Protective immunity in tuberculosis is dependent on the coordinated release of cytolytic effector molecules from effector T cells and the subsequent granule-associated killing of infected target cells. In this study, we investigated the expression of cytolytic (perforin and granzyme A) and antimicrobial (granulysin) molecules at the single-cell level in cryopreserved lung tissue from patients with chronic, progressive tuberculosis disease. Quantification of protein-expressing cells was performed by in situ imaging, while mRNA levels in the infected tissue were analyzed by real-time PCR. Persistent inflammation, including excessive expression of inducible nitric oxide synthase in CD68؉ macrophages and significant infiltration of CD3؉ , CD8 ؉ and CD4 ؉ T cells, was evident in tuberculosis lesions in all patients. However, despite the accumulation of CD3 ؉ T cells, perforin-and granulysin-expressing CD3 ؉ T cells were detected at two-to threefold-lower ratios in the tuberculosis lesions than in distal lung parenchyma and uninfected control lungs, respectively. This was evident at both the protein and mRNA levels. Moreover, perforin-and granulysin-expressing CD8 ؉ T cells were scarce in individual granulomas within the tuberculosis lesions. In contrast, significant up-regulation of granzyme A-expressing CD3 ؉ T cells was evident in the lesions from all patients. Confocal microscopy revealed coexpression of perforin and granulysin, primarily in CD8 ؉ T cells; however, this expression was lower in the tuberculosis lesions. These findings suggest that symptomatic, chronic tuberculosis disease is associated with insufficient up-regulation of perforin and granulysin coexpression in CD8؉ T cells at the local site of infection.

The transcriptional events that control T cell tolerance are still poorly understood. To investigate why tolerant T cells fail to produce interleukin (IL)-2, we analyzed the regulation of NF B-mediated transcription in CD4 ؉ T cells after tolerance induction in vivo. We demonstrate that a predominance of p50-p50 homodimers binding to the IL-2 promoter B site in tolerant T cells correlated with repression of NF B-driven transcription. Impaired translocation of the p65 subunit in tolerant T cells was a result from reduced activation of I B kinase and poor phosphorylation and degradation of cytosolic I Bs. Moreover, tolerant T cells expressed high amounts of the p50 protein. However, the increased expression of p50 could not be explained by activation-induced de novo synthesis of the precursor p105, which was constitutively expressed in tolerant T cells. We also demonstrate the exclusive induction of the I B protein B cell lymphoma 3 (Bcl-3) in tolerant T cells as well as its specific binding to the NF B site. These results suggest that the cellular ratio of NF B dimers, and thus the repression of NF B activity and IL-2 production, are regulated at several levels in tolerant CD4 ؉ T cells in vivo.Induction of T cell tolerance may result in either deletion or anergy, the latter being characterized by a block in proliferation and diminished expression of Th1 cytokines, particularly interleukin-2 (IL-2) 1 (1). Although T cell anergy has been described both in vitro and in vivo (2-5), recent studies have shown that rather than being functionally inert, the anergic T cells may adopt the role of regulatory T cells with a Tr1-like phenotype (6, 7). Importantly, anergic T cells may be functionally regulated at several different levels, e.g. in vivo tolerized T cells may neither produce IL-2 nor respond to signaling through the IL-2 receptor (8).Because the failure of tolerant T cells to produce IL-2 is a hallmark of anergy, it is particularly important to delineate the molecular explanation for this defect. Downstream of the T cell receptor (TCR), several intracellular signaling pathways converge at the level of transcriptional regulation of the IL-2 gene. AP-1, NFAT, and NF B families of transcription factors are essential in T cell activation as coordinators of IL-2 transcription (9) and tolerant CD4 ϩ T cell clones as well as in vivo tolerized T cells possess reduced AP-1 binding to the IL-2 promoter (10 -12). In addition, we have previously shown that CD4 ϩ T cells from mice tolerized in vivo express qualitative alterations in the composition of the NF B complex (13). Defective NF B expression is also a feature of tolerant B cells (14,15).The NF B family consists of five members, p65, c-rel, RelB, p105/p50, and p100/p52, and the NF B complex is formed by hetero-or homodimerization of these different proteins (16). p65, c-rel, and RelB are synthesized as mature proteins and contain transactivation domains, whereas p105/p50 and p100/ p52 are produced as inactive precursor proteins and lack transactivation domains. Activa...

Repeated exposures to both microbial and innocuous Ags in vivo have been reported to both eliminate and tolerize T cells after their initial activation and expansion. The remaining tolerant T cells have been shown to suppress the response of naive T cells in vitro. This feature is reminiscent of natural CD4+CD25+ regulatory T cells. However, it is not known whether the regulatory function of in vivo-tolerized T cells is similar to the function of natural CD4+CD25+ regulatory T cells. In this study, we demonstrate that CD4+CD25+ as well as CD4+CD25− T cells isolated from mice treated with superantigen three consecutive times to induce tolerance were functionally comparable to natural CD4+CD25+ regulatory T cells, albeit more potent. The different subpopulations of in vivo-tolerized CD4+ T cells efficiently down-modulated costimulatory molecules on dendritic cells, and their suppressive functions were strictly cell contact dependent. Importantly, we demonstrate that conventional CD4+CD25− T cells could also be induced to acquire regulatory functions by the same regimen in the absence of natural regulatory T cells in vivo, but that such regulatory cells were functionally different.