iNKT cells are activated by Mtb-infected mϕ.

<p>(A) iNKT cells were cultured either alone, with uninfected thioglycollate-elicited peritoneal mϕ, or H37Rv-infected mϕ for 24 hours. Cells were stained for CD69 and CD25 and mϕ were distinguished from iNKT cells by F4/80 staining. (B) Fold change in CD69 and CD25 MFI on iNKT cells cultured with uninfected or H37Rv-infected mϕ compared to iNKT cells alone. Supernatant was harvested at 24 hours and IFNγ measured by ELISA. Error bars indicate mean ± SEM. *P<.05, **P<.01, ***P<.001. (One-way ANOVA with Dunnet's post-test, compared to iNKT cells alone). Data are representative of eight independent experiments. Mϕ, macrophage; UI, uninfected; , MOI titration, 1.5∶1, 3∶1, 6∶1.</p

IFNγ-independent antimicrobial effector function of iNKT cells is independent of cytolytic activity.

<p>(A–D) CFU assay d1, d5, and/or d7 post-infection with H37Rv-infected WT mϕ (A, C), Fas^−/− mϕ (B), or TNFR1/2^−/− mϕ (D) with WT iNKT cells (A, B, D), IFNγ^−/− iNKT cells (B–D), or Prf^−/− iNKT cells (A) added on d1 post infection at a 1∶1 ratio. (C) H37Rv-infected mϕ were treated with 0.1–10 µM of caspase-3 inhibitor peptide (Z-DEVD-FMK) 2 hours prior to addition of iNKT cells. Error bars indicate mean ± SEM. *P<0.05, **P<0.01 (One-way ANOVA with Dunnet's post-test, compared to d5 or d7 untreated mϕ.) Data are representative of three (A, C, D) or two (B) independent experiments with three or more replicates.</p

iNKT cell mediated control is CD1d-dependent but does not require IL-12 or IL-18.

<p>(A) Colony forming unit (CFU) assay measuring Mtb bacterial growth in H37Rv-infected WT mϕ on d1 and d5 post-infection. iNKT cells, anti-IL-12p40, anti-IL-18 blocking or isotype control antibodies were added on d1 after infection. (B) CFU assay d1 and d5 post-infection for H37Rv-infected MyD88^−/− mϕ with iNKT cells added on d1. (C, E) CFU assay d1 and d5 post-infection for H37Rv-infected WT and CD1d^−/− mϕ with iNKT cells added on d1 at a ratio of 1∶1 (C) or HMNC at a ratio of 3∶1 (E). (D) Compiled data from 6 independent experiments as described in (C). (F) H37Rv-infected mϕ after 24 hours. CD1d MFI fold change over uninfected mϕ either without or with iNKT cells. Error bars indicate mean ± SEM. *P<0.05, **P<0.01, ***P<0.001 (One-way ANOVA with Dunnet's post-test, compared to d5 untreated mϕ). +++P<.001 (unpaired Student's t-test). Data are representative of two (A, B) six (C, D), and one (E) independent experiment(s) with three or more replicates, or more than 12 independent experiments (F).</p

The antimicrobial effector function of iNKT cells is a soluble factor.

<p>Transwell CFU assay for H37Rv-infected WT mϕ in a 24-well plate with either WT or IFNγ^−/− iNKT cells added directly (cis) or 0.4 µm transwell inserts with WT or IFNγ^−/− iNKT cells in the presence of uninfected WT mϕ (trans) added on d1. Error bars indicate mean ± SEM. *P<0.05, **P<0.01 (One-way ANOVA with Dunnet's post-test, compared to d5 untreated mϕ.) Data are representative of two independent experiments with four replicates each.</p

iNKT cells produce GM-CSF during Mtb infection in a CD1d-dependent manner and it is critical for controlling Mtb growth.

<p>(A, B) IFNγ^−/− iNKT cells added to uninfected or H37Rv-infected WT (A, B) and CD1d^−/− mϕ (B). Murine GM-CSF measured in supernatant harvested after 24 hours. (C) % CFU reduction calculated from CFU assays for H37Rv-infected WT mϕ with IFNγ^−/− iNKT cells added on d1 in the presence of anti-GM-CSF blocking antibody (10–50 µg/ml) or isotype control. (D) J3N.5 human iNKT cell clone added to uninfected or H37Rv-infected U937 cells in the presence of anti-human-CD1d blocking antibody or isotype control. Human GM-CSF measured in supernatant harvested after 24 hours. (E, F) Lung mononuclear cells from WT Mtb-infected mice were incubated with brefeldin A for four hours at 37°C and then stained. iNKT cells were identified as TCRβ⁺ and CD1d-tetramer⁺. Percentage (E) and number (F) of iNKT cells producing GM-CSF and IFNγ. (G) % GM-CSF⁺, % IFNγ⁺, and CD69 MFI for iNKT cells transferred iv into WT or CD1d^−/− Mtb-infected hosts and iNKT cells cultured for 20 hours in basic media. Lung mononuclear cells were treated and stained as in (E, F). Transferred iNKT cells were distinguished from host cells by eFluor 450 staining. Error bars indicate mean ± SEM. +P<.05, ++P<.01, +++P<.001 (unpaired Student's t-test, GM-CSF⁺ versus IFNγ⁺ for respective time points (E, F), WT versus CD1d^−/− hosts (B, G)). **P<0.01, ***p<0.001 (One-way ANOVA with Tukey post-test (C), and Dunnet's post-test (A, D), compared to uninfected mϕ). Data are representative of or compiled from four (A, C), two (B), and three (D, G) independent experiments with three or more replicates each or two independent experiments with 5 mice each (E, F). , MOI titration approximately 0.5∶1–3∶1 (A, B) and 2∶1–10∶1 (D).</p

GM-CSF is sufficient for inhibition of Mtb growth.

<p>CFU assay for H37Rv-infected murine WT mϕ treated with recombinant GM-CSF from 0.001–10 ng/ml concentration on d1. Error bars indicate mean ± SEM. *P<.05, **P<0.01 (One way ANOVA with Dunnet's post-test, compared to d5 untreated mϕ). Data are representative of four independent experiments.</p

iNKT cell mediated control is independent of IFNγ.

<p>(A) CFU for H37Rv-infected WT mϕ with WT and IFNγ^−/− iNKT cells added on d1 post-infection. CFU were measured on d0 (mϕ alone) and on d1, d3, d5 and d7 post-infection. (B) CFU assay d1 and d5 post-infection for H37Rv-infected IFNγR^−/− mϕ with WT and IFNγ^−/− iNKT cells added on d1. (C) IFNγ and nitrite, a stable breakdown product of NO₂, measured for H37Rv-infected WT mϕ with WT and IFNγ^−/− iNKT cells added on d1 post-infection. (D) Compiled data from 5 independent experiments of the CFU assay d1 and d5 post-infection with H37Rv-infected WT and CD1d^−/− mϕ with IFNγ^−/− iNKT cells added on d1. Error bars indicate mean ± SEM. *P<0.05, **P<0.01, ***P<.001 (One-way ANOVA with Dunnet's post-test, compared to d3, d5, or d7 untreated mϕ). ++P<.01 (Unpaired Student's t-test). Data are representative of or cumulative from 13 (d5) and four (d7) (A), two (B) three (C), or four (D) independent experiments with three or more replicates.</p

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 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