Evaluating human T cells specific for Mycobacterium tuberculosis antigens that are processed and presented by infected macrophages - We have only a basic understanding of how variation in human T cell responses elicited after Mycobacterium tuberculosis (Mtb) exposure and infection are linked to clinical outcomes. Emerging data in animal models and humans demonstrate that direct T cell recognition of Mtb-infected macrophages, the niche cell for Mtb infection, is central to a protective response. The premise of this proposal is that insight into which antigens are processed and presented by Mtb-infected macrophages, together with the composition, distribution, and kinetics of the T cell repertoire that target these antigens is vital to developing vaccines that prevent active tuberculosis (TB). In Uganda, our original study of household contacts (HHCs) of active pulmonary TB cases (since ’02) not only captured co-prevalent and incident TB but also new Mtb infection characterized by IFNg release assay (IGRA). Our current, follow-up TB HHC study focused on enrolling asymptomatic IGRA-negative contacts to identify persons undergoing IGRA conversion, i.e. “converters” (CVTR). HHCs with baseline and stable positive IGRAs in follow-up (i.e. LTBI), and “nonconverters” (NCVTRs), who remained IGRA negative, were also enrolled. Evaluating T cells and MF from peripheral blood and broncho-alveolar lavage (BAL) samples from the CVTRs form the basis of our proposed studies focusing on longitudinal immune responses. The experimental approach for this proposal builds on two complementary, ongoing lines of investigation. First, to quantify Mtb-derived peptide presentation in the context of diverse HLA alleles in Mtb-infected cells, we developed mass spectrometry approaches. Despite MHC-II allelic diversity, we found that peptides derived from CFP10, an Esx1 protein which is absent in the BCG vaccine, were presented by all MHC-II alleles analyzed while other antigens were found only in the context of certain MHC-II alleles. Second, using single-cell RNA sequencing (scRNAseq) with T antigen cell receptor (TCR) mapping, we found that Mtb-specific CD4+ T cell recognition of infected macrophages is variable and depends both on macrophage phenotype and T cell antigen specificity. Leveraging our team’s combined expertise, we will define the antigens presented by Mtb-infected macrophages and compare Mtb-specific T cell responses to these antigens at early vs. late time points after IGRA conversion, in peripheral blood and in the lungs. In Aim 1, we will use immunopeptidomics to identify Mtb antigens presented on Mtb-infected human macrophages. We will then enumerate the circulating CD4+ T cells specific for these antigens after Mtb exposure and IGRA conversion among CVTRs. In Aim 2, we will quantify direct T cell recognition of Mtb-infected macrophages, using scRNAseq to determine the TCR repertoire, antigen specificity, and function of T cells from BAL and peripheral blood, longitudinally, after IGRA conversion. In Aim 3 we will compare the capacities to control bacterial growth among human CD4+ T cells that target antigens presented by Mtb-infected monocyte-derived and alveolar macrophages vs. other immunodominant Mtb antigens. The antigens, TCRs, and T cell functions gained from this project will directly inform TB vaccine design.
