Granulysin-dependent killing of intracellular and extracellular Mycobacterium tuberculosis by Vgamma9/Vdelta2 T lymphocytes.

Contribution of Vgamma9/Vdelta2 T lymphocytes to immune protection against Mycobacterium tuberculosis is still a matter of debate. It was reported earlier that Vgamma9/Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis through a granule-dependent mechanism that results in killing of intracellular bacilli. This study found that Vgamma9/Vdelta2 T lymphocytes reduce the viability of both extracellular and intracellular M. tuberculosis. Granulysin and perforin, both detected in Vgamma9/Vdelta2 T lymphocytes, play a major role, which indicates that Vgamma9/Vdelta2 T lymphocytes directly contribute to a protective host response against M. tuberculosis infection.

Frequent recognition of BCRF1, a late lytic cycle protein of Epstein-Barr virus, in the HLA-B*2705 context: evidence for a TAP-independent processing.

Using a transient COS transfection assay, allowing a rapid estimation of the dominant CD8(+) T cell responses against a large number of HLA/viral protein combinations within polyclonal cell lines, we searched for HLA-B*2705-restricted CD8 T cell responses to Epstein-Barr virus (EBV) within T cell samples enriched for EBV-reactive cells. Among the 18 EBV proteins tested, only 2, the latent protein EBNA3A and the late lytic protein BCRF1 (viral IL-10), appeared dominant in the B27 context, as they triggered significant TNF and cytolytic responses in some donors. We provide evidence that the B27/BCRF1 epitope (RRLVVTLQC) is located in the signal sequence and that it can be presented in a TAP-independent manner. Using B27/BCRF1 monomeric complexes coated on immunomagnetic beads, we sorted out BCRF1-specific CD8 T cells from 8 of 15 HLA-B27(+) donors. This is, to our knowledge, the first demonstration of a recognition of BCRF1, suggesting that some immune control against EBV exists even during the late stage of the lytic cycle. This result also strengthens the unusual ability of HLA-B*2705 to present peptide in a TAP-independent manner.

egc, a highly prevalent operon of enterotoxin gene, forms a putative nursery of superantigens in Staphylococcus aureus.

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446-2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, psi ent1 and psi ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific V beta pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.

Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes.

INTRODUCTION: Epstein-Barr virus (EBV) is transmitted orally, replicates in the oropharynx and establishes life-long latency in human B lymphocytes. T-cell responses to latent and lytic/replicative cycle proteins are readily detectable in peripheral blood from healthy EBV-seropositive individuals. EBV has also been detected within synovial tissue, and T-cell responses to EBV lytic proteins have been reported in synovial fluid from a patient with rheumatoid arthritis (RA). This raises the question regarding whether T cells specific for certain viruses might be present at high frequencies within synovial fluid and whether such T cells might be activated or able to secrete cytokines. If so, they might play a 'bystander' role in the pathogenesis of inflammatory joint disease. OBJECTIVES: To quantify and characterize T cells that are specific for epitopes from EBV, cytomegalovirus (CMV) and influenza in peripheral blood and synovial fluid from patients with arthritis. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were obtained from patients with inflammatory arthritis (including those with RA, osteoarthritis, psoriatic arthritis and reactive arthritis). Samples from human leucocyte antigen (HLA)-A2-positive donors were stained with fluorescent-labelled tetramers of HLA-A2 complexed with the GLCTLVAML peptide epitope from the EBV lytic cycle protein BMLF1, the GILGFVFTL peptide epitope from the influenza A matrix protein, or the NLVPMVATV epitope from the CMV pp65 protein. Samples from HLA-B8-positive donors were stained with fluorescent-labelled tetramers of HLA-B8 complexed with the RAKFKQLL peptide epitope from the EBV lytic protein BZLF1 or the FLRGRAYGL peptide epitope from the EBV latent protein EBNA3A. All samples were costained with an antibody specific for CD8. CD4+ T cells were not analyzed. Selected samples were costained with antibodies specific for cell-surface glycoproteins, in order to determine the phenotype of the T cells within the joint and the periphery. Functional assays to detect release of IFN- or tumour necrosis factor (TNF)- were also performed on some samples. RESULTS: The first group of 15 patients included 10 patients with RA, one patient with reactive arthritis, one patient with psoriatic arthritis and three patients with osteoarthritis. Of these, 11 were HLA-A2 positive and five were HLA-B8 positive. We used HLA-peptide tetrameric complexes to analyze the frequency of EBV-specific T cells in PBMCs and SFMCs (Figs 1 and 2). Clear enrichment of CD8+ T cells specific for epitopes from the EBV lytic cycle proteins was seen within synovial fluid from almost all donors studied, including patients with psoriatic arthritis and osteoarthritis and those with RA. In donor RhA6, 9.5% of CD8+ SFMCs were specific for the HLA-A2 restricted GLCTLVAML epitope, compared with 0.5% of CD8+ PBMCs. Likewise in a donor with osteoarthritis (NR4), 15.5% of CD8+ SFMCs were specific for the HLA-B8-restricted RAKFKQLL epitope, compared with 0.4% of CD8+ PBMCs. In contrast, we did not find enrichment of T cells specific for the HLA-B8-restricted FLRGRAYGL epitope (from the latent protein EBNA3A) within SFMCs compared with PBMCs in any donors. In selected individuals we performed ELISpot assays to detect IFN- secreted by SFMCs and PBMCs after a short incubation in vitro with peptide epitopes from EBV lytic proteins. These assays confirmed enrichment of T cells specific for epitopes from EBV lytic proteins within synovial fluid and showed that subpopulations of these cells were able to secrete proinflammatory cytokines after short-term stimulation. We used a HLA-A2/GILGFVFTL tetramer to stain PBMCs and SFMCs from six HLA-A2-positive patients. The proportion of T cells specific for this influenza epitope was low (<0.2%) in all donors studied, and we did not find any enrichment within SFMCs. We had access to SFMCs only from a second group of four HLA-A2-positive patients with RA. A tetramer of HLA-A2 complexed to the NLVPMVATV epitope from the CMV pp65 protein reacted with subpopulations of CD8+ SFMCs in all four donors, with frequencies of 0.2, 0.5, 2.3 and 13.9%. SFMCs from all four donors secreted TNF after short-term incubation with COS cells transfected with HLA-A2 and pp65 complementary DNA. We analyzed the phenotype of virus-specific cells within PBMCs and SFMCs in three donors. The SFMC virus-specific T cells were more highly activated than those in PBMCs, as evidenced by expression of high levels of CD69 and HLA-DR. A greater proportion of SFMCs were CD38+, CD62L low, CD45RO bright, CD45RA dim, CD57+ and CD28- when compared with PBMCs. DISCUSSION: This work shows that T cells specific for certain epitopes from viral proteins are present at very high frequencies (up to 15.5% of CD8+ T cells) within SFMCs taken from patients with inflammatory joint disease. This enrichment does not reflect a generalized enrichment for the 'memory pool' of T cells; we did not find enrichment of T cells specific for the GILGFVFTL epitope from influenza A or for the FLRGRAYGL epitope from the EBV latent protein EBNA3A, whereas we found clear enrichment of T cells specific for the GLCTLVAML epitope from the EBV lytic protein BMLF1 and for the RAKFKQLL epitope from the EBV lytic protein BZLF1. The enrichment might reflect preferential recruitment of subpopulations of virus-specific T cells, perhaps based on expression of selectins, chemokine receptors or integrins. Alternatively, T cells specific for certain viral epitopes may be stimulated to proliferate within the joint, by viral antigens themselves or by cross-reactive self-antigens. Finally, it is theoretically possible that subpopulations of T cells within the joint are preferentially protected from apoptotic cell death. Whatever the explanation, the virus-specific T cells are present at high frequency, are activated and are able to secrete proinflammatory cytokines. They could potentially interact with synoviocytes and contribute to the maintenance of inflammation within joints in many different forms of inflammatory arthritis.

A global appraisal of immunodominant CD8 T cell responses to Epstein-Barr virus and cytomegalovirus by bulk screening.

Knowledge of the immunodominant responses to Epstein-Barr Virus (EBV) and human cytomegalovirus (HCMV) should help to generate cytotoxic T cell lines to these herpesviruses. Here we report on the analysis of CD8 T cell responses to EBV and HCMV in the blood of kidney transplant recipients undergoing viral reactivation (n = 16) and in healthy virus carriers (n = 10). We used a transient COS transfection assay that permits semi-quantitative estimation of CD8+ T cell responses against a larger number of HLA/viral protein combinations within polyclonal T cell lines and thus allows a rapid identification of major epitopes. From the comparison of these responses to those that we previously described in the synovial fluid of patients suffering from various forms of chronic arthritis (n = 32), it appears that EBV-specific T cells are mainly directed against a restricted set of immunodominant epitopes, primarily generated during the early lytic cycle and that both IE1 and pp65 are targets of the anti-HCMV response. We suggest that this method could be generally applied to the rapid identification of immunodominant T cell epitopes in viral and tumor immunity, and could help selecting HLA-peptide complexes that could be used to detect and sort specific T cell populations.

Frequent contribution of T cell clonotypes with public TCR features to the chronic response against a dominant EBV-derived epitope: application to direct detection of their molecular imprint on the human peripheral T cell repertoire.

In an attempt to provide a global picture of the TCR repertoire diversity of a chronic T cell response against a common Ag, we performed an extensive TCR analysis of cells reactive against a dominant HLA-A2-restricted EBV epitope (hereafter referred to as GLC/A2), obtained after sorting PBL or synovial fluid lymphocytes from EBV-seropositive individuals using MHC/peptide multimers. Although TCR beta-chain diversity of GLC/A2+ T cells was extensive and varied greatly from one donor to another, we identified in most cell lines several recurrent Vbeta subsets (Vbeta2, Vbeta4, and Vbeta16 positive) with highly conserved TCRbeta complementarity-determining region 3 (CDR3) length and junctional motifs, which represented from 11 to 98% (mean, 50%) of GLC/A2-reactive cells. While TCR beta-chains expressed by these subsets showed limited CDR1, CDR2, and CDR3 homology among themselves, their TCR alpha-chains comprised the same TCRAV region, thus suggesting hierarchical contribution of TCR alpha-chain vs TCR beta-chain CDR to recognition of this particular MHC/peptide complex. The common occurrence of T cell clonotypes with public TCR features within GLC/A2-specific T cells allowed their direct detection within unsorted PBL using ad hoc clonotypic primers. These results, which suggest an unexpectedly high contribution of public clonotypes to the TCR repertoire against a dominant epitope, have several implications for the follow-up and modulation of T cell-mediated immunity.

Characterization of CMVpp65-specific CD8+ T lymphocytes using MHC tetramers in kidney transplant patients and healthy participants.

BACKGROUND: Cytomegalovirus (CMV) is a ubiquitous herpesvirus that infects 50-90% of individuals in different populations. After primary infection, the virus persists latently in myeloid cells under the control of specific T-cells. Reactivation of CMV infection may cause lethal organ dysfunction and is frequently seen in immunosuppressed individuals. CD8+ cytotoxic T-cells (CTL) have a primary role in suppressing CMV reactivation, and the dominating CTL response is directed against pp65. METHODS: MHC tetramers, that is, complexes between HLA class I (or class II) molecules and antigenic peptides conjugated to fluorochromes allow the direct visualization of antigen-specific receptor-carrying T-cells using flow cytometry. We constructed a novel MHC tetramer for identification of CMVpp65-specific CD8+ T-cells using HLA-A2 molecules folded with the immunodominant NLVPMVATV peptide. RESULTS: The A2/pp65 tetramer specifically stained CMV-directed T-cell lines, and sorted cells showed CMV-specific cytotoxicity. High proportions (0.1-9%) of the CD8+ T-cells were A2/pp65 tetramer+ in healthy HLA-A2+ CMV carriers and in immunosuppressed kidney transplant patients with latent infection. Patients with reactivated CMV infection exhibited up to 15% A2/pp65 tetramer+ cells, which seemed to correlate with CMV load over time. A2/pp65 tetramer+ cells expressed T-cell activation markers. CONCLUSIONS: The construction of a novel A2/pp65 MHC tetramer enabled the design of a rapid and precise flow cytometric method allowing quantitative and qualitative analysis of CMV-specific T-cells. The number of A2/pp65 tetramer binding CTLs in blood may prove to be clinically relevant in assessing the immune response to CMV.

Vgamma9/Vdelta2 T lymphocytes reduce the viability of intracellular Mycobacterium tuberculosis.

An effective immune response against the intracellular pathogen Mycobacterium tuberculosis is strictly dependent on T cell activation. Although this protective response mainly depends on local release of pro-inflammatory cytokines by Th1 CD4(+) T cells, contribution of Vgamma9 / Vdelta2 T lymphocytes to immune protection against this pathogen is suggested by the antimycobacterial reactivity of this subset and its ability to produce large amounts of Th1 cytokines. Here we show that Vgamma9 / Vdelta2 T lymphocytes kill macrophages harboring live M. tuberculosis. The cytotoxic activity of Vgamma9 / Vdelta2 T lymphocytes was not MHC class I or class II restricted but was blocked by anti-TCR monoclonal antibodies, thus indicating that it involved specific interaction between the TCR and the target cell. The cytotoxicity of Vgamma9 / Vdelta2 T lymphocytes was not mediated by TNF-alpha or Fas-Fas ligand, but was shown to occur through a granule-dependent mechanism that resulted in reduction of the viability of intracellular bacilli. Perforin was shown to play an important role in killing of both infected macrophages and intracellular mycobacteria. These data strongly suggest that Vgamma9 / Vdelta2 T lymphocytes contribute to the host defense against M. tuberculosis infection.

Effects of alpha-galactosylceramide (KRN7000), interleukin-12 and interleukin-7 on phenotype and cytokine profile of human Valpha24+ Vbeta11+ T cells.

The alpha-galactosylceramide KRN7000 was reported to be presented by CD1d to natural killer (NK) T cells, cells that are thought to play an important role in the rejection of malignant tumours and in the regulation of several autoimmune diseases. Here we analysed human peripheral blood (PB) NK T cells (Valpha24+ Vbeta11+ T cells) before and after a short-term culture in the presence of KRN7000. KRN7000 strongly activated PB Valpha24+ Vbeta11+ T cells and, when stimulated, the vast majority of these cells expressed interferon-gamma (IFN-gamma). Exposure of these KRN7000-cultured Valpha24+ Vbeta11+ T cells to interleukin-12 (IL-12), but not to IL-7, resulted in a relative increase in IFN-gamma-expressing Valpha24+ Vbeta11+ T cells, compared with IL-4-expressing Valpha24+ Vbeta11+ T cells, indicating a shift towards a T-helper type 1 (Th1) phenotype. KRN7000 strongly up-regulated the expression of the cytotoxic molecule granzyme B (GrB) in Valpha24+ Vbeta11+ T cells. Although IL-7 resulted in a decrease in GrB levels in KRN7000-cultured Valpha24+ Vbeta11+ T cells, IL-12 increased GrB levels in both Valpha24+ Vbeta11+ T cells and in Valpha24+ Vbeta11+ T-cell clones and increased cytotoxicity against hCD1d-transfected HeLa cells. Our data provide further insight into the characteristics of human Valpha24+ Vbeta11+ T cells and indicate that KRN7000 is a potent activator of Valpha24+ Vbeta11+ T cells. Combined with the established anti-tumour effects of KRN7000 in mouse models, these results may support the use of KRN7000 as an anti-tumour agent in man.

3-Formyl-1-butyl pyrophosphate A novel mycobacterial metabolite-activating human gammadelta T cells.

Most human blood gammadelta T cells react without major histocompatibility complex restriction to small phosphorylated nonpeptide antigens (phosphoantigens) that are abundantly produced by mycobacteria and several other microbial pathogens. Although isopentenyl pyrophosphate has been identified as a mycobacterial antigen for gammadelta T cells, the structure of several other stimulating compounds with bioactivities around 1000-fold higher than isopentenyl pyrophosphate remains to be elucidated. This paper describes the structural identification of 3-formyl-1-butyl-pyrophosphate as the core of several non-prenyl mycobacterial phosphoantigens bioactive at the nM range. Recognition of this molecule by gammadelta T cells is very selective and relies on its aldehyde and pyrophosphate groups. This novel pyrophosphorylated aldehyde most probably corresponds to a metabolic intermediate of the non-mevalonate pathway of prenyl phosphate biosynthesis in eubacteria and algae. The reactivity to 3-formyl-1-butyl-pyrophosphate supports the view that human gammadelta T cells are physiologically devoted to antimicrobial surveillance.

Expansion of circulating V gamma 9/V delta 1 T cells in a patient with a syndrome of recurrent fever: evidence for an unusual antigen-driven process leading to selection of recurrent motifs within TCR junctional loops of diverse lengths.

Polyclonal expansions of human Vdelta1 T cells have been described in diverse physiopathological situations without strong TCR structural data for an antigen-driven selection. Here, we have analyzed the phenotype and TCR repertoire of gamma delta T cells obtained from the peripheral blood of a 19-year-old patient with a syndrome of recurrent fever, which accounted for up to 40% of CD3(+) T cells and expressed predominantly Vgamma9 and Vdelta1 TCR regions and a memory phenotype. Sequence analysis of Vdelta1-Jdelta1 transcripts derived from peripheral blood lymphocytes (PBL) indicated that, while Vdelta1-Jdelta1 junctional sequences were diverse in length, all but one contained several recurrent motifs at conserved positions from both the 5'- and 3'-ends of the complementarity-determining region (CDR)3 loop. Analysis of gamma delta T cell clones derived from patient PBL demonstrated that Vgamma9(+) but not Vgamma9(-) T cell clones frequently expressed Vdelta1 chains with these characteristics and unveiled a hierarchy between the constraints imposed on the 5'- vs. the 3' motifs of the Vdelta1 CDR3 loops. These results constitute the first strong evidence for a nominal antigen-driven selection of Vdelta1 T cells in vivo and also suggest that the hierarchy of the constraints imposed by antigens respectively on the length and amino acid composition of TCR CDR3 loops differs between alpha beta and gamma delta T cells.

Selection and long-term persistence of reactive CTL clones during an EBV chronic response are determined by avidity, CD8 variable contribution compensating for differences in TCR affinities.

Recent studies have suggested that the diversity of TCR repertoire after primary immunization is conserved in memory T cells and that a progressive narrowing of this repertoire may take place during recall infections. It now remains to be investigated which parameters determine the repertoire of the memory response and possibly restrict its diversity after subsequent antigenic challenges. To address this question, we took advantage of a panel of CD8+ T cell clones from the joint of a rheumatoid arthritis patient and selected for their reactivity against a single MHC/peptide complex. Characterization of both TCR chains documented a great diversity among those clones and the persistence of clonotypes over a 2-yr period. Strikingly, despite the observed repertoire heterogeneity, all clones displayed a narrow range of MHC/peptide density requirements in cytotoxicity assays (ED50 between 9 and 36 nM). TCR affinities were then indirectly estimated by blocking CD8 interaction with an anti-CD8 mAb. We found a wide range of TCR affinities among the different clonotypes that segregated with Vbeta usage. We thus propose that during an in vivo chronic response, a narrow range of avidity of the TCR-CD8 complex conditions long-term clonotype persistence, and that the level of CD8 contribution is adjusted to keep clonotypes with variable TCR affinities within this avidity window.

An enlarged subpopulation of T lymphocytes bearing two distinct gammadelta TCR in an HIV-positive patient.

Although T cell clone monospecificity is ensured by several allelic exclusion processes operating at either the genotypic or phenotypic levels, clones expressing two distinct alphabeta or gammadelta TCR have been described in several instances. Thus far, the origin of dual TCR-expressing cells and the homeostatic mechanisms controlling the size of this subset in the periphery remain poorly understood. In the course of a phenotypic analysis of gammadelta T cells in HIV-infected patients, we detected the presence of a T cell subset stained by both Vdelta2- and Vdelta3-specific mAb, which represented a large fraction (up to 16.5%) of gammadelta peripheral blood lymphocytes (PBL) in one HIV patient. The presence of two distinct functional delta chains on these cells was confirmed by phenotypic and molecular analysis of TCR transcripts expressed by Vdelta2+Vdelta3+ T cell clones derived from this patient. For 18 months, the absolute number of these cells varied similarly to the other PBL subsets, before becoming undetectable in blood samples. Moreover, most of these cells expressed CD8 receptors, which are classically found on activated, but not resting, gammadelta T cells. Taken together, these data suggest that dual TCR-expressing T cells are subjected to peripheral expansions and contractions presumably following antigen recognition, which would argue against a systematic counter-selection of these cells during peripheral antigen-driven responses.

Common intra-articular T cell expansions in patients with reactive arthritis: identical beta-chain junctional sequences and cytotoxicity toward HLA-B27.

Spondyloarthropathies constitute a group of autoimmune diseases of special interest because of their tight association with the MHC class I molecule HLA-B27 and the bacterial triggering of some clinical forms called reactive arthritis (ReA). One current hypothesis is the presentation by HLA-B27 of a so-called arthritogenic peptide to T cells. To better focus on the relevant T cell populations within the joint, we performed an extensive beta-chain T cell repertoire analysis of synovial fluid compared with PBL in seven patients, four of whom were characterized as having ReA triggered by Yersinia enterocolitica, Chlamydia trachomatis, or Shigella sonnei. Analysis of the size diversity of the beta-chain complementarity-determining region 3 (CDR3) allowed us to evaluate the degree of T cell clonality in the samples. Oligoclonal T cell expansions were frequently observed in the joint. In one patient, CDR3 amino acid sequences of major expansions using two different BV genes were identical. One dominant T cell expansion and several CDR3 amino acid sequences were identical in two different patients. Furthermore, one sequence was identical with a sequence reported independently in a Salmonella-induced ReA patient. Together, these data indicate a surprisingly high degree of conservation in the T cell responses in recent-onset ReA triggered by different micro-organisms. A CD8+ synovial line expressing shared clonotypes was established and reacted toward several B*2705 lymphoblastoid cell lines, therefore supporting a molecular mimicry phenomenon at the T cell level in the disease mechanism.

Frequent enrichment for CD8 T cells reactive against common herpes viruses in chronic inflammatory lesions: towards a reassessment of the physiopathological significance of T cell clonal expansions found in autoimmune inflammatory processes.

We recently evidenced a dramatic enrichment for T cells reactive against Epstein-Barr virus (EBV) within inflamed joints of two rheumatoid arthritis patients. To assess the generality of this phenomenon and its relevance to autoimmunity, we studied the responses of CD8 T cells from patients with either acute or chronic inflammatory diseases (rheumatoid arthritis: n = 18, ankylosing spondylitis: n = 5, psoriatic arthritis: n = 4, Reiter's syndrome: n = 3, arthrosis: n = 2, uveitis: n = 2, multiple sclerosis: n = 2, encephalitis: n = 1) against viral proteins derived from EBV and another common herpes virus, human cytomegalovirus (CMV). T cell responses against EBV and/or CMV epitopes were frequently observed within CD8 T cells derived from chronic inflammatory lesions, irrespective of their location (knee, eye, brain) and autoimmune features. In most cases, CD8 T cells derived from affected organs yielded stronger anti-viral T cell responses than CD8 T cells derived from patients' PBL, even in chronic inflammatory diseases devoid of autoimmune features or induced by defined bacterial agents. Taken together, these results suggest that the presence of virus-specific T cells within inflamed lesions of patients suffering from autoimmune diseases is a general phenomenon associated with chronic inflammation rather than the initiating cause of the autoimmune process. Since this phenomenon was sometimes associated with long-term T repertoire biases within inflamed lesions, the physiopathological significance of T cell clonal expansions found in a recurrent fashion within chronically inflamed autoimmune lesions should be interpreted with caution.

The mechanism of chromosome 7 inversion in human lymphocytes expressing chimeric gamma beta TCR.

Functional chimeric TCR chains, encoded by V gamma J gamma C beta or V gamma J beta C beta hybrid gene TCR, are expressed at the surface of a small fraction of alpha beta T lymphocytes in healthy individuals. Their frequency is dramatically increased in patients with ataxia-telangiectasia, a syndrome associated with inherited genomic instability. As the TCR gamma and beta loci are in an inverted orientation on chromosome 7, the generation of such hybrid genes requires at least an inversion event. Until now, neither the sequences involved in this genetic mechanism nor the number of recombinations leading to the formation of functional transcriptional units have been characterized. In this manuscript, we demonstrate that at least two rearrangements, involving classical recombination signal sequence and the V(D)J recombinase complex, lead to the formation of productive hybrid genes. A primary inversion 7 event between D beta and J gamma genic segments generates C gamma V beta and C beta V gamma hybrid loci. Within the C gamma V beta locus, secondary rearrangements between V gamma and J gamma or V gamma and J beta elements generate functional genes. Besides, our results suggest that secondary rearrangements were blocked in the C beta V gamma locus of normal but not ataxia-telangiectasia T lymphocytes. We also provide formal evidence that the same D beta-3' recombination signal sequence can be used in successive rearrangements with J gamma and J beta genic segments, thus showing that a signal joint has been involved in a secondary recombination event.

Diverse CD1d-restricted reactivity patterns of human T cells bearing "invariant" AV24BV11 TCR.

Human and murine natural T (NT) cells, also referred to as NK1.1+ or NK T cells, express TCR with homologous V regions (hAV24/BV11 and mAV14/BV8, respectively) and conserved "invariant" TCR AVAJ junctional sequences, suggesting recognition of closely related antigens. Murine NT cells recognize CD1-expressing cells and are activated in a CD1-restricted fashion by several synthetic alpha-glycosylceramides, such as alpha-GalCer. Here we studied the reactivity of human T cells against CD1d+ cells pulsed or not with alpha-GalCer and other related ceramides. CD1d-restricted recognition of alpha-GalCer was a general and specific feature of T cell clones expressing both BV11 and canonical AV24AJ18 TCR chains. Besides, human and murine NT cells showed the same reactivity patterns against a set of related glycosylceramides, suggesting a highly conserved mode of recognition of these antigens in humans and rodents. We also identified several AV24BV11 T cell clones self reactive against CD1+ cells of both hemopoietic and nonhemopoietic origin, suggesting the existence of distinct NT cell subsets differing by their ability to recognize self CD1d molecules.