Project Grant F32AI202776

INVESTIGATING THE MECHANISMS OF KLRG1-MEDIATED IMMUNE SUPPRESSION IN REGULATORY T CELLS - PROJECT SUMMARY DESPITE MAJOR ADVANCES IN CANCER IMMUNOTHERAPY, IMMUNE EVASION REMAINS A KEY BARRIER TO DURABLE RESPONSES. OUR LABORATORY AND OTHERS PREVIOUSLY IDENTIFIED THE INHIBITORY RECEPTOR KLRG1 AS A CRITICAL IMMUNE INHIBITORY CHECKPOINT THAT BINDS E-CADHERIN, AN EPITHELIAL MARKER COMMONLY RETAINED IN MANY AGGRESSIVE TUMORS THAT IS KNOWN TO CORRELATE WITH POOR OVERALL SURVIVAL IN CANCER PATIENTS. THOUGH THIS INTERACTION HAS BEEN CHARACTERIZED AT THE BIOCHEMICAL LEVEL, THE IN VIVO DYNAMICS OF LYMPHOCYTE KLRG1 SIGNALING IN THE CONTEXT OF E-CADHERIN+ MALIGNANCIES REMAIN POORLY UNDERSTOOD. PRELIMINARY EVIDENCE FROM OUR GROUP SHOWS THAT MELANOMA-INFILTRATING CD4+ FOXP3+ REGULATORY T CELLS (TREGS) STRONGLY UPREGULATE KLRG1 IN BOTH MICE AND HUMANS. TO DISSECT THE ROLE OF KLRG1 IN THE ANTI-TUMOR IMMUNE RESPONSE IN THIS POPULATION, OUR LABORATORY GENERATED A NOVEL KLRG1FL/FL MOUSE THAT ENABLES TREG-SPECIFIC KLRG1 DELETION WHEN CROSSED TO A STRAIN EXPRESSING A FOXP3-DRIVEN CRE RECOMBINASE. OUR INITIAL EXPERIMENTS REVEAL THAT DELETION OF KLRG1 FROM TREGS RESULTS IN SIGNIFICANTLY ENHANCED CONTROL OF E-CADHERIN+ B16 MELANOMA, SUGGESTING THAT KLRG1-E-CADHERIN INTERACTIONS DRIVE TREGS TOWARDS A SUPPRESSIVE, PRO-TUMOR STATE. FURTHERMORE, TREGS FROM CONDITIONAL KNOCKOUT (CKO) MICE DOWNREGULATE EXPRESSION OF KEY SUPPRESSIVE MARKERS SUCH AS ST2, CD120B, AND CD45RB, HIGHLIGHTING THE BROAD EFFECTS THAT KLRG1 SIGNALING HAS WITHIN THIS CELLULAR COMPARTMENT. IN THIS PROPOSAL, I WILL USE A COMBINATION OF IN VITRO, IN VIVO, AND IN SITU APPROACHES TO DISSECT THE ROLE OF KLRG1 SIGNALING IN TUMOR-INFILTRATING TREGS. IN AIM 1, I WILL PERFORM DIRECT KLRG1 STIMULATION ASSAYS PAIRED WITH IN VITRO SUPPRESSION EXPERIMENTS AND SINGLE-CELL CYTOKINE PROFILING TO REVEAL HOW KLRG1 SIGNALING MODULATES TREG EFFECTOR FUNCTIONS AND CYTOKINE SECRETION PROFILES. THIS WILL CLARIFY THE ROLE OF KLRG1 IN REGULATING TREG SUPPRESSIVE CAPACITY. IN AIM 2, I WILL USE MIXED BONE MARROW CHIMERAS USING KLRG1 CKO AND WT HEMATOPOIETIC CELLS TO DETERMINE WHETHER KLRG1 PROMOTES TREG POPULATION STABILITY AND TO TEST WHETHER THE PHENOTYPES OBSERVED IN TREG-SPECIFIC KLRG1 KO MICE ARISE VIA CELL-INTRINSIC PATHWAYS. LAST, IN AIM 3, I WILL USE SINGLE-CELL SPATIAL TRANSCRIPTOMICS USING THE 10X GENOMICS XENIUM PLATFORM TO MAP THE SPATIAL DISTRIBUTION AND MOLECULAR PHENOTYPES OF TREG SUBPOPULATIONS WITHIN TUMORS OF KLRG1 CKO AND WT MICE. I HYPOTHESIZE THAT LOSS OF KLRG1 SHIFTS THE BALANCE TOWARD LESS SUPPRESSIVE TREG SUBSETS AND PROMOTES INCREASED INFILTRATION AND ACTIVITY OF EFFECTOR LYMPHOCYTES, THEREBY ENHANCING ANTI-TUMOR IMMUNITY. IN SUMMARY, I AM CONFIDENT THAT THE COMBINATION OF CUTTING-EDGE RESEARCH INFRASTRUCTURE, ACADEMIC COLLABORATION, AND STRUCTURED CAREER DEVELOPMENT OPPORTUNITIES AT BROWN UNIVERSITY WILL PROVIDE ME WITH EXCEPTIONAL TRAINING AS I PREPARE FOR A CAREER AS A PRINCIPAL INVESTIGATOR. MY TRAINING PLAN WILL INCLUDE MULTIPLE INTERNATIONAL CONFERENCES PER YEAR, FREQUENT JOURNAL CLUBS AND SEMINARS, REGULAR MEETINGS WITH MY SPONSOR AND CO-SPONSOR, MENTORSHIP OF JUNIOR SCIENTISTS, AND BIOINFORMATICS COURSEWORK, WHICH WILL ALL BE KEY ACTIVITIES FOR MEETING MY FELLOWSHIP GOALS.