We evaluated the role of regulatory T cells (CD4 + CD25 + Foxp3 + cells, Tregs) in human Mycobacterium tuberculosis infection. Tregs were expanded in response to M. tuberculosis in healthy tuberculin reactors, but not in tuberculin-negative individuals. The M. tuberculosis mannose-capped lipoarabinomannan (ManLAM) resulted in regulatory T cell expansion, whereas the M. tuberculosis 19-kDa protein and heat shock protein 65 had no effect. Anti-IL-10 and anti-TGF-b alone or in combination, did not reduce expansion of Tregs. In contrast, the cyclooxygenase enzyme-2 inhibitor NS398 significantly inhibited expansion of Tregs, indicating that prostaglandin E2 (PGE2) contributes to Treg expansion. Monocytes produced PGE2 upon culturing with heat-killed M. tuberculosis or ManLAM, and T cells from healthy tuberculin reactors enhanced PGE2 production by monocytes. Expanded Tregs produced significant amounts of TGF-b and IL-10 and depletion of Tregs from PBMC of these individuals increased the frequency of M. tuberculosis-responsive CD4 + IFN-c cells. Culturing M. tuberculosis-expanded Tregs with autologous CD8 + cells decreased the frequency of IFN-c + cells. Freshly isolated PBMC from tuberculosis patients had increased percentages of Tregs, compared to healthy tuberculin reactors. These findings demonstrate that Tregs expand in response to M. tuberculosis through mechanisms that depend on ManLAM and PGE2.

The secreted Mycobacterium tuberculosis 10-kDa culture filtrate protein (CFP)10 is a potent T cell Ag that is recognized by a high percentage of persons infected with M. tuberculosis. We determined the molecular basis for this widespread recognition by identifying and characterizing a 15-mer peptide, CFP1071–85, that elicited IFN-γ production and CTL activity by both CD4+ and CD8+ T cells from persons expressing multiple MHC class II and class I molecules, respectively. CFP1071–85 contained at least two epitopes, one of 10 aa (peptide T1) and another of 9 aa (peptide T6). T1 was recognized by CD4+ cells in the context of DRB1*04, DR5*0101, and DQB1*03, and by CD8+ cells of A2+ donors. T6 elicited responses by CD4+ cells in the context of DRB1*04 and DQB1*03, and by CD8+ cells of B35+ donors. Deleting a single amino acid from the amino or carboxy terminus of either peptide markedly reduced IFN-γ production, suggesting that they are minimal epitopes for both CD4+ and CD8+ cells. As far as we are aware, these are the shortest microbial peptides that have been found to elicit responses by both T cell subpopulations. The capacity of CFP1071–85 to stimulate IFN-γ production and CTL activity by CD4+ and CD8+ cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.

IFN-γ production by T cells is pivotal for defense against many pathogens, and the proximal promoter of IFN-γ, −73 to −48 bp upstream of the transcription start site, is essential for its expression. However, transcriptional regulation mechanisms through this promoter in primary human cells remain unclear. We studied the effects of cAMP response element binding protein/activating transcription factor (CREB/ATF) and AP-1 transcription factors on the proximal promoter of IFN-γ in human T cells stimulated with Mycobacterium tuberculosis. Using EMSA, supershift assays, and promoter pulldown assays, we demonstrated that CREB, ATF-2, and c-Jun, but not cyclic AMP response element modulator, ATF-1, or c-Fos, bind to the proximal promoter of IFN-γ upon stimulation, and coimmunoprecipitation indicated the possibility of interaction among these transcription factors. Chromatin immunoprecipitation confirmed the recruitment of these transcription factors to the IFN-γ proximal promoter in live Ag-activated T cells. Inhibition of ATF-2 activity in T cells with a dominant-negative ATF-2 peptide or with small interfering RNA markedly reduced the expression of IFN-γ and decreased the expression of CREB and c-Jun. These findings suggest that CREB, ATF-2, and c-Jun are recruited to the IFN-γ proximal promoter and that they up-regulate IFN-γ transcription in response to microbial Ag. Additionally, ATF-2 controls expression of CREB and c-Jun during T cell activation.

IFN-γ is essential for resistance to many intracellular pathogens, including Mycobacterium tuberculosis. Transcription of the IFN-γ gene in activated T cells is controlled by the proximal promoter element (−73 to −48 bp). CREB binds to the IFN-γ proximal promoter, and binding is enhanced by phosphorylation of CREB. Studies in human T cell lines and in transgenic mice have yielded conflicting results about whether CREB is a positive or a negative regulator of IFN-γ transcription. To determine the role of CREB in mediating IFN-γ production in response to a microbial pathogen, we evaluated the peripheral blood T cell response to M. tuberculosis in healthy tuberculin reactors. EMSAs, chromatin immunoprecipitation, and Western blotting demonstrated that stimulation of PBMC with M. tuberculosis induced phosphorylation and enhanced binding of CREB to the IFN-γ proximal promoter. Neutralization of CREB with intracellular Abs or down-regulation of CREB levels with small interfering RNA decreased M. tuberculosis-induced production of IFN-γ and IFN-γ mRNA expression. In addition, M. tuberculosis-stimulated T cells from tuberculosis patients, who have ineffective immunity, showed diminished IFN-γ production, reduced amounts of CREB binding to the IFN-γ proximal promoter, and absence of phosphorylated CREB. These findings demonstrate that CREB positively regulates IFN-γ production by human T cells that respond to M. tuberculosis.

Background-Elevated homocysteine levels are associated with increased coronary risk, and it has been suggested that homocysteine screening may provide a method to identify high-risk patients for aggressive primary prevention. Methods and Results-Homocysteine was measured at baseline and after 1 year among 5569 participants in the Air Force/Texas Coronary Atherosclerosis Prevention Study (AFCAPS/TexCAPS), a randomized trial of lovastatin in the primary prevention of acute coronary events. The effects of homocysteine, LDL cholesterol, and lovastatin on risk were assessed over 5.2 years of trial follow-up. Median baseline homocysteine levels were significantly higher among study participants who subsequently had acute coronary events compared with those who did not (12.1 versus 10.9 mol/L, PϽ0.001). The relative risks of future events from lowest (referent) to highest quartile of homocysteine were 1.0, 1.6, 1.6, and 2.2 (PϽ0.001). These effects were similar among those allocated to lovastatin and those allocated to placebo and were modestly attenuated after adjustment for other traditional risk factors. As predicted, the subgroup of participants with elevated LDL cholesterol and elevated homocysteine levels were at high risk and benefited greatly from statin therapy (relative risk, 0.46; 95% CI, 0.29 to 0.75; number needed to treatϭ26). However, in contrast to findings in this trial for C-reactive protein, homocysteine evaluation did not help to define low LDL subgroups with different responses to lovastatin therapy. Conclusions-Although homocysteine predicted future coronary events in AFCAPS/TexCAPS, we found little evidence that homocysteine evaluation provided an improved method to target statin therapy among those with low-to-normal LDL cholesterol levels.

Previously we found that regulatory T cells (CD4+CD25+Foxp3+ cells, Tregs) expand in response to M.tuberculosis (M. TB) through mechanism that depend on prostaglandin E2 production. In the current study, we determined the role of NK cells in regulating Tregs expansion in human M. TB infection. M. TB whole cell lysate (TB lysate) resulted in regulatory T cell expansion (15.33±1.02% vs 1.5±0.22%, P<0.001). Addition of monokine-activated NK cells (IL-12, IL-15 and IL-18) markedly inhibited Treg expansion (6.2±1.2% vs 15±1.5%, P<0.001) but freshly isolated NK cells had no effect. NK cells activated with TB lysate-stimulated monocytes also reduced Tregs expansion (13.2±0.3% to 3.4±1.4%, P<0.001) confirming the physiological relevance of this effect. Reduced Tregs expansion was not due to inhibition of PGE2 production or because of IFN γ. Monokine activated NK cells lysed TB lysate-expanded Tregs, but not freashly isolated Tregs (% specific lysis of 21±3% vs 1±1% P<0.001), nor T regs depleted T cells. Anti-NKG2D and anti-NKp46 reduced the % specific lysis of expanded Tregs from 22±3% to 7±3% and 8±3%, respectively (P<0.001) but abs to CD16, DNAM-1, and 2B4 had no effect. NKG2D ligand, ULBP1 expression was markedly upregulated on expanded T regs, compared to T regs depleted cells ( 31±5% vs 2.1 ±0.4%, P=0.002) and freshly isolated Tregs (4.6±1.1%). Anti -ULBP1 significantly inhibited the NK mediated lysis of expanded Tregs (specific lysis of 27±4% vs 9±2%, P<0.001) but abs to ULBP2, ULBP3 and MICA/B had no effect. These finding suggest that NK cells reduce the frequency of M.TB-expanded T regs by direct lysis.