不同HBV感染状态患者外周血CD4~+CD25~+Tregs TCR CDR3谱系漂移特征分析

目的探讨慢性HBV携带者、慢性乙型肝炎(chronic hepatitis B,CHB)及急性乙型肝炎(acute hepatitis B,AHB)这3种不同HBV感染状态下患者外周血中CD4~+ CD25~+调节性T细胞(T regulatory cells,Tregs)T细胞受体(T cell receptor,TCR)β链各家族互补决定区3(complementarity-determining region 3,CDR3)谱系漂移特征。方法采集3例正常人、5例慢性HBV携带者、12例CHB及5例AHB患者外周抗凝静脉血,分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC);磁珠分选法分选CD4~+ CD25~+Tregs,提取总RNA,逆转录合成c DNA;根据TCRβ链24个可变区基因(TRBV)家族设计相应的上游引物,并在β链恒定区(BC)设计1条共用的FAM荧光标记下游引物及对照引物。PCR扩增出24个包含完整CDR3区的TRBV家族的PCR产物,电泳鉴定目的片段;PCR产物送上海基康用毛细管电泳法进行基因扫描;用Peak Scanner Software v1.0软件分析TRBV家族CDR3谱系特征。结果不同HBV感染状态下24个TRBV家族CDR3谱系图均呈现一个或者多个形态不一、数量不等的单峰、寡峰、偏峰谱型,其中一些家族表达极低或缺失,在1.5%琼脂糖凝胶电泳图上多数家族于预测范围大小处呈现1条模糊条带,部分家族出现清晰条带或无条带;对不同HBV感染者间各TRBV家族CDR3谱系偏移率进行比较发现:慢性HBV携带者中BV7谱系偏移率明显高于CHB及AHB患者(P均0.05),CHB患者中BV15谱系偏移率明显高于慢性HBV携带者及AHB患者(P均0.05),AHB患者中BV23谱系偏移率明显高于CHB及慢性HBV携带者(P均0.05)。CHB患者外周血CD4~+ CD25~+Tregs TRBV家族CDR3克隆增生总体异常率与HBV DNA载量呈正相关关系(r=0.576 5,P=0.049 8)。结论部分优势利用的TRBV家族可能在感染HBV后免疫耐受以及清除病毒中发挥重要作用。     

“荧光定量PCR溶解曲线法”监测人TCRβ链CDR3谱系漂移技术的建立

目的 建立"荧光定量PCR溶解曲线分析技术"监测人外周血T细胞TCR β链CDR3谱系漂移(单/寡/多克隆增生).方法 提取4例正常人、9例大肠癌患者外周血单个核细胞(peripheral blood mononuclear cell-PBMC)中的总RNA,逆转录成cDNA,以26个人TRBV基因家族设计上游引物,共同的TRBC基因设计下游引物,荧光定量PCR(FQ-PCR)扩增26个TRBV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生.结果 正常人外周血T细胞TCR β链26个家族CDR3表达频率不一致,各家族PCR产物的"溶解曲线谱型图"(melting curve spectratyping)呈现溶点不同的CDR3多态性,为多克隆增生的高斯分布;9例大肠癌患者的外周血TCR β链CDR3谱系的26个家族CDR3表达频率不一致,有的患者部分家族呈缺失状态,患者各家族PCR产物的"溶解曲线谱型图"上,多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族.结论 "荧光定量PCR溶解曲线分析TCR CDR3谱系漂移技术",方法稳定简便,能较好的监测正常人和临床样本外周血T细胞TCR β链CDR3谱系漂移(单/寡/多克隆增生).     

慢性乙型肝炎患者外周血CD8~+T细胞TCR Vβ基因亚家族克隆化的研究

目的:分析慢性乙型肝炎(CHB)患者CD8+T细胞TCR Vβ基因亚家族克隆化特征。方法:采用逆转录-聚合酶链反应(RT-PCR)扩增8例CHB患者外周血CD8+T细胞TCR Vβ基因22个亚家族的CDR3区,基因扫描技术对TCR Vβ亚家族的克隆化进行鉴定。结果:基因扫描显示所有8例CHB患者CD8+T细胞TCR Vβ基因亚家族均出现一个或一个以上单克隆或寡克隆增生。Vβ8、Vβ11、Vβ12出现单克隆增生的频率相对较高。8例健康者TCR Vβ基因亚家族均为多克隆。结论:CHB患者外周血CD8+T细胞TCR Vβ亚家族存在克隆性增生。     

采用荧光定量PCR溶解曲线法监测人TCR alpha链CDR3谱系漂移技术的初步探讨

目的:初步探讨荧光定量PCR溶解曲线分析技术监测人外周血T细胞TCR alpha链CDR3谱系漂移(单/寡/多克隆增生).方法:提取4例正常人、2例淋巴瘤型白血病患者PBMC中的总RNA,逆转录成cDNA,以32个人TCR alpha 链胚系可变区基因家族 (TRAV)设计上游引物,共同的TCR alpha 胚系链恒定区基因家族(TRAC)设计下游引物,荧光定量PCR(FQ-PCR)扩增32个TRAV基因各家族CDR3谱系,溶解曲线法分析各家族CDR3谱系的单/寡/多克隆增生.结果:正常人外周血T细胞TCR alpha链32个家族CDR3表达频率不一致,各家族PCR产物的溶解曲线谱型图(melting curve spectratyping)呈现熔点不同的CDR3多态性,为多克隆增生的高斯分布,2例淋巴瘤型白血病患者外周血T细胞TCR alpha链32个家族CDR3表达频率不一致,部分家族呈缺失状态,患者各家族PCR产物的溶解曲线谱型图上,多数家族为多克隆增生的高斯分布,但每个患者均出现数量不等的单克隆和寡克隆增生家族.结论:荧光定量PCR溶解曲线分析TCR alpha链CDR3谱系漂移技术方法稳定简便,能较好地监测正常人和临床样本外周血T细胞TCR alpha链CDR3谱系漂移(单/寡/多克隆增生).     

替比夫定对慢性乙型肝炎患者外周血CD4~+ CD25~+ CD127~(low) T和CD8~+ CD25~+ T细胞频率的影响及临床意义

目的探讨核苷类药物替比夫定对慢性乙肝患者(CHB)外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例的影响,并结合临床指标分析其临床意义。方法替比夫定抗病毒治疗22例CHB患者,在治疗前及治疗后3,6个月时,分别以流式细胞仪检测外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例,实时荧光定量RT-PCR检测Foxp3 mRNA的表达水平,荧光定量PCR检测血清HBV DNA水平,酶联免疫吸附法检测HBV标志物,全自动生化分析仪检测ALT水平。结果 CHB患者外周血中CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例显著高于对照组。替比夫定治疗3个月时,这两群细胞比例显著下降,Foxp3 mRNA的表达也显著下降;HBV DNA水平降至检测水平以下的CHB患者,其CD4+CD25+CD127lowT细胞也降至正常水平。治疗3、6个月时,HBeAg阴转率分别为9.1%和18.2%,发生HBeAg血清学转换者的CD4+CD25+CD127lowT细胞和CD8+CD25+T细胞比例均降至正常水平。结论替比夫定能快速有效抑制CHB患者的病毒复制...     

抗病毒治疗对CHB患者外周血TCRβ链CDR3谱系的影响

目的:分析慢性乙型肝炎(CHB)患者抗病毒治疗前后T淋巴细胞受体(TCR)β链各家族互补决定区3(CDR3)谱系的变化,了解抗病毒治疗前后T细胞应答的变化。方法:采用荧光定量PCR(FQ-PCR)溶解曲线法对11例CHB患者抗病毒治疗前后外周血单个核细胞(PBMC)T淋巴细胞受体β链可变区(TCR BV)基因各家族CDR3谱系进行分析,了解抗病毒治疗前后T细胞应答的变化。分析TCR CDR3谱系与CHB患者血清病毒载量的关系。结果:11例CHB患者抗病毒治疗后多个TCR CDR3谱系均出现了不同程度偏移。低病毒载量组(HBV DNA≤104拷贝/ml)治疗后谱型偏移率显著高于高病毒载量组(HBV DNA≥105拷贝/ml)。结论:CHB患者抗病毒治疗后单寡谱型偏移率与治疗前血清HBV DNA的水平相关。     

无症状HBV携带者外周血CD4~+TCR Vβ基因亚家族克隆化特征的研究

分析无症状HBV携带者(AsC)CD4+TCR Vβ基因家族克隆化特征。采用逆转录-聚合酶链反应(RT-PCR)扩增7例AsC外周血CD4+TCR Vβ基因22个亚家族的CDR3区,基因扫描技术对CD4+TCR Vβ亚家族的克隆化进行鉴定。结果基因扫描显示,7例AsC CD4+TCR Vβ基因亚家族均出现一个或一个以上单克隆或寡克隆增生;7例健康者CD4+TCR Vβ基因亚家族均呈正态分布。提示,AsC外周血CD4+TCR Vβ亚家族存在克隆性增生,这可能与AsC免疫耐受的形成有关。     

慢性乙型肝炎患者外周血CD4~+CD25~+Treg与CD4~+和CD8~+ T淋巴细胞亚群的相关研究

目的:探讨慢性乙型肝炎患者外周血中CD4+CD25+调节性T细胞的含量和CD4+CD8+T淋巴细胞亚群分布,两者之间相关性及与HBV的相关性。方法:采用流式细胞术检测50例慢性乙型肝炎患者和20例健康对照者外周血中CD4+CD25high、CD4+CD25+Foxp3+Treg细胞表达及CD3/CD4/CD8 T淋巴细胞亚群,荧光定量PCR法检测HBV DNA含量。结果:慢性乙型肝炎患者外周血中CD4+CD25highTreg明显高于健康对照组(P0.01),且随HBV DNA载量增加,患者外周血中CD4+CD25highTreg细胞的水平逐渐升高。慢性乙型肝炎患者外周血中CD4+CD25+Foxp3+Treg细胞也相应增高,且与CD4+CD25highTreg细胞的变化成正相关(r=0.890,P0.001)。与健康对照组比较,患者组CD4+T细胞百分率及CD4+/CD8+比值均降低,而CD3+T细胞和CD8+T细胞百分率差异无显著性(P0.05)。CD4+CD25highTreg细胞与HBV DNA取对数后成正相关(r=0.782,P0.001),与谷丙转氨酶(ALT)成正相关(r=0.432,P0.005);与CD3+、CD4+、CD8+T细胞水平及CD4+/CD8+比值均无相关性(P0.05)。CD3+、CD4+、CD8+T淋巴细胞及CD4+/CD8+比值与HBV DNA载量之间亦无相关性(P0.05)。结论:慢性乙型肝炎患者外周血中CD4+CD25+Treg细胞增高,且与HBV的复制水平及ALT增高具有一致性,而T细胞亚群是否可作为监测CHB患者免疫状态的指标需进一步探讨。     

CHB患者CD4+ T细胞ICOS表达及其与干扰素治疗的相互关系

目的 研究慢性乙型肝炎(CHB)患者经干扰素治疗前后外周血CD4+T细胞上可诱导共刺激分子( inducible costimulator,ICOS)表达水平的变化情况.方法 CHB患者56例,其中聚乙二醇(PEG)干扰素α2a治疗28例,拉米夫定治疗28例,以健康志愿者20例为正常对照组.采集两治疗组治疗前及治疗后24、48周的患者外周血,以流式细胞技术检测ICOS+ CD4+T细胞在外周血单个核细胞(peripheral blood mononuclear cells,PBMC)中的频数变化;采用real-time PCR法检测患者HBV DNA载量变化.结果 CHB患者CD4+T细胞ICOS表达水平明显高于正常对照者(P＜0.001).干扰素治疗后患者ICOS表达水平下降,同拉米夫定组差异有统计学意义(P＜0.05).干扰素治疗后患者ICOS改变值同HBV DNA载量变化值呈正相关(r=0.972,P＜0.001),而拉米夫定组则未发现相关性(r=-0.101,P=0.608).结论 CHB患者存在着细胞免疫紊乱,其CD4+T细胞ICOS的表达升高.干扰素可下调CD4+T细胞ICOS的表达,纠正细胞免疫偏移,发挥抗HBV作用.     

Lower proportion of CD19+IL-10+ and CD19+CD24+CD27+ but not CD1d+CD5+CD19+CD24+CD27+ IL-10+ B cells in children with autoimmune thyroid diseases

Abstract

Introduction: As it is generally known, regulatory B cells (Bregs) control inflammation and autoimmunity. The significance of Bregs in the population of children with autoimmune thyroid diseases (AITD) still offers plenty of potential to explore. The aim of this study was to estimate the expression of Bregs (phenotype CD19⁺CD24⁺CD27⁺IL-10⁺, CD19⁺IL-10⁺, CD1d⁺CD5⁺CD19⁺IL-10⁺ and CD1d⁺CD5⁺CD19⁺CD24⁺CD27⁺) in a paediatric cohort with AITD and in health controls.

Materials and methods: A total of 100 blood samples were obtained from 53 paediatric patients with Graves’ disease (GD) (N?=?12 newly diagnosed, mean age 12.5?±?3.5 and N?=?17 during methimazole therapy, mean age 12.7?±?4.4), Hashimoto’s thyroiditis (HT) (N?=?10 newly diagnosed, mean age 13.3?±?2.9 and N?=?10 during L-thyroxine therapy, mean age 13.7?±?3.4) and compared with healthy controls (C) (N?=?15, mean age 13.1?±?3.1). The expressions of the immune cell populations were analysed by four-color flow cytometry using a FASC Canto II cytometer (BD Biosciences).

Results: There was a decreasing tendency in the number of lymphocytes B producing IL-10 (B10) cells among all B lymphocytes and more widely, also among all lymphocytes, in each study group, as compared to C. We reported a reduction in IL-10 production in Bregs with the expression of CD19⁺CD24⁺CD27⁺IL-10 and CD1d⁺CD5⁺CD19⁺IL-10⁺ in both untreated and treated AITD.

Conclusions: Our data demonstrate that the reduction in the number of Bregs with CD19⁺CD24⁺CD27⁺IL-10⁺ and CD19⁺IL-10⁺ expression could be responsible for breaking immune tolerance and for AITD development in children.     

Circulating CD3+ CD4+ CD+ T lymphocytes in multiple sclerosis

Triple-antibody flow cytometry was used to search for distinctive populations of peripheral blood lymphocyte immunophenotypes in multiple sclerosis (MS). Using monoclonal antibodies to the cell surface markers CD3, CD4, and CD8, T cell subsets were quantified on a cohort of 31 MS patients (not treated with corticosteroids for at least 6 months), 30 healthy donors, and 14 patients with other autoimmune diseases (also corticosteroid treatment-free for at least 6 months). Untreated MS patients displayed a significantly greater population of CD3+CD4+CD8+ circulating T cells than healthy donors (P = 0.023). Patients with other autoimmune diseases displayed mean populations of CD3+CD4+CD8+ cells greater than normal donors and less than MS, but not significantly different from either. An additional 45 MS patients who had received corticosteroid therapy within the previous 6 months were phenotyped. Treatment of symptomatic MS with corticosteroids was associated with a smaller population of circulating CD3+CD4+CD8+ cells. Some MS patients have significantly greater numbers of peripheral blood T lymphocytes simultaneously expressing CD3, CD4, and CD8 surface markers than healthy donors and this population of cells may be reduced by corticosteroids treatment. This triple positive phenotype may be a manifestation of a systemic immune abnormality in MS.     

Expansion and differentiation of CD14+CD16(-) and CD14+ +CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3-10 days in X-VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 Ligand (Flt-3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt-3L, IL-3 and M-CSF for 7-14 days. These two step cultures resulted in up to a 600-fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16(-) and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16(-), the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA-DR, and CCR5. Both subpopulations secreted TNF and IL-12p40 but little or no IL-10. CD14++CD16+ monocytes released significantly more IL-12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.     

Circulating CD4+CD25+ and CD4+CD25+ T cells in myasthenia gravis and in relation to thymectomy

Studies in experimental animal models of human autoimmune diseases have revealed that CD4⁺CD25⁺ T regulatory (Tr) cells are of thymic origin and have potentials in preventing auto‐aggressive immunity. Myasthenia gravis (MG) is the best‐characterized autoimmune disease. Changes in the thymus are found in a majority of patients with MG. Thymectomy has beneficial effects on the disease severity and course in a substantial proportion of MG patients. But the occurrence and characteristics of Tr cells have not yet been defined in MG. We determined the frequencies and properties of circulating CD4⁺CD25⁺ versus CD4⁺CD25^– cells in MG patients and healthy controls (HCs), with special focus on the effect of thymectomy on CD4⁺CD25⁺ cells. CD4⁺CD25^high cells comprise only about 2% of blood lymphocytes in both MG patients and HCs. Frequencies of CD4⁺CD25^high cells were similar in MG patients irrespective of treatment with thymectomy. CD4⁺CD25⁺ cells in both MG patients and HCs are mainly memory T cells and are activated to a greater extent than CD4⁺CD25^– cells, as reflected by high levels of CD45RO and human leucocyte antigen (HLA)‐DR‐positive cells. In both MG patients and HCs, CD4⁺CD25⁺ cells also contained a high proportion of CD95‐expressing cells as possible evidence of apoptosis‐proneness. Upon stimulation with anti‐CD3/CD28 monoclonal antibodies, CD4⁺CD25⁺ cells responded more vigorously than CD4⁺CD25^– cells in MG, irrespective of treatment with thymectomy, as well as in HCs. Although CD4⁺CD25^– cells are mainly naïve T cells, in non‐thymectomized MG patients, they are activated to a greater extent as reflected by higher expression of HLA‐DR and CD95 on the surface compared to HCs. The data thus show that there is no deficiency of CD4⁺CD25⁺ cells in MG, nor is the proportion of CD4⁺CD25⁺ cells influenced by thymectomy.     

Prognostic Values of CD38+CD101+PD1+CD8+ T Cells in Pancreatic Cancer

Programmed death-1 (PD-1), a key immune checkpoint molecule, has been developed as an oncotherapy target for various carcinomas. However, treatment with anti-PD-1 elicited only a minimal effect in pancreatic ductal adenocarcinoma (PDAC). Subsequent studies revealed the existence of a subset of PD-1⁺ T cells coexpressing CD38 and CD101, representing a fixed dysfunctional subpopulation that are not able to be rescued by anti-PD-1 immunotherapy. However, whether this subpopulation of PD-1 expressing CD8⁺ T cells could be useful in predicting PDAC stage or prognosing survival is unknown. In this study, we used flow cytometry and immunofluorescence assay to analyze the expression of CD38 and CD101 in 183 clinical PDAC samples, including 84 of peripheral blood and 99 of surgical tissues. High coexpression of CD38/CD101 on peripheral PD-1⁺CD8⁺ T cells or tumor-infiltrating lymphocytes (TILs) was found to be most significantly correlated with Tumor/Node/Metastasis (T/N/M) classification and clinical stage, in contrast PD-1⁺CD8⁺ T cells could not correlate with T classification. CD38/CD101 co-repression on TILs also correlated with the poor survival in these PDAC patient samples. Our data suggest that CD38/CD101 might represent a more helpful biomarker than PD-1 alone for diagnosis and prognosis of PDAC.     

Human CD4+CD25+ regulatory T cells

In this report, we review studies of human CD4+CD25+ regulatory T cells (T-reg). Although lagging a few years behind the discovery of these cells in the mouse, the equivalent population of CD4+CD25+ regulatory T cells has also been isolated from human peripheral blood, thymus, lymph nodes and cord blood. In general, the characteristics of this T cell subset are strikingly similar between mouse and man. In the recent explosion of research reports on human CD4+CD25+ cells, although the majority of the characteristics ascribed to these cells appear to be consistent, contrasting results have been found primarily in regards to potential involvement of TGFbeta and production of IL-10. One explanation for this variability may reside in the fact that markedly different techniques are used to isolate human CD4+CD25+ T-reg cells and thus may result in the comparison of T-reg populations that differ in cellular composition and/or activation state. Another potential explanation for differences in human T-reg function may rest on the extreme variability of the culture conditions and TCR stimuli that have been used to test the functional properties of these cells in vitro. The strength of the TCR signal provided to the culture greatly affects the functional outcome of the co-culture and can result in the difference between suppression and full activation. Surprisingly, it appears that stronger stimulation has a greater and more rapid effect on the T-resp cell than on the T-reg cell as it causes T-resp cells to quickly become resistant to suppression. Thus, the details of in vitro culture conditions may at least partially account for disparate findings in regard to the functional characterization of human CD4+CD25+ cells. Here we review the evidence regarding the identification of human CD4+CD25+ regulatory T cells and their possible mechanism(s) of function.     

CD4+ CD45RA+ and CD4+ CD45RO+ T cells differ in their TCR-associated signaling responses.

Peripheral CD4+ T cells can be divided into two different functional populations based on the expression of distinct isoforms of the surface molecule CD45. We have investigated the differences in the proximal signaling induced by anti-CD3 monoclonal antibody in purified populations of "naive" CD45RA+ and "memory" CD45RO+ human CD4+ T cells. Expression of cell surface CD3, CD4 and CD28 was comparable between RA+ and RO+ cells. However, TCR-directed stimulation in the form of anti-CD3 produced markedly different patterns of intracellular signaling. Greater inositol triphosphate generation occurred in naive cells and the rise in intracellular free calcium was also substantially greater in naive than in memory cells. Cells with the naive phenotype were considerably more active in TCR-dependent tyrosine phosphorylation, both at an overall level and specifically in terms of TCR-zeta and ZAP-70 phosphorylation. Despite these differences in phosphorylation, the amounts of TCR-zeta, ZAP-70 and Ick were equivalent between the two subsets. These findings suggest that the TCR-dependent signaling is differentially regulated in naive and memory CD4+ T cells. This may be due to differences in the way that the two isoforms of the CD45 phosphatase regulate the activity of proximal kinases in the TCR signaling pathway, and could be an important means by which the unique functions of differentiated T cell populations are maintained.     

CD4+CD8+ murine intestinal intraepithelial lymphocytes

We have studied a population of CD4+ intestinal intraepithelial lymphocytes using two-color flow cytometric analyses, and in highly purified fluorescent-activated cell-sorted preparations. Although CD4+ T cells present within the epithelial immune compartment comprised only approximately 10-20% of the total intestinal epithelial lymphoid cells, an unusually high proportion of those CD4+ lymphocytes expressed a CD4+CD8+ phenotype which is rarely encountered in peripheral T cells. By comparison, CD4+ lymphocytes from spleen or lymph nodes existed exclusively as single-positive T cells. Analyses of CD4 and CD8 expression on lymphocytes from Peyer's patches, the lamina propria, and IEL indicated that CD4+CD8+ lymphocytes were unique to the IEL. Using CD4+CD8+ preparations obtained by fluorescent-activated cell sorting, CD4+CD8+ epithelial T cells were found also to express CD3 and Thy-1 surface markers. This heretofore undescribed extrathymic population of double-positive T cells constitutes a unique peripheral T cell subset which may be involved in intestinal T cell maturation and development, or could represent a highly specialized effector population.