ESAT-6 Inhibits Production of IFN-γ by Mycobacterium tuberculosis-Responsive Human T Cells

Abstract

The Mycobacterium tuberculosis early secreted Ag of 6 kDa (ESAT-6) is a potent Ag for human T cells and is a putative vaccine candidate. However, ESAT-6 also contributes to virulence in animal models, mediates cellular cytolysis, and inhibits IL-12 production by mononuclear phagocytes. We evaluated the effects of ESAT-6 and its molecular chaperone, culture filtrate protein of 10 kDa (CFP10), on the capacity of human T cells to produce IFN-γ and proliferate in response to TCR activation. Recombinant ESAT-6, but not CFP10, markedly inhibited IFN-γ production by T cells stimulated with M. tuberculosis or with the combination of anti-CD3 and anti-CD28, in a dose-dependent manner. ESAT-6 also inhibited T cell production of IL-17 and TNF-α but not IL-2. Preincubation of ESAT-6 with CFP10 under conditions that favor dimer formation did not affect inhibition of IFN-γ. ESAT-6 decreased IFN-γ transcription and reduced expression of the transcription factors, ATF-2 and c-Jun, which normally bind to the IFN-γ proximal promoter and stimulate mRNA expression. ESAT-6 inhibited T cell IFN-γ secretion through mechanisms that did not involve cellular cytotoxicity or apoptosis. ESAT-6, but not CFP10, bound to T cells and inhibited expression of early activation markers without reducing activation of ZAP70. We conclude that ESAT-6 directly inhibits human T cell responses to mycobacterial Ags by affecting TCR signaling pathways downstream of ZAP70.

The Mycobacterium tuberculosis Early Secreted Antigenic Target of 6 kDa Inhibits T Cell Interferon-γ Production through the p38 Mitogen-activated Protein Kinase Pathway

“…The cells were cultured in LB medium, and ESAT-6 expression was induced by the addition of isopropyl-D-1-thiogalactopyranoside. Recombinant ESAT-6 was purified, LPS was removed, and its purity was Ͼ95%, based on SDS-PAGE, followed by Coomassie Brilliant Blue staining and Western blotting with anti-ESAT-6 mAb (HYB 76-8), as described previously (14). Recombinant ESAT-6 contained Ͻ50 pg of LPS/mg of protein, by the QCL-1000 limulus amebocyte assay, and was resuspended in Hanks' balanced salt solution (2 mg/ml), aliquoted, and stored at Ϫ70°C until use.…”

“…To measure cell proliferation, purified CD3 ϩ cells were labeled with CFSE, as described previously (14), and treated with PK inhibitors and ESAT-6 prior to stimulation with anti-CD3 and anti-CD28, as outlined above. After 96 h, the cells were stained with PE-anti-CD8 (eBiosciences), and CFSE dilution was analyzed by flow cytometry.…”

“…Real-time PCR-Total RNA was extracted from 8 ϫ 10 5 CD3 ϩ cells with TRIzol reagent (Invitrogen), cDNA was synthesized, and IFN-␥ cDNA was quantified by real-time PCR, with 18 S ribosomal RNA as an endogenous control, as described previously (14). The relative quantity of IFN-␥ mRNA was calculated by the ⌬C t method (16).…”

Mycobacterium tuberculosis Lipoproteins Directly Regulate Human Memory CD4 + T Cell Activation via Toll-Like Receptors 1 and 2

“…ESAT-6 may directly inhibit gamma interferon (IFN-␥) secretion and T cell proliferation by interfering with T cell receptor (TCR) signaling (54). By directly controlling T cell function, M. tuberculosis molecules may influence development of protective responses.…”

Detection of Lipomannan in Cattle Infected with Bovine Tuberculosis

“…Noteworthy among them are LM and early secretory antigenic target 6, ESAT6. 1,[17][18][19][20] LM is an abundant lipoglycan component of the M. bovis cell envelope, and is secreted by the metabolically active bacterium during infection, which results in activation of the host immune response. 1,17,18 The amphiphile is well-conserved in evolution and like its counterpart secreted by M. tuberculosis, LAM, LM is known to be an immune activator.…”