CD4+CD25+ regulatory T cells in rheumatoid arthritis: Differences in the presence,phenotype, and function between peripheral blood and synovial fluid

Objective

In mice, CD4+CD25+ regulatory T cells play a pivotal role in preventing autoimmunity. Regulatory T cells are also present and functional in healthy humans. We investigated the presence, phenotype, and function of CD4+CD25+ regulatory T cells in peripheral blood (PB) and synovial fluid (SF) from patients with rheumatoid arthritis (RA).

Methods

The presence and phenotype of CD4+CD25+ regulatory T cells were determined by flow cytometry. Anergy and suppressive activity were assessed by culturing CD4+CD25− and CD4+CD25+ T cells with anti‐CD3 monoclonal antibodies and antigen‐presenting cells, followed by proliferation and cytokine detection.

Results

The percentage of CD4+CD25+ T cells in RA SF was significantly increased compared with that in RA PB, and both of these percentages were higher than that in PB from controls. The cells in RA PB were similar in phenotype and function to CD4+CD25+ regulatory T cells from controls. In SF, however, ∼40–50% of CD4+CD25+ T cells expressed an activated phenotype, i.e., CD69+, class II MHC+, OX‐40+, with high levels of CTLA‐4 and glucocorticoid‐induced tumor necrosis factor receptor. These synovial CD4+CD25+ T cells displayed an increased suppressive capacity compared with blood CD4+CD25+ T cells. However, this enhanced suppressive activity was counterbalanced, because activated responder T cells from SF were less susceptible to CD4+CD25+ T cell–mediated suppression than were responder cells from PB.

Conclusion

We demonstrate that CD4+CD25+ regulatory T cells are present and functional in patients with RA, with higher numbers of regulatory T cells with increased suppressive activity found in SF compared with PB. These findings suggest a negative feedback system that is active at the site of inflammation. The balance between activated responder and regulatory T cells appears to influence the extent of immunoregulation in RA.

     

Functional analysis of synovial fluid and peripheral blood T cells from patients with rheumatoid arthritis

Rheumatoid arthritis is a T cell-mediated autoimmune disease. The lack of knowledge of the involved target antigens severely hampers research on relevant T cells in patients. Here we describe the functional analysis of freshly isolated T cells from the peripheral blood and the site of the lesion (synovial fluid or synovial membrane) of patients with rheumatoid arthritis. Healthy donors and osteoarthritis patients served as controls. Using various polyclonal stimuli, we analyzed CD4⁺ T cells with respect to proliferation and their ability to produce lymphokines. Our data show that lesion-derived CD4⁺ T cells of patients with rheumatoid arthritis are severely defective in proliferation and lymphokine (interleukin-2, interleukin-4, tumor necrosis factor-, interferon-) production. This activation defect was most pronounced at lower cell densities and was present in both synovial fluid derived and synovial membrane derived CD4⁺ T cells of all patients tested. No difference was found between responses of synovial fluid derived CD4⁺ T cells from osteoarthritis patients and those observed with peripheral blood derived T cells from all groups. The observed defect in lesion-derived CD4⁺ T cells from rheumatoid arthritis patients was not due to the effect of inflammatory factors in the synovial fluid because preincubation with synovial fluid could not induce a similar defect in control T cells. Together, our data show a rheumatoid arthritis specific, general defect in the activation of lesion-derived CD4⁺ T cells.Abbreviations IFN Interferon - IL Interleukin - MNC Mononuclear cells - OA Osteoarthritis - PB Peripheral blood - PSA Phosphate-buffered saline+bovin serum albumin+NaN₃ - PIAlb Phosphate-buffered saline+0.38% trisodiumcitrate+10% human albumin - RA Rheumatoid arthritis - SF Synovial fluid - SM Synovial membrane - TNF Tumor necrosis factor     

Early rheumatoid arthritis is associated with a deficit in the CD4+CD25high regulatory T cell population in peripheral blood

OBJECTIVE: Our aim was to test the hypothesis that there is a deficit in the CD4+CD25high regulatory T-cell population in early rheumatoid arthritis (RA), either in size or functional activity. METHODS: Peripheral blood mononuclear cells were examined from subjects with early active RA who had received no previous disease-modifying therapy (n = 43), from individuals with self-limiting reactive arthritis (n = 14), from subjects with stable, well-controlled RA (n = 82) and from healthy controls (n = 72). The frequencies of CD4+CD25high T-cells were quantified using flow cytometry, and function was assessed by the ability to suppress proliferation of CD4+CD25- T-cells. Paired blood and synovial fluid was analysed from a small number of RA and reactive arthritis patients. RESULTS: There was a smaller proportion of CD4+CD25high T-cells in the peripheral blood of early active RA patients (mean 4.25%) than in patients with reactive arthritis or in controls (mean 5.90 and 5.30%, respectively, P = 0.001 in each case). Frequencies in stable, well-controlled RA (mean 4.63%) were not significantly different from early active RA or controls. There were no differences in suppressor function between groups. Higher frequencies of CD4+CD25high T-cells were found in synovial fluid than blood in both RA and reactive arthritis. CONCLUSIONS: These data demonstrate a smaller CD4+CD25high regulatory T-cell population in peripheral blood of individuals with early active RA prior to disease-modifying treatment. This may be a contributory factor in the susceptibility to RA and suggests novel approaches to therapy.     

Abnormality of CD4(+)CD25(+) regulatory T cells in idiopathic thrombocytopenic purpura

OBJECTIVES: The aim of this study was to explore the profile and function of CD4(+)CD25(+) regulatory T cells (Treg cells) in idiopathic thrombocytopenic purpura (ITP) patients. METHODS: Treg cell numbers were analyzed by flow cytometric analysis in peripheral blood mononuclear cells collected from healthy donors or patients with ITP. Quantification of cell proliferation was assayed by an enzyme-linked immunosorbent assay kit, based on the measurement of BrdU incorporation during DNA synthesis. RESULTS: The percentage of Treg cells was significantly decreased in ITP patients in active and non-remission state(5.79 +/- 1.22%) when compared with the patients in remission(11.63 +/- 4.56%) and to healthy subjects(12.68 +/- 3.59%). The suppressive activity of Treg cells in ITP patients was also found to be impaired. CONCLUSION: These results suggest that decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in ITP.     

Enrichment of differentiated CD45RBdim,CD27 – memory T cells in the peripheral blood,synovial fluid,and synovial tissue of patients with rheumatoid arthritis

Objective. To delineate in greater detail the phenotype of T cells that reside in the synovial tissue (ST) and synovial fluid (SF) of patients with rheumatoid arthritis (RA), in order to determine their precise differentiation status, and to determine whether the accumulation of these specific T cell subsets in these synovial compartments could be related to their capacity for transendothelial migration. Methods. Lymphocytes from normal subjects or from the peripheral blood (PB), ST, and/or SF of RA patients were phenotypically analyzed by flow cytometry. Normal PB CD4+ T cells were also characterized using an in vitro assay of transendothelial migration. Results. ST and SF were found to be enriched with memory (CD45RA-, CD45RO+, CD11a^bright, CD44^bright) and activated (CD69+) T cells. Moreover, ST and SF cells from RA patients were enriched in differentiated CD4+, CD45RB^dim, CD27- T cells, a subset of mature memory T cells that develops after prolonged antigenic stimulation. In addition, PB of some RA patients contained an increased number of CD4+, CD45RB^dim, CD27- T cells. The CD4+, CD11a^bright, CD44^bright memory T cells, which included the CD45RB^dim, CD27- more mature memory cells, exhibited an enhanced capacity for transendothelial migration that is likely to contribute to their enrichment in the rheumatoid synovium. Conclusion. RA patients manifest an increased number of mature memory T cells in the SF and ST, and some also have an increased number of these cells in PB that is likely to reflect chronic antigenic stimulation. The enrichment of these cells in the SF and ST reflects, in part, an enhanced capacity to migrate from the vascular space into inflamed tissue.     

乙型肝炎患者外周血CD4+ CD25+调节性T细胞表型与功能分析

目的 观察急、慢性乙型肝炎(AHB、CHB)患者外周血CD4+CD25 high调节性T细胞(Treg)的频率、表型和功能特点.方法 采集16例AHB急性发病期(发病后第1周)患者、72例CHB患者和32例健康人的外周血,检测Treg频率,并分析其表面CD45RO、CD45RA、HLA-DR、CD95和细胞内细胞毒T淋巴细胞相关抗原4(CTLA-4)的表达水平.应用实时荧光定量RT-PCR检测CD4+ CD25+、CD4+ CD25-、CD4+和CD4-等细胞亚群和外周血单个核细胞(PBMC)的FoxP3 mRNA表达量.通过MACS免疫磁珠分选Treg,并应用[3H]掺入法检测Treg抑制抗-CD3抗体和HBV抗原刺激的PBMC增殖能力,并观察Treg对HBV抗原或抗-CD3抗体刺激自体PBMC分泌IFNγ的影响.结果 CD4+CD25 high Treg高表达CD45RO、HLA-DR、CD95和细胞内CTLA-4,低表达CD45RA,并且较特异的高表达FoxP3 mRNA.乙型肝炎病人外周血Treg频率与健康对照(3.50±0.72)%比较无统计学差异,但CHB组(3.90±1.44)%显著高于AHB组(3.10±0.87)%,P＜0.05.Treg本身对于HBV抗原或抗-CD3抗体刺激没有明显的增殖反应和IFNγ分泌,但可抑制自体PBMC增殖和IFNγ分泌,其中对HBV抗原刺激引起的细胞反应抑制作用较强.结论 HBV感染者外周血Treg较特异地表达FoxP3分子,能抑制HBV抗原特异性细胞免疫反应,这对于深入阐明CHB发病机制具有重要意义.     

乙型肝炎患者外周血CD4+ CD25+调节性T细胞表型与功能分析

目的 观察急、慢性乙型肝炎(AHB、CHB)患者外周血CD4+CD25 high调节性T细胞(Treg)的频率、表型和功能特点.方法 采集16例AHB急性发病期(发病后第1周)患者、72例CHB患者和32例健康人的外周血,检测Treg频率,并分析其表面CD45RO、CD45RA、HLA-DR、CD95和细胞内细胞毒T淋巴细胞相关抗原4(CTLA-4)的表达水平.应用实时荧光定量RT-PCR检测CD4+ CD25+、CD4+ CD25-、CD4+和CD4-等细胞亚群和外周血单个核细胞(PBMC)的FoxP3 mRNA表达量.通过MACS免疫磁珠分选Treg,并应用[3H]掺入法检测Treg抑制抗-CD3抗体和HBV抗原刺激的PBMC增殖能力,并观察Treg对HBV抗原或抗-CD3抗体刺激自体PBMC分泌IFNγ的影响.结果 CD4+CD25 high Treg高表达CD45RO、HLA-DR、CD95和细胞内CTLA-4,低表达CD45RA,并且较特异的高表达FoxP3 mRNA.乙型肝炎病人外周血Treg频率与健康对照(3.50±0.72)%比较无统计学差异,但CHB组(3.90±1.44)%显著高于AHB组(3.10±0.87)%,P＜0.05.Treg本身对于HBV抗原或抗-CD3抗体刺激没有明显的增殖反应和IFNγ分泌,但可抑制自体PBMC增殖和IFNγ分泌,其中对HBV抗原刺激引起的细胞反应抑制作用较强.结论 HBV感染者外周血Treg较特异地表达FoxP3分子,能抑制HBV抗原特异性细胞免疫反应,这对于深入阐明CHB发病机制具有重要意义.     

CD4+ PD-1+ T cells accumulate as unique anergic cells in rheumatoid arthritis synovial fluid

OBJECTIVE: The PD-1 receptor, whose deficiency in mice causes autoimmune diseases such as arthritis, is considered to be a negative regulator of activated T cells and to play a crucial role in peripheral tolerance. To clarify the involvement of the PD-1 system in rheumatoid arthritis (RA), we investigated PD-1 expression on synovial fluid (SF) T cells from patients with RA. METHODS: FACS analysis for PD-1 was performed on SF T cells from 44 patients with RA and 6 with osteoarthritis (OA), and also on peripheral blood (PB) T cells from 12 RA patients and 7 healthy controls. Two-color analysis of cell surface PD-1 expression and the intracellular concentration of cytokine production was used to investigate CD4+ T cells from SF of patients with RA and PB from controls. RESULTS: Scarcely any PD-1 expression was detected on control PB T or OA SF T cells. In contrast, PD-1+ cells made up 20.9 +/- 8.6% (mean +/- SD) of RA SF T cells. In RA SF, PD-1 was expressed more predominantly on CD4+ T cells than on CD8+ T cells. As well as expressing CD45RO and CXCR3, CD4+ PD-1+ T cells were mostly CTLA-4 positive and CD26 negative, and were enriched in CD45RB(low) cells. Intracellular cytokine staining revealed that CD4+ PD-1+, but not CD4+ PD-1-, T cells produced interleukin 10 (IL-10), and that CD4+ PD-1+ T cells produced less IL-2 than CD4+ PD-1- T cells. CONCLUSION: PD-1+ T cells in RA SF are enriched, and phenotypic analysis suggests that these cells constitute a unique anergic T cell subset in RA SF.     

趋化因子受体CCR4在活动期类风湿关节炎患者外周血CD4+T细胞表达的研究

目的 探讨活动期类风湿关节炎(aRA)患者趋化因子受体CCR4在外周血CD4+T细胞的表达及其意义,并对该表达与血清重要细胞因子水平的相关性进行研究.方法流式细胞计数方法对12例aRA患者和10名健康对照者外周血CD4+T细胞表面CCR4的表达情况进行检测;酶联免疫吸附试验(ELISA)方法检测患者血清肿瘤坏死因子(TNF)-α、白细胞介素(IL)-10及干扰素(IFN)-γ的水平并记录患者相关临床及实验室资料.分析及评价aRA患者外周血淋巴细胞中CD4+CCR4+T细胞百分数(CD4+CCR4+T%)与TNF-α、IL-10、IFN-γ的血清水平及临床指标的相关性.结果 aRA组外周血CD4+CCR4+T%明显高于健康对照组(P＜0.01).aRA组血清TNF-α(P＜0.01)、IL-10(P＜0.01)及IFN-γ(P＜0.01)水平明显高于健康对照组.在检测的细胞因子中,aRA患者血清IL-10水平与CD4+CCR4+T%呈明显正相关关系(r＝0.66,P＜0.05).在临床指标中,CD4+CCR4+T%与aRA患者Lunsbury关节指数、血沉、C反应蛋白及血小板计数呈明显正相关关系(P＜0.05).结论 CCR4在CD4+T细胞趋化至病变关节过程中可能发挥重要作用;外周血CD4+CCR4+T%与血清IL-10水平密切相关;该百分数可以作为临床评价RA活动性的一个敏感指标.     

Critical role of CD4(+)CD25(+) regulatory T cells in preventing murine autoantibody-mediated thrombocytopenia

Autoimmune response suppression by regulatory T cells (Tregs) helps to maintain peripheral immune tolerance, and defects in this mechanism are thought to play a role in the pathogenesis of various autoimmune diseases. In patients with immune thrombocytopenia, naturally occurring CD4(+)CD25(+) Tregs are both functionally impaired and reduced in number. This?study was undertaken to investigate Tregs' role in preventing immune thrombocytopenia in mice. Treg-deficient mice were prepared by inoculation of Treg-depleted CD4(+)CD25(-) T?cells isolated from BALB/c mice into syngeneic nude mice intravenously. Platelet count, proportion of reticulated platelets, platelet-associated IgG, platelet-associated anti-platelet antibodies, and IgG anti-platelet antibody production in splenocyte cultures were examined by flow cytometry. Of 69 Treg-deficient mice, 25 (36%) spontaneously developed thrombocytopenia that lasted at least 5 weeks. The platelet-associated IgG level and proportion of reticulated platelets were elevated in the thrombocytopenic mice. Platelet eluates and splenocyte culture supernatants prepared from thrombocytopenic mice, but not from nonthrombocytopenic mice, contained IgG antibodies capable of binding to intact platelets. Simultaneous transfer of Tregs completely prevented the onset of thrombocytopenia, but Treg transfer after the onset of thrombocytopenia had no apparent effect. Treatment with IgG anti-cytotoxic T?lymphocyte-associated antigen 4 antibody canceled this Treg-governed suppressive effect.?In summary, these results indicate that Tregs play a critical role in preventing murine autoantibody-mediated thrombocytopenia by engaging cytotoxic T lymphocyte-associated antigen?4.     

Differences in responses of normal and rheumatoid arthritis peripheral blood T cells to synovial fluid and peripheral blood dendritic cells in allogeneic mixed leukocyte reactions

We examined the response of normal T cells to dendritic cells isolated from the synovial fluid (SF) of patients with either rheumatoid arthritis (RA) or seronegative spondylarthropathies (rheumatoid variants) and to dendritic cells from normal and RA peripheral blood (PB) in the allogeneic mixed leukocyte reaction. Despite the differences in the response kinetics, the stimulatory capacity of SF dendritic cells was similar to that of PB dendritic cells in a 7-day mixed leukocyte reaction. We also tested the responsiveness of normal and RA PB T cells to various allogeneic dendritic cells and found that RA PB T cells responded poorly to both rheumatoid variant SF dendritic cells and normal PB dendritic cells. However, when dendritic cells from RA SF were used as stimulators, the response of RA PB T cells was significantly greater than that of normal PB T cells (P less than 0.02). This difference in response was explained in part by a proliferation of the CD8 T cell subset. There was also a shift of low-intensity CD4+, CDw29+ cells to high-intensity CD4+, CDw29+ cells seen in RA PB T cells but not in normal PB T cells, by fluorescence-activated cell sorter analysis.     

强直性脊柱炎患者外周血CD4+CD25+调节性T细胞的研究

目的 观察强直性脊柱炎(AS)患者外周血CD4+CD25调节性T细胞(Treg)的数量、功能及肿瘤坏死因子(TNF)-a 抑制剂治疗的影响.方法 活动性AS患者25例,10例给予etanercept治疗12周,健康对照21名,分离外周血单个核细胞(PBMC),流式细胞术检测CD4+CD25high T细胞比例;实时定量聚合酶链反应检测FOXP3 mRNA表达;免疫磁珠法去除CD25+细胞,可溶性噻唑盐(WST-1)法检测T细胞增殖.结果 活动性AS组CD4+CD25high T细胞比例与对照组差异尤统计学意义,但FOXP3 mRNA表达明显低于对照组(P＜0.01),并与C反应蛋白(CRP)呈负相关(P＜0.01).两组的CD4+CD25+细胞体外均能抑制T细胞增殖(P均＜0.01). Etanercept治疗明显增加CD4+CD25highT细胞比例和FOXP3 mRNA表达(P均＜0.01),与CRP降低呈负相关(P＜0.05;P＜0.01).结论 AS患者外周血表达FOXP3的CD4+CD25+Treg细胞异常,可能参与AS发生和发展;Etanercept治疗上调Treg细胞,可能是抗TNF-α治疗的一个机制.     

Role of CD4(+) CD25(+) regulatory T cells in T helper 2 cell-mediated allergic inflammation in the airways

It has recently been shown that CD4(+) CD25(+) T cells are immunoregulatory T cells that prevent CD4(+) T cell-mediated organ-specific autoimmune diseases. To determine whether CD4(+) CD25(+) T cells downregulate Th2 cell-mediated allergic inflammation in the airways, we studied antigen-induced eosinophil recruitment in the airways in BALB/c Rag-2(-)(/-) mice transferred with CD4(+) CD25(+) T cell-depleted or unfractionated T cells from ovalbumin-specific TCR transgenic mice. Antigen-induced eosinophil recruitment into the airways was significantly decreased in the mice transferred with CD4(+) CD25(+) T cell-depleted splenocytes as compared with those transferred with unfractionated splenocytes. On the other hand, the depletion of CD4(+) CD25(+) T cells increased antigen-induced neutrophil and T cell recruitment in the airways of the mice. The depletion of CD4(+) CD25(+) T cells also decreased antigen-induced IL-4 and IL-5 production in the airways of the mice. Finally, the depletion of CD4(+) CD25(+) T cells prevented antigen-induced Th2 cell differentiation in vitro but increased the differentiation of Th1 cells. These results indicate that CD4(+) CD25(+) T cells modulate the Th1 and Th2 cell balance toward Th2 cells and thus upregulate Th2 cell-mediated allergic inflammation in the airways.     

Cord blood CD4(+)CD25(+)-derived T regulatory cell lines express FoxP3 protein and manifest potent suppressor function

CD4(+)CD25+ T regulatory (Treg) cells have been shown to critically regulate self and allograft tolerance in mice. Studies of human Treg cells have been hindered by low numbers present in peripheral blood and difficult purification. We found that cord blood was a superior source for Treg-cell isolation and cell line generation compared with adult blood. Cord blood CD4(+)CD25+ cells were readily purified and generated cell lines that consistently exhibited potent suppressor activity, with more than 95% suppression of allogeneic mixed lymphocyte reactions (MLRs) (29 of 30 donors). Cultured Treg cells blocked cytokine accumulation in MLRs, with a less robust inhibition of chemokine production. These cell lines uniformly expressed CD25, CD62L, CCR7, CD27, and intracellular cytotoxic T-lymphocyte antigen-4 (CTLA4). FoxP3 protein, but not mRNA, was specifically expressed. Upon restimulation with anti-CD3/CD28 beads, the cultured Treg cells produced minimal cytokines (interleukin-2 [IL-2], interferon-gamma [IFN-gamma], and IL-10) and preferentially expressed tumor growth factor-beta (TGF-beta) latency associated protein. Cytokine production, however, was restored to normal levels by restimulation with phorbol myristate acetate (PMA)/ionomycin. Cord blood-derived cultured suppressor cell function was predominantly independent of IL-10 and TGF-beta. These results demonstrate cord blood contains a significant number of Treg precursor cells capable of potent suppressor function after culture activation. Banked cord blood specimens may serve as a readily available source of Treg cells for immunotherapy.     

T gamma lymphocytes of peripheral blood and synovial fluid in rheumatoid arthritis: quantitative determination and qualitative analysis

The distribution of T gamma lymphocytes in the peripheral blood of one group of rheumatoid patients and in the synovial fluid in a second group was determined. The results were compared to those found for peripheral blood (PB) lymphocytes of normal subjects and for synovial fluid lymphocytes of osteoarthrosis and meniscitis patients. Besides recording percentage and absolute number, we also used cytofluorographic analysis to determine individual capacity of PB T gamma cells to bind heat-aggregated IgG (agg-IgG). The following results were found: 1) there is no significant difference between the percentage and absolute number of PB T gamma lymphocytes of rheumatoid arthritis (RA) patients and those of controls, 2) individual RA PB T gamma cells had a greater number and/or avidity of Fc receptor for IgG than those cells of controls, and 3) the percentage of RA T gamma lymphocytes in synovial fluid, revealed by IgG-EA ox rosetting, is significantly lower than that found in control patients. The factors that may determine a similar lymphocyte picture in RA are discussed.     

Basis of CTLA-4 function in regulatory and conventional CD4(+) T cells

CTLA-4 proteins contribute to the suppressor function of regulatory T cells (Tregs), but the mechanism by which they do so remains incompletely understood. In the present study, we assessed CTLA-4 protein function in both Tregs and conventional (Tconv) CD4(+) T cells. We report that CTLA-4 proteins are responsible for all 3 characteristic Treg functions of suppression, TCR hyposignaling, and anergy. However, Treg suppression and anergy only required the external domain of CTLA-4, whereas TCR hyposignaling required its internal domain. Surprisingly, TCR hyposignaling was neither required for Treg suppression nor anergy because costimulatory blockade by the external domain of CTLA-4 was sufficient for both functions. We also report that CTLA-4 proteins were localized in Tregs in submembrane vesicles that rapidly recycled to/from the cell surface, whereas CTLA-4 proteins in naive Tconv cells were retained in Golgi vesicles away from the cell membrane and had no effect on Tconv cell function. However, TCR signaling of Tconv cells released CTLA-4 proteins from Golgi retention and caused activated Tconv cells to acquire suppressor function. Therefore, the results of this study demonstrate the importance of intracellular localization for CTLA-4 protein function and reveal that CTLA-4 protein externalization imparts suppressor function to both regulatory and conventional CD4(+) T cells.