>> OKAY, GOOD AFTERNOON.

IT'S A SPECIAL PLEASURE FOR ME

POETIC INTRODUCE DAVEED

RAULET AS OUR SPEAKER TODAY AND

AS I WAS PREPARING THIS

INTRODUCTION, I REALIZE THAT WE

HAVE A SPECIAL ANNIVERSARY

BECAUSE 20 YEARS AGO, EXACTLY,

1991 HE PUBLISHED TWO LANDMARK

PAPERS ON NK CELLS AND SOME OF

THE ORIGINAL WORK ON NK CELL WAS

DONE BY DAVID AT THAT TIME AND

EVER SINCE.

SO THE FIRST PAPER WAS TO SHOW

THAT BONE MARROW FROM MICE

DEFICIENT MHC CLASS ONE.

SO THIS WAS THE BET ON TWO

MICROGLOBIN KNOCK OUT THAT THOSE

CELLS WERE REJECTED WHEN THEY

WERE INJECTED, AND MHC CLASS ONE

FOR RESISTANCE, SORT OF THE

STRONG SUPPORTED MISSING

HYPOTHESIS AND IT WAS A REAL

EXPLANATION UNTIL THEN.

AND ANOTHER PAPER, THE SAME ONE

HERE SHOWED THAT IN THE BETA TWO

M KNOCKOUT MICE THAT DON'T HAVE

CLASS ONE, THERE ARE NK CELLS.

AND THEY ARE NOT RESPONSIVE, SO

THE FORM OF EDUCATION SO THERE'S

NO--SO THERE'S SOME FORM OF

MAGNIFICATION AND IT WAS A NEW

CONCEPT.

SO THIS WAS IN 1991 AND THE

FIRST PAPER WAS FROM THE M. I.T.

AND SECOND PAPER FROM BERKLEY.

AND OVER THOSE 20 YEARS HE HAS

NEVER STOPPED MAKING

CONTRIBUTIONS TO OUR

UNDERSTANDING OF NK CELLS AND

I'LL MENTION, YOU KNOW JUST A

COUPLE EMPLOY HE HAS SHOWN THAT

EVEN IN WILD-TYPE MICE AND THE

SAME HOLDS TRUE IN HUMAN, THERE

ARE NK CELLS THAT DON'T HAVE

IMAGE RECEPTORS OR THEY MAY HAVE

AN INDIVIDUAL RECEPTOR FOR WHICH

THERE'S NO ENDOGENOUS CELLS MHC

CLASS ONE AND AGAIN, THESE

NKCELLS ARE NOT KILLING

INDISCRIMINATELY UNRESPONSIVE.

SO SOME IS FORM OF EDUCATION,

IT'S DONE CELL TRANSFER TO SHOW

IT'S NOT A DIFFERENTIATION OR

LINEAGE DIFFERENCE, IT'S

SOMETHING THAT CAN CHANGE

DEPENDING ON THE ENVIRONMENT.

SO THEY CAN BECOME RESPONSIVE

AND REQUIREMENT WHAT THEY DO

HAVE A CLASS ONE LIAISON GAPPED

FOR INDIVIDUAL RECEPTORS.

HE'S ALSO DONE WORK ON A MAJOR

ACTIVATION OF THE NK CELLS

TWO-D, HIS LAB GENERATED MICE

DEFICIENT IN THIS AND THOSE MICE

DEVELOP SPONTANEOUS TUMORS AND

THAT'S THE BEST EVIDENCE THERE

IS TO SHOW THAT NK CELLS HAVE A

ROLE IN TUMOR SURVEILLANCE.

IT WASN'T OBVIOUS BECAUSE NK

CELLS ARE NOT VERY GOOD AT

DEALING WITH ESTABLISHED TUMORS

BUT I THINK THIS WORKS SO THAT

THEY'RE REALLY IMPORTANT IN

PREVENTING CANCER TUMORS IN THE

FIRST PLACE.

AND HE'S DONE BEAUTIFUL WORK ON

THE REGULATION OF LIGANDS FOR

THE RECEPTORS AND DNA DAMAGE

INDUCES EXPRESSION OF THE

LIGANDS AND OF COURSE, THAT'S

IMPORTANT FAR TUMOR SURVEILLANCE

THROUGH NK CELLS SO I THINK I'LL

STOP AND I'LL LET HIM TAKE OVER.

I THINK HE WILL TAKE US ON A

TOUR BECAUSE THE TITLE IS INNATE

NK RECEPTORS AND THEIR LIGANDS,

REGULATION IN CANCER, INFECTION

AND INFLAMMATION DISEASE, SO

IT'S--THANK YOU.

>> IT'S REALLY GREAT TOW BE

HERE, ERIC, THAT WAS A NICE

INTRODUCTION, I HAVE A REALLY

TERRIFIC DAY TODAY.

NIH REALLY DOES HAVE GREAT

IMMUNOLOGY.

EVERYONE I TALKED TO HAS BEEN

REALLY INTERESTING, SO I'M A

LITTLE JEALOUS OF ALL THE STUFF

YOU HAVE GOING ON AROUND HERE

AND IT'S GREAT TO BE HERE.

SO, MY TITLE IS MORE GENERAL

THAN WHAT I'LL ACTUALLY TALK

ABOUT.

IT MAKE ITS SOUND LIKE I'M GOING

TO TALK ABOUT DIFFERENCES AND

I'LL BE FOCUSING ON USING THE

ONE THAT ERIC MENTIONED THE ONE

CALLED NKTTWO B AND I'D LIKE TO

TALK ABOUT THE ROLE IN THE

CONTEXT IN CANCER.

I'M ONLY GOING TO BARELY TOUCH

ON RINE FECKS BUT I WILL HAVE A

BIT TO SAY ON ITS ROLE IN

INFLAMMATORY DISEASE.

SO THIS SLIDE IS NOT NECESSARY

HERE AT NIH UNLESS WE'RE

FOCUSING ON NK CELLS AND OF

COURSE THEY PLAY A MAJOR ROLE IN

INFECTION BUT MUCH OF OUR WORK

FOCUSES ON THEIR ROLE IN CANCER.

SO, I THINK YOU ALL KNOW THAT NK

CELLS ARE REGULATED BY A

STIMLATTORY AND INHIBITORY

RECEPTORS AND THAT THE

ACTIVATION OF NK CELL SYSTEM

DETERMINED BY THE BALANCE OF

SIGNALING BETWEEN THESE TWO

TYPES OF RECEPTOR.

ONE OF THE--THE MAJOR CLASS OF

INHIBITORY RECEPTORS RECOGNIZES

MHC CLASS ONE MOLECULES AND

SIGNALS THROUGH THOSE RECEPTORS

THAT HELP TO PROTECT NORMAL

CELLS FROM BEING ATTACKED BY NK

CELLS.

BUT ACTIVATING THROUGH RECEPTOR

IS JUST AS IMPORTANT AND WE

THINK JUST AS REGULATED IN THE

SENSE IT CAN VARY UNDER

DIFFERENT CONDITIONS OF

INFECTION, UNDER DIFFERENT

CONDITIONS OF INFLAMMATION AND

IN CANCER CELLS, VERSES NORMAL

CELLS.

THAT'S WHAT I'M GOING TO FOCUS

ON AGAIN TODAY AND SPECIFICALLY

NKTWO D, THERE ARE NUMEROUS

ACTIVATING RECEPT ORS ON NKDTWO

DIS THE BEST CHARACTERIZE AT

THIS POINT BECAUSE WE KNOW MOST

ABOUT THE LIAISON--LIGANDS AND

THE ROLE OF THE NTWO KD CONTEXT.

SO IT TURNS OUT THAT MANY OF

THESE RECEPTORS RECOGNIZE

SELF-LIGANDS, ENCODE INDEED OUR

OWN GENOMES BUT SO THEY DON'T

RECOGNIZE THEM AND THE

REGULATION OF EXPRESSION OF

THESE SELF-LIGANDS VARIES

DEPENDING ON THE DISEASE STATUS

OF THE CELL AND WE CALL THAT

INDUCED CELL RECOGNITION SO

NKTWO D HAS BEEN STUDIED BY

NUMEROUS GROUPS AND I WON'T HAVE

TIME TO CREDIT EVERYONE BUT

ABOUT SPACE PLAYED A TIME EARLY

ON AND LEWIS' GROUPS AND OTHER

GROUP VS MADE MAJOR

CONTRIBUTIONS TO UNDERSTANDING

IT'S ROLE.

IT SIGNALS THROUGH SEVERAL

ADAPTER MOLECULES AND WELL,

PARTICULARLY DAP 10 IN BOTH

HUMANS AND MICE, AND IN MICE,

DAP 12 CAN ALSO PLAY A ROLL.

I WON'T REALLY SPEND ANY TIME

TALKING ABOUT HOW THAT WORKS BUT

BUT IT SIGNALS THROUGH SEVERAL

SIGNALING PATHWAYS AND INCLUDING

PTHREE CAIN ACE.

AND CYTOKINE PRODUCTION BUT IT'S

IMPORTANT TO POINT OUT AND I'LL

TOUCH ON THIS, THAT NKDTWO D IS

EXPRESSED BY NUMEROUS DIFFERENT

T-CELLS INCLUDING CDEIGHT

T-CELLS, ALL CDEIGHT T-CELLS IN

HUMANS IN FACT.

--THE ROLE IN T-CELLS IT

ENHANCES T-CELL ACTIVATION

THROUGH THE T-CELL RECEPTOR, SO

IT WILL THEREFORE COOPERATE WITH

ANTIGEN SPECIFIC SIGNALS IN

OTHER CASES, IT MAY SIGNAL

DIRECTLY AND I THINK WE'RE STILL

NOT--WE STILL DON'T HAVE ENOUGH

DATA UNDERSTAND THE COMPLETE

ROLE.

ONE OF THE STRIKING FEATURES

ABOUT NK2 D ARE THE NUMBER OF

LIGANDS THAT THE RECEPTOR

CHARACTERIZES AND WE HAVE BEEN

CHARACTERIZED BY NUMEROUS

GROUPS, INCLUDING AGAIN THE

SPACE LAB WHO IDENTIFIED THE

ULDP, THE MYC MOLECULES AND THE

OBPs WERE IDENTIFIEDED IN

SEATTLE BY, BY CONSTANCE GROUP

AND THE MOUSE LIGANDS

CHARACTERIZED BY A COUPLE GROUPS

AND OUR GROUP, AND OTHERS.

SO, THESE ARE ALL MHC 1

RELATIVES AND THERE'S A NUMBER

OF THEM.

EACH OF US HAS UP TO 10 OF THESE

THINGS OR SO, AND THEY'RE ALL

MHC RELATIVES.

THEY'RE--THE MAIN THEME WE THINK

OF THEIR REGULATION IS THAT

THEY'RE NOT EXPRESSED IN MOST

NORMAL CELLS BUT UPREGULATED IN

DISEASE CELLS AS I MENTIONED IF

YOU LOOK AT INDEPENDENT TUMORS

OR INFECTED CELLS YOU SEE 1 OR

MORE OF THE LIGANDS BEING

UPREGULATED AND IN TUMOR CELLS

IN PARTICULAR, IT'S A LITTLE

HARD TO PREDICT, YOU MAY SEE,

COVERT EXPRESSION OF SEVERAL

LIGANDS OR ONLY EXPRESSION OF 1

LIGAND.

NOW SOME YEARS AGO,

INVESTIGATED, THE POTENTIAL ROLE

OF THESE IN TUMOR REJECTION BY

SIMPLY IDENTIFYING A FEW TUMOR

CELL LINES THAT DIDN'T HAVE NK2

D LIGANDS, MOST OF THEM HAVE

LIGANDS NATURALLY AND THEN WE

INTRODUCE THEM BY TRANSDUCTION.

THIS IS THE B16 MELANOMA CELL

LINE AND THEN WE COMPARE THOSE

WITH CONTROL CELLS FOR WHETHER

THEY WOULD CAUSE SUBCUTANEOUS

AND GENETIC MICE AND AND IN CASE

OF THE CELLS EXPRESSIONING,

POWERFUL REJECTION IN THE TUMOR

SPACE WE DON'T GROW COMPARED TO

THE CONTROL TUMORS AND YOU COULD

REVERSE THIS INJECTION BY

INJECTING THE D2 K ANTIBODIES TO

BLOCK THE RECEPTOR INTERACTION,

THIS IS AN ANTIBODY THAT'S NOT

THE COMPLETE NK CELLS.

AND MORE RECENTLY, A POST DOC IN

THE LAB KNOCKED OUT THE NKD2

GENE AND THIS IS IF YOU COMPARE

THE SAME LIGAND EXPRESSING TUMOR

IN EITHER WILD-TYPE OR NKG2 D

KNOCK OUTS, AND WE WANT TO

UNDERSTAND THIS TO MORE NATURAL

MODELS AND WE SET THE TIES

SPONTANEOUS MODELS AND THIS

SHOWS THE DAT FRA THE TRAP

MODEL.

SO THIS IS THE MODEL OF

SPONTANEOUS CANCER, MICE

THAT--THAT OHM--TYPE OF THE

CANCER THAT'S DIFFERENTIATED AND

THE TUMORS ARE BIG AND THIS IS

THE WEIGHT OF THE AREA, IT'S TOO

BIG FOR A MOUSE TUMOR BUT IT

ARISES RELATIVELY LATE, IT WILL

EFFECTUALLY KILL ALL THE MICE,

BUT A FEW OF THE CANCERS ARISE

EARLY AND ARE MORE MALIGNANT AND

ARE ENORMOUS AND EXTREMELY

MALIGNANT CANCERS WHEN WE

COMPARE MICE THAT HAD NKG2 D,

WHAT WE OBSERVED WAS A

SIGNIFICANT INCREASE THESE EARLY

EXTREMELY AGGRESSIVE TYPES OF

CANCER.

THEY INCREASE BY I THINK ABOUT 3

FOLD IN THESE MICE.

SUGGESTING THAT NKG2 D IS

HOLDING THESE BACK IN THE

WILD-TYPE MICE.

WE ALSO SAW A PHENOTYPE IN THE

IMMUNO MYC MODEL WHERE THERE WAS

NYG2 D GOING BACK TO THE BASIS,

THERE WAS ANOTHER LINE OF

EVIDENCE OF NKG2 D'S ROLE AND

THAT WAS COMPARING THEM TO

LIGANDS IN THE TUMORS THAT AROSE

HERE AND IN THE TUMORS THAT ROSE

HERE IN THE KNOCK OUTS.

AND WHAT WE FOUND IS THAT THE

TUMORS THAT AROSE IN THE KNOCK

OUTS ALL EXPRESSED NKG2 D

LIGANDS AT THE RNA LEVEL AND THE

CELL SURFACE, THIS IS THE CASE

OF THE RAY 1 LIGAND SHOWN HERE.

BUT THE TUMORS THAT AROSE IN THE

WILD-TYPE MICE DIDN'T EXPRESS

NKG2 D, PROJECTING THEY HAD BEEN

SELECTED FOR A LOSS OF LIGANDS.

SOPHISTICATEDY WE PROPOSED THE

FOLLOWING SCHEME THAT REALLY,

THE EARLY TUMORS EXPRESSING NKG2

D SURFACE, SOME OF THEM ARE

ELIMINATED ALTOGETHER AND THAT'S

WHYRY HAVE THE LOWER INCIDENCE

IN THE WILD-TYPES BUT IN SOME

CASES A VARIANCE CAN GROW UP

THAT LACK THOSE AND THOSE OF

COURSE WILL GROW OUT BY

SELECTION AND WILL END UP

GROWING OUT TUMORS SO THAT CAN

EXPLAIN BOTH OF THOSE LINES OF

EFDZ.

--EVIDENCE.

NOW THERE'S A LOT MORE DATA ON

CANCER AND I HAVE MORE LATER IN

THE TALK BUT I WANTED TO TURN

FOR A TIME TO HAD RELATED

QUESTION OF WHETHER NKG2 D PLAYS

A ROLE AND PARTICULARLY IN

CHRONIC INFLAMMATION, AND WE

BELIEVE IT ARISES, EVOLVE FOR

THE PURPOSE OF HOST DEFENSE.

IT'S INTERESTING DINS DENTALLY

THAT TOM SPIES PUBLISHED A

FINDING THAT TUMORS MAY COMMAND

EAR THIS IN SOME CASES,

EXPRESSING IT, AND NOT THE

LIGANDS BUT THE RECEPTOR AND THE

ADAPTER MOLECULE AND HE THINKS

SERVES AS AN ORCHGA GENE THAT

ENABLES THEM TO GROW BETTER

BECAUSE IT ACTIVATES PI-3 KINASE

IN THE TUMORS.

WE HAVEN'T REALLY SEEN MUCH

EVIDENCE OF THAT IN THE MOUSE

SYSTEM, BUT IT'S AN INTERESTING

FINDING.

BUT THE FLIP SIDE OF THIS IS THE

POSSIBILITY THAT NKG2 D PLAY ACE

ROLE WHEN IT'S INAPPROPRIATELY

ACTIVATED IN CHRONIC

INFLAMMATION AND THERE'S SEVERAL

LINES OF EVIDENCE FOR THIS AND

IT'S NOT BEEN GENERALLY CLEAR IN

WHICH CELLS IT'S ACTING TO HAVE

THESE EASTS AND I THINK IT'S

STILL NOT CLEAR.

SO THE FIRST LINE OF EVIDENCE

DID COME FROM THE SPACE LAB,

LOOKING AT RHEUMATOID ARTHRITIS

PATIENTS WHERE THEY SIMPLY FOUND

EXPRESSION OF MYCA, HUMAN

LIGANDS FOR NKG2 D, AND CELLS

AND EVIDENCE THAT THEY WOULD CO

ACTIVATE EACH OTHER IN CULTURE,

AND SUGGEST THAT A POSSIBILITY

THAT THIS INTERACTION PLAYS A

ROLE IN THE PATHOLOGY ASSOCIATE

WIDE RA.

FOLLOWING A REPORT THAT IN

SILLIAC DISEASE THERE'S AN IL15

MEDIATED NKG2 D MEDIATED

ACTIVITY THAT SHE THINKS

UNDERLIES MUCH THE OF THE

PATHOLOGY IN THAT DISEASE.

THERE'S GENETIC EVIDENCE THAT I

THINK THIS IS ISN'T MEANT TO

BE--LINKAGE OF THE DISEASE TO

NKG2 D SUGGESTING A GENETIC LINE

OF EVIDENCE, AND THEN I'LL LOSE

FINANCIAL AND AND AND--I'D LIKE

TO SUMMARIZE FOR YOU WHAT WE

FOUND.

SO FIRST OF ALL SURPRISINGLY,

NKG2 D, AND 50 WE CROSS THE

ANYTHING OUT ALLELE BACK TO THE

NOD MOUSE EXPLTZ COMPARE IT

LITTER MATES, THIS WAS MATCHED

SO THIS WAS A PAINFUL THING TO

DO.

THE NKG2 D SITS IN THE MIDDLE OF

GENES, IT'S POLYMORPHIC SO YOU

HAVE TO WORRY WHEN YOU CROSS IT

ON TOO A DIFFERENT BACKGROUND

THAT YOU MIGHT END UP COMPARING

MICE THAT HAVE VASTLY DIFFERENT

NK GENE COMPLEXES BUT WE CONTROL

THAT BY COMPARING IT TO MICE

THAT HAVE THE SAME NK COMPLEX AS

THE KNOCK OUTS WHICH IS FROM

B-6.

HE'S SHOULD BE A PERFECTLY GOOD

COMPARISON AND WE REALLY SEE NO

CHANGE IN DISEASE INCIDENCE IN

EITHER FEMALES OR IN MALES.

WE ALSO LOOKED AT IT IN THE

CONTEXT OF THE MODEL OF TYPE 1

DIABETES, IN THE LAB, TRANSGENIC

LEVEL AND WE DEPARTMENT SEE ANY

CONTROL FOR NKG2 D EITHER.

SO THEN WE LOOK AT, E AND WE

HERE THE NEWS WAS MORE

INTERESTING, BECAUSE THERE HAD

BEEN EVIDENCE FROM GENETIC

LINKAGE OF NKG2 D, AND EAE IS A

MOUSE MODEL THAT DISEASE.

AND IT WAS A PARTIAL AND FAIRLY

MODEST PHENOTYPE AND IT'S LESS

WHEN WE LIMITED THE DISEASE BY

USING A LOW CONCENTRATION OF THE

ANTIGEN THAT INITIATES THE

DISEASE, THE MOCK PEPTIDE, WE

FOUND THAT THERE WAS A LOWER

SEVERITY OF THE DISEASE IN THE

KNOCKOUT MICE, BY THE WAY, THE

GENE IS CALLED KLRK 1.

THAT'S THE GENE.

THERE WAS A LOWER SEVERITY OF

THE DISEASE IN THE KNOCK OUTS

THAN THE WILD-TYPES.

AND THIS IS JUST 2 DIFFERENT

EXPERIMENTS.

HOWEVER, IT'S A RELATIVELY

MODEST EFFECT AND WE ONLY SAW IT

UNDER LIMITING CONDITIONS.

WHEN WE USE THE NORMAL DOSE OF

PEPTIDE COME IS ABOUT 5 TIMES

HIGHER, WE DIDN'T SEE ANY

PHENOTYPE.

NEVERTHELESS, THIS MAY BE--MAY

PLAY A ROLE UNDER SOME

CONDITIONS.

SO IT WAS REALLY IN THE--WHAT

TURNED OUT TO BE A TYPE 2 MODEL

OF DIABETES, AND THE ASSOCIATED

METABOLIC DISEASE ANDAT LO

SKEROSEIS WHERE WE'VE SEEN A

REALLY, A STRIKING PHENOTYPE AND

THIS WORK WAS DONE WITH NAJONG

AT PENN STATE UNIVERSITY AND HIS

STUDENT IS ACTUALLY A FORMER

POST DOCK IN MY LAB BUT HE MADE

THE INITIAL OBSERVATIONS HERE

AND THEN WE COLLABORATE WIDE HIM

TO DISCO MANY OF THE--DO MANY OF

THE FOLLOW UP EXPERIMENTS IN HIS

LAB AND I WANT TO TELL YOU ABOUT

THEM HERE.

THERE WAS WORK HERE SO I'M

ADMITTING THAT HERE AND

ACKNOWLEDGING IT HERE.

AND THIS IS WHAT--THIS IS A

MODEL OF WHERE WITH WHERE WE

WERE ORIGINALLY FOCUSING ONAT LO

SKEROSEIS BUT IT'S IN METABOLIC

DISEASE, IT'S BASICALLY MICE

WITH VERY HIGH CHOLESTEROL

BECAUSE THEY HAVE A MUTATION,

AND FED EYE HIGH FAT DIET, THEY

DEVELOPED METABOLIC DISEASE,

TYPE 2 DIABETES AND

ATHEROSCLEROSIS AND THE NOTION

IS THAT THE OXIDIZED LIPITTEDS

AND OTHER METABOLIC PRODUCTS IN

THESE CONDITIONS LEAD TO

INFLAMMATION AND SO CALLED

VICIOUS CIRCLE OF INFLAMMATION

BETWEEN THE ARTERYS AND MANY

ORGANS INCLUDING LIVER THAT

LEADS ULTIMATELY TO GREAT DEAL

OF PATHOLOGY, ACCUMULATION OF

MACROPHAGES AND THEATAT LO

SCLERATTIC PLAQUES AND

ATHEROSCLEROSIS, MANY OF YOU

PROBABLY KNOW MORE ABOUT THAT

DISEASE THAN I DO.

BUT NEVERTHELESS, I'LL TELL YOU

WHAT I FOUND HERE.

SO TO LOOK AT NKG2 D,--SO THE

FIRST INDICATION THERE MIGHT BE

A ROLE OF THIS IN THE DISEASE

CAME FROM NOS DISCOVERY THAT

THE--A SOLUBLE VERSION OF THE--1

OF THE HUMAN NKG2 D CALLED MYC A

WAS ELEVATE INDEED THE SERUM OF

THE TYPE 2 DIABETES PATIENTS

COMPARED TO HEALTHY CONTROLS.

AND THAT SUGGESTED, YOU KNOW THE

SIGNIFICANCE OF THE SHEDDING OF

THE LIGAND IN MY OPINION STILL

UNCLEAR, IT'S KNOWN THAT IT'S

SHED, BUT IT WAS CERTAINLY AN

INDICATION THAT LIGANDS WERE

EXPRESSED AT HIGH LEVELS IN THE

DISEASE AND THAT LED US TO

PURSUE THE QUESTION OF WHETHER

IT WAS PLAYING A ROLE IN THE

MOUSE MODEL.

SO WE--WE LOOKED AT APOE

KNOCKOUT MICE AS I MENTIONED.

THESE ARE THE HIGH CHOLESTEROL

MICE.

THEY'RE FED A WESTERN

DISCIPLINARY EXPET WE ASK

WHETHER NKG2 D LIGANDS ARE

EXPRESSED IN THE ATHLOSCLEROTIC

PLAQUES AND WE FOUND THAT ALL

THE 3 LIGANDS WERE ALL ELEVATED

AT THE MRNA LEVEL COMPARED TO

PLAQUE FREE AORTIC ARCHS AND

THAT SUGGESTS THE POSSIBILITY

THAT NKG2 D MIGHT BE PLAYING A

ROLE THERE.

SO THEN WE CROSSED, TO THE KNOCK

OUTS AND WE COMPARE THE

WILD-TYPES IN THE KNOCK OUTS AND

THAT SHOWED SAMPLE DATA THAT THE

WILD-TYPE MICE, YOU SEE

ACCUMULATE ALL THIS PLAQUE, IN

THE KNOCK OUTS NEED MUCH LESS

AND THIS SUMMARIZES I LITTLE

LESS AND IT'S QUITE A DRAMATIC

PHENOTYPE MUCH LESS DISEASE IN

THESE.

NOW WE ALSO LOOKED AT A SLIGHTLY

DIFFERENT MODEL AND INSTEAD OF

THE APV MODEL, THIS IS 1 WHICH

ABNORMALITIES BROIGATES THE HIGH

[INDISCERNIBLE] AND THESE

ATHEROSCLEROSIS, WE ALSO I DON'T

SHOW IT HERE BUT WE ALSO SHOW

THERE'S MUCH LESS

ATHEROSCLEROSIS IN THE 2 D KNOCK

OUT BACKGROUND, BUT WE'RE ALSO

CAPABLE IN THIS MODEL PARTICULAR

TO SHOW THAT SIMPLY

ADMINISTRATING THE NKG2 D

ANTIBODY DURING THAT PERIOD

CAUSED A MUCH LOWER INCIDENCE OF

DISEASE SUGGESTING THAT AGAIN WE

CAN EVEN BLOCK THE THERAPEUTIC

POTENTIAL THERE.

SO IN WITH THIS GENETIC EVIDENCE

AND BLOCK AID EVIDENCE THAT THE

NKG2 D PLAYS A ROLE IN THIS

DISEASE.

NOW I MENTIONED THERE WAS SOME

VICIOUS CIRCLE BETWEEN THE

ARTERYS AND LIVER INFLAMMATION

AND SO WE INVESTIGATED LIVER

INFLAMMATION IN THESE MICE AND

ALSO EVIDENCE OF A WIDE SPREAD

INFLAMMATION BY LOOKING AT SERUM

CYTOKINES AND IT WAS QUITE

STRIKE TAG THE AMOUNTS OF IL6

AND INTERFERON-GAMMA THAT YOU

COULD MEASURE IN THE SERUM OF

THESE MICE WAS DRAMATICALLY

LOWER IN MICE THAT LACKED NKG2 D

AND THAT LIVER ENZYMES, THERE'S

ELEVATED LIVER ENZYMES IN THESE

APOE MICE, IN THE WESTERN DIET

THERE WAS A SIGNIFICANT

REDUCTION IN THE KNOCKOUT MICE.

AND THEN FINALLY, A STRIKING

DIFFERENCE IN THE NUMBER OF

IMMUNE CELLS, THIS IS THE

ELEVATED LEVEL IN THE NK CELLS,

NKG2 D KNOCK OUTS AND THE APE.

AND THESE ARE REDUCED

DRAMATICALLY.

WE SEE IN FACT A REDUCTION IN

THE SERUM CHOLESTEROL, STILL

HIGHLY ELEVATED COMPARED TO WHAT

I'M SURE YOU WOULD LIKE TO HAVE,

IN THIS MODEL THEY GET EXTREMELY

HIGH LEVELS OF CHOLESTEROL, BUT

IN THE KNOCK OUTS WE SEE, WE SEE

A MUCH REDUCED LEVEL, WE REEE

DUCED LEVEL AND WE EVEN SEE A

REDUCED LEVEL OF GLUCOSE

SUGGESTING THE DIABETES IS

SOMEWHAT LESSEN SAID IN THIS

CONDITION.

SO IN TERMS OF THE MECHANISM, I

THINK WE DON'T KNOW THE COMPLETE

ANSWER BUT I'LL--BY ANY MEANS

BUT I'LL TELL YOU WHAT WE

SUSPECT IS GOING ON.

THE THERE'S PUBLISHED EVIDENCE

THAT SOME OF THESE METABOLITES

LIKE OXIDIZED LOW DENSITY

LIPOPROTEINS CAN IN FACT

ACTIVATE MACROPHAGES AND OTHER

CELLS IN VITRO AND IN FACT

THROUGH TOLL RECEPTORS.

AND SO, WE--WE SUSPECT THAT THIS

MAY BE OPERATIVE HERE AND MAY

SERVE TO INDUCE EXPRESSION OF

LIGANDS ON MACROPHAGES OR

DENDRITIC CELLS OF SOME TYPE.

AND WE DO, SEE SOME INDICATION

OF THAT, ALTHOUGH I WON'T--IT'S

NOT DRAMATIC BUT IF WE SIMPLY

TREAT MACROPHAGES INVITRO, WITH

GLUE MARIOUSICATION END PRODUCTS

WE SEE INDUCTION OF THESE CELLS

INVITRO.

AT THE SAME TIME WE CAN

CERTAINLY SHOW THAT THE

MACROPHAGES IN THE LIVER ARE

ACTIVATED WANT IT'S

INTRACELLULAR CYTOKINE STAINING

SHOWING LOTS OF IL6 POSITIVE

MACK WOPHAGES BUT INTERESTINGLY

THERE'S NO REDUCTION IN THE

PERCENTAGE OF ACTIVATED CELLS,

IN THE KNOCK OUTS BECAUSE WE

THINK THIS STEP IS INDEPENDENT

OF NKG2 D, BUT ACCORDING TO THE

MODEL, THE INTERACTION OF THESE

LIGANDS WITH CELLS, NKCELLS AND

T-CELLS AND WE'RE NOT AGAIN

SURE, WHICH ARE MOST IMPORTANT

YET, LEADS TO AN AMPPLIFICATION

STEP AND RECRUITMENT STEP WHERE

YOU INCREASE THE NUMBER OF

IMMUNE CELLS IN THE LOCAL

TISSUE, AND THIS LEADS TO MUCH

GREATER INFLAMMATION CONSISTENT

WITH THAT, AS I'VE SAID WE SEE

MUCH LESS, MANY FEWER IMMUNE

CELLS IN THE LESIONS IN THE NKG2

D KNOCK OUT AND IN FACT LOOKING

AT NKT SELLS WEEE A MUCH LOWER

DEGREE OF PROJECTION NEURONS OR

PIONS TANIOUS NKT CELL

ACTIVATION IN THE--OF THE CELLS

THAT THERE ARE IN THE KNOCK OUTS

COMPARED TO WILD-TYPES SO THAT'S

OUR GOING HYPOTHESIS FOR HOW

THIS MIGHT WORK.

SO LET ME SUMMARIZE WHAT I TOLD

YOU ABOUT THESE PATHOLOGIC

REACTIONS THAT WE SEE NO EFFECT

OF THE KNOCK OUT IN TYPE 1

DIABETES MODELS.

WE SEE A MODEST EFFECT IN THE

EACE MODEL BUT ONLY UNDER

LIMITING CONDITIONS BUT THEY SEE

A SUBSTANTIAL EFFECT IN THE

METABOLIC DISEASE

ATHEROSCLEROSIS MODEL.

HOW DOES IT WORK, YOU KNOW WE'RE

NOT SURE, BUT WE SUGGEST THAT

INFECTIONS, TRAUMA ABNORMAL

METABOLITES AND OTHER

DISSTRESSED TISSUE THAT MAY BE

AG GRI INTERSTATED BY FACTORS

PROMOTES EXPRESSION AND

APPROPRIATELY AND THESE MAY THEN

TRIGGER OR AT LEAST NOT

NECESSARILY TRIGGER THE DISEASE

BUT MAY AMPLIFY OTHER

INFLAMMATION EVENTS THAT ARE

ONGOING, WE THINK THEREFORE THAT

UNDERSTANDING HOW LIGANDS ARE

REGULATED IS IMPORTANT IN ORDER

TO UNDERSTAND THE ROLE OF NKG2

D, NOT ONLY IN INFLAMMATORY

DISEASE BUT ALSO AS A START WIDE

IN CANCER AS WELL.

SO LETNY TELL YOU ABOUT OUR WORK

IN THAT AREA, NEXT.

SO THIS HAS BEEN A LONG STANDING

SET OF INTEREST IN THE LAB, HOW

DISEASE ASSOCIATED STRESS,

EITHER ASSOCIATED WITH CANCER OR

WITH INFECTIONS OR IN THIS CASE

WITH OTHER DISEASE STATES CAN

INDUCE NKD2 G LIGANDS AND

PROMOTE EFFECTS SUCH AS

ELIMINATION OF TUMORS AND I

MENTIONED INFLAMMATION, I'M

GOING TO SUMMARIZE JUST IN THIS

1 KIND OF UGLY SLIDE, A LOT OF

THINGS AND THEN GO BACK THROUGH

A FEW, A FEW EXAMPLES OF OF

THINGS THAT ARE GOING ON AND

SHOW YOU NEW DATA ON THIS BUT IF

THIS IS THE BIOGENESIS OF NKD2 G

LIGANDS, ET CETERA, WE FIND

EVIDENCE FOR REGULATION OF

LIGAND EXPRESSION AT EVERY LEVEL

OF BIOGENESIS, TRANSCRIPTION,

I'LL TELL YOU NEW DATA ON THAT,

WOES 5 TAR TRANSLATION BUT WE

SUSPECT A ROLE AS WELL AS WELL,

AT THE LEVEL OF RNA TURNOVER, BY

ANOTHER STRESS PATHWAY AND AT

THE LEVEL OF PROTEIN TURNOVER BY

YET ANOTHER STRESS PATHWAY.

SO LET ME SUMMARIZE SOME OF THAT

WORK IN A BIT MORE DETAIL.

SO WHEN WE STARTED THIS WORK, WE

WERE INTERESTED IN HOW LIGAND

EXPRESSION MIGHT BE COORDINATED

WITH THE PROGRESSION OF TUMOR

GENESIS, BECAUSE WE KNOW THIS IS

A SO CALLED VOCAL GRAM

SUMMARIZING THE STEPS IN TUMOR

GENESIS FROM INITIAL MUTATIONS

AND GATE KEEPER TUMOR

SUPPRESSORS OR ONCA GENES THAT

LEAD TO INAPPROPRIATE

PROLIFERATION OF THE CELLS AND

THIS IS THOUGHT TO, YOU KNOW

TRIGGER BY POSSIBLY 2 PATHWAYS,

BY INCURRING DNA DAMAGE IN THESE

RAPIDLY REPLICATING CELLS OR BY

THE ACTIVATION OF THE P19

R-TUMOR SUPPRESSOR THAT THESE

LEAD THEN TO EXPRESSION OF THE

KEY TUMOR SUPPRESSOR,

PARTICULARLY P53 WHICH CAN

RESULT IN CELL CYCLE ARREST OR

PILOT PROJECTITOSEIN OF THE

TUMORS BUT OF COURSE TUMORS CAN

ACCUMULATE P53 AND ADVANCE

YAWBTD THAT AND OF COURSE

THERE'S MANY OTHER CHANGES THAT

MAY ALSO PARTICIPATE IN THIS, SO

WE FOCUSED ON THE ROLE OF THE

DNA DAMAGE RESPONSE AND I'LL

BRIEFLY SUMMARIZE THAT WORK

SINCE IT'S--WELL, SINCE SOME OF

ITS PUBLISHED AND I'M NOT QUITE

HAVING ENOUGH DATA TALK IN

DETAIL ABOUT THE REST OF THE

DATA AND THEN WE'VE ALSO FOUND

EVIDENCE FOR THE ROLE OF THESE

DIFFERENT PATHWAYS, THAT ALL

WOULD BE EXPECTED TO OCCUR IN

THE CONTEXT OF CANCER.

SO, LET ME SUMMARIZE THE DNA

DAMAGE RESPONSE, THIS WAS WORK

DONE BY, AND BASICALLY THE

STRATEGY WAS TO START WITH

RELATIVELY NORMAL CELLS AND ASK

WHETHER WITH THE DAMAGING

AGENTS, OR ANY KIND OF STRESS,

THEY UPREGULATE THE NKG2 D

LIGANDS, THE AGENTS WERE

PARTICULARLY APT AT INDUCING

NKG2 D LIGAND O SINGLE THE

FIBROBLASTS AND CULTURE AND

OTHER TYPES OF STRESS WERE NOT

GOOD @ IT IN THIS CONTEXT AND WE

IMPLICATED THE ATR CAIN

CAIN--KINASEIS, AND THESE ARE

ALSO LEADING TO P53 ACTIVATION.

THESE ACTIVATE A CASCADE THAT'S

RESPONSIBLE FOR CELL CYCLE

ARREST AND INDUCTION OF DNA

REPAIR FUNCTIONS AND AS WE NOW

SHOW INDUCTION OF NKG2 D

LIGANDS.

THIS SHOWS 1 OF MANY DIFFERENT

WAYS WE DID THIS EXPERIMENT OF

KNOCKING DOWN IN THIS CASE, THE

EXPRESSION OF ATM IN A TUMOR

CELL LINE AND WE COULD SHOW IN

THIS CASE, THAT THE CONSITTATIVE

EXPRESSION OF RAY 1 ON THIS

TUMOR CELL LINE DEPENDS ON ATM

ACTIVITY AND THIS IS ACTIVATE

INDEED THIS CELL LINE AND OTHER

ATM LINE ITS PLAYS AN IMPORTANT

ROLE.

THESE 2 KINASEIS RESPOND TO

DIFFERENT TYPES OF DNA DAMAGE.

SO WE COULD SEE IT BOTH INTERNAL

AUDIT DUCED DNA WHERE WE

ACTUALLY INDUCED DNA DAMAGE

PURPOSEFULLY BUT ALSO IN TUMOR

CELLS WHICH ARE KNOWN TO BE

SUBJECT TO ENDOGENOUS DNA DAMAGE

AND HAVE MANY CASES CONSITTATIVE

ACTIVATION OF THE DNA DAMAGE

RESPONSE.

SO WE KIND OF LIKE THIS RESULT

BECAUSE THE DNA DAMAGE RESPONSE

HAD ALREADY BEEN IMPLICATED AS A

BARRIER IN TUMOR GENESIS THAT

THIS IS 1, YET ANOTHER WAY IT

COULD IMPOSE A BARRIER BY

EXPRESSING IMMUNE CELLS COULD

SEE AND COME AND ELIM NAT THE

TUMORS.

NOW HOW DOES IT WORK, I'M NOT

GOING TO HAVE TIME TO SHOW YOU

THIS DATA BUT WE--WE FOUND THAT

IN FACT, IT ACTS NOT BY INDUCING

TRANSCRIPTION FOR EXAMPLE, BUT

IT ACTS BY STABILIZING THE

MESSENGER RTHIS,A BY PREVENTING

RNA TURN OR WHICH IS OTHERWISE

QUITE RAPID BY THE MESSENGER

RNA, AND AND THIS HAS SHOWN

EVIDENCE AND IRTHEIR AND THAT'S

REALLY INTERESTING BECAUSE THOSE

HAD SUGGESTED THAT THE TOLL

RECEPTOR STIMULATION COULD

ENHANCE NKG2 D LIGAND EXPRESSION

AND THIS MAY BE A LINKAGE

BETWEEN HOW CANCER INDUCES

LIGANDS AND HOW INFECTION K'S

INDUCE DISCIPLINARY GANDS

THROUGH THIS INNATE SENSE OR

IRTHEIR, AND WE'RE STILL NOT

SURE HOW THERE MUST BE

INTERMEDIATE STEPS IN HERE IN

TERMS OF HOW IT REGULATES MRNA

TURNOVER.

SO THAT JUST SUMMARIZES WHAT I

MENTION THAD IT'S ALREADY WELL

KNOWN, OF COURSE THE TLRs AND

NLRs AND INFECTIONS FOR

EXAMPLE CAN ALSO INDUCE RF3 AND

I MENTIONED THE RESULTS ON

OXIDIZED LDL, POETIC TENTSIALLY

INDUCING THE LIGANDS THAT MAY

ALSO WORK THROUGH A SIMILAR

MECHANISM.

SO WHAT ABOUT TRANSCRIPTION AND

TRANSLATION?

THAT'S SOMETHING THAT REALLY

VERY LITTLE IS KNOWN ABOUT HOW

THE GENES ARE REGULATED AND WHAT

KINDS OF STRESS INDUCES IT AND

THERE'S 2 RELATED LINES OF

EVIDENCE THAT I WANT TO MENTION.

1 CAME FROM ANALYZING THE

INDUCTION OF NKG2 D LIGANDS AND

AN INFECTED CELL.

SO THIS WAS A MOUSE THAT A

MEGALOVIRUS INFECTIONS WHERE THE

NKG2 D IS INDUCED.

WUBEFORE I MENTION THAT, LET ME

JUST POINT OUT THAT THE PI 3

KINASE PATHWAY IS SHOWN HERE AND

IT'S WELL KNOWN THAT IT'S

ACTIVATED IN CANCER, YOU

FREQUENTLY SEE MUTATIONS OR

ACTIVATING--ACTIVATING MUTATIONS

OR AMP PILOT PROJECTFICATION IN

THE P-110 ALPHA SUBUNIT OF PI3

KINASE OR IN P-10 WHICH

REGULATES THE PATHWAY OR AN AKT

ASK PI3 KINASE IS A TARGET FOR

SOME OF THE MODERN DRUGS THAT

ARE RECENTLY BEING DEVELOPED FOR

CANCER.

SO WHAT MARIA TACKA YAMAHA A

GRAD STUDENT SHOWED WAS THAT P1

ALPHA 10 INHIBITORS INHIBIT RAY

1 INFECT WIDE THE MEG LOW VIRUS,

THE STORY WAS PUBLISHED, AND

THAT LED US TO INVESTIGATE

WHETHER IT HAD A ROLE IN CANCER

CELLS AND INDEED IT SEEMS TO AND

ANY OF SEVERAL TUMOR CELL LINES

THAT EXPRESS THE LIGANDS

CONSITTATIVELY INHIBITORS EVER

PI3 CAIN ACE AND PARTICULARLY

THE P110 ALPHA CAUSE REDUCED

EXPRESSION OF CELL SURFACE, AND

SO WE THINK THAT THE PI3 CAIN

ACE HAS A ROLE AND IT TURNS OUT

MANY VIRUSES HAVE PART OF THEIR

LIFE CYCLES.

SO THIS JUST SUMMARIZES THAT

THERE, THERE'S WORK FROM THE

LAB, BY MARIA TOKE I DON'T MEANA

WHO'S A JOINT STUDENT WITH LAR.

SO HOW DOES THAT WORK WE THINK

AS WE MENTION THAT IT WORKS IN

PART THROUGH INDUCING

PROLIFERATION AND SUPPORTING

PROLIFERATION OF THE CELL SPES

MAY WORK BY PROTEIN TRANSLATION

AND I THINK THE EVIDENCE FOR

THIS IS STILL PRETTY WEAK SO WE

STILL HAVE MUCH TO LEARN ABOUT

EXACTLY HOW PI3 CAIN ICE AND HOW

IT DOES IS HELP SUPPORT

PROLIFERATION OF CELLS AND I'LL

SHOW YOU EVIDENCE, PROLIFERATION

ITSELF IS QUITE IMPORTANT FOR

NKG2 D LIGANDS SO THIS IS WORK

DONE BY A GRADUATE STUDENT IN

THE LAB.

JUST GRADUATED.

SHE HAD FOUND QUITE SURPRISING

TO US AT THE TIME THAT WHEN

PRIMARY FIBROBLASTS ARE SIMPLY

CULTURED INVITRO, FROM SAY TAIL

TISSUE FROM AN ADULT ANIMAL OR

FROM EARS OR THE PERO NEAL WALL

THEY INDUCE LIGANDS IN CULTURE

AND QUITE LEVELS IN FACT SO BY

DAY 6, ALL THE CELLS ARE QUITE

BRIGHT IN TERMS OF GRAY 1

EXPRESSION.

THIS INDUCTION OF RAY 1

EXPRESSION CAN BE INHIBITED SHE

FOUND BY ANYTHING THAT INICHITS

PROLIFERATION OF THE CELLS AND

WE'VE DONE THROUGH MANY

DIFFERENT INHIBITORS OF

PROLIFERATION.

THIS SUGGESTED A POSSIBLE LINK.

MORE OVER IF WE TOOK CELLS THAT

HAD ALREADY INDUCED THE LIGANDS

AND STARVED THEM OF SERUM WHICH

CAUSE THEM TO STOP

PROLIFERATING, WE INDUCE THE

LIGANDS FROM THE CELL SURFACE

AND THEN IF WE GIVE THEM THE

SERUM BACK FOR A FEW DAYS THEY

REEXPRESS IT, SO WE TOGGLE IT UP

AND DOWN BY ASKING THE CELLS TO

PROLIFERATE MORE OR LESS.

THIS SPONTANEOUS RAY 1 INDUCTION

IS IN FACT ASSOCIATED WITH

TRANSCRIPTIONAL ACTIVATION, WE

SY INDUCTION IN THESE CELLS AS

WE CULTURE THEM FOR SEVERAL DAYS

AND IF WE STARVE THE CELLS OF

SERUM, WE SEE RAPID LOSS OF RAY

1 MESSAGE FROM THE CELLS.

NOW, MANY PEOPLE, I AWIVE

COMPLAP TO MY SUANTS I SEE MANY

PAPERS WHERE STUDENTS SHOW

RNAs OR ROOTS INDUCED AND IT

GOES AWAY AND THEY CONCLUDE

TRANSIPGZAL REGULATION, BUT YOU

CAN GET THE SAME RESULT IF IT

WAS SUBJECT TO REGULATION AT THE

LEVEL OF RNA DEGRADATION.

SO, TO SHOW THAT IT'S REGULATED

AT THE LEVEL OF TRANSCRIPTION,

IT'S REALLY NECESSARY TO DO

ANOTHER ANOTHER EXPERIMENT, AND

I DON'T HAVE TIME TO SUMMARIZE

THIS BUT IT BASICALLY MEASURES

THE RATE AT WHICH THE MRNA AS

BEING LABELED AND BEING PRODUCED

IN THE NUCLEUS, AND THAT'S A

MEASURE OF TRANSCRIPTION.

AND SHE WAS ABLE TO SHOW THAT

YOU HIGH LEVELS OF TRANSCRIPTION

AND THE ABSENCE, THE

TRANSCRIPTION GOES DOWN AND IN

THE CONTROL, CONTROL SAMPLES.

SO, ARGUING THAT IN FACT THE

REGULATION HERE IS THEA THE

LEVEL OF TRANSCRIPTION.

NOW TO MAKE A LONG STORY SHORT,

WE BASICALLY ASK THE QUESTION

WELL, IF WE COULDN'T REALLY

SEPARATE THE EXPRESSION FROM

PROLIFERATION OF THE CELLS.

, THIS SUBJECT REGULATE

PROLIFERATION, AND AN IMPORTANT

AND THOSE ARE KNOWN TO DIRECTLY

REGULATE THE CELL CYCLE BY HOW

WELL THIS COMES OUT BUT BY

REGULATING PROTEINS INVOLVED IN

NNA SYNTHESIS AND OTHER EVENTS

IN TERMS OF CELL CYCLE ENTRY.

THEY'RE--THERE'S 3 OF THEM THAT

ARE--3 REDUNDANT EXAMPLES, WE

HAVE 1, TWORKS AND 3, THAT ARE

REDUNDANT FOR CONTROLLING

PROLIFERATION OF CELLS IN

CULTURE.

AND SOMETIMES THESE FACTORS

COOPERATE WITH NMYC IN THEIR

FUNCTIONS AND REGULATED BY THE

RETINAL BLASTOMA TUMOR

SUPPRESSOR.

SO, FIRST OF ALL EXAMINE THE

GENE AND FOUND EVIDENCE THAT THE

E2 F BINDING SITES IN THE

PROMOTER OF THE GENE, SHE THEN

SHOWED BY DOING A CHROMATIN

IMMUNOPRECIPITATION ASSAY SHE

COULD COAT-LIFE THAT THE E2 F1,

2, AND 3 ARE ASSOCIATED WITH THE

RAY 1 PROMOTER--RAE PROMOTER IN

LIVING CELLS AND THERE WAS MORE

ASSOCIATED IN PROLIFERATING

CELLS AND IT WENT DOWN 3 OR SO,

AND IT'S A 4 X TITRATION TYPE

EXPERIMENT.

SUGGESTING THAT E2 Fs WERE

ASSOCIATE WIDE THE GENE, AND

THERE WAS MORE ASSOCIATE WIDE

THE GENE WHEN THE CELLS WERE

PROLIFERATING THAN WHEN THEY

WERE NOT.

TRANSACTIVATEED RAE1, IF YOU

TRANSDUCE THESE INTO CELLS AND

LOOK AT EXPRESSION, THESE ARE

OTHERWISE STARVED OF SERUM SO

THEY DON'T HAVE SUPPORT FOR

ENDOGENOUS PROLIFERATION.

SHE COULD INDUCE, RAE 1

TRANSCRIPTION AS SIMILAR TO THE

EXTENT THAT SHE COULD INDUCE A

KNOWN TARGET FOR E-2 F GENES.

FINELY SHE DEVELOPED A

LUCIFERASE CONSTRUCT DRIVEN BY

THE E2 F REASONNION AND THE RAY

1 PROMOTER CONTAINED THOSE SET

SITES AND BY CO TRANSVECTING FOR

E2 F1 SHE COULD SHOW ACTIVATES,

IT ACTIVATES TRANSCRIPTION OF

THE RAY 1 PROMOTER, BUT THAT

APPROXIMATE YOU MUTATE BOTH OF

THOSE E2 F SITES YOU

ESSENTIALLY, HAVE YOU A STRONG

REDUCTION IN E2 F1

TRANSCRIPTION.

SO ALL THOSE TOGETHER MADE A

STRONG ARGUMENT THAT E2 Fs

WERE REGULATING TRANSCRIPTION OF

THE RAY 1 GENES.

SO RACE 1 GENES ARE REGULATED BY

FACTORS THAT PROXIMALLY CONTROL

PROLIFERATION.

WE BEGAN TO ASK THE OBVIOUS

QUESTION IS DO NORMAL

PROLIFERATING CELLS IN VIVO

UPREGULATE B-1 AND IT TURNED OUT

THAT THE FIRST DESCRIPTION OF

RAY 1 BY A JPS GROUP SOME YEARS

AGO, DEMONSTRATED THE EXPRESSION

IN THE EARLY EMBRYO.

IN NORMAL EARLY EMBRYOS AND THEN

IT PRETTY MUCH DISAPPEARED

AROUND THE TIME OF BIRTH AND SO

WE WENT BACK AND LOOKED, LOOKED

IN FOR EXAMPLE, PROLIFERATING

BRAIN CELLS BUT THERE'S AN

INTENSE AMOUNT OF PROLIFERATION

OCCURRING IN THE EARLY EMBRYO

AND THE OTHER NICE THING ABOUT

USING EMBRYONIC BRAIN CELL

SYSTEM THEY IMMEDIATELY

DISASSOCIATE WHEN YOU PUT THEM

INTO THE TEST TUBE AND WE CAN

EASILY DO FLOW CYTOMETRY ON THEM

AND IT WAS TURNED OUT IT WAS

EASY TO DEMONSTRATE THAT THEY

EXPRESS RACE 1, BUT AT THE

READILY DETECTED BY FLOW AND

ALSO WE COULD DETECT IT IN BY

STAINING OF EMBRYONIC DAY 14

BRAIN SECTIONS.

MOW WHAT WAS KIND OF SURPRISING

WAS THAT BY DAY 18 OF GESTATION,

THE STAINING HAD DISAPPEARED AND

THAT WAS A LITTLE SURPRISING

BECAUSE THE BRAIN DOESN'T STOP

PROLIFERATING AT DAY 18 BUT IT

DOES DECREASE.

THIS IS A BRDU INCORPORATION

EXPERIMENT TO LOOK AT DAY 14 OR

18, YOU CAN SEE THERE'S

CONSIDERABLY LEAF PROLIFERATION

AT 18.

THERE'S SOME AND IT'S SURPRISING

TO US THAT WE SEE DISAPPEARANCE

OF RACE 1 AND WE EXPECT THAT

MEANS A THRESH HOLD OR

PROLIFERATION THAT'S NECESSARY

TO INDUCE RACE 1 EXPRESSION.

WELL WHAT ABOUT IN OTHER

SCENARIOS OF PROLIFERATING CELLS

WEY DECIDED TO LOOK AT SKIN

WOUNDS SINCE WE KNEW THAT IT

PROLIFERATE INDEED HEALING SKIN

WOUNDS AND THIS IS AN EXPERIMENT

LOOKING AT HEALING SKIN WOUND

AND STAINING HERE IN RED FOR

BRDU, INCORPORATION, SO, THAT

TELLS YOU WHICH CELLS ARE

PROLIFERATING AND IN THE

EPIDERMIS HERE LABELED WITH THE

E, CAN YOU SEE LOTS OF

PROLIFERATING CELLS SO IN THE

EPIDERMIS, KERO10 O SIGHTS IN

THE EPIDERMAL LAYER AS THE

TISSUE HEALS.

SO WHAT SHE FOUND IS THAT IF YOU

EXIZE SAMPLES OF THESE AND LOOK

AT RACE 1 MESSAGE YOU SEE A

PRETTY SHARP INCREASE IN RACE 1

TRANSCRIPTS THIS THIS HEALING

SKID 1 TISSUE, COMPARED TO

UNWOUNDED SKIN, DAY 0 AND MORE

OVER BY STAINING SECTIONS AND

IMPUNE O FLUORESCENCE YOU CAN

SEE VERY NICE STAINING OF THE

SECTIONS WITH ANTIRAY 1 EPSILON

ANTIBODY IN THE HEALING WOUND

AND A MUCH THINNER LEVEL OF

STAINING IN THE NORMAL SKIN.

SO IT SUGGESTED THAT EVEN IN

THIS ADULT YOU ARE SEEING THE

EXPRESSION OF OF RACE 1.

IN THE BRAIN WE DON'T THINK

THERE ARE ANY NK CELLS SO IT'S

HARD--WE'RE NOT CLAIMS THAT THE

RACE 1 IS DOING ANYTHING IN THE

EMBRYONIC BRAIN BUT THIS IS AN

ADULT ANIMAL WHERE NK CELLS ARE

PROMINENT AND OF COURSE THERE

ARE MANY T-CELLS THAT ARE

EXPRESSING THE LIGANDS YOU MIGHT

EXPRESS TO HAVE A FUNCTION IN

THIS HEALING, THESE HEALING SKIN

WOUNDS.

SO, THIS LED US BACK TO THINKING

ABOUT GAMMADELTA T-CELLS BECAUSE

A POPULATION OF THEM LIVES IN

THE 69 AND WEND SCHECOLLEAGUES

HAD SHOWN THAT EVIDENCE THAT

THESE SUBSET OF GAMMADELTA CELLS

THAT LIVE IN THE SKIN SEEM TO

HAVE A ROLE IN WOUND HEALING AND

THESE EXPERIMENTS BASICALLY HOLE

PUNCHES ARE MADE IN THE SKIN OF

MICE AND YOU FOLLOW THE RATE OF

WHICH THE WOUNDS CLOSE AND

THAT'S SHOWN HERE IN THE

WILD-TYPE AND SHE FOUND EVIDENCE

THAT TCR DELTA KNOCK OUT WHICH

ANYTHING OUT ALL GAMMA T-CELLS

THE RATE OF WOUND CLOSING WAS

SLOWER SUGGESTING THAT

GAMMADELTA CELLS PLAY A ROLE IN

ACCELERATING WOUND HEALING IN

THESE ANIMALS.

MEAN WHILE MY FORMER STUDENT

INVESTIGATED THESE T-CELLS IN

THE SKIN.

THESE SKIN GAMMADELTA T-CELLS

INVITRO AND IN WENDY HAD ALREADY

SHOWN THAT YOU CAN GET THESE

T-CELLS TO KILL CULTURED KERRA

10 O SIGHTS BECAUSE PRESUMABLY

THEY'RE STRESSED BY BEING IN

CULTURE AND THAT THE--AND THE

CULTURE KERATIN O SIGHTS WERE

KILLED BY THEM IN CULT AND YOU

ARE WE COULD CORROBORATE THAT.

MIKE FOUND INTERESTINGLY

HOWEVER, THAT THIS KILLING WAS

COMPLETELY DEPENDENT ON NKG2 D,

IF WE LOOK TO T-CELLS FROM AN

NKG2 D KNOCK OUT OR FROM OR IF

WE PUT ADD THE NKG2 D ANTIBODIES

WE INHIBITED THE KILLING

COMPLETELY SO WE DON'T THINK

THAT NKG2 D ALONE IS MEDIATE

THANKSGIVING KILLING BUT THE

EVIDENCE TOGETHER SUGGESTS THAT

NKG2 D IS WORKING COOPERATIVELY

WITH THE STIMULUS TO ACTIVATE

THESE T-CELLS TO KILL, TO KILL

THESE KERATIN O SIGHTS IN

CULTURE AND SO HE WENT BACK AND

SAID, THIS IS ALL TRUE, MAYBE

NKG2 D HAS A ROLE IN WOUND

HEALING AS WELL, AND SHE'S DONE

THE EXPERIMENT--AND WHAT THEY

FOUND WAS THAT IN THE NKG2 D

KNOCK OUTS, EVEN THOUGH THEY DO

HAVE COMPLETELY NORMAL NUMBERS

OF SKIN BAMMA DELTA T-CELLS THIS

COULD HAVE A ROLE IN PROMOTER

WOUND HEALING REACTIONS SO LET

ME JUST SUMMARIZE, THESE DATA ON

REGULATION BY PROLIFERATIVE

SIGNALS THAT BY THE ACTION OF E2

F, THE PROLIFERATIVE SIGNALS

ACTIVATE TRANSCRIPTION OF THEM,

THE LIGANDS THAT WE BELIEVE IN

TUMOR CELLS THIS PROLIFERATIVE

COORDINATION IS REGULATION BY

OTHER STRESS SIGNAL SUCH AS DNA

DAMAGE RESPONSE AND THE--AND

OTHER PATHWAYS AND CANCER CELLS

AND PROBABLY INFECTED CELLS THAT

PROLIFERATING, AND INCLUDING

EMBRYONIC CELLS IN SKIN WOUNDS

AND EXPRESS, BASED ON WOUND

HEALING, AND THE LIGAND

INTERACTIONS ON THE 1 HAND

TARGET CANCER CELLS AND INFECTED

CELLS FOR ELEMINATION, BUT ALSO

REGULATE TISSUE PROCESSES SUCH

AS WOUND HEALING AND OTHER

CONTEXT.

OKAY.

SO TO SUMMARIZE WHAT I TOLD BUT

NKG2 D SUMMARIZE, THE RECENT

DATA, THE TRANSCRIPTION

REGULATED BY TRANSCRIPTION

FACTORS AS A RESULT OF HYPER

PROLIFERATION THAT PI3 CAIN ACE

MAY REGULATE TRANSLATION OF

INDIRECT EVIDENCE FOR

TRANSLATIONAL ROLE, PI3 CAIN ACE

IS KNOWN TO REGULATE TRANSLATION

OF MANY GENES.

THE DNA DAMAGE RESPONSE REGULATE

THE TURNOVER THE RACE 1 AND

MESSENGER RNAs AND WORK THAT I

DIDN'T HAVE TIME TO SHOW YOU 1

OF THE LIGANDS, MOLE 1 IN MICE

IS REGULATED AS A PROTEIN

TURNOVER LEVEL BITE THE HEAT

SHOCK STRESS RESPONSE.

AND SO, THESE ARE JUST SOME OF

THE PATHWAYS WE THINK THAT PLAY

A ROLE.

IN THE LAST FEW MINUTES I WANT

TO COME BACK TO CANCER AND

MENTION SOME OTHER DATA THAT

SUGGESTS HOW OTHER KINDS OF

TUMOR BARRIERS TO TUMOR GROWTH

MAY INTERACT WITH SIGNALS THAT

INDUCE NKD2 G LIGANDS TO HELP

PREVENT TUMORS.  THIS IS A PAPER

OF A COUPLE YEARS AGO NOW BY

SCOTT LOW'S LAB AND WHAT HE AND

OTHER VS SHOWN IS THAT, YOU KNOW

IT'S KNOWN THAT TUMORS ARE THE

INFLUENCED AND CAN SOMETIMES A

STATE OF SENESCENS WHERE THEY

STOP DRAWING AND THEY--RESINENT

IN THE TISSUE FOR A WHILE AND

THEY DISAPPEAR.

IT WAS SHOWN IN THESE

EXPERIMENTS THAT SIN ESTIMATE

THAD ENS IS INDUCED BY THE P53

TUMOR SUPPRESSOR.

SO WHAT THEY DID HERE WAS THEY

TOOK NORMAL LIVER CELLS, THEY

TRANSDUCED THEM WITH THE

RETROVIRUS ENCODING ACTIVATED

RAS, AND THEY TRANSDUCE WITH A

REGULATED SH--SHRNA, BUT PUT

THEM IN MICE TO PLAY TUMORS, AND

YOU TURN IT ON BY ADDING O

SILLIN.

DOCKS, AND THEY SEE TUMOR

REGRESSION, THAT'S SHOWN HERE.

, AND STOP GROWING AND AND THIS

SHOWS DISAPPEARANCE OF THE TUMOR

CELLS SEEMS TO DEPEND ON THE

IMMUNE CELLS, OR MACROPHAGES OR

THE CESS ENS ESTIMATE THAD

DISAPPEARS.

NOW I DON'T UNDERSTAND WHAT'S

GOING ON WITH THE NUTRIFILLS OR

MACROPHAGES BUT THAT'S AN

INTERESTING THING TO PURSUE.

SO WE DECIDED TO ASK ABOUT, IT'S

BEEN DOING THIS WORK.

AND I SHOULD POINT OUT, FOCUS ON

THE INNATE RESPONSE.

WE USE RAG MICE TO ACHIEVE THE

SAME OUTCOME WHEN WE CAM PAIR

EITHER RAGS OR RAGS THAT LACK

NKG 2 D AND WERE COMPLETED OF NK

CELLS WITH AN ANTIBODY.

AND WE DID EXACTLY THE SAME

EXPERIMENT.

WE INTRODUCED TUMORS THEY GO TAR

10 DAYS THROUGHOUT AND THEN WE

TURN P53 ON AND WE ASK WHAT

HAPPENS IN THE CONTROLS WE

EXPECT THE TUMOR TO GO AWAY AND

THE QUESTION IS WHAT HAPPENS IN

THE KNOCK OUT.

SO WHAT'LL EXPECTATIONS FOUND IS

THAT WE COULD REPRODUCE WHO LOW

HAD SHOWN, AND THIS DISAPPEARED

WITH THIS RATE AND WHEN WE

DEPLETED NK CELLS AND RAD, THEY

DEPLETED AT A SLOWER RATE.

SO IT ARGUES AGAIN THAT IT PLAYS

A ROLE IN THE DEPLETION.

INTERESTINGLY WITH AN NKG2 D

KNOCK OUT HOST, WE GET THE SAME

RESULT SUGGESTING THAT NKG2 D IS

HELPING TO MEDIATE THIS

SENESCENT TUMOR AND ALEX DID

THIS EXPERIMENT BLOCKING THE

ANTIBODY TO GET THE SAME RESULT.

THE TUMORS BY THE WAY GROW AND

BASICALLY THE WAY WE DO THIS

EXPERIMENT WE LOAD THEM UP WITH

LOTS OF TUMOR CELLS SO THE

TUMORS ARE NOT GROWING AT

DRAMATICALLY DIFFERENT RATES IN

THESE 3 SCENARIOS AND INITIALLY

AND THAT'S TO--WE WANT TO AVOID

DIFFERENCES BETWEEN COMPARING

REALLY TINY TUMORS WITH REALLY

LARGE TUMORS IN THE DIFFERENT

CONTEXT.

SO I MIGHT SAY WELL, MAYBE P53

INDUCES THE LIGANDS BUT WE

DIDN'T THINK THAT NKG2 D; WE

NEVER FOUND ANY EVIDENCE BUT

THERE ARE SOME REPORTS OF THAT

IN THE LAT RATTURE BUT IT'S

RELATIVELY MODEST INDUCTION AND

RELATIVELY SPORADIC.

AND INDEED WE DON'T SEE MUCH

EVIDENCE FOR IT HERE.

WE'RE LOOKING HERE AT MINUS

THIS--THIS IS DONE EXVIVO

LOOKING AT TUMORS WITHOUT DOCK

OR WITH DOCKS WE SEE THE TUMORS

STILL GFP POSITIVE BECAUSE THE

SHRNA ENCODES WE SEE RACE 1

EXPRESSION SIMILARLY AT DAY 4.

AND BY DAY 7 IF ANYTHING THERE'S

LESS RACE 1 WITH THE TUMORS AND

* TO SCALE, AND THE P53

SCENARIO, LESS RAY 1 NOT MORE,

AND INDEED WE LOOK AT LOTS OF

DIFFERENT LIGANDS AND THIS IS

REALLY WHERE A LOT OF THE

ELIMINATION IS OCCURRING AROUND

DAY 4 AND WE DON'T SEE MUCH

DIFFERENCE IN RACE 1 OR CD155

WHICH IS AND THE MOLECULES ICAM

1 OR 2.

THERE'S SOME DIFFERENCES BUT

THEY'RE NOT DRAM THEICALLY

DIFFERENT AND THE ADAPTER OF THE

LIGAND FOR TRAILER, THE LIGAND

PROFESS, WE SEE AGAIN NOT REALLY

DRAMATIC DIFFERENCES SO WE DON'T

THINK YOU CAN ASCRIBE THE

DEPLETION OF THE SENESCENT CELLS

TO INCREASED EXPRESSION OF THESE

DIFFERENT TARGETING MOLECULES.

AND IN FACT, IF YOU LOOK INVITRO

AT THE SUSEPTIBILITY OF THESE

CELLS, THIS IS DONE BY

TURNING--ADDING DOX OR NOT,

MAKING THE CELLS SIN ESTIMATE

THADENT AND THEN COMPARING

SENSITIVITY TO THE INVITRO.

NEVERTHELESS, THE SENSITIVITY IN

BOTH, P53 THAT'S BECAUSE THE

TUMOR CELLS ALL EXPRESS, THEIR

NK SENSITIVE AND NKG2 D PLAYS A

BIG ROLE BUT P53 DOESN'T

REGULATE THAT SENSITIVITY TO

KILL IT.

, AND THEY'RE SUSEPTIBLE, P53

INDUCTION ENHANCES NEITHER

INDUCTION OF LIGANDS OR

SUSEPTIBILITY TO NK CELLS, THE

OTHER QUESTIONS ARE THE OTHER

PROCESSES OPERATIVE IN THE P53

ION CONDITION THAT ENABLE

NK CELLS TO ENABLE THESE CELLS.

WHY ARE THEY ELIM NATTED BETTER?

WHAT WE FOUND IS THAT THE

INFILTRATION OF LYMPHOCYTES AND

NK CELL INTO THE TUMORS.

SO IN THE P53 CONDITION, WE SEE

MANY MORE IMMUNE CELLS

INFILTRATING INTO THE TUMORS

THAN IN THE P53 CONDITION.

AND A BIGGER FRACTION OF THOSE

INFILTRATING CELLS ARE NKCELLS.

SO WE THINK IT'S THE ACTUAL

RESILIENCE CRUITMENT OF THE

CELLS THAT'S LARGELY DIFFERENT.

NOW, THIS HARKENED BACK TO

RESULTS FROM JUDY CAMP SPECIE

AND OTHERS AND SHY DEFINED THIS

SENESCENT PHENOTYPE THAT'S

SENESCENT TUMORS AND OTHER CELLS

IN THAT MATTER, BEGIN TO SECRETE

A SERIES OF WHAT SHE CALLED OF

FAJTORS THAT INDUCE A WHOLE

SERIES OF PROCESSES IN SENESCENT

TUMORS AND OTHERS TYPES OF SIN

ESTIMATE THADENT SALs AND SHE

DEFINES A GENE EXPRESSION

PATTERN AND THE SIN ESTIMATE

THADENT TUMORS AND FOUND MANY

INTERESTING CYTOKINES AND CHEMO

KIPES BEING INDUCED IN SENESCENT

TUMORS AND SHE PROPOSE THAD MAY

MIGHT BE INVOLVE INDEED IMMUNE

CLEARANCE AND I THINK THAT'S

CORRECT AND AMONG THE FACTORS

THAT WE THINK ARE RELL VABT ARE

THESE INDUCED CHEMOKINES, CXCL8,

CCL3 AND 5 THAT MAY ALL BE

INVOLVED IN RECRUITING IMMUNE

CELLS INTO THE TUMORS.

NOW YOU MIGHT ASK, IS SUSPICIOUS

53 DEPENDENT RECRUITMENT NKG2 D

DEPENDENT AND WE THINK IT'S NOT

MOSTLY NKG2 D DEPENDENT IN THE

P53 CONDITION WE FIND LESS

RICRUITMENT IN THE NKG2 D KNOCK

OUTS THAN IN THE WILD-TYPES BUT

THERE'S STILL A LOT OF

RECRUITMENT, SO THE SENESCENT

IMINIZATION IS LARGELY DEPENDENT

BUT THE SUSEPTIBILITY OF THE

CELLS IS NOT ENHANCED MUCH WE

THINK THE ELIMINATION OF OF

TUMORS ELIMINATE SIN ESTIMATE

THADENT CELLS.

I'M FINISHING HERE.

EARLY IN TUMOR GENESIS AND

INDUCE EXPRESSION OF NKG2 D

LIGANDS AND THAT'S BY A POST

TRANSCRIPTIONAL MECHANISM, AND

PROLIFERATIVE SIGNALS, AND I

SHOULD MENTION BY THE WAY THAT

OTHER NKG 2 D LIGANDS WE LOOKED

AT THEM ALL AND H60 ARE NOT

INDUCED BY--NOT AT LEAST NOT

NEARLY AS DRAMATICALLY INDUCED

BY THE ARRAY 1 FAMILY SO NOT ALL

LIGANDS ARE NECESSARILY

SUSEPTIBILITY TO THIS TYPE OF

REGULATION THAT THE PI-3 KINASE

PATHWAY ACTIVATES INDUCES NKG2 D

LIGANDS AND IT STABLE ICES 1 OF

THE LIGAND ASTERISKS THE PROTEIN

LEVEL, WE THINK THAT THE

COLLECTION OF STRESSES ARE

COORDINATED TO INDUCE SOME NKG2

D LIGANDS MAYBE A BAR CODE THAT

THE COLLECTION OF DIFFERENT

KINDS OF STRESSES AND

PROLIFERATION AND OTHER EVENTS

MAY WORK TOGETHER TO INDUCE

LIGANDS OPERATING GLOBALLY MALLY

IN THE MOST DANGEROUS TYPES OF

CELLS LIKE CANCER CELLS AND

FINALLY THE SIN ESTIMATE THAD

ENS ASSOCIATED CELLS MAY

FACILITATE NKG2 D ELIMINATION

PROCESSS AND FINALLY THE

PATHOLOGICAL CONDITIONS THAT

EXIST IN METABOLIC DISEASE MAY

TRIGGER NKG2 D AMPLIFICATION

INDUCTION ANDATLER O SCLEROSIS.

I'D LIKE TO END THERE AND THANK

MY COLLEAGUES FOR THEIR WORK AND

I MENTIONED MOST OF THE PEOPLE

AS I WENT ALONG AND THE POST

DOCK IN THE LAB WHO KNOCKED OUT

THE NKG-2 GENE AND MADE THOSE

STUDIES EXPOBL TO THE ANALYSIS

OF TUMOR GENESIS AND THE NKG2 D

KNOCK OUTS, POST DOC WHEN SHOWED

THE ROLE OF THE DNA DAMAGE

RESPONSE.

AKNEELA DID THE WORK ON THE SIN

ESTIMATE THADENT CELLS I

MENTIONED MIKE WANG DID WORK ON

THE GAMMADELTA T CELLS AND

ACTIVATING THOSE CELLS, WE HAVE

A FORMER GRADUATE STUDENT WHO

DID THE WORK ON PROLIFERATION

AND THE LIGAND TRANSCRIPTION

MARIA OTHER WORKS ON THE KINASE

PATHWAY AND WE HAVE OUR

COLLABORATOR THE ON THE DISEASE

IFAT LO SCLEROSE AND I GUESS I

DIDN'T HAVE TIME TO TELL BUT THE

OTHER WORK IN THE LAB, THANK YOU

FOR YOUR ATTENTION AND I'LL TAKE

ANY QUESTIONS.

[ APPLAUSE ]

>> SO, ASSUME YOU LOOKEDDA THOSE

BECAUSE THAT'S ANOTHER PLACE

THAT WEND SCHEOTHERS CELLS

MAINTAINING THE BARRIER,.

>> YOU KNOW WE HAVEN'T--WITH

WE'VE ONLY DONE--WE HAVEN'T DONE

GOOD EXPERIMENTS LOOKING AT IT,

IT'S BEEN ON THE LIST, BUT I

WILL POINT OUT THAT THOM'S LAB

ORIGINALLY REPORT THAD THE

LIGANDS, HUMAN LIGANDS ARE

EXPRESSED IN THE NORMAL GUT

EPITHELIUM SO THAT MAY BE AN

EXAMPLE FROM THESTST STUDIES OF

THESE LIGANDS THAT WOULD BE

CONSISTENT WITH IT, I THINK THE

NOTION THERE IS THAT IT MIGHT BE

INDUCED BY, YOU KNOW FLORA OR

SOMETHING LIKE THAT.

BUT IN FACT, IT MAY BE

PROLIFERATION IS PLAYING A ROLE

THERE AS WELL.

>> AND IN TERMS OF YOUR IRF 1

STORY AND TRANSLATION AND RNA

STABILITY, WHAT'S THE ROLE OF

SORT OF AN INVERSE ROLE OF

MICRORNASSIZE THAT INHIBITING

THE MICRORNAs OR OTHER

MECHANISM?

>> I SHOULD POINT OUT THAT OBER

MENDEL SON'S LAB HAS THE ROLE OF

REGULATING HUMAN NKG2 D LIGANDS.

WE HAVEN'T--WE REALEE DON'T KNOW

WHETHER IT'S MICRORNA REGULATED.

WE JUST DON'T KNOW FOR SURE.

YOU KNOW WE CERTAINLY LOOKED FOR

TARGETS AND DONE SOME OF THOSE

KINDS OF EXPERIMENTS AND I

ATTEND TO SUSPECT IT'S NOT

INVOLVED BECAUSE THE--THE

AMOUNT--RANGE OF REGULATION IS

LARGER THAN 1 OFTEN SEES FOR

MICRORNA REGULATION BUT WE'RE

NOT SURE ABOUT THE ROLE OF MICRO

RNAs.

THE ATHEROSCLEROSIS MODEL YOU'VE

SHOWN THAT THE KNOCK OUT HAS A

STRONG PROTECTIVE ROLE AND YOU

COULD SEE THE SAME WHEN YOU

BLOCK WITH ANTIBODIES RIGHT, BUT

IN THE TYPE 1 DIABETES, YOU SHOW

THAT THE KNOCK OUT HAS NO

PARTICULAR EFFECT.

THERE WAS--

>> IN THE TYPE 1 DIABETES.

>> TYPE 1.

>> THERE WAS A LENIER AND JEFF

WHERE AT A BLOCK THE NKG2 D AND

IF I REMEMBER THEY HAD A STRONG

PROTECTIVE EFFECT.

SO WHAT WAS THE DIFFERENCE IN

THE SYSTEMS OR HOW DO YOU TEST

THE ANTIBODIES?

>> WE HAVE DONE SOME EXPERIMENTS

NOW LOOKING AT ANTIBODIES AND WE

SHOW FAR DON'T SEE ANY

CONTROLLER OF BANKING OF THE

DISEASE--BLOCKING OF THE

DISEASE.

SO I DON'T UNDERSTAND WHY THE

RESULTS ARE DIFFERENT.

ALL I KNOW IS WE--THE KNOCK OUT

EXPERIMENTS WE'VE DONE

EXTENSIVELY AND WE HAVEN'T SEEN

ANYTHING.

THE ANTIBODY EXPERIMENTS, I

WOULD SAY IS STILL PRELIMINARY

BUT IT'S EXPERIMENTS WE HAVEN'T

DONE MUCH AND I DON'T HAVE AN

EXPLANATION FOR THE DISCREPANCY.

DIFFERENCES IN FLORA, THAT'S

WHAT YOU CAN ALWAYS SAY.

>> WHEN YOU LOOK AT THE

AGGRESSIVE TUMOR CELLS, DO THEY

EXPRESS RACE 1 AT A HIGHER LEVEL

THAN THE RACE 1 EXPRESS IN

NORMAL PROLIFERATING SKIN CELLS?

THERE WOULDN'T BE ANY SELECTION

AGAINST IT, IT'S NOT THAT HIGHLY

EXPRESSEDDA AT LEAST AT THE

TIME--AT THE TIME WE EXTRACT THE

TUMOR.

THE PROBLEM OF COURSE IS THAT--I

MEAN IT'S THERE AND WE CAN SEE

IT.

BUT, THE PROBLEM WITH THOSE

EXPERIMENTS OF COURSE IS THAT

YOU LOOK ONCE THE TUMOR IS A

MASSIVE TUMOR, YOU'RE

NOT--PROBABLY LOOKING AT THE

RIGHT TIME WHEN THE IMMUNE

SYSTEM MIGHT BE ACTING ON THE

TUMOR WHICH WOULD BE MUCH

EARLIER.

SO WE--WE'VE FOR YEARS NOW BEEN

TRYING TO DO THAT KIND OF TIME

STUDY, AND WE STILL DON'T HAVE

REALLY VERY GOOD RESULTS ON

THAT, I'M AFRAID.

BUT WE DON'T HAVE ANY EVIDENCE

THAT THEY EXPRESS MORE NKG2 D

LIGAND FIST THAT'S YOUR

QUESTION, NO.

>> IN SCOTT LOW'S EXPERIMENT,

DISCIPLINARY BED CORRECTLY YOU

EITHER DEPLETE NUTRIFELLS OR

MACROPHAGES, YOU GET THE

EQUIVALENT RESULT ON THE

NKCELLS.

>> YES, HE REPORT THAD.

>> HAVE YOU BEEN ABLE TO REPEAT

THAT.

>> DOES THAT SUGGEST YOU THROW

THE PART OF THE INTERACTION FOR

IT TO WORK?

>> THAT'S A GOOD QUESTION.

HE DIDN'T REALLY PROVE THEY'RE

ACTS IN THE SAME PATHWAY, IF YOU

WILL.

AND HE NEVER COMBINED THE 3, AND

WE HAVEN'T--WE HAVEN'T REPEAT

THAD PART OF THE STORY.

SO, I DON'T KNOW WHETHER THEY'RE

ACTING COOPERATIVELY WITH NKG2 D

IN A COOPERATIVELY, OR SEPARATE

MODE OF ELIMINATION OR NOT, BUT

IF YOU DEPLETED ALL 3, THE SIN

ESTIMATE THADENT THINGS WOULD

GROW A BIT.

>> BUT WITH YOUR IMMUNE

INFILTRATION YOU'RE SEEING A LOT

OF MACROPHAGES AND NUTRIFILLS IN

ADDITION TO NK CELLS.

>> WE SEE A LOT OF THOSE, WE DO

SEE A LOT OF MYELOID CELLS.

IT MAY PLAY A ROLE, I'M NOT

TRYING TO DIMINISH THAT.

>> TWO QUESTIONS.

1 IS THAT THE T-CELL PEOPLE

SHOULD ASK THIS QUESTION, DID

YOU LOOK AT THE CD8 T-CELL

PHENOTYPE IN YOUR KNOCKOUT MICE,

WOULD IT BE POSSIBLE THAT THE

AUTOIMMUNE MILD AUTOIMMUNE IN

THE EACE THAT COULD CONTRIBUTE

TO THE EXTRA LESS ACTIVE CD8

T-CELLS, PARTICULARLY

AUTOREACTIVE T-CELLS?

SECOND QUESTION, IS THAT, WHAT

STARTS OFF RAE 1?

IT'S UBIQUITOUS.

RAE 1 EXPRESSION IS CONTROLLED

RATHER TIGHT AND SO THERE MUST

BE SOME GENES THAT NEGAATIVELY

SUPPRESS EXPRESSION

TRANSCRIPTION.

SO YOUR FIRST QUESTION WAS CD8

THEAV CELL PHENOTYPE.

>> WE'VE BEEN SPENDING EFFORT

NOW LOOKING AT CD8 T-CELL

FUNCTION USING THE KNOCK OUTS

AND YOU KNOW THEY'RE

SURPRISINGLY NORMAL IN ASSAYS OF

YOU KNOW VIRUS RESPONSES TO

VIRUSES THAT WE'VE LOOKED AT AND

THINGS LIKE THAT, AND WE'RE NOT

LOOKING IN THE CONTEXT OF COURSE

OF TUMORS AND I WOULD SAY WE

DON'T REALLY HAVE ANY CLEAR

RESULTS SO I CAN'T TELL WHAT

YOU'S WRONG WITH THE CD8S.

IN OUR VERY EARLY EXPERIMENTS IN

TUMORS DID SUGGEST THAT NKG2 D

PLAYS AN AMPLIFYING ROLE IN

T-CELL RECEPT SPONSES BUT IT'S

KNOTS NECESSARY FOR CDT-CELL

ACTIVATION IN SOME CONTEXT

INFECTION ON FOR EXAMPLE OR THE

LIKE.

YOU KNOW WHETHER IT HAS

ANY--WHETHER THE EACE RESULTS

ARE EXPLAINED BY DEFECTIVE CD8

FUNCTION, WE JUST DON'T KNOW

WHAT CELLS IT'S ACTING IN AND IN

THAT MODEL SO I JUST CAN'T

DIAGONAL YOU THE ANSWER TO THAT.

IT'S A GOOD QUESTION.

WE DON'T HAVE A GOOD CONDITIONAL

KNOCK OUT AND IF WE DID, THERE

IT'S STILL NOT THAT EASY TO

TARGET THINGS TO NK CELLS.

BECAUSE THERE'S JUST--JUST

APPEARING IN GOOD CREE MICE FOR

NK CELLS.

WE DON'T HAVE A CONDITIONAL

KNOCK OUT IN EITHER CASE.

YOUR SECOND QUESTION WAS ON

WHETHER HOW RAE 1 IS PREVENTED?

>> IT'S SURPRISING WE TAKE THESE

NORMAL CELLS AND WE GET THEM TO

PROLIFERATE AND THEY TURN ON RAE

1.

SO I'M NOT SURE IN ALL CELL

TYPES THERE ARE OTHER MECHANISMS

TO SUPPRESS RAE 1 EXPRESSION.

THAT'S ACTUALLY A QUESTION,

SINCE REALLY BACKED BY OUR

RESULTS.

OT OTHER HAND, THERE ARE--YOU

KNOW I DON'T THINK THAT THERE'S

ALTHOUGH I THINK WE'RE GOING

BACK TO REVISIT THIS NOW.

I DON'T THINK THAT THERE'S

REALLY STRONG NKT 2 D LIGAND IN

THE CONTEXT OF LYMPHOCYTES FOR

EXAMPLE WHICH WOULD CAUSE A REAL

DISASTER.

AND SO, I DO ACTUALLY LIKE THE

IDEA THAT AT LEAST IN CERTAIN

CELL TYPES AND CERTAIN CONTEXT,

THERE WOULD?

FACT BE AS YOU SUGGEST WAYS TO

REPRESS RAE 1 EXPRESSION.

BUT WE DON'T KNOW WHAT THEY ARE.

>> SO I GUESS 1 OF THE PLAGUING

THEY THINKS DAVID IS THAT THE

HUMAN AND THE MOUSE ARE SO

DIFFERENT IN TERMS OF THEIR NKD2

D LIGANDS--

>> ARE THEY?

[LAUGHTER]

>> THOU THEY'RE DIFFERENTIALLY

EXPRESSED AND SO FORTH, SO THE

QUESTION IS THE MOUSE HAS AN

ARRAY OF THINGS THAT SERVE AS

LIGAND BUS THE INDUCTION OR

EXPRESSION SEEMS TO BE SO

DIFFERENT.

>> WHAT'S OF THE EVIDENCE FROM--

>> IN TERMS OF GENOTOXIC STRESS

VERSES HEAT SHOCK STRESS, ET

CETERA SO I GUESS THE QUESTION

IS, WHAT ARE THE PARALLELS THAT

YOU SEE WITH YOU HUMAN LIGANDS

VERSES THE ANTIPARALLELS.

>> WELL, THE GENOTOXIC STRESS

HAS BEEN SHOWN IN BOTH MOUSE AND

HUMAN FOR SURE.

THAT SEEMS TO BE OPERATIVE THERE

IN BOTH CASES.

YOU KNOW HEAT SHOCK STRESS, I

DON'T KNOW IF ANYONE'S--WE DON'T

SEE MUCH EVIDENCE FOR ROLE, TOM

HAD REPORTED THAT MYC'S ARE

REGULATED TRANSCRIPTIONALLY, AND

IT'S TRUE THAT WE DON'T FIND

THAT BUT WE'RE NOT THE ONLY 1S

WHO DON'T FIND THAT SO I DON'T

KNOW, YOU KNOW I DON'T

REALLY--I'M NOT REALLY CONVINCE

TREAD'S DISCI'S CREPANCY THERE.

THE PROLIFERATION RESULTS I

DON'T THINK THERE'S REASON TO

BELIEVE THAT'S NOT TRUE FOR SOME

OF THE HUMAN LIGANDS.

THE ULBPs AND THE SUB60 GROUPS

ARE PRETTY SIMILAR IN MOST OTHER

RESECTS SO I DON'T REALLY EXPECT

THEM TO BE THAT DIFFERENT.

I GUESS WE'LL SEE.

>> THANK YOU.

>> THANK YOU.