Project Grant R21AI185410

UNLEASHING CD8+ T CELLS FOR TB DEFENSE - PROJECT SUMMARY THIS R21 APPLICATION IS BASED ON EXCITING AND NOVEL PRELIMINARY DATA SUPPORTING THE HYPOTHESIS THAT THE ORPHAN NUCLEAR RECEPTOR NR4A1 RESTRAINS THE PROTECTIVE FUNCTION OF CD8+ T CELLS IN TUBERCULOSIS (TB). WE FOUND THE NR4A1-/- MICE CONTROL PULMONARY INFECTION WITH MYCOBACTERIUM TUBERCULOSIS (MTB) SIGNIFICANTLY BETTER THAN WILDTYPE (WT) MICE. A SIMILAR EFFECT WAS OBSERVED IN WT MICE TREATED WITH THE NR4A1 ANTAGONIST DIM-C. IN AN ADOPTIVE TRANSFER EXPERIMENT, NR4A1-/- CD8+ T CELLS PROTECTED RAG1-/- MICE FROM AEROSOL MTB INFECTION BETTER THAN TRANSFERRED CD8+ T CELLS FROM NR4A1-/- OR WT DONORS. THIS IS REMARKABLE SINCE PRIOR STUDIES FOUND LITTLE OR NO PROTECTION FROM TRANSFERRED WT CD8+ T CELLS, WHICH WAS CONFIRMED IN OUR EXPERIMENT. IMMUNOHISTOCHEMISTRY OF LUNG TB LESIONS SHOWED GREATER INFILTRATION OF CD8+ T CELLS IN NR4A1-/- MICE COMPARED TO WT. ANALYSIS OF BULK RNA SEQUENCING DATA FROM PURIFIED LUNG CD8+ T CELLS FROM MTB-INFECTED MICE IDENTIFIED UPREGULATION OF GENES LINKED TO CELLULAR INFILTRATIVE CAPACITY AND CYTOTOXICITY IN NR4A1-/- COMPARED TO WT HOSTS. IF TRUE, THESE FINDINGS WILL ADVANCE THE FIELD OF CELLULAR IMMUNITY IN TB AND OFFER TRANSLATION POTENTIAL FOR NR4A1 INHIBITION AS HOST-DIRECTED THERAPY. EXPRESSION OF NR4A1 IS INDUCED BY T CELL RECEPTOR SIGNALING, WHICH IS THE PREDOMINANT MECHANISM FOR REGULATING ITS ACTIVITY. WE FOUND THAT NR4A1 MRNA IS INCREASED IN THE LUNGS OF MTB-INFECTED MICE, RAISING THE FUNDAMENTAL QUESTION WHETHER NR4A1-REGULATED T CELL TOLERANCE SUPPORTS A PATHOGEN-PERMISSIVE ENVIRONMENT IN TB. THE GOAL OF THIS R21 APPLICATION IS TO CONFIRM AND EXTEND KEY FINDINGS OF THE TB PHENOTYPING, ADOPTIVE TRANSFER, AND DIM-C TREATMENT EXPERIMENTS, AND TO ENHANCE THESE STUDIES WITH MORE DETAILED OUTCOME MEASURES. WE ALSO PLAN TO PERFORM SINGLE CELL RNA SEQUENCING OF LUNG LEUKOCYTES AND SPATIAL TRANSCRIPTOMIC STUDIES IN MTB-INFECTED NR4A1-/- AND WT MICE AS A FIRST STEP TOWARDS IDENTIFYING AND PRIORITIZING MECHANISTIC HYPOTHESES ON WHICH TO BASE A FUTURE RO1 PROPOSAL. ACCORDINGLY, WE DESCRIBE A RESEARCH PLAN THAT IS FEASIBLE TO COMPLETE WITHIN TWO YEARS WITH THE DELIVERABLES OF AN INITIAL PUBLICATION AND A COMPETITIVE RO1 APPLICATION.