Role of Tim3 in Mediating T Cell Exhaustion During Chronic Mycobacterium Tuberculosis Infection

Mycobacterium tuberculosis infection is one of the leading causes of mortality worldwide. One third of the population is estimated to be infected, however only 5-10% of those individuals can transmit the disease. While T cell immunity initially limits mycobacterium growth, it is unclear why T cell immunity fails to sterilize the infection and prevent subsequent recrudescence. One hypothesis is T cell exhaustion is mediating the failure of T cell immunity late during infection. Here we show the development of T cell exhaustion during chronic infection, and that the inhibitory receptor T cell-immunoglobulin and mucin domain containing 3 (TIM3) mediates the development of T cell exhaustion. TIM3 accumulates on the surface of T cells throughout the course of infection and there is a subsequent decrease in effector cytokine production, such as IL-2, TNFα, and IFNγ. Furthermore, antibody blockade of TIM3 restores T cell function and improves bacterial control. Our results show that TIM3 is mediating T cell exhaustion during chronic TB infection and leading to suboptimal bacterial control

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

Comprehensive immunophenotyping of solid tumor-infiltrating immune cells reveals the expression characteristics of LAG-3 and its ligands

BackgroundImmune cell expression profiling from patient samples is critical for the successful development of immuno-oncology agents and is useful to understand mechanism-of-action, to identify exploratory biomarkers predictive of response, and to guide treatment selection and combination therapy strategies. LAG-3 is an inhibitory immune checkpoint that can suppress antitumor T-cell responses and targeting LAG-3, in combination with PD-1, is a rational approach to enhance antitumor immunity that has recently demonstrated clinical success. Here, we sought to identify human immune cell subsets that express LAG-3 and its ligands, to characterize the marker expression profile of these subsets, and to investigate the potential relationship between LAG-3 expressing subsets and clinical outcomes to immuno-oncology therapies.MethodsComprehensive high-parameter immunophenotyping was performed using mass and flow cytometry of tumor-infiltrating lymphocytes (TILs) and peripheral blood mononuclear cells (PBMCs) from two independent cohorts of samples from patients with various solid tumor types. Profiling of circulating immune cells by single cell RNA-seq was conducted on samples from a clinical trial cohort of melanoma patients treated with immunotherapy.ResultsLAG-3 was most highly expressed by subsets of tumor-infiltrating CD8 T central memory (TCM) and effector memory (TEM) cells and was frequently co-expressed with PD-1. We determined that these PD-1+ LAG-3+ CD8 memory T cells exhibited a unique marker profile, with greater expression of activation (CD69, HLA-DR), inhibitory (TIM-3, TIGIT, CTLA-4) and stimulatory (4-1BB, ICOS) markers compared to cells that expressed only PD-1 or LAG-3, or that were negative for both checkpoints. In contrast to tumors, LAG-3 expression was more limited in circulating immune cells from healthy donors and solid tumor patients. Additionally, we found abundant expression of the LAG-3 ligands MHC-II and galectin-3 in diverse immune cell types, whereas FGL1 and LSECtin were minimally expressed by immune cells in the tumor microenvironment (TME). Lastly, we found an inverse relationship between baseline and on-treatment levels of circulating LAG3 transcript-expressing CD8 memory T cells and response to combination PD-1 and CTLA-4 blockade in a clinical trial cohort of melanoma patients profiled by scRNAseq.ConclusionsThese results provide insights into the nature of LAG-3- and ligand-expressing immune cells within the TME, and suggest a biological basis for informing mechanistic hypotheses, treatment selection strategies, and combination immunotherapy approaches to support continued development of dual PD-1 and LAG-3 blockade

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

TIM3 impairs clearance of <i>M</i>. <i>tuberculosis</i>.

<p>(A) Bacterial load in the lungs and spleens of WT BALB/c and TIM3^-/- mice at wk4 post-infection. (B) Percent survival in <i>M</i>. <i>tuberculosis</i> infected WT and TIM3^-/- mice. (C) Therapeutic protocol for TIM3 blockade in C57BL/6J mice. Beginning at week 10 post-infection, chronically infected mice were treated every third day for two weeks with isotype-matched control antibody or anti-TIM3 mAb. (D) Bacterial load in the lungs of C57BL/6J mice treated with isotype-matched control antibody or anti-TIM3 mAb. (E) Bacterial load in the lungs of TCRα^-/- mice treated with murine IgG1 control antibody (mIgG1) or anti-TIM3 mAb. No Tx, No treatment. Data is representative of 5 (A), 1 (B), 2 (D), independent experiments. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 for: (A) student’s t-test; (B) Log-rank (Mantel-Cox) test; (D) one-way Anova with Dunnett’s post-test. Bars represent mean ± SEM.</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

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

Two distinct subsets of TIM3 expressing T cells exhibit divergent molecular signatures.

<p>(A) CD4⁺ and CD8⁺ T cell populations sorted based on their TIM3 and PD1 expression was subjected to nanostring codeset and gene expression analyses. Heatmap of differentially expressed genes by TIM3^–PD1^–, TIM3^–PD1⁺, TIM3⁺PD1^– and TIM3⁺PD1⁺ CD4⁺ or CD8⁺ populations are shown. Blue indicates low relative expression and red, high relative expression. #1 and #2 indicate data from two independent experiments. (B) Fold expression of genes were normalized with respect to highest value among the four TIM3/PD1-expressing populations. Value of 100 indicates preferential expression of a gene set to a particular TIM3/PD1 expressing population and allows assessing population-specific gene patterns. Data is representative of 2 independent experiments.</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