Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes.
Specificity of T cells in synovial fluid: high frequencies of CD8(+) T cells that are specific for certain viral epitopes.
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DOI：
10.1186/ar80
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发表时间：
2000
期刊：
影响因子：
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通讯作者：
Callan MF
中科院分区：
文献类型：
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作者：
Tan LC;Mowat AG;Fazou C;Rostron T;Roskell H;Dunbar PR;Tournay C;Romagné F;Peyrat MA;Houssaint E;Bonneville M;Rickinson AB;McMichael AJ;Callan MF
慢性炎症性关节炎滑膜液中CD8+ T细胞占主导地位。众所周知，这些CD8+ T细胞通常是克隆或少克隆扩增的，但它们的特异性及其与关节疾病发病机制的相关性尚不清楚。我们发现多达15.5%的滑膜CD8+ T细胞可能对Epstein-Barr病毒裂解周期蛋白的单个表位具有特异性。与外周细胞相比，关节内的病毒特异性T细胞表现出活化和分化标志物的表达增加，并在刺激下保持其分泌促炎细胞因子的功能。因此，这些活化的病毒特异性CD8+ T细胞可以通过细胞间接触或细胞因子网络与滑膜细胞相互作用，并在关节炎患者的炎症维持中发挥“旁观者”作用。eb病毒（EBV）经口传播，在口咽部复制，并在人B淋巴细胞中建立终身潜伏期。在健康的ebv血清阳性个体的外周血中，t细胞对潜伏和溶解/复制周期蛋白的反应很容易检测到。EBV也在滑膜组织中被检测到，并且在一例类风湿关节炎（RA）患者的滑膜液中报道了对EBV裂解蛋白的t细胞反应。这就提出了一个问题，即滑膜液中是否存在特定病毒的T细胞，以及这些T细胞是否可能被激活或能够分泌细胞因子。如果是这样，它们可能在炎症性关节疾病的发病机制中扮演“旁观者”的角色。量化和表征关节炎患者外周血和滑液中EBV、巨细胞病毒（CMV）和流感表位特异性T细胞。从炎性关节炎患者（包括RA、骨关节炎、银屑病关节炎和反应性关节炎）中提取外周血单核细胞（PBMCs）和滑液单核细胞（SFMCs）。来自人白细胞抗原(HLA)- a2阳性供体的样品用荧光标记的HLA- a2四聚体与EBV裂解周期蛋白BMLF1的GLCTLVAML肽表位、流感A基质蛋白的GILGFVFTL肽表位或CMV pp65蛋白的NLVPMVATV表位络合进行染色。来自HLA-B8阳性供体的样品用荧光标记的HLA-B8与EBV裂解蛋白BZLF1的RAKFKQLL肽表位或EBV潜伏蛋白EBNA3A的FLRGRAYGL肽表位络合的四聚体进行染色。所有样品均用CD8特异性抗体染色。未分析CD4+ T细胞。选定的样品用细胞表面糖蛋白特异性抗体染色，以确定关节内和周围T细胞的表型。还对一些样品进行了功能测定，以检测IFN-γ或肿瘤坏死因子(TNF)-α的释放。第一组15例患者包括10例RA患者、1例反应性关节炎患者、1例银屑病关节炎患者和3例骨关节炎患者。其中11例HLA-A2阳性，5例HLA-B8阳性。我们使用hla -肽四聚体复合物来分析pbmc和sfmc中ebv特异性T细胞的频率（图1和2）。在研究的几乎所有供者的滑液中，包括银屑病关节炎和骨关节炎患者以及RA患者，都可以观察到EBV裂解周期蛋白特异性CD8+ T细胞的明显富集。在供体RhA6中，9.5%的CD8+ SFMCs对HLA-A2限制性GLCTLVAML表位具有特异性，而CD8+ PBMCs的这一比例为0.5%。同样，在患有骨关节炎（NR4）的供体中，15.5%的CD8+ SFMCs对hla - b8限制性RAKFKQLL表位具有特异性，而CD8+ PBMCs的这一比例为0.4%。相比之下，我们没有发现SFMCs中与任何供体的PBMCs相比，富集了针对hla - b8限制性FLRGRAYGL表位（来自潜伏蛋白EBNA3A）的T细胞。在选定的个体中，我们使用ELISpot法检测SFMCs和PBMCs在体外与EBV裂解蛋白的肽表位短暂孵育后分泌的IFN-γ。这些实验证实了滑膜液中eb病毒裂解蛋白的表位特异性T细胞的富集，并表明这些细胞亚群在短期刺激后能够分泌促炎细胞因子。我们使用HLA-A2/GILGFVFTL四聚体对6例HLA-A2阳性患者的pbmc和sfmc进行染色。在所有研究的供体中，对这种流感表位特异性的T细胞比例很低（<0.2%），我们没有发现SFMCs中有任何富集。我们仅从第二组4名hla - a2阳性RA患者中获得SFMCs。HLA-A2与CMV pp65蛋白NLVPMVATV表位的四聚体在所有四名供者中与CD8+ SFMCs亚群发生反应，频率分别为0.2、0.5、2.3和13.9%。所有四名供者的SFMCs与转染HLA-A2和pp65互补DNA的COS细胞短期孵育后分泌TNF。我们分析了三名供者的PBMCs和SFMCs中病毒特异性细胞的表型。SFMC病毒特异性T细胞比pbmc中的T细胞活性更高，CD69和HLA-DR的表达水平较高。与PBMCs相比，SFMCs中CD38+、CD62L低、CD45RO亮、CD45RA暗、CD57+和CD28-的比例更高。这项工作表明，在取自炎症性关节疾病患者的SFMCs中，对病毒蛋白的某些表位具有特异性的T细胞以非常高的频率（高达CD8+ T细胞的15.5%）存在。这种富集并不反映T细胞“记忆池”的普遍富集；我们没有发现来自甲型流感的GILGFVFTL表位或来自EBV潜伏蛋白EBNA3A的FLRGRAYGL表位的T细胞特异性富集，而我们发现来自EBV裂解蛋白BMLF1的GLCTLVAML表位和来自EBV裂解蛋白BZLF1的RAKFKQLL表位的T细胞特异性富集。这种富集可能反映了病毒特异性T细胞亚群的优先招募，可能是基于选择素、趋化因子受体或整合素的表达。或者，对某些病毒表位具有特异性的T细胞可以被病毒抗原本身或交叉反应性自身抗原刺激在关节内增殖。最后，从理论上讲，关节内的T细胞亚群可能优先受到凋亡细胞死亡的保护。无论如何解释，病毒特异性T细胞以高频率存在，被激活并能够分泌促炎细胞因子。它们可能潜在地与滑膜细胞相互作用，并在许多不同形式的炎性关节炎中维持关节内的炎症。
CD8+ T cells dominate the lymphocyte population in synovial fluid in chronic inflammatory arthritis. It is known that these CD8+ T cells are often clonally or oligoclonally expanded, but their specificity and their relevance to the pathogenesis of joint disease has remained unclear. We found that as many as 15.5% of synovial CD8+ T cells may be specific for a single epitope from an Epstein-Barr virus lytic cycle protein. The virus-specific T cells within the joint showed increased expression of markers of activation and differentiation compared with those in the periphery, and retained their functional capacity to secrete proinflammatory cytokines on stimulation. These activated, virus-specific CD8+ T cells could therefore interact with synoviocytes, either by cell-cell contact or by a cytokine network, and play a 'bystander' role in the maintenance of inflammation in patients with arthritis. 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. 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. 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. 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. 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.