Our recent advances in the use of fluorescent fitness reporter Mtb strains in the murine TB model has afforded us a unique appreciation of the role of macrophage lineages in both the control and promotion of Mtb growth. Moreover, the extension of these observations through the successful development of Dual RNA-seq protocols for characterization of infected cells isolated directly from the murine lung has provided a further understanding of how host nutritional immunity is central to the control of bacterial growth, at least in early infection. We propose building on our collaboration with Dr. Henry Mwandumba in Malawi and extending these observations through an ex vivo Mtb challenge model with human bronchoalveolar lavage (BAL) cells to functionally phenotype the macrophage lineages present in the human lung airways. Furthermore, we recently demonstrated the persistence of transcriptionally-active HIV-1 genomes in the alveolar macrophages of ART- naïve and ART-suppressed donors in Malawi and believe that we can take advantage of this unique human subjects cohort to characterize the impairment of lung immunity known to occur in people living with HIV-1 that renders them hypersusceptible to both TB and other lower respiratory tract infections. Our hypothesis is that the functional and phenotypic typing of Mtb-infected human lung macrophage subsets from healthy and HIV-1-infected volunteers will generate testable models for immune-mediated control of Mtb growth that will inform future vaccine development programs. Specific Aim 1: Assessment of anti-Mtb immune function in a BAL ex vivo challenge model. This aim will be overseen by Dr. Mwandumba in Malawi. We will use our fluorescent readouts of bacterial fitness to quantify and optimize anti-microbial activities in human BAL cell cultures from HIV-1 uninfected and infected donors challenged with Mtb ex vivo. Specific Aim 2. Utilization of SILAC labeling and single cell RNA-seq to identify soluble modulators of Mtb host macrophage function in HIV-1 negative and HIV-1 positive donors. This aim will be overseen by Dr. Russell at Cornell University on human macrophages and BAL samples from Malawi. We will perform (i) Proteomic analysis of released effector proteins by metabolic labeling studies (SILAC) of secreted proteins from human BAL cells pulsed ex vivo in Malawi. (ii) We will perform single cell (scRNA-seq) RNA-seq analysis of Mtb-challenged BAL cell populations from HIV-1 uninfected and infected donors to identify macrophage- dependent pathways of immune control of Mtb growth and their impairment by HIV-1. Specific Aim 3. The use of Loss of function and Gain of function approaches to assess candidate genes and pathways in successful control of intracellular Mtb infection. We will use siRNA and synthetic mRNA to manipulate host HMDMs and BAL macrophages to validate candidate genes/pathways involved in restriction of bacterial growth, and how it is compromised in the HIV-1 lung environment.