Th1 cells are critical for containment of Mycobacterium tuberculosis infection, but little else is known about the properties of protective CD4 T cell responses. In this study, we show that the pulmonary Th1 response against M. tuberculosis is composed of two populations that are either CXCR3hi and localize to lung parenchyma or are CX3CR1hiKLRG1hi and are retained within lung blood vasculature. M. tuberculosis–specific parenchymal CD4 T cells migrate rapidly back into the lung parenchyma upon adoptive transfer, whereas the intravascular effectors produce the highest levels of IFN-γ in vivo. Importantly, parenchymal T cells displayed greater control of infection compared with the intravascular counterparts upon transfer into susceptible T cell–deficient hosts. Thus, we identified a subset of naturally generated M. tuberculosis–specific CD4 T cells with enhanced protective capacity and showed that control of M. tuberculosis correlates with the ability of CD4 T cells to efficiently enter the lung parenchyma rather than produce high levels of IFN-γ.

IFN-γ–producing CD4 T cells are required for protection against Mycobacterium tuberculosis (Mtb) infection, but the extent to which IFN-γ contributes to overall CD4 T cell-mediated protection remains unclear. Furthermore, it is not known if increasing IFN-γ production by CD4 T cells is desirable in Mtb infection. Here we show that IFN-γ accounts for only ~30% of CD4 T cell-dependent cumulative bacterial control in the lungs over the first six weeks of infection, but >80% of control in the spleen. Moreover, increasing the IFN-γ–producing capacity of CD4 T cells by ~2 fold exacerbates lung infection and leads to the early death of the host, despite enhancing control in the spleen. In addition, we show that the inhibitory receptor PD-1 facilitates host resistance to Mtb by preventing the detrimental over-production of IFN-γ by CD4 T cells. Specifically, PD-1 suppressed the parenchymal accumulation of and pathogenic IFN-γ production by the CXCR3+KLRG1-CX3CR1- subset of lung-homing CD4 T cells that otherwise mediates control of Mtb infection. Therefore, the primary role for T cell-derived IFN-γ in Mtb infection is at extra-pulmonary sites, and the host-protective subset of CD4 T cells requires negative regulation of IFN-γ production by PD-1 to prevent lethal immune-mediated pathology.

SUMMARY Recent data indicate that the differentiation state of Th1 cells determines their protective capacity against tuberculosis. Therefore, we examined the role of Th1 polarizing factors in the generation of protective and non-protective subsets of Mtb-specific Th1 cells. We find IL-12/23p40 promotes Th1 cell expansion and maturation beyond the CD73+CXCR3+T-betdim stage, and T-bet prevents deviation of Th1 cells into Th17 cells. Nevertheless, IL-12/23p40 and T-bet are also essential for the production of a prominent subset of intravascular CX3CR1+KLRG1+ Th1 cells that persists poorly and can neither migrate into the lung parenchyma nor control Mtb growth. Furthermore, T-bet suppresses development of CD69+CD103+ tissue resident phenotype effectors in lung. In contrast, Th1 cell-derived IFNγ inhibits the accumulation of intravascular CX3CR1+KLRG1+ Th1 cells. Thus, although IL-12 and T-bet are essential host survival factors, they simultaneously oppose lung CD4 T cell responses at several levels, demonstrating the dual nature of Th1 polarization in tuberculosis.

Protection against Mycobacterium tuberculosis (Mtb) infection requires CD4 T cells to migrate into the lung and interact with infected macrophages. In mice, less-differentiated CXCR3+ CD4 T cells migrate into the lung and suppress growth of Mtb, while CX3CR1+ terminally-differentiated Th1 cells accumulate in the blood vasculature and do not control pulmonary infection. Here we examine CD4 T cell differentiation and lung homing during primary Mtb infection of rhesus macaques. Mtb-specific CD4 T cells simultaneously appeared in the airways and blood ~21–28 days post-exposure, indicating that recently primed effectors are quickly recruited into the lungs after entering circulation. Mtb-specific CD4 T cells in granulomas display a tissue-parenchymal CXCR3+CX3CR1−PD-1hiCTLA-4+ phenotype. However, most granuloma CD4 T cells are found within the outer lymphocyte cuff, and few localize to the myeloid cell core containing the bacilli. Using the intravascular stain approach, we find essentially all Mtb-specific CD4 T cells in granulomas have extravasated across the vascular endothelium into the parenchyma. Therefore, it is unlikely that lung-homing defects introduced by terminal differentiation limit the migration of CD4 T cells into granulomas following primary Mtb infection of macaques. However, intralesional positioning defects within the granuloma may pose a major barrier to T cell-mediated immunity during tuberculosis.

Mucosal-associated invariant T (MAIT) cells are potential targets of vaccination and host-directed therapeutics for tuberculosis, but the role of MAIT cells during Mycobacterium tuberculosis (Mtb) infection in vivo is not well understood. Here we find that following Mtb infection MAIT cells mount minimal responses, and MAIT cell-deficient MR1 −/− mice display normal survival. Preinfection expansion of MAIT cells through 5-OP-RU vaccination fails to protect against subsequent Mtb challenge. In fact, 5-OP-RU vaccination delays Mtb-specific CD4 T cell priming in lung-draining lymph nodes, and conversely MR1 deficiency or blockade accelerates T cell priming. The MAIT cell-mediated delay in T cell priming is partly dependent on TGF-β. Surprisingly, 5-OP-RU treatment during chronic infection drives MAIT cell expansion and an IL-17A-dependent reduction in bacterial loads. Thus, during early infection MAIT cells directly contribute to the notoriously slow priming of CD4 T cells, but later during infection MAIT cell stimulation may be an effective host-directed therapy for tuberculosis.

Mycobacterium tuberculosis infection (Mtb) is the leading cause of death due to a single infectious agent and is among the top ten causes of all human deaths worldwide. CD4 T cells are essential for resistance to Mtb infection, and for decades it has been thought that IFNγ production is the primary mechanism of CD4 T-cell-mediated protection. However, IFNγ responses do not correlate with host protection, and several reports demonstrate that additional anti-tuberculosis CD4 T-cell effector functions remain unaccounted for. Here we show that the tumour-necrosis factor (TNF) superfamily molecule CD153 (encoded by the gene Tnfsf8) is required for control of pulmonary Mtb infection by CD4 T cells. In Mtb-infected mice, CD153 expression is highest on Mtb-specific T helper 1 (T1) cells in the lung tissue parenchyma, but its induction does not require T1 cell polarization. CD153-deficient mice develop high pulmonary bacterial loads and succumb early to Mtb infection. Reconstitution of T-cell-deficient hosts with either Tnfsf8 or Ifng CD4 T cells alone fails to rescue mice from early mortality, but reconstitution with a mixture of Tnfsf8 and Ifng CD4 T cells provides similar protection as wild-type T cells. In Mtb-infected non-human primates, CD153 expression is much higher on Ag-specific CD4 T cells in the airways compared to blood, and the frequency of Mtb-specific CD153-expressing CD4 T cells inversely correlates with bacterial loads in granulomas. In Mtb-infected humans, CD153 defines a subset of highly polyfunctional Mtb-specific CD4 T cells that are much more abundant in individuals with controlled latent Mtb infection compared to those with active tuberculosis. In all three species, Mtb-specific CD8 T cells did not upregulate CD153 following peptide stimulation. Thus, CD153 is a major immune mediator of host protection against pulmonary Mtb infection and CD4 T cells are one important source of this molecule.

Boosting immune cell function by targeting the coinhibitory receptor PD-1 may have applications in the treatment of chronic infections. Here, we examine the role of PD-1 during Mycobacterium tuberculosis (Mtb) infection of rhesus macaques. Animals treated with anti–PD-1 monoclonal antibody developed worse disease and higher granuloma bacterial loads compared with isotype control–treated monkeys. PD-1 blockade increased the number and functionality of granuloma Mtb-specific CD8 T cells. In contrast, Mtb-specific CD4 T cells in anti–PD-1–treated macaques were not increased in number or function in granulomas, expressed increased levels of CTLA-4, and exhibited reduced intralesional trafficking in live imaging studies. In granulomas of anti–PD-1–treated animals, multiple proinflammatory cytokines were elevated, and more cytokines correlated with bacterial loads, leading to the identification of a role for caspase 1 in the exacerbation of tuberculosis after PD-1 blockade. Last, increased Mtb bacterial loads after PD-1 blockade were found to associate with the composition of the intestinal microbiota before infection in individual macaques. Therefore, PD-1–mediated coinhibition is required for control of Mtb infection in macaques, perhaps because of its role in dampening detrimental inflammation and allowing for normal CD4 T cell responses.

Mucosal-associated invariant T cells (MAITs) are positioned in airways and may be important in the pulmonary cellular immune response against infection, particularly prior to priming of peptide-specific T cells. Accordingly, there is interest in the possibility that boosting MAITs through tuberculosis (TB) vaccination may enhance protection, but MAIT responses in the lungs during tuberculosis are poorly understood. In this study, we compared pulmonary MAIT and peptide-specific CD4 T cell responses in-infected rhesus macaques using 5-OP-RU-loaded MR-1 tetramers and intracellular cytokine staining of CD4 T cells following restimulation with an -derived epitope megapool (MTB300), respectively. Two of four animals showed a detectable increase in the number of MAIT cells in airways at later time points following infection, but by ∼3 weeks postexposure, MTB300-specific CD4 T cells arrived in the airways and greatly outnumbered MAITs thereafter. In granulomas, MTB300-specific CD4 T cells were ∼20-fold more abundant than MAITs. CD69 expression on MAITs correlated with tissue residency rather than bacterial loads, and the few MAITs found in granulomas poorly expressed granzyme B and Ki67. Thus, MAIT accumulation in the airways is variable and late, and MAITs display little evidence of activation in granulomas during tuberculosis in rhesus macaques.