Benaroya Research Institute At Virginia Mason

IMMUNE TOLERANCE NETWORK

REGULATION OF PULMONARY INFLAMMATION BY LEUKOCYTES AND EXTRACELLULAR MATRIX

TYPE 1 DIABETES TRIALNET CLINICAL NETWORK HUB

DEFINING THE ROLE OF ALTERED CYTOKINE SIGNALING PATHWAYS ON AUTOIMMUNITY

SYSTEMS IMMUNOLOGY PROFILING OF RESPIRATORY VIRAL INFECTIONS IN VULNERABLE POPULATIONS - SUMMARY/ABSTRACT – OVERALL ACUTE RESPIRATORY VIRAL INFECTIONS (ARVI) ARE THE MOST FREQUENTLY OCCURRING GLOBAL ILLNESS PRODUCING SIGNIFICANT MORBIDITY AND MORTALITY, PARTICULARLY IN VULNERABLE POPULATIONS. CHILDREN SUFFER HIGHER FREQUENCIES OF ARVI AND OFTEN EXPERIENCE RE-INFECTIONS. COMMON CHRONIC DISEASES OF CHILDHOOD, MOST NOTABLY ASTHMA BUT ALSO ALLERGIES (ATOPY) AND OBESITY, CAN PREDISPOSE TO INCREASED SEVERITY OF ARVI. SIMILARLY, ADULTS WITH CHRONIC INFLAMMATORY DISEASES OR ON IMMUNOSUPPRESSION SUFFER SIGNIFICANT CONSEQUENCES FROM ARVI. ADULTS WITH RHEUMATOID ARTHRITIS (RA) HAVE AN INCREASED RISK FOR INFECTION AND RESPIRATORY MUCOSAL INFLAMMATION MAY CONTRIBUTE TO AUTOIMMUNE DISEASE SEVERITY. THE GOAL OF THIS RESEARCH PROGRAM IS TO UNDERSTAND THE MOLECULAR AND CELLULAR IMMUNE SIGNATURES OF THE VULNERABLE HOST RESPONSE TO ARVI TO IDENTIFY NOVEL THERAPIES AND INDIVIDUALS AT RISK FOR CLINICAL COMPLICATIONS. THE PROGRAM INCLUDES A DETAILED SYSTEMS IMMUNOLOGY ASSESSMENT OF ACUTE AND LONG-TERM AIRWAY AND ADAPTIVE SYSTEMIC IMMUNE RESPONSES TO NATURALLY OCCURRING ARVI. THE FIRST PROJECT WILL IDENTIFY HOW ASTHMA, ATOPY, AND OBESITY LEAD TO MALADAPTIVE IMMUNE RESPONSES TO ARVI IN PEDIATRIC SUBJECTS. THE SECOND PROJECT WILL EXAMINE HOST RESPONSE TO ARVI IN ADULTS WITH RA. RA IS A DISEASE PROVOKED BY ENVIRONMENTAL STIMULI LIKE RESPIRATORY INFECTIONS AND RA PATIENTS HAVE BASELINE IMMUNE DIFFERENCES. THESE PROJECTS ARE COMPLEMENTARY AND SYNERGISTIC BY UTILIZING SIMILAR SAMPLE TYPES AND TIMING OF SAMPLE COLLECTION, AND COMMON CLINICAL ENDPOINTS. THE INDIVIDUAL PROJECTS BENEFIT FROM SHARED MULTI-OMICS APPROACHES THROUGH A GENOMICS CORE FOR THE SAMPLE PROCESSING AND GENERATION OF AIRWAY HOST TRANSCRIPTOME, PROTEOME, EPITHELIAL METHYLATION, AND VIRAL QUANTITY AND EXPRESSION DATA, ALONG WITH HOST GENETICS. THERE IS ALSO A SHARED ADAPTIVE PHENOTYPING CORE FOR THE GENERATION OF HIGH DIMENSIONAL CYTOMETRY DATA TO BROADLY CHARACTERIZE IMMUNE CELL PHENOTYPES AND FOR DETAILED IDENTIFICATION OF ANTIGEN-SPECIFIC CELLS. THIS WILL ALLOW FOR DIRECT COMPARISONS TO BE MADE BETWEEN THE ADULT AND PEDIATRIC COHORTS TO IDENTIFY COMMON AND DIVERGENT RESPONSES TO ARVI. IN THE OVERALL, THE FIRST SPECIFIC AIM IS TO DETERMINE SIMILAR AND DIVERGENT HOST RESPONSES TO ARVI CONSIDERING THE PEDIATRIC ALLERGY/ASTHMA (PROJECT 1) AND ADULT RA (PROJECT 2) COHORTS. THE SECOND SPECIFIC AIM IS TO CONSIDER THESE HOST RESPONSES IN THE CONTEXT OF OTHER LARGE PUBLICLY-FUNDED STUDIES OF VIRAL INFECTION THROUGH META-ANALYSES. THE FINAL SPECIFIC AIM WILL BE TO DEVELOP PREDICTIVE SPATIOTEMPORAL MODELS OF HOW MUCOSAL AND SYSTEMIC IMMUNE RESPONSES TO ARVI INFLUENCE CLINICAL OUTCOMES. OUR RESEARCH PROGRAM WILL PRODUCE NOVEL MECHANISTIC INSIGHTS INTO THE DIVERSITY AND COMMONALITY OF HUMAN IMMUNE RESPONSES TO ACUTE RESPIRATORY VIRUSES AND USE CUTTING- EDGE METHODS TO IDENTIFY POTENTIAL THERAPIES.

EPITHELIAL CONTROL OF RESPONSES TO ALLERGEN CHALLENGE AND VIRAL EXACERBATION

NORTHWEST CLINICAL CENTER FOR TYPE 1 DIABETES - TRIALNET

ALLERGEN T CELL EPITOPES AND PHENOTYPES IN PEANUT ALLERGY AND IMMUNOTHERAPY

MECHANISMS OF IL-6 MEDIATED T CELL PATHOGENESIS IN AUTOIMMUNITY

BLOOD TRANSCRIPTIONAL BIOMARKER PROFILES FOR CATEGORY B PATHOGENS

DEFINING THE FEATURES OF T CELL RESPONSE TO TUMOR AND SELF-ANTIGENS AS PREDICTORS OF RESPONSE TO CHECKPOINT THERAPY

DEFINING THE FUNCTIONAL IMPACT OF T1D GENES IN MOUSE AND MAN: A UNIFIED STRATEGY

HARNESSING ENGINEERED T REGULATORY CELLS TO PROMOTE BETA CELL HEALTH IN T1D - SUMMARY TYPE 1 DIABETES (T1D) IS CHARACTERIZED BY THE DESTRUCTION OF SS-CELLS, DRIVEN THROUGH AUTOIMMUNE ATTACK DIRECTED AT THE PANCREATIC ISLET. IN CONCERT WITH SS-CELL DESTRUCTION, SS-CELL STRESS AND INJURY CONTRIBUTE TO DISEASE BY INITIATING ENZYMATIC ACTIVITIES THAT COMPROMISE SS-CELL FUNCTION AND GENERATE NEOANTIGENS. THE INVESTIGATORS OF THIS PROJECT WILL INTEGRATE KNOWLEDGE OF CLINICAL T1D, SS-CELL STRESS, ISLET REACTIVE T CELLS, REGULATORY T CELLS (TREGS) AND GENE ENGINEERING TO CREATE TREGS THAT WILL HOME TO THE ISLET AND SUPPRESS AUTOIMMUNITY AND INFLAMMATION IN A MANNER THAT WILL CREATE AN ENVIRONMENT THAT WILL ALLOW SS-CELL RECOVERY AND PROMOTE SS-CELL HEALTH. THESE ENGINEERED TREGS (ENGTREGS) WILL BE GENERATED UTILIZING HOMOLOGY-DIRECTED REPAIR (HDR)-BASED GENE EDITING TO MEDIATE CONSTITUTIVE EXPRESSION OF FOXP3 COMBINED WITH LENTIVIRAL TRANSDUCTION OF T CELL RECEPTOR (TCR) SEQUENCES SPECIFIC TO ANTIGENS PRESENTED IN THE ISLET DURING PERIODS OF SS-CELL STRESS. FURTHER, WE PROPOSE DEVELOP ENGTREGS WITH THE ABILITY TO CO-DELIVER A PAYLOAD (WITH OR WITHOUT TCR ENGAGEMENT) THAT WILL FUNCTION TO SUPPRESS ONGOING INFLAMMATION AND/OR PROMOTE SS-CELL SURVIVAL AND GROWTH. AIM 1 WILL GENERATE STRESSED-ISLET-SPECIFIC CD4+ AND CD8+ ENGTREGS VIA GENE EDITING AND, IN PARALLEL, CONFER SPECIFICITY TO THE PANCREATIC ISLET VIA EXPRESSION OF TCRS THAT RECOGNIZE CITRULLINATED OR DEAMIDATED SS-CELL-DERIVED NEOEPITOPES. THE CENTRAL HYPOTHESIS FOR AIM 1 IS THAT TARGETING NEOANTIGENS GENERATED DURING PERIODS OF SS-CELL STRESS WILL ENHANCE THE TARGETED DELIVERY OF ENGTREGS TO THE SITE OF TISSUE INJURY. NEOEPITOPE DISCOVERY STUDIES WILL IDENTIFY TCR SEQUENCES RESTRICTED TO NOVEL SS-CELL STRESS NEOEPITOPES. FUNCTIONAL STUDIES WILL ASSESS STRESSED-ISLET-SPECIFIC ENGTREGS ACTIVATION IN THE PRESENCE OF SS-CELL STRESS AS WELL AS IN THE PRESENCE OF CD4+ AND CD8+ EFFECTOR T CELLS. AIM 2 WILL GENERATE ISLET-SPECIFIC CD4+ AND CD8+ ENGTREGS THAT MEDIATE TARGETED IMMUNE SUPPRESSION AND CO-DELIVER AN ISLET-PROTECTIVE THERAPEUTIC CARGO. IN PARALLEL, WE WILL DEVELOP ENGTREGS THAT RELEASE THEIR CARGO UPON TCR RECOGNITION OF ANTIGEN. THE PROPOSED STUDIES INTEGRATE PRIMARY HUMAN CELL STUDIES WITH RELEVANT T1D MURINE MODELS, FACILITATING MORE RAPID IDENTIFICATION OF PROMISING CANDIDATE ENGINEERED T CELL PRODUCTS. THE PROPOSED STUDIES ARE DIRECTLY RESPONSIVE TO RFA-DK-21-005 WITH ITS REQUEST TO 1) ENGINEER ANTIGEN-SPECIFIC TREG CELLS THAT CAN HOME TO THE PANCREATIC ISLET OR PANCREATIC DRAINING LYMPH NODES AND INHIBIT EFFECTOR T CELLS IN THESE COMPARTMENTS; 2) ENGINEER ISLET- HOMING SYNTHETIC SUPPRESSOR CELLS, SUCH AS CD4+ T CELLS ENGINEERED TO LOCALLY PRODUCE FACTORS THAT DAMPEN INFLAMMATION, INACTIVATE EFFECTOR T CELLS, OR PROMOTE ISLET TISSUE REPAIR; AND 3) ENGINEER T CELLS THAT PRODUCE FACTORS WITH TROPHIC EFFECTS ON SS-CELLS TO PROMOTE FUNCTION, IMMUNOPROTECTION AND/OR REPLICATION.

BCAP REGULATION OF TLR7/9 SIGNALING IN LUPUS

TSLP AND THE PATHOPHYSIOLOGY OF ASTHMA

MECHANISMS FOR HLA-DQ MEDIATED DISEASE PROTECTION AND SUSCEPTIBILITY

BRI CENTER FOR INFLAMMATION AND TISSUE REPAIR CHANGING THE WAY WE HEAL

TARGETING THE IL-2/REGULATORY T CELL AXIS FOR AUTOIMMUNE DISEASE PREVENTION IN REALISTIC ANIMAL MODELS

VIRGINIA MASON COMMUNITY CLINICAL ONCOLOGY PROGRAM

IRF5 AND MACROPHAGE ACTIVATION SYNDROME IN SYSTEMIC JUVENILE IDIOPATHIC ARTHRITIS

MECHANISTIC AND FUNCTIONAL DISSECTION OF INFLAMMAGING IN DOWN SYNDROME - PROJECT SUMMARY/ABSTRACT THE IMMUNE SYSTEM BECOMES FUNCTIONALLY IMPAIRED WITH AGING (INFLAMMAGING), IMPACTING RISK AND OUTCOME OF INFECTION, VACCINATION, CANCER, CARDIOVASCULAR DISEASE AND AUTOIMMUNITY. INDIVIDUALS WITH DOWN SYNDROME (DS) EXHIBIT CLINICAL, CELLULAR AND BIOCHEMICAL FEATURES OF INFLAMMAGING, INCLUDING INCREASED AUTOIMMUNITY, DECREASED NAÏVE T CELLS AND INCREASED PRO-INFLAMMATORY CYTOKINES. WE DEVELOPED A NOVEL UNBIASED APPROACH TO ANALYZE IMMUNE ARCHITECTURE IN PEOPLE WITH DS AND QUANTITATIVELY SHOWED THAT PEOPLE WITH DS EXHIBIT ADVANCED IMMUNE AGING BEGINNING IN CHILDHOOD DRIVEN IN PART BY DYSREGULATION OF NAÏVE CD4+ T CELLS. THIS APPROACH SHOWED SHARED FEATURES OF ADVANCED IMMUNE AGING IN DS AND OTHER AUTOIMMUNE DISEASE, SUGGESTING FUNCTIONAL RELEVANCE AND OVERLAP. THEREFORE, UNDERSTANDING (I) HOW ADVANCED IMMUNE AGING EXPLAINS IMPAIRED FUNCTIONAL IMMUNE RESPONSES IN DS, AND (II) HOW DYSREGULATION OF NAÏVE CD4+ T CELLS CONTRIBUTES TO ADVANCED IMMUNE AGING, WILL HELP US DEVELOP NOVEL THERAPEUTIC STRATEGIES TO ADDRESS IMMUNE DEFECTS IN DS. MOREOVER, THIS UNDERSTANDING WILL HELP US IDENTIFY SUBSETS OF THE GENERAL POPULATION WHO MAY BENEFIT FROM SIMILAR THERAPEUTIC STRATEGIES. THIS ALSO ESTABLISHES A BASIS TO UNDERSTAND WHICH GENES ON CHROMOSOME 21 DRIVE ADVANCED IMMUNE AGING, WITH ATTENDANT MECHANISTIC AND TRANSLATIONAL IMPLICATIONS. WE HYPOTHESIZE THAT ADVANCED IMMUNE AGING IMPAIRS IMMUNE RESPONSES BY DYSREGULATING NAÏVE CD4+ T CELLS VIA CELL-INTRINSIC AND CELL-EXTRINSIC PATHWAYS. WE PROPOSE KEY EXPERIMENTS HERE TO ADVANCE OUR UNDERSTANDING OF IMMUNE AGING AND IMMUNE RESPONSE IN DS. OUR SPECIFIC AIMS ARE: 1: DISSECTING MECHANISTIC FEATURES OF NAÏVE CD4+ T CELL DYSREGULATION IN DS-INFLAMMAGING. WE WILL LEVERAGE A NOVEL DS BIOREPOSITORY THAT I HELPED ESTABLISH AT BRI TO EXAMINE HOW DS IMPACTS CD4+ T CELL BIOLOGY IN THE ABSENCE OF CONFOUNDING IMMUNE DISORDERS. WE WILL USE IN VITRO ASSAYS COUPLED WITH RNASEQ TO IDENTIFY AND TEST CANDIDATE MECHANISMS. WE WILL STUDY INDIVIDUALS WITH MOSAIC DS TO PROVIDE UNIQUE INSIGHT TO DISSECT THE CELL-INTRINSIC ROLES OF TRISOMY 21 IN CD4+ T CELL DYSREGULATION IN VIVO. 2: INTERROGATE PHENOTYPIC AND FUNCTIONAL FEATURES OF DS-INFLAMMAGING IN VIVO. WE WILL USE OUR IMMUNE CELLULAR CLOCK TO QUANTITATIVELY UNDERSTAND HOW INFLAMMAGING IMPACTS VACCINE RESPONSE IN PEOPLE WITH DS AND CONTROLS. WE WILL ALSO EXAMINE ROBUSTNESS OF OUR DS-INFLAMMAGING FINDINGS IN AN INDEPENDENT COHORT AND FOLLOW OUR ORIGINAL COHORT LONGITUDINALLY TO EVALUATE TEMPORAL PROGRESSION OF DS-INFLAMMAGING.

IMMUNE CHECKPOINTS IN ACUTE RESPIRATORY DISTRESS SYNDROME (IC-ARDS)

T CELLS PROMOTING TRANSITIONS TOWARD AUTOIMMUNITY - SUMMARY AUTOIMMUNE DISEASE RESULTS FROM A COMBINATION OF IMMUNOLOGIC MISSTEPS INVOLVING MULTIPLE GENES, CELL TYPES AND ENVIRONMENTAL FACTORS. IN THE PROPOSED STUDIES, WE WILL STUDY HOW THESE FACTORS PROMOTE THE TRANSITION FROM SUB-CLINICAL AUTOIMMUNITY TO AUTOIMMUNE DISEASE BY COMPARING INDIVIDUALS AT RISK FOR TYPE 1 DIABETES, A DISEASE WITH A WELL-DEFINED NATURAL HISTORY OF SUB-CLINICAL AUTOIMMUNITY, AND INDIVIDUALS WITH DOWN SYNDROME (DS), A CONDITION WITH EXTREMELY HIGH RISK OF DEVELOPING AUTOIMMUNE DISEASE. THE CENTRAL HYPOTHESIS IS THAT THE NAÏVE T CELL COMPARTMENT OF AT-RISK INDIVIDUALS IS ENRICHED FOR POTENTIALLY PATHOGENIC PRECURSORS “POISED” TO TRANSITION TO AUTOREACTIVE EFFECTOR T CELLS, AND THE FAILURE OF REGULATORY MECHANISMS TO RESTRAIN THIS PROCESS ULTIMATELY RESULTS IN AUTOIMMUNE DISEASE. THE APPROACH LEVERAGES ACCESS TO CROSS SECTIONAL AND LONGITUDINAL COHORTS AT HIGH RISK OR KNOWN TO PROGRESS TO AUTOIMMUNITY; EXPERTISE IN THE GENETICS AND AUTOANTIGEN-SPECIFIC T CELLS IN THE CONTEXT OF AUTOIMMUNITY AND EXPERTISE IN SINGLE CELL MULTI-MODAL ANALYSES. TOGETHER, THIS EXPERTISE WILL ALLOW US TO PROBE T CELL STATES IN A MANNER NOT YET APPLIED TO AUTOIMMUNITY. IN OUR FIRST TWO AIMS, WE WILL DEEPLY CHARACTERIZE T CELLS ACROSS PLATFORMS FROM AT RISK SUBJECTS AND ADDRESS THE ROLE OF EPIGENETICS. IN AIMS 3 AND 4 WE ADDRESS THE ROLE OF EXTERNAL TRIGGERS USING LONGITUDINAL COHORTS PROSPECTIVELY TRACKING TRIGGER EVENTS AND DETERMINE WHETHER AUTOIMMUNE FEATURES ARE EXAGGERATED IN SUBJECTS ACQUIRING A SECOND AUTOIMMUNE DISEASE AIM 1 WILL DETERMINE WHETHER PATHOGENIC PRECURSORS ARE PRESENT IN THE NAÏVE T CELL COMPARTMENT OF INDIVIDUALS AT RISK FOR AUTOIMMUNE DISEASE COMPARED TO NON-RISK INDIVIDUALS. AIM 2 WILL IDENTIFY CELL TYPES THAT HARBOR THE GENETIC RISK FOR DISEASE TRANSITIONS AND IDENTIFY UNDERLYING PATHWAYS THAT SUPPORT PATHOGENIC PRECURSOR T CELLS. AIM 3 WILL IDENTIFY EXTERNAL FACTORS THAT CONTRIBUTE TO THE TRANSITION OF PATHOGENIC PRECURSORS TO EFFECTORS, INCLUDING A PROSPECTIVE LONGITUDINAL HIGH-RISK COHORT WITH INTENSE AT HOME SAMPLE COLLECTION TO TRACK IMMUNE VARIATION BRIDGING TRIGGER EVENTS. AIM 4 WILL DETERMINE WHETHER FEATURES SEEN IN INDIVIDUALS WHO TRANSITION TO T1D ARE EXAGGERATED IN INDIVIDUALS WHO DEVELOP ADDITIONAL AUTOIMMUNE DISEASES. ULTIMATELY, THE KNOWLEDGE GAINED FROM THIS WORK WILL CONTRIBUTE TO STAGING OF DISEASE, SELECTIVE THERAPIES AND ESTABLISH BIOMARKERS TO DETERMINE RISK OF PROGRESSION AND RESPONSE TO THERAPY.

IL-33 AND FOOD ALLERGY

COMMENSAL-SPECIFIC T CELL FUNCTION IN SKIN WOUND REPAIR - PROJECT SUMMARY OUR UNDERSTANDING OF IMMUNITY LARGELY STEMS FROM MODELS OF INFECTION WITH PATHOGENIC MICROBES. HOWEVER, THE VAST MAJORITY OF MICROBIAL-IMMUNE ENCOUNTERS OCCUR AS A SYMBIOTIC RELATIONSHIP WITH THE COMMENSAL MICROBIOTA. RECENTLY, THE CONTRIBUTION OF COMMENSAL-SPECIFIC T CELLS TO HOST PHYSIOLOGY HAS RECEIVED SIGNIFICANT ATTENTION. THESE COMMENSAL-SPECIFIC RESPONSES NOT ONLY CONTROL MICROBIOTA CONTAINMENT BUT ALSO PROMOTE ANTIMICROBIAL DEFENSES VIA THEIR ACTION ON BOTH INNATE AND EPITHELIAL CELLS. LOCAL TUNING OF KERATINOCYTES BY COMMENSAL-SPECIFIC T CELLS ALSO CONTRIBUTES TO TISSUE REPAIR FOLLOWING SKIN INJURY. CONVERSELY, ABERRANT IMMUNITY TO COMMENSAL MICROBES HAS BEEN PROPOSED TO UNDERLIE PATHOLOGIES OF BARRIER TISSUES, INCLUDING ATOPIC DERMATITIS AND INFLAMMATORY BOWEL DISEASE. A BETTER UNDERSTANDING OF THE PROPERTIES AND FUNCTIONS OF COMMENSAL-SPECIFIC T CELL RESPONSES IS THEREFORE FUNDAMENTAL TO STUDIES OF TISSUE IMMUNITY IN HEALTH AND DISEASE. OUR LONG-TERM GOAL IS TO BETTER UNDERSTAND HOW COMMENSAL-SPECIFIC T CELL RESPONSES CONTRIBUTE TO BARRIER TISSUE HOMEOSTASIS, AND THE OBJECTIVE IN THIS APPLICATION IS TO INVESTIGATE THE MECHANISMS REGULATING CYTOKINE PRODUCTION DURING SKIN INJURY AND WOUND REPAIR. OUR RATIONALE FOR THE PROPOSED WORK IS THAT UNCOVERING THESE MECHANISMS HAS THE POTENTIAL TO TRANSLATE INTO NEW THERAPEUTIC APPROACHES. OUR CENTRAL HYPOTHESIS IS THAT COMMENSAL-SPECIFIC T CELLS ARE SENTINELS OF THE SKIN TISSUE AND CONTRIBUTE TO WOUND REPAIR THROUGH RAPID PRODUCTION OF TYPE-2 CYTOKINES IN RESPONSE TO TISSUE INJURY. IN THIS PROPOSAL, WE WILL FOCUS ON TWO MECHANISMS, THE POST-TRANSCRIPTIONAL REGULATION OF IL-5 AND IL-13 CYTOKINE PRODUCTION BY COMMENSAL-SPECIFIC T THROUGH RNA-BINDING PROTEINS AND INDUCTION OF THE INTEGRATED STRESS RESPONSE. BASED ON STRONG PRELIMINARY DATA, WE WILL TEST THREE SPECIFIC AIMS: (1) UNDERSTAND THE KEY PROXIMAL AND DISTAL IL-18R-SIGNALING PATHWAYS THAT TRIGGER POISED TYPE-2 IMMUNITY IN COMMENSAL-SPECIFIC CD8+ T CELLS. WE HAVE RECENTLY FOUND THAT IL-18 ACTING DIRECTLY ON CD8+ T CELLS CAN TRIGGER RAPID PRODUCTION OF IL-5 AND IL-13. WE WILL TEST THE HYPOTHESIS THAT PATHWAYS UNIQUE TO IL-18R, BUT NOT OTHER IL-1R FAMILY MEMBERS TRIGGERS POISED TYPE-2 IMMUNITY IN COMMENSAL-SPECIFIC CD8+ T CELLS. (2) DETERMINE THE ROLE OF UNTRANSLATED REGIONS (UTR) OF IL5 AND IL13 MRNA IN POISED TYPE-2 IMMUNITY IN COMMENSAL-SPECIFIC CD8+ T CELLS. WE HAVE IDENTIFIED MULTIPLE RNA-BINDING PROTEIN MOTIFS IN THE UTR OF IL5 AND IL13 MRNA. WE WILL TEST THE CONTRIBUTION OF THESE REGULATORY ELEMENTS TO POISED TYPE-2 IMMUNITY IN COMMENSAL-SPECIFIC CD8+ T CELLS. (3) UNDERSTAND THE CONTRIBUTION OF THE INTEGRATED STRESS RESPONSE TO POST-TRANSCRIPTIONAL REGULATION OF IL5 AND IL13 MRNA IN COMMENSAL-SPECIFIC CD8+ T CELLS AND THE CONTRIBUTION TO WOUND REPAIR. OUR APPROACH IS INNOVATIVE AS IT INVESTIGATES NEW MECHANISMS OF IMMUNITY UNIQUE TO COMMENSAL-SPECIFIC T CELL RESPONSES. THE PROPOSED WORK IS SIGNIFICANT BECAUSE IT WILL ESTABLISH NEW INSIGHTS INTO THE INTERACTION AND COMMUNICATION BETWEEN COMMENSAL MICROBES AND IMMUNE CELLS IN THE SKIN MICROENVIRONMENT AND IDENTIFY POTENTIAL TARGETS FOR THERAPEUTIC INTERVENTION IN CONDITIONS OF CHRONIC NON-RESOLVING WOUNDS AND SKIN INFECTION.

REGULATION OF CUTANEOUS IMMUNITY AND TISSUE-REPAIR BY A SPECIALIZED POPULATION OF CD4+ T CELLS

DEFINING MECHANISMS OF CD8 T CELL EXHAUSTION IN T1D

DEEP ANALYSIS OF EPITOPE SPECIFIC CD4+ T CELLS BEFORE THE ONSET OF T1D - PROJECT SUMMARY/ABSTRACT TYPE 1 DIABETES (T1D) IS AN IMMUNE-MEDIATED DISEASE IN WHICH FUNCTIONAL Β CELLS ARE LOST, LEADING TO INSULIN DEPENDENCE. HLA CLASS II GENOTYPES ARE THE DOMINANT GENETIC RISK FACTOR, SUGGESTING A KEY ROLE FOR CD4+ T CELLS. ISLET-REACTIVE T CELLS PRODUCE INFLAMMATORY CYTOKINES AND SUPPORT FORMATION AND AFFINITY MATURATION AUTOANTIBODIES, THE APPEARANCE OF WHICH ACCURATELY PREDICTS DISEASE RISK. AT-RISK INDIVIDUALS DEVELOP ADDITIONAL SPECIFICITIES DURING PROGRESSION. THEREFORE, EPITOPE OCCURS AND MAY BE AN INDICATOR OF IMPENDING DISEASE ONSET. OUR PRELIMINARY STUDIES SHOW THAT EXPANDED NUMBERS OF CD4+ T CELLS THAT TARGET SELF-ANTIGENS ARE PRESENT IN INDIVIDUALS WITH ESTABLISHED T1D. SPECIFICALLY, OUR PRELIMINARY DATA ENABLED US TO DEFINE AN ARRAY OF SELF-EPITOPES THAT ARE DISEASE RELEVANT AND RECOGNIZED IN INDIVIDUALS WITH TWO OF THE HIGHEST RISK HLA CLASS II GENOTYPES. THESE INCLUDE NEWLY DISCOVERED CONVENTIONAL AND NEOEPITOPES FROM GAD65 AND OTHER BETA CELL ANTIGENS. IT IS BELIEVED THAT SELF-REACTIVE T CELL RESPONSES EVOLVE OVER TIME AND THAT BETA CELL INJURY ELICITS NEOEPITOPE FORMATION. HOWEVER, THESE PHENOMENA HAVE NOT BEEN DIRECTLY STUDIED, LEAVING A SIGNIFICANT KNOWLEDGE GAP. GIVEN THEIR IMPORTANT ROLE IN ORCHESTRATING AUTOIMMUNITY, UNDERSTANDING THE CD4+ T CELL RESPONSES IS A TOPIC OF GREAT IMPORTANCE. INDEED, THE APPROVAL OF TEPLIZUMAB AS A THERAPY TO DELAY THE PROGRESSION OF T1D INCREASES THE RATIONALE FOR UNDERSTANDING AND MONITORING EPITOPE SPREADING IN AT-RISK INDIVIDUALS. OUR WORK WILL TEST THE HYPOTHESIS THAT IN AT-RISK SUBJECTS THERE ARE PIVOTAL SHIFTS IN THE NUMBER, PHENOTYPE, AND TRANSCRIPTIONAL PROGRAMS OF AUTOREACTIVE CD4+ T CELLS AND AN EXPANSION OF DISTINCT TCR SEQUENCES AND THAT THESE CHANGES REVEAL IMPORTANT PATHWAYS THAT DRIVE DISEASE PROGRESSION. OUR FIRST AIM WILL CHARACTERIZE LONGITUDINAL CHANGES IN THE NUMBER AND PHENOTYPE OF AUTOREACTIVE T CELLS IN AT-RISK SUBJECTS BY PERFORMING HIGH DIMENSIONAL FLOW CYTOMETRY ANALYSIS USING A ROBUST SURFACE MARKER PANEL COMBINED WITH SECOND GENERATION HLA CLASS II TETRAMER REAGENTS. OUR SECOND AIM WILL UTILIZE THE 10X GENOMICS PLATFORM TO GENERATE RICH WHOLE GENOME RNASEQ DATA SETS ON ANTIGEN SPECIFIC T CELLS SORTED FROM IAA POSITIVE AT-RISK INDIVIDUALS. THIS WILL ALLOW DEEP MOLECULAR PROFILING OF AUTOREACTIVE T CELLS TO IDENTIFY IMMUNE CELL SUBSETS THAT CONTRIBUTE TO T1D PROGRESSION AND DEEP ANALYSIS OF TCR SEQUENCES AND THEIR STRUCTURAL FEATURES, TRACKING THEIR PHENOTYPE TRAJECTORIES OVER TIME AND THE EMERGENCE OF KEY T CELL TRANSCRIPTIONAL STATES IN AT-RISK SUBJECTS. IN TOTAL, OUR PROPOSED STUDIES WILL ADVANCE OUR UNDERSTANDING OF T1D, PROVIDING IMPORTANT NEW INSIGHTS ABOUT THE T CELL SPECIFICITIES, PHENOTYPES, AND FUNCTIONAL ATTRIBUTES THAT DRIVE THE DEVELOPMENT OF T1D THE IMMUNOLOGIC CHANGES AND MECHANISMS THAT PROMOTE AUTOIMMUNE PROGRESSION.

CONTROL OF CD8+ T CELL ACTIVATION AND DIFFERENTIATION BY THE SIGNALING ADAPTOR BCAP

REGULATORY T CELLS MAINTAIN IMMUNE HOMEOSTASIS THROUGH TRANSLATION CONTROL - PROJECT SUMMARY THE IMMUNE SYSTEM NEEDS TO DISTINGUISH BETWEEN SELF AND NON-SELF, AND BETWEEN PATHOGEN AND COMMENSAL. IN PART IT DOES THROUGH THE FUNCTION OF REGULATORY T CELLS, A SPECIALIZED SUBSET OF CD4 T CELLS THAT ARE CRITICAL FOR MAINTAINING IMMUNE HOMEOSTASIS, AS WELL AS MAINTAINING TOLERANCE TOWARD SELF-ANTIGENS. THEIR IMPORTANCE TO MAINTAINING PERIPHERAL TOLERANCE WAS ILLUSTRATED FROM EXPERIMENTS OF NATURE IN BOTH HUMANS (IPEX SYNDROME) AND MICE (SCURFY MUTATION) WHERE LOSS OF THIS SUBSET OF T CELLS RESULTS IN THE ONSET OF FATAL AUTOIMMUNITY. TREG DYSFUNCTION HAS ALSO BEEN FOUND TO PLAY A ROLE IN AUTOIMMUNE DISEASES SUCH AS TYPE 1 DIABETES AND MULTIPLE SCLEROSIS. TREGS HAVE THE ABILITY TO POTENTLY SUPPRESS CD4 EFFECTOR T CELLS (TEFF) EITHER DIRECTLY OR THROUGH THE MODULATION OF ANTIGEN PRESENTING CELLS (MAINLY DCS) TO ULTIMATELY SUPPRESS ACTIVATION, PROLIFERATION, AND SUBSEQUENT EFFECTOR FUNCTIONS OF TEFF CELLS. SEVERAL MECHANISMS HAVE BEEN PROPOSED FOR TREG-MEDIATED SUPPRESSION, INCLUDING RELEASE OF SUPPRESSIVE CYTOKINES AND EXPRESSION OF INHIBITORY RECEPTORS. HOWEVER, VERY LITTLE IS KNOWN MECHANISTICALLY AS TO THE EFFECT OF SUPPRESSION ON TEFF CELL FUNCTION. WE FOUND THAT TREGS PROMOTED A SIGNIFICANT DROP IN OVERALL TRANSLATIONAL ACTIVITY OF CD4 TEFF CELLS. RATHER THAN INDUCING A GLOBAL, NON-SPECIFIC TRANSLATIONAL SHUTDOWN IN TEFF CELLS, TREGS INDUCED A SPECIFIC REMODELING OF THE TRANSLATOME, WITH SEVERAL MRNAS WHOSE TRANSCRIPTION WAS UNAFFECTED NO LONGER PRESENT ON RIBOSOMES. THESE DATA PROVIDE NOVEL INSIGHTS INTO TREG FUNCTION AND IDENTIFY NEW PATHWAYS THAT ARE POTENTIALLY AVAILABLE FOR THERAPEUTIC INTERVENTION. THE GOAL OF THE STUDIES IN THIS PROPOSAL ARE TO DETERMINE THE MOLECULAR MECHANISMS THAT UNDERLIE TREG-MEDIATED TRANSLATOME REMODELING IN TEFFS, AND TO ASK WHETHER THIS MECHANISM IS ALTERED IN HUMAN AUTOIMMUNE DISEASE. TO ACHIEVE THIS WE WILL: DETERMINE THE ROLE OF RNA AND RNA-BINDING PROTEINS IN TREG-MEDIATED SUPPRESSION OF TRANSLATION (AIM 1) AND DETERMINE THE ROLE OF TRANSLATION CONTROL IN HUMAN TREG FUNCTION (AIM 2).

ENGINEERED ATRIUM: NEW AUTOLOGOUS CELLS FOR HEART REPAIR

PLASMACYTOID DENDRITIC CELLS AND IGA AUTOANTIBODIES IN SLE - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS AN AUTOIMMUNE DISEASE THAT PREDOMINANTLY AFFECTS WOMEN AND IS MORE PREVALENT AMONG NON-WHITE INDIVIDUALS, INCLUDING THOSE OF BLACK, HISPANIC AND ASIAN ANCESTRIES. DISEASE IN SLE CAN TARGET A VARIETY OF ORGANS, INCLUDING THE SKIN, JOINTS, KIDNEYS, LUNG, BRAIN, AND CARDIOVASCULAR SYSTEM, WHICH CAN MAKE DIAGNOSIS DIFFICULT. SLE IS CHARACTERIZED BY THE BREAKDOWN OF TOLERANCE TO NUCLEAR ANTIGENS; THUS INDIVIDUALS WITH SLE HAVE CIRCULATING ANTI-NUCLEAR ANTIBODIES (ANAS) THAT RECOGNIZE COMPLEX ANTIGENS SUCH AS NUCLEIC ACIDS, AND DNA-ASSOCIATED OR RNA-ASSOCIATED PROTEINS, INCLUDING HISTONES OR RIBONUCLEOPROTEIN (RNP) SUBUNITS. THESE ANAS FORM NUCLEIC ACID-CONTAINING IMMUNE COMPLEXES (ICS) THAT ARE RECOGNIZED BY AND TRIGGER INFLAMMATORY RESPONSES FROM FC RECEPTOR (FCR)-EXPRESSING IMMUNE CELLS. IN THIS WAY, ANA-ICS FACILITATE NUCLEIC ACID INTERNALIZATION AND DELIVERY TO ENDOSOMES WHERE THE RNA AND DNA SENSING TOLL-LIKE RECEPTORS (TLR) 7 AND 9 RESIDE, THEREBY ALLOWING ABERRANT RESPONSES TO AUTOLOGOUS NUCLEIC ACIDS IN SLE. PLASMACYTOID DCS (PDCS) ABUNDANTLY EXPRESS TLR7 AND TLR9, AND THUS ARE KEY MEDIATORS OF RESPONSES TO NUCLEIC ACID-CONTAINING ICS BY PRODUCING LARGE QUANTITIES OF TYPE I IFNS, CRUCIAL CYTOKINES IN SLE. WHILE MOST STUDIES HAVE FOCUSED ON HOW IGG ISOTYPE ANAS CONTRIBUTE TO SLE PATHOGENESIS, WE HAVE DISCOVERED A CRITICAL ROLE FOR IGA ISOTYPE AUTOANTIBODIES IN PDC RESPONSES TO NUCLEIC ACID-CONTAINING ICS IN SLE. WE FOUND THAT MOST INDIVIDUALS WITH SLE HAVE A BROAD ARRAY OF IGA AUTOANTIBODIES TO ANTIGENS TO WHICH THEY ALSO HAVE IGG. WE SHOWED THAT PDCS EXPRESS THE IGA- BINDING FCΑRI IN ADDITION TO THE IGG-BINDING FCΓRIIA. USING AN INNOVATIVE IN VITRO SYSTEM EMPLOYING SERUM FROM INDIVIDUALS WITH SLE TO GENERATE ICS WITH POTENT PDC STIMULATORY FUNCTION, WE FOUND A NOVEL AND CRITICAL FUNCTION FOR IGA1 ANTIBODIES IN PDC TYPE I IFN PRODUCTION IN RESPONSE TO RNA-CONTAINING RNPS. FCΑR AND FCΓRIIA ACTED SYNERGISTICALLY TO INDUCE TYPE I IFN PRODUCTION BY FACILITATING INTERNALIZATION OF ICS, AND PDCS FROM INDIVIDUALS WITH SLE HAD HIGHER IC INTERNALIZATION AND FCΑRI EXPRESSION THAN THOSE FROM HCS. THUS, WE HAVE UNCOVERED AN IMPORTANT ROLE OF IGA1 AND FCΑR IN PDC RESPONSES IN SLE THAT WE WILL FURTHER STUDY HERE. THE PREMISE OF THIS PROPOSAL IS THAT IGA ANTI-NUCLEAR AUTOANTIBODIES ARE IMPORTANT CONTRIBUTORS TO SLE PATHOGENESIS THROUGH ENHANCEMENT OF PDC TYPE I IFN PRODUCTION IN RESPONSE TO NUCLEIC ACID-CONTAINING ICS. HERE WE WILL USE PRIMARY HUMAN SAMPLES FROM INDIVIDUALS WITH SLE ALONG WITH NOVEL RESEARCH TOOLS WE HAVE DEVELOPED TO 1) DETERMINE THE MECHANISMS UNDERLYING THE POTENT SYNERGY BETWEEN IGA1 AND IGG IN SM/RNP ICS, AND 2) DETERMINE HOW IGA AUTOANTIBODIES TO NUCLEIC ACID-ASSOCIATED ANTIGENS RELATE TO SLE. TOGETHER THESE STUDIES WILL LEAD TO A BETTER MECHANISTIC UNDERSTANDING OF HOW IGA AND IGA-PRODUCING B CELLS CONTRIBUTE TO TYPE I IFN PRODUCTION IN SLE.

CLONAL EXPANSION: A MARKER OF DISEASE ACTIVITY IN STAGE 1 T1D - SUMMARY TYPE 1 DIABETES (T1D) IS AN IMMUNE MEDIATED DISEASE IN WHICH IMMUNE CELLS DESTROY INSULIN PRODUCING BETA CELLS OF THE PANCREAS. YET, T1D PATHOGENESIS STARTS EVEN BEFORE DAMAGE TO BETA CELLS CAN BE DETECTED AS OVERT DYSGLYCEMIA WITH THE APPEARANCE OF TWO OR MORE ISLET-SPECIFIC AUTOANTIBODIES (AAB), DEFINED AS STAGE 1 T1D. YET, AAB NUMBER AND TYPE CAN BE UNSTABLE OVERTIME IN STAGE 1. IN THE PROPOSED STUDIES, WE DETERMINE WHETHER ISLET ANTIGEN REACTIVE T AND B CELL CLONAL EXPANSION IS A STABLE EARLY BIOMARKER OF DISEASE ACTIVITY IN STAGE 1 T1D. THE CENTRAL HYPOTHESIS IS THAT ISLET ANTIGEN REACTIVE CLONAL EXPANSION OF PATHOGENIC CELL SUBSETS IS INDICATIVE OF DISEASE ACTIVITY IN STAGE 1 T1D AND DISEASE PROGRESSION TO STAGE 2 OR 3 OCCURS WHEN CLONAL EXPANSION WITHIN PATHOGENIC CELLS EXCEEDS CLONAL EXPANSION WITHIN PROTECTIVE CELLS. WE WILL ADDRESS THIS QUESTION BY INVESTIGATING WELL ANNOTATED CROSS SECTIONAL AND LONGITUDINAL SAMPLES FROM THE TRIALNET PATHWAY TO PREVENTION STUDY. THE CROSS SECTIONAL COHORT WILL INCLUDE AGE AND HLA MATCHED STAGE 1 INDIVIDUALS WITH STABLE VERSUS VARIABLE AAB IN THE PRIOR YEAR, VARIABLE AAB WILL BE DEFINED AS INCREASED AAB NUMBER OR CHANGE IN AAB AS A LONGITUDINAL MEASURE OF DISEASE ACTIVITY. THE LONGITUDINAL COHORT WILL COMPARE PAIRED SAMPLES FROM STAGE 1 INDIVIDUALS WHO DO OR DO NOT PROGRESS TO STAGE 2 OR 3 T1D. THE APPROACH LEVERAGES OUR EXPERTISE IN ISLET ANTIGEN SPECIFIC T AND B CELL IDENTIFICATION, CELLULAR IMMUNOLOGY OF T1D, AND SYSTEMS BIOLOGY SINGLE CELL MULTI- MODEL ANALYSES. TOGETHER, THESE EXPERTISE WILL ALLOW US TO DEFINE CLONAL EXPANSION IN PROTECTIVE AND PATHOGENIC CELL SUBSETS OF STAGE 1 INDIVIDUALS AND EXPLORE THE FUNCTIONAL NATURE OF THE EXPANDING CELLS. SPECIFICALLY, IN AIM 1 WE WILL DETERMINE THE DEGREE OF PATHOGENIC ISLET ANTIGEN REACTIVE CLONAL EXPANSION THAT MARKS DISEASE ACTIVITY AND PROGRESSION USING SINGLE CELL T CELL RECEPTOR (TCR)- OR B CELL RECEPTOR (BCR)-, CITE-, AND RNA-SEQ IN CROSS SECTIONAL COHORTS DEFINED BY STAGE OF DISEASE AND STABILITY OF AAB COMPOSITION. IN AIM 2 WE WILL DETERMINE THE FUNCTIONAL QUALITY OF CLONALLY EXPANDED CELLS IN EARLY T1D BY ASSESSING CHANGES IN THE TRANSCRIPTOME AND EPIGENOME OVER TIME OF PATHOGENIC AND PROTECTIVE POPULATIONS SELECTED BASED ON THE DEGREE OF CLONAL EXPANSION, ASSOCIATION WITH OUTCOME, AND ENRICHMENT IN IAR CELLS. ULTIMATELY, THE KNOWLEDGE GAINED FROM THIS WORK WILL IDENTIFY STABLE DISEASE ACTIVITY MARKERS EARLY IN DISEASE THAT MAY GUIDE TIMING OF TREATMENT, EXPAND OUR UNDERSTANDING OF IMMUNITY AT STAGE 1 T1D, AND IDENTIFY PATHOGENIC AND PROTECTIVE FEATURES THAT MAY BE TARGETED THERAPEUTICALLY.

REPROGRAMMING OF TISSUE STRUCTURAL CELLS BY CUTANEOUS CD4+ T CELLS - PROJECT SUMMARY/ABSTRACT ALTHOUGH LONG THOUGHT TO BE A UNIQUE FEATURE OF ADAPTIVE CELLS SUCH AS T AND B CELLS, IT IS NOW CLEAR THAT TISSUE STRUCTURAL CELLS CAN ALSO HARBOR ‘MEMORY’ OF PRIOR INFLAMMATORY RESPONSES IN THE FORM OF STABLE EPIGENETIC MODIFICATIONS THAT ALTER THEIR TRANSCRIPTIONAL POTENTIAL AND BEHAVIOR FOLLOWING SUBSEQUENT TISSUE DAMAGE, AND THIS IS REFERRED TO AS ‘INFLAMMATORY MEMORY’. THIS HIGHLIGHTS KEY GAPS IN OUR KNOWLEDGE OF T CELL-TISSUE CROSS- TALK THAT THIS PROPOSAL SEEKS TO ADDRESS. THE PREMISE OF THIS PROPOSAL IS THAT CUTANEOUS T CELLS REPROGRAM LOCAL KERATINOCYTES AND FIBROBLASTS DURING INFLAMMATION, THEREBY ALTERING THEIR TRANSCRIPTIONAL AND EPIGENETIC LANDSCAPE, FUNCTION, AND RESPONSES TO SUBSEQUENT STIMULI, AND THAT THIS INFLUENCES THE COURSE AND RESOLUTION OF INFLAMMATORY SKIN DISEASE. TO FILL THESE KEY KNOWLEDGE GAPS, WE WILL EXAMINE THE CROSS-TALK BETWEEN T CELL AND STRUCTURAL CELLS IN INNOVATIVE CELL CULTURE SYSTEMS TO MONITOR CHANGES IN CELL BEHAVIOR AND FUNCTION, AND MECHANISTICALLY LINK THEM TO TRANSCRIPTIONAL AND EPIGENETIC REPROGRAMMING BY T CELL-DERIVED CYTOKINES. WE WILL ALSO LEVERAGE AN ESTABLISHED AND HIGHLY MANIPULATABLE MURINE MODEL OF T CELL-DEPENDENT SKIN INFLAMMATION TO EXAMINE THE DEVELOPMENT OF INFLAMMATORY MEMORY IN KCS AND FIBS IN VIVO, TO MECHANISTICALLY IDENTIFY IMPORTANT CYTOKINES AND EPIGENETIC MEDIATORS, AND TO ASSESS THE FUNCTIONAL CONSEQUENCES ON SUBSEQUENT INFLAMMATORY AND TISSUE-REPAIR RESPONSES. FINALLY, WE WILL ANALYZE SIGNATURES OF INFLAMMATORY MEMORY IN TISSUE SAMPLES FROM PATIENTS WITH THE COMMON SKIN INFLAMMATORY DISEASES PSORIASIS AND ATOPIC DERMATITIS, AND ASSESS EPIGENETIC REPROGRAMMING IN A PRECLINICAL MODEL AS A NEW THERAPEUTIC MODALITY TO RESET INFLAMMATORY MEMORY AND BREAK THE INFLAMMATORY CYCLE IN THE SKIN OF THESE PATIENTS.

LITAF REGULATION OF MEMBRANE REPAIR AND INFLAMMATION - PROJECT SUMMARY MEMBRANE DAMAGE BY MECHANICAL OR BIOCHEMICAL STRESS, LEADS TO CELL DEATH AND ACTIVATION OF INNATE IMMUNE INFLAMMATORY PATHWAYS, AND CONTRIBUTES TO THE PATHOLOGY OF MANY INFLAMMATORY CONDITIONS. FURTHERMORE, PATHOGENS CAN SECRETE PORE-FORMING TOXINS (PFT) TO PROMOTE INFECTION AND DISRUPT IMMUNITY, AND ENDOGENOUS PORE-FORMING PROTEINS SUCH AS GASDERMIN D AND MLKL HAVE BEEN SHOWN TO CONTRIBUTE TO INFLAMMATORY SIGNALING AND SECRETION OF CYTOKINES. CELLS HAVE EVOLVED MULTIPLE MECHANISMS TO REPAIR MEMBRANE DAMAGE AND MAINTAIN CELLULAR HOMEOSTASIS, BUT OUR UNDERSTANDING OF HOW DAMAGE IS SENSED AND LINKED TO REPAIR REMAINS INCOMPLETE. WE HAVE RECENTLY DEVELOPED A TRANSPOSON-BASED FORWARD GENETIC SCREENING APPROACH, WHICH WE HAVE USED TO IDENTIFY GENES THAT PROMOTE RESISTANCE TO CELL DEATH INDUCED BY S. AUREUS Α-TOXIN. WE IDENTIFIED THE LYSOSOMAL MEMBRANE PROTEIN LITAF AS A CELL-AUTONOMOUS INHIBITOR OF CELL DEATH. IN PRELIMINARY DATA, WE SHOW THAT LITAF PROMOTES SEQUESTRATION OF DAMAGED MEMBRANES INTO VESICLES THROUGH THE ACTIVATION OF THE ESCRT MACHINERY. WE HYPOTHESIZE THAT LITAF ACTS AS AN EFFECTOR OF CELLULAR DEFENSE AGAINST PORE-FORMING PROTEINS, LINKING SENSING OF MEMBRANE DAMAGE TO EFFECTOR MECHANISMS OF REPAIR. IN THIS APPLICATION, WE PROPOSE TO TEST THIS HYPOTHESIS BY: (1) IDENTIFYING THE MECHANISMS OF LITAF ACTIVATION AND FUNCTION; (2) DETERMINING THE ROLE OF THIS PATHWAY IN LUNG INFLAMMATION AND INFECTION; (3) TESTING WHETHER LITAF REGULATES INNATE IMMUNE SIGNALING, INFLAMMASOME ACTIVATION AND INFLAMMATORY CELL DEATH IN MACROPHAGES. THIS RESEARCH IS OF HIGH SIGNIFICANCE AS IT WILL PROVIDE A DEEPER UNDERSTANDING OF CELLULAR DEFENSE MECHANISMS AGAINST MEMBRANE DAMAGE, AND OF THE BALANCE BETWEEN CELL SURVIVAL AND INFLAMMATORY CELL DEATH. IDENTIFYING STRATEGIES TO COUNTERACT MEMBRANE DAMAGE AND PREVENT CELL DEATH WILL CONTRIBUTE TO UNDERSTANDING AND TREATING THE PATHOLOGY OF A WIDE RANGE OF INFECTIOUS AND INFLAMMATORY DISEASES.

PHENOTYPIC ANALYSIS OF ISLET ANTIGEN-SPECIFIC EFFECTOR T CELLS IN PRE-DIABETIC SUBJECTS

ENGINEERING VASCULAR REPLACEMENTS FOR STRENGTH AND ELASTICITY

EMERGENCE OF DEVELOPMENTAL AND GENOMIC COMPLEXITY IN A BASAL VERTEBRATE

REGULATION OF DENDRITIC CELL INFLAMMATORY RESPONSES

CONTROL OF TUMOR GROWTH AND METASTASIS BY THE CYTOKINE TSLP

REGULATION OF PATHOGENIC T CELLS IN EAE

FUNCTIONAL SPECIALIZATION OF FOXP3+ REGULATORY T CELLS

INDUCTION AND SIGNATURE OF PATHOGENIC T CELLS IN ALLERGY

REGULATION OF INFLAMMATORY SIGNALING DURING THE INNATE IMMUNE RESPONSE

A FOXP3 COMPLEX THAT CONTROLS HUMAN REGULATORY T CELL FUNCTION

TSLP AND CONTACT HYPERSENSITIVITY

REGULATION OF TSLP-MEDIATED SKIN INFLAMMATION

BCAP/PI3K REGULATION OF INNATE IMMUNITY TO LISTERIA MONOCYTOGENES

MOLECULAR MECHANISMS OF TH17 PLASTICITY IN MS

GENOMICS RESOURCES AND INFRASTUCTURE FOR THE ZEBRAFISH

INTERPLAY BETWEEN PATHOGENIC AND REGULATORY T CELLS IN EAE

IMPACT OF THE AUTOIMMUNITY ASSOCIATED PTPN22 1858T

HOMING AND HOMEOSTASIS OF REGULATORY T CELLS

C-SKI AND THE REGULATION OF CD4 T CELL-MEDIATED AUTOIMMUNITY AND TOLERANCE

TYPE 1 DIABETES IN ACUTE PANCREATITIS CONSORTIUM, PACIFIC NORTHWEST CLINICAL CENTER: IMMUNE PATHOGENESIS OF POST-PANCREATITIS T1D

IDENTIFICATION OF HOST DRUG DEVELOPMENT TARGETS IN INFLUENZA USING TRANSPOSON MUTAGENESIS

INVESTIGATING GENETIC AND EPIGENETIC CONTROL OF T CELL FUNCTION IN AUTOIMMUNITY - SUMMARY ~24 MILLION PEOPLE IN THE UNITED STATES HAVE AN AUTOIMMUNE DISEASE, IMPACTING QUALITY OF LIFE AND LONGEVITY OF THE AFFECTED INDIVIDUALS. WHILE THESE DISEASES HAVE A GENETIC COMPONENT, AS DETERMINED FROM HIGH INCIDENCE IN MONOZYGOTIC TWINS, WE STILL KNOW VERY LITTLE ABOUT HOW GENETICS DRIVES RISK AND PROGRESSION FOR EACH DISEASE. GENOME-WIDE ASSOCIATION STUDIES HAVE IDENTIFIED HUNDREDS OF AUTOIMMUNE DISEASE-ASSOCIATED REGIONS OF THE GENOME, BUT THERE IS OFTEN TIGHT LINKAGE DISEQUILIBRIUM BETWEEN CAUSAL AND NON-CAUSAL VARIANTS IN THESE REGIONS, AND MOST DISEASE-ASSOCIATED GENETIC VARIANTS ARE IN NON-CODING REGIONS. THUS, DETERMINING THE VARIANTS THAT PROMOTE DISEASE AND THEIR EFFECTS ON DISEASE-RELEVANT CELL TYPES IS CHALLENGING. ONE CELL TYPE THAT IS IMPLICATED IN MANY AUTOIMMUNE DISEASES IS THE T CELL. IN RESPONSE TO SELF-ANTIGEN, AUTOREACTIVE T CELLS CLONALLY EXPAND AND MIGRATE TO TARGET TISSUES, WHERE THEY CAUSE TISSUE DESTRUCTION. NON-CODING GENETIC VARIANTS ASSOCIATED WITH AUTOIMMUNE DISEASES ARE ENRICHED WITHIN THE ACCESSIBLE CHROMATIN OF T CELLS, SUGGESTING THAT DISEASE-CAUSAL VARIANTS ALTER ENHANCERS THAT MAY AFFECT T CELL FUNCTION, MAKING THEM MORE PATHOGENIC. WE RECENTLY CREATED A METHODOLOGY TO IDENTIFY LIKELY CAUSAL VARIANTS THROUGH TESTING VARIANTS FOR WHETHER THEY ALTER REGULATORY REGION ACTIVITY IN ALLELE-SPECIFIC REPORTER ASSAYS AND HAVE USED THIS METHODOLOGY IN A T CELL LINE TO DISCOVER HUNDREDS OF PUTATIVELY CAUSAL VARIANTS ACROSS THE GENOME. THE NEXT AND PERHAPS MOST DAUNTING STEP IS TO CONNECT VARIANTS TO THEIR EFFECT ON THE FUNCTION OF DISEASE-RELEVANT CELL TYPES. IN THIS PROPOSAL, WE AIM TO IDENTIFY VARIANTS IN NON-CODING REGULATORY REGIONS THAT ALTER T CELL PROLIFERATION AND MIGRATION. FIRST, WE WILL USE A NOVEL CRISPR-INTERFERENCE SCREEN TO IDENTIFY VARIANT REGULATORY REGIONS THAT REGULATE T CELL PROLIFERATION AND MIGRATION TOWARD CHEMOKINES FOUND IN INFLAMED TISSUES. NEXT, WE WILL USE A SINGLE-CELL SCREENING APPROACH IN PRIMARY T CELLS TO IDENTIFY THE GENES MODULATED BY VARIANT REGULATORY REGIONS. WE WILL THEN DETERMINE VARIANT REGULATORY REGIONS THAT ACT SYNERGISTICALLY OR IN AN EPISTATIC MANNER ON T CELL FUNCTION, THEREBY IDENTIFYING A RELATIONSHIP BETWEEN SEPARATE GENETIC RISK LOCI AND THEIR EFFECTS ON T CELL FUNCTION. FINALLY, WE WILL DETERMINE WHETHER VARIANTS THAT INFLUENCE T CELL FUNCTION ARE ALSO ASSOCIATED WITH THE PREVALENCE OF AUTOREACTIVE T CELLS AND DISEASE SEVERITY USING A LARGE MULTIPLE SCLEROSIS COHORT. IF SUCCESSFUL, THIS WORK WILL TAKE THE FIRST LEAP IN DIRECTLY LINKING HUNDREDS OF RISK LOCI TO A CELLULAR FUNCTION IN AN AUTOIMMUNE DISEASE-RELEVANT CELL TYPE AND IT WILL PROVIDE INSIGHT INTO HOW GENETIC RISK PROMOTES DISEASE. OUR FINDINGS MAY THEREFORE IDENTIFY THERAPEUTICALLY TARGETABLE PATHWAYS FOR TREATMENT OF AUTOIMMUNE DISEASES.

REGULATION OF INTESTINAL IMMUNITY AND REPAIR BY INTEGRINS - PROJECT SUMMARY ULCERATIVE COLITIS (UC) IS A CHRONIC INFLAMMATORY BOWEL DISEASE (IBD) OF THE COLON THAT AFFECTS ROUGHLY ONE MILLION AMERICANS. UC IS GENERALLY NONFATAL AND OFTEN OCCURS IN THE PRIME OF LIFE, POTENTIALLY RESULTING IN DECADES OF SUFFERING, DISABILITY AND LOST PRODUCTIVITY. THE UNDERLYING MECHANISMS THAT LEAD TO DISEASE ARE POORLY UNDERSTOOD, AND THERE ARE NO CURRENT MEDICATIONS THAT CAN ‘CURE’ UC. RECENTLY ALMOST ALL PATIENTS WITH UC (BUT ALMOST NONE WITH CROHN’S DISEASE OR NO IBD) HAVE BEEN FOUND TO MAKE HIGH TITER ANTIBODIES TO THE INTEGRIN ΑVΒ6. FURTHERMORE, ANTI-ΑVΒ6 AUTOANTIBODIES PRECEDED CLINICAL DIAGNOSIS OF UC BY UPTO 10 YEARS, AND WERE ASSOCIATED WITH INCREASED ADVERSE OUTCOMES, SUGGESTING THEY MAY REPRESENT A FUNDAMENTAL PREREQUISITE OR CAUSE OF THE PATHOGENESIS OF UC. A MAJOR ROLE FOR ΑVΒ6 IS THE ACTIVATION OF THE CYTOKINE TGF-Β, A MAJOR REGULATOR OF INTESTINAL BARRIER FUNCTION AND MUCOSAL IMMUNE RESPONSES. WE HAVE SPENT MANY YEARS INVESTIGATING THE ROLE OF ΑV INTEGRINS IN ACTIVATING TGF-Β TO PROMOTE INTESTINAL IMMUNITY AND MAINTAIN HOMEOSTASIS, AND WE HAVE FOUND THAT MICE IN WHICH ΑVΒ6 IS SELECTIVELY DELETED IN THE INTESTINAL EPITHELIUM DEVELOP MORE SEVERE INTESTINAL INFLAMMATION IN A COLITIS MODEL, AND THIS IS ASSOCIATED WITH CHANGES IN EPITHELIAL CELL GENE EXPRESSION SIMILAR TO THOSE SEEN IN UC. BASED ON THESE DATA WE HYPOTHESIZE THAT INTESTINAL EPITHELIAL ΑVΒ6 ACTIVATES TGF-Β FOR AUTOCRINE AND PARACRINE SIGNALING TO PROMOTE INTESTINAL IMMUNE HOMEOSTASIS, AND THAT DISRUPTION OF ΑVΒ6 FUNCTION RESULTS IN ‘PRE-CLINICAL’ UC IN MICE AND HUMANS. IN THIS PROJECT, WE WILL MAKE USE OF UNIQUE MOUSE MODELS, HUMAN EPITHELIAL CELL AND ORGANOID CULTURES, AND UC PATIENT SAMPLES TO TEST THESE HYPOTHESES AND DETERMINE THE ROLE OF ΑVΒ6 AUTOANTIBODIES IN IBD. SUCCESSFUL COMPLETION OF THESE EXPERIMENTS WILL DEFINE A ROLE FOR EPITHELIAL CELLS AS A NEXUS FOR REGULATION OF TGF- Β ACTIVATION IN THE INTESTINE, AND PROVIDE A CRITICAL UNDERSTANDING OF THE REGULATION OF THIS CYTOKINE IN HEALTH AND DISEASE.

TRANSPOSON MUTAGENESIS FOR HOST-TARGET AND DRUG DISCOVERY IN INFECTIOUS DISEASE

INVESTIGATION OF T CELL RESPONSES TO IMMUNOTHERAPY USING CLASS II TETRAMERS

FOXP3 ISOFORM EXPRESSION AND REGULATION OF AUTOANTIBODY PRODUCTION - PROJECT SUMMARY SYSTEMIC LUPUS ERYTHEMATOSUS (SLE) IS A MULTI-SYSTEMIC AUTOIMMUNE DISEASE THAT ARISES FROM A COMBINATION OF GENETIC FACTORS, DYSREGULATED IMMUNE RESPONSES AND ENVIRONMENTAL TRIGGERS. SLE-LIKE PHENOTYPES, INCLUDING ELEVATED ANTI-NUCLEAR IGG AUTOANTIBODY TITERS, KIDNEY NEPHRITIS, SPLENOMEGALY AND REDUCED SURVIVAL HAVE BEEN REPORTED IN SEVERAL ANIMAL MODELS OF SLE, BUT FEW OF THESE MODELS REQUIRE ENVIRONMENTAL FACTORS TO DRIVE DISEASE. THEREFORE, STUDIES OF CAUSATIVE ENVIRONMENTAL FACTORS HAVE USED MOUSE MODELS THAT ALREADY HAVE LETHAL SLE DISEASE PRIOR TO EXPOSURE. CONSEQUENTLY, THEY CANNOT BE USED TO DETERMINE HOW THESE FACTORS CAN INDUCE FLARES IN PATIENTS WITH LATENT SLE. THEREFORE, A MOUSE MODEL OF MODERATE SLE IS REQUIRED TO APPROPRIATELY REPLICATE SLE FLARES THAT ARE INDUCED BY ENVIRONMENTAL TRIGGERS. REGULATORY T (TREG) CELLS PLAY A CENTRAL ROLE IN MAINTAINING IMMUNE SYSTEM HOMEOSTASIS AND MODULATING IMMUNE RESPONSES. FOXP3 IS A MASTER REGULATOR OF TREG DEVELOPMENT AND FUNCTION. FOXP3 MUTATIONS IN PATIENTS WITH IPEX (IMMUNODYSREGULATION, POLYENDOCRINOPATHY, ENTEROPATHY, X-LINKED SYNDROME), WHICH RESULT IN A DEFICIENCY IN TREGS, RESULT IN LETHAL AUTOIMMUNITY, SIMILAR TO THE DISEASE OBSERVED IN FOXP3 DEFICIENT MICE. WHILE HIGHLY CONSERVED IN BOTH AMINO ACID SEQUENCE AND GENE STRUCTURE, ONE DIFFERENCE BETWEEN HUMANS AND MICE IS THAT THE HUMAN FOXP3 GENE ENCODES TWO MAJOR ALTERNATIVELY SPLICED ISOFORMS: A FULL-LENGTH VERSION THAT USES ALL 10 EXONS (FOXP3FL, THE ONLY ISOFORM IN MICE) AND A SHORTER ISOFORM LACKING EXON 2 (FOXP3∆E2). RECENT STUDIES HAVE SHOWN THAT TREGS FROM PATIENTS WITH SOME AUTOIMMUNE DISEASES EXPRESS INCREASED LEVELS OF THE ∆E2 ISOFORM COMPARED TO THOSE FROM HEALTHY DONORS. CONSISTENT WITH THIS FINDING, WE HAVE FOUND THAT TREGS FROM SLE PATIENTS HAVE INCREASED EXPRESSION OF THE FOXP3∆EX2 ISOFORM. TO STUDY THE ROLE OF THE ∆E2 ISOFORM IN TREG FUNCTION WE GENERATED A NEW MOUSE STRAIN WITH FOXP3 EXON 2 DELETION. INTERESTINGLY, WE FOUND THAT FOXP3∆E2 MICE DEVELOP HALLMARK FEATURES OF SLE, INCLUDING ANTI-DNA AND ANTI-NUCLEAR AUTOANTIBODIES, INCREASED NUMBER AND SIZE OF SPONTANEOUS GERMINAL CENTERS AND KIDNEY DEPOSITION OF ANTIBODY COMPLEXES, BY 4-5 WEEKS OF AGE. OUR CENTRAL HYPOTHESIS IS THAT EXPRESSION OF THE ∆E2 ISOFORM OF FOXP3 LEADS TO TREG DYSFUNCTION AND LOSS OF TOLERANCE. TO TEST THIS HYPOTHESIS WE WILL FIRST DETERMINE THE ROLE OF FOXP3∆EX2-EXPRESSING TREGS IN GERMINAL CENTER FUNCTION, FOCUSING ON THE RELATIONSHIP BETWEEN TFH AND TFR CELLS. NEXT, WE WILL ASSESS THE RESPONSE TO UVB LIGHT IN FOXP3∆E2 MICE. FINALLY, WE WILL EXAMINE THE EXPRESSION OF FOXP3∆E2 IN TREGS FROM COHORTS OF SLE AND MCTD PATIENTS TO DETERMINE THE POTENTIAL ROLE OF THIS ISOFORM AND DISEASE EXPRESSION. THESE STUDIES WILL PROVIDE INSIGHTS INTO THE ROLE OF FOXP3∆E2 TREG FUNCTION AND PREDISPOSITION TO SLE.

DENDRITIC CELL CONTROL OF INTESTINAL T CELL RESPONSES

SPECIALIZATION AND TARGETING OF CD4+ TISSUE RESIDENT MEMORY T CELLS IN EAE - PROJECT SUMMARY MULTIPLE SCLEROSIS (MS) IS AN AUTOIMMUNE DISEASE OF THE CENTRAL NERVOUS SYSTEM (CNS) CHARACTERIZED BY DEMYELINATION, AXONAL LOSS, AND PROGRESSIVE DISABILITY. SIMILARLY, ITS ANIMAL MODEL, EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) IS MARKED BY CNS INFLAMMATION AND DEMYELINATION. CIRCULATING CD4+ T HELPER (TH) SUBSETS HAVE LONG BEEN IMPLICATED IN THE PATHOGENESIS OF MS AND EAE. RECENTLY, T CELLS WITH TISSUE RESIDENCE CAPACITIES AND CHARACTERISTICS HAVE BEEN IDENTIFIED IN CNS AND THE CEREBROSPINAL FLUID (CSF) THERE IS A PAUCITY OF INFORMATION REGARDING THE SPECIALIZATION AND FUNCTIONS OF THESE OF MS PATIENTS BUT CELLS DURING CNS AUTOIMMUNITY. THE STUDY OF CD4+ TISSUE RESIDENT MEMORY (TRM) T CELLS IN THE CONTEXT OF EAE HAS BEEN IN PART HAMPERED BY THE LACK OF APPROPRIATE MODELS AND TOOLS TO TRACK AND ELIMINATE THESE CELLS INDEPENDENTLY OF OTHER CIRCULATING MEMORY AND EFFECTOR T CELLS. WE HAVE DEVELOPED NOVEL TOOLS AND MODELS TO FURTHER CHARACTERIZE AND MODULATE CD4+ TRM DURING EAE. WE HYPOTHESIZE THAT CNS CD4+ TRMS FORM A RESERVOIR OF AUTOREACTIVE T CELLS IN THE CNS WHICH SUSTAINS DISEASE AND IS POORLY TARGETED BY DISEASE MODIFYING THERAPIES DIRECTED AGAINST CIRCULATING T CELLS. IN THIS PROPOSAL, WE WILL USE THESE NOVEL TOOLS TO SPECIFICALLY: 1) ESTABLISH THE DIVERSITY AND SPECIALIZATION OF CD4+ TRMS DURING EAE, 2) DETERMINE THE MECHANISMS PROMOTING THE MAINTENANCE OF CD4+ TRMS IN THE CNS, AND 3) DETERMINE HOW TO DISRUPT THE TISSUE RESIDENT PROGRAM IN TRM AND NEUTRALIZE OR ELIMINATE THEM IN THE CNS. THE COMPLETION OF THIS PROPOSAL WILL HELP US UNDERSTAND HOW TISSUE RESIDENT MEMORY T CELLS PROMOTE SUSTAINED AUTOIMMUNITY AND MAY LEAD TO THE DEVELOPMENT OF NOVEL THERAPIES FOR MS AND ASSOCIATED AUTOIMMUNE DISEASES OF THE CNS.

GERMLINE SEQUENCE RESOURCES & ANALYSES IN A VERTEBRATE MODEL THAT UNDERGOES PGR

RECOGNIZING VIRULENT PATHOGENS

BUILD TO LEAD ? BUILDING PARTNERSHIPS TO LINK THE EXPOSOME TO AUTOIMMUNE DISEASE (ADMIN SUPP) - SUMMARY WE ARE REQUESTING AN ADMINISTRATIVE SUPPLEMENT FOR 1R21AR084042 “BUILD TO LEAD – BUILDING PARTNERSHIPS TO LINK THE EXPOSOME TO AUTOIMMUNE DISEASE”. THE ADMINISTRATIVE SUPPLEMENT STUDIES ARE WITHIN THE SCOPE OF THE ACTIVE PARENT GRANT, WHICH IS FOCUSED ON ESTABLISHING COLLABORATIVE TEAMS TO PARTICIPATE IN THE EXPOSOME IN AUTOIMMUNE DISEASES COLLABORATING TEAMS (EXACT) NETWORK. ALL EXACT NETWORK SITES WILL STUDY HOW ENVIRONMENTAL EXPOSURES INFLUENCE AUTOIMMUNE DISEASE SUSCEPTIBILITY, ONSET AND OUTCOMES. THIS ADMINISTRATIVE SUPPLEMENT WILL PROVIDE AN OPPORTUNITY TO ESTABLISH ADDITIONAL COLLABORATIONS, EXPAND THE AUTOIMMUNE EXPOSOME SUMMITS PROPOSED IN THE PARENT GRANT AND EXPAND HARMONIZATION OF EXPOSOME DATA COLLECTION TO ALL MEMBERS OF THE EXACT NETWORK. THE ADMINISTRATIVE SUPPLEMENT HAS THREE SPECIFIC AIMS. SPECIFIC AIM 1 PROPOSES TO ESTABLISH ADDITIONAL COLLABORATIONS WITH EXPERTS IN ENVIRONMENTAL EXPOSURES AND TO INCREASE THE INVOLVEMENT OF THE EXPERT CONSULTANTS IN THE PARENT GRANT. SPECIFIC AIM 2 PROPOSES TO EXPAND THE AUTOIMMUNE EXPOSOME SUMMITS AND RELATED WORKING AND ADVISORY GROUPS TO INCLUDE ADDITIONAL KEY STAKEHOLDERS WITH EXPERTISE BEYOND THE PARENT GRANT BUT CRITICAL FOR THE EXACT NETWORK. THIS COULD INCLUDE EXPERTS IN PEDIATRIC AUTOIMMUNE DISEASES AND SOCIAL DETERMINANTS OF HEALTH. SPECIFIC AIM 3 PROPOSES TO EXPAND THE HARMONIZATION OF EXPOSOME DATA COLLECTION TO THE EXACT NETWORK. THIS WILL INCLUDE A LANDSCAPE ANALYSIS OF EXPOSOME DATA ACROSS THE NETWORK, IDENTIFICATION AND SELECTION OF OPTIMAL PROCESSING PROTOCOLS FOR EXPOSURE-ASSOCIATED BIOSPECIMENS AND DEFINING SHARED PRIORITIES FOR EXISTING DATA TO BE HARMONIZED IN FUTURE EXACT NETWORK STUDIES. NOTABLY, ALL OF THE PROPOSED WORK WILL BE DONE IN CONSULTATION WITH NIH AND THE EXACT NETWORK AND WILL BE FOUNDATIONAL FOR FUTURE EXACT NETWORK STUDIES. THE PROPOSED WORK IS WITHIN THE SCOPE OF THE PARENT AWARD AS IT FOCUSES ON ESTABLISHING COLLABORATIVE TEAMS FOR STUDYING THE ROLE OF THE EXPOSOME IN THE DEVELOPMENT AND PROGRESSION OF AUTOIMMUNE DISEASE.

IN-DEPTH ANALYSIS OF CITRULLINE-SPECIFIC CD4T CELL IN RHEUMATOID ARTHRITIS

IN VIVO ASSESSMENT OF T CELL KINETICS IN INDIVIDUALS AT RISK FOR TYPE 1 DIABETES

DETERMINING THE MOLECULAR BASIS FOR DIFFERENT RATES OF T1D PROGRESSION

AIRMAX AS SENSITIVE MEASURE OF BETA CELL FUNCTION IN AT-RISK INDIVIDUALS

GENETIC VARIANTS GUIDING PATHOGENICITY OF COLITOGENIC T CELLS - SUMMARY ~2.4 MILLION PEOPLE IN THE UNITED STATES HAVE INFLAMMATORY BOWEL DISEASES (IBDS). WHILE THESE SEVERE AND DEBILITATING DISEASES HAVE A GENETIC COMPONENT, AS DETERMINED FROM CONCORDANCE STUDIES IN MONOZYGOTIC TWINS, WE STILL KNOW VERY LITTLE ABOUT HOW GENETICS DRIVES RISK AND PROGRESSION FOR EACH DISEASE. GENOME-WIDE ASSOCIATION STUDIES HAVE IDENTIFIED HUNDREDS OF IBD-ASSOCIATED REGIONS OF THE GENOME, BUT THERE IS OFTEN TIGHT LINKAGE DISEQUILIBRIUM BETWEEN CAUSAL AND NON-CAUSAL VARIANTS IN THESE REGIONS, AND MOST DISEASE-ASSOCIATED GENETIC VARIANTS ARE IN NON-CODING REGIONS. THUS, DETERMINING THE VARIANTS THAT PROMOTE DISEASE AND THEIR EFFECTS ON DISEASE-RELEVANT CELL TYPES IS CHALLENGING. ONE CELL TYPE THAT IS IMPLICATED IN IBD IS THE PATHOGENIC TH17 CELL. TH17 CELLS NORMALLY MAINTAIN HOMEOSTASIS OF THE COLONIC LAMINA PROPRIA, BUT IN THE CONTEXT OF IBD, PATHOGENIC TH17 CELLS CAUSE UNWANTED INFLAMMATION. NON-CODING GENETIC VARIANTS ASSOCIATED WITH IBD ARE HIGHLY ENRICHED WITHIN THE ACCESSIBLE CHROMATIN OF PATHOGENIC TH17 CELLS COMPARED TO OTHER T CELL TYPES. THUS, WE HYPOTHESIZE THAT DISEASE-CAUSAL VARIANTS ALTER CIS-REGULATORY ELEMENT (CRE) ACTIVITY IN PATHOGENIC TH17 CELLS THAT MAY AFFECT THEIR DIFFERENTIATION AND FUNCTION, MAKING THEM MORE PATHOGENIC. WE RECENTLY DISCOVERED THAT WE CAN IDENTIFY LIKELY CAUSAL VARIANTS THROUGH TESTING THEIR ALLELE-SPECIFIC EFFECTS ON REGULATORY REGION ACTIVITY IN MASSIVELY PARALLEL REPORTER ASSAYS (MPRAS). OUR MPRAS IN T AND B CELL LINES AND PRIMARY T CELLS IDENTIFY PUTATIVE CAUSAL VARIANTS THAT ARE HIGHLY CELL TYPE AND STATE DEPENDENT. THUS, WE WILL PERFORM MPRAS ON IBD GWAS VARIANTS, IDENTIFIED IN EAST ASIAN AND EUROPEAN INDIVIDUALS, USING HUMAN PATHOGENIC TH17 CELLS TO DISCOVER PUTATIVELY CAUSAL IBD VARIANTS ACROSS THE GENOME. WE WILL THEN CONNECT VARIANTS TO THEIR EFFECTS ON PATHOGENIC TH17 CELL MIGRATION AND FUNCTION USING CRISPR-INTERFERENCE (CRISPRI) SCREENS IN PRIMARY T CELLS. NEXT, WE WILL USE A SINGLE-CELL SCREENING APPROACH IN PATHOGENIC TH17 CELLS TO IDENTIFY THE GENES THAT VARIANT CRES CONTROL. WE WILL THEN DETERMINE GENE REGULATORY NETWORKS THAT VARIANT CRES ACT ON TO BEGIN TO DEFINE IMPORTANT DISEASE PATHWAYS. IN CONCERT WITH OUR STUDIES IN HUMAN T CELLS, WE WILL USE IN VIVO CRISPRI SCREENS TO TEST CONSERVED VARIANT CRE EFFECTS ON MIGRATION AND FUNCTION OF PATHOGENIC TH17 CELLS IN A MURINE MODEL OF IBD, THUS EXPLORING ADDITIONAL PHYSIOLOGICAL AVENUES OF VARIANT EFFECTS IN THE IN VIVO SETTING. IF SUCCESSFUL, THIS WORK WILL TAKE THE FIRST LEAP IN DIRECTLY LINKING HUNDREDS OF IBD RISK LOCI TO THE FUNCTION OF A DISEASE-RELEVANT CELL TYPE AND IT WILL PROVIDE INSIGHT INTO HOW GENETIC RISK PROMOTES DISEASE. OUR FINDINGS MAY THEREFORE IDENTIFY THERAPEUTICALLY TARGETABLE PATHWAYS FOR TREATMENT OF IBD.

T CELL SPECIFICITY AND PHENOTYPES IN PRECLINICAL TYPE 1 DIABETES

EXPLORING IN VIVO TREG FUNCTION IN T1D THROUGH THE LENS OF EXPANDED TREGS - PROJECT SUMMARY/ABSTRACT A CRITICAL BARRIER TO OPTIMALLY TREATING TYPE 1 DIABETES (T1D), AN AUTOIMMUNE DISEASE IN WHICH THE ISLET BETA CELLS ARE DESTROYED BY IMMUNE CELLS, IS UNDERSTANDING HOW AUTOIMMUNITY IS REGULATED IN VIVO. SEVERAL LINES OF EVIDENCE SUGGEST THAT DEFECTIVE CD4+FOXP3+ REGULATORY T CELLS (TREG) LIKELY CONTRIBUTE TO THE LOSS OF TOLERANCE IN T1D. YET, LESS IS KNOWN ABOUT HOW HUMAN TREG FUNCTION IN VIVO. IN THE SANFORD T-REX STUDY IN WHICH ADOLESCENTS DIAGNOSED WITH T1D WERE TREATED WITH A SINGLE DOSE OF POLYCLONAL AUTOLOGOUS IN VITRO EXPANDED TREG (EXPTREG), WE FOUND THAT A LOWER DEGREE OF IN VITRO TREG EXPANSION SIGNIFICANTLY CORRELATED WITH BETTER PRESERVATION OF C- PEPTIDE (A BIOMARKER OF INSULIN SECRETION AND BETA CELL FUNCTION) A YEAR AFTER TREATMENT. THIS CORRELATION COULD NOT BE EXPLAINED BY AGE, EXPTREG PHENOTYPE OR IN VITRO EXPTREG SUPPRESSIVE FUNCTION. HOWEVER, WE DID IDENTIFY AN EXPTREG GENE SIGNATURE THAT CORRELATED WITH BETTER C-PEPTIDE PRESERVATION AND THIS EXPTREG SIGNATURE WAS CONSISTENTLY EXPRESSED OVER TIME WITHIN INDIVIDUALS. FURTHER, LOWER- AND HIGHER- EXPTREG DIFFERED PHENOTYPICALLY AND TRANSCRIPTIONALLY BY SIGNATURES IMPLICATING METABOLIC, HOMING AND SUPPRESSIVE FUNCTIONS. TOGETHER, THESE DATA SUGGEST THAT INTRINSIC FEATURES OF AN INDIVIDUAL’S TREG MAY CONTRIBUTE TO THE EXTENT OF IN VITRO TREG EXPANSION. THEY ALSO SUGGEST THAT STRONG ACTIVATION AND EXPANSION CAN DIFFERENTIALLY AMPLIFY OR ALTER THE STATE OF TREGS, LEADING TO CHANGES IN HOMING AND FUNCTION THAT MAY IMPACT CLINICAL RESPONSE. BASED ON THESE FINDINGS, WE HYPOTHESIZE THAT TREG PROLIFERATIVE CAPACITY IS DRIVEN BY THE ACTIVATION AND METABOLIC STATE OF TREG RESULTING IN DIFFERENTIAL IN VITRO FOLD EXPANSION, HOMING POTENTIAL AND IN VIVO SUPPRESSIVE FUNCTION THAT IMPACTS CLINICAL OUTCOME. WE WILL TEST THIS HYPOTHESIS BY LEVERAGING EXISTING PRIMARY HUMAN SAMPLES FROM BOTH THE T-REX CLINICAL TRIAL AND THE BENAROYA RESEARCH INSTITUTE REGISTRY AND REPOSITORY THAT INCLUDES INDIVIDUALS WITH KNOWN DEGREE OF IN VITRO TREG EXPANSION AND KNOWN C-PEPTIDE DECLINE. IN AIM1, WE WILL IDENTIFY HOW ACTIVATION STATES OF PRE- AND POST- EXPANSION TREG AND LONGITUDINAL TREG IN T-REX PARTICIPANTS CONTRIBUTE TO PROLIFERATIVE CAPACITY AND OUTCOME USING CELLULAR, TRANSCRIPTOMIC AND EPIGENETIC ASSAYS. IN AIM 2 WE WILL DETERMINE HOW METABOLIC SHIFTS DURING TREG IN VITRO FOLD EXPANSION ALTER TREG SUPPRESSIVE FUNCTION, THEREBY IMPACTING CLINICAL OUTCOME. IN AIM 3, WE WILL COMPARE THE IN VIVO SUPPRESSIVE FUNCTION OF LOWER- VERSUS HIGHER-EXPTREG FROM CLINICAL SAMPLES USING A XENOGENEIC GRAFT VERSUS HOST DISEASE (GVHD) MOUSE MODEL IN ADDITION TO ASSESSING IN VIVO EXPTREG HOMING AND FUNCTION USING THE ASSAYS FROM AIMS 1 AND 2 AND A NOVEL IN VITRO ASSAY OF CELL TRAFFICKING TO PANCREATIC ISLETS. SUCCESSFUL COMPLETION OF THESE AIMS WILL REVEAL MECHANISMS REGULATING TREG PROLIFERATIVE CAPACITY AND IN VIVO FUNCTION THAT IMPACT CLINICAL OUTCOME. UNDERSTANDING THESE MECHANISMS WILL GUIDE DEVELOPMENT OF NEXT GENERATION TREG ACTIVATION AND EXPANSION PROTOCOLS FOR TREG THERAPIES AND HELP TAILOR THE TREG EXPANSION PROCESS TO AN INDIVIDUAL’S BASELINE TREG SIGNATURE.

MECHANISMS OF COMMENSAL- SPECIFIC CD8+ T CELL DIFFERENTIATION, RESTRAINT AND DYSREGULATION IN INTESTINAL INFLAMMATION - PROJECT SUMMARY OUR UNDERSTANDING OF IMMUNITY LARGELY STEMS FROM MODELS OF INFECTION WITH PATHOGENIC MICROBES. HOWEVER, THE VAST MAJORITY OF MICROBIAL-IMMUNE ENCOUNTERS OCCUR AS A SYMBIOTIC RELATIONSHIP WITH THE COMMENSAL MICROBIOTA. RECENTLY, THE CONTRIBUTION OF COMMENSAL-SPECIFIC T CELLS TO HOST PHYSIOLOGY HAS RECEIVED SIGNIFICANT ATTENTION. THESE COMMENSAL-SPECIFIC RESPONSES NOT ONLY CONTROL MICROBIOTA CONTAINMENT BUT ALSO PROMOTE IMMUNE TOLERANCE WITHIN THE GASTROINTESTINAL TRACT. WHILE COMMENSAL-SPECIFIC CD4+ T CELL RESPONSES IN THE LAMINA PROPRIA HAVE DOMINATED MODELS OF MUCOSAL IMMUNE REGULATION, THESE ARE VASTLY OUTNUMBERED BY CD8+ INTRAEPITHELIAL LYMPHOCYTES WITHIN THE EPITHELIUM. HOW CD8+ T CELL RESPONSES TO GUT MICROBIOTA ARE PRIMED, DIFFERENTIATE AND FUNCTION UNDER HOMEOSTASIS HAS NOT BEEN ADDRESSED. CONVERSELY, ABERRANT IMMUNITY TO COMMENSAL MICROBES HAS BEEN PROPOSED TO UNDERLIE PATHOLOGIES OF BARRIER TISSUES, INCLUDING INFLAMMATORY BOWEL DISEASE (IBD), WHERE COMMENSAL-SPECIFIC T CELLS ACCUMULATE IN BLOOD AND INTESTINAL TISSUES OF AFFLICTED PATIENTS. A BETTER UNDERSTANDING OF THE PROPERTIES AND FUNCTIONS OF COMMENSAL-SPECIFIC T CELL RESPONSES IS THEREFORE FUNDAMENTAL TO STUDIES OF TISSUE IMMUNITY IN HEALTH AND DISEASE. OUR LONG TERM GOAL IS TO BETTER UNDERSTAND HOW COMMENSAL-SPECIFIC T CELL RESPONSES CONTRIBUTE TO BARRIER TISSUE HOMEOSTASIS, AND THE OBJECTIVE IN THIS APPLICATION IS TO INVESTIGATE THE MECHANISMS REGULATING INDUCTION OF COMMENSAL-SPECIFIC CD8+ T CELLS IN HOMEOSTASIS AND HOW THEY BECOME DYSREGULATED IN IBD. OUR RATIONALE FOR THE PROPOSED WORK IS THAT UNCOVERING THESE MECHANISMS HAS THE POTENTIAL TO TRANSLATE INTO NEW THERAPEUTIC APPROACHES. OUR CENTRAL HYPOTHESIS IS THAT COMMENSAL-SPECIFIC CD8+ T CELLS DEVELOP AS FUNCTIONALLY RESTRAINED INTRAEPITHELIAL LYMPHOCYTES (IEL) UNDER HOMEOSTASIS, BUT THAT PERTURBATION OF LOCAL IMMUNE REGULATION WITHIN THE INTESTINAL EPITHELIUM, IN THE CASE OF PATIENTS WITH ULCERATIVE COLITIS, BY AUTOANTIBODY-MEDIATED BLOCKADE OF INTEGRIN AVB6 RESULTS IN ABERRANT CD8+ EFFECTOR T CELL RESPONSES IN IBD. BASED ON STRONG PRELIMINARY DATA, WE WILL TEST THREE SPECIFIC AIMS: (1) DETERMINE KEY ANTIGEN-PRESENTING CELLS (APC) PRIMING SFB-SPECIFIC CD8⍺Β+ IEL. (2) IDENTIFY HOW CELL-INTRINSIC PATHWAYS DRIVE DIFFERENTIATION, MAINTENANCE AND RESTRAINT OF SFB-SPECIFIC CD8⍺Β+ PIEL. (3) DETERMINE HOW PATHOGENIC KLRG1+EOMES+ CD8+ T CELLS ARISE AND CONTRIBUTE TO INFLAMMATION IN MURINE MODELS OF ULCERATIVE COLITIS OUR APPROACH IS INNOVATIVE AS IT INVESTIGATES NEW MECHANISMS OF IMMUNITY UNIQUE TO COMMENSAL-SPECIFIC CD8+ T CELL RESPONSES. THE PROPOSED WORK IS SIGNIFICANT BECAUSE IT WILL ESTABLISH NEW INSIGHTS INTO THE INTERACTION AND COMMUNICATION BETWEEN COMMENSAL MICROBES AND IMMUNE CELLS IN THE GUT ENVIRONMENT AND IDENTIFY POTENTIAL TARGETS FOR THERAPEUTIC INTERVENTION IN CONDITIONS OF CHRONIC INTESTINAL INFLAMMATION.

MUCOSAL AND SYSTEMIC CIRCUITS REGULATING TREG DIFFERENTIATION AND FUNCTION

TSLP RECEPTOR IN THE REGULATION OF CELL DEVELOPMENT

HLA SUSCEPTIBILITY GENES IN RHEUMATOID ARTHRITIS

INVESTIGATING THE ROLE OF IL-6 SIGNALING IN TEFF RESISTANCE AND T1D DEVELOPMENT

EXTRACELLULAR PROTEINS AS SIGNALS IN ENDOTHELIAL GROWTH

INTESTINAL DENDRITIC CELL MODULATION OF REGULATORY T CELL FUNCTION IN IBD

HARNESSING AUTOLOGOUS STEM CELLS TO RECONSTRUCT SKELETAL MUSCLE WITH INNERVATION POTENTIAL

ACTIVATION INDUCED GENERATION OF HUMAN REGULATORY T CELLS

DEFINING THE CD4 T CELL RESPONSE TO IMMUNIZATION WITH GAD

HOMING AND FUNCTION OF REGULATORY T CELLS SPECIFIC FOR ISLET ANTIGENS

PATIENT ORIENTED RESEARCH & MENTORING IN LIVER DISEASES

HOX CLUSTERS, HEMATOPOIESIS AND GENOME ENABLEMENT IN THE ANTARCTIC NOTOTHENOID FISHES

GENOME-WIDE PROFILING OF IMMUNE RESPONSIVENESS IN PATIENTS WITH PRIMARY IMMUNODEF

IMPACT OF THE SLE GENE BANK1 ON AUTOPHAGY AND PLASMABLAST DIFFERENTIATION IN LUPUS

NOVEL MINOR HISTOCOMPATIBILITY ANTIGENS DETERMINED BY CODING DELETION POLYMORPHIS

NONCANONICAL AUTOPHAGY AND TOLL-LIKE RECEPTOR SIGNALING IN SLE

THE ROLE OF THE CYTOKINE TSLP IN FOOD ALLERGY

DISSECTING HOW DYRK1A-GP130 AXIS DRIVES IMMUNE DYSREGULATION IN DOWN SYNDROME AND BEYOND - PROJECT SUMMARY/ABSTRACT AUTOIMMUNITY IS A MAJOR DRIVER OF CHRONIC MORBIDITY IN OVER 15 MILLION AMERICANS, REPRESENTING SIGNIFICANT UNMET MEDICAL NEED. INDIVIDUALS WITH DOWN SYNDROME (DS, TRISOMY 21) ARE AT ESPECIALLY INCREASED RISK (4-100X) OF AUTOIMMUNITY, SUGGESTING A CONTRIBUTORY ROLE OF GENES ON CHROMOSOME 21. WE IDENTIFIED THE CHROMOSOME 21- ENCODED KINASE DYRK1A (STUDIED ALMOST EXCLUSIVELY IN THE CONTEXT OF NEURODEVELOPMENT) AS A CANDIDATE DRIVER OF AUTOIMMUNITY RISK. BETTER UNDERSTANDING THE UNDERLYING MECHANISMS MAY HIGHLIGHT DYRK1A AS A DRUGGABLE TARGET TO TREAT OR PREVENT AUTOIMMUNITY IN PEOPLE WITH DS AND SUBSETS OF PEOPLE WITHOUT DS. WE SHOWED THAT DYRK1 REGULATES DIFFERENTIATION OF PRO-AUTOIMMUNITY TH17 CELLS. WE FURTHER UNCOVERED THAT THIS WORKS AT LEAST IN PART BY A NOVEL REGULATORY ROLE OF DYRK1A ON SURFACE EXPRESSION OF THE IL-6 RECEPTOR SUBUNITS GP130 AND IL-6R. THESE FINDINGS HIGHLIGHT A MUCH BROADER ROLE FOR DYRK1A IN AUTOIMMUNE BIOLOGY THAN PREVIOUSLY APPRECIATED BECAUSE (I) IL-6 IS A PLEIOTROPIC CYTOKINE THAT DRIVES AUTOIMMUNITY THROUGH MULTIPLE MECHANISMS INCLUDING BUT NOT LIMITED TO TH17 DIFFERENTIATION; AND (II) GP130 MEDIATES SIGNALING OF MANY CYTOKINES OTHER THAN IL-6 INCLUDING (IMMUNE-RELEVANT) IL-11 AND IL-27. THEREFORE, THE DYRK1A-GP130 AXIS HAS BROAD POTENTIAL TO IMPACT BIOLOGY RELEVANT TO AUTOIMMUNITY AND BEYOND. WE HYPOTHESIZE THAT DYRK1A REGULATES CELLULAR AND FUNCTIONAL RESPONSE TO IL-6 AND OTHER GP130 FAMILY CYTOKINES IN PEOPLE AND MURINE MODELS OF DS. WE PROPOSE KEY EXPERIMENTS HERE TO ADVANCE OUR UNDERSTANDING OF THE DYRK1A- GP130 AXIS (I) IN PEOPLE WITH AND WITHOUT DS, (II) IN CELL TYPES BEYOND NAÏVE CD4+ T CELLS AND (III) IN MURINE MODELS OF DS. PILOT HIGH-RISK HIGH-REWARD APPROACHES INCLUDE FIRST-IN-KIND IMMUNE STUDIES OF INDIVIDUALS WITH DYRK1A SYNDROME. OUR SPECIFIC AIMS ARE: SPECIFIC AIM 1. DEFINE HOW DYRK1A REGULATES HUMAN GP130 SIGNALING. THESE STUDIES WILL DEFINE HOW THE DYRK1A-GP130 AXIS IS FUNCTIONALLY ALTERED AND LAY KEYSTONE DATA TO RATIONALLY EXPAND THE SCOPE OF FOLLOW-UP STUDIES. NOTABLY, WE INCLUDE PEOPLE WITH DS AND DYRK1A SYNDROME WHO NATURALLY OVER-/UNDER-EXPRESS DYRK1A RESPECTIVELY. THIS FIRST-IN-KIND ALLELIC SERIES ALLOWS UNPRECEDENTED TRANSLATIONALLY RELEVANT STUDIES OF DYRK1A IN IMMUNOBIOLOGY, INCLUDING NOVEL STUDIES IN IMMUNE AGING AND AUTOIMMUNE-POISED STATE. SPECIFIC AIM 2. VALIDATE ALTERED DYRK1A/GP130 AXIS IN MURINE MODELS OF DS. THESE STUDIES TEST THE HYPOTHESIS THAT THE DP16 MODEL OF DS SHOWS SIMILAR DYSREGULATION OF THE DYRK1A-GP130 AXIS AS SEEN IN PEOPLE WITH DS. WE FURTHER DEMONSTRATE UNIQUE UTILITY OF THE MURINE MODEL, INCLUDING LEVERAGING NOVEL GENETIC MODELS TO DEMONSTRATE THE CAUSAL ROLE OF DYRK1A AND MECHANISTICALLY DISSECTING T CELL GENERATION BEYOND TH17. THIS WILL GENERATE FIRST-IN-KIND DATA THAT THE DP16 MODEL CAN AND SHOULD BE USED TO STUDY DS- IMMUNODYSREGULATION.

INHIBITING CNS RESIDENT MEMORY T CELLS DRIVING NEUROINFLAMMATION - PROJECT SUMMARY MULTIPLE SCLEROSIS (MS) IS A CHRONIC, AUTOIMMUNE-MEDIATED DISEASE OF THE CENTRAL NERVOUS SYSTEM CHARACTERIZED BY DEMYELINATION, AXONAL LOSS, AND PROGRESSIVE DISABILITY. EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) IS AN ANIMAL MODEL WHICH SHARES SPECIFIC PARTICULARITIES WITH MS, HAS HELPED DEVELOP CURRENT DISEASE MODIFYING THERAPIES (DMTS) USED TO TREAT MS PATIENTS AND HAS BEEN INSTRUMENTAL TO UNDERSTAND MECHANISMS BEHIND DISEASE DEVELOPMENT AND PROGRESSION. WHILE CURRENT DISEASE MODIFYING THERAPIES HAVE BEEN BENEFICIAL TO IMPROVE THE HEALTH OF MS PATIENTS, A SIGNIFICANT PROPORTION OF THEM CONTINUE TO EXPERIENCE RELAPSES, AND DISEASE TEND TO WORSEN OVER TIME SUGGESTING THE INFLAMMATORY AND AUTOIMMUNE RESPONSE COULD BE SUSTAINED IN THE CNS. MEMORY CD4+T CELLS AND B CELLS HAVE BEEN IMPLICATED IN THE PATHOGENESIS OF MS. WHILE MOST MEMORY T CELLS CAN BE SAMPLED IN THE BLOOD BECAUSE THEY CIRCULATE BETWEEN LYMPHOID TISSUES, BLOOD AND NON-LYMPHOID TISSUES; AND THEIR EGRESS FROM LYMPHOID TISSUES AND MIGRATION TO THE CNS CAN BE STOPPED BY DMTS, A SMALL FRACTION OF MEMORY T CELLS CALLED TISSUE RESIDENT MEMORY T CELLS (TRM) HAVE UNIQUE PROPRIETIES AND RESIDES IN NON- LYMPHOID TISSUES. T CELLS WITH TRM PHENOTYPE HAVE BEEN IDENTIFIED IN THE CSF AND CNS OF INDIVIDUAL WITH FROM MS. WE HYPOTHESIZE THAT CNS CD4+ TRMS FORM A RESERVOIR OF AUTOREACTIVE T CELLS IN THE CNS WHICH SUSTAINS DISEASE AND IS POORLY TARGETED BY DISEASE MODIFYING THERAPIES DIRECTED AGAINST CIRCULATING T CELLS. USING A NEWLY DEVELOPED TRM DEPENDENT MODEL OF EAE AND A TARGETED CRISPR SCREEN, WE WILL: 1) IDENTIFY MOLECULAR CUES THAT INHIBIT TRM MAINTENANCE IN THE CNS DURING EAE, 2) VALIDATE INDIVIDUALLY THE EFFECT OF SELECTED GENES ON TRM MAINTENANCE AND EAE PROGRESSION. OUR APPROACH IS POISED TO IDENTIFY NOVEL INHIBITORY GENES FOR TRM AND THEREFORE COULD PROVIDE THE BASIS FOR THE DEVELOPMENT OF NEW MS THERAPIES WHICH COULD WORK INDEPENDENTLY OR IN CONJUNCTION WITH CURRENT DMTS. IN ADDITION, OUR FINDINGS WILL PROVIDE NOVEL FUNDAMENTAL INFORMATION REGARDING TRM AND MEANS TO INHIBIT THEM.

TRANSLATIONAL REPROGRAMMING BY REGULATORY T CELLS

FOXP3DEX2 ISOFORM EXPRESSION LEADS TO TREG DYSFUNCTION AND SLE - PROJECT SUMMARY REGULATORY T (TREG) CELLS PLAY A CENTRAL ROLE IN MAINTAINING IMMUNE SYSTEM HOMEOSTASIS AND MODULATING IMMUNE RESPONSES. FOXP3 IS A MASTER REGULATOR OF TREG DEVELOPMENT AND FUNCTION. FOXP3 MUTATIONS IN PATIENTS WITH IPEX, WHICH RESULT IN A DEFICIENCY IN TREGS, RESULT IN LETHAL AUTOIMMUNITY, SIMILAR TO THE DISEASE OBSERVED IN FOXP3 DEFICIENT MICE. WHILE HIGHLY CONSERVED IN BOTH AMINO ACID SEQUENCE AND GENE STRUCTURE, ONE DIFFERENCE BETWEEN HUMANS AND MICE IS THAT THE HUMAN FOXP3 GENE ENCODES TWO MAJOR ALTERNATIVELY SPLICED ISOFORMS: A FULL LENGTH VERSION THAT USES ALL 10 EXONS (FOXP3-FL, THE ONLY ISOFORM IN MICE) AND A SHORTER ISOFORM LACKING SEQUENCES SHOWN TO BE IMPORTANT IN REGULATING TH17 DIFFERENTIATION. RECENT STUDIES HAVE SHOWN THAT TREGS FROM PATIENTS WITH SOME AUTOIMMUNE DISEASES EXPRESS INCREASED LEVELS OF THE E2 ISOFORM COMPARED TO THOSE FROM HEALTHY DONORS. CONSISTENT WITH THIS FINDING, WE HAVE FOUND THAT TREGS FROM SLE PATIENTS HAVE INCREASED EXPRESSION OF THE FOXP3EX2 ISOFORM. TO STUDY THE ROLE OF THE E2 ISOFORM IN TREG FUNCTION WE GENERATED A NEW MOUSE STRAIN WITH FOXP3 EXON 2 DELETION. INTERESTINGLY, WE FOUND THAT FOXP3E2 MICE DEVELOP HALLMARK FEATURES OF SLE, INCLUDING ANTI- DNA AND ANTI-NUCLEAR AUTOANTIBODIES, INCREASED NUMBER AND SIZE OF SPONTANEOUS GERMINAL CENTERS AND KIDNEY DEPOSITION OF ANTIBODY COMPLEXES, BY 4-5 WEEKS OF AGE. HOWEVER, THESE MICE DO NOT DEVELOP FULL-BLOWN DISEASE AND HAVE A NORMAL LIFE SPAN. OUR CENTRAL HYPOTHESIS IS THAT THE REGION ENCODED BY EXON 2 OF FOXP3 GENE IS CRITICAL FOR NORMAL TREG IDENTITY AND FUNCTION. TO TEST THIS HYPOTHESIS WE WILL TAKE 2 APPROACHES. FIRST, WE WILL DETERMINE THE LEVELS OF FOXP3-FL AND -EX2-EXPRESSING TREGS IN HEALTHY HUMAN SUBJECTS AND SUBJECTS WITH AUTOIMMUNE DISEASE. THESE STUDIES WILL BE COMPLEMENTED WITH STUDIES USING MICE CONTAINING VARYING RATIOS OF TREGS EXPRESSING EACH ISOFORM. SECOND, WE TEST THE HYPOTHESIS THAT TREGS EXPRESSING FOXP3-EX2 HAVE A REDUCED ABILITY TO REGULATE EFFECTOR T CELL ACTIVATION, USING BOTH HUMAN TREG CLONES AND A MOUSE TRANSFER COLITIS MODEL. TOGETHER THESE STUDIES WILL ALLOW US TO GAIN IMPORTANT INFORMATION ON THE EXPRESSION OF FOXP3EX2 AND THE FUNCTION OF TREGS EXPRESSING THIS ISOFORM.

CONTROL OF CD8+ T CELL MIGRATION AND ACTIVATION BY FLIGHTLESS-1 - PROJECT SUMMARY ADAPTIVE IMMUNE SURVEILLANCE DEPENDS ON THE ABILITY OF T CELLS TO SUCCESSFULLY MIGRATE THROUGH SECONDARY LYMPHOID TISSUES AND FORM IMMUNE-SYNAPSES WITH ANTIGEN-PRESENTING CELLS. PRECISE AND DYNAMIC ORGANIZATION OF THE ACTIN CYTOSKELETON IS ESSENTIAL FOR BOTH OF THESE PROCESSES. IN MIGRATING T CELLS RAPID REORGANIZATION OF THE ACTIN CYTOSKELETON IS ESSENTIAL FOR CELL POLARIZATION AND CHEMOTACTIC RESPONSES REQUIRED FOR TISSUE ENTRY AND PROPER MICRO-ENVIRONMENTAL POSITIONING. DURING T CELL ACTIVATION, CHANGES IN THE ACTIN CYTOSKELETON CONTROL PROPER FORMATION OF THE IMMUNOLOGICAL SYNAPSE, A HIGHLY ORGANIZED CELLULAR STRUCTURE THAT ALLOWS T CELLS TO PROPERLY INTEGRATE SIGNALS FROM THE T CELL RECEPTOR WITH THOSE FROM CO-STIMULATORY MOLECULES SUCH AS CD28 AND INTEGRINS SUCH AS LFA-1 (ALSS2). FLIGHTLESS-1 (FLII) WAS INITIALLY IDENTIFIED IN DROSOPHILA AS AN ACTIN MODIFYING PROTEIN THAT CONTROLS ACTIN MYOFIBRIL STRUCTURE IN THE MUSCLES THAT CONTROL FLIGHT. FLII CONTAINS AN N-TERMINAL LEUCINE-RICH REPEAT (LRR) DOMAIN THAT FACILITATES PROTEIN-PROTEIN INTERACTION AND HAS BEEN IMPLICATED IN CONTROL OF RAS ACTIVATION OF ERK/MAPK SIGNALING, RAC1 ACTIVATION AND PI3K SIGNALING. THE FLII C-TERMINUS ENCODES 6 GELSOLIN-RELATED DOMAINS THAT CAN INTERACT WITH ACTIN AND REGULATE ACTIN FILAMENT ASSEMBLY/DISASSEMBLY. BASED ON ITS UNIQUE DOMAIN STRUCTURE, WE HYPOTHESIZE THAT FLII ACTS AS A KEY REGULATOR OF CD8+ T CELL HOMEOSTASIS AND FUNCTION BY LINKING CHANGES IN THE ACTIN CYTOSKELETON DURING CELL MIGRATION AND ACTIVATION WITH SPATIAL CONTROL OF VARIOUS SIGNALING CASCADES. WE WILL USE STATE-OF-THE-ART CELLULAR AND MOLECULAR TECHNIQUES TO STUDY FLII FUNCTION IN PHYSIOLOGICALLY RELEVANT AND INNOVATIVE MOUSE MODELS. COMPLETION OF THESE STUDIES WILL PROVIDE IMPORTANT NEW INSIGHTS INTO A NOVEL AND COMPLETELY UNCHARACTERIZED SIGNALING HUB THAT REGULATES CD8+ T CELL-MEDIATED IMMUNITY.

T CELL-MEDIATED CONTROL OF DERMAL FIBROBLAST GENE PROGRAMS AND DYSFUNCTION IN SCLERODERMA - PROJECT SUMMARY/ABSTRACT SYSTEMIC SCLEROSIS (SSC), ALSO KNOWN AS SCLERODERMA, IS AN IDIOPATHIC DISEASE OF THE CONNECTIVE TISSUE CHARACTERIZED BY FIBROSIS OF THE SKIN AND UNDERLYING ORGANS. IT HAS THE HIGHEST MORTALITY OF ALL RHEUMATIC CONDITIONS AND NO CURE. T CELLS PREDOMINATE IN LESIONAL SSC SKIN AND PATIENT SERUM IS ENRICHED IN T CELL CYTOKINES. IMMUNOSUPPRESSION THAT LIMITS T CELL FUNCTION CAN DECREASE SYMPTOMS OF FIBROSIS, BUT ALSO INCREASES PATIENT SUSCEPTIBILITY TO INFECTION. AN ARRAY OF FUNCTIONALLY DISTINCT T CELLS ARE IN CLOSE-CONTACT WITH FIBROBLASTS IN HEALTHY SKIN, AND DYSREGULATED T CELL-FIBROBLAST CROSSTALK IS THOUGHT TO BE A MAJOR DRIVER OF DISEASE PATHOLOGY. HOWEVER, WHILE T CELLS ARE IMPLICATED IN SSC PROGRESSION, NO STUDIES HAVE PERFORMED A RIGOROUS MECHANISTIC DISSECTION OF T CELL-DIRECTED CHANGES TO FIBROBLAST GENE PROGRAMS IN HEALTHY AND FIBROTIC SKIN. WE PREVIOUSLY IDENTIFIED A NOVEL POPULATION OF CUTANEOUS CD4+CLA+CD103+ T CELLS IN THE BLOOD AND SKIN THAT CO-PRODUCE IL-22 AND IL-13 IMPLICATING A ROLE IN SKIN HOMEOSTASIS. WE NOW FIND THAT THE FREQUENCY OF THESE AND OTHER CUTANEOUS T CELLS ARE ALTERED IN SSC, THOUGH THEIR FUNCTION IN AFFECTED PATIENT SKIN REMAINS UNEXPLORED. OUR NOVEL PRELIMINARY DATA SHOW THAT DIFFERENT CUTANEOUS T CELL POPULATIONS PROMOTE DISTINCT FIBROBLAST GENE SIGNATURES AND DEMONSTRATE A STRONG CD103+ T CELL-DEPENDENT EFFECT ON A NEWLY IDENTIFIED SSC-ASSOCIATED FIBROBLAST SUBSET. WE THEREFORE HYPOTHESIZE THAT CD103+ T CELL ACTIVITY IS ESSENTIAL FOR FIBROBLAST HOMEOSTASIS AT THE STEADY STATE AND THAT NORMAL T CELL-FIBROBLAST INTERACTIONS ARE DISRUPTED IN SSC, LEADING TO FIBROBLAST DYSFUNCTION. THIS PROPOSAL WILL USE SINGLE- CELL SPATIAL TRANSCRIPTOMICS AND INNOVATIVE ORGANOTYPIC SKIN CULTURE TO FILL CRITICAL KNOWLEDGE GAPS AND OVERCOME EXISTING TECHNICAL HURDLES IN THE FIELD. COMPLETION OF THESE AIMS WILL BUILD A FOUNDATION FOR THE GENERATION OF EXCITING NEW HYPOTHESIS AND WILL ADVANCE A NOVEL ORGANOTYPIC CULTURE MODEL WITH THE POTENTIAL TO RECAPITULATE IN VIVO FIBROBLAST HETEROGENEITY ASSOCIATED WITH DISEASE AND TISSUE DYSFUNCTION.

T CELL EPIGENOMIC DRIVERS OF DISEASE FLARES IN MULTIPLE SCLEROSIS - PROJECT SUMMARY RELAPSING REMITTING MULTIPLE SCLEROSIS (RRMS) IS AN AUTOIMMUNE DISEASE IN WHICH CD4 T CELLS PLAY A CENTRAL ROLE. STUDYING THE EPIGENOME OF CD4 T CELLS IN RRMS THROUGH THE INTEGRATION OF GENOME-WIDE ANALYSIS OF DNA METHYLATION, OPEN CHROMATIN, AND HISTONE MODIFICATIONS WITH GWAS VARIANTS AND RNA SEQUENCING (RNA-SEQ) DATA HAS THE POTENTIAL TO ELUCIDATE THE MOLECULAR PROCESSES THAT POTENTIATE DISEASE ACTIVITY. THE STUDIES PROPOSED IN THIS APPLICATION WILL EXAMINE THE EPIGENOMIC LANDSCAPE OF CD4 T CELLS IN A WELL CHARACTERIZED COHORT OF INDIVIDUALS WITH RRMS, USING LONGITUDINAL SAMPLES TO ALLOW AN ASSESSMENT OF HOW THE EPIGENOME IS ALTERED IN THE CONTEXT OF DISEASE FLARE AND REMISSION. THE CENTRAL HYPOTHESIS IS THAT DIFFERENCES IN THE EPIGENOMIC LANDSCAPE OF CD4 T CELLS DURING FLARE AND REMISSION WILL IDENTIFY FACTORS THAT PROMOTE DISEASE FLARES AND THE RESULTING CHANGES IN T CELL FUNCTION. THE STUDIES IN SPECIFIC AIM 1 WILL DETERMINE HOW THE EPIGENOMIC AND TRANSCRIPTOMIC LANDSCAPES OF T CELLS CHANGE DURING DISEASE FLARE COMPARED TO REMISSION IN A LONGITUDINAL RRMS COHORT. WE WILL PROFILE CHROMATIN ACCESSIBILITY (ATAC-SEQ) AND THE TRANSCRIPTOME (RNA-SEQ) IN TOTAL CD4, MEMORY CD4, TH17 AND REGULATORY T CELLS (TREGS). ATAC-SEQ DATA WILL BE ANALYZED USING AN ANALYTIC PIPELINE INTEGRATING CAPTURE HI- C DATA, TRANSCRIPTION FACTOR FOOT PRINTING AND MS GENETIC RISK. THE RNA-SEQ DATASET WILL THEN BE INTEGRATED TO IDENTIFY GENE EXPRESSION CHANGES RELATED TO EPIGENOMIC ALTERATIONS DURING DISEASE FLARE. FINDINGS IN THE LONGITUDINAL COHORT WILL BE VALIDATED IN AN INDEPENDENT CROSS-SECTIONAL COHORT INCLUDING INDIVIDUALS FLARING AND INDIVIDUALS IN REMISSION. THE STUDIES IN SPECIFIC AIM 2 WILL INVESTIGATE THE IMPACT OF DIFFERENTIAL CHROMATIN ACCESSIBILITY AT THE ZFP36L2 LOCUS ON CD4 T CELL LINEAGE AND FUNCTION. THEY WILL DETERMINE WHETHER THE CHANGES IN CHROMATIN ACCESSIBILITY AT THE ZFP36L2 LOCUS DURING DISEASE FLARE ALTER ZFP36L2 GENE EXPRESSION IN ALL CD4 T CELLS OR ONLY SPECIFIC T CELL SUBSETS USING PRIMEFLOW. CRISPR EDITING TO EITHER OPEN OR CLOSE THE CHROMATIN AT THE ZFP36L2 LOCUS WILL BE USED TO DIRECTLY CONFIRM THE REGULATION OF ZFP36L2 EXPRESSION BY THE ENHANCER. THE EFFECT OF ZFP36L2 CHROMATIN ACCESSIBILITY ON CD4 T CELLS LINEAGE AND FUNCTION WILL ALSO BE EVALUATED USING SAMPLES FROM RRMS SUBJECTS WITH OPEN OR CLOSED CHROMATIN AT THE ZFP36L2 LOCUS. COLLECTIVELY, THESE STUDIES WILL ADVANCE OUR UNDERSTANDING OF HOW CHANGES IN CHROMATIN ACCESSIBILITY IN THE CONTEXT OF DISEASE ACTIVITY CONTRIBUTE TO THE PATHOGENICITY OF T CELLS IN RRMS AND MAY ALSO REVEAL DRIVERS OF DISEASE DEVELOPMENT.

IDENTIFYING AUTOIMMUNE ASSOCIATED GENES IN PATROLLING MONOCYTES THAT PROMOTE LUPUS NEPHRITIS - PROJECT SUMMARY LUPUS NEPHRITIS (LN) IS SEEN IN ~50% OF INDIVIDUALS WITH THE CHRONIC AUTOIMMUNE DISEASE SYSTEMIC LUPUS ERYTHEMATOSUS AND IS A SERIOUS CAUSE OF MORBIDITY IN THIS DISEASE, WITH ~10% OF SLE CASES LEADING TO END STAGE RENAL DISEASE WITHIN 5 YEARS OF DIAGNOSIS. LN OCCURS DISPROPORTIONATELY IN WOMEN OF NON-EUROPEAN ANCESTRY, INCLUDING AFRICAN-AMERICANS, HISPANICS AND ASIANS, AND DESPITE MANY YEARS OF STUDY, THERE ARE FEW TARGETED TREATMENTS. THE RECENT APPROVAL OF THE FIRST TWO SPECIFIC LN THERAPEUTICS, BELIMUMAB AND VOCLOSPORIN TARGETING ADAPTIVE IMMUNE CELLS (T AND B CELLS), WAS AN IMPORTANT MILESTONE. HOWEVER, THESE THERAPEUTICS ONLY HAVE ~40% EFFICACY. THUS, A MAJOR CHALLENGE IN THE FIELD IS TO IDENTIFY THERAPIES THAT TREAT OR PREVENT LN IN ALL PATIENTS. THERE IS GROWING EVIDENCE THAT INNATE IMMUNE CELLS ALSO CONTRIBUTE TO LN. IN PARTICULAR, NON-CLASSICAL, PATROLLING MONOCYTES HAVE RECENTLY BEEN IMPLICATED IN GLOMERULONEPHRITIS, SUGGESTING THAT A NOVEL INNATE IMMUNE MECHANISM THROUGH RECRUITMENT OF PATROLLING MONOCYTES TO THE KIDNEY CONTRIBUTES TO LN, AND THAT INTERFERING WITH ACCUMULATION OF PATROLLING MONOCYTES EARLY IN DISEASE COULD BE THERAPEUTICALLY EFFICACIOUS. THERE ARE NO EXISTING THERAPIES THAT SPECIFICALLY TARGET PATROLLING MONOCYTE ACCUMULATION IN THE KIDNEY, OR OTHER ORGANS, THUS, A BETTER UNDERSTANDING OF THE GENES AND MECHANISMS THAT DRIVE THIS PROCESS HOLDS PROMISE FOR IDENTIFYING NEW THERAPEUTIC TARGETS FOR LN AND IS THE FOCUS OF THIS PROPOSAL. INFORMED BY HUMAN LUPUS GENETIC RISK LOCI, WE WILL DEFINE KEY GENES INVOLVED IN PATROLLING MONOCYTE ACCUMULATION IN THE KIDNEY IN LN. THOUGH MANY GENETIC RISK VARIANTS HAVE BEEN IDENTIFIED AS ASSOCIATED WITH INCREASED RISK FOR SLE, THE FUNCTION OF MOST GENES REGULATED BY THESE VARIANTS HAVE NOT BEEN SYSTEMATICALLY ASSESSED IN INNATE IMMUNE CELLS SUCH AS MONOCYTES. WE HYPOTHESIZE THAT GENES ASSOCIATED WITH GWAS RISK VARIANTS MAY SERVE AS A RICH SOURCE OF REGULATORS OF PATROLLING MONOCYTE ACCUMULATION IN GLOMERULAR CAPILLARIES AND THEREFORE OF LN. TO UNDERSTAND HOW GENETIC RISK LOCI CONTRIBUTE TO PATROLLING MONOCYTE ACCUMULATION IN LN, WE WILL USE AN IN VIVO CRISPR SCREEN USING THE TLR7.1 MOUSE MODEL OF LUPUS- LIKE DISEASE WITH VALIDATION IN ADDITIONAL MOUSE LUPUS MODELS. UPON COMPLETION, WE WILL ILLUMINATE GENES AND PATHWAYS THAT MAY BE TARGETED BY NOVEL THERAPEUTIC INTERVENTIONS, WHICH COULD BE USED PRIOR TO THE ONSET OF KIDNEY NEPHRITIS. ADDITIONALLY, RISK HAPLOTYPES CORRELATED WITH IDENTIFIED GENES COULD HELP PREDICT THE EFFICACY OF SUCH THERAPEUTIC APPROACHES AS WELL AS RISK FOR LN.

MECHANISMS OF IL-2-MEDIATED IMMUNE TOLERANCE - PROJECT SUMMARY FOXP3+ REGULATORY T CELLS (TR) ARE ESSENTIAL FOR ESTABLISHING AND MAINTAINING IMMUNE TOLERANCE, AND MANIPULATING TR ACTIVITY IS AN ATTRACTIVE NEW THERAPEUTIC STRATEGY FOR TREATING AUTOIMMUNE AND INFLAMMATORY DISEASES. THE DEVELOPMENT, HOMEOSTASIS AND FUNCTION OF TR DEPENDS ON THE CYTOKINE IL-2, AND TR CONSTITUTIVELY EXPRESS THE HIGH AFFINITY IL-2 RECEPTOR WHICH ALLOWS THEM TO COMPETE FOR LIMITING IL-2 PRODUCED BY ACTIVATED CD4+ T CELLS. AS A STRATEGY FOR INCREASING TR ABUNDANCE AND FUNCTION TO TREAT AUTOIMMUNE DISEASE, WE HAVE DEVELOPED A NOVEL IL-2 ‘MUTEIN’ THAT IS HIGHLY TR SELECTIVE, POTENTLY EXPANDS TR IN VIVO, AND ARRESTS ONGOING AUTOIMMUNITY AND INDUCES DURABLE DISEASE PROTECTION IN NOD MICE. INTERESTINGLY, WE HAVE SHOWN THAT IN BOTH MICE AND HUMANS, IL-2 MUTIEN TREATMENT IS ASSOCIATED WITH PRONOUNCED EXPANSION OF A SUBSET OF HIGHLY ACTIVATED TR CHARACTERIZED BY EXPRESSION OF ACTIVATION MARKERS ASSOCIATED WITH T CELL RECEPTOR STIMULATION. FURTHERMORE, EXPANDED TR EXPRESS HIGH LEVELS OF THE IMMUNOSUPPRESSIVE MOLECULE CTLA4, AND IL-2 MUTEIN TREATMENT INHIBITS THE ACTIVATION OF DENDRITIC CELLS (DCS) AND THEIR SURFACE EXPRESSION OF THE KEY CO-STIMULATORY LIGANDS CD80 AND CD86 THAT ARE REQUIRED FOR FULL EFFECTOR T CELL ACTIVATION. BASED ON THESE RESULTS, WE HYPOTHESIZE THAT IL-2 MUTEIN TREATMENT PROMOTES TR/DC INTERACTION, AND THAT THIS RESULTS IN SYNERGISTIC IL-2 AND TCR SIGNALING THAT DRIVES THE PROLIFERATION AND EXPANSION OF HIGHLY ACTIVATED TR THAT INHIBIT DC FUNCTION AND PREVENT THE ACTIVATION, DIFFERENTIATION AND FUNCTION OF AUTOREACTIVE T CELLS. IN THIS PROPOSAL WE USE A NUMBER OF INNOVATIVE METHODS TO TEST THIS HYPOTHESIS, AND THESE EXPERIMENTS WILL PROVIDE A COMPREHENSIVE MECHANISTIC UNDERSTANDING OF HOW IL-2 MUTEINS FUNCTION TO PROMOTE TR EXPANSION AND INDUCTION OF IMMUNE TOLERANCE. THIS HAS IMPORTANT IMPLICATIONS FOR THE TRANSLATION OF IL-2 MUTEINS INTO THERAPEUTIC USE, AND WILL PROVIDE IMPORTANT NEW INSIGHTS IN THE BASIC BIOLOGY OF IL-2-MEDIATED CONTROL OF TR HOMEOSTASIS AND FUNCTION.

AUTOREACTIVE CD4 TSCM DEVELOPMENT IN TYPE 1 DIABETES - PROJECT SUMMARY AUTOIMMUNE TYPE 1 DIABETES (T1D) RESULTS FROM T CELL-MEDIATED DESTRUCTION OF PANCREATIC BETA CELLS IN THE ISLET, LEADING TO LIFELONG DEPENDENCE ON EXOGENOUS INSULIN AND RISK OF LIFE-THREATENING GLYCEMIC EVENTS. AS A RESULT, DELETION OR IMMUNE DEVIATION OF ISLET-AUTOREACTIVE T CELLS IS A MAJOR THERAPEUTIC GOAL IN THE PREVENTION AND TREATMENT OF T1D. THE EXISTENCE OF MEMORY T CELLS WITH STEM CELL PROPERTIES (TSCM) RAISES THE POSSIBILITY THAT THEY SERVE AS AN ONGOING RESERVOIR OF AUTOREACTIVE T CELLS IN T1D. HOWEVER, LITTLE IS KNOWN ABOUT THE PRESENCE OF ISLET-SPECIFIC CD4 TSCM CELLS IN T1D. WE HAVE MADE THE NOVEL OBSERVATION THAT THERE IS AN INCREASED FREQUENCY OF TSCM WITHIN THE CD4 T CELL COMPARTMENT IN PERIPHERAL BLOOD FROM T1D PATIENTS COMPARED WITH HEALTHY DONORS (HD). IMPORTANTLY, WE ALSO DETECTED INCREASED ISLET-SPECIFIC CD4 T CELLS WITH A TSCM PHENOTYPE IN THE PERIPHERAL BLOOD OF T1D SUBJECTS RELATIVE TO HD, BOTH IMMEDIATELY AND UP TO 7 YEARS FOLLOWING DIAGNOSIS. TRANSCRIPT ANALYSIS OF ISLET-SPECIFIC CD4 T CELLS SHOWED INCREASED EXPRESSION OF TCR, WNT-SS-CATENIN, AND IL-7 SIGNALING GENE SETS IN CELLS FROM T1D SUBJECTS COMPARED TO HD, PATHWAYS KNOWN TO PLAY A CENTRAL ROLE IN CD4 TSCM DEVELOPMENT. FURTHER STUDY IS REQUIRED TO CONFIRM THAT ISLET-SPECIFIC CD4 TSCM IN T1D HAVE FUNCTIONAL TSCM PROPERTIES AND TO DETERMINE THE MECHANISM LEADING TO INCREASED TSCM CELL FREQUENCY IN T1D. WE HYPOTHESIZE THAT ISLET-SPECIFIC CD4 TSCM IN T1D HAVE THE CAPACITY FOR SELF-RENEWAL AND DIFFERENTIATION INTO CENTRAL AND EFFECTOR MEMORY T CELLS, AND ARE INCREASED IN T1D DUE TO ELEVATED TCR, WNT-SS-CATENIN, AND/OR CYTOKINE SIGNALING. WE WILL ADDRESS OUR HYPOTHESIS IN TWO SPECIFIC AIMS. AIM 1 WILL INVESTIGATE WHETHER ISLET-SPECIFIC CD4 TSCM CELLS IN T1D SUBJECTS HAVE PHENOTYPIC AND FUNCTIONAL PROPERTIES OF A MEMORY STEM CELL POPULATION. WE WILL EVALUATE THE TRANSCRIPT PROFILE, TELOMERE LENGTH, SURVIVAL, AND THE POTENTIAL FOR SELF-RENEWAL AND DIFFERENTIATION OF ISLET-SPECIFIC CD4 TSCM COMPARED TO TOTAL CD4 NAÏVE, TSCM, CENTRAL MEMORY (TCM) AND EFFECTOR MEMORY (TEM) POPULATIONS IN PERIPHERAL BLOOD FROM T1D PATIENTS. THE CHARACTERISTICS OF CD8 TSCM CELLS WILL SERVE AS A REFERENCE. AS A CONTROL FOR DISEASE, WE WILL COMPARE TRANSCRIPT PROFILES AND FUNCTION OF TOTAL CD4 NAÏVE, TSCM, TCM, AND TEM POPULATIONS IN PERIPHERAL BLOOD FROM MATCHED HD. AIM 2 WILL TEST THE HYPOTHESIS THAT SIGNALING PATHWAYS SUPPORTING THE DEVELOPMENT OF CD4 TSCM ARE ELEVATED IN T1D SUBJECTS VERSUS HD, CONTRIBUTING TO INCREASED ISLET-SPECIFIC CD4 TSCM CELLS IN T1D. WE WILL COMPARE TCR, WNT-SS-CATENIN, AND IL-7R PATHWAY EXPRESSION AND SIGNALING IN CD4 NAÏVE T CELLS FROM T1D SUBJECTS AND HD. IN PARALLEL, WE WILL DETERMINE IF THESE PATHWAYS CONTRIBUTE TO SUPERIOR DIFFERENTIATION OR SURVIVAL OF CD4 TSCM CELLS IN T1D SUBJECTS VS. HD IN RESPONSE TO ISLET PEPTIDES OR POLYCLONAL STIMULATION IN THE PRESENCE OR ABSENCE OF WNT INHIBITORS/AGONISTS AND IL-7. THE PROPOSED STUDY IS APPROPRIATE FOR THE R21 MECHANISM BECAUSE IT IS AN EXPLORATORY INVESTIGATION OF A NOVEL AUTOREACTIVE CELL TYPE IN T1D. THE RESULTS WILL OPEN NEW AREAS OF INQUIRY FOR TARGETING AUTOREACTIVITY TO PREVENT AND TREAT T1D.

CHARACTERIZATION OF E. COLI-SPECIFIC T CELLS IN CROHN'S DISEASE - PROJECT SUMMARY/ABSTRACT SEVERAL LINES OF EVIDENCE HAVE IMPLICATED T CELLS IN THE PATHOGENESIS OF CROHN'S DISEASE, AN UNCONTROLLED INFLAMMATORY CONDITION OF THE INTESTINES IN WHICH IMMUNE CELLS OVERREACT TO THE BACTERIA THAT LIVE THERE. PREVIOUS STUDIES OF ANTIGEN-NONSPECIFIC T CELLS IN CROHN'S DISEASE HAVE NOT IDENTIFIED OVERALL DIFFERENCES IN THEM THAT WOULD EXPLAIN THIS OVER REACTIVITY. HOWEVER, WE HAVE RECENTLY IDENTIFIED CD4 T CELLS OF THE IMMUNE SYSTEM THAT CAN REACT TO A PEPTIDE FROM PROTEIN ANTIGEN (OMPC) MADE BY ONE SUCH BACTERIA (E. COLI), TO WHICH ANTIBODIES ARE COMMONLY SEEN ONLY IN PEOPLE WITH CROHN'S DISEASE. WE FOUND THAT THESE OMPC-SPECIFIC CELLS MAKE IL-10 UNLESS THEY COME FROM CROHN'S DISEASE PATIENTS, AND HYPOTHESIZE THAT THIS DEFECT PLAYS A CENTRAL ROLE IN THE INFLAMMATION OF CROHN'S DISEASE. IL-10 IS A CYTOKINE THAT CLEARLY PLAYS A CENTRAL ROLE IN LIMITING INFLAMMATION IN THE INTESTINES, BECAUSE MICE WITHOUT THE IL-10 GENE AND HUMANS BORN WITH A MUTATION IN THE RECEPTOR FOR IL-10 BOTH QUICKLY DEVELOP SEVERE ENTEROCOLITIS, RESEMBLING CROHN'S DISEASE. THE OVERALL GOAL OF THE STUDIES PROPOSED HERE IS TO DETERMINE WHY THESE GUT FLORA ANTIGEN-SPECIFIC T CELLS FAIL TO MAKE IL-10 IN CROHN'S DISEASE, AS A MECHANISM BY WHICH TOLERANCE TO GUT FLORA IS LOST IN THIS CONDITION. THE NOVEL APPROACH IS TO INTEGRATE SINGLE CELL GENE EXPRESSION AND EPIGENETIC ANALYSES IN THESE OMPC-SPECIFIC T CELLS WE CAN ISOLATE WITH MHC-II TETRAMERS. THE HYPOTHESIS WILL BE ADDRESSED IN TWO SPECIFIC AIMS WITH GENOME-WIDE EXPRESSION DIFFERENCES CORRELATED WITH IL-10 EXPRESSION IN AIM 1, AND AN EPIGENETIC BASIS FOR FAILED IL-10 EXPRESSION IN CROHN'S DISEASE TO BE REVEALED IN AIM 2. TOGETHER THESE STUDIES WILL ADVANCE OUR UNDERSTANDING OF ABNORMAL IL-10 REGULATION BY GUT MICROBIAL ANTIGEN-SPECIFIC T CELLS IN CROHN'S DISEASE, AND PROVIDE THE FOUNDATION FOR DETERMINING HOW SUCH A DEFECT CONTRIBUTES TO DISEASE PATHOGENESIS.

DEVELOPMENT AND FUNCTIONS OF TISSUE RESIDENT MEMORY T CELLS DURING EAE - PROJECT SUMMARY MULTIPLE SCLEROSIS (MS) IS AN AUTOIMMUNE DISEASE OF THE CENTRAL NERVOUS SYSTEM (CNS) CHARACTERIZED BY DEMYELINATION, AXONAL LOSS, AND PROGRESSIVE DISABILITY. THE DISEASE CAN FOLLOW A RELAPSING REMITTING OR A MORE CHRONIC COURSE. SIMILARLY, EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE) MODELS ARE CHARACTERIZED BY CNS INFLAMMATION AND DEMYELINATION, AND EACH RECAPITULATE SOME ASPECTS OF MS. ALTHOUGH THERE IS A WEALTH OF KNOWLEDGE REGARDING THE ASSOCIATION OF DIFFERENT CIRCULATING T HELPER (TH) SUBSETS WITH MULTIPLE SCLEROSIS (MS), THERE IS A PAUCITY OF INFORMATION REGARDING THE CHARACTERISTICS AND FUNCTIONS OF TISSUE RESIDENT MEMORY T CELLS (TRM) WHICH HAVE BEEN RECENTLY IDENTIFIED IN THE CENTRAL NERVOUS SYSTEM (CNS) AND THE CEREBROSPINAL FLUID (CSF) OF MS PATIENTS. TO BEGIN TO ADDRESS THIS GAP, WE HAVE USED A NEWLY DEVELOPED MOUSE STRAIN TO IDENTIFY, TRACK, CHARACTERIZE AND ELIMINATE TRMS DURING THE COURSE OF EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE). WE PROPOSE THAT AUTOREACTIVE CNS CD4+ TRM EXPRESS A UNIQUE SET OF MARKERS THAT DISTINGUISH THEM FROM OTHER CIRCULATING CENTRAL MEMORY T CELLS AND PLAY AN IMPORTANT ROLE IN DISEASE PROGRESSION. USING OUR NEWLY DEVELOPED TOOLS, WE WILL CHARACTERIZE THE DISTRIBUTION, KINETIC AND CHARACTERISTICS OF CD4+ TRM CELLS DURING EAE, ESTABLISH WHETHER THEY RECIRCULATE AND PARTICIPATE IN DISEASE PROGRESSION AND RELAPSES DURING EAE. THE COMPLETION OF THIS PROPOSAL WILL HELP US UNDERSTAND HOW MEMORY T CELLS PROMOTE CHRONIC AUTOIMMUNITY AND MAY LEAD TO THE DEVELOPMENT OF NOVEL THERAPIES FOR MS.

FOXP3 ISOFORMS AND IGE-MEDIATED UVB-INDUCED SKIN INFLAMMATION EXPRESSION - PROJECT SUMMARY ULTRAVIOLET B LIGHT (UVB; 280-315NM) IS A COMMON ENVIRONMENTAL TRIGGER THAT CAN INDUCE SKIN INFLAMMATION AND FLARES IN SEVERAL AUTOIMMUNE DISEASES. UVB LIGHT IS AN ESTABLISHED TRIGGER FOR SKIN FLARES IN SLE PATIENTS, AND REGIONAL SPIKES IN ATMOSPHERIC UVB INTENSITY CORRELATE WITH FLARE FREQUENCY AMONG SLE PATIENTS WITHIN THAT GEOGRAPHIC REGION. IN MICE, IGE ANTIBODIES ARE ELEVATED AFTER UVB EXPOSURE, AND PREVIOUS WORK SHOWS THAT SKIN-REACTIVE IGE ALSO PLAYS A KEY ROLE IN REMOVING DAMAGED KERATINOCYTES AFTER CARCINOGEN EXPOSURE. HOWEVER, THE ROLE OF SELF-REACTIVE IGE HAS NOT BEEN STUDIED IN PHOTOSENSITIVITY REACTIONS IN AUTOIMMUNITY. REGULATORY T (TREG) CELLS PLAY A CENTRAL ROLE IN MAINTAINING IMMUNE SYSTEM HOMEOSTASIS AND MODULATING IMMUNE RESPONSES. FOXP3 IS A MASTER REGULATOR OF TREG DEVELOPMENT AND FUNCTION. FOXP3 MUTATIONS IN PATIENTS WITH IPEX (IMMUNODYSREGULATION, POLYENDOCRINOPATHY, ENTEROPATHY, X-LINKED SYNDROME), WHICH RESULT IN A DEFICIENCY IN TREGS, RESULT IN LETHAL AUTOIMMUNITY, SIMILAR TO THE DISEASE OBSERVED IN FOXP3 DEFICIENT MICE. WHILE HIGHLY CONSERVED IN BOTH AMINO ACID SEQUENCE AND GENE STRUCTURE, ONE DIFFERENCE BETWEEN HUMANS AND MICE IS THAT THE HUMAN FOXP3 GENE ENCODES TWO MAJOR ALTERNATIVELY SPLICED ISOFORMS: A FULL-LENGTH VERSION THAT USES ALL 10 EXONS (FOXP3FL, THE ONLY ISOFORM IN MICE) AND A SHORTER ISOFORM LACKING EXON 2 (FOXP3E2). RECENT STUDIES HAVE SHOWN THAT TREGS FROM PATIENTS WITH SOME AUTOIMMUNE DISEASES EXPRESS INCREASED LEVELS OF THE E2 ISOFORM COMPARED TO THOSE FROM HEALTHY DONORS. CONSISTENT WITH THIS FINDING, WE HAVE FOUND THAT TREGS FROM SLE PATIENTS HAVE INCREASED EXPRESSION OF THE FOXP3E2 ISOFORM. TO STUDY THE ROLE OF THE E2 ISOFORM IN TREG FUNCTION WE GENERATED A NEW MOUSE STRAIN WITH FOXP3 EXON 2 DELETION. INTERESTINGLY, WE FOUND THAT FOXP3E2 MICE DEVELOP HALLMARK FEATURES OF SLE, INCLUDING ANTI-DNA AND ANTI-NUCLEAR AUTOANTIBODIES, INCREASED NUMBER AND SIZE OF SPONTANEOUS GERMINAL CENTERS AND KIDNEY DEPOSITION OF ANTIBODY COMPLEXES, BY 4-5 WEEKS OF AGE. IMPORTANTLY, THESE MICE DISPLAY A MARKED INCREASE IN CIRCULATING IGE AND DEVELOP IGE-SPECIFIC AUTOANTIBODIES AGAINST SKIN ANTIGENS, INCLUDING KERATINS 2 AND 14. IN ADDITION, THESE MICE HAVE IGE DEPOSITS IN THE SKIN, WHICH INCREASE DRAMATICALLY FOLLOWING UVB IRRADIATION. OUR CENTRAL HYPOTHESIS IS FOXP3DE2-EXPRESSING TREGS FAIL TO REGULATE GERMINAL CENTER RESPONSES AND PROMOTE IGE AUTOANTIBODIES. TO TEST THIS HYPOTHESIS, WE WILL FIRST DETERMINE THE ROLE OF FOXP3EX2-EXPRESSING TREGS IN GERMINAL CENTER FUNCTION, FOCUSING ON THE RELATIONSHIP BETWEEN TFH AND TFR CELLS. NEXT, WE WILL ASSESS THE ROLE OF AUTOREACTIVE IGE IN UVB-MEDIATED SKIN INFLAMMATION IN THESE MICE. THESE STUDIES WILL PROVIDE INSIGHTS INTO THE ROLE OF FOXP3E2 TREG FUNCTION AND THE DEVELOPMENT AND PROGRESSION OF INFLAMMATORY SKIN DISEASE.

IGA-CONTAINING IMMUNE COMPLEXES IN PLASMACYTOID DENDRITIC CELL ACTIVATION IN SLE

REGULATION OF TFH FUNCTION IN AUTOIMMUNITY BY TSLP - PROJECT SUMMARY GERMINAL CENTERS (GCS) ARE DYNAMIC IMMUNE MICROARCHITECTURES THAT EXPAND IN SECONDARY LYMPHOID ORGANS DURING INFECTION OR IMMUNIZATION. GCS CAN ALSO DEVELOP IN THE ABSENCE OF OVERT IMMUNIZATION OR DETECTABLE ADVENTITIOUS INFECTION (CALLED SPONTANEOUS GCS, SPT-GCS). AS OPPOSED TO INDUCED GCS THAT FORM DURING IMMUNIZATION OR ANTI-PATHOGEN RESPONSES, SPT-GCS DEVELOP IN RESPONSE TO ENDOGENOUS ANTIGENS AND CONTRIBUTE, IN PART, TO CHRONIC AUTOIMMUNITY. SPT-GCS ARE ENLARGED AND MORE FREQUENT IN AUTOIMMUNE-PRONE MICE IN THE ABSENCE OF DETECTABLE PATHOGENS OR OVERT IMMUNE CHALLENGE. SPT-GCS EXHIBIT A LINEAR CORRELATION WITH NUCLEAR-REACTIVE AUTOANTIBODY TITERS IN LUPUS-PRONE MICE AND MANY GC B CELLS ISOLATED FROM SPT-GCS DEVELOPED IN SLE-PRONE MICE ARE AUTOREACTIVE. BOTH TFH CELLS AND CGC B CELLS WITHIN THE SPT-GCS PLAY ESSENTIAL ROLES IN REGULATING HIGH-AFFINITY AUTOANTIBODY PRODUCTION. SPT-GCS HAVE ALSO BEEN DETECTED IN PATIENTS WITH SEVERAL DIFFERENT AUTOIMMUNE DISEASES INCLUDING SYSTEMIC LUPUS ERYTHEMATOSUS (SLE), RHEUMATOID ARTHRITIS (RA), MULTIPLE SCLEROSIS (MS), SJOGREN’S SYNDROME (SS) AND TYPE 1 DIABETES (T1D). FOR EXAMPLE, PEDIATRIC SLE PATIENTS HAVE ELEVATED NUMBERS OF CIRCULATING PRE- GC B CELLS AND ADULT SLE PATIENTS EXHIBIT INCREASED CIRCULATING TFH CELLS. THESE DATA INDICATE THE IMPORTANT ROLES THAT SPT-GCS PLAY IN PATHOGENIC AUTOANTIBODY PRODUCTION. WHILE THE CORRELATION OF SPT-GCS WITH AUTOIMMUNITY IS CLEAR, THE FACTORS THAT CONTROL THEIR DEVELOPMENT REMAIN OBSCURE. WE HAVE FOUND THAT THE CYTOKINE THYMIC STROMAL LYMPHOPOIETIN (TSLP) PLAYS A SIGNIFICANT ROLE IN THE DEVELOPMENT OF SPT-GCS. TSLP- AND TSLPR-DEFICIENT MICE HAVE DRAMATICALLY REDUCED NUMBERS OF SPT- GCS, AND THOSE THAT EXIST APPEAR TO BE VESTIGIAL. CONSISTENT WITH THIS OBSERVATION, WE HAVE ALSO FOUND THAT TSLP SIGNALING IS CRITICAL FOR THE DIFFERENTIATION OF TFH. TAKEN AS A WHOLE, THESE DATA DEMONSTRATE AN IMPORTANT ROLE FOR TSLP IN SPT- AND INDUCED-GC FORMATION AND FUNCTION. WE WILL TEST THIS HYPOTHESIS BY DETERMINING THE ROLE OF TSLP IN TFH DEVELOPMENT AND FUNCTION (AIM 1), AND DETERMINE THE ROLE OF TFH-SPECIFIC TSLP SIGNALING IN AUTOIMMUNE PRONE MICE.

REGULATION OF TLR SIGNALING IN ANTI-COMMENSAL B CELL RESPONSES AND MUCOSAL INFLAMMATION - PROJECT SUMMARY MUCOSAL SURFACES HOUSE NUMEROUS COMMENSAL AND SYMBIOTIC BACTERIA, VIRUSES AND OTHER MICROORGANISMS, WHICH ESTABLISH MUTUALLY BENEFICIAL INTERACTIONS WITH THEIR HOST. THIS MICROBIAL COLONIZATION RELIES ON COMPLEX INTERACTIONS WITH THE INNATE AND ADAPTIVE IMMUNE SYSTEMS, INCLUDING THE GENERATION OF ANTIBODIES AGAINST COMMENSAL BACTERIA ANTIGENS. THERE IS A PRESSING NEED TO UNDERSTAND HOW ADAPTIVE IMMUNE RESPONSES TO COMMENSALS ARE REGULATED, AND HOW FAILURE OF THESE MECHANISMS LEAD TO INFLAMMATION AND IMMUNE PATHOLOGY. RECENT STUDIES HAVE IDENTIFIED A REQUIREMENT FOR TOLL-LIKE RECEPTOR (TLR) SIGNALING IN B CELLS IN GENERATING ANTI- COMMENSAL ANTIBODIES. WE HAVE PREVIOUSLY SHOWN THAT THE CELL SURFACE RECEPTOR INTEGRIN AVSS3 AND COMPONENTS OF THE AUTOPHAGY PATHWAY REGULATE TLR SIGNALING IN B CELLS TO PREVENT OVEREXPANSION OF AUTOREACTIVE CELLS AND AUTOIMMUNITY. IN PRELIMINARY STUDIES, WE HAVE SHOWN THAT B CELL-SPECIFIC AV-KNOCKOUT MICE (AV-CD19 MICE) HAVE INCREASE NUMBERS OF SPONTANEOUS GERMINAL CENTERS IN THE INTESTINE AND ARE MORE SUSCEPTIBLE TO INFLAMMATORY COLITIS. BASED ON THESE DATA, WE HYPOTHESIZE THAT AVSS3 REGULATES B CELL RESPONSES TO COMMENSAL BACTERIA TO MAINTAIN DEFENSE AGAINST INFECTION BUT PREVENT OVERACTIVE INFLAMMATORY RESPONSES. IN THIS APPLICATION WE PROPOSE TO TEST THIS HYPOTHESIS BY: (1) MEASURING ANTI-COMMENSAL ANTIBODIES AND REPERTOIRES IN AV-CD19 AND CONTROL MICE, AND FOLLOWING B CELL RESPONSES AFTER BACTERIAL COLONIZATION; (2) DETERMINING THE MECHANISM OF INCREASED SUSCEPTIBILITY TO DSS COLITIS IN AV-CD19 MICE. THIS RESEARCH IS HIGHLY SIGNIFICANT AS IT WILL PROVIDE IMPORTANT INSIGHTS INTO MECHANISMS OF REGULATION OF ANTI-COMMENSAL ANTIBODY RESPONSES, AND IF SUCCESSFUL, WILL ESTABLISH A NEW PARADIGM LINKING DYSREGULATED TLR SIGNALING IN B CELLS TO SUSCEPTIBILITY TO COLITIS.

SINGLE CELL TRANSCRIPTOME ANALYSIS OF ISLET ANTIGEN REACTIVE MEMORY CD4+ T CELLS IN ESTABLSHED T1D

PRIORITIZING AUTOIMMUNE-ASSOCIATED GENETIC VARIANTS THAT ALTER REGULATORY ELEMENT ACTIVITY IN B CELLS - SUMMARY GENOME-WIDE ASSOCIATION STUDIES HAVE DISCOVERED HUNDREDS OF GENETIC LOCI THROUGHOUT THE GENOME THAT ASSOCIATE WITH AUTOIMMUNE DISEASES. HOWEVER, THE CAUSAL GENETIC VARIANTS THAT DRIVE AUTOIMMUNE DISEASE IN EACH LOCUS ARE FOR THE MOST PART UNKNOWN. IT IS DIFFICULT TO IDENTIFY THE CAUSAL VARIANT(S) IN EACH LOCUS BECAUSE 1) THERE IS OFTEN TIGHT LINKAGE DISEQUILIBRIUM BETWEEN CAUSAL VARIANTS AND TENS TO HUNDREDS OF NON-CAUSAL VARIANTS, MAKING IT DIFFICULT TO DETERMINE WHICH VARIANT IS INDEED CAUSAL, AND 2) BECAUSE MOST GENETIC ASSOCIATIONS OCCUR IN NON-CODING REGIONS, WHERE THEIR ACTIONS ON DISEASE-RELEVANT GENES AND CELL TYPES ARE HARD TO DETERMINE. HOWEVER, VARIANTS ARE ENRICHED IN NON-CODING CANDIDATE CIS-REGULATORY REGIONS THAT MAY MODULATE GENE EXPRESSION OF DISEASE-RELEVANT GENES. AUTOIMMUNE GENETIC VARIANTS ENRICH HIGHLY IN B CELL ACCESSIBLE CHROMATIN, INDICATING THAT VARIANT MODULATION OF CIS-REGULATORY ACTIVITY IN THIS CELL TYPES MAY BE IMPORTANT FOR DISEASE PROGRESSION. ONE WAY TO DETERMINE THE VARIANTS THAT CAN ALTER CIS-REGULATORY ACTIVITY IS THROUGH TESTING THEM IN MASSIVELY PARALLEL REPORTER ASSAYS, WHICH CAN TEST THOUSANDS OF VARIANTS IN ONE ASSAY FOR THEIR ABILITY TO ALTER REPORTER EXPRESSION IN AN ALLELE-SPECIFIC FASHION. THIS APPROACH IS HIGHLY EFFECTIVE, AS WE HAVE FOUND VARIANTS THAT ALTERED REPORTER EXPRESSION ENRICH UP TO 58-FOLD FOR CAUSAL VARIANTS (ACCORDING TO STATISTICAL FINE-MAPPING). IN THIS PROPOSAL, WE WILL APPLY MPRA IN B CELLS TO TEST ~18,000 VARIANTS ASSOCIATED WITH TYPE 1 DIABETES, MULTIPLE SCLEROSIS, INFLAMMATORY BOWEL DISEASE, RHEUMATOID ARTHRITIS, AND PSORIASIS FOR THEIR ABILITY TO ALTER REPORTER EXPRESSION. TO ENSURE B CELL MPRA HITS ARE RELEVANT FOR AUTOIMMUNE DISEASE, WE WILL ASSESS WHETHER THEY ENRICH FOR CAUSAL VARIANTS ACCORDING TO STATISTICAL FINE-MAPPING. WE WILL RANK VARIANTS ACCORDING TO THEIR ALLELIC BIAS EFFECT SIZE AND CHOOSE 3 HIGH EFFECT VARIANTS FOR FURTHER MECHANISTIC FOLLOW-UP. FOR EACH OF THE 3 VARIANTS, WE WILL BASE EDIT VARIANT ALLELES INTO PRIMARY NAÏVE B CELLS FROM HEALTHY DONORS AND OBSERVE EFFECTS ON LOCAL B CELL GENE EXPRESSION (WITHIN 1 MB OF VARIANT), CYTOKINE SECRETION, AND MARKERS OF B CELL DIFFERENTIATION. FOR 1-2 VARIANTS WHERE WE FIND BASE EDITING TO ALTER B CELL PHENOTYPES, WE WILL IDENTIFY HOMOZYGOUS RISK AND NON-RISK ALLELE CARRIERS FOR BOTH HEALTHY SUBJECTS AND SUBJECTS WITH AUTOIMMUNE DISEASE WITHIN THE BENAROYA RESEARCH INSTITUTE BIOREPOSITORY. WE WILL ASSESS WHETHER VARIANTS CORRELATE WITH ALTERED B CELL PHENOTYPES, TESTING WHETHER RISK VS. NON-RISK VARIANT CARRIERS IN HEALTH AND DISEASE HAVE DIFFERENCES IN THE PERCENTAGE OF NAÏVE, MEMORY, PLASMA- BLASTS, AND PLASMA CELL B CELL POPULATIONS, AND CORRESPONDING DIFFERENCES IN GENE EXPRESSION THAT MAY HIGHLIGHT DISEASE-RELEVANT PATHWAYS DOWNSTREAM OF THE VARIANT. UPON COMPLETION OF THIS STUDY, WE WILL HAVE EXHAUSTIVELY TESTED VARIANTS FOR THEIR ABILITY TO ALTER CIS-REGULATORY ACTIVITY IN MPRA IN B CELLS AND FOLLOWED UP ON HIGH EFFECT VARIANTS TO DETERMINE THEIR BIOLOGICAL RELEVANCE TO DISEASE. MECHANISTIC DISSECTION OF THESE PRIORITIZED RISK VARIANTS COULD PROVIDE A WEALTH OF TARGETABLE PATHWAYS FOR WHICH TO DESIGN NEW THERAPEUTICS FOR AUTOIMMUNITY.

MECHANISMS OF SUPPRESSION OF EFFECTOR T CELLS IN EAE

MODULATION OF PATHOGENIC T CELLS IN EAE - PROJECT SUMMARY MULTIPLE SCLEROSIS (MS) IS A DISEASE CHARACTERIZED BY INFLAMMATION, DEMYELINATION, AND NEURODEGENERATION OF THE CENTRAL NERVOUS SYSTEM (CNS). HUMAN GENETIC STUDIES AND ADOPTIVE TRANSFER EXPERIMENTS IN EXPERIMENTAL AUTOIMMUNE ENCEPHALOMYELITIS (EAE), AN ANIMAL MODEL OF MS POINT TO CD4+ T HELPER CELLS AS IMPORTANT ACTORS IN THE DEVELOPMENT AND PROGRESSION OF CNS SPECIFIC AUTOIMMUNITY. IL-2 IS A CYTOKINE CRITICAL FOR MAINTAINING T CELL HOMEOSTASIS, GROWTH, AND PROLIFERATION. IL-2 IS PREDOMINANTLY PRODUCED BY ACTIVATED CD4+ T CELLS IN LYMPHOID ORGANS. HOWEVER, THIS PRODUCTION IS LOST IN MOST CELLS AND ONLY SUSTAINED IN A UNIQUE FRACTION OF CD4+ T CELLS. WE HAVE DEVELOPED A NOVEL TOOL WHICH ALLOW US TO TRACK, ELIMINATE AND STUDY IL-2+ T CELLS DURING THE DEVELOPMENT AND PROGRESSION OF EAE. HERE, WE WILL DETERMINE THE IDENTITY, CHARACTERISTICS AND MEANS TO MODULATE IL-2+ T CELLS IN THE CONTEXT OF EAE DEVELOPMENT AND PROGRESSION AND ESTABLISH WHETHER STAT1 LIMITS IL-2+ T CELLS AND THEIR PATHOGENIC FUNCTIONS. THE COMPLETION OF THESE STUDIES WILL PROVIDE NEW INSIGHTS ON THE FUNCTION OF IL-2+ CELLS DURING PROLONGED DISEASE PROGRESSION, ESTABLISH WHETHER THE ENHANCE STAT1 SIGNALING CAN LIMIT THE PATHOGENIC FUNCTION OF THESE CELLS, AND IDENTIFY MARKERS OF PATHOGENIC IL-2+ T CELLS AND PATHWAYS THAT CAN BE FURTHER STUDIED AND TARGETED TO LIMIT THE ACTIVITY OF THESE CELLS IN THE CONTEXT OF CNS AUTOIMMUNITY.

DCS AS CLINICAL TARGETS OF ANTI-INTEGRIN THERAPY IN IBD - PROJECT SUMMARY/ABSTRACT ANTI-INTEGRIN Α4Β7 THERAPY (VEDOLIZUMAB) HAS DEMONSTRATED OUTSTANDING SAFETY AND EFFICACY FOR THE TREATMENT OF INFLAMMATORY BOWEL DISEASE (IBD), A RELATIVELY COMMON, CHRONIC, INCURABLE PAIR OF IDIOPATHIC IMMUNE-MEDIATED DISEASES OF THE INTESTINES (CROHN’S DISEASE AND ULCERATIVE COLITIS) THAT TYPICALLY STRIKE IN YOUTH TO EARLY ADULTHOOD TO PRODUCE LIFELONG SUFFERING AND DISABILITY. HOWEVER, FOR UNKNOWN REASONS, MANY IBD PATIENTS DO NOT RESPOND TO EVEN THIS THERAPY. WE RECENTLY GENERATED EVIDENCE THAT THIS THERAPY SELECTIVELY AFFECTS THE INTESTINAL MIGRATION OF DENDRITIC CELLS (DC) RATHER THAN T CELLS, AS HAD BEEN WIDELY PRESUMED. WE THEREFORE SEEK TO DEMONSTRATE THAT DIRECTLY OBSERVING THE NATURE OF THESE DC, AND THEIR RESPONSE TO VEDOLIZUMAB, WILL IMPROVE OUR CURRENTLY LIMITED CLINICAL TOOLS FOR PREDICTING AND OPTIMIZING THE EFFICACY OF THIS THERAPY. ADDITIONALLY, BY DEMONSTRATING THAT DC ARE THE ACTUAL TARGET OF THIS PROFOUNDLY SPECIFIC YET EFFECTIVE THERAPY, WE HAVE IMPLICATED THEM DIRECTLY IN THE PATHOGENESIS OF IBD, WHICH TO DATE REMAINS POORLY UNDERSTOOD. WE THEREFORE PROPOSE TO PERFORM A DETAILED ANALYSIS ON DC FROM THE BLOOD AND COLONIC MUCOSA OF IBD PATIENTS TO CORRELATE THEIR PHENOTYPE AND DIVERSITY WITH THE PRESENCE OR ABSENCE OF INFLAMMATION, AND MORE SPECIFICALLY HOW VEDOLIZUMAB INDUCES REMISSION IN IBD. BY DOING SO, WE WILL NOT ONLY IMPROVE OUR EXISTING TOOLS FOR HELPING IBD PATIENTS, BUT ALSO REVEAL FOR NEW STRATEGIES TO TREAT OR EVEN CURE THIS SIGNIFICANT PUBLIC HEALTH PROBLEM.

CHARACTERIZING CIRCULATING AND VISCERAL T CELLS SPECIFIC FOR THE AUTOANTIGEN INTEGRIN ΑVΒ6 IN ULCERATIVE COLITIS - PROJECT SUMMARY/ABSTRACT THE INTEGRIN HETERODIMER ΑVΒ6 EXPRESSED ON INTESTINAL EPITHELIAL CELLS HAS RECENTLY BEEN DISCOVERED AS THE TARGET OF AUTOANTIBODIES UNIQUELY FOUND IN NEARLY EVERY PATIENT WITH ULCERATIVE COLITIS (UC), EVEN YEARS BEFORE DISEASE ONSET. WE PROPOSE TO FIND AND CHARACTERIZE THE T CELLS ULTIMATELY RESPONSIBLE FOR LETTING B CELLS MAKE THESE ANTIBODIES, AND THUS REVEAL A NOVEL INSIGHT INTO HOW IMMUNE SELF-TOLERANCE TO A MUCOSAL AUTOANTIGEN IS LOST IN UC, OR CONVERSELY MAINTAINED IN PEOPLE WITHOUT UC. TO THIS END, WE WILL ADAPT THE ACTIVATION INDUCED MARKER (AIM) ASSAY THAT WE HAVE USED EXTENSIVELY IN THE STUDY OF TYPE 1 DIABETES TO NOW EVALUATE UC. THIS WILL ALLOW US FOR THE FIRST TIME TO EMPLOY THIS ASSAY ON CELLS FROM THE MESENTERIC LYMPH NODES OF HUMANS, WHERE MUCH OF THE T CELL-B CELL INTERACTIONS NECESSARY FOR GUT-CENTRIC ANTIBODY PRODUCTION OCCUR, AND LEVERAGE DATA THUS GENERATED INTO AN ANALYSIS OF THE COLONIC MUCOSA ITSELF. WE ARE ABLE TO DO THIS BECAUSE WE UNIQUELY HAVE A BANK OF CRYOPRESERVED LIVE MLN AND COLON SAMPLES FROM THE SURGICAL RESECTIONS OF DOZENS OF PEOPLE WITH UC AND CROHN'S DISEASE, IN ADDITION TO CRYOPRESERVED LIVE BLOOD CELLS AND SERUM FROM HUNDREDS OF OTHER UC PATIENTS AND HEALTHY CONTROLS. THE AIM ASSAY IS DESIGNED TO INDEPENDENTLY EVALUATE PRO-INFLAMMATORY AND REGULATORY T CELLS. IN ADDITION TO FINDING AND IMMUNOPHENOTYPING AUTOANTIGEN-SPECIFIC T CELLS BY FLOW CYTOMETRY, THE AIM ASSAY WILL ALLOW US TO ISOLATE THESE CELLS FOR SINGLE-CELL ANALYSES OF THEIR GENE EXPRESSION PROFILES AND UNIQUE T CELL RECEPTOR SEQUENCES. THESE IN TURN SERVE AS A UNIQUE IDENTIFIER WITH WHICH TO TRACK AUTOANTIGEN- SPECIFIC T CELLS THROUGHOUT THE BODY, INCLUDING IN THE COLON, WHERE AUTOREACTIVE T CELLS MAY PLAY A DIRECT ROLE IN PROMOTING OR SUPPRESSING PATHOLOGY.

TSLP AND CHILDHOOD ASTHMA

GENERATING TOLERANCE TO ANTIBODY-BASED DRUGS

FUNCTION OF A NOVEL SUBSET OF DENDRITIC CELLS IN EAE

CONTROL OF REGULATORY T CELL DIFFERENTIATION AND FUNCTION

FUNCTION OF THE TREM2 R47H VARIANT ASSOCIATED WITH RISK OF ALZHEIMER'S DISEASE

IMPACT OF THE AUTOIMMUNITY ASSOCIATED PTPN22 1858T

INVESTIGATING THE ROLE OF HEMOPHAGOCYTIC MACROPHAGES IN MALARIA

GENOMICS RESOURCES AND INFRASTUCTURE FOR THE ZEBRAFISH

THE ROLE OF IRON IN THE PATHOGENESIS OF NAFLD

A FOXP3 COMPLEX THAT CONTROLS HUMAN REGULATORY T CELL FUNCTION

IDENTIFICATION OF HOST DRUG DEVELOPMENT TARGETS IN INFLUENZA USING TRANSPOSON MUTAGENESIS

BCAP CONTROL OF LUPUS PATHOGENESIS VIA PLASMACYTOID DC TYPE I IFN PRODUCTION

HOMING AND HOMEOSTASIS OF REGULATORY T CELLS

MECHANISTIC STUDY OF PEANUT-SPECIFIC T CELLS PRE AND POST ORAL IMMUNOTHERAPY

EVOLUTION AND DEVELOPMENT OF VERTEBRATE HOX14 GENES

ZINC TRANSPORTER-8-SPECIFIC T CELL RESPONSES IN THE PREDICTION OF TYPE 1 DIABETES

BCAP REGULATION OF PDC IFNA PRODUCTION IN LUPUS

EXPLORING NOVEL DISEASE MECHANISMS IN RA LINKED TO HLA CLASS II RISK

UNDERSTANDING REGULATORY DEFECTS T1D: NATURAL HISTORY AND IMPACT OF THERAPY

LINKING GENETIC VARIATION IN THE PTPN2 GENE TO AUTOIMMUNE DISEASE SUSCEPTIBILITY

USING B CELL FEATURES TO EXPLAIN HETEROGENEITY IN THE RATE OF T1D PROGRESSION

REGULATION OF CELL FUNCTION BY MATRICELLULAR HEVIN

ISOGENIC MODELING OF IMMUNE-BETA CELL INTERACTIONS, ALTERATIONS IN BETA CELL PHENOTYPE, AND VULNERABILITY TO CYTOTOXIC KILLING - PROJECT SUMMARY/ABSTRACT BECAUSE IT IS UNSAFE TO ACCESS HUMAN PANCREATIC ISLETS FROM LIVING DONORS, SURROGATE EXPERIMENTAL SYSTEMS ARE NEEDED TO ANSWER IMPORTANT QUESTIONS ABOUT THE MECHANISMS THROUGH WHICH INSULIN-PRODUCING Β CELLS ARE DESTROYED IN INDIVIDUALS WHO DEVELOP TYPE 1 DIABETES (T1D). PROTOCOLS FOR DIFFERENTIATING INDUCED PLURIPOTENT STEM CELLS (IPSCS) INTO ISLET-LIKE CLUSTERS (SC-ISLETS) PROVIDE A REPLENISHABLE SOURCE OF BETA CELLS AND ARE A PROMISING ALTERNATIVE MEANS FOR MODELLING INTERACTIONS BETWEEN HUMAN ISLET ENDOCRINE CELLS AND IMMUNE CELLS. HOWEVER, CURRENTLY AVAILABLE BIOMIMETIC SYSTEMS ARE NOT ABLE TO MAINTAIN THE LONG-TERM VIABILITY OF SC-ISLETS AND ARE NOT ISOGENIC AND THEREFORE, UNABLE TO ACCURATELY MODEL AUTOIMMUNE INTERACTIONS. TO MEET THIS NEED, THIS PROJECT WILL DEVELOP A VASCULARIZED 3D BIOMIMETIC MICROPHYSIOLOGICAL SYSTEM (MPS) THAT WILL ALLOW FULLY ISOGENIC MODELLING OF INTERACTIONS BETWEEN ISLETS AND IMMUNE CELLS IN PROLONGED CULTURE. OUR BASIS FOR THIS MODEL SYSTEM IS A PROVEN PERFUSION-CAPABLE MICROFLUIDIC SKIN-ON-CHIP PLATFORM. THIS PLEXIGLASS-BASED CHAMBER SYSTEM HAS AN OPEN WELL ON THE TOP, WHICH IS READILY ADAPTABLE TO CREATE AN IDEAL SYSTEM FOR CULTURING SC-ISLETS. A MICROCHANNEL NETWORK WITHIN THE CHAMBER PROMOTES THE FORMATION OF A VASCULAR NETWORK IN A SUPPORTING MATRIX. THE SYSTEM HAS BEEN DESIGNED WITH INLET AND OUTLET PORTS FOR PERFUSING ENDOTHELIAL CELLS, MEDIUM, CYTOKINES, OR IMMUNE CELLS. FURTHERMORE, THE SYSTEM IS CONFIGURED TO ALLOW LIVE IMAGING AND REMOVAL OF SC-ISLETS AND IMMUNE CELLS FROM THE SYSTEM FOR DOWNSTREAM ANALYSIS. WE PREDICT THAT THIS APPROACH WILL OVERCOME SOME OF THE DESCRIBED LIMITATIONS OF EXISTING SC-ISLET CULTURE SYSTEMS AND WILL ALLOW MECHANISTIC INTERROGATION OF MECHANISMS THAT PROMOTE SUSTAINED AUTOIMMUNITY AND PATHOLOGIC INTERACTIONS BETWEEN SC-ISLETS AND AUTOREACTIVE T CELLS. WE WILL FULLY IMPLEMENT THIS SYSTEM AND DEMONSTRATE ITS SUITABILITY FOR STUDYING INTERACTIONS BETWEEN HUMAN SC-ISLETS AND AUTOREACTIVE T LYMPHOCYTES AND THEN UTILIZE IT TO ASK SPECIFIC QUESTIONS ABOUT THE EFFECTS OF INFLAMMATORY STRESS ON SC-ISLET PHENOTYPE. IMPORTANTLY, OUR EXPERIMENTS WILL UTILIZE T CELL LINES AND T CELL RECEPTOR SEQUENCES OBTAINED FROM PANCREATIC ORGAN DONORS WITH T1D, AS THESE REPRESENT THE MOST RELEVANT T CELLS FOR MECHANISTIC STUDIES. SPECIFICALLY, WE WILL INVESTIGATE THE EFFECTS OF INFLAMMATORY STRESS ON ISLET PHENOTYPE, FUNCTION AND INTERACTIONS WITH AUTOREACTIVE T CELLS, FIRST USING 3D CULTURES (SUITABLE FOR MODELING SHORT-TERM INFLAMMATORY STRESS) AND THEN IN THE ISLET-ON-CHIP SYSTEM (SUITABLE FOR SHORT AND LONG-TERM INFLAMMATORY STRESS). THIS WILL ENABLE US TO TEST THE HYPOTHESIS THAT INFLAMMATORY STRESS ALTERS BETA CELL PHENOTYPE AND DRIVES INCREASED IMMUNE PERCEPTION DURING THE DEVELOPMENT OF T1D. IN ADDITION, WE WILL UTILIZE THE ISLET-ON-CHIP SYSTEM TO INVESTIGATE THE ROLE THAT THE MEMBRANE REPAIR PATHWAY PLAYS IN DICTATING THE VULNERABILITY OF BETA CELLS TO IMMUNE ATTACK. WE ANTICIPATE THAT MODELING INTERACTIONS BETWEEN SC-ISLETS AND CYTOTOXIC T CELLS WILL REVEAL A CRUCIAL ROLE OF THE MEMBRANE REPAIR PATHWAY IN DETERMINING VULNERABILITY TO IMMUNE ATTACK. THESE INSIGHTS ARE LIKELY TO SUGGEST NOVEL PATHWAYS THAT CAN BE LEVERAGED TO TREAT T1D.

LITAF REGULATION OF CELL DEATH AND INFLAMMATORY RESPONSES - PROJECT SUMMARY MEMBRANE DAMAGE BY MECHANICAL OR BIOCHEMICAL STRESS, LEADS TO CELL DEATH AND ACTIVATION OF INNATE IMMUNE INFLAMMATORY PATHWAYS, AND CONTRIBUTES TO THE PATHOLOGY OF MANY INFLAMMATORY CONDITIONS. FURTHERMORE, PATHOGENS CAN SECRETE PORE-FORMING TOXINS (PFT) TO PROMOTE INFECTION AND DISRUPT IMMUNITY, AND ENDOGENOUS PORE-FORMING PROTEINS SUCH AS GASDERMIN D AND MLKL HAVE BEEN SHOWN TO CONTRIBUTE TO INFLAMMATORY SIGNALING AND SECRETION OF CYTOKINES. CELLS HAVE EVOLVED MULTIPLE MECHANISMS TO REPAIR MEMBRANE DAMAGE AND MAINTAIN CELLULAR HOMEOSTASIS, BUT OUR UNDERSTANDING OF HOW DAMAGE IS SENSED AND LINKED TO REPAIR REMAINS INCOMPLETE. WE HAVE RECENTLY DEVELOPED A TRANSPOSON-BASED FORWARD GENETIC SCREENING APPROACH, WHICH WE HAVE USED TO IDENTIFY GENES THAT PROMOTE RESISTANCE TO CELL DEATH INDUCED BY S. AUREUS A-TOXIN. WE IDENTIFIED THE LYSOSOMAL MEMBRANE PROTEIN LITAF AS A CELL-AUTONOMOUS INHIBITOR OF CELL DEATH. IN PRELIMINARY DATA, WE SHOW THAT LITAF PROMOTES SEQUESTRATION OF DAMAGED MEMBRANES INTO VESICLES THROUGH THE ACTIVATION OF THE ESCRT MACHINERY. WE HYPOTHESIZE THAT LITAF ACTS AS AN EFFECTOR OF CELLULAR DEFENSE AGAINST PORE-FORMING PROTEINS, LINKING SENSING OF MEMBRANE DAMAGE TO EFFECTOR MECHANISMS OF REPAIR. IN THIS APPLICATION, WE PROPOSE TO TEST THIS HYPOTHESIS BY: (1) IDENTIFYING THE MECHANISMS OF LITAF ACTIVATION AND FUNCTION; (2) DETERMINING THE ROLE OF THIS PATHWAY IN LUNG INFLAMMATION AND INFECTION; (3) TESTING WHETHER LITAF REGULATES INNATE IMMUNE SIGNALING, INFLAMMASOME ACTIVATION AND INFLAMMATORY CELL DEATH IN MACROPHAGES. THIS RESEARCH IS OF HIGH SIGNIFICANCE AS IT WILL PROVIDE A DEEPER UNDERSTANDING OF CELLULAR DEFENSE MECHANISMS AGAINST MEMBRANE DAMAGE, AND OF THE BALANCE BETWEEN CELL SURVIVAL AND INFLAMMATORY CELL DEATH. IDENTIFYING STRATEGIES TO COUNTERACT MEMBRANE DAMAGE AND PREVENT CELL DEATH WILL CONTRIBUTE TO UNDERSTANDING AND TREATING THE PATHOLOGY OF A WIDE RANGE OF INFECTIOUS AND INFLAMMATORY DISEASES.

EVALUATING THE ROLE OF HIGH-DOSE INFLUENZA VACCINE IN PEOPLE WITH DOWN SYNDROME UNDER AGE 65 - PROJECT SUMMARY/ABSTRACT THE GOAL OF THIS PLANNING GRANT IS TO PREPARE FOR A CLINICAL TRIAL TESTING OUR HYPOTHESIS THAT HIGH-DOSE INFLUENZA VACCINE (FLUZONE) SAFELY IMPROVES VACCINE RESPONSE IN PEOPLE WITH DOWN SYNDROME (DS) < 65 YEARS OLD. OUR TEAM OF CLINICIANS, SCIENTISTS, AND MEMBERS OF THE DS COMMUNITY WILL WORK TOGETHER TO FINALIZE CLINICAL TRIAL DESIGN, COMPLETE START UP ACTIVITIES, AND DESIGN THE MECHANISTIC AND EPIDEMIOLOGICAL STUDIES NEEDED TO LEARN WHETHER AND HOW HIGH-DOSE INFLUENZA VACCINE IS MORE EFFECTIVE THAN STANDARD IMMUNIZATION. THE PLANNED CLINICAL TRIAL ADDRESSES AN URGENT PUBLIC HEALTH NEED; PEOPLE WITH DS ARE AT INCREASED RISK OF DEATH FROM RESPIRATORY INFECTIONS, EXHIBIT ADVANCED IMMUNE AGING, AND SHOW AN IMPAIRED RESPONSE TO INFLUENZA IMMUNIZATION COMPARED TO PEOPLE WITHOUT DS ACROSS ALL AGES. HIGH-DOSE INFLUENZA VACCINE SAFELY IMPROVES RESPONSE IN PEOPLE WITHOUT DS, PROVIDING A STRONG RATIONALE FOR TESTING IN DS. IMPORTANTLY, IT IS ALREADY STANDARD OF CARE IN PEOPLE > 65 YEARS OF AGE. THE PROPOSED RANDOMIZED-CONTROLLED CLINICAL TRIAL WILL COMPARE SAFETY AND EFFICACY OF HIGH-DOSE TO STANDARD- DOSE INFLUENZA VACCINE IN 111 INDIVIDUALS WITH DS. THE PRIMARY ENDPOINT WILL BE VACCINE-SPECIFIC HEMAGGLUTININ INHIBITION RESPONSES IN SERUM AT 2 WEEKS POST-VACCINATION. RE-RANDOMIZATION WITHIN EACH ARM IN THE SECOND YEAR WILL ASSESS REPRODUCIBILITY AND QUANTIFY ANY PRIMING EFFECT OF HIGH DOSE IMMUNIZATION. OUR R34 SPECIFIC AIMS ARE: 1: DEVELOP A PROTOCOL AND COMPLETE STUDY START-UP ACTIVITIES TO PREPARE FOR A RANDOMIZED-CONTROLLED CLINICAL TRIAL OF HIGH- VS. STANDARD-DOSE INFLUENZA IMMUNIZATION IN DS. OUR PARTNERSHIP BETWEEN THE BENAROYA RESEARCH INSTITUTE AND BAYLOR COLLEGE OF MEDICINE BRINGS EXPERTISE IN DS CLINICAL CARE, DS IMMUNOLOGY, CLINICAL TRIAL DESIGN, VACCINE STUDIES, AND EXPERIENCE ENROLLING DIVERSE COHORTS. OUR TEAM INCLUDES EXPERTISE IN INFLUENZA ASSAYS AND EPIDEMIOLOGY, AND IN DEVELOPING COLLABORATIVE RESEARCH ACTIVITIES WITH PEOPLE WITH DS (CO-RESEARCHERS) AND THEIR FAMILIES. OUR TEAM, INCLUDING CO-RESEARCHERS, WILL: FINALIZE PROTOCOL PROCEDURES, EDUCATIONAL MATERIALS, AND CONSENT AND ASSENT DOCUMENTS; OBTAIN REGULATORY APPROVALS; PREPARE CASE REPORT FORMS; AND DEVELOP PLANS FOR DATA AND SAFETY MONITORING, SITE TRAINING, AND STATISTICAL ANALYSIS. 2: FINALIZE MECHANISTIC APPROACHES TO COMPLEMENT THE PRIMARY CLINICAL TRIAL. EMBEDDED STUDIES WITHIN THE RANDOMIZED TRIAL ARE PLANNED TO PROVIDE DEEP MECHANISTIC INSIGHTS INTO MOLECULAR PATHWAYS INVOLVED IN RESPIRATORY INFECTIONS AND RESPONSE TO VACCINATION, INCLUDING A MECHANISTIC PILOT CHALLENGE WITH LIVE ATTENUATED INFLUENZA VIRUS VACCINE (LAIV). WE WILL FINALIZE SAMPLING METHODS, TIMEPOINTS, AND ASSAYS DURING THE R34 PERIOD. 3: CONDUCT DESCRIPTIVE EPIDEMIOLOGY OF FLU VACCINATION AND FLU OUTCOMES AND PLAN OUR SIMULATION STUDY TO COMPLEMENT THE PRIMARY CLINICAL TRIAL. WE HAVE MEDICAID AND MEDICARE CLAIMS DATA FOR ALL ADULTS WITH DOWN SYNDROME IN THE US FROM 2011-2022 (N>130,000). IN THE R34 PHASE WE WILL DESCRIBE DEMOGRAPHIC AND FORMULATION PATTERNS IN FLU VACCINE RECEIPT, AND FLU-RELATED HOSPITALIZATION AND MORTALITY. WE WILL PLAN A SIMULATION STUDY TO MODEL THE IMPACT OF THE HIGH DOSE VACCINE AT THE POPULATION, INCORPORATING U01 TRIAL OUTCOMES.

PRIORITIZING AND CHARACTERIZING T CELL-RELEVANT GENETIC VARIANTS ASSOCIATED WITH AUTOIMMUNE DISEASES - PROJECT SUMMARY GREATER THAN 8% OF THE UNITED STATES POPULATION SUFFERS FROM AUTOIMMUNE DISEASE, BUT, DUE TO COMPLEX NON- MENDELIAN INHERITANCE, THE GENETIC DETERMINANTS OF AUTOIMMUNE DISEASE ARE DIFFICULT TO PARSE. TO BEGIN TO ADDRESS THIS PROBLEM, GENOME-WIDE ASSOCIATION STUDIES (GWAS) HAVE IDENTIFIED THOUSANDS OF GENETIC VARIANTS THAT TRACK WITH DISEASE, ALLOWING THE FIELD OF AUTOIMMUNITY TO FOCUS ON KEY DISEASE-CAUSAL REGIONS OF THE GENOME. HOWEVER, THE EXACT CAUSAL GENETIC VARIANTS FOR MOST OF THESE ASSOCIATIONS REMAIN UNIDENTIFIED, AND THUS THE GENES AND PATHWAYS THEY ALTER REMAIN POORLY UNDERSTOOD. TO TACKLE THIS PROBLEM, I FIRST WILL ENRICH FOR LIKELY CAUSAL VARIANTS FOR DISEASES IN WHICH T CELLS ARE KNOWN TO BE PATHOGENIC, INCLUDING MULTIPLE SCLEROSIS, TYPE I DIABETES, RHEUMATOID ARTHRITIS, PSORIASIS, AND INFLAMMATORY BOWEL DISEASE. I WILL USE A HIGH-THROUGHPUT APPROACH TO TEST 20,000 VARIANTS FOR ALLELIC SKEW IN REPORTER EXPRESSION. FURTHERMORE, FOR 4 HIGHLY IMPORTANT GWAS LOCI (EACH WITH MORE THAN 10 DISEASE ASSOCIATIONS), I WILL SCREEN FOR REGULATORY REGIONS THAT ALTER GENE EXPRESSION AND THE DISEASE-ASSOCIATED VARIANTS THAT LIE WITHIN THESE REGIONS. WITH THESE TWO APPROACHES (AND OTHER GENOMIC DATA, SUCH AS CHROMATIN ACCESSIBILITY AND ALLELE-SPECIFIC TRANSCRIPTION FACTOR CHIP-SEQ), I WILL PRIORITIZE VARIANTS FOR ENGINEERING IN THE GENOMES OF PRIMARY CELLS, AND DETERMINE THE EFFECTS OF THESE ENGINEERED ALLELES ON EXPRESSION, ACTIVATION, AND POLARIZATION OF T CELLS. THIS PROJECT WILL RESULT IN EXHAUSTIVE CHARACTERIZATION OF VARIANTS ASSOCIATED TO 5 IMPORTANT AUTOIMMUNE DISEASES, ELUCIDATION OF THE REGULATORY ARCHITECTURE OF 4 HIGHLY IMPORTANT DISEASE LOCI, AND EXPERIMENTAL VALIDATION OF 10 PUTATIVELY CAUSAL VARIANTS THROUGH EDITING THEM INTO THE GENOME OF PRIMARY T CELLS. THIS WORK WILL PROVIDE AN EXTENSIVE RESOURCE FOR GWAS FOLLOW-UP STUDIES, HELP BRING THE FIELD CLOSER TO UNDERSTANDING THE PATHWAYS AND REGULATORY ARCHITECTURE INVOLVED IN DISEASE, AND INFORM APPROACHES FOR IDENTIFYING NEW TARGETED THERAPEUTICS FOR AUTOIMMUNITY.

CIRCULATING AND MUCOSAL PREDICTORS AND EFFECTS OF THERAPEUTIC INTERLEUKIN-23 BLOCKADE IN CROHN'S DISEASE - PROJECT SUMMARY/ABSTRACT SINCE ITS DISCOVERY 20 YEARS AGO, THE CYTOKINE INTERLEUKIN (IL)-23 HAS INCREASINGLY BEEN IMPLICATED IN THE PATHOGENESIS OF IMMUNE MEDIATED DISEASES, SUCH AS CROHN’S DISEASE (CD). CONSEQUENTLY, FOUR MONOCLONAL ANTIBODIES THAT BLOCK IL-23 ARE CURRENTLY APPROVED CD THERAPIES, INCLUDING RISANKIZUMAB. ALTHOUGH SUPPRESSION OF PATHOGENIC TH17 CELLS HAS BEEN WIDELY CITED AS THE MECHANISM BY WHICH IL-23 BLOCKADE CONTROLS DISEASE, THERE IS A PAUCITY OF DATA TO INDICATE THAT THIS IS HOW SUCH THERAPY WORKS, AND A FEW OTHER IMMUNE CELL POPULATIONS EXPRESSING THE IL-23 RECEPTOR COULD INSTEAD BE ITS TARGET. WE THEREFORE PROPOSE TO STUDY HOW RISANKIZUMAB AFFECTS NOT ONLY TH17 CELLS, BUT ALSO MUCOSA-ASSOCIATE INVARIANT T (MAIT) CELLS ΓΔ T CELLS AND (IN THE COLON) TYPE 3 INNATE LYMPHOID CELLS (ILC3S). IN ADDITION TO QUANTIFYING THESE CELLS, WE WILL STUDY THEIR GENE EXPRESSION TO DETECT PHENOTYPIC DIFFERENCES IN TREATED PATIENTS, AND IN THE CASE OF T CELLS, TRACK THEIR CLONAL EXPANSION AND DELETION THROUGH THEIR UNIQUE T CELL RECEPTOR SEQUENCES. IN COLON SAMPLES, WE WILL USE A COMBINATION OF SINGLE CELL SEQUENCING OF SORT-ENRICHED IMMUNE CELL POPULATIONS AND SPATIAL TRANSCRIPTOMICS TO CHARACTERIZE CELLS IN SITU, AT THE SITE OF DISEASE, AND DETERMINE HOW IL-23 BLOCKADE AFFECTS THEIR MICROENVIRONMENT IN VIVO. BY CONTRASTING RESULTS IN PATIENTS WHO DO OR DO NOT RESPOND THERAPEUTICALLY TO IL-23 BLOCKADE, WE WILL REVEAL VALUABLE INSIGHTS INTO HOW THIS TREATMENT SUCCEEDS OR FAILS IN CD, IN THE PROCESS IDENTIFYING PREDICTIVE BIOMARKERS TO GUIDE TREATMENT DECISIONS, AND POTENTIALLY IDENTIFYING FUTURE MOLECULAR TARGETS WITH WHICH TO PREVENT TREATMENT FAILURE.

SERUM BIOMARKERS ASSOCIATED WITH PHENOTYPIC EXPRESSION OF HEMOCHROMATOSIS.

DEVELOPMENTAL AND GENOMIC STUDIES ON THE AGNATHA VLR SYSTEM

REGULATION OF GERMINAL CENTERS BY THYMIC STROMAL LYMPHOPOIETIN IN MICE AND HUMANS

INFLAMMATION-ASSOCIATED ANEMIA AND THE ROLE OF MONOCYTE-DERIVED INFLAMMATORY HEMOPHAGOCYTES IN A MODEL OF BLOOD-STAGE MALARIA

ALPHA V INTEGRIN REGULATION OF B CELL TOLERANCE - PROJECT SUMMARY THERE IS A FUNDAMENTAL NEED TO UNDERSTAND HOW THE IMMUNE SYSTEM DISCRIMINATES SELF-ANTIGENS AND REGULATES POTENTIALLY HARMFUL AUTOIMMUNE RESPONSES. INCREASING EVIDENCE FROM GENETICS AND FUNCTIONAL STUDIES INDICATE THAT INNATE IMMUNE SIGNALING IN RESPONSE TO PRODUCTS CAUSED BY CELL DEATH IS A MAJOR ETIOLOGICAL FACTOR IN AUTOIMMUNE AND CHRONIC INFLAMMATORY DISEASE. OUR LONG-TERM GOAL IS TO BETTER UNDERSTAND HOW IMMUNE TOLERANCE TO ANTIGENS DERIVED FROM APOPTOTIC CELLS AND OTHER SELF-ANTIGENS IS MAINTAINED AND HOW THIS CAN BREAK DOWN IN AUTOIMMUNE DISEASES. THE OBJECTIVE IN THIS APPLICATION IS TO UNDERSTAND HOW AV INTEGRINS AND COMPONENTS OF THE AUTOPHAGY PATHWAY CONTRIBUTE TO B CELL TOLERANCE. OUR CENTRAL HYPOTHESIS IS THAT AV- MEDIATED ACTIVATION OF AUTOPHAGY COMPONENTS REGULATES TLR SIGNALING IN B CELLS, LIMITING CELL PROLIFERATION, DIFFERENTIATION AND PRODUCTION OF AUTOANTIBODIES. THE RATIONALE FOR THIS GRANT IS THAT THE PROPOSED WORK WILL DEFINE THE ROLE OF AV-MEDIATED AUTOPHAGY IN B CELL TOLERANCE, AND DEVELOP BETTER MODELS FOR AUTOIMMUNE DISEASE. FURTHERMORE, THIS WORK HAS THE POTENTIAL TO TRANSLATE INTO NEW THERAPEUTIC APPROACHES THROUGH THE USE OF EXISTING COMPOUNDS THAT TARGET AV AND AUTOPHAGY. BASED ON STRONG PRELIMINARY DATA, OUR HYPOTHESIS WILL BE TESTED IN THREE SPECIFIC AIMS: (1) DETERMINE HOW AV REGULATES DEVELOPMENT OF AUTOREACTIVE B CELLS AND LUPUS- LIKE AUTOIMMUNITY IN MICE. AV-KNOCKOUT MICE CROSSED WITH AN AUTOREACTIVE BCR HEAVY CHAIN TRANSGENIC MOUSE STRAIN WILL BE USED TO FOLLOW DEVELOPMENT OF AUTOREACTIVE B CELLS, AND UNDERSTAND HOW AV REGULATES TOLERANCE AND RESPONSE TO SELF-ANTIGEN. (2) DETERMINE HOW AV-MEDIATED REGULATION OF TYPE I-IFNS REGULATES B CELL TOLERANCE. WE WILL TEST HOW INCREASED TYPE I IFN PRODUCTION BY B CELLS AND PLASMACYOTID DCS CONTRIBUTE TO B CELL ACTIVATION. (3) TEST THE HYPOTHESIS THAT NON-CANONICAL AUTOPHAGY REGULATES B CELL TLR SIGNALING TO PROMOTE TOLERANCE. WE WILL ANALYZE THE ROLE OF RUBICON IN AUTOREACTIVE B CELL ACTIVATION, AND EVALUATE A POTENTIAL NEW COMPONENT OF NON-CANONICAL AUTOPHAGY, ATG16L2 OUR APPROACH IS INNOVATIVE AS IT FOCUSES ON A NOVEL ROLE FOR INTEGRINS AND AUTOPHAGY COMPONENTS IN REGULATING IMMUNE SIGNALING IN B CELLS. THE PROPOSED WORK IS SIGNIFICANT BECAUSE IT WILL ESTABLISH A NEW PARADIGM FOR B CELL RECOGNITION OF POTENTIAL SELF-ANTIGENS IN IMMUNE TOLERANCE AND PROVIDE A MECHANISM BY WHICH THIS OCCURS. ULTIMATELY THIS HAS THE POTENTIAL TO CHANGE OUR UNDERSTANDING OF HOW IMMUNE TOLERANCE IS MAINTAINED AND HOW AUTOREACTIVE B CELLS MAY ESCAPE CONTROL TO PROMOTE AUTOIMMUNITY.

MHC PEPTIDE TETRAMER ASSAY VALIDATION FOR CD4+ T CELLS IN MS

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

BUILDING TOOLS TO STUDY COMMENSAL-SPECIFIC CD8+ T CELLS IN THE SMALL INTESTINE - PROJECT SUMMARY OUR UNDERSTANDING OF IMMUNITY LARGELY STEMS FROM MODELS OF INFECTION WITH PATHOGENIC MICROBES. HOWEVER, THE VAST MAJORITY OF MICROBIAL-IMMUNE ENCOUNTERS OCCUR AS A SYMBIOTIC RELATIONSHIP WITH THE COMMENSAL MICROBIOTA. RECENTLY, THE CONTRIBUTION OF COMMENSAL-SPECIFIC T CELLS TO HOST PHYSIOLOGY HAS RECEIVED SIGNIFICANT ATTENTION. THESE COMMENSAL-SPECIFIC RESPONSES NOT ONLY CONTROL MICROBIOTA CONTAINMENT BUT ALSO PROMOTE MICROBIAL DIVERSITY WITHIN THE GASTROINTESTINAL TRACT. CONVERSELY, ABERRANT IMMUNITY TO COMMENSAL MICROBES HAS BEEN PROPOSED TO UNDERLIE PATHOLOGIES OF BARRIER TISSUES, INCLUDING INFLAMMATORY BOWEL DISEASE. A BETTER UNDERSTANDING OF THE PROPERTIES AND FUNCTIONS OF COMMENSAL-SPECIFIC T CELL RESPONSES IS THEREFORE FUNDAMENTAL TO STUDIES OF TISSUE IMMUNITY IN HEALTH AND DISEASE. OUR LONG TERM GOAL IS TO BETTER UNDERSTAND HOW COMMENSAL-SPECIFIC T CELL RESPONSES CONTRIBUTE TO BARRIER TISSUE HOMEOSTASIS, AND THE OBJECTIVE IN THIS APPLICATION IS TO DEVELOP NOVEL TRANSGENIC MOUSE MODELS TO ENABLE INVESTIGATION OF THE MECHANISMS REGULATING INDUCTION AND MAINTENANCE OF COMMENSAL-SPECIFIC PLASMA CELL RESPONSES IN THE GUT. OUR RATIONALE FOR THE PROPOSED WORK IS THAT UNCOVERING THESE MECHANISMS HAS THE POTENTIAL TO TRANSLATE INTO NEW THERAPEUTIC APPROACHES. OUR CENTRAL HYPOTHESIS IS THAT COMMENSAL-SPECIFIC T CELLS ARE SENTINELS OF THE GUT TISSUE AND CONTRIBUTE TO INTESTINAL HOMEOSTASIS BY LIMITING TRANSLOCATION OF COMMENSAL MICROBES. IN THIS PROPOSAL, WE WILL DEVELOP TWO TECHNICALLY INNOVATIVE APPROACHES TO GENERATE COMMENSAL-SPECIFIC CD8+ T CELL RECEPTOR TRANSGENIC MICE, AND IDENTIFY THE MICROBIAL ANTIGENS THEY RECOGNIZE. BASED ON STRONG PRELIMINARY DATA, WE WILL CONDUCT TWO SPECIFIC AIMS: (1) DEVELOP SEGMENTED FILAMENTOUS BACTERIA-SPECIFIC CD8+ T CELL RECEPTOR TRANSGENIC MICE AND (2) IDENTIFY THE ANTIGENS RECOGNIZED BY SEGMENTED FILAMENTOUS BACTERIA-SPECIFIC CD8+ T CELLS. OUR APPROACH IS INNOVATIVE AS IT INVESTIGATES NEW MECHANISMS OF IMMUNITY UNIQUE TO COMMENSAL-SPECIFIC T CELL RESPONSES. THE PROPOSED WORK IS SIGNIFICANT BECAUSE IT WILL ESTABLISH NEW INSIGHTS INTO THE INTERACTION AND COMMUNICATION BETWEEN COMMENSAL MICROBES AND IMMUNE CELLS IN THE GUT ENVIRONMENT AND IDENTIFY POTENTIAL TARGETS FOR THERAPEUTIC INTERVENTION IN CONDITIONS OF CHRONIC INTESTINAL INFLAMMATION.

EPIGENETIC CONTROL OF THE T-BET TRANSCRIPTOME BY FOXP3 IN TREGS EXPRESSING T-BET

NOVEL INSIGHT INTO STABILITY AND CHANGE IN A BASAL VETEBRATE GENOME

REQUIREMENT OF HIF2A SIGNALING IN TH2 CELL DIFFERENTIATION AND ALLERGIC ASTHMA - PROJECT SUMMARY ASTHMA AFFECTS OVER 300 MILLION PEOPLE GLOBALLY, YET THERE A FEW THERAPIES AVAILABLE FOR PATIENTS WITH SEVERE ASTHMA. TH2 CELLS AND THEIR CYTOKINES, PRIMARILY IL-4 AND IL-13, ARE RESPONSIBLE FOR AIRWAY INFLAMMATION AND HYPERRESPONSIVENESS IN ALLERGIC ASTHMA. LYMPH NODE TH2 CELLS PRIMARILY EXPRESS IL-4 WHILE TISSUE TH2 CELLS EXPRESS IL-13. HOWEVER, IT IS CURRENTLY UNCLEAR HOW TH2 CELLS SENSE AND RAPIDLY ADAPT TO THE INFLAMMATORY TISSUE MILIEU, ALTERING THEIR CYTOKINE EXPRESSION AND PROMOTING LUNG TISSUE PATHOLOGY. HYPOXIA INDUCIBLE FACTOR EXPRESSION ASSOCIATED WITH INCREASED INFLAMMATION IN ALLERGIC ASTHMA AND MAY REPRESENT A KEY TISSUE-DERIVED SIGNAL CAPABLE OF LICENSING TH2 CELLS. SUPPORTING THIS, DURING INTESTINAL HELMINTH INFECTION, WE IDENTIFIED SELECTIVE TRANSCRIPTIONAL UPREGULATION OF THE HYPOXIA INDUCIBLE FACTOR (HIF)2Α-ENCODING GENE EPAS1 IN INTESTINAL TH2 CELLS. IN ADDITION, EXPERIMENTAL EXPOSURE TO HYPOXIC CONDITIONS, OR SELECTIVE HIF2Α ACTIVATION IN CD4+ T CELLS PROMOTED TH2 CELL DIFFERENTIATION BOTH IN VITRO AND IN VIVO. AS SUCH, WE HYPOTHESIZE THAT HIF2Α IS REQUIRED FOR TH2 CELL TERMINAL DIFFERENTIATION AND PROMOTION OF INFLAMMATION IN ALLERGIC ASTHMA DISEASE. THE REQUIREMENT OF HIF2Α IN TH2 CELL TERMINAL DIFFERENTIATION WILL BE DETERMINED BY DELETING HIF2Α IN TH2 CELLS DURING HOUSE DUST MITE-INDUCED (HDM) AIRWAY INFLAMMATION IN VIVO AND IN DIFFERENTIATING TH2 CELLS IN VITRO. THE MECHANISM OF HIF2Α-INDUCED TH2 DIFFERENTIATION WILL BE MEASURED THROUGH EVALUATION OF HIF2Α-MEDIATED CHANGES IN GENE EXPRESSION, EPIGENETIC REMODELING, AND HIF2Α GENOMIC BINDING SITES IN TH2 CELLS. THE REQUIREMENT OF HIF2Α IN THE DEVELOPMENT OF ALLERGIC ASTHMA DISEASE WILL BE TESTED BY DELETING HIF2Α IN T CELLS AFTER SENSITIZATION OR CHALLENGE WITH HDM AND MEASURING THE DEVELOPMENT OF PATHOLOGY AND AIRWAY HYPERRESPONSIVENESS. FINALLY, HIF2Α WILL BE EVALUATED AS A THERAPEUTIC TARGET IN TREATING ALLERGIC ASTHMA THROUGH TREATMENT OF MICE WITH A HIF2Α INHIBITOR DURING HDM SENSITIZATION OR CHALLENGE. THESE STUDIES WILL UNCOVER THE ROLE OF HIF2Α IN TH2 CELL DIFFERENTIATION AND THE DEVELOPMENT OF ALLERGIC ASTHMA. OUR FINDINGS WILL ADVANCE OUR UNDERSTANDING OF THE SIGNALS SHAPING TH2 CELL RESPONSES IN INFLAMED TISSUES AND IDENTIFY A NOVEL THERAPEUTIC TARGET FOR SUPPRESSING TH2 CELL RESPONSES IN ALLERGIC ASTHMA AND TH2 CELL-INDUCED AIRWAY INFLAMMATION.

IDENTIFICATION OF NOVEL MYELIN-ASSOCIATED CD4+ T CELL AUTOANTIGENS TARGETED IN MS USING A HIGH-THROUGHPUT GENE SYNTHESIS