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 xenobiotic-activated nuclear receptors PXR (pregnane X receptor) and CAR (constitutive androstane receptor) and the vitamin D 3 -activated nuclear receptor VDR regulate steroid and xenobiotic metabolism by inducing the phase I cytochrome P450 monooxygenases, phase II conjugating transferases, and the phase III transporters, which mediate the efflux of water-soluble lipid metabolites from cells. Metabolic stress due to the deviant expression of steroid-and xenobioticmetabolizing enzymes is known to have severe health consequences including accelerated aging, and increased expression of these enzymes is associated with extended longevity (Gachon et al, 2006;McElwee et al, 2004). Information on the similarities and dissimilarities in drug metabolism between the young and old, as may be uncovered by studying aging regulation of the genes relevant to steroid and xenobiotic metabolism, is likely to have clinical significance. In this report, we examined the VDR-and PXR-mediated gene induction of the phase II sulfotransferase Sult2A1 in the livers of 4-month and 20-month old mice. Sult2A1 converts bile acids, steroids and a number of drugs to the corresponding sulfated metabolites, which are readily eliminated from the body due to increased water solubility. In RT-PCR assay, aging did not change the induction of Sult2A1 mRNAs by the hormonally active vitamin D 3 and the catatoxic synthetic steroid PCN (pregnenolone-16α-carbonitrile). Chromatin immunoprecipitation (ChIP) from liver nuclei showed that aging had no effect on the activity of an IR0 enhancer in the Sult2A1 chromatin to recruit VDR, RXR-α (retinoid X receptor) and PXR in mice injected with D 3 or PCN. Thus, mice in late life are as competent as those in early life in responding to the hormonal and xenobiotic signaling for Sult2A1 induction. This is the first report describing the role of aging in the functional response of an enhancer in the liver chromatin to the nuclear receptor-dependent signaling.