The human lung and lung-draining lymph node response to Mycobacterium tuberculosis

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Project summary/Abstract This project is focused on gaining conceptual and mechanistic insights into immune mediators that contribute to the development of protective immunity against Mycobacterium tuberculosis (Mtb) in human lung and lung- draining lymph nodes. Although tuberculosis (TB) typically manifests as a pulmonary disease, thoracic lymph nodes are the most common sites of extrapulmonary Mtb infection. Mtb can be reactivated in lymph nodes, leading to the formation of granulomas and Mtb dissemination to the lungs and other organs. Yet, the lymph node response to Mtb in humans remains understudied. A key feature of Mtb pathogenesis is granuloma formation; finely tuned immune responses within these structures determine localized infection outcomes. Here, we aim to perform a comparative assessment of the immune (specifically, T cell) responses within human pulmonary and lymph node granulomas that are successful and unsuccessful in eliminating Mtb. To accomplish this, we propose to characterize specimens with histological evidence of a granulomatous reaction that (a) show negative Acid-Fast-Bacilli (AFB) stain but test positive for the presence of TB-DNA by PCR (reflective of sterilizing immunity or a successful immune response at the granuloma level), and (b) show positive AFB stain (representing an unsuccessful host effort). Studies in animal models have shown that T cells are important in controlling the clinical course of TB and that the T cell response to Mtb antigens is initiated in thoracic lymph nodes and not the lungs. Thus, first, we will test the hypothesis that differences between sterilizing immunity and unsuccessful responses stem from differences in specific effector T cell phenotypes and functions, and their spatial organization in the granulomas. Indeed, our preliminary data show that specific CD8+ T cell responses associate with Mtb clearance at the granuloma level. Our next goal is to use this knowledge to guide the selection of appropriate antigens to re-elicit this response through vaccination. To accomplish this, we will establish tissue- mimetic organoids from lymph nodes, which are the staging grounds for cellular immunity following immunization. We will test the hypothesis that these protective T cell responses that are naturally elicited in granulomas can be induced in lymph node organoids from TB-naïve donors by specific Mtb (not BCG) antigens. Finally, we will employ a combination of T cell receptor sequencing and machine-learning algorithms to identify the precise protective epitopes. Collectively, we expect our results to generate important new insight into the determinants of immune defense in TB, identify candidates for new TB vaccines, and create opportunities for the management of the global burden of tuberculosis.