Most infections with Mycobacterium tuberculosis (Mtb) manifest as a clinically asymptomatic, contained state, known as latent tuberculosis infection, that affects approximately one-quarter of the global population. Although fewer than one in ten individuals eventually progress to active disease, tuberculosis is a leading cause of death from infectious disease worldwide. Despite intense efforts, immune factors that influence the infection outcomes remain poorly defined. Here we used integrated analyses of multiple cohorts to identify stage-specific host responses to Mtb infection. First, using high-dimensional mass cytometry analyses and functional assays of a cohort of South African adolescents, we show that latent tuberculosis is associated with enhanced cytotoxic responses, which are mostly mediated by CD16 (also known as FcγRIIIa) and natural killer cells, and continuous inflammation coupled with immune deviations in both T and B cell compartments. Next, using cell-type deconvolution of transcriptomic data from several cohorts of different ages, genetic backgrounds, geographical locations and infection stages, we show that although deviations in peripheral B and T cell compartments generally start at latency, they are heterogeneous across cohorts. However, an increase in the abundance of circulating natural killer cells in tuberculosis latency, with a corresponding decrease during active disease and a return to baseline levels upon clinical cure are features that are common to all cohorts. Furthermore, by analysing three longitudinal cohorts, we find that changes in peripheral levels of natural killer cells can inform disease progression and treatment responses, and inversely correlate with the inflammatory state of the lungs of patients with active tuberculosis. Together, our findings offer crucial insights into the underlying pathophysiology of tuberculosis latency, and identify factors that may influence infection outcomes.

To permit the recognition of antigens, T cells generate a vast diversity of T cell receptor (TCR) sequences. Upon binding of the TCR to an antigen–MHC complex, T cells clonally expand to establish an immune response. To study antigen-specific T cell clonality, we have developed a method that allows selection of rare cells, based on RNA expression, before in-depth scRNA-seq (named SELECT-seq). We applied SELECT-seq to collect both TCR sequences and then transcriptomes from single cells of peripheral blood lymphocytes activated by a Mycobacterium tuberculosis (Mtb) lysate. TCR sequence analysis allowed us to preferentially select expanded conventional CD8+ T cells as well as invariant natural killer T (iNKT) cells and mucosal-associated invariant T (MAIT) cells. The iNKT and MAIT cells have a highly similar transcriptional pattern, indicating that they carry out similar immunological functions and differ considerably from conventional CD8+ T cells. While there is no relationship between expression profiles and clonal expansion in iNKT or MAIT cells, highly expanded conventional CD8+ T cells down-regulate the interleukin 2 (IL-2) receptor alpha (IL2RA, or CD25) protein and show signs of senescence. This suggests inherent limits to clonal expansion that act to diversify the T cell response repertoire.

Background: Once considered primarily a disorder of lipid deposition, coronary artery disease is an incurable, life-threatening disease that is now also characterized by chronic inflammation notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. Methods: We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. Results: In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin , indicating that they are primarily antigen-experienced, memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4<CD8), exhibiting clonal expansion of specific TCRs. Interestingly, we found that these plaque T cells had TCRs specific for influenza, coronavirus, and other viral epitopes, which share sequence homologies to proteins found on smooth muscle cells and endothelial cells, suggesting potential autoimmune-mediated T-cell activation in the absence of active infection. To better understand the potential function of these activated plaque T cells, we then interrogated their transcriptome at the single-cell level. Of the 3 T-cell phenotypic clusters with the highest expression of the activation marker HLA-DRA identified by the Seurat algorithm, 2 clusters express a proinflammatory and cytolytic signature characteristic of CD8 cells, while the other expresses AREG (amphiregulin), which promotes smooth muscle cell proliferation and fibrosis, and, thus, contributes to plaque progression. Conclusions: Taken together, these findings demonstrate that plaque T cells are clonally expanded potentially by antigen engagement, are potentially reactive to self-epitopes, and may interact with smooth muscle cells and macrophages in the plaque microenvironment.

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