] CELL THAT YOU ASPIRATE INTO A GLASS [INDISCERNIBLE] THAT CONNECTS TO A PRESSURE REGULATOR SO YOU CAN APPLY VERY GENTLE SUNGS. ON THE OTHER SIDE ANOTHER [INDISCERNIBLE] CONNECTS ASPIRATED T CELLS. THIS [INDISCERNIBLE] IS IN A PA. YOU CAN USE THE COMPUTER PROGRAM TO DRIVE IT BACK AND FORTH. THAT'S WHAT YOU SAW IN THE VIDEO. SOMEBODY ASKED ME TO DO, HOW DID YOU GET TO DO THAT IN THE BEGINNING. SCOTT CHESTER IS THE STUDENT THAT DID THAT IN THE BEGINNING. WHEN I DESCRIBED THAT IDEA WITH THEM I THOUGHT NOBODY WOULD DO THAT BECAUSE THERE'S NO [INDISCERNIBLE]. HE SAID, I'LL DO IT. HE'S ACTUALLY MANUALLY USING THE MANUAL MANIPULATE OR AND MOVING IT BACK AND FORTH. EACH CELL APPEAR TWO HUNDRED TIMES. NOW WE GET 50. IT'S KIND OF GETTING DOWN THE HILL BUT BEFORE IT WAS LIKE TWO HUNDRED TIMES AND IT WAS DONE THE FIRST PAPER. IT'S A BINARY OUTCOME. YOU CAN SEE ADHESION WHEN YOU STRETCH THE RED CELL OR YOU DON'T SEE ADHESION WHEN THE RED CELL IS NOT STRETCHED. HERE, YOU CAN GET BETTER, UM, TO DO THIS. SO NOW YOU CAN PUT A GLASS BEAD ON THE APEX OF THE RED CELL AND YOU CAN TRACK THE MOTION OF THAT BEAD SO IF THE RED CELL GETS STRETCHED, YOU SEE A SPIKE ON THE DATA SLIDE. MAYBE I SHOULD PLAY AGAIN. WHY IS IT DOING THIS? NOT SUPPOSED TO SHOW YOU THAT. [LAUGHTER] NOW THERE WAS AN ADHESION, FORCE IN THE ACCESS, NOW NO ADHESION, NO FORCE. NOW, LET'S SEE. SO NOW, ON THE SURFACE OF A RED BLOOD CELL, YOU COULD [INDISCERNIBLE] RED BLOOD CELL, PUT IN [INDISCERNIBLE] AND THEN USE THE NIH TET MER FACILITY TO SUPPORT THIS WALK [INDISCERNIBLE]. YOU CAN MUTATE THAT DOMAIN TO ABOLISH THE CD 8 BINDING OR YOU CAN ORDER OR MUTATE THE BINDING SITE. TO [INDISCERNIBLE] HAS FOUR BIDING SITES THEN YOU CAN MUTATE THAT TO CORRECT FORMATION OF DIMERS OR TRY MERS HERIMERS HERE. ONLY TWO BIDING SITES TO THE ONLY MONOMER YOU CAN HAVE. SO THE BINARY OUTCOME, YOU DO IT TWO HUNDRED TIMES WHEN SCOTT DOES IT, AND HERE YOU LOOK AT THE BINARY SCORES, ZERO OR ONE. HERE ARE THE ONES AND THE ZEROS. THIS IS WHEN YOU HAVE CONTROL ON THE CELL. IF YOU THEN HAVE IgG WHEN WE STUDY Fc RECEPTOR. WITH FIVE-SECOND THIS IS RUNNING ADHESION FREQUENCY NOW, IT FLUCTUATE WHEN THE SMALL NUMBER STATISTIC WAS LOW NUMBER ACCOUNTS, BUT THEN GOES OUT TO TWO HUNDRED, IT STABILIZE. SO THAT FREQUENCY ESTIMATE THE LIKELIHOOD OF BIDING FROM THE BINARY OUTCOME. YOU CAN SEE THAT IS SPECIFIC COMPARED TO THE BSA AND THEN IT DEPENDS ON THE CONTACT TIME. TEN SECOND VERSUS FIVE-SECOND HERE, YOU ALLOW THE TWO CELL SIT TOGETHER BEFORE YOU SEPARATE THEM. NOW YOU PLOT THE LIKELIHOOD OF FREQUENCY ON THE Y AXIS. [INDISCERNIBLE] VERSES THE CONTACT TIME. YOU GET DATA LIKE THAT YOU MODEL THE ADHESION KI NE KINETICS -- REMEMBER, THAT'S A MODEL AND IT'S A SMALL NUMBER STATISTIC. SO YOU DERIVE A SARCASTIC COURT PART OF THIS DETERMINE NIS TICK MODEL FROM THIS AND THAT'S WHAT IT IS. SO YOU HAVE HERE THE DENSITY OF THE MOLECULES FROM THE T CELL T DENSITY OF THE MOLECULE YOU PLACE ON THE RED CELL, MEASURE THEM BY FLOW CYTOMETRY AND THEN YOU FIT WITH THAT MODEL TO THIS IS WHAT YOU MEASURE, THIS IS WHAT YOU CONTROL [INDISCERNIBLE] AND THE TWO FITTING PARAMETER ARE THE AC -- COUNTER AREA, HOW HOT YOU SQUASH THE RED CELL ON TO THE T CELL AND THE KA IS WHAT WE CALL THE TWO DIMENSIONAL AFFINITY AND THERE'S OFF RATE HERE AND YOU GET THIS NUMBER. BECAUSE THESE TWO NUMBERS ARE PUT TOGETHER AND WE DON'T REALLY KNOW HOW THE DETAIL COUNTER AREA BETWEEN THE RED CELL MEMBRANE AND THE KIND OF RUFFLE T CELL SURFACE WOULD BE. WE LUMPED THEM TOGETHER. [INDISCERNIBLE]. HERE'S ANOTHER ASSAY THAT WE DEVELOPED TEN YEARS AFTER THAT CALLED THE THERMAL FUNCTIONUATION METHOD. IT'S POWERFUL BUT A LOT HEART R HEARTER -- HARDER. WE PUT A GLASS BEAD ON THE APEX OF THE RED CELL. YOU CAN THINK OF THIS AS THE SPRING. THE MOVEMENT OF THIS IN THE X AXIS HERE TELLS YOU THE FORCE [INDISCERNIBLE] ON IT. SO UH NOW YOU GOT A T CELL HERE. WHEN THERE WAS A MOLECULE ABOUND BETWEEN A MC FOLLOW MOL KUL AND THE T CELL RECEPTOR HERE, IT'S LIKE A MOLECULE OR SPRING. SO NOW THESE TWO SPRINGS ARE ACTUALLY IN PARALLEL. THE SYSTEM SPRING, IT'S ACTUALLY THE SUM OF THE TWO. NOW TT CELL IS VERY SOFT, SO YOU CAN ACTUALLY SEE THE BEAD IS MOVING BACK AND FORTH BY THERMAL FRUNGSUATION. IF YOU ANCHOR THE BEAD WITH ANOTHER MOLECULAR SPRING TO THE T CELL T FLUCTUATION IS GOING TO [INDISCERNIBLE] CONTINUALLY. YOU LOOK AT THE T CELL AND SAY THE FRAKTUATION WAS REDUCED. MAYBE THERE'S A FORMATION [INDISCERNIBLE]. BEST WAY TO DO IT IS TO LOOK AT MOVIES. SO THIS IS [INDISCERNIBLE]. SO THE Y AXIS POSITION OF THE BEAD TX AXIS IS TIME. THIS IS ACTUALLY THERMAL FLUCTUATION, THAT'S PUSH AND RETURN BACK TO THE KNOWN POSITION. YOU SEE SOMETHING HAPPEN COMING UP PRETTY SOON. YOU SEE THE MEAN POSITION SHIFTED UPWARDS AND THEN THE FLUCTUATION AMPLITUDE GREATLY REDUCED. ALL RIGHT. THE WAY TO LOOK AT FLUCTUATION IS, UM, TO LOOK AT THE STANDARD DEVIATION. SO I'M GOING TO -- NOW THIS IS REALTIME AND YOU'RE GOING SEE THE FIRST PANEL IS WHAT YOU SAW. THIS PANEL IS [INDISCERNIBLE] OF THIS FLUCTUATION, AND THIS IS THE HIS-SELF GRAM OF THIS STANDARD DEVIATION. WHEN YOU HAPPEN HERE, WHEN YOU THINK THERE IS ABOUT STANDARD DEVIATION IS LOW, AND THE HIS TOE GRAM OF THESE GUYS ARE HERE, THE HIS TOE GRAM OF THESE GUYS ARE HERE SO THE TWO POPULATION CAN CLEARLY BE DISTINCT. . THAT'S HOW YOU DO IT. LET'S HAVE SOME FUN WITH BIOLOGY. SO NOW -- OH, BEFORE THAT. SO IF WE KNOW WHEN THE BOND IS FORM AN WE KNOW WHEN THE BOND IS [INDISCERNIBLE], THE DURATION WHERE YOU HAVE THE BOND IS A LIFETIME. THE FASTER THE OFF RATE, THE SHORTER IS THAT LIFETIME. THE OTHER STATISTIC IS WHEN A BAND IS [INDISCERNIBLE], HOW LONG DO YOU HAVE TO WAIT TO OBSERVE THE NEXT BOUND FORMATION. IF THE ON-RATE IS HIGH, YOU DON'T HAVE TO WAIT LONG. IF YOU HAVE A SLOW ON RATE, YOU TO WAIT A LOT LONGER. SO YOU JUST LOOK AT THE STATISTICS OF THOSE AND IT'S FOR THIS CASE A SINGLE EXP POE NEN SHALL DECAY. YOU TAKE THE [INDISCERNIBLE] THE EXPONENTIAL, IT'S A STRAIGHT LINE AND THIS IS OT 1 T CELL INTERRUPTED WITH THE OVARIAN PEPTIDE [INDISCERNIBLE], THIS IS A G 4 MUTANT. YOU SEE THE OFF RATE IS ACTUALLY DIFFERENT. THE SLOPE IS THE OFF RATE. NOW, FIRST COMMENT I WANT TO MAKE IS THIS [INDISCERNIBLE] ASSAYS ARE HIGHLY SENSITIVE. THIS IS A PAPER PUBLISHED THE [INDISCERNIBLE] THIS YEAR BY [INDISCERNIBLE]. THEY LOOK AT POLLY CARNAL T CELL THAT IS RESPONSIVE TO A PEPTIDE DERIVED FROM MULTIPLE SCLEROSIS. COMPARED TO THE TETRAMER STAINING AND WHAT IS MEASURABLE BY OUR 2D METHOD -- OF COURSE THESE ARE ALL SPECIFIC, I DIDN'T SHOW YOU THE NON-SPECIFIC CONTROL -- THERE WAS MUCH GREATER FRACTION OF CELLS THAT YOU CAN DETECT BY OUR METHOD COMPARED TO THE TETRAMER STAINING IN CENTRAL NERVE SYSTEM, SPLEEN AND ALSO IN LUNG. NOW, SO THE REASON BEING THAT OUR ASSAY IS SINGLE-MOLECULE BASE. IF YOU BELIEVE THAT FOR A T CELL TO GET TRIGGER, AT LEAST ONE MHC BINDS TO ONE TCR IS REQUIRED THEN WE CAN DETECT THAT. SECONDLY, WE CAN PLACE MANY, MANY MORE MHC MOLECULE ON THE RED CELL MEMBRANE THAN A TYPICAL APC, TEN TIMES MORE EVEN HUNDRED TIMES MORE. SO WHATEVER THE T CELL WILL SEE IN VIVO WHEN THEY [INDISCERNIBLE] APC, WE CAN CERTAINLY DETECT THAT, AND EVEN MORE SENSITIVE. ALL RIGHT. SO LIKE IN THIS CASE, THIS IS THE TETRAMER [INDISCERNIBLE] POPULATION. WE DON'T HAVE TO P PUT MANY MORE MHC MOLECULE ON THE REST [INDISCERNIBLE] IF IT'S A TET TRI MER [INDISCERNIBLE] TET TRI MER -- THIS THE TE TRI MER [INDISCERNIBLE] CELLS ARE ACTUALLY MAKING CYTOWINES -- THIS IS A PAPER THAT WE PUBLISHED LAST YEAR IN NATURE THAT REPORTING HOW THE 2D AND 3 D PARAMETER COMPARE. COLUMN ON THE LEFT IS 2D ON THE RIGHT IS 3 D. THIS IS AFFINITY, ON RATE AND OFF RATE. FIRST OF ALL, YOU LOOKED AT THE DYNAMIC RANGE. THIS MATCHED THE SAME NUMBER OF LOCKS. THEY ARE NOT OF THE SAME UNIT SO YOU CAN'T COMPARE THE ABSOLUTE VALUE, BUT YOU CAN COMPARE THE RELATIVE SPEND. SO THIS IS ABOUT A ONE LOCK VARIATION, THIS IS THREE LOCK VARIATION. TWO ORDER OF [INDISCERNIBLE] GREATER DIE DYNAMIC RANGE. IF YOU ROOK LOOK AT THE NUMBER IT'S ACTUALLY COME RABLT TO [INDISCERNIBLE] COMPARABLE. WE GOT FOUR LOCKS VARIATION HERE. IT'S ACTUALLY VERY. THE MOLECULE THAT I STUDIED A LOT IS SELECT SELECTIN FAMILY OF MOLECULES AND IT'S VERY FAST. IT'S ACTUALLY COMPARABLE OR EVEN FASTER. THIS GUYS HERE THAN P SELECTIN. THIS IS THE MOST CONTROVERSIAL RESULT THAT A LOT OF PEOPLE P ASK ME AND I'M GOING SHOW YOU LATER SOME RESULT THAT THIS IS NOT NECESSARY UNIVERSAL OBSERVATION. IN THE 3 D MEASUREMENT, THERE ARE A LOT OF THEM NOW. I WOULD SAY THE SLOWER THE OFF RATE OR LONGER THE HALF LIFE OF THE PEP MHC BOUND WITH [INDISCERNIBLE]. WE GOT IT IN THE OPPOSITE TREND. THEN YOU FEEL COMPARE THE NUMBER. SO OFF RATE HAS THE UNIT OFF ONE PER UNIT TIME AND IT'S THE SAME ON BOTH 2D AND 3 D. YOU COMPARE THE NUMBER. LOOK AT THIS NUMBER AND THAT NUMBER. IN EXTREME CASES, A THOUSAND FOLDS DIFFERENCE. ALSO IT'S VERY, VERY FAST, THESE GUYS ARE VERY, VERY SLOW. HOW FAST? IT'S ACTUALLY FASTER THAN THE OFF RATE OF [INDISCERNIBLE] IS THE FATTEST OFF RATE OF THREE SELECTINS DISASSOCIATE FROM PSGR ONE. THE 2 PARAMETER WE HAVE T CELL RESPONSIVE MAY BE BETTER THAN THE 3 D PARAMETER. LET ME SUMMARIZE WHAT WE'VE DESCRIBED SO FAR. MECHANICAL BASE METHOD HAVE DEVELOP IN SIDE SHOW ANALYSIS OF CROSS [INDISCERNIBLE] AT THE T CELL SURFACE. THESE METHODS ARE FAR MORE SENSITIVE THAN THE SPR AND TETRAMER TECHNOLOGIES. 2D AND 3 D PROGRAM TERS CAN BE SUBSTANTIALLY DIFFERENT. THE 2D PARAMETERS SEEM BETTER CORRELATE WITH THE T CELL RESPONSIVENESS THAN THE 2D PARAMETERS. 3 D PARAMETERS. NOW I'LL MOVE TO THE NEXT TOPIC LOOKING AT THREE MOLECULES. HERE IS THE CORECEPTOR. THE DATA I SHOWED YOU BEFORE WAS USING A MUTANT [INDISCERNIBLE] MO MOLECULE. NOW WE USE THE WILD TYPE [INDISCERNIBLE]. HOW TO HANDLE [INDISCERNIBLE] INTERACTION? THAT'S THE QUESTION. UM, THIS IS THE MODZ L THAT WE HAVE DESCRIBED FOR SINGLE SPECIES BIDING, BUT NOW WHAT HAPPENED YOU MAY HAVE MULTIPLE SPECIES LABEL BID THE SUBSCRIPT, I. YOU GET -- THIS IS THE AFTERNOON NUMBER OF BOUNDS THIS INTERACTION WILL FORM BASED ON THE SYMBOL MOD. YOU GET I SPECIES ONE TWO THREE FOUR. THE THEORY WAS ACTUALLY -- AND WE PUBLISHED THAT. THE AVERAGE NUMBER OF BANDS ARED A TI, POWERBILITY OF ADDS HEEGS ARE NOT ADDITIVE. IT CAN WORK ON INDEPENDENT BINDING. WE HAVE DONE DIFFERENT SPECIES OF MOLECULES THAT DO NOT CROSS TALK. I COULD DO ALSO COMPETITIVE BIDING. THIS IS PUBLISHED ELEVEN YEARS AGO AND FOR, LET'S SAY WE TEST IT WITH IgG ONE AND TWO, BIND IT TO Fc GAMMA RECEPTOR. -- THEN NOW YOU MIX THEM INTO DUR ROE SPECIES. FIRST, YOU PROTECT IF YOU MIX THEM TWO TO ONE RATIO OR TWO TO THREE RATIO BASED ON THE PARAMETER YOU -- WHERE THE DATA ARE SUPPOSED TO DO THEN YOU DO THE EXPERIMENT. DATA ACTUALLY FORCE MOSTLY INTO TA 95% [INDISCERNIBLE], AND THEN THE FIT IS ACTUALLY WELL WITHIN THAT [INDISCERNIBLE]. IT WORKS. NOW, TAKE THAT AND YOU THINK ABOUT THE CD 8, NOW BRING THAT INTO THE PICTURE. ALL RIGHT. SO HERE'S THE PARAMETER THAT MEASURE IN 3 D. -- THIS IS WHAT WENESS 2007, 2D. IT'S MUCH LOWER, HUNDRED FOLDS LOWER THAN THE AGONIST PEPTIDE INTERACTION COMPARABLE TO SOME OF THE WEAK PEPTIDE INTERACTION WITH TCR. THIS ON RATE, THERE'S OFF RATE. YOU WOULD PREDICT THAT WHEN YOU HAVE THIS INTERACTION YOU WILL NOT SEE THE INTERACTION BY THE CD 8 BECAUSE SUCH LOWER AFFINITY. EVEN IF YOU GET MAYBE TWOFOLDS OR THREEFOLDS HIGHER DENSITY OF CD #, IT WOULD NOT COMPENSATE FOR A HUNDRED FOLD DIFFERENT IN THE AFFINITY. SO THAT'S WHAT THE MODEL PREDICT. NOW, SO WHEN YOU HAVE MULTIPLE SPECIES INTERACTION ON T CELL SURFACE, UM, THERE ARE ACTUALLY, UM, MANY THINGS THAT YOU CANNOT JUST ASSUME. THE MODEL WE HAVE IS INDEPENDENT BIDING, CONCURRENT INDEPENDENT BIDING, RIGHT? WHAT IF THE BIDING IS NOT CON CURRENT, IT'S SEQUENTIAL? WHAT IF THE CORROBORATIVE? YOU COULD GET COMPETITIVE OR INHIBITORY OR SYNERGISTIC BIDING. IN F IF THE MOLECULES CROSS TALK -- [INDISCERNIBLE] BY T CELL RECEPTOR -- THEN THE CD 8 COMES IN AND INFORMS A TRI MOLECULAR COMPLEX. IT'S ONE BIG EXPERIMENT. IF YOU USE THE KNOWN PEPTIDE YOU DON'T SEE ANY BIDING AT THE DENSITY OF [INDISCERNIBLE]. NOW THAT WAS STILL BY CD 8. BECAUSE OF THE LOW AFFINITY OF CD 8 AT THE DENSITY USED, YOU DON'T SEE ANYBODY, BUT IF YOU CHANGING THAT WITH AGONIST -- THIS IS AGONIST TO THE Fc, F FIGHT T CELL RECEPTOR, THEN YOU SEE SOMETHING LIKE THAT. THIS IS A [INDISCERNIBLE]. YOU SEE A ROUGHLY TWO STEP, WE CALL IT TWO STAGE KINETICS. IF YOU USE AN ANTICD 8 ANTIBODY TO BLOCK THE -- THE SECOND STATION IS D 8-DEPENDENT. WAIT A MINUTE, CD 8 BY ITSELF DOESN'T BIND. YOU CAN ALSO USE ANTITCR ANTIBODY, AND THE OOENT TCR ANTIBODY WOULD BLOCK THESE THINGS TO THE GROUND. WITHOUT THE TCR BIDING, THE CD 8 PER SE WOULD NOT GIVE YOU ANYTHING. THAT'S THE F FIVE SYSTEM AND IT'S A DBMHC AND YOU CAN REPEAT THE EXPERIMENT. IT'S THE SAME THING DATA, BUT YOU CAN NOW USE A MUTANT KB THAT ABOLISH THE CD 8 BIDING WITHOUT USING ANTIBODY. WORRY ABOUT THE ANTIBODY DOING SOMETHING FUNNY. Y NOW, IF YOU USE A MUTANT MHC MOLECULE THAT ABOLISH TCR BIDING BUT ALLOWS T CELL RECEPTOR BIDING -- WE KNOW THAT'S ONLY A FLAT CURVE BECAUSE ABOLISH THE STAGE, RIGHT? IF YOU USE 100% NO PEPTIDE BUT IT'S A WILD TIME [INDISCERNIBLE] THAT ALLOWS CD 8 BINDING AT THAT DENSITY, YOU DON'T SEE ANYTHING. NOW YOU MIX THE TWO. YOU MIX THE TWO TO ALLOW TCR TO BIND THIS GUY AND CD 8 TO BIND THAT GUY. WELL, YOU STILL GET A MORE OR LESS CURVE. IT DOESN'T REALLY GIVE YOU THE SECOND STAGE. SO WAIT A MINUTE, YOU HAVE CD 8 BIDING SITE, IT JUST NOT IN THE SAME MHC MOLECULE. SO IT ARGUES THAT CD 8 HAS TO BIDE TO THE SAME MHC MOLECULE THAT ENGAGED THE T CELL RECEPTOR. OKAY. UM, IF YOU TIE TRAITED THE MHC DENSITY GOING FROM THE 22 PER SQUARE MILE MOLL QUME TO ABOUT TEN TO ABOUT THREE, YOU GET THIS TWO-STAGE CURVE. AT THIS LEVEL, EVEN THE AGONIST PEPTIDE BINDING IS REALLY, REALLY LOW, AND YOU WILL GET MOST OF THE TIME NO BIDING AND WHEN YOU GET DO GET BIDING, ALMOST 99% OF THE TIME IT'S A SINGLE MHC ENGAGEMENT. YOU ARE STILL ABLE TO TRIGGER THE SECOND STAGE. WE DO THE EXPERIMENT BY REPEATEDLY BRINGING THE RED CELL IN TOUCH WITH THE T CELL, AND YOU WORRY AT HOME MAYBE ONE TOUCH YOU KIND OF ACTIVATE A T CELL AND THEN WHEN YOU DO THIS REPEAT TOUCHES, GOD KNOWS YOU KIND OF AROUSE THE T CELL TO THE EXTEND IT SPITS SOME STRANGE CHARACTERISTIC, BUT IF YOU ONLY DO A SINGLE TOUCH [INDISCERNIBLE] YOU DO STATISTIC THAT WAY, YOU GET THE SAME. IT'S NOT REQUIRE FOR REPEAT TOUCHES. IF YOU USE PT TWO TO INHIBIT THE CIRCUMFAM KINASE, YOU ABOLISH THE SECOND STAGE. THAT ARGUES THAT THE COOPERATIVE BIDING OP BETWEEN T CELL RECEPTOR AND CD 8 TO THE SAME MHC MOLECULE REQUIRES SOME SIGNALING. SO WE COME BACK TO THIS PROPOSED MECHANISM. SO WE SAY, OKAY, IF YOU DO THIS ONE, WE ACTUALLY MAKE THE MEASUREMENT AND WE GET THESE NUMBERS. NOW, IF YOU LOOK AT THIS ONE, WE MAKE THE MEASUREMENT, DO THAT. THEN WHEN YOU GO FROM HERE TO HERE, THERE IS A EQUATION THAT ALLOWS YOU TO DO TRI MOLECULAR ACTION AS A TRI MOLECULAR BOUND, BUT THERE WAS A ONE-SECOND DELAY. WHERE DOES IT COME FROM? WELL, IF YOU THINK ABOUT THE CDA AND TCR, THEY BIDE THROUGH THEIR EXCEL LAR DOMAIN [INDISCERNIBLE] ACROSS [INDISCERNIBLE] SPACE TO THE OTHER APC. THEY WERE ALSO INTERCELLAR INTERACTIONS BETWEEN THE TWO VIA DOCKING MOLECULES AND SIGNALING AND ALL THAT. THAT'S NOT REALLY BEING MODELLED HERE, AND THAT COULD PICK UP SOME TIME. SO THE [INDISCERNIBLE] MEASURE SYSTEM A LOT MORE COMPLEX THAN WHAT YOU PUT NIT THE VEHICLE MACHINE. NOW UH, SO THE LAST THING HERE IS ACTUALLY IT WAS SAYING THAT THE TWO STAGE BIDING FOR YA TWO STAGE DISCRIMINATION. SO IF YOU LOOK AT THE INCREMENT, SO YOU CAN ACTUALLY MEASURE THE NUMBER OF BOUNDS -- OOPS, GO BACK. SO WHAT YOU HAVE HERE IS THE ADHESION ABILITY WHEN YOU HAVE BOTH THE T CELL RECEPTOR AND CD 8 CONTRIBUTING AND YOU LOOK AT THE PAM -- PANEL OF PEPTIDES. NOW, UH -- WHOOPS. THINK I GOT THESE SLIDE REVERSED. ANYWAY. SO THIS IS ONE MORE THING BEFORE I GOT -- SO THIS SLIDE WAS TRYING TO SHOW YOU THAT THE SECOND STAGE BIDING IS VERY [INDISCERNIBLE] AND REVERSIBLE, AND LET ME SEE -- SO WHAT DO IS ALTERNATE THE [INDISCERNIBLE] BETWEEN THE SHORT ONE AND THE LONG ONE. FROM THE TWO STAGE BINDING [INDISCERNIBLE] -- WHEREAS THE LONG CONTACT TIME IS A FIVE-SECOND YOU DO HAVE THE CONTRIBUTION OF THE CD 8. NOW, IF BY ALTERNATING SHORT VERSUS LONG AND THEN I SORT THEM SEPARATELY INTO THE GROUP OF SHORT CONTACT TIME AND GROUP OF LONG CONTACT TIME AND DO THE STATISTICS. IT'S LIKE THIS. SO IT'S A LONG THING SO LET'S SPEED IT UP. SO YOU DON'T HAVE TO, UM -- SO YOU CAN EITHER DO [INDISCERNIBLE] CONTACT TIME 50 TIMES YOU GET ADHESION FREQUENCY WHICH IS HERE, AND THEN YOU DO A CONCERN TURN 50 TIMES OF LONG CONTACT TIME, YOU GET ADHESION HERE AND DIFFERENT HERE. BUT YOU ALSO ALTERNATING BETWEEN SHORT AND LONG AND YOU STILL GET THIS, WHICH MEANS THAT THE SECOND STAGE BINDING REQUIRES SIGNALING AND WHEN YOU A FIVE-SECOND CONTACT, YOU TRIGGER THAT [INDISCERNIBLE] ENABLE CD 8 BINDING. THEN SWITCH TO A SHORT CONTACT TIME IN A SECOND TIME. THAT SIGNAL GONE AWAY. IT'S NO LONGER BEING ABLE TO GET THE CD 8 TO BIND IF THE CONTACT TIME IS SHORT. IT GET RETRIGGERED AGAIN IF YOU SWITCH TO A LONG CONTACT TIME. IT'S VERY RAPID, TRANSIENT AND REVERSIBLE. SO, COMING BACK TO THIS POINT OF TWO STAGE BINDING PROVIDES TWO STAGE DISCRIMINATION. WHAT WE DO HERE IS TO LOOK AT THE INCREMENT WITHOUT CD 8 CONTRIBUTION. HERE IS THE NUMBER OF BOUNDS PER DENSITY OF MHC MOLECULE. NORMALIZE AGAINST MHC MOLECULE AND ALSO ONLY LOOKING AT THE INCREMENT, AND THIS IS THE PEPTIDE THAT IN THE ORDER OF INCREASE RESPONSE. SO YOU SEE FOR THE AGNIST THE INCREASE IS ACTUALLY BIGGER FOR THE MORE POTENT PEP SITIDE, AND THIS IS SOMEWHAT ADDITION TO THE PREVIOUS [INDISCERNIBLE] LIKE THE CD 8 BUT STAY THERE TO HELP WEAK PEPTIDE AND FOR THE STRONG L LIGAND, YOU DON'T REALLY NEED THE HELP OF CD 8, AND THAT'S TRUE TOO, BUT IT ALSO AMPLIFIES THE SIGNAL IN THIS CONTENT. ALL RIGHT. SO LET ME CONCLUDE FOR THE SECOND PIECE. SO CD 8 POSITIVE T CELL BINDS [INDISCERNIBLE] IN TWO STAGES. THE FIRST STAGE IS T CELL RECEPTOR DOMINANT. THE SECOND STAGE IS CD 8 DEPENDENT. AND THE TWO STAGE IS THERE WAS A ONE SECOND DELAY TIME BETWEEN THE TWO STAGES. THE SECOND STAGE CAN BE TRIGGER BY A SINGLE PEPTIDE [INDISCERNIBLE] REQUIRED CIRCUMFAMILY KINASETIVITY, MEDIATE BID A COOPERATIVE BINDING OF T CELL RECEPTOR AND CD 8 TO THE SAME PEPTIDE MHC MOLECULE. THE PEP SIDE BINDING IS TRANSIENT AND REVERSIBLE AND THE TWO STAGE BINDING FOR Y TWO STAGE ANTIGEN DISCRIMINATION. SO ALL OF THESE ARE PUBLISHED RESULT, AND I THOUGHT THAT I'M GOING TO SHOW A FEW SLIDES OF UNPUBLISHED RESULT TO SAY THINGS ARE NOT THAT SIMPLE. HERE IS A CD 4 POSITIVE T CELL THAT A LOT OF PEOPLE ASK ME ABOUT; HAVE YOU DONE THAT? WE'VE DONE SOME. NOW, JUST LOOK AT THIS IS THE RANGE OF DIFFERENT PEPTIDES. THE HOMO GLOBIN PEPTIDE IS THE MOST STRONG THEN YOU GET WEAK PEPTIDES, RIGHT? SO THE FIRST OF ALL, THE AFFINITY RANGE IS ONE LOCK LOWER COMPARED TO THE AGONIST HERE WITH THE OVER AGONIST, BUT STILL THIS IS KIND OF ONE LOCK HERE, NOT SPEND THREE LOCK, BUT IT STILL BEATS THE [INDISCERNIBLE]? THIS IS BERN BERN AGAIN, THIS IS 2D MEASUREMENT. THE ON WAY WAS GOING THIS WAY WAS OUR ON WAY WAS GOING THIS WAY. NOW THE THIRD THING IS, THERE WAS THIS CONTROVERSIAL THING THAT ON THE OFF RATE THE 3D PARAMETER WERE THE MORE POTENT THE PEPTIDE, THE SLOW TER OFF RATE AND WE GOT IT REVERSE, NOW IT'S NOT REVERSE ANYMORE. WELL, I DON'T KNOW, I MEAN, LOOK AT THIS DATA, HOW GOOD IS IT? THE 2D PARAMETER STILL CALL IT WITH THE RESPONSE. WELL, IF YOU LOOK AT THIS, HOW CAN YOU EXPECT IT CALL A RESPONSE? NOT VERY WELL. SO THE 2 D PARAMETER NEVERTHELESS CORRELATE THEIR RESPONSE BETTER. SO HERE IS ACTUALLY IN PRESS IT'S CHRIS GARCIA'S LAB AND WE CONTRIBUTED TO MAKE THIS 2D MEASUREMENT USING [INDISCERNIBLE] CELLS. SO THIS IS A 42 F [INDISCERNIBLE] TCR, CD 8 POSITIVE. THE PEPTIDE RANGE FROM WEAK TO STRONG AND IT'S AFIN THETY ON RATE AND OFF RATE. AGAIN, HERE, YOU GET THE POSITIVE SLOPE. NOW, THEN YOU LOOK AT 3D. THESE THREE HAVE A BIGGER RANGE, BUT THAT GUY, HERE, IS TOTALLY, THEY CALL IT CRAZY PEPTIDE. YOU CAN SEE WHY. THEN, LET'S SEE -- OOPS, WHAT HAPPENED? UM, SO THE OFF RATE, IT'S ACTUALLY 3D MEASUREMENT, THEY WERE ABLE TO ONLY MEASURE THIS ONE, OKAY. THE OTHERS ARE TOO FAST TO MEASURE. NOW, IF IT'S TOO FAST TO MEASURE, THEN THE SLOPE IS GOING TO BE LIKE THIS, WHICH MEANS THE MORE POTENT THE PEPTIDE, THE FASTER THE [INDISCERNIBLE], EVEN BY 3 D MEASUREMENT. OKAY. NOW, SO IF YOU -- HERE'S THE DATA OF THE 2D AFFINITY IN THIS PANEL. HERE'S WHAT YOU USE A MHC MOLECULE, MAKE A TETRAMER TO STAIN T CELL AND YOU MEASURE THE MEAN FLORENCE, THEY MATCH UP PRETTY WELL, WHICH MEANS IT DOESN'T MATCH THIS GUY. SO [INDISCERNIBLE] MEASUREMENT DOES NOT NECESSARILY MATCH TETRAMER STAINING, NOT IN THIS GUY, BUT IF YOU MAKE A TETRAMER OF T CELL RECEPTOR, NOW -- AND USE IT TO STAIN APC -- THIS DATA IS THE SAME AS THAT DATA AND THEN THE TETRAMER T CELL RECEPTOR IN APC NOW MATCHES THIS. YOU CAN INTERPRET IT WHAT IT WILL BE WANT TO. NOW, SO THIS IS THE MICHAEL [INDISCERNIBLE] BEEN USING [INDISCERNIBLE]. IS THE VEHICLE MEASUREMENT, RIGHT? SO IF YOU TAKE THE SOLUBLE T CELL RECEPTOR AND YOU PUT IT IN THE RED BLOOD CELL AND MAKE 2D MEASUREMENT, IT'S NOT [INDISCERNIBLE] RED BLOOD CELL BUT NEVERTHELESS IT'S 2D. PURIFYING MOLECULE 2D VERSES [INDISCERNIBLE]. THE [INDISCERNIBLE] 2D MATCHES THIS, NOT THIS. NOW. OKAY. SO, OF COURSE, IF YOU TREAT THE T CELL WITH THINGS LIKE MBCD AND DAY BAY TA CD THAT YOU SWAP [INDISCERNIBLE] YOU SEE 2D PARAMETER CHANGE. WE GOT A LOT OF CRITICISM SO IT MANNERS THE MEASUREMENT IS NOT INTRINSIC. OF COURSE, IF YOU TREAT THE VEHICLE FLOW CELL WITH THESE GUYS AND YOU RUN THE VEHICLE MEASUREMENT, YOU'RE NOT GOING TO SEE ANY DIFFERENCE, RIGHT? HERE IS [INDISCERNIBLE] BOTH EXPRESS ON CELL [INDISCERNIBLE]. THEY ARE NOT THE SAME. SO EVEN WITH THE PHYSIOLOGICAL [INDISCERNIBLE] OR MANGE NEEZ THAT CHANGE AFFINITY T AFFINITY RANGE CHANGE. THIS IS 15 A HUNDRED FOLD THIS IS ONLY 120 FOLD BETWEEN THESE TWO. NOT ONLY THE ABSOLUTE VALUE ON NOT THE SAME, THE CHANGE IN RESPOND TO AN AGONIST IS NOT THE SAME, SO IT'S KREG LAR CONTENT REALLY DETERMINES A LOT OF THE 2D PARAMETERS. GOOD OR BAD. HERE'S ANOTHER ONE, I THINK IT WAS [INDISCERNIBLE] MARTIN. IF YOU MAKE A MONOMERIC [INDISCERNIBLE] OR USE THE WILD [INDISCERNIBLE] TO ENABLE MULL MERIC MOLECULE, YOU SEE THIS TWO-STAGE, THAT'S THE TRANSITION AT ONE SECOND DELAY. THE TWO LEVELS WHEN YOU COMPARE THE MONOMER AND MULTIMER IS THE SAME BUT THE TRAN SIXTH TAKES PLACE EARLIER FOR MONOMER, YOU HAVE THOUGHT THE OPPOSITE AND I QUESTION [INDISCERNIBLE] WHO DID THIS AND HE SWEAR TO ME THAT IT'S TRUE. SO I HAVE TO TAKE HIS WORD. NOW THIS IS LOOKING AT MEMORY T CELL VERSUS NATIVE T CELL FROM THE PERIPHERAL OR SPLEEN. IT'S NORMALIZED AGAINST THE T CELL RECEPTOR MHC BECAUSE THE [INDISCERNIBLE] EXPRESS A LOT LOWER [INDISCERNIBLE] BUT YOU NORMALIZE IT AND YOU SEE THIS IS THE TWO-STAGE YOU SEE BEFORE. ON THE [INDISCERNIBLE] IT COMES BACK A LOT BIGGER JUMP, AND, OF COURSE, THAT'S, UM, DEPENDENT ON THE CD 8. THE CD 8 CONTRIBUTION OCCURS NOT ONLY WHEN IT JUMPS UP, BUT EARLY ON, BUT EARLY ON. SO THAT'S ALL OF THIS IS JUST SAYING THAT MOLECULAR INTERACTION AT THE CELL SURFACE ARE FAR MORE COMPLEX THAN BINDING OF PURIFYING MOLECULE, UNIVERSAL [INDISCERNIBLE] PARAMETER MAY NOT EXIST. ALTHOUGH YOU SEE A REASON NATURE PAPER CLAIM THAT [INDISCERNIBLE] COMPUTATIONAL WAYS TO RELATE THEM. IN [INDISCERNIBLE] ANALYSIS ENABLE US TO PROBE SELF-SERVICE MOLECULE [INDISCERNIBLE] NATIVE MEMBRANE ENVIRONMENT. ON TO FINISH BY ACKNOWLEDGING PEOPLE THAT HELP US ALONG THE WAY. I HAVE COLLABORATED WITH [INDISCERNIBLE] AND LINDSEY EDWARDS AND JOE SABATINO WITH STUDENTS IN BRIANS'S LACK THAT WORK WAS. THE F 5 T CELL WAS PROVIDED BY [INDISCERNIBLE] AT CDC AND HIS STUDENT JULIE. CHRIS GARCIA, HIS POST DOC PROVIDE US WITH THE MOLECULES FOR THE 43 Fc SYSTEM AND THE MEMORY T CELL WORK WAS DONE BY LUCACHUR. FUNDING FROM THE NIH, AND IN MY LAB SCOTT CHESTER AND WILLIAMS; THEY DEVELOPED THE ADHESION FREQUEN FREQUENCY ASSAY AN [INDISCERNIBLE]. CHIN DEVELOPED THE ADHESION FREQUENCY ASSAY. JENNY, JOHN, VERONICA, JOHN DID THE WORK IN THE NATURE PAPER AND HE ALSO DO THE WORK OF THE MONOMER VERSUS DIMER TWO STAGE PAPER. AND HONG DID THE CD 4 POSITIVE T CELL WORK. THANK YOU VERY, VERY MUCH. [APPLAUSE] >> COUPLE HANDS ARE RAISING UP. >> [LOW AUDIO]. >> DID I SHOW ANY DATA? >> [LOW AUDIO]. >> THE AFFINITY I SHOW. >> [LOW AUDIO]. >> WAS IN THE NATURE PAPER. SO YOU KNOW -- WELL, IF YOU LOOK TO THAT, THE OFF RATE WAS AFFECT BID [INDISCERNIBLE] OR THAT BY HOW MUCH EFFECTOR OF TWO. AND THAT ACTUALLY WAS DEPENDING ON HOW MANY POINTS YOU MEASURE IN THE TRANSIENT PHASE OF THE CURVE. WE NOW COME BACK AND DO A MORE RIGOROUS ANALYSIS. OUR CURRENT VIEW IS THAT THE OFF RATE IS NOT AFFECTED BY THOSE THINGS. >> [LOW AUDIO]. >> NO, NO, NO. IT WAS ON THE PAPER. YOU READ OUR PAPER BUT NOT ON THE SLIDE. I TOOK IT OUT. [LAUGHTER] >> [LOW AUDIO]. >> NO, I SHOW OFF RATE, BUT THE OFF RITE DEPENDENT ON PHARMACOLOGICAL [INDISCERNIBLE]. THAT WAS OPT PAPER BUT NOT ON THE TALK. >> [LOW AUDIO]. >> NO. THEY'RE NOT THE SAME IN TWO OR THREE DIMENSION. THEY'RE NOT, BUT THE 2D OFF RATE IS NOT AFFECTED BY PHARMACOLOGICAL TREATMENT. THAT'S WHAT I THOUGHT YOU WERE ASKING. >> [LOW AUDIO]. >> SO IT'S HARD FOR ME TO UNDERSTAND TOO. SO WE HAVE STUDY OTHER MOLECULAR ACTIONS. IN THE CASE OF SELECTIN, WE ACTUALLY FIND AGREEMENT BETWEEN THE VEHICLE MEASURED OFF RATE AND THE THERMAL FLUCTUATION MEASURED OFF RATE. NOW, IN THE CASE OF LFA ONE, THERE'S DIFFERENT. I CAN'T EXPLAIN IT TO YOU. MAYBE WHEN YOU MAKE A SOLUBLE MOLECULE -- AND I KNOW WHEN SAY THAT THE CHEMISTS ARE GOING TO KILL ME -- BUT WE HAVE ACTUALLY PUBLISHED THAT MEMBRANE ANCHOR OF THE MOLECULE AWAY FROM THE BINDING POCKET ACTUALLY AFFECTS BINDING. DON'T ASK ME WHY AND I WAS TALKING TO DAVID EARLIER, TALKING ABOUT MHC MOLECULE FOLDING AND THERE WAS -- FOLDING WAS AFFECTED BY THINGS OCCUR DISTANCE. >> [LOW AUDIO]. >> RIGHT. >> [LOW AUDIO]. >> SO THIS KIND OF 3D DATES BACK TO GEORGE BELL IN A SCIENCE PAPER IN 1978, AND HE HAS A GROUP IN LOS ALMOS, BRIAN GOLD STOOEN, THEY ACTUALLY STARTED OUT LOOKING AT 2D BINDING IN THE SAME CELL SURFACE. SO THEY DON'T THINK THERE'S ANY DIFFERENCE, RIGHT, IN THE THEORETICAL TREATMENT. EXPERIMENTALLY THERE WAS A LOT OF DIFFERENCE BECAUSE WHEN YOU HAVE THE ANTIBODY BINDING TO MOLECULE IN THE SAME SURFACE, ONE LEG BEHIND, SECOND LEG BEHIND THAT KIND OF THING, THE MOLECULE ARE STILL NOT TETHERED TO A ENTITY THAT A THOUSAND DOLLARS BIGGER. IF YOU THINK ABOUT SCOOP DYEING AND THEN YOU GET RACKED BY A ROPE TIED UP WITH A BOAT UP THERE TO DO [INDISCERNIBLE], IT'S A DIFFERENT THING. THERE WAS A [INDISCERNIBLE] METHOD TO EXPERIMENTALLY MEASURE THIS INTERACTION OF THE SAME MEMBRANE AND THAT'S REALLY CRITICAL TO UNDERSTAND HOW SIGNALING EVEN OCCUR. >> [LOW AUDIO]. >> I GUESS THE METHOD THAT MARK DAVIS HAD PUBLISHED COULD BE ADAPTED TO DO THAT, BUT THAT'S A HOT METHOD. YOU CAN'T SCALE IT, RIGHT? EVERY SINGLE MOLECULE ENTITY YOU HAVE TO MAKE IT STRAP AND TEST IT AND MAKE IT TO WALK. >> SO MAYBE I MISSED SOMETHING, BUT, UM, YOU'RE LOOKING AT INTERACTIONS AS BILL WAS JUST SAYING BETWEEN A MOLECULE, TWO MOLECULES THAT ARE ON TWO DIFFERENT SURFACES THAT ARE COMING TOGETHER, RIGHT? >> MM-HMM. >> NOW, DOESN'T -- ISN'T THAT STRONGLY AFFECTED BY THE DENSITY OF THE MOLECULES IN THE SURFACE THAT ARE MULTIBALANCING OF THE TWO SURFACE. >> IT DOES. >> SO IF YOU HAD JUST ONE MOLECULE ON EACH SURFACE AND YOU COULD MEASURE THE STRENGTH OF THAT INTERACTION, THAT WOULD BE, UM, THE SINGLE, SORT OF MONO VA LENT INTERACTION, BUT AS SOON AS YOU HAVE MULTIPLE COPYINGS OF A MOLECULE ON ONE SURFACE OR THE OTHER OR BOTH, NOW YOU HAVE ALL THESE EFFECTS OF MULTIVA LENT IS I WHERE YOU HAVE, IT'S LIKE, YOU KNOW, TWO ARMS OF ANTIBODY WHERE ONE ARM COMES OFF OR THE OTHER ARM COMES OFF AND YOU ONLY MEASURE AN ASSOCIATION WHEN BOTH ARMS COME OFF. HOW DO YOU TAKE THAT FWOO INTO ACCOUNT IN THIS SYSTEM? >> THE SIMPLEST WAY POSSIBLE, THAT'S A SHORT ANSWER. YOU'RE ABSOLUTELY CORRECT. I STRESS WE ARE MEASURING ADHESION KINETICS. WE INTERRUPT ADHESION KINETICS USING RECEPTOR LEGION BINDING KINETICS. THAT'S A MODEL AND YOU CAN CONSTRUCT DIFFERENT MODELS TO FIT THE DATA. THE SIMPLEST MODEL IS ASSUME THAT THE MOLECULES ARE DISBURSE LIKE IDEAL SOLUTION. EVEN THEY ARE PRESENTED BUT THEY'RE CONSTRAINED BY TWO SURFACES, BUT THAT CONSTRAINT IS NOT AS STRONG AS THE TWO FAB TOR ANTIBODY THAT LINK I Fc. SO YOU CAN NAK [INDISCERNIBLE] AS THE WILD TYPE. SO THEN YOU HAVE TWO MHC MOLECULES SITTING ON THE SAME [INDISCERNIBLE] FORMING A DIMER OR MAYBE EVEN A TRI MER. ALL YOU CAN USE A [INDISCERNIBLE] AND ONE OF THE [INDISCERNIBLE] BINDS TO THE REST [INDISCERNIBLE] THE OTHER ONLY OTHER SITE THAT ALLOW YOU TO DOCK INTO A MHC MOLECULE. NOW, THE TWO MHC MOLECULE ARE SITTING ON TWO DIFFERENT [INDISCERNIBLE]. THEY ARE LINKED TO A DIFFERENT SURFACE PROTEINS AND THEY COULD MOVE AROUND. SO THE SPECIAL ORGANIZATION OF THE MOLECULE ON THE CELL MEMBRANE. >> MAKES A BIG DERNS. ALSO THE MOBILITY. THE OTHER POINT I WAS GOING TO MAKE IS IF THERE'S A LOT OF MOBILITY IN THE MEMBRANE YOU CAN GET CLUSTERING, IF THERE ISN'T, YOU MAY NOT GET THE CLUSTERING AND THAT WOULD MAKE A HUGE DIFFERENCE. >> THAT'S CORRECT. ON ONE HAND YOU CONTINUE KNOW WHAT YOU'RE MEASURING, ON THE OTHER HAND THAT PROVIDE YOU OPPORTUNITY TO LOOK INTO THOSE ISSUES THAT YOU WOULDN'T BE ABLE TO DO IT WITH THE VEHICLE. >> THANK YOU.