Early T cell responses in tuberculosis

University Of Pittsburgh At Pittsburgh, Pittsburgh PA

Investigators

Abstract

PROJECT SUMMARY Tuberculosis (TB) remains the deadliest infectious disease worldwide, causing more than 1.5 million deaths per year. The causative bacteria, Mycobacterium tuberculosis (Mtb), is a slow growing intracellular pathogen that predominantly affects the lungs. BCG, an attenuated strain of Mycobacterium bovis, is the only vaccine in use and offers limited protection against pulmonary TB and protection that wanes through adolescence. To make progress in new vaccine development, we need a better understanding of the immune responses to Mtb in relevant model systems. Notably, contributions of unconventional T cells (CD1-restricted, MR1-restricted, and gd T cells) to containment of acute Mtb infection are not well understood. In this proposal, I outline a characterization of these niche T cell subsets using a non-human primate model of Mtb infection. Non-human primates recapitulate the disease states observed in humans, have immune systems that mirror humans, and can be challenged with a low dose of Mtb. This work is therefore critical for building on transgenic rodent studies and contextualizing human peripheral blood analysis that has limited correlation with lung granulomas. Our results will inform vaccine design using unconventional antigen classes. The first aim of this project will characterize the frequency and functionality of these populations in early granulomas relative to uninvolved lung tissue. Based on prior work in our group, 4 weeks is the earliest that lesions can be grossly identified and isolated. PET CT guided necropsies enable excision of individual granulomas for immunological analyses. Unconventional T cell populations will be identified using loaded tetramers and antibodies targeting conserved TCR regions. In the second aim, I will use T cells isolated from bronchoalveolar lavages (BAL) before and after Mtb challenge and lung tissue excised at necropsy to perform FucoID, a method of identifying T cells that recognize Mtb infected macrophages. This will demonstrate the abundance of Mtb-reactive T cells and, in combination with tetramer staining, the expansion of unconventional subsets following infection. In Aim 3, I will use sections of uninvolved lung lobes to develop an ex vivo granuloma organoid model. The lung sections will be incubated with Mtb and tracked over time to monitor the formation of cellular aggregates and early granuloma structures. This model will provide the TB field with a tool to study early disease dynamics without the prohibitive cost and facility requirements for in vivo experiments, while also enabling analysis at earlier timepoints (i.e. days) post-inoculation than can be feasibly done using in vivo systems. This work directly addresses open questions in the field of TB immunology, including the presence, localization and functions of unconventional T cells in acute TB, which will guide antigen selection and platforms in future vaccine development. The aims provide experimental and analytical training prospects via existing collaborations as I become an independent investigator. This work will be performed at the University of Pittsburgh, where I have access to ABSL-3 facilities, with mentorship from Dr. JoAnne Flynn, a leading expert in TB and NHP immunology and Dr. Philana Ling Lin, a pediatric ID physician and expert in NHP models of SIV/TB co-infection.