TIM3 Mediates T Cell Exhaustion during Mycobacterium tuberculosis Infection

While T cell immunity initially limits Mycobacterium tuberculosis infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of M. tuberculosis infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic M. tuberculosis infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic M. tuberculosis infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis

Human and murine clonal CD8+ T cell expansions arise during tuberculosis because of TCR selection

The immune system can recognize virtually any antigen, yet T cell responses against several pathogens, including Mycobacterium tuberculosis, are restricted to a limited number of immunodominant epitopes. The host factors that affect immunodominance are incompletely understood. Whether immunodominant epitopes elicit protective CD8+ T cell responses or instead act as decoys to subvert immunity and allow pathogens to establish chronic infection is unknown. Here we show that anatomically distinct human granulomas contain clonally expanded CD8+ T cells with overlapping T cell receptor (TCR) repertoires. Similarly, the murine CD8+ T cell response against M. tuberculosis is dominated by TB10.44-11-specific T cells with extreme TCRß bias. Using a retro genic model of TB10.44-11-specific CD8+ Tcells, we show that TCR dominance can arise because of competition between clonotypes driven by differences in affinity. Finally, we demonstrate that TB10.4-specific CD8+ T cells mediate protection against tuberculosis, which requires interferon-? production and TAP1-dependent antigen presentation in vivo. Our study of how immunodominance, biased TCR repertoires, and protection are inter-related, provides a new way to measure the quality of T cell immunity, which if applied to vaccine evaluation, could enhance our understanding of how to elicit protective T cell immunity.This work was supported by the Portuguese Foundation for Science and Technology individual fellowship (CNA) www.fct.pt, a National Institutes of Health Grant R01 AI106725 (SMB) www.nih.gov, and a Center for AIDS Research Grant P30 AI 060354 (SMB) www.nih.gov. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

TIM3 Mediates T Cell Exhaustion during <i>Mycobacterium tuberculosis</i> Infection

<div><p>While T cell immunity initially limits <i>Mycobacterium tuberculosis</i> infection, why T cell immunity fails to sterilize the infection and allows recrudescence is not clear. One hypothesis is that T cell exhaustion impairs immunity and is detrimental to the outcome of <i>M</i>. <i>tuberculosis</i> infection. Here we provide functional evidence for the development T cell exhaustion during chronic TB. Second, we evaluate the role of the inhibitory receptor T cell immunoglobulin and mucin domain–containing-3 (TIM3) during chronic <i>M</i>. <i>tuberculosis</i> infection. We find that TIM3 expressing T cells accumulate during chronic infection, co-express other inhibitory receptors including PD1, produce less IL-2 and TNF but more IL-10, and are functionally exhausted. Finally, we show that TIM3 blockade restores T cell function and improves bacterial control, particularly in chronically infected susceptible mice. These data show that T cell immunity is suboptimal during chronic <i>M</i>. <i>tuberculosis</i> infection due to T cell exhaustion. Moreover, in chronically infected mice, treatment with anti-TIM3 mAb is an effective therapeutic strategy against tuberculosis.</p></div

Cytokine expression in antigen-specific CD4⁺ and CD8⁺ T cells is diminished following chronic <i>M</i>. <i>tuberculosis</i> infection.

<p>(A) Representative flow cytometry data showing the frequency of ESAT6-tetramer⁺ CD4⁺ T cells after <i>M</i>. <i>tuberculosis</i> infection. At each time point, lung cells were stimulated in vitro with the ESAT6_1-15 peptide or anti-CD3/CD28 mAbs to measure IFNγ and TNF expression. (B) The frequency of TB10.4-tetramer⁺ CD8⁺ T cells after <i>M</i>. <i>tuberculosis</i> infection. At each of time point, lung cells were stimulated in vitro with the TB10.4₄₋₁₁ peptide or anti-CD3/CD28 mAbs and IFNγ and TNF production was measured. (C) Representative flow cytometry data of ESAT6-tetramer CD4⁺ T cells at d19 or d84 post infection. IL-2, IFNγ, and TNF production after stimulation with ESAT6_1-15 peptide. (D) The fraction of ESAT-specific CD4⁺ T cells that make IL-2, IFNγ, and TNF on d19 (unfilled), w12 (striped), or w17 (filled) post infection. (E) The fraction of the number of cytokines being produced by ESAT6-specefic CD4⁺ T cells. (F) The fraction of CD4⁺ T cells producing IL-2, IFNγ, and TNF on d19 (unfilled), w12 (striped), or w17 (filled) post infection. (G) The percentage of IFNγ-producing CD4⁺ and CD8⁺ T cells that also make TNF over the course of infection. (H) The fraction of ESAT6-specific CD4⁺ T cells and bacterial burden in the lungs as d19, w12, and w17 post infection. All data is representative of three independent experiments with at least five mice per time point. *p<0.05, **p<0.01, ***p<0.001, one-way anova compared. Bars represent mean ± SEM. The “background” cytokine production, defined as cytokine production that occurs in the absence of specific stimulation was subtracted for each sample before calculations or normalizations were performed.</p

TIM3 blockade improves T cell function and disease outcome.

<p>(A) Representative contour plots for PD1 and TIM3 expression on pulmonary CD4⁺ and CD8⁺ T cells in susceptible C3HeB/FeJ mice, 4 and 12 weeks after <i>M</i>. <i>tuberculosis</i> infection. (B) Emergence of TIM3⁺PD1⁺, TIM3⁺PD1^- and TIM3^-PD1⁺ CD4⁺ and CD8⁺ T cells populations in C3HeB/FeJ mice following <i>M</i>. <i>tuberculosis</i> infection. Frequency of CD4⁺ or CD8⁺ T cells that are positive or negative for TIM3 and PD1 expression at different times post <i>M</i>. <i>tuberculosis</i> infection is plotted. Each point represents the mean ± SEM of 5 mice per strain per time point, and is representative of 2–3 independent experiments. (C) Protocol for TIM3 blockade in C3HeB/FeJ mice. C3HeB/FeJ mice were treated every third day for two weeks with isotype-matched control antibody or anti-TIM3 mAb, starting 4 weeks after <i>M</i>. <i>tuberculosis</i> infection. (D) Data from a representative experiment shows the bacterial loads in lung and spleen. (E) Cumulative results from all blocking experiments performed in C3HeB/FeJ mice representing 26 mice/group from six independent experiments. Each point represents lung CFU from an individual mouse. p<0.0001 by unpaired t-test after log₁₀ transformation. (F) The Δlog₁₀ protection [control CFU—treatment CFU] from eight independent experiments. Black circles, C57BL/6 experiments; white circles, C3HeB/FeJ experiments. (G) Production of IFNγ, TNF and IL-2 by CD4⁺ and CD8⁺ T cells from the lungs of infected C3HeB/FeJ mice that had been treated as described above. T cells were stimulated in vitro with ESAT6_53-71 or CFP10_32-39 peptides (recognized by CD4⁺ or CD8⁺ T cells, respectively) or anti-CD3/28 mAbs. Data is from 12–13 mice from three independent experiments tested by unpaired t-test: *, p<0.05; **, p<0.01; ***, p<0.001; ****, <0.0001. Bars represent median.</p

Distinct TIM3-expressing T cells display discrete functions.

<p>(A) CD4⁺ or CD8⁺ T cells from the lungs of mice 45 weeks after <i>M</i>. <i>tuberculosis</i> infection were stimulated with anti-CD3/CD28 mAbs in vitro and their expression of TIM3 and PD1, and production of IFNγ and TNF analyzed by flow cytometry. Representative gating showing the cytokine production by each of the TIM3/PD1-expressing T cell populations. (B) Frequency of TIM3/PD1-expressing CD4⁺ or CD8⁺ T cells that produce IFNγ⁺TNF⁺ or IL-10. (C) Expression of the inhibitory receptors PD1, LAG-3 or 2B4 by TIM3-expressing T cells that produce IFNγ or TNF. Data in A, B and C is representative of 3 independent experiments with 5–8 mice per time point per experiment. Bars represent mean ± SEM.</p

TB10.4-specific CD8+ T cells are selected during infection.

<p>(<b>a</b>) Frequency distribution of CDR3β amino acid length of TB10.4_4-11-specific CD8⁺ T cells from the lungs of <i>M</i>. <i>tuberculosis</i> infected C57BL/6J mice. (<b>b</b>) Consensus analysis of the CDR3β amino acid sequence of TB10.4_4-11-specific CD8⁺ T cells with 14 amino acids in length. (<b>c</b>) VDJ DNA rearrangements for the public CDR3β CASSLDRENSDYTF found in four different C57BL/6J mice, showing Vβ (black), N (red), Dβ (blue), and Jβ (black) sequences. The count and frequency for each sequence in the respective lung is also shown. The box highlights the nucleotides that encode the conserved aspartic acid (Asp, “D”) and arginine (Arg, “R”) residues. (<b>d</b>) Frequency of human TCRs containing the “DREN” motif among normal PBMC or CD8⁺ T cells from TB patients. Each point represents a unique clonotype and their corresponding CDR3β amino acid sequences are shown for some. (<b>e</b>) Ratio of unique amino acid clones to nucleotide sequences in T cells from naïve and infected C57BL/6J mice. TCR sequences were analyzed from uninfected ‘B6 spleen’ (n = 3 mice); infected ‘Mtb lung’ (TB10.4_4-11-specific CD8⁺ T cells; n = 6 mice); or the following subsets of sequences: ‘frequent’ (>1% of the TB10-specific sequences) or ‘shared sequences’ (TB10.4_4-11-specific TCRs present in at least 2 mice). ****, p < 0.05 by one-way ANOVA and Holm-Sidak’s multiple comparison test.</p

Retrogenic T cell priming and acquisition of effector functions.

<p>(<b>a</b>) Kinetic analysis of frequency (filled circles) and number (opened circles) of activated (CD44^HiCD62L^Lo) Rg cells in the draining LN (left panel) and lung (right panel) following adoptive transfer into mice infected with <i>M</i>. <i>tuberculosis</i>. (<b>b</b>) Kinetic analysis of frequency of divided Rg cells in the draining LN, lung and spleen following adoptive transfer into mice infected with <i>M</i>. <i>tuberculosis</i>. (<b>c</b>) Kinetic analysis of frequency of IFNγ-producing Rg cells in the draining LN (left panel) and lung (right panel) following adoptive transfer into mice infected with <i>M</i>. <i>tuberculosis</i>. Data are representative from two (b) or three (a, c) independent experiments, each with 5 mice per group. (<b>a,c</b>) One way ANOVA with Dunnett’s post test to compare differences over time (vs. day 7 [<b>a</b>] or d11 [<b>c</b>]) time points. P<0.05 indicated by asterisks (phenotype or IFNγ) or hash marks (cell numbers). (<b>b</b>) One way ANOVA with Tukey’s post test to compare differences in proliferation between lung, LN and spleen; p<0.05 indicated by asterisks.</p