Frequencies of circulating IL-17-producing CD4+CD161+ T cells and CD4+CD161+ T cells correlate with disease activity in rheumatoid arthritis

Abstract

Objective. Rheumatoid arthritis (RA) is a common autoimmune disease that is primarily driven by effector T cells, particularly Th17 cells, which are mainly contained within CD4+CD161+ T cells. Thus, we aimed to explore whether the frequencies of circulating IL-17-producing CD4+CD161+ T cells and CD4+CD161+ T cells were correlated with RA disease activity.

Methods. The surface phenotype and cytokine production of blood were analyzed by flow cytometry in 52 RA patients and 17 healthy controls. The disease activity was evaluated by the 28-joint disease activity score.

Results. The frequencies of circulating IL-17-producing CD4+CD161+ T cells and CD4+CD161+ T cells were increased in RA patients, and they were elevated in patients with active disease status compared to patients with low disease status. Furthermore, their frequencies were positively correlated with disease activity parameters. Receiver operating characteristic curve analysis revealed that IL-17-producing CD4+CD161+ T cell levels were able to distinguish disease activity with 60.7 % sensitivity and 87.5 % specificity, while CD4+CD161+ T cell levels showed 92.9 % sensitivity and 66.7 % specificity.

Conclusion. These results support the hypothesis that Th17 cells are involved in the pathogenesis of RA and suggest that circulating CD4+CD161+ T cells are a potential biomarker of RA disease activity.     

Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena

BACKGROUND: The expressions of CD95 (Fas/APO-1) and Bcl-2 are determinants of apoptosis in normal lymphocytes, and abnormalities in their expressions might contribute to the induction of autoimmunity. In this study, we examined the expressions of CD95 and Bcl-2 on freshly isolated T and B cells from patients with autoimmune hepatitis (AIH) or chronic hepatitis C associated with autoimmune phenomena (CH-C(AI)). METHODS: The CD95 and Bcl-2 expressions within CD4+ T, CD8+ T, and CD19+ B cell subsets were analysed by two-colour flow cytometry. RESULTS: The surface expression of CD95 was significantly high in both the CD4+ T and CD8+ T cell subsets derived from the patients with AIH and those with CH-C(AI), compared with expression in patients with CH-C and normal subjects. The increase in CD95 expression was associated with the phenotypic conversion of naive CD45RO- to primed CD45RO+ CD4+ T cells. Bcl-2 was detected in the vast majority of peripheral T and B cells. There was no significant difference in the percentage of Bcl-2-positive cells in the CD4+ T cell, CD8+ T cell and CD19+ B cell subsets among the patient groups and normal subjects. CONCLUSIONS: These results indicate that an increase in CD4+ T cells expressing CD45RO and CD95 marks an important subset of AIH and CH-C(AI) patients. These expanded CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen-specific or non-specific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes might play a role in the induction of autoimmunity in AIH and CH-C(AI).     

The immunosuppressive effects of CD4+CD25+ regulatory T cells on dendritic cells in patients with chronic hepatitis B

The characteristics and functions of CD4⁺CD25⁺ regulatory T cells (Tregs) have been well defined in murine and human systems. However, the interaction or crosstalk between CD4⁺CD25⁺ Tregs and dendritic cells (DCs) remains controversial. In this study, the effects of chronic hepatitis B (CHB) CD4⁺CD25⁺ Tregs on the maturation and function of monocyte‐derived DCs were examined. The results showed that CD4⁺CD25⁺ render the DCs inefficient as antigen‐presenting cells (APCs) despite prestimulation with CD40 ligand. This effect was marginally reverted by applying neutralizing antibodies (Abs) to IL‐10 and TGF‐β. There were an increased IL‐10 and TGF‐β secretion and reduced expression of costimulatory molecules in DC. Thus, in addition to a direct suppressor effect on CD4⁺T cells, CD4⁺CD25⁺ may modulate the immune response through DCs in CHB patients.     

结核性胸膜炎患者胸液中CD4~+ CD25~+ FoxP3~+调节T细胞抑制抗结核免疫

目的研究结核性胸膜炎患者胸液中CD4+CD25+FoxP3+调节T细胞是否增多,这些调节T细胞是否抑制结核的特异细胞免疫反应。方法使用细胞分离、流式细胞分析及体外细胞培养作细胞增殖及增殖抑制等实验方法,对15例结核性胸膜炎患者及17例健康正常人群胸液及外周血白细胞中CD4+CD25+FoxP3+调节T细胞的量及特征作研究。结果结核性胸膜炎患者胸液中CD4+CD25+FoxP3+调节T细胞明显高于患者及健康人群外周血。在体外,结核性胸膜炎患者胸液中单核细胞对BCG刺激产生γ-干扰素(IFN-γ)的能力明显强于患者及健康人群外周血中单核细胞;把这些调节T细胞从胸液单核细胞中清除,增强了结核患者胸液单核细胞对BCG刺激产生IFN-γ;从结核患者胸液分离的这些调节T细胞能抑制结核患者Th1细胞产生IFN-γ。结论结核性胸膜炎患者胸液CD4+CD25+FoxP3+调节T细胞增多,抑制结核性胸膜炎患者Th1细胞免疫反应,从而参与了结核性胸膜炎的发病。     

Interaction with activated monocytes enhances cytokine expression and suppressive activity of human CD4+CD45ro+CD25+CD127low regulatory T cells

Objective

Despite the high frequency of CD4+ T cells with a regulatory phenotype (CD25+CD127^low FoxP3+) in the joints of patients with rheumatoid arthritis (RA), inflammation persists. One possible explanation is that human Treg cells are converted into proinflammatory interleukin‐17 (IL‐17)–producing cells by inflammatory mediators and thereby lose their suppressive function. The aim of this study was to investigate whether activated monocytes, which are potent producers of inflammatory cytokines and are abundantly present in the rheumatic joint, induce proinflammatory cytokine expression in human Treg cells and impair their regulatory function.

Methods

The presence and phenotype of CD4+CD45RO+CD25+CD127^low T cells (memory Treg cells) and CD14+ monocytes in the peripheral blood (PB) and synovial fluid (SF) of patients with RA were investigated by flow cytometry. Memory Treg cells obtained from healthy control subjects underwent fluorescence‐activated cell sorting and then were cocultured with autologous activated monocytes and stimulated with anti‐CD3 monoclonal antibodies. Intracellular cytokine expression, phenotype, and function of cells were determined by flow cytometry, enzyme‐linked immunosorbent assay, and proliferation assays.

Results

In patients with RA, the frequencies of CD4+CD45RO+CD25+CD127^low Treg cells and activated CD14+ monocytes were higher in SF compared with PB. In vitro–activated monocytes induced an increase in the percentage of IL‐17–positive, interferon‐γ (IFNγ)–positive, and tumor necrosis factor α (TNFα)–positive Treg cells as well as IL‐10–positive Treg cells. The observed increase in IL‐17–positive and IFNγ‐positive Treg cells was driven by monocyte‐derived IL‐1β, IL‐6, and TNFα and was mediated by both CD14+CD16− and CD14+CD16+ monocyte subsets. Despite enhanced cytokine expression, cells maintained their CD25+FoxP3+CD39+ Treg cell phenotype and showed an enhanced capacity to suppress T cell proliferation and IL‐17 production.

Conclusion

Treg cells exposed to a proinflammatory environment show increased cytokine expression as well as enhanced suppressive activity.

     

Autoimmunity and its association with regulatory T cells and B cell subsets in patients with common variable immunodeficiency

Background

Common variable immunodeficiency (CVID) is one of the most prevalent symptomatic primary immunodeficiencies (PIDs), which manifests a wide clinical variability such as autoimmunity, as well as T cell and B cell abnormalities.

扩张型心肌病患者CD4~+CD25~+Foxp3~+ T细胞的检测及意义

目的:探讨扩张型心肌病(DCM)患者外周血CD4+CD25+Foxp3+T细胞的水平及意义。方法:采用流式细胞术检测DCM患者30例及健康对照组20例外周血CD4+CD25+T细胞和CD4+CD25+Foxp3+T细胞的比例。结果:DCM患者外周血CD4+CD25+T细胞占CD4+T细胞的比例为(8.53±1.64)%,显著低于健康对照组的(11.4±2.17)%,P0.01;DCM患者CD4+CD25+Foxp3+T细胞占CD4+T细胞比例为(0.99±0.54)%,显著低于健康对照组的(1.55±0.55)%,P0.01;且DCM患者心功能越差,CD4+CD25+Foxp3+T细胞占CD4+T细胞的比例越低。结论:DCM患者调节性T细胞比例的减少,可能打破了自身免疫耐受,发生了针对心肌抗原的自身免疫反应,参与了DCM的发病。     

IL-17-producing CD4+ T cells, pro-inflammatory cytokines and apoptosis are increased in low risk myelodysplastic syndrome

Immunological responses are increasingly recognised as being important in the initiation and progression of myelodysplastic syndrome (MDS). Indeed, autoimmune diseases commonly occur in association with MDS, particularly in subtypes with a low risk of leukaemic transformation. This study showed for the first time that the numbers of CD3⁺ CD4⁺ IL-17 producing T cells (Th17) were markedly increased in low risk MDS compared with high risk MDS ( P  < 0·01). An inverse relationship between the numbers of Th17 cells and naturally occurring CD4⁺CD25^high FoxP3⁺ regulatory T cells (Tregs) were also described. The Th17:Tregs ratio was significantly higher in low risk disease ( P  < 0·005) compared with high risk MDS and was correlated with increased bone marrow (BM) apoptosis ( P  < 0·01). Tregs from MDS patients suppressed interferon-γ (IFN-γ) secretion by effector CD4⁺ T cells but had no effect on interleukin (IL)-17 production. In addition, the serum levels of IL-7, IL-12, RANTES and IFN-γ are significantly elevated in low risk MDS, while inhibitory factors, such as IL-10 and soluble IL-2 receptor, are significantly higher in high risk disease. The 'unfavourable' Th17:Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and the improved response to immune suppression in patients with low risk MDS compared to those with high risk disease.     

CD4+ CD25+ 调节性T细胞与自身免疫性甲状腺疾病

CD4+ CD25+ 调节性T细胞为新近发现的一群功能成熟的T细胞亚群.其特征性表达叉头盒蛋白3(Foxp3)分子,专职免疫无能和免疫抑制,在维持外周免疫耐受,防止自身免疫性疾病发病中起着极为关键的作用.CD4+ CD25+ 调节性T细胞在自身免疫性甲状腺疾病(AITD)发病中的作用引起了人们的关注.动物实验发现CD4+ CD25+ 调节性T细胞存在与否决定了实验动物是否发生实验性自身免疫性甲状腺炎(EAT)和Graves病.人体研究发现CD4+ CD25+ 调节性T细胞数目和功能异常与人AITD发生密切相关.这些研究结果提示,CD4+ CD25+ 调节性T细胞可能在AITD发病中起重要作用.     

Regulatory T cells and CD20+ B cells in pediatric very severe aplastic anemia: possible clinical markers for evaluating the therapeutic efficacy and prognosis

ABSTRACT

Objectives: To investigate the immune status of children with very severe aplastic anemia (VSAA), and evaluate the frequencies of CD20⁺ B cells and Regulatory T cells (Tregs) as potential markers for evaluating the therapeutic efficacy and prognosis.

Methods: We systematically analyzed CD20⁺ B cells and Tregs using Flow Cytometry in 36 children with VSAA (14 newly diagnosed cases and 22 cases in remission after therapy with HDIVIG?+?r-ATG?+?CSA).

Results: In newly diagnosed VSAA patients, the percentage of CD20⁺ B cells was higher than that in healthy children (P?<?.01), whereas the percentage of Tregs was lower than that in healthy children (P?<?.001). After treatment with HDIVIG?+?r-ATG?+?CSA, the percentage of CD20⁺ B cells in peripheral blood was decreased obviously, and the percentage of Tregs was significantly increased.

Conclusion: There is a moderate negative correlation between the percentage of Tregs and CD20⁺ B cells in our study. Our results shed light on the roles of Tregs and CD20⁺ B cells as therapeutic efficacy and prognostic markers of pediatric VSAA. Moreover, the mechanism underlying the decrease of blood Tregs and increase of CD20⁺ B cells in pediatric VSAA patients have been discussed, indicating that Tregs may suppress B cell responses.     

支气管哮喘大鼠CD4+CD25+T细胞的变化及地塞米松干预作用的研究

目的 研究支气管哮喘(简称哮喘)大鼠模型支气管肺泡灌洗液(BALF)、血液、脾脏CD4+CD25+T细胞的变化,及地塞米松对CD4+CD25+T细胞的影响.方法 50只SD大鼠随机分为5组,空白对照(A)组,哮喘(B)组,地塞米松1(C)组、地塞米松2(D)组,地塞米松3(E)组.A组第l天给予腹腔注射生理盐水l ml,第15～21天每天给予生理盐水雾化.B、C、D、E组用卵蛋白建立哮喘大鼠模型,第1天,每只大鼠腹腔注射抗原l ml(卵蛋白1 mg+灭活百日咳杆菌9×106个+氢氧化铝干粉100 mg)混悬液,第15～21天给予1%的卵蛋白雾化30 min,C、D、E组于雾化后分别给予腹腔注射地塞米松0.2 mg/kg、1 mg/kg、2 mg/kg.采用流式细胞仪检测的方法 ,观察大鼠体内BALF、外周血、脾脏CD4+CD25+T细胞的变化及使用不同剂量地塞米松后对其的影响.结果 B组BALF、外周血、脾脏CD4+CD25+T细胞表达占CD4+T细胞的百分比分别是(42.21±5.62)%、(12.69±2.70)%、(11.15±1.05)%,A组结果 分别是(18.76±5.85)%、(6.21±1.73)%、(7.85±2.13)%.B组与A组比较,差异均具有统计学意义(P＜0.01,P＜0.01,P＜0.05);C组、D组、E组BALF中CD4+CD25+T细胞占CD4+T细胞的百分比表达分别是(10.49±4.03)%、(13.28±5.12)%、(7.51±5.39)%,显著低于A组和B组,(P＜0.05,P＜0.01);外周血中,C组(6.03±1.43)%、D组(4.88±0.95)%与A组(6.21±1.73)%比较,差异无统计学意义,E组(3.49±0.62)%与C组、A组比较,差异有统计学意义(P＜0.05).脾脏中,C组(7.25±1.82)%、D组(8.63±3.18)%与A组(7.85±2.13)%比较,差异无统计学意义,E组(3.38±1.37)%与C组、D组、A组比较,差异有统计学意义(P＜0.05).结论 CD4+CD25+T细胞在哮喘大鼠体内有明显的优势表达,可能是哮喘发病的机制之一.地塞米松可以抑制CD4+CD25+T细胞的表达.BALF内CD4+CD25+T细胞的变化与外周血和脾脏的变化具有一致性,监测外周血或脾脏CD4+CD25+T细胞变化可了解肺部情况.     

Myeloma cells act as tolerogenic antigen‐presenting cells and induce regulatory T cells in vitro

Regulatory T cells (Tregs) are essential for maintenance of self‐tolerance; however, tumor cells can exploit the tolerance to escape the immune system. We investigated the Tregs frequency in patients with multiple myeloma (MM) and in those with monoclonal gammopathy of undetermined significance (MGUS), and found that CD4⁺FoxP3⁺ and CD8⁺FoxP3⁺ Tregs were significantly increased in patients with MM and correlated with the active phase. Both Tregs subsets were expanded in cocultures of CD3⁺ lymphocytes and fresh CD138⁺ MM plasma cells or RPMI8226 and U266 cell lines and functioned as natural (n) and inducible (i) Tregs insofar as they inhibited the proliferation of stimulated CD3 lymphocytes via contact‐dependent and contact‐independent pathways. Induction of Tregs by MM plasma cells required a contact‐dependent pathway, implying antigen recognition by T cells. MM plasma cells acted as immature and tolerogenic antigen‐presenting cells (APCs), in that they displayed low CD80/CD86 expression associated with a phagocytic activity. By acting as immature APCs, MM plasma cells plausibly expand (n)Tregs and (i)Tregs both through conversion of CD3⁺FoxP3^? into CD3⁺FoxP3⁺ T cells and proliferation of CD3⁺FoxP3⁺ T cells, which may suppress the anti‐MM immune response.     

CD147 (Basigin/Emmprin) identifies FoxP3+CD45RO+CTLA4+-activated human regulatory T cells

Human CD4(+)FoxP3(+) T cells are functionally and phenotypically heterogeneous providing plasticity to immune activation and regulation. To better understand the functional dynamics within this subset, we first used a combined strategy of subcellular fractionation and proteomics to describe differences at the protein level between highly purified human CD4(+)CD25(+) and CD4(+)CD25(-) T-cell populations. This identified a set of membrane proteins highly expressed on the cell surface of human regulatory T cells (Tregs), including CD71, CD95, CD147, and CD148. CD147 (Basigin or Emmprin) divided CD4(+)CD25(+) cells into distinct subsets. Furthermore, CD147, CD25, FoxP3, and in particular CTLA-4 expression correlated. Phenotypical and functional analyses suggested that CD147 marks the switch between resting (CD45RA(+)) and activated (CD45RO(+)) subsets within the FoxP3(+) T-cell population. Sorting of regulatory T cells into CD147(-) and CD147(+) populations demonstrated that CD147 identifies an activated and highly suppressive CD45RO(+) Treg subset. When analyzing CD4(+) T cells for their cytokine producing potential, CD147 levels grouped the FoxP3(+) subset into 3 categories with different ability to produce IL-2, TNF-α, IFN-γ, and IL-17. Together, this suggests that CD147 is a direct marker for activated Tregs within the CD4(+)FoxP3(+) subset and may provide means to manipulate cells important for immune homeostasis.     

Elevated levels of activated CD4 T cells in common variable immunodeficiency: association with clinical findings

BackgroundCommon variable immunodeficiency (CVID) is a very heterogeneous syndrome defined by impaired immunoglobulin production. The primary defect remains unknown, but many reports describe peripheral blood T and B lymphocyte dysfunctions in a substantial proportion of CVID patients. Immunophenotypic alterations on memory B lymphocytes correlate with clinical findings. A B-cell-oriented classification principle of the patients has been proposed.Methods and resultsWe investigated the expression of activation surface molecules on CD4 and CD8 T-cells from 14 patients with CVID, 6 non-CVID hypogammaglobulinemic patients with recurrent infections, 47 asymptomatic HIV-positive patients without AIDS defining conditions and 23 healthy subjects. Lymphocyte subsets were analysed by three-colour flow cytometry. Monoclonal panel: CD38-FITC/HLADR-PE/CD4 or CD8-PerCP. In CVID patients serum levels of CD4 T-cells co-expressing the activation marker HLA-DR [CD4 + DR + (34 %), CD4 + CD38 + DR + (18 %)] were significantly elevated compared with controls. Significant increases in CD8 + DR + (54%), CD8 + CD38 + (43 %) and CD8 + CD38 + DR + (29 %) T-cells were observed in comparison with healthy controls. CVID patients with splenomegaly, lower pre-infusion IgG levels (< 600 mg/dl), autoimmune or lymphoproliferative conditions demonstrated even higher levels of CD4 + CD38 + DR + T cells (22, 22, 21 and 21 % respectively) compared with other CVID patients (13, 13, 15 and 15 % respectively).ConclusionThese findings indicate a state of ongoing T lymphocyte activation which is associated with clinical findings frequently observed in CVID.     

Autologous stem cell transplantation for autoimmunity induces immunologic self-tolerance by reprogramming autoreactive T cells and restoring the CD4+CD25+ immune regulatory network

Despite a rapidly accumulating clinical experience with autologous stem cell transplantation (ASCT) as a treatment for severe refractory autoimmune disease, data on the mechanisms by which ASCT induces immune tolerance are still very scarce. In this study it is shown that ASCT restores immunologic self-tolerance in juvenile idiopathic arthritis (JIA) via 2 mechanisms. First, ASCT induces a restoration of the frequency of FoxP3 expressing CD4+CD25bright regulatory T cells (Tregs) from severely reduced numbers before ASCT to normal levels after ASCT. This recovery is due to a preferential homeostatic expansion of CD4+CD25+ Tregs during the lymphopenic phase of immunereconstitution, as measured by Ki67 and CD44 expression, and to a renewed thymopoiesis of naive mRNA FoxP3 expressing CD4+CD25+ Tregs after ASCT. Second, using artificial antigen-presenting cells to specifically isolate self-reactive T cells, we demonstrate that ASCT induces autoimmune cells to deviate from a proinflammatory phenotype (mRNA interferon-gamma [IFN-gamma] and T-bet high) to a tolerant phenotype (mRNA interleukin-10 [IL-10] and GATA-3 high). These data are the first to demonstrate the qualitative immunologic changes that are responsible for the induction of immune tolerance by ASCT for JIA: the restoration of the CD4+CD25+ immune regulatory network and reprogramming of autoreactive T cells.     

Decrease in CD4+CD25+ and CD8+CD28+ T cells in interstitial pneumonitis associated with rheumatic disease

Abstract

The expression of CD25 or CD28 on T cells was examined in patients with rheumatic diseases associated with interstitial pneumonitis (IP), in order to investigate the conditions of CD4⁺CD25⁺ regulatory T cells and CD8⁺CD28^? suppressor T cells. Fifty-five patients with various rheumatic diseases and 23 normal controls were enrolled. CD4⁺CD25⁺ T cells of patients with IP were significantly decreased in comparison with non-IP patients, and the ratio of CD8⁺CD28^? T cells in patients with IP was significantly higher than that in non-IP patients or normal controls. These results for CD8⁺CD28^? T cells were in accord with the decrease in CD8⁺CD28⁺ T cells, and may be related to activation-induced CD8⁺CD28⁺ T-cell death. Thus, the abnormality of CD4⁺CD25⁺ regulatory T cells may be related to the pathogenesis of IP, and the survival and activation of CD8⁺ T cells.     

心肌梗死患者CD4~+CD25~+Foxp3~+ T细胞检测及意义

目的探讨心肌梗死患者外周血CD4+CD25+Foxp3+T细胞的水平及意义。方法采用流式细胞分析法,检测20例急性心肌梗死患者(AMI组)、21例陈旧性心肌梗死患者(OMI组)和36例健康体检者(对照组)外周血CD4+CD25+Foxp3+T细胞水平。结果 AMI组、OMI组的CD4+CD25+T细胞/CD4+T细胞比例分别为(7.20±1.96)%和(7.55±1.77)%均低于对照组的(8.81±1.50)%(P0.05);CD4+CD25+Foxp3+T细胞/CD4+T细胞比例AMI组为(1.42±0.38)%和OMI组为(1.46±0.55)%均比对照组(1.75±0.58)%低(P0.05)。结论 CD4+CD25+调节性T细胞比例降低可能打破了外周免疫耐受,参与了心肌梗死患者动脉粥样硬化的发生发展。