IL-2协同抗CD45RB抗体调节Treg/Th17细胞的比例并延长小鼠移植皮肤存活时间

目的:研究IL-2在抗CD45RB抗体诱导免疫耐受中对Treg/Th17细胞分化的影响,进一步阐明抗CD45RB抗体诱导免疫耐受的机制。方法:用免疫磁珠分选C57BL/6小鼠脾脏中的CD4+T细胞,在抗CD45RB抗体与IL-2的作用下培养72小时后,流式检测Treg/Th17细胞的变化。以BALB/c小鼠为供体,C57BL/6小鼠为受体建立同种异基因皮肤移植模型,分别给予抗CD45RB抗体及IL-2等治疗,术后1、3、5、7、9天取受体鼠脾细胞,动态检测Treg/Th17细胞的变化;术后第9天取移植皮肤HE染色观察炎性细胞的浸润情况;观察并记录移植皮肤的存活时间。结果:CD4+T细胞在IL-2联合抗CD45RB抗体的作用下培养72小时后,Treg比例升高,Th17细胞比例下降;IL-2联合抗CD45RB抗体治疗后明显延长小鼠移植皮肤的存活时间。结论:IL-2可以明显增强抗CD45RB抗体诱导免疫耐受的形成,使Treg细胞上调,下调Th17细胞,有利于免疫耐受的形成。     

人参皂苷衍生物AD-1对CD4^(+)T细胞亚群的调控及对DSS诱导的实验性结肠炎小鼠的影响北大核心CSCD

目的:探讨新型人参皂苷衍生物AD-1对体外分化CD4^(+)T细胞亚群的影响及对DSS诱导的实验性结肠炎小鼠CD4^(+)T细胞亚群的调控作用。方法:采用磁珠阴性选择法获得正常小鼠幼稚CD4^(+)T细胞,定向分化为Th1、Th2、Th17细胞、Treg,流式细胞术检测AD-1对其分化的影响。3%DSS三次循环法建立小鼠实验性结肠炎模型,AD-1药物干预后,流式细胞术、HE染色检测结肠、脾脏、肠系膜淋巴结中Th1、Th2、Th17细胞、Treg所分泌的细胞因子和特异性转录因子。结果:细胞活化后,早期标志物CD69无显著变化;AD-1下调Th1和Th17细胞分泌的细胞因子IFN-γ和IL-17A表达,上调Th2细胞分泌的细胞因子IL-4和Treg转录因子Foxp3及表面标志物CD25表达。AD-1可通过下调脾脏和肠系膜淋巴结中Th1和Th17细胞表达及上调Th2和Treg表达改善DSS诱导的小鼠实验性结肠炎。结论:AD-1对CD4^(+)T细胞活化无影响,但可抑制Th1和Th17细胞分化,促进Th2细胞和Treg分化,同时通过调节CD4^(+)T细胞亚群改善DSS诱导的小鼠实验性结肠炎。     

HMGB1通过TLR4促进心肌缺血损伤并调节脾脏CD4~+、CD8~+T细胞和Th17细胞比例

目的探讨高迁移率族蛋白B1(HMGB1)通过Toll样受体4 (TLR4)调控心肌缺血损伤及对脾脏组织CD4~+T细胞、CD8~+T细胞及Th17细胞亚群的影响。方法 C57BL/6野生型小鼠(WT)和TLR4基因敲除(TLR4-/-)小鼠各30只,随机分为对照组、异丙肾上腺素诱导心肌缺血(ISO)组和ISO联合重组HMGB1(r HMGB1)组。采用心脏超声检查小鼠心功能,应用HE染色和天狼星红染色观察心肌组织病理学变化;原位末端转移酶标记技术(TUNEL)检测心肌细胞凋亡指数,流式细胞术检测脾脏组织中CD4~+T细胞、CD8~+T细胞和Th17细胞亚群的比例。结果与对照组相比,ISO组小鼠诱发心脏功能损害、心肌组织坏死和纤维化、心肌细胞凋亡,脾脏组织中CD4~+T淋巴细胞比例、CD4~+/CD8~+T细胞比值和Th17细胞比例升高;与ISO组小鼠相比,ISO联合r HMGB1组心脏功能损害、心肌组织坏死和纤维化、心肌细胞凋亡状况均加重,脾脏组织中CD4~+T淋巴细胞比例、CD4~+/CD8~+T细胞比值和Th17细胞比例更高;与ISO联合r HMGB1组野生型小鼠相比,ISO联合r HMGB1组TLR4-/-小鼠心脏功能损害、心肌组织坏死和纤维化和心肌细胞凋亡减轻,脾脏组织CD4~+T淋巴细胞比例、CD4~+/CD8~+T细胞比值和Th17细胞比例显著降低。结论 HMGB1通过TLR4诱发心肌缺血时心肌组织损伤,并上调脾脏组织CD4~+T细胞比例、CD4~+/CD8~+T细胞比值和Th17细胞比例,从而可能促进心肌组织炎症损伤。     

辐射损伤后T细胞亚群的免疫重建特点及中药山茱萸的调节作用

目的:探讨小鼠辐射损伤后T细胞亚群免疫重建特点及山茱萸的调节作用。方法:采用X射线2.6 Gy单次全身照射建立小鼠辐射损伤模型及山茱萸实验模型,分别在辐射前后,检测小鼠血常规变化;应用流式细胞术检测CD3+、CD4+和CD8+T细胞及其Th1、Tc1、Th2、Th17和Treg亚群的比例。结果:辐射后第3天,外周血和脾脏中淋巴细胞总数及T细胞(包括CD4+、CD8+)比例显著降低(P0.05);T细胞在辐射后第5天开始重建,其中以CD8+T细胞为主,CD4+T细胞在第8天恢复重建。但T细胞分泌IFN-γ能力(Th1/Tc1)低于正常对照组(P0.05)。相反,Th2、Th17、Treg亚群比例显著增高(P0.05)。与照射组相比,山茱萸处理小鼠Th1亚群比例明显上调(P0.05),Th2、Th17、Treg亚群的比例或数量显著下降(P0.05)。结论:X射线单次照射后,CD8+T细胞免疫重建早于CD4+T细胞,但IFN-γ分泌能力较弱。山茱萸可显著上调Th1比例,抑制Th2、Th17、Treg增殖,改善辐射诱导T细胞亚群失衡,增强辐射损伤后Th1类细胞亚群的免疫重建优势。     

美沙拉嗪对DSS诱导小鼠溃疡性结肠炎模型Th1、Th17及Treg细胞亚群的影响

 目的:观察葡聚糖硫酸钠（DSS）诱导小鼠溃疡性结肠炎（UC）模型中辅助性T细胞（Th1、Th17亚群）及调节性T细胞（Treg）细胞亚群的变化,探讨美沙拉嗪（MSLZ）治疗UC的免疫学机制。方法: 采用流式细胞分析术检测DSS诱导的小鼠UC模型结肠组织及外周血单个核细胞中白细胞介素17（IL-17）、γ-干扰素（IFN-γ）及核转录因子Foxp3的表达,并检测MSLZ预治疗对小鼠UC 模型Th1、Th17和Treg亚群的影响。结果: 在DSS诱导的小鼠UC模型中,其外周血单个核细胞（PBMC）中CD3+T细胞高表达IL-17、IFN-γ及Foxp3,肠黏膜单个核细胞（LPMC）中CD3+T细胞高表达IFN-γ和Foxp3,但IL-17的表达与对照组无差异。进一步发现UC模型小鼠LPMC中Th17、Th1和Treg均显著高于对照组,但PBMC中只有Treg高于对照组。MSLZ预治疗能显著下调UC 模型小鼠PBMC和LPMC中Th17、Th1和Treg细胞亚群。结论: DSS诱导的小鼠 UC模型中CD4+T细胞亚群Th1、Th17及Treg细胞显著升高,提示CD4+T细胞亚群在UC发病中起重要作用,美沙拉嗪可能通过调节Th1、Th17及Treg细胞亚群发挥抗炎及治疗UC作用。     

丙酮酸激酶M2缺失不影响TCRαβ~+T细胞的增殖、活化、分化和抗肿瘤能力

为探究丙酮酸激酶M2(pyruvate kinase M2, PKM2)在TCRαβ~+T细胞中的功能,研究制备了TCRαβ~+T细胞条件性敲除PKM2小鼠。FACS检测发现PKM2缺失不影响小鼠外周淋巴器官中TCRαβ~+CD4~+与CD8~+T细胞百分比、增殖能力及活化潜能。TCRαβ~+T细胞特异性PKM2~(-/-)小鼠可在结肠组织中形成正常的Th1与Th17亚群,也可在体内外诱导产生正常的Th1与Th17。PKM2缺失同样不影响MC38移植瘤生长,也不改变移植瘤中TCRαβ~+CD4~+/CD8~+T细胞比例和T细胞IFN-γ的表达。因而,TCRαβ~+T细胞特异性PKM2~(-/-)小鼠具有正常的T细胞功能亚群。Western blotting表明,PKM2~(-/-)TCRαβ~+CD4~+与CD8~+T细胞中PKM1表达上调。据此,作者推测PKM1部分代偿了PKM2在TCRαβ~+T细胞中的生理功能。     

β2 GP1诱导的EAPS小鼠Treg/Th17细胞比率变化的研究

目的:观察EAPS疾病进展过程中小鼠外周血Th17细胞与CD4+CD25+调节性T(Treg)细胞的比率变化。方法:以重组人β2糖蛋白1(rhβ2GP1)主动免疫BALB/c小鼠建立EAPS模型,免疫12周后检测外周血浆抗β2糖蛋白1抗体(anti-β2GP1)、抗心磷脂抗体(a CA)、IL-17、IL-2、IL-6、TGF-β、活化部分凝血活酶时间(APTT)和血小板计数(PLT)及流产率。流式细胞术(FCM)检测小鼠PBMC中CD4+CD25+Treg及Th17细胞的比率。结果:与对照组相比,模型小鼠anti-β2GP1、a CA、IL-17、IL-2、IL-6水平明显升高,APTT延长,TGF-β降低,PLT升高,流产率提高,差异有统计学意义(P<0.05)。PBMC中CD4+CD25+Treg细胞频率8周前与对照组相比差异无统计学意义(P>0.05),12周后逐渐减少,明显低于对照组(P<0.05);Th17细胞频率逐渐升高,明显高于对照组(P<0.05);CD4+CD25+Treg/Th17比值明显低于对照组(P<0.05)。结论:EAPS小鼠外周血Th17/Treg细胞比率失衡可能在EAPS的发生发展中起重要作用。     

Bcl3基因敲除对小鼠脾脏免疫细胞组成及抗肿瘤能力的影响

目的观察Bcl3基因敲除对小鼠脾脏免疫细胞的组成及抗肿瘤能力的影响。方法使用CRISPR/Cas9基因编辑技术建立Bcl3基因敲除小鼠(Bcl3-/-), 血常规检验和流式细胞术检测Bcl3-/-小鼠的免疫细胞组成;建立B16F10黑色素瘤肺转移小鼠模型, 记录肺部肿瘤结节数和小鼠生存时间, 对比野生型(wild type, WT)小鼠和Bcl3-/-小鼠的抗肿瘤能力。结果 Bcl3-/-小鼠成功繁育成品系, 子代基因敲除纯合小鼠无胚胎致死现象, 且生长正常, 与WT小鼠相比外观、生长发育、繁育性能未见明显差异;主要脏器无明显异常, 但脾脏肿大且脾脏免疫细胞总数明显增加(P<0.05);血小板计数和中性粒细胞计数及百分比均显著低于WT小鼠;CD19+B细胞比例无明显改变, CD3+T细胞比例显著增加, 同时T细胞亚群(CD4+、CD8+、Treg)比例均呈明显上升趋势(P<0.05);固有免疫细胞中NK细胞(NK1.1+)和中性粒细胞(Gr1+)比例下降(P<0.05), DC(CD11b+)比例无明显变化;Bcl3-/-荷瘤小鼠的肺部可见大量由黑色素瘤细胞形成的肿瘤...     

成人哮喘患者Treg/Th17细胞失衡与呼出气一氧化氮的相关性研究

目的:探讨成人哮喘患者Treg/Th17细胞失衡与呼出气一氧化氮(FeNO)的相关关系。方法:募集成人哮喘患者17例,慢性咳嗽非哮喘患者16例。所有患者均检测FeNO值,并用ELISA法检测血浆IL-17的表达水平,流式细胞术检测外周血中Treg、Th17细胞占CD4~+T淋巴细胞比例。比较两组患者FeNO水平的差异及其与Treg/Th17的相关关系。结果:与慢性咳嗽组相比,哮喘组血浆IL-17浓度升高(P<0.05)、外周血Th17/CD4~+T细胞比值升高(P<0.001)、FeNO水平升高(P<0.001)、Treg/Th17细胞的比值降低(P<0.001),而Treg/CD4~+细胞比值无明显变化(P>0.05)。哮喘组FeNO值与外周血Treg/CD4~+T细胞比值无明显相关性,而与Th17细胞占CD4~+T淋巴细胞比例呈正相关(r=0.663,P=0.01),与Treg/Th17细胞比值呈负相关(r=-0.757,P=0.002)。结论:哮喘患者FeNO水平升高与Treg/Th17失衡有关,主要与Th17细胞表达升高有关。     

白芍总苷对过敏性紫癜小鼠Treg/Th17免疫平衡及JAK1/STAT3信号通路蛋白的影响北大核心CSCD

目的:基于蛋白酪氨酸激酶1/信号转导因子3(JAK1/STAT3)信号通路探讨白芍总苷对过敏性紫癜小鼠Treg/Th17免疫平衡的影响。方法:动物实验分为对照组、模型组、实验组、抑制剂组,模型组、实验组、抑制剂组小鼠尾静脉注射印度墨水及灌胃麦角蛋白复制过敏性紫癜模型,实验组、抑制剂组小鼠每日灌胃0.1 g/kg白芍药总苷和AG490,连续处理4周。ELISA测定各组小鼠血清IL-10及IL-17含量;流式细胞术检测各组小鼠血清Treg、Th17变化;Western blot检测各组小鼠皮肤和肾脏组织p-JAK1、p-STAT3表达。结果:与对照组相比,模型组、实验组、抑制剂组小鼠血清Treg占CD4+T细胞比例和IL-10含量明显下降,Th17占CD4+T细胞比例、IL-17含量以及小鼠皮肤和肾脏组织p-JAK1、p-STAT3表达明显升高(均P<0.05);与模型组相比,实验组、抑制剂组小鼠血清Treg占CD4+T细胞比例和血清IL-10含量明显升高,Th17占CD4+T细胞比例、IL-17含量以及小鼠皮肤和肾脏组织p-JAK1、p-STAT3表达明显降低(均P<0.05)。结论:白芍总苷可通过抑制JAK2/STAT3信号通路调控过敏性紫癜小鼠Treg/Th17免疫平衡。     

Competition between colitogenic Th1 and Th17 cells contributes to the amelioration of colitis

Th17 cells and Th1 cells coordinate to play a critical role in the formation of inflammatory bowel diseases. To examine how Th17 and Th1 cells are regulated at inflammatory sites, we used Th1‐dominant CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? and Th1/Th17‐mixed IL‐10^?/? mice. Interestingly, not only did colitic RAG‐2^?/? mice that were parabiosed with WT mice show significant amelioration of colitis, but amelioration of disease was also observed in those parabiosed with colitic IL‐10^?/? mice. To assess the interference between Th1 and Th17 colitogenic T cells, we co‐transferred colitogenic CD4⁺ T cells from the lamina propria (LP) of CD4⁺CD45RB^high T cell‐transferred RAG‐2^?/? mice and IL‐10^?/? mice into RAG‐2^?/? mice. Surprisingly, the co‐transferred RAG‐2^?/? mice showed a vast cellular infiltration of LP CD4⁺ T cells similar to that seen in RAG‐2^?/? mice re‐transferred with the cells from colitic RAG‐2^?/? mice alone, but the co‐transferred RAG‐2^?/? mice did not have the wasting symptoms, which are also absent in RAG‐2^?/? mice transferred with cells from colitic IL‐10^?/? mice alone. Furthermore, the percentages of Th1 and Th17 cells originating from IL‐10^?/? mice and those of Th1 cells originating from colitic RAG‐2^?/? mice were all significantly decreased in the co‐transferred mice as compared with the singly‐transferred paired RAG‐2^?/? mice, suggesting that Th1 and Th17 cells are in competition, and that their orchestration results in a merged clinical phenotype of the two types of murine colitis.     

B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用

 目的: 探讨B淋巴细胞在抗CD45RB抗体诱导的移植免疫耐受中的作用。方法: 抗CD45RB抗体对BALB/c裸鼠进行预处理后制备脾脏单细胞悬液,与BALB/c小鼠T淋巴细胞和C57BL/6小鼠脾细胞混合培养,流式细胞术分析Th1、Th2、Treg和Tm淋巴细胞。以B6.μMT^-/-小鼠为受体、BALB/c小鼠为供体建立皮肤移植模型,移植后向受体鼠腹腔注射抗CD45RB单抗,监测脾淋巴细胞CD3⁺CD45RB^hi细胞比例。在混合淋巴培养过程中加入抗CD45RB单抗,分离B细胞,建立以BALB/c小鼠为供体、B6.μMT^-/-小鼠为受体的心脏移植模型,通过尾静脉注射B细胞给B6.μMT^-/-小鼠,观察受体鼠生存期和B细胞分布。结果: 在裸鼠体内用抗CD45RB抗体处理过的B淋巴细胞,与T淋巴细胞混合培养时,可使Treg和Th2淋巴细胞比例明显升高,Th1淋巴细胞的比例明显下降,Tm细胞无明显变化。在体内B淋巴细胞缺失的情况下,抗CD45RB抗体依然能够降低T细胞表面CD45RB的表达,与对照组B淋巴细胞存在组相比,抗CD45RB抗体对T淋巴细胞表面CD45RB下调更为快速,但最终CD3⁺CD45RB^hi T细胞比例无明显变化。体外抗CD45RB抗体处理过的B淋巴细胞可以延长受体鼠的生存时间。B6.μMT^-/-鼠在接受抗CD45RB抗体处理的B细胞并进行同种异体心脏移植后,B细胞可向胸腺迁移。结论: 在抗CD45RB抗体诱导的免疫耐受中,B淋巴细胞可能通过介导各T淋巴细胞亚群比例发挥着重要作用,且在中枢耐受中也起到一定作用,但是仅靠B淋巴细胞无法形成完全耐受。     

IL‐2 is positively involved in the development of colitogenic CD4+ IL‐7Rαhigh memory T cells in chronic colitis

IL‐2 and IL‐7 share a common γ‐chain receptor and are critical for T‐cell homeostasis. We aimed to clarify the reciprocal roles of IL‐2 and IL‐7 in the development and persistence of chronic colitis. We performed a series of adoptive transfers of IL‐2^?/? CD4⁺CD45RB^high T cells into RAG‐2^?/? mice and assessed the role of IL‐2 in the induction of IL‐7Rα on colitogenic CD4⁺ T cells and the development of chronic colitis. RAG‐2^?/? mice transferred with WT but not with IL‐2^?/? CD4⁺CD45RB^high T cells developed Th1/Th17‐mediated colitis. Consistently, re‐expression of IL‐7Rα was severely impaired on IL‐2^?/? but not on WT CD4⁺ T cells from the transferred mice. To exclude a contribution of the preclinical autoimmunity of IL‐2^?/?mice, WT Ly5.1⁺ or IL‐2^?/? Ly5.2⁺ CD4⁺CD45RB^high T cells from GFP mice previously transplanted with the same number of WT and IL‐2^?/? BM cells were transferred into RAG‐2^?/? mice. RAG‐2^?/? mice transferred with IL‐2^?/?‐derived CD4⁺CD45RB^high T cells did not develop colitis, but their splenic CD4⁺ T cells changed from effector‐memory to central‐memory type. These results show that IL‐2 is critically involved in the establishment and maintenance of IL‐7‐dependent colitogenic memory CD4⁺IL‐7Rα^high T cells.     

Host-dependent control of early regulatory and effector T-cell differentiation underlies the genetic susceptibility of RAG2-deficient mouse strains to transfer colitis

De novo differentiation of CD4⁺Foxp3⁺ regulatory T cells (induced (i) Tregs) occurs preferentially in the gut-associated lymphoid tissues (GALT). We addressed the contribution of background genetic factors in affecting the balance of iTreg, T helper type 1 (Th1), and Th17 cell differentiation in GALT in vivo following the transfer of naive CD4⁺CD45RB^high T cells to strains of RAG2-deficient mice with differential susceptibility to inflammatory colitis. iTregs represented up to 5% of CD4⁺ T cells in mesenteric lymph nodes of less-susceptible C57BL/6 RAG2^−/− mice compared with <1% in highly susceptible C57BL/10 RAG2^−/− mice 2 weeks following T-cell transfer before the onset of colitis. Early Treg induction was correlated inversely with effector cell expansion and the severity of colitis development, was controlled primarily by host and not T-cell-dependent factors, and was strongly associated with interleukin-12 (IL-12)/23 production by host CD11c⁺CD103⁺ dendritic cells. These data highlight the importance of genetic factors regulating IL-12/23 production in controlling the balance between iTreg differentiation and effector-pathogenic CD4⁺ T-cell expansion in lymphopenic mice and indicate a direct role for iTregs in the regulation of colonic inflammation in vivo.     

TGF-beta1 production by CD4+ CD25+ regulatory T cells is not essential for suppression of intestinal inflammation

Naturally occurring CD4+ CD25+ regulatory T cells (Treg) are potent suppressors of CD4+ and CD8+ T cell responses in vitro and inhibit several organ-specific autoimmune diseases. While most in vitro studies suggest that CD4+ CD25+ Treg cells adopt a cytokine-independent but cell contact-dependent mode of T cell regulation, their precise mechanism of suppression in vivo remains largely unknown. Here we examine the functional contribution of Treg cell-derived TGF-beta1 and effector T cell responsiveness to TGF-beta in CD4+ CD25+ T cell-mediated suppression of inflammatory bowel disease (IBD). We show that CD4+ CD25+ Treg cells from either TGF-beta1+/+ or neonatal TGF-beta1-/- mice can suppress the incidence and severity of IBD as well as colonic IFN-gamma mRNA expression induced by WT CD4+ CD25- effector T cells. Furthermore, TGF-beta-resistant Smad3-/- CD4+ CD25+ Treg cells are equivalent to WT Treg cells in their capacity to suppress disease induced by either WT or Smad3-/- CD4+ CD25- effector T cells. Finally, anti-TGF-beta treatment exacerbates the colitogenic potential of CD4+ CD25- effector T cells in the absence of CD4+ CD25+ Treg cells. Together, these data demonstrate that in certain situations CD4+ CD25+ T cells are able to suppress intestinal inflammation by a mechanism not requiring Treg cell-derived TGF-beta1 or effector T cell/Treg cell responsiveness to TGF-beta via Smad3.     

Involvement of 4-1BB (CD137)-4-1BBligand interaction in the modulation of CD4 T cell-mediated inflammatory colitis

4-1BB ligand (4-1BBL) expressed on antigen-presenting cells interacts with 4-1BB on activated T cells (especially CD8+ cells) and co-stimulates the latter to secrete cytokines and to proliferate. The role of 4-1BB-4-1BBL interaction was studied here in a model of colitis based on naive CD4+ T cell transfer to SCID mice, a disease model in which CD8 cells do not take part. We found that CD4+ T cells from 4-1BB-deficient mice, after transfer in SCID mice, proliferated more rapidly compared to wild-type CD4+ T cells. Mice reconstituted with naive CD4+ T cells from 4-1BB-deficient mice developed colitis, however, with a mixed Th1/Th2 response, in contrast to the Th1-type response in mice reconstituted with wild-type naive CD4+ T cells. Importantly, this altered cytokine response did not temper colitis severity. Although it has been reported previously that 4-1BB co-stimulation may contribute to regulatory T cell functioning, we found that CD4+CD25+ regulatory T cells from 4-1BB-deficient mice were perfectly able to prevent naive CD4+ T cell-induced colitis. In conclusion, our data provide evidence that 4-1BB-4-1BBL interaction modulates the effector CD4+ T cell-driven immune response and cytokine production in experimental colitis without affecting regulatory T cell function.     

CVB3诱导的病毒性心肌炎小鼠模型中CD4~+T细胞亚群格局及其作用

本研究主要关注BALB/c小鼠感染柯萨奇病毒B3型(CVB3)后CD4+T细胞亚群格局的变化及其对病毒性心肌炎发病的贡献度。CVB3腹腔感染小鼠后第7d,通过小鼠体重下降率、心肌损伤血清学指标肌酸激酶(CK)活性以及心肌组织病理学改变等多项指标证实小鼠心肌炎模型的成功建立。经Real-time PCR检测感染第7d脾脏CD4+T细胞亚群主要细胞因子IFN-γ、IL-4、IL-17A、IL-17F及转录因子Foxp3表达情况;以流式细胞术检测了CD4+T细胞各亚群比例,并通过多元线性回归分析法评估了各T细胞亚群对病毒性心肌炎发病的影响。结果显示,与对照组相比,CVB3感染小鼠脾脏IL-17A、IL-17F、IFN-γ表达均显著上升(分别约为12、8.5、5倍),而IL-4和Foxp3表达无明显变化。CVB3感染小鼠脾脏中CD4+IL-17+Th17细胞的上调幅度最为明显,约2.75倍,其次为IFN-γ+Th1细胞,上调约2.27倍,而Th2和Treg细胞无明显变化。进一步统计学分析显示,Th17对病毒性心肌炎发病的贡献度最高(1.808),Th1次之,贡献度为1.581,而Th2和Treg对病毒性心肌炎发病无显著影响,提示CD4+T细胞亚群格局变化与病毒性心肌炎发病密切相关,其中以Th17对心肌炎发病的贡献度尤为显著。     

Evaluation of the immunoregulatory activity of intraepithelial lymphocytes in a mouse model of chronic intestinal inflammation

Intraepithelial lymphocytes (IELs) represent the first line of lymphocyte defense against the intestinal bacteria. Although previous studies have demonstrated a protective role of IELs in the development of colitis, the data supporting a regulatory role for IELs are limited. The objective of this study was to examine the suppressive activity of IELs in vitro and in vivo using a mouse model of chronic small and large bowel inflammation. Adoptive transfer of CD8α(+) IELs isolated from small intestines of wild-type (WT) mice into TCR βxδ-deficient (TCR βxδ(-/-)) recipients did not prevent or delay the onset of the disease induced by WT CD4(+)CD45RB(high) T cells. On the contrary, we observed a more rapid onset of wasting and clinical signs of intestinal inflammation when compared with animals injected with CD4(+)CD45RB(high) T cells alone. Histopathological scores of small and large bowel did not differ significantly between the two groups. Transfer of IELs alone did not produce any pathological changes. Real-time PCR analysis of intestinal tissues showed up-regulation of message for T(h)1- and macrophage-derived cytokines in colon and small bowel. Using Foxp3-GFP reporter mice, we were unable to detect any Foxp3(+) cells within the CD8α(+) IELs but did find a small population of Foxp3(+)CD4(+) IELs in the small and large bowel. Using in vitro suppression assay, we found that neither TCRαβ(+)CD8αα(+), TCRαβ(+)CD8αβ(+) nor TCRγδ(+)CD8αα(+) IELs were capable of suppressing CD4(+) T-cell proliferation. Taken together, our data do not support an immunoregulatory role for CD8α(+) IELs in a mouse model of small and large bowel inflammation.