PROJECT SUMMARY: A third of the world’s population is infected with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis (TB). The human immune system is highly successful at controlling Mtb and immunocompetent individuals who are infected have a 5-10% lifetime chance of experiencing active (symptomatic) TB. This level of protection is mediated by cytokine-based cell-cell communication in granulomas, the lesions associated with TB. Factors that dysregulate the protective Th1 responses, including downregulation of the macrophage activat- ing cytokines IFNγ and TNF, can promote active TB. HIV significantly increases the risk of developing TB and HIV-Mtb coinfected individuals experience a 5-15% annual risk of developing active TB and TB is a leading killer in this population. This elevated risk occurs in individuals who have normal range peripheral CD4+ T cell numbers and well controlled virus loads. Studies in SIV-Mtb coinfected nonhuman primates (NHPs), which are the best available model of HIV-Mtb coinfection, show that granulomas from coinfected and Mtb-only animals contain similar numbers of Mtb-specific T cells. These data suggest that virus-associated T cell-independent factors promote TB in SIV/HIV+ individuals. Macrophages may be the key to understanding the pathology of HIV-Mtb coinfection. Macrophages can support Mtb replication but are also the primary anti-Mtb effector cell in granulo- mas. Our preliminary data using RNAscope indicates that macrophages in NHP granulomas are infected with SIV and transcriptional analysis of granulomas from SIV+ and SIV- monkeys shows that coinfected granulomas have increased expression of type 1 interferon (IFN1) transcripts. IFN1s are induced by viral infection but are also associated with exacerbated TB, and based on these data, we hypothesize that viral infection induces IFN1- regulated immunity in granuloma macrophages and this suppresses macrophage antimycobacterial immunity, thus promoting TB. This hypothesis cannot be tested in humans and murine models are experimentally tractable but have significantly different immunobiology, HIV susceptibility, and TB presentation than humans. To over- come these obstacles, we will use highly multiplexed cyclic IHC and RNAscope on FFPE NHP granulomas to identify the relationship between IFN1, SIV infection, and macrophage anti-Mtb activity in SIV/Mtb coinfection. These studies will be done on a unique set of NHP granulomas with known Mtb and SIV loads, and we will use computational image analysis and machine learning to identify SIV+/- cells, how SIV affects cellular organization, IFN1 expression, and macrophage activation. To test our findings from these studies, we will use lung granuloma organoids composed of lung tissue from Mtb-infected macaques, in combination with fluorescent protein ex- pressing Mtb, SIV and IFN1 inhibition. These studies will identify relationships between SIV-regulated IFN1 ex- pression and Mtb survival in coinfec...