Project Grant R21AI183511

CLONAL ANALYSIS OF NATURALLY OCCURRING IL-10-PRODUCING TR1 CELLS - ABSTRACT THE DEVELOPMENT OF EFFECTIVE THERAPIES FOR THE TREATMENT OF T CELL-DEPENDENT AUTOIMMUNE DISEASES REQUIRES A LUCID UNDERSTANDING OF THE MECHANISMS BY WHICH T CELL TOLERANCE IS ESTABLISHED AND CONFERRED. IN MICE AND HUMANS, THE T CELL REPERTOIRE HARBORS SELF-REACTIVE CONVENTIONAL T CELLS WITH PATHOGENIC POTENTIAL, NECESSITATING EXTRINSIC REGULATION BY FOXP3-EXPRESSING REGULATORY T CELLS AND OTHER MECHANISMS. IN THIS REGARD, RESEARCH OVER THE PAST THREE DECADES HAS IDENTIFIED A UNIQUE CD4+ REGULATORY T CELL POPULATION, TERMED "TR1" CELLS, THAT IS CHARACTERIZED BY LACK OF FOXP3 EXPRESSION, PRODUCTION OF THE SUPPRESSIVE CYTOKINE IL-10, AND POTENT REGULATORY ACTIVITY CAPABLE OF PREVENTING OR STIFLING AUTOIMMUNE REACTIONS IN MURINE MODELS. IT HAS BEEN PROPOSED THAT TR1 CELLS SERVE AS A "FAILSAFE" IN SCENARIOS IN WHICH TOLERANCE MEDIATED BY FOXP3+ TREG CELLS IS INCOMPLETE. IMPORTANTLY, NUMEROUS STUDIES HAVE DEMONSTRATED THAT SUPPRESSIVE, ANTIGEN-SPECIFIC TR1-LIKE CELLS CAN BE GENERATED IN VITRO AND IN VIVO, GALVANIZING CONSIDERABLE INTEREST IN HARNESSING TR1 CELLS FOR THE CLINICAL TREATMENT OF HUMAN AUTOIMMUNE DISEASE. DESPITE THIS GREAT INTEREST, THERE REMAIN FUNDAMENTAL ASPECTS OF TR1 BIOLOGY THAT HAVE EVADED ELUCIDATION. IN THIS STUDY, WE WILL PERFORM CLONAL ANALYSIS OF NATURALLY OCCURRING TR1 (NTR1) CELLS IN MICE. WE WILL UTILIZE A TWO-TIERED STRATEGY PAIRING TCR REPERTOIRE PROFILING OF TR1-PHENOTYPE CELLS WITH CLONAL ANALYSES OF TCR "RETROGENIC" T CELLS EXPRESSING TR1-BIASED TCRS. THE OBJECTIVES OF THIS PROPOSAL ARE TO DEFINE THE NATURE OF THE TCR REPERTOIRE EXPRESSED BY NTR1 CELLS AND DEFINE THE DIFFERENTIATION TRAJECTORY, REACTIVITY, AND TRANSCRIPTIONAL PROFILE OF THESE CLONES IN VIVO AND IN VITRO. WE WILL TEST THE HYPOTHESIS THAT NTR1 CELL DIFFERENTIATION IS INITIATED IN THE THYMUS FOLLOWING THE RECOGNITION OF WIDESPREAD SELF-ANTIGENS, LEADING TO THE ESTABLISHMENT OF A STABLE PERIPHERAL POOL OF NTR1 CELLS EXPRESSING A DISTINCT TCR REPERTOIRE. IN AIM 1, WE WILL DEFINE THE TCR REPERTOIRE EXPRESSED BY NATURALLY OCCURRING TR1 CELLS. THIS WILL PROVIDE KEY INSIGHTS REGARDING THE DIVERSITY AND NATURE OF ANTIGENS THAT DIRECT NTR1 DIFFERENTIATION, THE STABILITY OF NTR1 CELLS, AND THE CLONAL RELATIONSHIP BETWEEN NTR1 CELLS AND OTHER T CELLS SUBSETS. IN AIM 2 WE WILL DETERMINE WHETHER NTR1 CELLS ARE REACTIVE TO WIDESPREAD SELF-ANTIGENS, REGIONAL SELF-ANTIGENS, OR ANTIGENS DERIVED FROM COMMENSAL MICROBIOTA, AND WILL DEFINE WHETHER NTR1 CELL DIFFERENTIATION IS TRIGGERED IN THE THYMUS OR PERIPHERY. IN ADDITION, WE WILL USE RNA SEQUENCING TO DEFINE UNIQUE SIGNATURES THAT DISTINGUISH NTR1 CELLS FROM OTHER REGULATORY AND CONVENTIONAL T CELL SUBSETS. OUR STUDIES WILL YIELD THE FIRST CLONAL ANALYSES OF NATURALLY OCCURRING IL-10-PRODUCING TR1 CELLS - AN IMPORTANT REGULATORY T CELL POPULATION FOR WHICH FUNDAMENTAL ASPECTS OF THEIR ANTIGEN SPECIFICITY, DIFFERENTIATION, LINEAGE STABILITY, AND FUNCTION REMAIN UNDEFINED. ULTIMATELY, THESE ADVANCES WILL BE CRUCIAL FOR THE DEVELOPMENT OF NEW STRATEGIES TO MOBILIZE NTR1 CELLS FOR THE TREATMENT OF HUMAN AUTOIMMUNE DISEASE.