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. T cell receptor 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 interleukin-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.
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.
Mycobacterium tuberculosis cell wall glycolipid, Lipoarabinomannan, can inhibit CD4+ T cell activation by down-regulating 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 antigen encounter, yet remain viable and hyporesponsive. We tested whether LAM-induced inhibition of CD4+ T cell activation resulted in CD4+ T cell anergy. The presence of LAM during primary stimulation of P25TCR-Tg murine CD4+ T cells with M. tuberculosis Ag85B peptide resulted in decreased proliferation and IL-2 production. P25TCR-Tg CD4+ T cells primed in the presence of LAM also exhibited decreased response upon re-stimulation with Ag85B. The T cell anergic state persisted after the removal of LAM. Hypo-responsiveness to re-stimulation was not due to apoptosis, generation of FoxP3-positive regulatory T cells or inhibitory cytokines. Acquisition of the anergic phenotype correlated with up-regulation of GRAIL (gene related to anergy in lymphocytes) 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 pre-treatment abrogated LAM induced hypo-responsiveness. In addition, exogenous IL-2 reversed defective proliferation by down-regulating GRAIL expression. These results demonstrate that LAM up-regulates 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.
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