Vitiligo is a common skin disease characterized by the presence of well circumscribed, depigmented, milky white macules devoid of identifiable melanocytes. Although the detection of circulating anti-melanocytic antibodies and of infiltrating lymphocytes at the margin of lesions supports the view that vitiligo is an autoimmune disorder, its etiology remains unknown. In particular, it is still a matter of debate whether the primary pathogenic role is exerted by humoral or cellular abnormal immune responses. In this study, the presence of specific cytotoxic T lymphocyte responses against the melanocyte differentiation antigens Melan-A/MART1, tyrosinase, and gp100 in vitiligo patients have been investigated by the use of major histocompatibility complex/peptide tetramers. High frequencies of circulating melanocyte-specific CD8+ T cells were found in all vitiligo patients analyzed. These cells exerted anti-melanocytic cytotoxic activity in vitro and expressed skin-homing capacity. In one patient melanocyte-specific cells were characterized by an exceptionally high avidity for their peptide/major histocompatibility complex ligand. These findings strongly suggest a role for cellular immunity in the pathogenesis of vitiligo and impact on the common mechanisms of self tolerance.

CD8+ T cells are thought to play an important role in protective immunity to tuberculosis. Although several nonprotein ligands have been identified for CD1-restricted CD8+ CTLs, epitopes for classical MHC class I-restricted CD8+ T cells, which most likely represent a majority among CD8+ T cells, have remained ill defined. HLA-A*0201 is one of the most prevalent class I alleles, with a frequency of over 30% in most populations. HLA-A2/Kb transgenic mice were shown to provide a powerful model for studying induction of HLA-A*0201-restricted immune responses in vivo. The Ag85 complex, a major component of secreted Mycobacterium tuberculosis proteins, induces strong CD4+ T cell responses in M. tuberculosis-infected individuals, and protection against tuberculosis in Ag85-DNA-immunized animals. In this study, we demonstrate the presence of HLA class I-restricted, CD8+ T cells against Ag85B of M. tuberculosis in HLA-A2/Kb transgenic mice and HLA-A*0201+ humans. Moreover, two immunodominant Ag85 peptide epitopes for HLA-A*0201-restricted, M. tuberculosis-reactive CD8+ CTLs were identified. These CD8+ T cells produced IFN-γ and TNF-α and recognized Ag-pulsed or bacillus Calmette-Guérin-infected, HLA-A*0201-positive, but not HLA-A*0201-negative or uninfected human macrophages. This CTL-mediated killing was blocked by anti-CD8 or anti-HLA class I mAb. Using fluorescent peptide/HLA-A*0201 tetramers, Ag85-specific CD8+ T cells could be visualized in bacillus Calmette-Guérin-responsive, HLA-A*0201+ individuals. Collectively, our results demonstrate the presence of HLA class I-restricted CD8+ CTL against a major Ag of M. tuberculosis and identify Ag85B epitopes that are strongly recognized by HLA-A*0201-restricted CD8+ T cells in humans and mice. These epitopes thus represent potential subunit components for the design of vaccines against tuberculosis.

SummaryWe previously showed that H-2Ka-restricted cytotoxic T lymphocyte (CTL) clones specific for a single nonapeptide derived from the Plasraodium berghei circumsporozoite (PbCS) protein displayed T cell receptors (TCRs) of highly diverse primary structure. We have now analyzed the TCR repertoire of CTLs that recognize a peptide derived from the human dass I major histocompatibility complex (MHC) molecule HLA-Cw3 in association with the same murine class I MHC molecule H-2K a. We first sequenced the TCR o~ and/3 genes of the CTL clone Cw3/1.1 and, based on this genomic analysis, the TCR c~ and/3 cDNA junctional regions of 23 independent H-2K drestricted CTL clones specific for HLA-Cw3. The results show that the TCR chains display very limited heterogeneity, both in terms of Vet, Jo~, V~ and J/3 segments, and in terms of length and sequence of the CDR3 cx and/3 loops. The TCR repertoire used in vivo was then analyzed by harvesting CTL populations from the peritoneal cavity of immune mice. The peritoneal exudate lymphocytes (PELs) displayed HLA-Cw3-specific cytolytic activity in the absence of any stimulation in vitro. Remarkably, most of these freshly isolated PELs expressed TCRs that shared the same structural features as those from HLA-Cw3-reactive CTL clones. Thus, our results show that a peptide from HLA-Cw3 presented by H-2K d selects CTLs that bear TCILs of very limited diversity in vivo. When taken together with the high diversity of the TCRs specific for the PbCS peptide, these findings suggest that natural tolerance to self peptides presented by class I MHC molecules may substantially reduce the size of the TCR repertoire of CTLs specific for antigenic peptides homologous to self.

Slltmmsr~Allelic exclusion of lymphocyte antigen receptor chains has been hypothesized as a mechanism developed by the immune system to ensure efficient lymphocyte repertoire selection and tight control of lymphocyte specificity. It was effectively shown to be operative for both the immunoglobulin (Ig) and the T cell receptor (TCR) B chain genes. Our present observations suggest that close to 1% of human T lymphocytes escape this allelic control, and express two surface TCK B chains with distinct superantigenic reactivities. Since this high frequency of dual ~ chain expressors did not result in any dramatic immune dysregulations, these results question the need for a mechanism ensuring clonal monospecificity through allelic exclusion.T mphocyte antigen receptors are composed of two vari-1./ able glycoprotein subunits, the Ig heavy and light chains on B cells and the TCK ot and/3 or y and ~ chains on T cells. The genes coding for Ig and TCK chains are formed through somatic rearrangement of V, D, and J elements (for reviews see references I and 2). Given its diploid nature, any lymphocyte clone could theoretically express up to four distinct combinations of antigen receptor chains on its surface. Since donal plurispecificity, possibly resulting from expression of multiple antigen receptors on the same cell, is expected to lead to immune dysregulations (that is, autoimmunity), control processes ensuring allelic exclusion of antigen receptor chains were postulated more than a decade ago, and their existence demonstrated through elegant Ig and TCK transgenic mouse models (3-5).More recently it has become clear that the stringency of aUelic exclusion greatly differs from one antigen receptor chain gene to another. In the case of TCK/~ chain genes, expression of any productively rearranged gene prevents further rearrangement in the B locus, a process referred to as "genotypic" exclusion (6). Such a genotypic control is illustrated by the arrest of endogenous TCR B gene rearrangements in mice carrying a functional or truncated B transgene (5, 7). However, similar transgenic studies have failed to demonstrate a dramatic influence of functional ol transgenes on the occurrence of rearrangements within endogenous TCK o~ loci (8-12). Moreover, studies performed on normal T cells have revealed the expression of distinct productive cz transcripts (13) or even distinct surface cz chains (14) on a fraction of murine and human T cell clones.Although lack of aUelic exclusion of ol chains can result in a violation of the "one cell, one receptor" rule, current ontogenic models suggest that dual ol chain expression would have limited physiological consequences. Experiments with normal and transgenic T cells suggest that a developing lymphocyte will keep on rearranging its ot loci until it produces a TCR o~ chain able to pair with the available TCK ~ chain and form a TCK showing sufficient affinity for selfMHC-peptide complexes (12,13,15). The corollary to this hypothesis, which proposes a close coupling between arrest of TCK cz gene...

Antigen-specific T-cell responses may be described by combining three categories: (i) the specificity and effector functions of a T-cell population, (ii) the quantity of T-cell responses (i.e., the number of responding T cells within the CD4/CD8 population), and (iii) the "quality" of T cells (defined by the T-cell receptor [TCR] structure). Several methods to measure T-cell responses are now available including evaluation of T-cell precursors using limiting dilution, the enzyme-linked immunospot assay, ex vivo TCR variable (v)-segment analysis determined by flow cytometry, and TCR-CDR3 length analysis (spectratyping), as well as identification of peptide-specific T cells using major histocompatibility complex (MHC) class I tetramers containing appropriate peptides. Until now, only a limited set of MHC-peptide complexes have been available as tetramer complexes. We demonstrate that CD8 ؉ or CD4 ؉ T cells in patients with cancer can be molecularly defined using a combination of spectratyping (TCR structure and "molecular composition") plus the implementation of an antibody panel directed against 21 individual VB TCR chains ("quantity" of T-cell families). This approach is instrumental in defining and comparing the magnitudes of CD4 ؉ or CD8 ؉ T-cell responses over time in individual patients, in comparing the TCR VA and VB repertoire in different anatomic compartments, and in comparing the TCR VA-VB diversity with that in normal healthy controls. This method provides the means of objectively defining and comparing the TCR repertoire in patients undergoing vaccination protocols and underlines the necessity to calibrate the TCR-CDR3 analysis with a qualitative assessment of individual TCR VB families.