During tuberculosis, lung myeloid cells have two opposing roles: they are an intracellular niche occupied by Mycobacterium tuberculosis, and they restrict bacterial replication. Lung myeloid cells from mice infected with yellow-fluorescent protein expressing M. tuberculosis were analyzed by flow cytometry and transcriptional profiling to identify the cell types infected and their response to infection. CD14, CD38, and Abca1 were expressed more highly by infected alveolar macrophages and CD11c Hi monocyte-derived cells compared to uninfected cells. CD14, CD38, and Abca1 "triple positive" (TP) cells had not only the highest infection rates and bacterial loads, but also a strong interferon-γ signature and nitric oxide synthetase-2 production indicating recognition by T cells. Despite evidence of T cell recognition and appropriate activation, these TP macrophages are a cellular compartment occupied by M. tuberculosis long-term. Defining the niche where M. tuberculosis resists elimination promises to provide insight into why inducing sterilizing immunity is a formidable challenge.

CD4 T cell help prevents CD8 T cell exhaustion and promotes control of Mycobacterium tuberculosis infectionGraphical abstract Highlights d CD4 T cell help promotes CD8 T cell effector functions and prevents exhaustion d Synergy between CD4 and CD8 T cells promotes survival during murine tuberculosis d Helped, but not helpless, CD8 T cells restrict intracellular mycobacterial growth d Protection mediated by CD8 T cells

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CD8 T cells provide limited protection against Mycobacterium tuberculosis (Mtb) infection in the mouse model. As Mtb causes chronic infection in mice and humans, we hypothesize that Mtb impairs T cell responses as an immune evasion strategy. TB10.4 is an immunodominant antigen in people, nonhuman primates, and mice, which is encoded by the esxH gene. In C57BL/6 mice, 30–50% of pulmonary CD8 T cells recognize the TB10.4 4−11 epitope. However, TB10.4-specific CD8 T cells fail to recognize Mtb-infected macrophages. We speculate that Mtb elicits immunodominant CD8 T cell responses to antigens that are inefficiently presented by infected cells, thereby focusing CD8 T cells on nonprotective antigens. Here, we leverage naturally occurring polymorphisms in esxH , which frequently occur in lineage 1 strains, to test this “decoy hypothesis”. Using the clinical isolate 667, which contains an EsxH A10T polymorphism, we observe a drastic change in the hierarchy of CD8 T cells. Using isogenic Erd.EsxH A10T and Erd.EsxH WT strains, we prove that this polymorphism alters the hierarchy of immunodominant CD8 T cell responses. Our data are best explained by immunodomination, a mechanism by which competition for APC leads to dominant responses suppressing subdominant responses. These results were surprising as the variant epitope can bind to H2-K b and is recognized by TB10.4-specific CD8 T cells. The dramatic change in TB10.4-specific CD8 responses resulted from increased proteolytic degradation of A10T variant, which destroyed the TB10.4 4-11 epitope. Importantly, this polymorphism affected T cell priming and recognition of infected cells. These data support a model in which nonprotective CD8 T cells become immunodominant and suppress subdominant responses. Thus, polymorphisms between clinical Mtb strains, and BCG or H37Rv sequence-based vaccines could lead to a mismatch between T cells that are primed by vaccines and the epitopes presented by infected cells. Reprograming host immune responses should be considered in the future design of vaccines.

20Immune responses following Mycobacterium tuberculosis (Mtb) infection or vaccination 21 are frequently assessed by measuring T cell recognition of crude Mtb antigens, 22 recombinant proteins, or peptide epitopes. We previously showed that not all Mtb-23 specific T cells recognize Mtb-infected macrophages. Thus, an important question is 24 what proportion of T cells elicited by Mtb infection recognize Mtb-infected macrophages. 25We answer this question by developing a modified elispot assay using viable Mtb-26 infected macrophages, a low multiplicity of infection and purified T cells. In C57BL/6 27 mice, CD4 and CD8 T cells were classically MHC restricted. Comparable frequencies of 28 T cells that recognize Mtb-infected macrophages were determined using interferon-γ 29 elispot and intracellular cytokine staining, and lung CD4 T cells more sensitively 30 recognized Mtb-infected macrophages than lung CD8 T cells. Compared to the numbers 31 of Mtb antigen-specific T cells for antigens such as ESAT-6 and TB10.4, low frequencies 32 of pulmonary CD4 and CD8 T cells elicited by aerosolized Mtb infection recognize Mtb-33 infected macrophages. Finally, we demonstrate that BCG vaccination elicits T cells that 34 recognize Mtb-infected macrophages. We propose that the frequency of T cells that 35 recognize infected macrophages could correlate with protective immunity and may be an 36 alternative approach to measuring T cell responses to Mtb antigens. 37with the failure of T cells specific for those antigens to recognize Mtb-infected 64 macrophages 4 . Importantly, following aerosol infection, Mtb disseminates to the 65 mediastinal lymph node, where T cells are first primed by dendritic cells, which then 66 expand and traffic to the lung 9, 10 . We speculate that there may be a mismatch in the 67 antigens presented (or cross-presented) by uninfected DC in the lymph nodes and 68 antigens presented by infected macrophages in the lung. Thus, T cells primed in the lymph 69 nodes during natural infection may not necessarily recognize antigens presented by Mtb-70 infected macrophages in the lung 11 . Regardless of the mechanism, we wondered whether 71 the inability of some T cells to recognize Mtb-infected macrophages might explain why the 72 number of antigen-specific T cells may not necessarily correlate with vaccine-induced 73 protection. 74To assess T cell recognition of Mtb-infected macrophages we developed a 75 modified elispot assay based on interferon (IFN)-g spot forming cells (SFC). Using a low 76 multiplicity of infection (MOI), we quantify the frequency of T cells that recognize Mtb-77 infected macrophages during primary infection in mice. We find that an unexpectedly low 78 frequency of ex vivo CD8 and CD4 T cells recognizes Mtb-infected macrophages. We 79 demonstrate that majority of the T cells from C57BL/6 mice that recognize Mtb-infected 80 macrophages are conventionally MHC-restricted T cells. Our data shows that CD4 T 81 cells efficiently detect Mtb-infected macrophages at a lower MOI, whereas CD8 T cells 82 on...