全葡聚糖颗粒对DC诱导CD4+T细胞向Th17细胞分化的影响

目的研究全葡聚糖颗粒(whole glucan particle,WGP)对DC诱导CD4~+T细胞向Th17细胞分化的影响。方法采用重组小鼠FMS样酪氨酸激酶3配体(Flt3L)法诱导C57BL/6小鼠骨髓来源树突状细胞(BMDC)。用MACS磁珠分选法对OT-Ⅱ小鼠的脾脏和淋巴结中CD4~+T细胞进行纯化,加入特异性抗原卵清蛋白(OVA),与DC共培养。实验分组为:CON组、WGP组、TGP-β组、WGP+TGF-β组,培养5 d后,利用流式细胞术(FCM)检测各组中CD4~+T细胞增殖和Th17细胞变化情况,ELISA检测各组细胞上清液中IL-17A的含量,Q-PCR检测各组细胞中Th17细胞核转录因子维甲酸孤儿受体-γt(retinoid-related orphan receptor-γt,ROR-γt)mRNA表达水平。结果 TGF-β可以促进DC诱导CD4~+T细胞向Th17分化,而经WGP激发后,T细胞显著下调ROR-γt的转录水平,并减少对IL-17A的分泌。结论 WGP显著抑制TGF-β诱导CD4+T细胞向Th17细胞分化。     

白介素10抑制幼稚T细胞分化为辅助性T细胞17的研究

目的:研究白介素10(interleukin,IL-10)在辅助性T细胞17(helper T cell,Th17)体外诱导分化中的作用。方法:分选小鼠脾脏CD4+幼稚T(nave T)细胞,在经典的Th17诱导条件中加入不同浓度的IL-10中和抗体(0、1、5、10μg/ml NA-IL-10),流式细胞术检测各组CD4+IL-17+细胞的生成比率,ELISA法检测各组培养上清中Th17特异细胞因子IL-17A和IL-17F的浓度,qRT-PCR及Western Blot检测各组细胞中表达转录因子维甲酸相关孤核受体γt(retinoid-related orphan nuclear receptor gamma t,Rorγt)的表达水平。结果:在经典的诱导CD4+nave T细胞分化为Th17的过程中,IL-10的浓度随诱导天数的增加而逐渐上升。加入NA-IL-10后,IL-17+细胞的诱导比例随NA-IL-10浓度的增加而逐渐上升,在10μg/ml的NA-IL-10条件下,诱导生成的IL-17+细胞可达22.1%±4.5%,与无NA-IL-10组8.1%±1.9%相比差异有统计学意义(P0.05)。10μg/ml NA-IL-10组IL-17A和IL-17F的浓度以及Rorγt的表达水平均明显高于无NA-IL-10组(P0.05)。结论:降低培养条件中IL-10的水平能够有效地促进Th17的体外诱导分化,也进一步验证了IL-10对Th17分化的抑制作用。     

红霉素可抑制弹性蛋白肽诱导的CD4~+T细胞向Th17细胞分化

目的探讨红霉素对弹性蛋白肽暴露下CD4+T细胞向Th17细胞分化的影响。方法从小鼠脾脏经免疫磁珠分选出CD4+T细胞,随机分为空白对照组、弹性蛋白肽组和弹性蛋白肽加红霉素干预组。弹性蛋白肽组和弹性蛋白肽联合红霉素处理组加入终浓度30μg/mL的弹性蛋白肽,弹性蛋白肽联合红霉素处理组在弹性蛋白肽处理的基础上,另外加入100μg/mL的红霉素。3组细胞在无血清培养液培养24h,用流式细胞术检测Th17的百分比,荧光定量PCR检测维甲酸相关孤儿核受体γt(RORγt)mRNA表达,Western blot法检测核因子κB(p65)[NF-κB(p65)]及信号转导子和转录激活子3(STAT3)蛋白相对含量。结果弹性蛋白肽组CD4+IL-17+细胞的百分比为(11.32±2.34)%、对照组为(5.21±1.36)%;qRT-PCR发现RORγt mRNA表达比对照组明显增加,Western blot法检测发现NF-κB(p65)和STAT3蛋白表达比对照组增强。与弹性蛋白肽组比较,弹性蛋白肽联合红霉素处理组的CD4+IL-17+细胞的百分比、RORγt mRNA表达、NF-κB(p65)和STAT3蛋白表达明显减少。结论红霉素可抑制弹性蛋白肽诱导CD4+T细胞向Th17分化。     

miR-126敲减增强小鼠CD4~+T细胞体内活性并促进其向Th1细胞分化

目的观察miR-126基因敲减(KD)小鼠体内CD4+T细胞活性的变化并探讨其意义。方法利用磁珠活化细胞分选技术(MACS)分选miR-126KD小鼠脾脏CD4+CD62L+T细胞,实时定量PCR检测细胞内miR-126成熟体的表达水平;荧光激活细胞分选术(FACS)检测CD4+T细胞表面活化标志CD69、CD62L和CD44分子以及Ki-67的表达变化;膜联素Ⅴ/碘化丙啶(annexinⅤ/PI)双染色法结合流式细胞术检测CD4+T细胞的凋亡情况;实时定量PCR检测CD4+T细胞功能相关细胞因子白细胞介素4(IL-4)、IL-10、IL-12、转化生长因子β(TGF-β)、肿瘤坏死因子α(TNF-α)、γ干扰素(IFN-γ)的mRNA水平变化。结果与野生型(WT)小鼠组相比,miR-126KD小鼠组CD4+T细胞内miR-126成熟体的表达显著下调;FACS结果显示miR-126KD小鼠CD4+T细胞表达CD62L的比例明显减少,而表达CD69、CD44以及Ki-67细胞的比例显著增加,CD4+T细胞的体内凋亡比例显著减少;CD4+T细胞中IL-4、IL-10的mRNA水平明显降低,而IL-12、TGF-β、TNF-α以及IFN-γ的mRNA水平显著增加。结论 miR-126KD小鼠体内CD4+T细胞的活性显著增强并促进其向Th1细胞分化。     

CD4~+CD25~+调节性T细胞/Th17细胞失衡与婴幼儿脓毒症

目的 观察不同免疫状态下婴幼儿脓毒症CD4~+CD25~+Foxp3~(high)岫调节性T细胞(Tr)/Th17细胞的变化,探讨婴幼儿脓毒症适应性免疫紊乱可能的机制.方法 婴幼儿脓毒症48例,健康同龄儿童对照组26例.用流式细胞术榆测CD14~+单核细胞HLA-DR表达率,CD4~+CD25~+Foxp3~(high)Tr 比例及Th17细胞比例;用ELISA法检测细胞因子IL-1β、IL-6、IL-10、TNF-α、TGF-β及IL-17A等浓度,计算IL-10/TNF-α比值;实时荧光定量PCR(real-time PCR)检测CD4~+T细胞Foxp3、ROR-γt mRNA表达及IL-17A mRNA表达.以CD14~+单核细胞HLA-DR表达＞或＜30%为阈值,将患儿分为免疫激活组(DR-H组)和免疫抑制组(DR-L组).结果 DR-L组IL-10/TNF-α比值明显高于DR-H组(P＜0.05)及对照组(P＜0.05).CD4~+CD25~+Foxp3~(high) Tr细胞比例及转录因子Foxp3基因表达DR-L组明显高于对照组及DR-H组(P＜O.05).Th17细胞比例、IL-17A血浓度、Th17细胞IL-17A基因表达及转录因子ROR-γt基因表达DR-H组及DR-L组均明显高于对照组(P＜0.05),两组之间差异无统计学意义(P＞0.05).DR-H组和DR-L组Th17细胞主要分化调控因子IL-6、IL-1β血清浓度明显高于正常对照组(P＜0.05),两组问IL-6、IL-1β浓度差异无统计学意义(P＞0.05),DB-L组CD4~+CD25~+Foxp3~(high) Tr细胞主要调节因子TGF-β血浓度明显高于DR-H组及对照组(P＜0.05).结论 Th17持续过度活化可能是导致脓毒症前炎症细胞因子/趋化因子持续增高的原因之一;CD4~+CD25~+Foxp3~(high) Tr细胞/Th17细胞失衡可能参与脓毒症混合性拮抗反应综合征(MARS)的发生发展,婴幼儿脓毒症细胞因子微环境变化可能是导致CD4~+CD25~+Foxp3~(high) Tr细胞/Thl7细胞失衡的原因之一.     

Galectin-9对活化的CD4+T细胞免疫调节作用的机制研究

目的 研究Galectin-9对活化的CD4+T细胞的免疫调节作用,并进一步探讨其作用机制.方法 获取野生型C57BL/6小鼠淋巴细胞,利用MACS分选CD4+ na(i)ve T细胞,给予anti-CD3 (2.5 μg/ml)抗体、anti-CD28(5μg/ml)抗体和IL-2(100 ng/ml)刺激,培养3d.将活化的CD4+T细胞分为3组:正常对照组、Galectin-9组和Galectin-9+α-乳糖组.利用CFSE检测T细胞增殖情况,并动态观察细胞形态变化;检测CD4+ CD69+T细胞、Th1、Th2和Th17细胞的比例;并利用ELISA检测淋巴细胞分泌IFN-γ 、IL-4、IL-10、IL-12、IL-17A和TGF-β1等细胞因子的水平;利用Western blot检测T-bet、GATA-3和ROR-yt等T细胞分化调控蛋白的变化.结果 与正常对照组和Galectin-9+α-乳糖组相比,Galectin-9组于2h时开始出现细胞形态改变,同时CD4+ CD69+T细胞、Th1和Th17细胞表达减少(P＜0.05),但Th2细胞无明显变化;培养上清液中IFN-γ、IL-12、IL-17A和TGF-β1的表达水平降低(P＜0.05),而IL-4和IL-10等Th2型细胞因子水平无明显变化;同时T-bet和ROR-γt的表达水平减少(P＜0.05).结论 Galectin-9抑制活化CD4+T细胞的Th1和Th17型免疫应答,而对Th2型免疫应答无影响,免疫调节的机制可能与其在转录水平影响Th1和Th17特定转录因子的表达有关.     

茯苓多糖对系统性红斑狼疮患者Th17/Treg平衡的影响

目的:研究茯苓多糖对系统性红斑狼疮(SLE)患者外周血辅助性T细胞17(Th17)/调节性T细胞(Treg)平衡的免疫调节作用。方法:选取45例SLE患者和35例健康对照者,应用磁珠分选法分离外周血CD4~+ T细胞,流式细胞术检测CD4~+ T细胞中Th17和Treg细胞的比例。用茯苓多糖分别处理健康对照者及患者的CD4~+ T细胞,MTT法检测细胞活力以测定茯苓多糖毒性,ELISA检测细胞中白细胞介素17(IL-17)、IL-6、IL-10及转化生长因子β(TGF-β)的含量,RT-q PCR和Western blot法分别测定维甲酸相关孤儿受体γt(RORγt)与叉头框蛋白P3(Foxp3)的mRNA和蛋白表达水平。结果:与健康对照组相比,SLE患者的Th17细胞比例显著升高,Treg细胞比例明显降低(P0.05)。用100μg/L的茯苓多糖处理SLE患者CD4~+ T细胞,与空白对照组相比,IL-17和IL-6的含量显著降低,IL-10和TGF-β的含量明显上升(P0.05);RORγt的mRNA和蛋白表达显著下降,同时Foxp3的表达在mRNA和蛋白水平上明显增加(P0.05);并且Th17/Treg的比值降低(P0.05)。结论:茯苓多糖可以通过升高Treg并降低Th17细胞的比例,对SLE起到一定的治疗作用。     

PCPA对于外周血CD4~+T细胞Th1/Th2分化失衡影响的初步研究

目的研究PCPA暴露对于外周血CD4+T细胞向Th1/Th2分化的影响及其分子机制。方法分离并培养人外周血CD4+T细胞。采用MTT、流式细胞术、QRT-PCR和酶联免疫吸附试验法(ELISA)对PCPA组和对照组细胞中Th1、Th2亚型的代表细胞因子IFN-γ、IL-4进行检测。结果 PHA刺激48 h后,ELISA检测细胞培养上清显示,PCPA组上清中IFN-γ分泌水平明显高于对照组(P<0.05),而IL-4分泌量较对照组略有增加;RT-PCR检测显示,PCPA组IFN-γ基因表达高于对照组1.69倍,IL-4基因表达没有显著改变;流式细胞技术对胞内因子检测显示,PCPA组IFN-γ+T细胞比例(46.1%)明显高于对照组(32.6%),2组间有统计学意义(P<0.05),而IL-4+T细胞比例与对照组无显著变化(48.1%和46.5%)。PCPA组的IFN-γ+T细胞与IL-4+T细胞比值是对照组的1.53倍。结论 PCPA暴露(40μmol/L)导致CD4+T细胞LSD1的H3K4(2m)脱甲基化酶活性被抑制,引起Th1/Th2分化平衡向Th1方向的"漂移"。     

子痫前期患者外周血树突状细胞对Th1/Th17细胞分化的影响

目的 观察正常妊娠妇女和子痫前期患者外周血单个核细胞(PBMC)来源的树突状细胞(DC)对Th1、Th17细胞分化的影响.方法 实验组为子痫前期疾病患者32例;对照组为正常妊娠妇女20例.分离正常妊娠组和子痫前期组PBMC,经贴壁获得单核细胞,加GM-CSF,IL-4和LPS培养诱导为成熟DC,流式细胞术检测表面分子标志CD14,CD80,CD83,CD86的表达,ELISA检测培养上清液中IL-23的含量.磁珠分选出CD4+T淋巴细胞,与正常妊娠孕妇外周血单核细胞来源的树突状细胞(N-DC)共同培养,同时添加细胞因子IL-2,或与子痫前期患者外周血单核细胞来源的树突状细胞(P-DC)、IL-2共同培养;或与N-DC共同培养,同时添加细胞因子IL-1p、IL-6;或与P-DC共同培养,并添加细胞因子IL-1β、IL-6.以上各组培养到第6天用流式细胞术检测CD4+ IFN-γ+ Th1、CD4+ IL-17+ Th17细胞比例.结果 P-DC中CD83、CD80、CD86的表达高于N-DC,差异有统计学意义(P＜0.05).P-DC与不同的细胞因子共同作用,促使CD4+T细胞分化为Th1、Th17细胞的能力高于N-DC,差异有统计学意义(P＜0.01).结论 子痫前期患者外周血DC表型和功能的改变可能与患者免疫失衡有一定关系.     

1型糖尿病患儿CD4~+T细胞亚群变化初探

目的 探讨CD4~+细胞亚群[Th1、Th2、CD4~+CD25~+Foxp3~+调节性T细胞(Tr)及Th17细胞]在1型糖尿病(TIDM)患儿免疫发病机制中的作用.方法 新诊断TIDM患儿20例,同年龄对照组(Ctrl组)20例.用流式细胞术检测外周血Th1、Th2、Tr及Th17细胞比例.荧光定量PCR(real-time PCR)检测Th1、Th2、Tr、Th17细胞转录因子T-bet、GATA-3、Foxp3、ROR-γt、IFN-、IL-4、IL-10、IL-17A、CTLA-4、GITR等细胞因子和负性调节因子mRNA表达;应用酶联免疫吸附方法(ELISA)检测IFN-γ、IL-4、TGF-β、IL-6血浆水平.结果 (1)与正常对照组相比,TIDM患儿Th1细胞比例明显增高(P<0.01),Th2细胞比例明显降低(P<0.01),Tr和Th17细胞比例与正常对照组相比无明显差别(P>0.05).(2)Th1细胞转录因子及细胞因子T-bet、IFN-γ较正常对照组明显升高(P<0.01);Th2细胞转录因子及细胞因子GATA-3、IL-4明显降低(P<0.01);Tr细胞转录因子Foxp3表达与正常对照组相比差异无统计学意义(P>0.05),Tr细胞相关细胞因子及负性调节因子IL-10、CTLA-4及GITR基因表达明显低于对照组(P<0.01);Th17细胞转录因子ROR-γt及细胞因子IL-17A基因表达与对照组相比无明显差异(P>0.05);(3)TIDM患儿外周血IFN-γ浓度明显增高,IL-4明显降低,TGF-β、IL-6浓度无明显改变(P>0.05).结论 TIDM患儿Th1/Th2失衡,加上Tr细胞抑制功能缺陷,可能导致TIDM严重细胞免疫功能紊乱.     

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.     

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.     

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.     

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.     

CD25+CD4+ regulatory T-cells in cancer

Regulatory T-cells (T_reg) protect the host from autoimmune disease by suppressing self-reactive immune cells. As such, T_reg may also block antitumor immune responses. Recent observations by us and others showed that the prevalence of T_reg is increased in cancer patients, particularly in the tumor environment. Our studies in a mouse pancreas cancer model suggest that the tumor actively promotes the accural of T_reg through several mechanisms involving activation of naturally occurring T_reg as well as conversion of non-T_reg into T_reg. Our studies focus on further defining these mechanisms with the ultimate goal of designing strategies that block T_reg-mediated suppression in cancer patients.     

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.