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CD8 T-cell immunity

A greater understanding of how the immune system responses to Mtb is needed to develop an effective vaccine. Although both CD4 and CD8 T cell responses are generated during infection, and their arrival into lungs effectively contains Mtb replication, CD4 T cells are thought to play a more important role in the protection. This is supported by the higher risk in AIDS patients to develop active tuberculosis, and the early mortality in experimental mice deficient in CD4 T cells [1]. In contrast, CD8 T cell’s role in the protective immunity is less appreciated. Nevertheless, evidence highlighting the importance of CD8 T cells are emerging. In non-human primate (NHP) studies, CD8 depletion compromised BCG-induced control [2], and their cytotoxicity functions were corelated with Mtb restriction in granulomas.

Although CD4 T cell mediated protection has shown to be indispensable, we hypothesized that CD8 T cells also played a crucial part against Mtb. The Behar lab has dedicated to understanding the roles CD8 T cells played during infection and made significant progresses that have implications for future vaccine design. Recently, we found CD8 T cells specific for TB10.44-11, a dominate epitope occurred in infection, didn’t efficiently recognize infected macrophages [3]. This may be the underlining mechanism that vaccines designed to induce strong Mtb-specific responses didn’t confer protection. We and others suggested that recognition of infected cells could be a criterion for screening vaccine candidates before clinical trial [4]. Moreover, we showed that to generate optimal CD8 T cell responses, CD4 T cells were essential. Through comparing Mtb-specific CD8 T cell responses in WT vs. MHC II KO mice, we revealed that helped CD8 T cells were effector cells with cytotoxicity functions. In contrast, helpless CD8 T cells were exhausted. Importantly, helped CD8 T cells mediated better Mtb control than helpless CD8 T cells. Since current vaccines are evaluated mainly on their ability to generate CD4 T cell immunity, we suggest that vaccines eliciting both CD4 and CD8 T cell responses are likely to be successful.

Currently, the Behar lab has identified a portion of polyclonal CD8 T cells do recognize infected cells [5]. We’re trying to understand the transcriptional signatures of CD8 T cells after recognizing infected cells, and to discover new antigens expressed by infected cells that are recognized. We believe these are important questions to tackle in order to uncover the role of CD8 T cells in protective immunity and to design better vaccines.

  1. Mogues, T., et al., The relative importance of T cell subsets in immunity and immunopathology of airborne Mycobacterium tuberculosis infection in mice. J Exp Med, 2001. 193(3): p. 271-80.
  2. Chen, C.Y., et al., A critical role for CD8 T cells in a nonhuman primate model of tuberculosis. PLoS Pathog, 2009. 5(4): p. e1000392.
  3. Yang, J.D., et al., Mycobacterium tuberculosis-specific CD4+ and CD8+ T cells differ in their capacity to recognize infected macrophages. PLoS Pathog, 2018. 14(5): p. e1007060.
  4. Nyendak, M., et al., Adenovirally-Induced Polyfunctional T Cells Do Not Necessarily Recognize the Infected Target: Lessons from a Phase I Trial of the AERAS-402 Vaccine. Sci Rep, 2016. 6: p. 36355.
  5. Patankar, Y.R., et al., Limited recognition of Mycobacterium tuberculosis-infected macrophages by polyclonal CD4 and CD8 T cells from the lungs of infected mice. Mucosal Immunol, 2020. 13(1): p. 140-148.

T cell recognition to Mtb infected macrophages

Following virulent Mycobacterium tuberculosis (Mtb) infection in mice, a robust T cell response is primed and recruited to the lung. There, T cells restrict Mtb growth, which leads to a plateau in the bacillary burden. Despite T cell dependent clearance of most bacilli, sterilization is never achieved. Under optimal conditions, the best vaccines in the murine model provide only an additional 30-fold CFU reduction. Why does a relatively small, but biologically important subpopulation of bacteria persist in the face of otherwise effective T cell immunity? Our overarching hypothesis is that bacteria survive despite robust T cell responses because of a failure in T cell surveillance and effector function. For bacteria that infect mammalian cells, immune clearance relies on CD4 and CD8 T cells that recognize antigens sampled in endosomes (by MHC II) or the cytosol (by MHC I). Non-MHC restricted T cells (e.g., NKT, MAIT cells) also sample cellular compartments. A major scientific goal is to identify microbial antigens for use in vaccines. In the past, microbial antigens were identified based on their ability to be recognized by T cells. Immunization with such antigens would often elicit T cell responses, but few were protective. The frequent failure of microbial antigens to induce protective T cell responses was rationalized by a probabilistic view of immunology: “some antigens are protective, some are not”. While this may be a truism, we now know that intracellular pathogens evolved to evade detection by the immune system and avoid T cell recognition. Mtb has co-evolved with us for 15,000 years and has numerous mechanisms to evade detection and avoid killing by our immune system. Our inability to predict which Mtb antigens will be presented by infected cells is a major gap in selecting candidate antigens for vaccines. We are building upon our previous work at the host-pathogen interface to determine: 1) which T cells recognize infected macrophages; 2) which antigens are recognized; 3) how T cells restrict Mtb growth, and 4) how Mtb evades elimination.

T-cell exhaustion

An estimated one-fourth of the world’s population is latently infected with Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Within this group, only a small percentage develop active and contagious disease, and in both humans and mouse models, the mechanisms underlying this progression to symptomatic TB remain unclear. Many factors contribute to the efficacy of the immune system in eliminating pathogens, but literature demonstrates a requirement for CD4+ T-cells in order to mount a protective response against Mtb. Accordingly, one possible explanation for bacterial recrudescence and the progression of latent to active TB is CD4+ T-cell exhaustion. However, T-cell exhaustion has primarily been studied in the context of CD8+ T-cells in chronic viral infection and cancer models. As such, our lab uses mouse models of TB to study the CD4+ T-cell response in chronic Mtb infection to determine the phenotypic and transcriptional characteristics of CD4+ T-cell exhaustion and the mechanisms that mediate it.

We previously demonstrated that dysfunctional PD-1+ TIM-3+ double positive T-cells accumulate in the lungs of chronically infected C57Bl/6 mice, and these T-cells exhibit a transcriptional signature associated with T-cell exhaustion (Jayaraman et al., 2016). We are currently working to more rigorously characterize exhausted antigen-specific T-cells using C7 TCR transgenic mice with CD4+ T-cells specific to ESAT-61-15 (C7 T-cells) and NGS platforms. We also previously presented that susceptible C3HeB/FeJ mice succumbed to infection earlier and had a higher lung bacterial burden than TIM-3 KO mice on the same background. Furthermore, blockade of TIM-3 in C57Bl/6 mice results in a lower lung bacterial burden compared to isotype controls, suggesting that the inhibitory receptor, TIM-3, plays a role in mediating T-cell exhaustion during TB (Jayaraman et al., 2016). However, the mechanism through which TIM-3 promotes bacterial survival and mediates exhaustion remains an active question in the lab.

A newer arm of this project has stemmed from the discovery that TIM-3 also regulates innate immunity. Recent studies have found links between the mutations in TIM-3 that prevent its expression on the cell surface and increased IL-1b and inflammation, resulting in the human disease SPTCL. Currently, we hypothesize that TIM-3 negatively regulates inflammasome activation, and we predict that disruption of TIM-3:ligand interactions will disinhibit the inflammasome, lead to IL-1b and IL-18 secretion, and secondarily affect TNF and type I IFN production. As these cytokines all affect the outcome of TB and other infections, we expect that the TIM-3/inflammasome axis will regulate innate immunity to TB.

Innate immunity including Efferocytosis

Efferocytosis, the process of clearing dead cells by phagocytes, is crucial to maintenance of homeostasis. Defective efferocytosis is often the underlying cause of several chronic inflammatory diseases. Despite profound advances in research on mechanisms of apoptotic cell recognition and uptake, several key areas regarding the role of efferocytosis in host defense is poorly understood. It has been seen in some infectious diseases, such as tuberculosis (TB), that the infected cells can undergo alternative fates of apoptosis or necrosis, that can lead to beneficial or detrimental outcomes for the host. My research encompasses studying the role of efferocytosis in the outcome of disease using TB as a disease model. Tuberculosis, caused by Mycobacterium tuberculosis (Mtb), is the leading cause of death caused by a single infectious agent. Despite TB being a preventable and curable disease, approximately 1.5 million deaths are caused by TB annually. Alveolar macrophages (AM) are considered the first line of defense against Mtb as they are the first cells that the invading-Mtb encounters. However, Mtb has been hypothesized to exploit AM as a protective niche for survival and replication. To control the bacteria, the host induces apoptosis of infected cells but virulent Mtb strains suppress host-induced apoptosis of infected cells which instead undergo necrosis resulting in pathogenesis. It has been shown in vitro that of infected macrophages, and not apoptosis per se, leads to better Mtb control. However, neither the exact mechanistic role of AM in the pathogenesis of Mtb infection, nor the role of efferocytosis in Mtb control in vivo, has been demonstrated. “Eat-me signals” (e.g., exposed phosphatidylserine on cell surface), receptors (e.g., MerTK) that recognize “eat-me” molecules, and “don’t-eat-me” signals (e.g. CD47) determine efferocytosis efficiency. Studies show that CD47 is upregulated in cancer and atherosclerosis through unknown mechanism. Role of CD47 in efferocytosis by AM in Mtb pathogenesis is a relatively unventured field but shows considerable promise.

This project aims to look at the effects of efferocytosis on the outcome of Mtb infection in vivo. There is some preliminary data by Yu-Jung Lu (graduate student) with MerTK KO mice showing that the mice deficient in MerTK (receptor bringing about efferocytosis) show increased bacterial burden and higher number and frequencies of CD4 and CD8 T cells compared to their wildtype counterparts. Tasfia Rakib (graduate student) is now trying to look at efferocytosis and further investigate the mechanism by which CD47 is upregulated in virulence strains of Mtb, causing detrimental effects on the host.

Vaccine development

Studies in different geographic regions and ethnic populations have produced disparate estimates of protection conferred by BCG against TB. BCG efficacy is estimated to be greater than 75% in some populations. In contrast, no significant protection is detected in regions where TB remains endemic. Such variable efficacy could be due to differences in the BCG strain, its preparation, the environment, or the host genetic background. While the role of genetic variation in BCG strains and previous exposure to environmental mycobacteria have been investigated extensively, the role of host genetic variation has been more difficult to quantify. Many studies find that the immunological response to mycobacterial infection and BCG vaccination is heritable. However, the relationship between these immunological markers and vaccine efficacy is unknown and difficult to address in natural populations. Using the CC panel, we have identified host genetic diversity as crucial factor that affects BCG efficacy. As in human meta-analyses of BCG efficacy, when these genetically diverse mice were considered as a single population, BCG has a modest protective effect. However, when assessed by genotype, we found some lines to be protected, others that were not protected, and a few in which vaccination exacerbated disease. These observations indicate that host genetic variation limits BCG efficacy, and suggests that current efforts to develop vaccines that are effective in C57BL/6 mice (e.g., a single genotype), could fail in a genetically diverse population. Thus, this project seeks to determine the genetic and immunological determinants of vaccine-induced immunity in genetically diverse populations with the ultimate goal of improving the mouse model for pre-clinical vaccine development.