利用RNA干扰诱导产生的CD8+CD28-抑制性T淋巴细胞的免疫学特性

目的 探讨通过RNA干扰技术诱导产生的CD8+ CD28-抑制性T淋巴细胞(Ts细胞)的免疫学特性.方法 取SD大鼠骨髓,培养分离树突状细胞(DC),设计、合成主要组织相容性复合物(MHC)Ⅰ类小片段干扰RNA(siRNA),以MHC Ⅰ siRNA转染DC.先以Wistar大鼠肠系膜淋巴组织液刺激转染MHCI siRNA的DC,然后将DC与从SD大鼠脾脏分离得到的CD8+T淋巴细胞共同培养,通过磁珠法分离出Ts细胞.分别在由SD大鼠脾脏淋巴细胞(反应细胞)和Wistar大鼠肠系膜淋巴组织细胞(刺激细胞)组成的混合淋巴细胞培养体系中加入数量不等的Ts细胞,检测反应细胞增殖情况;分别以Wistar大鼠肠系膜淋巴组织细胞和卵白蛋白(OVA)刺激SD大鼠脾脏淋巴细胞,然后再按不同比例加入Ts细胞,检测各组脾脏淋巴细胞的增殖情况;在由SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入可溶性重组白细胞介素2(rrIL-2),观察IL-2对Ts细胞功能的影响;采用实时定量聚合酶链反应(PCR)测定Ts细胞中转化生长因子β(TGF-β和γ干扰素(IFN-γ)mRNA的表达,流式细胞仪和实时PCR检测Ts细胞上CD25分子的表达.结果 Ts细胞对SD大鼠脾脏淋巴细胞和Wistar大鼠肠系膜淋巴组织细胞之问的混合淋巴细胞反应(MLR)具有抑制作用,但对于SD大鼠脾脏淋巴细胞和OVA之间的MLR则无抑制作用.在SD大鼠脾脏淋巴细胞、Wistar大鼠肠系膜淋巴组织液和Ts细胞组成的混合淋巴细胞培养体系中加入rrIL-2后,SD大鼠脾脏细胞的增殖并无明显增加(P＞0.05).与CD8+CD28+T淋巴细胞和CD8+ T淋巴细胞比较,Ts细胞的TGF-β和IFN-γ mRNA的表达量明显升高(P＜0.01,P＜0.05),而CD25的表达量明显降低(P＜0.05).结论 采用经MHC I siRNA干扰的DC能够诱导CD8+T淋巴细胞产生CD8+ CD28-Ts细胞;Ts细胞在体外具有免疫抑制特性,其免疫抑制作用不被外源性IL-2所逆转,且其免疫调节作用具有抗原特异性.     

西罗莫司和环孢素A对Th17细胞和调节性T淋巴细胞分化影响的比较

目的 比较西罗莫司(SRL)和环孢素A(CsA)对体外诱导CD4+T淋巴细胞向Th17和调节性T淋巴细胞分化的影响.方法 使用抗CD3和抗CD28单克隆抗体(简称抗CD3单抗和抗CD28单抗)刺激CD4+T淋巴细胞,并在培养体系中分别加入转化生长因子β(TGF-β);TGF-β+白细胞介素6(IL-6);TGF-β+IL-6+SRL;TGF-β+IL-6+CsA.72 h后用流式细胞术检测细胞内Foxp3和IL-17的表达水平,观察CD4+T淋巴细胞的分化情况及SRL和CsA对CD4+T淋巴细胞体外分化的影响.结果 在经抗CD3单抗和抗CD28单抗刺激后,TGF-β可诱导Foxp3+细胞(调节性T淋巴细胞,Treg)的增殖与分化,而TGF-β+ID6可诱导Th17细胞的增殖与分化.在培养体系中加人SRL和CsA,均可使Th17细胞的增殖与分化减少;SRL可促进Treg的增殖与分化,而CsA抑制Treg的增殖与分化.结论 SRL可以促进CD4+T淋巴细胞向Treg增殖与分化,而抑制Th17细胞的增殖与分化;CsA既抑制Treg的增殖与分化,又抑制Th17细胞的增殖与分化.     

OX40信号调节抑制小鼠CD4+CD25+调节性T淋巴细胞Foxp3的表达

目的 探讨共刺激信号OX40对体外诱导的小鼠CD4+ CD25+适应性调节性T淋巴细胞(iTreg)的Foxp3表达的影响.方法 制备C57BL/6小鼠淋巴细胞悬液,经免疫磁珠法分选,获得CD4+ CD25-静息T淋巴细胞,与抗CD3单克隆抗体、抗CD28单克隆抗体、转化生长因子β1、白细胞介素2共孵育,诱导产生Foxp3+ iTreg.在此基础上,于培养体系中加入OX40激动型抗体及其对照抗体,利用流式细胞仪分析研究OX40信号刺激对iTreg Foxp3表达的影响.结果 C57BL/6小鼠淋巴结中CD4+ CD25+天然调节性T淋巴细胞(Treg)比例为(5.0±0.4)％,体外诱导培养的CD4+CD25+ Treg比例为(71.8±13.4)％,其中Foxp3阳性表达占(74.9±1.9)％.OX40激动型抗体组CD4+ CD25+ Treg细胞比例为(80.0±1.6)％,其中Foxp3表达水平为(59.2±0.7)％;OX40激动型抗体对照抗体组CD4+ CD25+ Treg细胞比例为(86.0±1.4)％,其中Foxp3表达水平为(70.0±0.8)％,两组间差异有统计学意义(P＜0.05).结论 静息T淋巴细胞可以在体外诱导培养获得高纯度iTreg;OX40信号刺激可以显著抑制CD25+ iTreg细胞Foxp3的表达.     

小鼠补体调节蛋白对CD4+T淋巴细胞的调控作用及其机制

目的 研究小鼠补体调节蛋白Crry对CD4+T淋巴细胞的调控作用及诱导同种移植免疫低反应性的机制.方法 分离C57BL/6小鼠脾淋巴细胞,用免疫磁珠法分选出CD4+T淋巴细胞后,将CD4+T淋巴细胞分为A、B、C、D、E和F组,分别用抗小鼠CD3、CD28、Crry、CD3/CD28、CD3/Crry和CD3/CD28/Crry抗体共刺激通路与CD4+T淋巴细胞进行反应,采用噻唑蓝(MTT)法检测各组CD4+T淋巴细胞的增殖情况,并采用酶联免疫吸附试验检测CD4+T淋巴细胞培养上清中白细胞介素2(IL-2)、γ干扰素(γ-IFN)、IL-4和IL-10的水平;另外,以BALB/c小鼠和C57BL/6小鼠的脾细胞分别作为刺激细胞和反应细胞,建立同种混合淋巴细胞反应(MLR)体系并加入抗小鼠Crry抗体,通过岍法观察Crry对MLR的影响.结果 D、E、F组的CD4+T淋巴细胞均出现明显增殖,增殖活性显著高于A、B、C组(P<0.05),其中F组显著高于D组和E组(P<0.05),D组和E组间增殖活性的差异无统计学意义.D组CD4+T淋巴细胞经抗CD3/CD28抗体共刺激后,培养上清中γ-IFN和IL-2的水平显著升高,与A、B、C和E组比较,差异均有统计学意义(P<0.05),但与F组的差异无统计学意义;E组CD4+T淋巴细胞经抗CD3/Crry抗体共刺激后,IL-4的水平显著升高,与A、B、C、D组比较,差异均有统计学意义(P<0.05),但显著低于F组(P<0.05);各组间IL-10水平的差异无统计学意义.Crry可以明显抑制MLR中的细胞增殖(P<0.05).结论 补体调节蛋白Crry能刺激CD4+T淋巴细胞的增殖,并使其IL-4的表达升高及抑制IL-2和γ-IFN的表达,从而诱导同种移植免疫低反应性.     

RNAi对大鼠T淋巴细胞CD28和OX40基因表达的联合抑制作用

目的 通过RNAi技术干扰T细胞CD28、CD134基因表达后,诱导获得抑制性T细胞(Ts);探讨Ts免疫学特性.方法 设计针对目标基因的siRNA,转染大鼠T淋巴细胞,FCM检测CD28、CD134水平,混合淋巴细胞反应(MLR)检测转染后的T淋巴细胞对异体淋巴细胞的增殖能力的影响,逆转录-聚合酶链反应(RT-PCR)及酶联免疫吸附试验(ELISA)法检测细胞因子水平.结果 siRNA转染大鼠T淋巴细胞后,抑制CD28、CD134分子的表达,siRNA转染24 h后,siRNA组CD28、CD134表达受到抑制[转染后及转染前表达分别为(22.35±4.37)%及(34.76±3.51)%(P<0.05)],T淋巴细胞分泌细胞因子IL-10水平增高,转染组及未转染组表达分别为(77.15±12.60)ng/L及(37.56±5.93)ng/L(P<0.01).而转染组及未转染组的IL-2表达分别为(2.79±0.51)及(4.35±1.11)ng/L(P<0.05);干扰素(IFN)-γ表达分别为(277.15±14.8)、(682.7±53.5)ng/L(P<0.05).结论 siRNA可以特异性抑制大鼠T淋巴细胞共刺激分子CD28、CD134基因表达,抑制了IL-2、IFN-γ的表达,提高了IL-10细胞因子表达水平,从而产生了免疫耐受效应.Ts细胞具有抗原特异性,而且在外源性rrIL-2存在时,不能逆转Ts细胞的功能.     

CD4+CD25-T淋巴细胞转化为CD4-CD8-调节T细胞的体外实验研究

目的 探讨体外诱导和纯化CD4+ CD25-T淋巴细胞(effector T cell,Teff)转化为CD4-CD8-双阴性调节T细胞(double negative regulatory T cell,DN Treg)的最适条件.方法 采用免疫磁珠分选方法提取C57BL/6小鼠的CD4+ CD25-T淋巴细胞、DBA/2小鼠的成熟树突状细胞共培养,加入不同剂量的IL-2,通过流式细胞检测CD4-CD8-T细胞的转化比例并确定最适条件,免疫磁珠阴性选择分选提纯转化的CD3+ CI4-CD8-T细胞,流式细胞仪检测转化的CD4-CD8-调节T细胞对CD4+ CD25-效应T细胞增殖抑制情况.结果 CD4+ CD25-T淋巴细胞与DBA/2小鼠的树突状细胞共培养6d后检测CD4-CD8-调节T细胞的转化比率为6.21％ ±2.03％,实验组加入不同浓度IL-2的转化率:A组(25 ng/ml)为14.77％±2.15％,B组(50 ng/ml)为21.29％±2.68％,C组(75 ng/ml)为43.45％ ±4.45％,D组(100 ng/ml)为28.59％±3.05％,IL-2浓度在75 ng/ml时,转化获得率最高(C组与对照组、实验A、B、D组比较分别t=10.700,8.288,6.158,3.932,均P＜0.05);分离提纯CD4-CD8-双阴性调节细胞纯度达到98.10％,CD4-CD8-双阴性调节细胞与CFSE染色的CD4+ CD25-T淋巴细胞、小鼠树突状细胞共培养6d,实验组增殖指数为1.15明显低于对照组2.07.结论 小鼠CD4+ CW25-T淋巴细胞在体外,成熟树突状细胞刺激下可转化为CD4-CD8-双阴性调节T细胞,IL-2可显著提高其转化率.     

恒河猴外周血调节性T淋巴细胞的分离纯化及鉴定

目的建立恒河猴外周血CD4+CD25+调节性T淋巴细胞(regulatory T cells,Tregs)快速有效的分离纯化方法。方法 4～5岁健康恒河猴10只,雌性4只,雄性6只,体重5～8 kg;于大隐静脉抽取外周血,每只抽取8 mL。密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cell,PBMC),分别采用非人灵长类Tregs分离试剂盒的生物素标记的混合抗体和磁珠标记的抗生物素抗体阴性分选,以及大鼠抗人CD4-活化蛋白C(activatedprotein C,APC)和抗APC多选试剂盒中的磁珠标记的抗APC抗体阳性分选出CD4+T淋巴细胞,比较两种方法获得细胞的得率、活性及纯度;选择得率、活性和纯度较高的分选方法获得的CD4+T淋巴细胞,用磁珠标记的抗人CD25抗体进行阳性分选,获得CD4+CD25+Tregs,流式细胞仪检测纯度、活性及FoxP3表达水平,以及Tregs对刀豆蛋白(concanavalinA,ConA)刺激的自体CD4+CD25-效应性T淋巴细胞(effective T cells,Teffs)增殖的抑制功能。结果 CD4+T淋巴细胞阳性分选和阴性分选后,细胞活性均达95%左右,差异无统计学意义(P>0.05),但阳性分选后CD4+T淋巴细胞得率和纯度均显著高于阴性分选(P<0.05)。后续CD4+CD25+Tregs分选采用阳性分选法获得的CD4+T淋巴细胞。经双阳性分选收集的目的细胞中CD4+CD25+Tregs占76.2%±8.6%,活细胞比例为93.3%±4.7%,FoxP3阳性细胞比例为74.2%±6.9%。混合培养后Tregs均对ConA刺激的Teffs的增殖有抑制作用。结论免疫磁珠双阳性分选法能有效分选出恒河猴外周血中有功能的CD4+CD25+Tregs。     

血必净促进内毒素/脂多糖刺激调节性T淋巴细胞凋亡并介导辅助性T淋巴细胞漂移的作用

目的 了解血必净注射液促进LPS刺激CD4+CD25+调节性T淋巴细胞(Treg细胞)凋亡过程及介导辅助性T淋巴细胞(Th)漂移的调节作用.方法 免疫磁珠法分选获得大鼠脾脏CD4+CD25+Treg细胞,分为常规培养对照组、抗CD3/CD28组、抗CD3/CD28+LPS组、抗CD3/CD28+血必净组和抗CD3/CD28+LPS+血必净组,培养3 d后应用流式细胞术检测Treg细胞凋亡率及叉头翼状螺旋转录因子3(Foxp3)表达.将CD4+CD25+Treg细胞与CD4+CD25+T淋巴细胞1:1培养,伴刀豆球蛋白A刺激68 h,检测上清液中Th1分泌的γ干扰素(IFN-γ)、Th2分泌的IL-4、Th17分泌的IL-17水平.结果 抗CD3/CD28+LPS+血必净组Treg细胞凋亡率为(45.1±2.7)%,明显高于抗CD3/CD28+LPS组[(29.4±1.6)%,P＜0.01];2组Foxp3平均荧光强度分别为95±9、140±18,差异有统计学意义(P＜0.01).同时,抗CD3/CD28+LPS+血必净组IFN-γ分泌水平显著高于抗CD3/CD28+LPS组(P＜0.01),IL-4则呈相反变化(P＜0.05),抗CD3/CD28+LPS+血必净组IFN-γ/IL-4较对照组升高(P＜0.01);抗CD3/CD28+血必净组IL-17分泌水平较抗CD3/CD28组明显下降(P＜0.05).结论 CD4+CD25+Treg细胞活化介导了Th1向Th2功能性极化;血必净对LPS诱导的T淋巴细胞免疫功能有重要调节作用,可促进CD4+CD25+Treg细胞凋亡并介导Th2向Th1漂移,从而缓解细胞免疫抑制状态.     

IKK2dn转染树突状细胞诱导产生的CD4+CD25-T细胞的筛选及鉴定

目的 探讨从转染IKK2dn并负载供者抗原的未成熟树突状细胞(imDC)诱导产生的调节性T细胞(Treg)中筛选CD4+ CD25 - Treg的方法,并进行鉴定.方法 Lewis大鼠骨髓源性imDC,转染IKK2dn后负载供者BN大鼠抗原,与Lewis大鼠T细胞进行体外混合淋巴细胞反应(MLR)诱导产生Treg,用免疫磁珠法(MACS)筛选出CD4+ CD25 -T细胞,流式细胞仪(FCM)检测细胞纯度.加入CD4+ CD25 -T细胞行再次MLR检测其抑制T细胞增殖的作用.结果 经MACS筛选,CD4+ CD25 -T细胞纯度为(95.78±1.25)％.再次MLR结果显示CD4+ CD25 -T细胞组的吸光度值为(0.106±0.006),低于BN抗原组(0.189±0.007)、Adv0-CD4+T细胞组(0.419±0.014)及第三方供者抗原组(0.200±0.008),差异有统计学意义(P＜0.05).结论 转染IKK2dn并负载抗原的imDC诱导产生的Treg,经MACS筛选可以获得高纯度的CD4+CD25-T细胞,对同种T细胞增殖具有针对供者的特异性免疫抑制作用.     

人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制

目的 揭示人单核细胞共刺激分子在异种免疫反应中的表达及其作用机制.方法 从猪的主动脉分离血管内皮细胞(PEC)并培养扩增;从人单个核细胞(PBMC)中纯化CD4+T淋巴细胞和单核细胞.建立PEC和人PBMC混合培养体系,培养后收集细胞,然后加入荧光标记的单克隆抗体,通过流式细胞术检测CD14+单核细胞表面共刺激分子表达情况.为了检测淋巴细胞增殖反应以及阻断共刺激分子对PEC免疫反应的作用,在PEC和人PBMC混合培养体系中分别加入抗CD154、CD80和CD86单克隆抗体.在培养的最后24 h加入同位素,于培养结束后收集细胞并经同位素计数仪进行检测.纯化的单核细胞经PEC刺激后与CD4+T淋巴细胞共培养来研究这些单核细胞诱导CD4+T淋巴细胞的增殖以及阻断共刺激分子的作用.结果 PEC和人PBMC混合培养后可检测到PBMC对异种PEC的高度免疫增殖反应;流式细胞术检测到PBMC中的CD14+单核细胞表面无CD40和CD80的表达,但表达CD86,经PEC刺激后,CD14+单核细胞膜表面显著上调CD40和CD80蛋白分子的表达,CD86表达上调.与未经刺激的单核细胞相比较,经PEC刺激后的单核细胞和CD4+T淋巴细胞共培养后可诱导CD4+T淋巴细胞明显增殖,抗人CD154、CD80、CD86单克隆抗体可以阻断CD4+T淋巴细胞对PEC的增殖反应.结论 人CD14+单核细胞在异种免疫反应过程的间接抗原提呈和共刺激信号传导中发挥重要作用,通过上调其共刺激分子的表达与CD4+T淋巴细胞共刺激分子CD154和CD28相互作用形成第二信号,并诱导CD4+T淋巴细胞对PEC的增殖反应;阻断共刺激分子可抑制异种细胞免疫反应.     

Induction of xenoreactive CD4+ T-cell anergy by suppressor CD8+CD28- T cells

BACKGROUND: The underlying mechanism of immune suppression mediated by regulatory T cells is not completely understood. In previous studies we have shown that antigen-specific human T suppressor cells (Ts) can be generated in vitro by multiple rounds of stimulation with allogeneic, xenogeneic, or antigen-pulsed autologous antigen-presenting cells (APC). Human Ts express the CD8+CD28- phenotype and require specific recognition of MHC class I/peptide complexes on the surface of APC to block proliferation of T helper cells (Th). The aim of the present study was to explore the activation requirements of Ts as well as the nature of Th unresponsiveness to xenogeneic (swine) antigens induced by Ts. METHODS AND RESULTS: We investigated whether specific antigenic stimulation of Ts is required for their ability to inhibit early activation of xenoreactive Th (up-regulation of CD40 ligand). Flow cytometry studies indicated that Ts function required specific recognition of MHC class I on the surface of the stimulating APC. However, neither proliferation nor protein synthesis was required for the ability of Ts to inhibit Th. Ts drastically reduced the capacity of xenoreactive Th cells to produce interleukin (IL)-2 in response to the specific APC, without affecting their surface expression of IL-2 receptor. The suppressor effect that Ts exerted on Th proliferation could not be circumvented by CD40 ligation on the surface of the APC but could be reversed by the addition of exogenous IL-2. CONCLUSION: These data indicate that Ts induce anergy of xenoreactive human Th cells upon specific recognition of MHC class I antigens. Hence, Ts may prevent the activation of T cell-mediated immune responses against xenogeneic transplants.     

Tolerance to rat heart grafts induced by intrathymic immunomodulation is mediated by indirect recognition primed CD4+CD25+ Treg cells

BACKGROUND: In a rat model (PVG.R8-to-PVG.1U) disparate for one class I antigen, RT.1Aa, we previously demonstrated that intrathymic immunomodulation with donor antigens resulted in prolonged survival of cardiac allografts that underwent chronic rejection. However, long-term survivors developed a regulatory cell population that prevented both acute and chronic rejection when adoptively transferred into secondary graft recipients. The purpose of this study was to characterize these regulatory cells with particular emphasis on CD4+CD25+ Treg cells. METHODS: Spleens, lymph nodes, and peripheral blood lymphocytes of secondary tolerant recipients were characterized using antibodies to various T cell markers in flow cytometry. In vitro MLR and in vivo adoptive transfer experiments were conducted to investigate the involvement of CD4+CD25+ T cells in the observed tolerance. The presence of various cytokines in the sera of graft recipients and MLR culture supernatants was tested using ELISA. RESULTS: Tolerant recipients compared with naive rats had substantially higher percentages of CD4+CD25+ T cells in the spleen (28+/-3% vs. 11+/-5%) and blood (23+/-6% vs. 9+/-4%). Tolerant animals also had higher levels of serum IL-10 than naive and rejecting animals. CD4+CD25+ T cells from secondary long-term graft survivors inhibited donor-specific proliferative responses in vitro that was associated with high IL-10 production. Importantly, depletion of CD4+CD25+ T cells from splenocytes of tolerant rats abrogated their ability to transfer tolerance to tertiary graft recipients. CONCLUSIONS: Our data demonstrate that cardiac allograft tolerance in this model is mediated by CD4+CD25+ Treg cells primed by indirect recognition and is associated with high levels of IL-10.     

RATG对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响

目的 探讨兔抗人胸腺细胞多克隆抗体(RATG)在体外对CD4+细胞和CD8+细胞共刺激分子基因表达和细胞因子分泌的影响.方法 从正常成人外周血单个核细胞中分离和纯化CD4+细胞和CD8+细胞,加入RATG,37℃下培养.分别于24、48和72 h收集培养上清液和细胞,以不处理的细胞为正常对照,正常兔Ig处理的细胞作为阴性对照.采用实时聚合酶链反应技术检测培养细胞的细胞毒性T淋巴细胞相关抗原4(CTLA4)、CD154、Foxp3、OX40、γ干扰素(IFN-γ)、白细胞介素2(IL-2)、IL-10和IL-2受体(CD25)的基因表达水平,应用Multiplex检测技术测定培养上清液中IFIN-γ、IL-2、IL_4和IL-10的水平.结果 与正常CD4+细胞比较,加入RATG的CD4+细胞培养24 h,其CTLA-4、CD154、Foxp3、OX40、IFN-γ、IL-2、IL-10和CD25基因转录表达均上调,阴性对照CD4+细胞则无这些基因转录表达的上调.处理48 h后,CD4+细胞的CD154和IL-2的基因转录表达呈现下调现象,而CTLA4、Foxp3、0X40、IFN-γ、IL-10和CD25的基因转录水平均较24 h时明显降低.处理72 h后,CD4+细胞的CTLA4、Foxp3、OX40和CD25的基因转录表达再次呈现高水平,CD154和IFN-γ的基因转录表达上调表达不明显,而IL-2和IL-10的基因转录表达则呈现明显的下调.RATG处理的CD4+细胞培养上清液中的IFN-γ、IL-2、IL-4和IL-10浓度显著增加,以处理24 h的水平最高,而阴性对照者未测出.与正常CD8+细胞相比较,加入RATG处理的CD8+细胞培养24 h,其CTLA4、Foxp3、OX40、IFN-γ,、IL-2、IL-10和CD25的基因转录表达呈现明显上调,而CD154基因转录表达稍有下调.RATG处理48 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ和CD25基因转录表达仍维持在高水平,CD154基因转录表达仅仅呈现低水平的上调,IL-10基因转录表达水平显著下降,而IL-2的基因转录表达则明显下调.处理72 h,CD8+细胞的CTLA4、Foxp3、OX40、IFN-γ、IL-10和CD25的基因转录表达仍维持在高水平,CD154基因转录表达则呈现下调,而IL-2基因转录表达的下调更为显著.阴性对照CD8+细胞则未呈现这些基因转录的上调现象.RATG处理的CD8+细胞培养上清液中IFN-γ、IL-2和IL-10显著增多,以处理24 h的浓度最高,IL-4浓度升高的幅度较小,而正常CD8+细胞和阴性对照CD8+细胞几乎检测不到IFN-γ、IL-2、IL-4和IL-10.结论 在体外,RATG可以刺激CD4+细胞和CD8+细胞上调多种促进免疫抑制的共刺激分子基因表达,促进其分泌与免疫调节相关的IFN-γ、IL-2、IL-4和IL-10.     

外源性IL-2对小鼠CD4^＋CD25^＋调节性T细胞影响的实验研究

目的：探讨IL-2对CD4＋CD25＋调节性T细胞（Tregs）的增殖及功能的影响。方法：提取B6小鼠脾脏细胞,流式细胞仪分离CD4＋CD25＋Tregs,将新鲜分离的CD4＋CD25＋Tregs与抗CD3单克隆抗体、同种同系抗原递呈细胞（APCs）及外源性IL-2共同培养,测定其增殖活性;并检测体外扩增后的CD4＋CD25＋Tregs的免疫抑制活性及其Foxp3的表达。结果：与外源性IL-2共同培养的CD4＋CD25＋Tregs增殖程度强烈,与对照组比较,差异有统计学意义（P〈0.05）;体外扩增的CD4＋CD25＋Tregs抑制CD4＋CD25-T细胞增殖活性的能力与新鲜分离的CD4＋CD25＋Tregs相似（P〉0.05）。体外扩增的CD4＋CD25＋Tregs的Foxp3表达与新鲜分离的CD4＋CD25＋Tregs亦相似（P〉0.05）。结论：外源性IL-2能够消除CD4＋CD25＋Tregs的无反应状态,且体外扩增的CD4＋CD25＋Tregs保持了其抑制活性。     

Requirements for the induction of allospecific CD8+ suppressor T cells in the rat primary mixed lymphocyte response. CD4+, CD45R+ T cells, or supernatant factor

We examined the requirements for the induction of the MLR-generated allospecific CD8+ suppressor T cells in the rat. Depleting the responder population of CD4+ T cells before initiating the primary MLR abrogates the generation of day-5 CD8+ T suppressor effectors. Readdition of at least 10% CD4+ T cells to the CD4+ depleted primary MLR reconstitutes suppressor cell generation. Using the anti-CD45R monoclonal antibody OX22, we also show that the T suppressor inducer cells are CD4+ CD45R+. Using a dual chamber Transwell culture system, which allows cells to be co-incubated without direct cell-to-cell contact, we show that a soluble factor/s, produced during the course of the primary MLR, is capable of inducing naive CD8+ T cells to become suppressor effectors but only when these CD8 T cells are in direct contact with allogeneic stimulators. Allospecificity is conferred by the stimulator cells and not by the suppressor-inducer factor. The supernatant of day-5 primary MLR is also capable of inducing antigen-specific suppressor effectors from naive CD8+ T cells, and also only in the presence of allogeneic stimulator cells. Recombinant human IL-2, in doses that are up to five times the amount present in the supernatant cultures, is unable to induce suppressor-effector cells from naive CD8+ T cells. We conclude that, to become allospecific suppressor effectors, naive CD8+ T cells require contact with allogeneic stimulator cells and either CD4+ CD45R+ suppressor inducer cells or suppressor inducer factor/s produced during the course of the primary MLR.     

A novel mechanism of action for anti-thymocyte globulin: induction of CD4+CD25+Foxp3+ regulatory T cells

T cell-depleting agents are being tested as part of clinical tolerance strategies in humans with autoimmunity and transplantation. The immunosuppressive activity of anti-thymocyte globulin (ATG) has been thought to result primarily from depletion of peripheral lymphocytes. Herein is reported for the first time that ATG but not anti-CD52 mAb (alemtuzumab) or the IL-2R antagonists causes rapid and sustained expansion of CD4+CD25+ T cells when cultured with human peripheral blood lymphocytes. These cells display enhanced expression of the regulatory markers glucocorticoid-induced TNF receptor, cytotoxic T lymphocyte-associated antigen-4 (CTLA-4), and forkhead box P3 and efficiently suppress a direct alloimmune response of the original responder lymphocytes. It is interesting that the cells do not suppress memory responses to the recall antigen mumps. Ex vivo expansion of regulatory T cells is due mainly to conversion of CD4+CD25- into CD4+CD25+ T cells and to a lesser degree to proliferation of natural CD4+CD25+ T cells. The induction of regulatory T cells depends on production of Th2 cytokines in the generating cultures. These novel data suggest that ATG not only may promote expansion/generation of regulatory T cells but also may be useful in future ex vivo expansion of these cells for cellular therapy in autoimmunity and clinical transplantation.     

Observational support for an immunoregulatory role of CD3+CD4+CD25+IFN-gamma+ blood lymphocytes in kidney transplant recipients with good long-term graft outcome.

There is evidence that interferon-gamma (IFN-gamma)-dependent interactions of dendritic cell (DC), T regulatory (Treg), and T suppressor (Ts) subpopulations contribute to allograft acceptance. We measured DC subsets, CD3+CD4+CD25+ (Treg phenotype) and CD3+CD8+CD28(-) (Ts phenotype) peripheral blood lymphocytes (PBL) expressing Foxp3, Th1 or Th2 cytokines, peripheral T- and B-cell counts, and plasma cytokines in 33 kidney transplant recipients with a serum creatinine of < or =1.8 mg/dl and 32 recipients with a serum creatinine of > or =2.0 mg/dl more than 100 days post-transplant. Cell subsets were measured in whole blood using four-color flow cytometry. Patients with increased creatinine had less frequently detectable CD3+CD4+CD25+IFN-gamma+ PBL than patients with good graft function (P = 0.017). In patients with good graft function, CD3+CD4+CD25+IFN-gamma+ PBL were associated with high Foxp3+, IL-2+, IL-12+, IL-4+, and IL-10+ CD3+CD4+CD25+ T PBL (P < 0.001), low CD3+CD8+CD28(-)Foxp3+ (P = 0.002), CD3+CD4+DR+ (P = 0.002), CD3+CD8+DR+ T (P = 0.005) and CD19+ B PBL (P = 0.005), and low lineage(-)HLA-DR+CD11c+CD123(-) DC1 (P = 0.006). Patients with impaired graft function did not show these associations. Additional flow cytometric analysis confirmed strong co-expression of IFN-gamma and Foxp3 by CD4+CD25+ PBL particularly in patients with good graft function. Our data support an immunoregulatory role of CD3+CD4+CD25+Foxp3+IFN-gamma+ cells in a subgroup of transplant recipients with good graft acceptance.     

移植抗原特异性转基因CD8+T细胞对白细胞介素2的依赖性

目的研究白细胞介素2（IL-2）在调节移植抗原特异性转基因CD8^＋T细胞介导的免疫排斥反应中的作用。方法将经荧光染科CFSE标记后的C57BL／6小鼠和2CTg小鼠（CD4敲除鼠）淋巴细胞分别植入经致死剂量γ射线照射过的两组DBA／2J小鼠体内，检测CD4^＋与CD8^＋T细胞在体内分裂增殖的时相，并用胞浆内IL-2标志染色方法测定活化后T细胞表达IL-2的能力。以Balb／c小鼠为供者，糖尿病2CTg小鼠和2C Tg-IL-2KO小鼠（IL-2敲除鼠）为受者，进行胰岛细胞移植。观察CD8^＋ T细胞在介导移植排斥中的作用。结果DBA／2J小鼠输注了C57BL／6小鼠的淋巴细胞后，CD4^＋与CD8^＋T细胞分裂增殖均非常明显，前者表达大量IL-2，后者则不表达。DBA／2J小鼠输注了2CTg小鼠的淋巴细胞后。在完全没有CD4^＋T细胞存在的情况下，CD8^＋T细胞仍明显分裂增殖并大量表达IL-2。2CTg和2CTg—IL-2KO小鼠移植胰岛细胞后，前者迅速发生排斥反应，胰岛移植物的平均存活时间仅为8d，而后者胰岛移植物的平均存活时间〉50d。结论CD8^＋T细胞在产生和利用IL-2时有很大的可塑性。CD4^＋T细胞存在时，CD8^＋T细胞能有效利用CD4^＋T细来源的IL-2进行分裂增殖，在缺乏CD4^＋T细胞时，则利用自身来源的IL-2进行分裂增殖；移植抗原特异性CD8^＋T细胞的效应功能完全依赖于IL-2，排斥反应由CD8^＋T细胞介导时，阻断IL-2／IL-2受体通路可诱导移植物长期存活。     

Expansion of CD25+CD4+ regulatory T cells by natural mature dendritic cells

Because of the anergy of CD25+CD4+ regulatory T cells, it is unclear how the number of these regulatory T cells is sustained and expanded in normal physiologic circumstances. In the present study, we examined the effect of natural allogeneic mature dendritic cells (DCs) on the proliferation and function of CD25+CD4+ T cells. Our data showed that natural allogeneic mature DCs stimulated CD25+CD4+ T-cell growth vigorously, whereas immature DCs had little effect on the proliferation of CD25+CD4+ T cells. After expansion by mature DCs, CD25+CD4+ T cells maintained their expression of Foxp3 and suppressed the proliferation of CD25- CD4+ T cells similar to freshly isolated CD25+CD4+ T cells. Our results introduce a potentially critical role played by natural allogeneic mature DCs, which exist in normal physiologic circumstances, in controlling CD25+CD4+ regulatory T-cell expansion and function.