类风湿关节炎小鼠关节聚集的T细胞克隆型变化的分析

目的 :通过对类风湿性关节炎 (RA)小鼠模型发病不同时期的四肢足关节聚集的T细胞克隆型的分析 ,了解T细胞克隆型与RA病变的关系。方法 :用RT PCR/SSCP法 ,对一种自发性RA小鼠模型 (SKG小鼠 )发病初期与后期四肢足关节聚集的T细胞克隆型进行比较分析。结果 :在发病后期 ,四肢足关节中一致的Vβ2和Vβ8.2T细胞克隆型均明显升高 (分别为 72 .3%和 6 0 .2 % )。结论 :TCR为Vβ2和Vβ8.2的T细胞克隆 ,可能对于SKG小鼠RA的发展起重要作用     

T cells accumulating in the inflamed joints of a spontaneous murine model of rheumatoid arthritis become restricted to common clonotypes during disease progression

Although a number of studies have revealed that T cells expand clonally in the joints of patients suffering from rheumatoid arthritis (RA), the kinetics of T cell clonality in multiple joints of an individual throughout progression of the disease is not known. By employing a TCR beta chain gene-specific RT-PCR and subsequent single-strand conformation polymorphism, which enables us to monitor T cell clonality, we analyzed transgenic mice (Tg) carrying the human T cell leukemia virus type I env-pX region. These mice spontaneously develop destructive progressive arthritis similar to RA as they age. In the early stage, the majority of accumulating T cell clones differed in each of four affected feet analyzed. However, in the advanced stage, many of the clones were common to all four feet. The total number of distinct clones gradually decreased as the disease progressed. When splenocytes from arthritic elder Tg were adoptively transferred into either nude mice or young Tg, the clones common to all four feet of the donor were detected again in four feet of the recipients. These findings suggest that, as arthritis progresses, the T cell clones accumulating in the arthritic joints are gradually restricted to certain common clonotypes, some of which are arthrotropic.     

类风湿关节炎小鼠关节浸润的T细胞克隆型分析

目的 :对一种自发性类风湿关节炎 (RA)小鼠 (SKG小鼠 )四肢足关节浸润的T细胞克隆型进行分析 ,以了解T细胞克隆型与RA的关系。方法 :采用RT PCR与单链构象多态性分析 (SSCP)相结合的方法。结果 :在SKG小鼠的四肢足关节Vβ2和Vβ8 2T细胞克隆型的一致率比较高 (分别为 6 8 1%和 5 9 2 % ) ,并且在被检测的所有小鼠的四肢足关节中都具有一致的Vβ2和Vβ8 2克隆型 (10 0 % )。DNA测序表明在SSCP中显示的来源于不同关节样品的共同的DNA带是相同的T细胞克隆型。T细胞转移试验表明关节聚集的T细胞克隆与RA的病变形成有关。结论 :TCR为Vβ2和Vβ8 2的T细胞可能在SKG小鼠的RA中具有重要作用。     

利用II型胶原变构肽抑制类风湿关节炎患者T细胞活化的研究

为了研究II型胶原(CII)变构肽对类风湿性关节炎(RA)患者外周血T细胞激活的抑制作用,以固相法合成CII263-272原型肽及3条变构肽,计算机模拟分析变构肽与HLA-DR4分子的结合能力3。H掺入法检测61例RA患者外周血单个核细胞对CII263-272原型肽及变构肽的T细胞增殖反应。ELISA法检测CII263-272原型肽及变构肽刺激下外周血T细胞IL-2及IFN-γ的分泌。竞争抑制试验观察CII变构肽对原型肽介导T细胞激活的抑制作用。计算机模拟结果发现,CII263-272原型肽可与HLA-DR4分子结合,在此基础上替换TCR结合位点267位谷氨酰胺、270位赖氨酸和271或265位甘氨酸为丙氨酸,不影响该多肽与HLA-DR4分子的结合能力。CII变构肽在RA患者外周血T细胞具增殖中有明显低反应性,下调IL-2和IFN-γ的产生,并且可特异性抑制CII原型肽诱导的T细胞活化(P<0.05或P<0.01)。以上结果提示,替换CII263-272多肽中与T细胞受体结合的氨基酸形成的CII变构肽可抑制RA患者外周血T细胞活化,可能在本病的免疫治疗中有重要意义。     

HLA-dependent peripheral T cell receptor (TCR) repertoire formation and its modification by rheumatoid arthritis (RA)

Our previous studies have disclosed that the peripheral T cell receptor β (TCRB) gene repertoires of RA monozygotic twins were similar. This suggested that the TCRBV repertoire is controlled primarily by genetic factors. Here, we examine how the combination of HLA and presence of RA influence the peripheral TCRB repertoire. Peripheral blood mononuclear cells from six pairs of healthy monozygotic twins, six pairs of monozygotic twins discordant for RA, and nine siblings of a large family, including three RA patients, were examined for their TCRB gene repertoires. Among healthy twins and siblings, the BV repertoires between HLA-identical pairs were significantly more similar than those of HLA-non-identical pairs. When RA-affected members were included, the repertoires of the HLA-identical pairs discordant for RA were dissimilar compared with those of healthy pairs. TCRBV–BJ combination repertoire analysis of CD4 and CD8 T cell subsets from the twins showed that the dissimilarity was primarily confined to CD8 T cells in the healthy identical twins, whereas it was seen in both CD4 and CD8 T cell subsets in the RA-discordant twins. These results suggest (i) the presence of RA modifies the genetically controlled TCR repertoire of peripheral T cells, and (ii) the RA-associated alterations appear to occur more frequently in CD4 T cells than in CD8 T cells.     

类风湿关节炎患者外周血CD4+T细胞亚群的分析

目的 研究类风湿性关节炎(RA)患者不同时期外周血Treg和Th1、Th2、Th17细胞以及中性粒细胞上CD64的表达水平,及它们与RA的活动性指标和自身抗体的相关性.方法 采集78例RA患者和21例健康人外周静脉血,采用流式细胞术检测淋巴细胞亚群(Treg,Th1、Th2、Th17细胞)的变化.结果 RA的CD3+ CD4+T细胞和CD4+ CD25+T细胞增多,活动期RA组Treg比率高于缓解期RA组和健康对照组,缓解期RA组Th2细胞减少,RA中Th1,TH17细胞与健康对照组相比无统计学意义,Treg和Th1、Th2、Th17细胞与RA的活动性指标(ESR,CRP和PLT)均无关联.结论 CD4+T细胞亚群数量异常可能与RA疾病发展有关.     

T cell interactions in active rheumatoid arthritis: insights from the human autologous mixed lymphocyte reaction as a model of T cell activation cascade.

The autologous mixed lymphocyte reaction (AMLR) represents the activation, proliferation and differentiation of T cells in response to signals from autologous non-T cells. Using monoclonal anti-Leu8 antibody to isolate subpopulations of human CD4+ and CD8+ T cells, we have investigated the role of these subpopulations in the T cell activation cascade during the course of AMLR. In normal subjects, CD4+Leu8+ cells are necessary for the initiation of the AMLR response, and sequentially lead to activation and proliferation of both CD4+Leu8- cells and CD8+Leu8+ cells. The activated CD8+Leu8+ cells, in turn, induce CD8+Leu8- cells to generate proliferation of the latter cells. Soluble mediators could be involved in the T cell activation cascade induced by the AMLR. Patients with active rheumatoid arthritis have a profound defect in the AMLR. Further analysis indicates that rheumatoid arthritis CD8+ T cells are markedly defective as responding cells in the AMLR. The impaired AMLR response by CD8+ cells cannot be reconstituted with AMLR-derived supernatants from normal T cells. The data suggest that the defective CD8+ T cell function may contribute to the pathogenesis of the disease.     

T细胞与类风湿性关节炎

类风湿性关节炎(RA)是一种慢性、炎症性自身免疫性疾病,发病机制非常复杂,至今尚未阐明.尽管多种免疫细胞和分子被证实参与了RA的关节炎症反应和组织破坏,但抗原特异性T细胞的激活始终被认为是RA发病起始和进展的中心环节.不同亚群T细胞在RA发病中发挥不同作用,以往认为,自身抗原诱导的促炎性Th1细胞活化是RA发病的主要因...     

Simultaneous analysis of T cell clonality and cytokine production in rheumatoid arthritis using three-colour flow cytometry

In this study we examined the cytokine production by T cells and TCRVbeta subsets in peripheral blood (PB) and synovial fluid (SF) from six RA patients and PB from 10 normal subjects, using three-colour flow cytometry. In two RA subjects we assessed T cell clonality by RT PCR using TCRBV family-specific primers and analysed the CDR3 (complementarity determining region 3) length by GeneScan analysis. A high percentage of IFN-gamma- and IL-2- producing cells was observed among the PB T cells in both the RA patients and normal controls and among the SF T cells in RA patients. In contrast, the percentage of T cells producing IL-4 and IL-5 was small among PB T cells in both RA patients and normal controls and among SF T cells in RA patients. There was no significant difference in the production of IFN-gamma, IL-2 and IL-5 between the two compartments (PB and SF); however, there were significantly more IL-4-producing cells in SF. Molecular analysis revealed clonal expansions of four TCRBV families in SF of two of the RA patients studied: TCRBV6.7, TCRBV13.1 and TCRBV22 in one and TCRBV6.7, TCRBV21.3 and TCRBV22 in the second. These expansions demonstrated cytokine expression profiles that differed from total CD3+ cells, implying that T cell subsets bearing various TCR-Vbeta families may have the potential to modulate the immune response in RA patients.     

Characterization of immunogenic properties of polyclonal T cell vaccine intended for the treatment of rheumatoid arthritis

Two-staged technology for obtaining polyclonal T cell vaccine intended for the treatment of rheumatoid arthritis is described. Stage 1 includes antigen-dependent cultural selection of patient’s T cells and stage 2 consists in their reproduction in the needed amounts by nonspecific mitogenic stimulation. T cell vaccination induces an effective specific anti-idiotypic immune response against T cells reactive to joint antigens. Vaccine therapy significantly reduces plasma level of IFN-γ and increases IL-4 level. The results indicate immunological efficiency and safety of polyclonal T cell vaccine in patients with rheumatoid arthritis. __________ Translated from Kletochnye Tehnologii v Biologii i Medicine, No. 4, pp. 221–225, October, 2007     

Limiting-dilution analysis of T cell reactivity to mycobacterial antigens in peripheral blood and synovium from rheumatoid arthritis patients.

Limiting-dilution analysis (LDA) was used to quantify the frequency of Mycobacterium bovis BCG- and 65-kD-reactive T cells in paired samples of peripheral blood and synovial tissue from patients with rheumatoid arthritis. The frequency of BCG-reactive T cells detected in the peripheral blood of patients ranged from 1/585 to 1/7639 versus a control frequency range of 1/480 to 1/6773. The frequency of such cells in the synovium was found to be much lower than it was in peripheral blood; in fact, in 80% of patients synovial BCG-reactive T cells were not detected. The frequency of 65-kD-reactive cells in the peripheral blood of each individual was lower than the frequency of BCG-reactive cells (range 1/3738 to 1/55,324), as would be expected. However, no synovial 65-kD-reactive cells were detected from any of the patients studied. The LDA assay for the 65-kD antigen was consistent with the single hit model, that for BCG was not. The relatively high proportion of mycobacterial-reactive precursors seen in the peripheral blood of non-vaccinated individuals may reflect a population of cells induced either by natural environmental exposure to mycobacteria or, given the highly conserved nature of heat shock proteins across phylogeny, by some other infection. The results also suggest that the frequent finding of reactivity to proteins such as the 65-kD heat shock protein contained within BCG may not be a generalized phenomenon in rheumatoid synovium.     

Lack of T cell oligoclonality in enzyme-digested synovial tissue and in synovial fluid in most patients with rheumatoid arthritis.

The dominant presence of specific T-cell populations in the rheumatoid joint as detected by Southern blot analysis of T cell receptor (TCR) gene rearrangements would indicate local antigen recognition and T cell proliferation. We therefore studied TCR beta chain gene rearrangements using a C beta 2 probe in paired samples of T cell populations from synovial tissue and peripheral blood (n = 6) as well as synovial fluid (n = 16) and peripheral blood (n = 18) of patients with rheumatoid arthritis (RA). Peripheral blood mononuclear cells from healthy donors (n = 7) served as a control. T cells were studied directly after isolation or after non-specific expansion with OKT3 monoclonal antibody (MoAb) and T cell growth factor (TCGF). DNA samples were digested with EcoRI and HindIII to detect rearrangements to C beta 1 and C beta 2, respectively. Extra bands were detected in all EcoRI-digested DNA samples prepared from both freshly isolated and non-specifically expanded T cell populations of patients and healthy donors, possibly representing 'common' (V-) D-J rearrangements. Dominant rearrangements were found in only two out of 16 synovial fluid T cell populations (one freshly isolated and one expanded) and not in peripheral blood or synovial tissue derived T cell populations. No extra bands were detected in HindIII-digested DNA samples. To investigate the effect of in vitro culture techniques on rearrangement patterns we studied DNA samples prepared from synovial tissue T cells obtained both by outgrowth from tissue with TCGF or by enzyme digestion and subsequent expansion either with TCGF or with OKT3 MoAb and TCGF. Whereas the latter T cell population yielded 'common' rearrangements, the former T cell populations yielded different dominant rearrangements. These data indicate that oligoclonality of the T cell populations in synovial tissue and synovial fluid of patients with RA is a rare event. The data also show the influence of in vitro culture techniques on the result of TCR gene rearrangement analysis.     

Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.     

Analysis of T cell receptors in rheumatoid arthritis: the increased expression of HLA-DR antigen on circulating gamma delta+ T cells is correlated with disease activity.

The phenotypic characteristics of peripheral blood T cells, isolated from 37 rheumatoid arthritis (RA) patients and 17 healthy controls were determined with special emphasis on gamma delta+ T cells and CD4-CD8- alpha beta+ T cells. Two- and three-colour automated flow cytometry analyses were performed using a panel of MoAbs directed against differentiation antigens and T cell receptor molecules. The results demonstrated: (i) no significant difference between the percentages of CD4-CD8- alpha beta+ T cells in patients and controls; (ii) a significant decrease of the gamma delta+ T cell level in the peripheral blood of RA patients relative to controls; (iii) phenotypic abnormalities of circulating gamma delta+ T cells in RA patients suggestive of an activation status in vivo. These abnormalities included a significant reduction in the density of the T cell differentiation antigen CD3 and an increase in the expression of HLA-DR antigen. The level of circulating HLA-DR+/gamma delta+ T cells was significantly higher in patients with active disease. HLA-DR+/gamma delta+ T cells were also present in the synovial fluid obtained from three patients with an active disease. In addition, preliminary experiments showed that the activated gamma delta+ T cells were predominantly V delta 1. Taken together, these data support the involvement of gamma delta+ T cells in the pathogenesis of RA.     

In vitro differentiation of peripheral blood T cells towards a type 2 phenotype is impaired in rheumatoid arthritis (RA)

We have examined the capacity of peripheral blood T cells from RA patients to be polarized in vitro towards a type 1 (T1) or a type 2 (T2) phenotype. Peripheral blood T cells from RA patients and from healthy donors were primed by 1 week of culture with soluble OKT3 in the presence of polarizing cytokines. The recovered T cells were restimulated and their cytokine secretion profile determined. Priming of T cells from RA patients in the presence of recombinant (r)IL-2 plus rIL-12 induced a shift towards a T1 pattern, characterized by increased production of interferon-gamma, that was more pronounced than in the case of healthy donors. Conversely, priming of T cells from RA patients in the presence of IL-4 failed to induce a shift towards a T2 profile after 1 week, whereas it induced T cells from healthy donors to acquire such a profile characterized by heightened production of IL-4, IL-5 and IL-13. However, a T2 polarization profile emerged in T cells from RA patients that were primed in the presence of rIL-4 and subsequently maintained in culture in rIL-2 alone for 1 or 2 additional weeks. We conclude that in vitro differentiation of peripheral T cells towards a type 2 phenotype is impaired in RA. Nevertheless, conditions required to drive peripheral T cells towards a type 2 phenotype were established. Administration of autologous polyclonal T cells expressing a type 2 cytokine secretion profile is proposed as a therapeutic strategy in RA.     

Effects of the novel immunosuppressant FTY720 in a murine rheumatoid arthritis model

We investigated the effects and mechanisms by which FTY720 (FTY) inhibits arthritis development in the SKG mouse rheumatoid arthritis (RA) model. FTY (1 mg/kg/day) administration suppressed the progression of laminarin-induced arthritis in SKG mice. FTY treatment decreased IL-6 and TNF-α expression in synovial fibroblast cells and the number of inflammatory cells overall. Bone destruction was also suppressed by treatment with FTY. The numbers of CD4⁺ and CD8⁺ T cells were significantly increased in the thymus and decreased in the spleen in FTY-treated SKG mice. FTY enhanced IL-4 production by CD4⁺ T cells stimulated by allogeneic spleen cells and inhibited prostaglandin E₂ (PGE₂) production by a TNF-α-stimulated synovial fibroblast cell line. These results indicate that FTY can inhibit arthritis in SKG mice via sequestration of autoimmune CD4⁺ T cells in the thymus, enhancement of Th2 immune responses, and inhibition of PGE₂ production by synovial cells.     

Early activation of invariant natural killer T cells in a rheumatoid arthritis model and application to disease treatment

Invariant NKT (iNKT) cells are a distinctive subtype of CD1d‐restricted T cells involved in regulating autoimmunity and capable of producing various T helper type 1 (Th1), Th2 and Th17 cytokines. Activation of iNKT cells by their exogenous ligand α‐galactosylceramide (α‐GalCer) exerts therapeutic effects in autoimmune diseases such as rheumatoid arthritis (RA). However, the pathophysiological role of iNKT cells in RA, in the absence of exogenous stimulation, is incompletely understood. We investigated the potential pathophysiological effects of iNKT cells in mice with collagen‐induced arthritis (CIA), a model of RA. We found that iNKT cells underwent activation only in the early phases of the disease (6 days post‐induction). In the liver, but not the spleen or lymph nodes, this early activation led to the release of interleukins ‐4, ‐17A and ‐10 and of interferon‐γ; and an increased CD69 expression. Importantly, clinical and histological signs of arthritis were improved by the functional blockade of iNKT cells by a monoclonal antibody to CD1d at the early phase of the disease. This improvement was associated on day 6 post‐induction with decreased expression of co‐stimulatory molecules (CD80, CD86, CD40) on splenic dendritic cells and macrophages, whereas regulatory T‐cell suppressive effects and proportions were not modified. Taken in concert, these findings suggest that iNKT cells are activated early in the course of CIA and contribute to the pathogenesis of arthritis. Therefore, iNKT‐cell activation may be a valid treatment target in RA.     

High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints.

The CD30 is a surface molecule expressed by Th2-type lymphokine-producing T cells upon activation. CD30-expressing activated T cells release a soluble form of the molecule, which can be detectable both in vitro and in vivo. In the present study, high levels of soluble CD30 were found in peripheral blood and synovial fluid from patients with RA. However, CD30+ CD3+ cells, either CD4+ or CD8+, were significantly present in synovial fluid, but not in peripheral blood, of RA patients. Serum values of soluble CD30 were higher in active than inactive RA patients and directly correlated with rheumatoid factor serum titres. These data strongly support an involvement of CD30+ T cells in the immune processes of rheumatoid synovitis, and may suggest a relationship between Th2-type cytokine-secreting T cells and the pathological response in RA.     

Oral GABA treatment downregulates inflammatory responses in a mouse model of rheumatoid arthritis

Current treatments for rheumatoid arthritis (RA) have long-term side effects such that new treatments are needed that can safely help manage the disease. There is a growing appreciation that GABA receptors (GABA-Rs) on immune cells provide new targets that can be used to modulate immune cell activity. Here, we show for the first time that activation of peripheral GABA-Rs can inhibit the development of disease in the collagen-induced arthritis (CIA) mouse model of RA. Mice that received oral GABA had a reduced incidence of CIA, and those mice that did develop CIA had milder symptoms. T cells from GABA-treated mice displayed reduced proliferative responses to collagen and their APC had a reduced ability to promote the proliferation of collagen-reactive T cells. Thus, GABA downregulated both T-cell autoimmunity and APC activity. Collagen-reactive T cells from GABA-treated mice displayed reduced recall responses in the presence of GABA ex vivo, indicating that GABA consumption did not desensitize these cells to GABA. GABA-treated mice had reduced collagen-reactive IgG2a, but not IgG1 antibodies, consistent with reduced Th1 help. The levels of serum anti-collagen IgG2a antibodies were correlated significantly with the CIA disease scores of individual mice. Our results suggest that activation of peripheral GABA-Rs may provide a new modality to modulate T cell, B cell, and APC activity and help ameliorate RA and other inflammatory diseases.