Role of Granulocyte-Macrophage Colony-Stimulating Factor Production by T Cells during Mycobacterium tuberculosis Infection

ABSTRACT Mice deficient for granulocyte-macrophage colony-stimulating factor (GM-CSF−/−) are highly susceptible to infection with Mycobacterium tuberculosis, and clinical data have shown that anti-GM-CSF neutralizing antibodies can lead to increased susceptibility to tuberculosis in otherwise healthy people. GM-CSF activates human and murine macrophages to inhibit intracellular M. tuberculosis growth. We have previously shown that GM-CSF produced by iNKT cells inhibits growth of M. tuberculosis. However, the more general role of T cell-derived GM-CSF during infection has not been defined and how GM-CSF activates macrophages to inhibit bacterial growth is unknown. Here we demonstrate that, in addition to nonconventional T cells, conventional T cells also produce GM-CSF during M. tuberculosis infection. Early during infection, nonconventional iNKT cells and γδ T cells are the main source of GM-CSF, a role subsequently assumed by conventional CD4+ T cells as the infection progresses. M. tuberculosis-specific T cells producing GM-CSF are also detected in the peripheral blood of infected people. Under conditions where nonhematopoietic production of GM-CSF is deficient, T cell production of GM-CSF is protective and required for control of M. tuberculosis infection. However, GM-CSF is not required for T cell-mediated protection in settings where GM-CSF is produced by other cell types. Finally, using an in vitro macrophage infection model, we demonstrate that GM-CSF inhibition of M. tuberculosis growth requires the expression of peroxisome proliferator-activated receptor gamma (PPARγ). Thus, we identified GM-CSF production as a novel T cell effector function. These findings suggest that a strategy augmenting T cell production of GM-CSF could enhance host resistance against M. tuberculosis

Path-seq identifies an essential mycolate remodeling program for mycobacterial host adaptation

The success of Mycobacterium tuberculosis (MTB) stems from its ability to remain hidden from the immune system within macrophages. Here, we report a new technology (Path-seq) to sequence miniscule amounts of MTB transcripts within up to million-fold excess host RNA Using Path-seq and regulatory network analyses, we have discovered a novel transcriptional program for in vivo mycobacterial cell wall remodeling when the pathogen infects alveolar macrophages in mice. We have discovered that MadR transcriptionally modulates two mycolic acid desaturases desA1/desA2 to initially promote cell wall remodeling upon in vitro macrophage infection and, subsequently, reduces mycolate biosynthesis upon entering dormancy. We demonstrate that disrupting MadR program is lethal to diverse mycobacteria making this evolutionarily conserved regulator a prime antitubercular target for both early and late stages of infection

EspA Acts as a Critical Mediator of ESX1-Dependent Virulence in Mycobacterium tuberculosis by Affecting Bacterial Cell Wall Integrity

Mycobacterium tuberculosis (Mtb) requires the ESX1 specialized protein secretion system for virulence, for triggering cytosolic immune surveillance pathways, and for priming an optimal CD8+ T cell response. This suggests that ESX1 might act primarily by destabilizing the phagosomal membrane that surrounds the bacterium. However, identifying the primary function of the ESX1 system has been difficult because deletion of any substrate inhibits the secretion of all known substrates, thereby abolishing all ESX1 activity. Here we demonstrate that the ESX1 substrate EspA forms a disulfide bonded homodimer after secretion. By disrupting EspA disulfide bond formation, we have dissociated virulence from other known ESX1-mediated activities. Inhibition of EspA disulfide bond formation does not inhibit ESX1 secretion, ESX1-dependent stimulation of the cytosolic pattern receptors in the infected macrophage or the ability of Mtb to prime an adaptive immune response to ESX1 substrates. However, blocking EspA disulfide bond formation severely attenuates the ability of Mtb to survive and cause disease in mice. Strikingly, we show that inhibition of EspA disulfide bond formation also significantly compromises the stability of the mycobacterial cell wall, as does deletion of the ESX1 locus or individual components of the ESX1 system. Thus, we demonstrate that EspA is a major determinant of ESX1-mediated virulence independent of its function in ESX1 secretion. We propose that ESX1 and EspA play central roles in the virulence of Mtb in vivo because they alter the integrity of the mycobacterial cell wall

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

iNKT cell production of GM-CSF controls Mycobacterium tuberculosis

Invariant natural killer T (iNKT) cells are activated during infection, but how they limit microbial growth is unknown in most cases. We investigated how iNKT cells suppress intracellular Mycobacterium tuberculosis (Mtb) replication. When co-cultured with infected macrophages, iNKT cell activation, as measured by CD25 upregulation and IFNγ production, was primarily driven by IL-12 and IL-18. In contrast, iNKT cell control of Mtb growth was CD1d-dependent, and did not require IL-12, IL-18, or IFNγ. This demonstrated that conventional activation markers did not correlate with iNKT cell effector function during Mtb infection. iNKT cell control of Mtb replication was also independent of TNF and cell-mediated cytotoxicity. By dissociating cytokine-driven activation and CD1d-restricted effector function, we uncovered a novel mediator of iNKT cell antimicrobial activity: GM-CSF. iNKT cells produced GM-CSF in vitro and in vivo in a CD1d-dependent manner during Mtb infection, and GM-CSF was both necessary and sufficient to control Mtb growth. Here, we have identified GM-CSF production as a novel iNKT cell antimicrobial effector function and uncovered a potential role for GM-CSF in T cell immunity against Mtb.This work was supported by National Institutes of Health (NIH) R01HL080330 to SMB, T32AR007530 supporting ACR, the American Lung Association postdoctoral research training fellowship, RT-123085-N, and Harvard University Center for AIDS Research (CFAR) Scholar Award, an NIH funded program, P30 AI060354, to PJ, and a PhD fellowship from Fundacao para a Ciencia e Tecnologia (Portugal) to CNA. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A contained Mycobacterium tuberculosis mouse infection model predicts active disease and containment in humans

Previous studies have identified whole-blood transcriptional risk and disease signatures for Tuberculosis (TB); however, several lines of evidence suggest that these signatures primarily reflect bacterial burden, which increases prior to symptomatic disease. We found that the peripheral blood transcriptome of mice with contained Mycobacterium tuberculosis infection (CMTB) has striking similarities to that of humans with active TB and that a signature derived from these mice predicts human disease with comparable accuracy to signatures derived directly from humans. A set of genes associated with immune defense are upregulated in CMTB mice but not in humans with active TB suggesting that their upregulation is associated with bacterial containment. A signature comprised of these genes predicts both protection from TB disease and successful treatment at early time points where current signatures are not predictive. These results suggest that detailed study of the CMTB mouse model may enable identification of biomarkers for human TB

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

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

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