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

doi: 10.1186/ar80. Epub 2000 Feb 7.

A G Mowat, C Fazou, T Rostron, H Roskell, P R Dunbar, C Tournay, F Romagné, M A Peyrat, E Houssaint, M Bonneville, A B Rickinson, A J McMichael, M F Callan

Affiliations

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

L C Tan et al. Arthritis Res. 2000.

Abstract

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.

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Figures

Figure 1

Figure 1

Staining of paired samples of peripheral blood and synovial fluid with human leucocyte antigen (HLA)-peptide tetrameric complexes. Paired samples of peripheral blood mononuclear cells (PBMCs; left column) and synovial fluid mononuclear cells (SFMCs; right column) from donors (a) RhA6, (b, c) RhA5 and (d) RhA7 were stained with a monoclonal antibody that was specific for CD8 and with (a, b) the A2/GLCTLVAML tetramer or (c, d) the B8/RAKFKQLL tetramer. The percentage frequency of CD8+ T cells that reacted with the tetrameric complexes is shown; 50 000 live cells were included in the analysis.

Figure 2

Figure 2

T cells specific for the Epstein-Barr virus (EBV) lytic protein epitopes GLCTLVAML and RAKFKQLL are enriched within synovial fluid. Paired samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were stained with (a) the A2/GLCTLVAML tetramer or (b) the B8/RAKFKQLL tetramer together with a monoclonal antibodies specific for CD8. The percentage of CD8+ T cells that react with the relevant tetrameric complex is shown. L, left knee; R, right knee.

Figure 3

Figure 3

Frequency of IFN-γ secreting antigen-specific T cells detected using an ELISpot assay. ELISpot assays to detect IFN-γ secreted by T cells following short-term incubation with (a) the GLCTLVAML peptide or (b) the RAKFKQLL peptide were performed on paired samples of peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs). The number of spots detected per 106 mononuclear cells is shown.

Figure 4

Figure 4

Expression of CD69 and human leucocyte antigen (HLA) DR by B8-RAKFKQLL-specific T cells in peripheral blood and synovial fluid from patient RhA7. Samples of peripheral blood mononuclear cells (dashed lines) and synovial fluid mononuclear cells (continuous lines) were stained with the B8/RAKFKQLL tetramer, with anti-CD8 and with monoclonal antibodies specific for CD69 or HLA-DR; 200 000 live cells were analyzed, and expression of CD69 and HLA-DR by the populations of CD8+ B8/RAKFKQLL tetramer reactive cells is shown. FITC, fluorescein isothiocyanate

Figure 5

Figure 5

The phenotype of RAKFKQLL-specific T cells in peripheral blood and synovial fluid from patient RhA7. Samples of peripheral blood mononuclear cells (PBMCs; left column) and synovial fluid mononuclear cells (SFMCs; right column) were stained with anti-CD8, the B8/RAKFKQLL tetramer and with monocloncal antibodies specific for (a) CD38, (b) CD62L, (c) CD45RA, (d) CD45RO, (e) CD57 and (f) CD28. The percentage frequency of CD8+ tetramer reactive cells that stain with the phenotypic markers are shown; 200000 live cells were included in the analysis. FITC, fluorescein isothiocyanate.

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References

    1. Rickinson AB, Kieff E. Epstein-Barr virus. Virology, 3rd ed. Edited by Fields BN, Knipe DM, Howley PM. Philadelphia: Lippincott-Raven, 1996. pp. 2397–2446.
    1. Massuci MG, Ernberg I. Epstein-Barr virus: adaptation to a life within the immune system. Trends Microbiol. 1994;2:125–130. - PubMed
    1. Sixbey JW, Nedrud JG, Raab-Traub N, Hanes RA, Pagano JS. Epstein-Barr virus replication in oropharyngeal epithelial cells. . N Engl J Med. 1984;310:1225–1230. - PubMed
    1. Tierney RJ, Steven N, Young LS, Rickinson AB. Epstein-Barr virus latency in blood mononuclear cells: analysis of viral gene transcription during primary infection and in the carrier state. J Virol. 1994;68:7374–7385. - PMC - PubMed
    1. Murray RJ, Kurilla MG, Brooks JM, et al. Identification of target antigens for the human cytotoxic T cell response to Epstein-Barr virus (EBV): implications for the immune control of EBV-positive malignancies. J Exp Med. 1992;176:157–168. - PMC - PubMed

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