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Although a role for TLR2 on T cells has been indicated in prior studies, in vivo stimulation of TLR2 on T cells by Mtb and its impact on Mtb infection has not been tested. Furthermore, it is not known if the enhanced susceptibility to Mtb of Tlr2 gene knockout (ko) mice is due to its role in macrophages, on T cells or both. To address TLR2 on T cells, we generated Tlr2fl/flxCd4cre/cre mice, which lack expression of TLR2 on both CD4 and CD8 T cells, to study the in vivo role of TLR2 on T cells after aerosol infection with virulent Mtb. Deletion of TLR2 in CD4+ and CD8+ T cells reduces their ability to be co-stimulated by TLR2 ligands for cytokine production. These include both pro- (IFN-g, TNF-a) and anti-inflammatory cytokines (IL-10). Deletion of TLR2 in T cells did not affect early control but did result in decreased late control of Mtb in the lungs of infected mice. This suggests that T cell co-stimulation by mycobacterial TLR2 ligands in vivo is important for control of infection during the chronic phase of Mtb infection in the lung.

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4+ T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4+ T-cell differentiation and function, we conducted a gene expression analysis of murine naive CD4+ T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naive CD4+ T-cells co-stimulated via TLR2 showed a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up-regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Under non-polarizing conditions, TLR2 engagement on CD4+ T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4+ T-cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells. This article is protected by copyright. All rights reserved

Mycobacterium tuberculosis utilizes multiple mechanisms to evade host immune responses, and inhibition of effector CD4+ T cell responses by M. tuberculosis may contribute to immune evasion. TCR signaling is inhibited by M. tuberculosis cell envelope lipoglycans, such as lipoarabinomannan and lipomannan, but a mechanism for lipoglycans to traffic from M. tuberculosis within infected macrophages to reach T cells is unknown. In these studies, we found that membrane vesicles produced by M. tuberculosis and released from infected macrophages inhibited the activation of CD4+ T cells, as indicated by reduced production of IL-2 and reduced T cell proliferation. Flow cytometry and Western blot demonstrated that lipoglycans from M. tuberculosis–derived bacterial vesicles (BVs) are transferred to T cells, where they inhibit T cell responses. Stimulation of CD4+ T cells in the presence of BVs induced expression of GRAIL, a marker of T cell anergy; upon restimulation, these T cells showed reduced ability to proliferate, confirming a state of T cell anergy. Furthermore, lipoarabinomannan was associated with T cells after their incubation with infected macrophages in vitro and when T cells were isolated from lungs of M. tuberculosis–infected mice, confirming the occurrence of lipoarabinomannan trafficking to T cells in vivo. These studies demonstrate a novel mechanism for the direct regulation of CD4+ T cells by M. tuberculosis lipoglycans conveyed by BVs that are produced by M. tuberculosis and released from infected macrophages. These lipoglycans are transferred to T cells to inhibit T cell responses, providing a mechanism that may promote immune evasion.

Little is known about whether pathogen invasion of neural tissue is affected by immune-based mechanisms in endothelial cells. We examined the effects of endothelial cell CD40 on Toxoplasma gondii invasion of the retina and brain, organs seeded hematogenously. T. gondii circulates in the bloodstream within infected leukocytes (including monocytes and dendritic cells) and as extracellular tachyzoites. After T. gondii infection, mice that expressed CD40 restricted to endothelial cells exhibited diminished parasite loads and histopathology in the retina and brain. These mice also had lower parasite loads in the retina and brain after intravenous (i.v.) injection of infected monocytes or dendritic cells. The protective effect of endothelial cell CD40 was not explained by changes in cellular or humoral immunity, reduced transmigration of leukocytes into neural tissue, or reduced invasion by extracellular parasites. Circulating T. gondii-infected leukocytes (dendritic cells used as a model) led to infection of neural endothelial cells. The number of foci of infection in these cells were reduced if endothelial cells expressed CD40. Infected dendritic cells and macrophages expressed membrane-associated inducible Hsp70. Infected leukocytes triggered Hsp70-dependent autophagy in CD40(+) endothelial cells and anti-T. gondii activity dependent on ULK1 and beclin 1. Reduced parasite load in the retina and brain not only required CD40 expression in endothelial cells but was also dependent on beclin 1 and the expression of inducible Hsp70 in dendritic cells. These studies suggest that during endothelial cell-leukocyte interaction, CD40 restricts T. gondii invasion of neural tissue through a mechanism that appears mediated by endothelial cell anti-parasitic activity stimulated by Hsp70.

Bacteria can utilize a mammalian host siderophore to usurp host iron; however, the host can respond by down-regulating siderophore expression and up-regulating expression of an inhibitory siderophore-binding protein., Competition for iron influences host–pathogen interactions. Pathogens secrete small iron-binding moieties, siderophores, to acquire host iron. In response, the host secretes siderophore-binding proteins, such as lipocalin 24p3, which limit siderophore-mediated iron import into bacteria. Mammals produce 2,5-dihydroxy benzoic acid, a compound that resembles a bacterial siderophore. Our data suggest that bacteria use both mammalian and bacterial siderophores. In support of this idea, supplementation with mammalian siderophore enhances bacterial growth in vitro. In addition, mice lacking the mammalian siderophore resist E. coli infection. Finally, we show that the host responds to infection by suppressing siderophore synthesis while up-regulating lipocalin 24p3 expression via TLR signaling. Thus, reciprocal regulation of 24p3 and mammalian siderophore is a protective mechanism limiting microbial access to iron.

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4(+) T cells and upregulate TCR-triggered IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4(+) T-cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T-cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4(+) T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T-cell TLR2 in priming of CD4(+) T cells in vivo, naive MTB Ag85B-specific TCR transgenic CD4(+) T cells (P25 TCR-Tg) were adoptively transferred into Tlr2(-/-) recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3 Cys-SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN-γ-secreting P25 TCR-Tg T cells 1 week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4(+) T cells increases MTB Ag-specific responses and may contribute to protection against MTB infection.

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on CD4+ T cells and up-regulate T-cell receptor (TCR) triggered Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4+ T cell differentiation and function, we conducted a gene expression analysis of murine naïve CD4+ T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4+ T- cells co-stimulated via TLR2 show a significant up-regulation of Il9 mRNA compared to cells co-stimulated via CD28. Under TH9 differentiation, we observed up regulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants in the presence of TLR2 agonist both in polyclonal and Ag85B cognate peptide specific stimulations. Human CD4+ T cells also showed increased in both IL9 mRNA expression and IL-9 cytokine secretion in response to TLR2 agonist under polarized condition. Under non-polarizing conditions, TLR2 engagement on CD4+ T-cells had minimal effect on IL-9 secretion and TH9 differentiation, likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation. We also report that, TLR2 signaling in CD4+ T-cells increased expression of transcription factors BATF and PU.1, known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells.

Objective: Infection by MTB or exposure to MTB constituents is associated with intense microbial stimulation of the immune system, through both antigenic and TLR components, and induction of a milieu that is rich in proinflammatory/ anti-inflammatory cytokines. Here, we addressed the basis of induced regulatory T-cell (iT-reg) expansion in response to MTB stimulation, in the absence of prior T cell antigen responsiveness. Methods: PBMC from HIV-1 un-infected TST negative and TST positive control subjects were stimulated by virulent MTB H37Rv lysate (L), a French press preparation of MTB that includes all bacterial components. Phenotype of MTB H37RvL induced iT-reg was assessed using immunostaining and flow cytometry. Functional capacity of iT-reg was assessed using 3H-Thymidine incorporation and IFNγ production of non-adherent T cells (NAC) in the presence or absence of iT-reg in corresponding culture supernatants in response to TCR stimulation. Realtime PCR was used to assess IDO and FoxP3 mRNA expression. Results: The capacity of MTB H37RvL to induce CD4+CD25hi+ Foxp3+ T-cells in PBMC from TST negative subjects was robust (p

Chemical regulation of macrophage function is one key strategy for developing host-directed adjuvant therapies for tuberculosis (TB). A critical step to develop these therapies is the identification and characterization of specific macrophage molecules and pathways with a high potential to serve as drug targets. Using a barcoded lentivirus-based pooled short-hairpin RNA (shRNA) library combined with next generation sequencing, we identified 205 silenced host genes highly enriched in mycobacteria-resistant macrophages. Twenty-one of these “hits” belonged to the oxidoreductase functional category. NAD(P)H:quinone oxidoreductase 1 (NQO1) was the top oxidoreductase “hit”. NQO1 expression was increased after mycobacterial infection, and NQO1 knockdown increased macrophage differentiation, NF-κB activation, and the secretion of pro-inflammatory cytokines TNF-α and IL-1β in response to infection. This suggests that mycobacteria hijacks NQO1 to down-regulate pro-inflammatory and anti-bacterial functions. The competitive inhibitor of NQO1 dicoumarol synergized with rifampin to promote intracellular killing of mycobacteria. Thus, NQO1 is a new host target in mycobacterial infection that could potentially be exploited to increase antibiotic efficacy in vivo. Our findings also suggest that pooled shRNA libraries could be valuable tools for genome-wide screening in the search for novel druggable host targets for adjunctive TB therapies.

Immune evasion is required for Mycobacterium tuberculosis to survive in the face of robust CD4+ T cell responses. We have shown previously that M. tuberculosis cell wall glycolipids, including mannose capped lipoarabinomannan (ManLAM), directly inhibit polyclonal murine CD4+ T cell activation by blocking ZAP-70 phosphorylation. We extended these studies to antigen-specific murine CD4+ T cells and primary human T cells and found that ManLAM inhibited them as well. Lck and LAT phosphorylation also were inhibited by ManLAM without affecting their localization to lipid rafts. Inhibition of proximal TCR signaling was temperature sensitive, suggesting that ManLAM insertion into T cell membranes was required. Thus, M. tuberculosis ManLAM inhibits antigen-specific CD4+ T cell activation by interfering with very early events in TCR signaling through ManLAM’s insertion in T cell membranes.

Our recent microarray analysis of infected human alveolar macrophages (AMs) found serine protease inhibitor 9 (PI-9) to be the most prominently expressed of a cluster of apoptosis-associated genes induced by virulent Mycobacterium tuberculosis. In the current study, we show that induction of PI-9 occurs within hours of infection with M. tuberculosis H37Rv and is maintained through 7 days of infection in both AMs and blood monocytes. Inhibition of PI-9 by small inhibitory RNA decreased M. tuberculosis–induced expression of the antiapoptotic molecule Bcl-2 and resulted in a corresponding increase in production of caspase 3, a terminal effector molecule of apoptosis. Further, PI-9 small inhibitory RNA mediated a significant reduction in the subsequent survival of M. tuberculosis within AMs. Thus PI-9 induction within human mononuclear phagocytes by virulent M. tuberculosis serves to protect these primary targets of infection from elimination by apoptosis and thereby promotes intracellular survival of the organism.

Mtb regulates many aspects of the host immune response, including CD4+ T lymphocyte responses that are essential for protective immunity to Mtb, and Mtb effects on the immune system are paradoxical, having the capacity to inhibit (immune evasion) and to activate (adjuvant effect) immune cells. Mtb regulates CD4+ T cells indirectly (e.g., by manipulation of APC function) and directly, via integrins and TLRs expressed on T cells. We now report that previously uncharacterized Mtb protein Rv2468c/MT2543 can directly regulate human CD4+ T cell activation by delivering costimulatory signals. When combined with TCR stimulation (e.g., anti-CD3), Rv2468c functioned as a direct costimulator for CD4+ T cells, inducing IFN-γ secretion and T cell proliferation. Studies with blocking antibodies and soluble RGD motifs demonstrated that Rv2468c engaged integrin VLA-5 (α5β1) on CD4+ T cells through its FN-like RGD motif. Costimulation by Rv2468c induced phosphorylation of FAKs and Pyk2. These results reveal that by expressing molecules that mimic host protein motifs, Mtb can directly engage receptors on CD4+ T cells and regulate their function. Rv2468c-induced costimulation of CD4+ T cells could have implications for TB immune pathogenesis and Mtb adjuvant effect.

Knockout of lprG results in decreased virulence of Mycobacterium tuberculosis (MTB) in mice. MTB lipoprotein LprG has TLR2 agonist activity, which is thought to be dependent on its N-terminal triacylation. Unexpectedly, here we find that nonacylated LprG retains TLR2 activity. Moreover, we show LprG association with triacylated glycolipid TLR2 agonists lipoarabinomannan, lipomannan and phosphatidylinositol mannosides (which share core structures). Binding of triacylated species was specific to LprG (not LprA) and increased LprG TLR2 agonist activity; conversely, association of glycolipids with LprG enhanced their recognition by TLR2. The crystal structure of LprG in complex with phosphatidylinositol mannoside revealed a hydrophobic pocket that accommodates the three alkyl chains of the ligand. In conclusion, we demonstrate a glycolipid binding function of LprG that enhances recognition of triacylated MTB glycolipids by TLR2 and may affect glycolipid assembly or transport for bacterial cell wall biogenesis.

Vdelta2+ T cells, the major circulating T-cell receptor-gammadelta-positive (TCR-gammadelta+) T-cell subset in healthy adults, are involved in immunity against many microbial pathogens including Mycobacterium tuberculosis. Vdelta2+ T cells recognize small phosphorylated metabolites (phosphoantigens), expand in response to whole M. tuberculosis bacilli, and complement the protective functions of CD4+ T cells. CD4+ CD25(high) Foxp3+ T cells (Tregs) comprise 5-10% of circulating T cells and are increased in patients with active tuberculosis (TB). We investigated whether, in addition to their known role in suppressing TCR-alphabeta+ lymphocytes, Tregs suppress Vdelta2+ T-cell function. We found that depletion of Tregs from peripheral blood mononuclear cells increased Vdelta2+ T-cell expansion in response to M. tuberculosis (H37Ra) in tuberculin-skin-test-positive donors. We developed a suppression assay with fluorescence-activated cell sorting-purified Tregs and Vdelta2+ T cells by coincubating the two cell types at a 1 : 1 ratio. The Tregs partially suppressed interferon-gamma secretion by Vdelta2+ T cells in response to anti-CD3 monoclonal antibody plus interleukin-2 (IL-2). In addition, Tregs downregulated the Vdelta2+ T-cell interferon-gamma responses induced by phosphoantigen (BrHPP) and IL-2. Under the latter conditions there was no TCR stimulus for Tregs and therefore IL-2 probably triggered suppressor activity. Addition of purified protein derivative (PPD) increased the suppression of Vdelta2+ T cells, suggesting that PPD activated antigen-specific Tregs. Our study provides evidence that Tregs suppress both anti-CD3 and antigen-driven Vdelta2+ T-cell activation. Antigen-specific Tregs may therefore contribute to the Vdelta2+ T-cell functional deficiencies observed in TB.

Mycobacterium tuberculosis cell wall glycolipid, lipoarabinomannan, can inhibit CD4+ T cell activation by downregulating the phosphorylation of key proximal TCR signaling molecules: Lck, CD3ζ, ZAP70, and LAT. Inhibition of proximal TCR signaling can result in T cell anergy, in which T cells are inactivated following an Ag encounter, yet remain viable and hyporesponsive. We tested whether mannose-capped lipoarabinomannan (LAM)-induced inhibition of CD4+ T cell activation resulted in CD4+ T cell anergy. The presence of LAM during primary stimulation of P25 TCR-transgenic murine CD4+ T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25 TCR-transgenic CD4+ T cells primed in the presence of LAM also exhibited decreased response upon restimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hyporesponsiveness to restimulation was not due to apoptosis, generation of Foxp3-positive regulatory T cells, or inhibitory cytokines. Acquisition of the anergic phenotype correlated with upregulation of gene related to anergy in lymphocytes (GRAIL) protein in CD4+ T cells. Inhibition of human CD4+ T cell activation by LAM also was associated with increased GRAIL expression. Small interfering RNA–mediated knockdown of GRAIL before LAM treatment abrogated LAM-induced hyporesponsiveness. In addition, exogenous IL-2 reversed defective proliferation by downregulating GRAIL expression. These results demonstrate that LAM upregulates GRAIL to induce anergy in Ag-reactive CD4+ T cells. Induction of CD4+ T cell anergy by LAM may represent one mechanism by which M. tuberculosis evades T cell recognition.

The pathological hallmark of the host response to Mycobacterium tuberculosis is the granuloma where T cells and macrophages interact with the extracellular matrix (ECM) to control the infection. Recruitment and retention of T cells within inflamed tissues depend on adhesion to the ECM. T cells use integrins to adhere to the ECM, and fibronectin (FN) is one of its major components. We have found that the major M. tuberculosis cell wall glycolipid, phosphatidylinositol mannoside (PIM), induces homotypic adhesion of human CD4+ T cells and T cell adhesion to immobilized FN. Treatment with EDTA and cytochalasin D prevented PIM-induced T cell adhesion. PIM-induced T cell adhesion to FN was blocked with mAbs against α5 integrin chain and with RGD-containing peptides. α5β1 (VLA-5) is one of two major FN receptors on T cells. PIM was found to bind directly to purified human VLA-5. Thus, PIM interacts directly with VLA-5 on CD4+ T lymphocytes, inducing activation of the integrin, and promoting adhesion to the ECM glycoprotein, FN. This is the first report of direct binding of a M. tuberculosis molecule to a receptor on human T cells resulting in a change in CD4+ T cell function.

Mycobacterium tuberculosisresides in phagosomes inside macrophages. In this study, we analyzed the kinetics and location ofM. tuberculosispeptide-major histocompatibility complex class II (MHC-II) complexes inM. tuberculosis-infected human macrophages.M. tuberculosispeptide-MHC-II complexes were detected with polyclonal autologousM. tuberculosis-specific CD4+T cells or F9A6 T hybridoma cells specific forM. tuberculosisantigen (Ag) 85B (96-111). Macrophages processed heat-killedM. tuberculosismore rapidly and efficiently than liveM. tuberculosis. To determine whereM. tuberculosispeptide-MHC-II complexes were formed intracellularly, macrophages incubated with heat-killedM. tuberculosiswere homogenized, and subcellular compartments were separated on Percoll density gradients analyzed with T cells. In THP-1 cells,M. tuberculosisAg 85B (96- 111)-DR1 complexes appeared initially in phagosomes, followed by MHC class II compartment (MIIC) and the plasma membrane fractions. In monocyte-derived macrophages,M. tuberculosispeptide-MHC-II complexes appeared only in MIIC fractions and subsequently on the plasma membrane. Although phagosomes from both cell types acquired lysosome-associated membrane protein 1 (LAMP-1) and MHC-II, THP-1 phagosomes that support formation ofM. tuberculosispeptide-MHC-II complexes had increased levels of both LAMP-1 and MHC-II. Thus,M. tuberculosisphagosomes with high levels of MHC-II and LAMP-1 and MIIC both have the potential to form peptide-MHC-II complexes fromM. tuberculosisantigens in human macrophages.

γδ T cells represent 1%–5% of circulating human T cells [1, 2]. Vγ9+Vδ2+ γδ T cells (Vδ2+ T cells), the dominant circulating γδ T cell subset in adults, are activated in response to intracellular pathogens, such as Mycobacterium tuberculosis [3–7]. Vδ2+ T cells secrete interferon (IFN)–γ and have cytotoxic T lymphocyte and natural killer cell–like effector function. Vδ2+ T cells recognize small organic phosphate antigens (phosphoantigens) and other nonpeptidic molecules. The tuberculosis antigens found inside M. tuberculosis bacilli were among the first phosphoantigens to be purified [8, 9]. Others include isopentenyl pyrophosphate (IPP) [10, 11] and 3-formyl-1-butyl pyrophosphate [12]. The synthetic analogue bromohydrin pyrophosphate (BrHPP) activates Vδ2+ T cells at nanomolar concentrations [13, 14]. Phosphoantigens are produced by many pathogens and are recognized in a non–major histocompatibility complex–restricted manner. Thus, Vδ2+ T cells can provide a link between innate and adaptive immunity to a wide range of pathogens. In primates, Vδ2+ T cell responses increase during primary mycobacterial infection and increase further on challenge after vaccination with bacille Calmette-Guerin (BCG) [15, 16]. BCG vaccination of adults increases Vδ2+ T cell responses [17]. Vδ2+ T cells are activated in vitro by M. tuberculosis [18]. Studies investigating total γδ or Vδ2+ T cell function in peripheral blood and lungs of patients with tuberculosis, their household contacts, and healthy subjects have provided contradictory results. Some studies demonstrated increased numbers of γδ T cells in peripheral blood of patients with tuberculosis [19, 20], and others demonstrated decreased γδ T cell responses to M. tuberculosis antigens in patients with tuberculosis [21–27]. This variability was likely due to the different antigens used to activate γδ T cells, the stage of infection or disease, and the age of the subjects. Alterations in the γδ T cell repertoire and its function also have been described in HIV-1–positive patients and simian immunodeficiency virus–infected primates [28–32]. Vδ2+ T cell activation is dependent on interleukin (IL)–2 produced by CD4+ T cells, which is diminished in HIV-1 infection [33]. Vδ2+ T cells lyse HIV-1–infected cells and block entry of HIV-1 via β-chemokine secretion [34]. Therefore, decreased Vδ2+ T cell function may contribute not only to the loss of anti–HIV-1 immunity but also to diminished immune protection against M. tuberculosis. Most studies of γδ T cell function have applied multicolor flow cytometry to enumerate Vδ2+ T cells in mixed populations of peripheral-blood mononuclear cells (PBMCs) stimulated with crude microbial antigens and rely on IL-2 produced by CD4+ T cells for Vδ2+ T cell expansion. Flow-cytometric assays are not optimal for field studies in settings of endemicity for tuberculosis. A growing literature has validated the use of whole-blood assays to measure immune function in population-based studies of M. tuberculosis infection and disease [35–38]. We have developed a simple whole-blood assay that measures IFN-γ secretion in response to the phosphoantigen BrHPP in the presence of IL-2 to assess Vδ2+ T cell function independent of CD4+ T cell responses. This assay was used to assess Vδ2+ T cell responses in M. tuberculosis– and HIV-1–positive patients in the United States and Uganda.

Mycobacterium tuberculosis (Mtb) cell wall glycolipid mannose‐capped lipoarabinomannan (ManLAM) inhibits CD4+ T‐cell activation by inhibiting proximal T‐cell receptor (TCR) signaling when activated by anti‐CD3. To understand the impact of ManLAM on CD4+ T‐cell function when both the TCR–CD3 complex and major costimulator CD28 are engaged, we performed label‐free quantitative MS and network analysis. Mixed‐effect model analysis of peptide intensity identified 149 unique peptides representing 131 proteins that were differentially regulated by ManLAM in anti‐CD3‐ and anti‐CD28‐activated CD4+ T cells. Crosstalker, a novel network analysis tool identified dysregulated translation, TCA cycle, and RNA metabolism network modules. PCNA, Akt, mTOR, and UBC were found to be bridge node proteins connecting these modules of dysregulated proteins. Altered PCNA expression and cell cycle analysis showed arrest at the G2M phase. Western blot confirmed that ManLAM inhibited Akt and mTOR phosphorylation, and decreased expression of deubiquitinating enzymes Usp9x and Otub1. Decreased NF‐κB phosphorylation suggested interference with CD28 signaling through inhibition of the Usp9x‐Akt‐mTOR pathway. Thus, ManLAM induced global changes in the CD4+ T‐cell proteome by affecting Akt‐mTOR signaling, resulting in broad functional impairment of CD4+ T‐cell activation beyond inhibition of proximal TCR–CD3 signaling.

Mycobacterium tuberculosis is the etiologic agent of human tuberculosis and is estimated to infect one-third of the world's population. Control of M. tuberculosis requires T cells and macrophages. T-cell function is modulated by the cytokine environment, which in mycobacterial infection is a balance of proinflammatory (interleukin-1 [IL-1], IL-6, IL-8, IL-12, and tumor necrosis factor alpha) and inhibitory (IL-10 and transforming growth factor β [TGF-β]) cytokines. IL-10 and TGF-β are produced by M. tuberculosis -infected macrophages. The effect of IL-10 and TGF-β on M. tuberculosis -reactive human CD4 + and γδ T cells, the two major human T-cell subsets activated by M. tuberculosis , was investigated. Both IL-10 and TGF-β inhibited proliferation and gamma interferon production by CD4 + and γδ T cells. IL-10 was a more potent inhibitor than TGF-β for both T-cell subsets. Combinations of IL-10 and TGF-β did not result in additive or synergistic inhibition. IL-10 inhibited γδ and CD4 + T cells directly and inhibited monocyte antigen-presenting cell (APC) function for CD4 + T cells and, to a lesser extent, for γδ T cells. TGF-β inhibited both CD4 + and γδ T cells directly and had little effect on APC function for γδ and CD4 + T cells. IL-10 down-regulated major histocompatibility complex (MHC) class I, MHC class II, CD40, B7-1, and B7-2 expression on M. tuberculosis -infected monocytes to a greater extent than TGF-β. Neither cytokine affected the uptake of M. tuberculosis by monocytes. Thus, IL-10 and TGF-β both inhibited CD4 + and γδ T cells but differed in the mechanism used to inhibit T-cell responses to M. tuberculosis .

Tuberculosis (TB) is the leading cause of mortality among those infected with human immunodeficiency virus (HIV-1) worldwide. HIV-1 load and heterogeneity are increased both locally and systemically in active TB. Mycobacterium tuberculosis (MTB) infection supports HIV-1 replication through dysregulation of host cytokines, chemokines, and their receptors. However the possibility that mycobacterial molecules released from MTB infected macrophages directly interact with CD4(+) T cells triggering HIV-1 replication has not been fully explored. We studied the direct effect of different MTB molecules on HIV-1 replication (R5-tropic strain Bal) in anti-CD3- stimulated CD4(+) T cells from healthy donors in an antigen presenting cell (APC)-free system. PIM6, a major glycolipid of the mycobacterial cell wall, induced significant increases in the percent of HIV-1 infected T cells and the viral production in culture supernatants. In spite of structural relatedness, none of the other three major MTB cell wall glycolipids had significant impact on HIV-1 replication in T cells. Increased levels of IFN-γ in culture supernatants from cells treated with PIM6 indicate that HIV-1 replication is likely dependent on enhanced T cell activation. In HEK293 cells transfected with TLR2, PIM6 was the strongest TLR2 agonist among the cell wall associated glycolipids tested. PIM6 increased the percentage of HIV infected cells and viral particles in the supernatant in a T-cell-based reporter cell line (JLTRg-R5) transfected with TLR1 and TLR2 but not in the cells transfected with the empty vector (which lack TLR2 expression) confirming that PIM6-induced HIV-1 replication depends at least partially on TLR2 signaling.

We have previously demonstrated that mycobacterial lipoproteins engage TLR2 on human CD4(+) T cells and upregulate TCR-triggered IFN-γ secretion and cell proliferation in vitro. Here we examined the role of CD4(+) T-cell-expressed TLR2 in Mycobacterium tuberculosis (MTB) Ag-specific T-cell priming and in protection against MTB infection in vivo. Like their human counterparts, mouse CD4(+) T cells express TLR2 and respond to TLR2 costimulation in vitro. This Th1-like response was observed in the context of both polyclonal and Ag-specific TCR stimulation. To evaluate the role of T-cell TLR2 in priming of CD4(+) T cells in vivo, naive MTB Ag85B-specific TCR transgenic CD4(+) T cells (P25 TCR-Tg) were adoptively transferred into Tlr2(-/-) recipient C57BL/6 mice that were then immunized with Ag85B and with or without TLR2 ligand Pam3 Cys-SKKKK. TLR2 engagement during priming resulted in increased numbers of IFN-γ-secreting P25 TCR-Tg T cells 1 week after immunization. P25 TCR-Tg T cells stimulated in vitro via TCR and TLR2 conferred more protection than T cells stimulated via TCR alone when adoptively transferred before MTB infection. Our findings indicate that TLR2 engagement on CD4(+) T cells increases MTB Ag-specific responses and may contribute to protection against MTB infection.

We have recently demonstrated that mycobacterial ligands engage Toll like receptor 2 (TLR2) on human CD4+ T cells and up-regulate T-cell receptor (TCR) triggered-Th1 responses in vitro and in vivo. To better understand the role of T-cell expressed TLR2 on CD4+ T cell differentiation and function, we conducted a gene expression analysis of naïve CD4+ T-cells stimulated in the presence or absence of TLR2 co-stimulation. Unexpectedly, naïve CD4+ T-cells co-stimulated via TLR2 show a significant up-regulation of IL-9 mRNA compared to cells co-stimulated via CD28. To further evaluate the impact of T-cell expressed TLR2 on TH9 differentiation, naïve CD4+ T cells were polyclonally stimulated under TH9 polarizing conditions with or without a TLR2 agonist. Upregulation of TH9 differentiation, evidenced by increases in both percent of IL-9 secreting cells and IL-9 in culture supernatants, was observed in cells differentiated in the presence of TLR2 agonist. Also, engagement of T-cell TLR2 up-regulated the percent of IL-9 secreting cells in Ag85B-specific CD4+ T-cells stimulated with cognate peptide in TH9 polarizing conditions. On the other hand, in the absence of TH9-polarizing conditions, TLR2 engagement on CD4+ T-cells had minimal effect on IL-9 secretion and TH9 differentiation. This is likely due to a prominent effect of TLR2 signaling on IFN-γ secretion and TH1 differentiation under non-polarizing conditions. Here we also report that, TLR2 signaling in CD4+ T-cells increased expression of transcription factors BATF and PU.1, which are known to positively regulate TH9 differentiation. These results reveal a novel role of T-cell expressed TLR2 in enhancing the differentiation and function of TH9 T cells.

Intracellular pathogens, such as Mycobacterium tuberculosis, reside in the phagosomes of macrophages where antigenic processing is initiated. Mycobacterial antigen-MHC class II complexes are formed within the phagosome and are then trafficked to the cell surface. Interferon-γ (IFN-γ) and interleukin-10 (IL-10) influence the outcome of M. tuberculosis infection; however, the role of these cytokines with regard to the formation of M. tuberculosis peptide-MHC-II complexes remains unknown. We analysed the kinetics and subcellular localization of M. tuberculosis peptide-MHC-II complexes in M. tuberculosis-infected human monocyte-derived macrophages (MDMs) using autologous M. tuberculosis-specific CD4(+) T cells. The MDMs were pre-treated with either IFN-γ or IL-10 and infected with M. tuberculosis. Cells were mechanically homogenized, separated on Percoll density gradients and manually fractionated. The fractions were incubated with autologous M. tuberculosis -specific CD4(+) T cells. Our results demonstrated that in MDMs pre-treated with IFN-γ, M. tuberculosis peptide-MHC-II complexes were detected early mainly in the phagosomal fractions, whereas in the absence of IFN-γ, the complexes were detected in the endosomal fractions. In MDMs pre-treated with IL-10, the M. tuberculosis peptide-MHC-II complexes were retained in the endosomal fractions, and these complexes were not detected in the plasma membrane fractions. The results of immunofluorescence microscopy demonstrated the presence of Ag85B associated with HLA-DR at the cell surface only in the IFN-γ-treated MDMs, suggesting that IFN-γ may accelerate M. tuberculosis antigen processing and presentation at the cell membrane, whereas IL-10 favours the trafficking of Ag85B to vesicles that do not contain LAMP-1. Therefore, IFN-γ and IL-10 play a role in the formation and trafficking of M. tuberculosis peptide-MHC-II complexes.

The success of Mycobacterium tuberculosis as a pathogen relies on its ability to regulate the host immune response. M. tuberculosis can manipulate adaptive T cell responses indirectly by modulating antigen-presenting cell (APC) function or by directly interacting with T cells. Little is known about the role of M. tuberculosis molecules in direct regulation of T cell function. Using a biochemical approach, we identified lipoproteins LprG and LpqH as major molecules in M. tuberculosis lysate responsible for costimulation of primary human CD4 + T cells. In the absence of APCs, activation of memory CD4 + T cells with LprG or LpqH in combination with anti-CD3 antibody induces Th1 cytokine secretion and cellular proliferation. Lipoprotein-induced T cell costimulation was inhibited by blocking antibodies to Toll-like receptor 2 (TLR2) and TLR1, indicating that human CD4 + T cells can use TLR2/TLR1 heterodimers to directly respond to M. tuberculosis products. M. tuberculosis lipoproteins induced NF-κB activation in CD4 + T cells in the absence of TCR co-engagement. Thus, TLR2/TLR1 engagement alone by M. tuberculosis lipoprotein triggered intracellular signaling, but upregulation of cytokine production and proliferation required co-engagement of the TCR. In conclusion, our results demonstrate that M. tuberculosis lipoproteins LprG and LpqH participate in the regulation of adaptive immunity not only by inducing cytokine secretion and costimulatory molecules in innate immune cells but also through directly regulating the activation of memory T lymphocytes.

Immune evasion is required forMycobacterium tuberculosisto survive in the face of robust adaptive CD4+T-cell responses. We have previously shown thatM. tuberculosiscan indirectly inhibit CD4+T cells by suppressing the major histocompatibility complex class II antigen-presenting cell function of macrophages. This study was undertaken to determine ifM. tuberculosiscould directly inhibit CD4+T-cell activation. Murine CD4+T cells were purified from spleens by negative immunoaffinity selection followed by flow sorting. Purified CD4+T cells were activated for 16 to 48 h with CD3 and CD28 monoclonal antibodies in the presence or absence ofM. tuberculosisand its subcellular fractions. CD4+T-cell activation was measured by interleukin 2 production, proliferation, and expression of activation markers, all of which were decreased in the presence ofM. tuberculosis. Fractionation identified thatM. tuberculosiscell wall glycolipids, specifically, phosphatidylinositol mannoside and mannose-capped lipoarabinomannan, were potent inhibitors. Glycolipid-mediated inhibition was not dependent on Toll-like receptor signaling and could be bypassed through stimulation with phorbol 12-myristate 13-acetate and ionomycin. ZAP-70 phosphorylation was decreased in the presence ofM. tuberculosisglycolipids, indicating thatM. tuberculosisglycolipids directly inhibited CD4+T-cell activation by interfering with proximal T-cell-receptor signaling.

Direct regulation of T cell function by microbial ligands through Toll-like receptors (TLR) is an emerging area of T cell biology. Currently either immunomagnetic cell sorting (IMACS) or fluorescence-activated cell sorting (FACS), are utilized to isolate T-cell subsets for such studies. However, it is unknown to what extent differences in T cell purity between these isolation techniques influence T cell functional assays. We compared the purity, response to mitogen, activation requirements, and response to TLR ligands between human CD4(+) T cells isolated either by IMACS (IMACS-CD4(+)) or by IMACS followed by FACS (IMACS/FACS-CD4(+)). As expected, IMACS-CD4(+) were less pure than IMACS/FACS-CD4(+) (92.5%+/-1.4% versus 99.7%+/-0.2%, respectively). Consequently, IMACS-CD4(+) proliferated and produced cytokines in response to mitogen alone and had lower activation requirements compared to IMACS/FACS-CD4(+). In addition IMACS-CD4(+) but not IMACS/FACS-CD4(+) responses were upregulated by the TLR-4 ligand lipopolysaccharide (LPS). On the other hand, TLR-2 and TLR-5 engagement induced costimulation in both IMACS-CD4(+) and highly purified IMACS-/FACS-CD4(+). Altogether these results indicate that small differences in cell purity can significantly alter T cell responses to TLR ligands. This study stresses the importance of a stringent purification method when investigating the role of microbial ligands in T cell function.

Summary Active tuberculosis (TB) is associated with prolonged suppression of Mycobacterium tuberculosis (MTB)-specific immune responses, but mechanisms involved are understood incompletely. We investigated a potential role for CD4+CD25+ regulatory T cells in depressed anti-MTB immunity by evaluating serially CD4 cell phenotype and interferon (IFN)-γ production by mononuclear cells from patients with TB. At diagnosis, frequencies of CD4+CD25+ T cells were increased in blood from TB patients compared to healthy purified protein derivative (PPD)-positive controls (with a history of prior TB exposure), and remained elevated at completion of therapy (6 months). By contrast, expression of another activation marker, CD69, by CD4 T cells was increased at diagnosis, but declined rapidly to control levels with treatment. Among CD4+CD25+ T cells from TB patients at diagnosis those expressing high levels of CD25, probably representing regulatory T cells, were increased 2·9-fold when compared to control subjects, while MTB-stimulated IFN-γ levels in whole blood supernatants were depressed. A role for CD4+CD25+ T cells in depressed IFN-γ production during TB was substantiated in depletion experiments, where CD25+-depleted CD4 T cells produced increased amounts of IFN-γ upon MTB stimulation compared to unseparated T cells. At follow-up, IFN-γ production improved most significantly in blood from TB patients with high baseline frequencies of CD4+CD25+ T cells (more than threefold higher than controls for both total and CD25hi+ CD4 T cells), who also had a significant drop in frequencies of both total and ‘regulatory’ CD4+CD25+ T cells in response to treatment. Expansion of CD4+CD25+ regulatory T cells during active TB may play a role in depressed T cell IFN-γ production.

Mycobacterium tuberculosis survives in macrophages in the face of acquired CD4 + T-cell immunity, which controls but does not eliminate the organism. Gamma interferon (IFN-γ) has a central role in host defenses against M. tuberculosis by activating macrophages and regulating major histocompatibility complex class II (MHC-II) antigen (Ag) processing. M. tuberculosis interferes with IFN-γ receptor (IFN-γR) signaling in macrophages, but the molecules responsible for this inhibition are poorly defined. This study determined that the 19-kDa lipoprotein from M. tuberculosis inhibits IFN-γ-regulated HLA-DR protein and mRNA expression in human macrophages. Inhibition of HLA-DR expression was associated with decreased processing and presentation of soluble protein Ags and M. tuberculosis bacilli to MHC-II-restricted T cells. Inhibition of HLA-DR required prolonged exposure to 19-kDa lipoprotein and was blocked with a monoclonal antibody specific for Toll-like receptor 2 (TLR-2). The 19-kDa lipoprotein also inhibited IFN-γ-induced expression of FcγRI. Thus, M. tuberculosis , through 19-kDa lipoprotein activation of TLR-2, inhibits IFN-γR signaling in human macrophages, resulting in decreased MHC-II Ag processing and recognition by MHC-II-restricted CD4 T cells. These findings provide a mechanism for M. tuberculosis persistence in macrophages.

A hallmark of M. tuberculosis infection is the ability of most (90-95%) healthy adults to control infection through acquired immunity, in which antigen specific T cells and macrophages arrest growth of M. tuberculosis bacilli and maintain control over persistent bacilli. In addition to CD4+ T cells, other T cell subsets such as, gammadelta, CD8+ and CD1-restricted T cells have roles in the immune response to M. tuberculosis. A diverse T cell response allows the host to recognize a wider range of mycobacterial antigens presented by different families of antigen-presenting molecules, and thus greater ability to detect the pathogen. Macrophages are key antigen presenting cells for T cells, and M. tuberculosis survives and persists in this central immune cell. This is likely an important factor in generating this T cell diversity. Furthermore, the slow growth and chronic nature of M. tuberculosis infection results in prolonged exposure to antigens, and hence further T cell sensitization. The effector mechanisms used by T cells to control M. tuberculosis are poorly understood. To survive in macrophages, M. tuberculosis has evolved mechanisms to block immune responses. These include modulation of phagosomes, neutralization of macrophage effector molecules, stimulating the secretion of inhibitory cytokines, and interfering with processing of antigens for T cells. The relative importance of these blocking mechanisms likely depends on the stage of M. tuberculosis infection: primary infection, persistence, reactivation or active tuberculosis. The balance of the host-pathogen interaction in M. tuberculosis infection is determined by the interaction of T cells and infected macrophages. The outcome of this interaction results either in control of M. tuberculosis infection or active disease. A better understanding of this interaction will result in improved approaches to treatment and prevention of tuberculosis.

Vγ9Vδ2+T cells (γδ T cells) are activated byMycobacterium tuberculosisand recognize mycobacterial nonpeptide phosphoantigens. The role of antigen-presenting cells in the processing and presentation of phosphoantigens to Vγ9Vδ2+T cells is not understood. We analyzed the role of macrophages for activation of γδ T cells by a new synthetic phosphoantigen bromohydrin pyrophosphate (BrHPP) andM. tuberculosis. Macrophages greatly increased γδ T-cell activation by both BrHPP andM. tuberculosis. Fixation of macrophages before infection demonstrated that uptake ofM. tuberculosiswas required for presentation to γδ T cells. Antigens ofM. tuberculosisremained stably associated with macrophage surface and were not removed by paraformaldehyde fixation or washing. Macrophages processedM. tuberculosisfor γδ T cells through a brefeldin A-insensitive pathway, suggesting that transport through the endoplasmic reticulum and Golgi complex of a putative presenting molecule is not important in the early processing ofM. tuberculosisantigens for γδ T cells. Processing ofM. tuberculosiswas not eliminated by chloroquine, indicating that processing of γδ antigens is not dependent on acidic pH in the lysosomes. Chloroquine treatment of BrHPP-pulsed macrophages increased activation of γδ T cells. Ammonium chloride treatment of macrophages did not increase reactivity of γδ T cells to BrHPP, indicating that the effect of chloroquine was independent of pH changes in endosomes. Chloroquine, by inhibiting membrane traffic, may increase association and retention of phosphoantigens with cell surface membrane molecules on macrophages.

For MTB to survive in the face of robust innate and adaptive immune responses multiple immune evasion mechanisms are required. These mechanisms and the MTB molecules responsible for them are beginning to be elucidated. Apart from interfering with the innate and adaptive immune functions of macrophages, recent studies suggest that MTB also can directly modulate CD4+ T cell function. The current study was undertaken to identify MTB molecules and mechanisms for direct inhibition of CD4+ T cell activation. Murine CD4+ T cells were purified from spleens by a combination of negative selection with mAb-coated beads and flow sorting. Purified T cells were activated for 24–48 hrs. with plate-bound anti-CD3 and soluble anti-CD28 mAbs with whole bacilli or subcellular fractions of MTB. Multiple measures of activation were used including IL-2 production by ELISA, proliferation by thymidine incorporation, and cell surface receptor expression by flow cytometry. MTB bacilli decreased T cell activation by all three measures. Fractionation identified a lipophilic fraction of the MTB cell wall as a major inhibitor of anti-CD3/CD28 mediated activation. T cell metabolic activity was unchanged in the presence of the lipophilic fraction. Activation of CD4+ T cells by PMA/ionomyocin was not affected by MTB. Thus lipophilic molecules in the MTB cell wall directly inhibit CD3/CD28 mediated CD4+ T cell activation by interfering with proximal T cell signaling events.

Background Multiple toll-like receptors (TLRs) are expressed in cells of the monocytic lineage, including microglia, which constitute the major reservoir for human immunodeficiency virus (HIV) infection in the brain. We hypothesized that TLR receptor mediated responses to inflammatory conditions by microglial cells in the central nervous system (CNS) are able to induce latent HIV proviruses, and contribute to the etiology of HIV-associated neurocognitive disorders. Results Newly developed human microglial cell lines (hµglia), obtained by immortalizing human primary microglia with simian virus-40 (SV40) large T antigen and the human telomerase reverse transcriptase, were used to generate latently infected cells using a single-round HIV virus carrying a green fluorescence protein reporter (hµglia/HIV, clones HC01 and HC69). Treatment of these cells with a panel of TLR ligands showed surprisingly that two potent TLR3 agonists, poly (I:C) and bacterial ribosomal RNA potently reactivated HIV in hμglia/HIV cells. LPS (TLR4 agonist), flagellin (TLR5 agonist), and FSL-1 (TLR6 agonist) reactivated HIV to a lesser extent, while Pam3CSK4 (TLR2/1 agonist) and HKLM (TLR2 agonist) only weakly reversed HIV latency in these cells. While agonists for TLR2/1, 4, 5 and 6 reactivated HIV through transient NF-κB induction, poly (I:C), the TLR3 agonist, did not activate NF-κB, and instead induced the virus by a previously unreported mechanism mediated by IRF3. The selective induction of IRF3 by poly (I:C) was confirmed by chromatin immunoprecipitation (ChIP) analysis. In comparison, in latently infected rat-derived microglial cells (hT-CHME-5/HIV, clone HC14), poly (I:C), LPS and flagellin were only partially active. The TLR response profile in human microglial cells is also distinct from that shown by latently infected monocyte cell lines (THP-1/HIV, clone HA3, U937/HIV, clone HUC5, and SC/HIV, clone HSCC4), where TLR2/1, 4, 5, 6 or 8, but not for TLR3, 7 or 9, reactivated HIV. Conclusions TLR signaling, in particular TLR3 activation, can efficiently reactivate HIV transcription in infected microglia, but not in monocytes or T cells. The unique response profile of microglial cells to TLR3 is fundamental to understanding how the virus responds to continuous microbial exposure, especially during inflammatory episodes, that characterizes HIV infection in the CNS. Electronic supplementary material The online version of this article (doi:10.1186/s12977-017-0335-8) contains supplementary material, which is available to authorized users.