+1 Frameshifting as a Novel Mechanism to Generate a Cryptic Cytotoxic T Lymphocyte Epitope Derived from Human Interleukin 10

Recent data indicate that some cytotoxic T cells (CTLs) recognize so-called cryptic epitopes, encoded by nonprimary open reading frame (ORF) sequences or other nonclassical expression pathways. We describe here a novel mechanism leading to generation of a cryptic CTL epitope. We isolated from the synovial fluid of a patient suffering from a Reiter's syndrome an autoreactive T cell clone that recognized cellular IL-10 in the HLA-B*2705 context. The minimal IL-10 sequence corresponding to nucleotides 379–408 was shown to activate this clone, upon cotransfection into COS cells with the DNA encoding HLA-B*2705, but the synthetic peptide deduced from this sequence did not stimulate the clone. Using a site-directed mutagenesis approach, we found that this clone recognized a transframe epitope generated by an internal +1 frameshifting in the IL-10 sequence and so derived partly from ORF1, partly from ORF2. We defined that +1 frameshifting was induced by a specific heptamer sequence. These observations illustrate the variety of mechanisms leading to generation of cryptic epitopes and suggest that frameshifting in normal cellular genes may be more common than expected

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-γ or tumour necrosis factor (TNF)-α 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-γ 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

Up-regulation of cytolytic functions of human Vδ2− γδ T lymphocytes through engagement of ILT2 expressed by tumor target cells

International audienceAbstract In humans, the majority of peripheral blood γδ T cells expresses Vγ9Vδ2 T-cell receptors (TCR) and recognize nonpeptidic phosphorylated antigens. In contrast, most tissue-derived γδ T cells, which are located mainly in spleen and epithelia, preferentially use Vδ1 or Vδ3 chains paired with diverse Vγ chains to form their TCR. Our knowledge about the antigenic specificity and costimulation requirements of human Vδ2− γδ T cells remains limited. In an attempt to address this important issue, we characterized the specificity of a monoclonal antibody (mAb 256), screened for its ability to specifically inhibit cytolytic responses of several human Vδ2− γδ T-cell clones against transformed B cells. We show that mAb 256 does not target a TCR ligand but blocks key interactions between non-TCR molecules on effector γδ T cells and ILT2 molecule, expressed by tumor targets. In line with the previously reported specificity of this NK receptor for classic and nonclassic major histocompatibility complex (MHC) class I molecules, blockade of MHC class I/ILT2 interactions using MHC class I- or ILT2-specific mAbs and ILT2-Fc molecules inhibited tumor-induced activation of Vγ8Vδ3 T-cell clones. Therefore, this study describes a new cytotoxic T lymphocyte activation pathway involving MHC class I engagement on γδ T cells

A novel nucleotide-containing antigen for human blood γδ T lymphocytes

International audienceThe stimulation of human gamma delta T cells by mycobacteria occurs through recognition of four distinct nonpeptide phosphorylated antigens termed TUBag1-4. Among these latter, TUBag4 has already been biochemically characterized as a gamma-X derivative of 5'-deoxythymidine triphosphate (Constant, P., Davodeau, F., Peyrat, M. A., Poquet, Y., Puzo, G., Bonneville, M. and Fourni?J.-J., Science 1994. 264: 267). However, despite chemical synthesis of weakly stimulatory nucleotide-containing analogs, these mycobacterial compounds remained the sole nucleotide-containing antigens actually isolated from natural sources. Here, we present the complete isolation of the TUBag3 antigen from Mycobacterium fortuitum and demonstrate that this nonpeptide molecule contains a 5'-UTP nucleotide moiety. On selected V gamma 9/V delta 2 clones, T cell responses can be triggered with nanomolar concentrations of TUBag3. Like crude mycobacterial extracts, this purified nucleotide conjugate elicits a strong polyclonal response of gamma delta PBL from healthy donors. Furthermore, we present evidence that this compound is distinct from the recently synthesized gamma-isopentenyl 5'-UTP, a nucleotide conjugate of isopentenyl pyrophosphate that was found to be stimulatory for human gamma delta T cells (Tanaka, Y., Morita, C.T., Tanaka, Y., Nieves, E., Brenner, M. B. and Bloom, B. R., Nature 1995. 375: 155). Since it appears that both mycobacterial nucleotide antigens are molecules structurally related to peculiar precursors of nucleic acid synthesis, we propose that TUBag-reactive T cells might be specifically devoted to surveillance of proliferating cells