PROJECT SUMMARY / ABSTRACT T cells are required for protection against intracellular infections such as leprosy and tuberculosis; however, the specific mechanisms by which they contribute to host immunity remain unresolved. Leprosy, caused by the intracellular pathogen Mycobacterium leprae (mLEP), provides an accessible model to investigate human immune responses to intracellular bacterial infection. The spectrum of clinical manifestations of leprosy correlates with the immune response to mLEP. Patients with tuberculoid leprosy (T-lep) develop protective immunity and eliminate the infection, whereas those with lepromatous leprosy (L-lep) sustain a progressive infection. The dynamics of T cell immunity can be studied in the context of the reversal reaction (RR), in which patients upgrade from L-lep to T-lep. We established that in patients with leprosy, T cell subsets differ dramatically between the blood and lesions, indicating the importance of studying the immune response at the site of disease. During the first 38 years of this R01, we discovered new mechanisms by which CD4+ and CD8+ T cells engender antimicrobial activity against mLEP in infected human macrophages (MØs), which we also showed to be relevant in Mycobacterium tuberculosis infection. These include 1) cytolytic T lymphocyte release of the antimicrobial protein granulysin; 2) Th1 cell secretion of IFN- which activates vitamin D-dependent induction of the antimicrobial proteins cathelicidin and DEFB4 in MØs; and 3) Th17 cell release of the antimicrobial protein IL-26, which enters myeloid cells targeting intracellular bacteria. None of these mechanisms exist in mice, necessitating the study of human disease. By integrating scRNA-seq and spatial transcriptomics, we identified an ecosystem of T cells, MØs, keratinocytes, fibroblasts, and endothelial cells that together express 66 genes involved in the antimicrobial response in leprosy. We also discovered that TREM2 MØs are a major MØ subpopulation in the disseminated L-lep form of leprosy, representing the mLEP- infected foamy MØs that are unable to eliminate the pathogen. We hypothesize that T cell-dependent antimicrobial mechanisms can overcome immune suppression in TREM2 MØs and thus eliminate mLEP. Our specific aims are to: Aim 1: Elucidate the mechanisms by which T cells mediate an antimicrobial response to intracellular mycobacteria, Aim 2: Define mechanisms by which TREM2 MØs contribute to the pathogenesis of progressive infection in leprosy; and, Aim 3: Identify immune pathways that can augment antimicrobial responses in leprosy, including in CD8+ T cells and TREM2 MØs. The proposed studies will provide insights into the mechanisms of pathogenesis and host defense in intracellular bacterial infections.