Mst1和WASp共同调控调节性T细胞外周稳态及抑制功能

目的以Mst1 KO、WASp KO、Mst1-WASp DKO和野生型模型小鼠为研究对象,探究Mst1和WASp共同作用对调节性T细胞(Treg)发育和功能影响及作用机制。方法使用流式细胞仪检测胸腺和脾脏中T细胞各亚群特别是Treg细胞的比例、数量及其相关分子的表达,并通过构建DKO骨髓嵌合小鼠验证表型变化是否为自发性。结果与野生型小鼠相比,DKO小鼠胸腺的T细胞各亚群比例无明显变化,而脾脏中T细胞各亚群细胞数均降低,除活化T细胞和Treg的比例升高外,其他各T细胞亚群比例均降低。DKO小鼠脾脏Treg中的NRP-1表达减少,CTLA-4的表达增加。骨髓嵌合动物模型中Treg的检测结果与上述一致。结论 Mst1与WASp以细胞固有方式共同影响Treg细胞外周稳态和功能,其机制可能涉及NRP-1和CTLA-4。     

BTLA对调节性T细胞发育及功能的影响

目的研究B和T淋巴细胞弱化因子(B and T lymphocyte attenuator,BTLA)对调节性T细胞(regulatory T cell,Treg)发育和功能的影响。方法构建在Treg中特异性敲除BTLA基因的小鼠模型,使用流式细胞术检测该模型小鼠中枢及外周各淋巴器官中T细胞外周环境稳态、T细胞的活化及细胞因子的产生、Treg的发育及其功能相关分子的表达,并通过流式细胞术分选Treg及Naive细胞体外培养,探究Treg细胞在体外的抑制作用。结果相比于野生型小鼠,Treg中选择性敲除BTLA的小鼠胸腺、脾脏、淋巴结以及肠系膜淋巴结的T细胞外周环境稳态、T细胞的活化及细胞因子的产生、Treg的发育及其功能相关分子的表达均未发现明显差异;体外的Treg细胞抑制功能试验发现Treg的抑制功能较野生型亦无明显差异。结论在Treg中特异性敲除BTLA对于Treg的发育和功能表达并无明显影响。     

转录因子IRF8抑制Th17细胞分化并减轻T细胞免疫介导的小鼠结肠炎

 目的：转录因子干扰素调节因子（interferon regulatory factor, IRF）家族与Th17的发育密切相关,近年来发现Th17细胞在炎症性肠病的发病中发挥重要作用,本研究探讨IRF8对Th17发育及T细胞转染免疫介导的小鼠实验性肠炎的影响。方法：(1)采用流式细胞术分选野生型（WT）或IRF8全基因敲除(IRF8 -/-)小鼠脾脏和淋巴结的naive CD4 +T细胞(CD4 + CD62L +CD44 low),在Th1、Th2或Th17极化的条件下培养,采用流式细胞术检测Th1、Th2和Th17的比例。(2)建立实验性肠炎模型：采用免疫磁珠法分选WT或IRF8 -/-小鼠中的脾脏和淋巴结中CD4 +CD25 +Treg,WT小鼠的CD4 + CD45RB hi T细胞单独或者分别联合WT或IRF8 -/-小鼠的CD4 +CD25 +Treg腹腔注射给RAG1 -/-小鼠;WT或IRF8 -/-小鼠的naive CD4 + CD45RB hi T细胞腹腔注射给RAG1 -/-小鼠;观察上述小鼠每周体重的变化,第5周时处死小鼠,进行结肠炎病理评分和肠系膜淋巴结T淋巴细胞亚群检测。结果：(1)IRF8 -/-较WT的naive CD4 +T细胞在极化条件下向Th17细胞分化更明显(P<001),而对Th1和Th2细胞的分化无影响(P>0.05)。(2)CD4 + CD45RB hi T细胞转染给RAG1 -/-小鼠,IRF8 -/-较WT供体鼠引起的RAG1 -/-小鼠体重显著降低(P<0.05),结肠炎评分显著增高（P<0.05）,且肠系膜淋巴结中IL-17 +CD4 +细胞比例明显增高(P<0.01),而 IFN-γ +CD4 + 和 Foxp3 +CD4 +细胞比例无影响(P>0.05);IRF8 -/-小鼠的CD4 +CD25 +Treg对WT小鼠CD4 + CD45RB hi T细胞转染给RAG1 -/-小鼠诱发的免疫介导的结肠炎显示出正常的免疫抑制作用。结论：转录因子IRF8基因敲除促进CD4 +T细胞向Th17细胞分化,促进转染naive CD4 +T细胞诱导的实验性结肠炎的发生,IRF8基因敲除小鼠Treg细胞免疫抑制功能正常。     

Treg细胞参与LIGHT缺失小鼠对利什曼原虫感染的敏感性

目的探讨LIGHT对利什曼原虫(L.major)感染模型中CD4+T细胞亚群免疫应答的影响。方法采用LIGHT KO和野生型(WT)小鼠,经小鼠脚掌注射L.major建立感染模型。从感染第2周至24周连续观察被感染脚掌的病损程度,并测定感染24周小鼠体内寄生虫载量。其次,取感染第7天小鼠引流淋巴结(d LN)细胞,运用3H-Td R掺入方法检测T细胞体外刺激的增殖情况,并且利用ELSIA检测体外刺激后d LN细胞IL-12和IFN-γ的表达水平。最后,通过流式细胞术检测感染第7天小鼠脾脏和d LN中Treg水平。结果 LIGHT KO小鼠被感染脚掌和d LN的寄生虫载量均显著高于WT对照小鼠。感染早期LIGHTKO小鼠d LN中T细胞增殖水平,以及IL-12和IFN-γ的表达水平均显著低于WT对照小鼠。感染早期LIGHT KO小鼠中脾脏和d LN中的Treg细胞百分率相对WT小鼠均显著升高。结论 LIGHT可能通过抑制Treg细胞的产生,促进Th1细胞的分化,从而增强小鼠抗L.major感染的能力。     

颗粒蛋白前体通过CTLA4调控调节性T细胞的发育和功能

目的探讨颗粒蛋白前体(PGRN)对调节性T细胞发育和功能的影响及分子机制。方法在野生型和PGRN敲除小鼠模型中,采用流式细胞术检测小鼠中枢及外周各淋巴器官中T细胞各亚群的比例及调节性T细胞比例及其功能相关分子的表达;采用RT-PCR检测调节性T细胞中CTLA4的表达;最后采用调节性T细胞体外抑制试验验证其功能变化。结果与野生型小鼠相比,PGRN敲除小鼠脾脏、胸腺和淋巴结中调节性T细胞的比例均升高,特别发现在脾脏调节性T细胞中CTLA4在基因和蛋白表达水平均显著升高。体外功能试验发现PGRN敲除小鼠的脾脏调节性T细胞的抑制功能也较野生型增强。结论 PGRN在调节性T细胞的发育和功能中扮演重要角色,其机制可能涉及CTLA4。     

Balb/c小鼠PD-1敲除后抑制疟原虫生长及其机制初探

目的探讨PD-1 KO(knock out)对约氏疟原虫P.y17XL增殖的影响及其可能的作用机制。方法感染约氏疟原虫P.y17XL后,比较WT和PD-1 KO小鼠的存活率及其体内的原虫血症;然后,流式细胞技术检测约氏疟原虫P.y17XL感染能否诱导WT小鼠巨噬细胞、DC、中性粒细胞和活化的CD4+T细胞表面PD-1表达;最后比较感染约氏疟原虫P.y17XL的WT和PD-1 KO小鼠的脾脏CD4+T细胞功能以及血清中的抗体水平。结果与WT小鼠相比,PD-1 KO小鼠的疟原虫增殖明显受到抑制,而且存活率也明显高于感染的WT小鼠;约氏疟原虫P.y17XL感染能诱导WT小鼠巨噬细胞、DC、中性粒细胞和CD4+T细胞表面PD-1的表达;虽然WT和PD-1 KO小鼠的脾脏CD4+T细胞功能没有显著差别,但是PD-1 KO小鼠的血清总IgG水平在感染后第6、8天显著高于WT小鼠。结论 PD-1 KO后能提高感染小鼠的存活率,增强其清除红内期疟原虫的能力;其机制可能与PD-1 KO小鼠血清中抗体水平升高有一定的关系。     

TNF-α缺失减少抗李斯特菌CD8~+T细胞应答并增加效应记忆细胞形成

目的探讨TNF-α在李斯特菌(Listeria monocytogenes,LM)感染模型中对CD8~+T细胞应答的影响。方法采用尾静脉注射重组OVA的李斯特菌株感染TNF-α基因敲除(TNF-αKO)小鼠和野生型(WT)对照小鼠建立系统性感染模型。运用流式细胞术检测不同时相点TNF-αKO小鼠和WT小鼠中内源性和外源性特异性CD8+T细胞应答反应情况。采用体内细胞杀伤实验和检测细菌滴度的方法评估TNF-αKO小鼠和WT小鼠感染后记忆期CD8~+T细胞的效应功能。接着分析记忆期和增殖期CD8~+T细胞的KLRG-1和CD127等分子的表达变化。结果与WT对照小鼠相比,TNF-αKO小鼠中CD8~+T细胞应答减少,记忆细胞增多;TNF-αKO小鼠中记忆CD8~+T细胞的效应功能正常;TNF-αKO小鼠记忆CD8~+T细胞中含有更多比例的KLRG-1+CD127-CD62L-效应记忆细胞,而第7天TNF-αKO小鼠特异性CD8~+T细胞的KLRG-1和CD127的表达与对照组无明显差别。结论TNF-αKO小鼠初次应答降低,形成更多记忆细胞,且效应记忆细胞增多。其机制与早期效应CD8~+T细胞分化无关,可能是TNF-αKO小鼠更多的KLRG-1+CD127-细胞在应答收缩期存活所致。     

TNFSF14对STZ诱发Ⅰ型糖尿病的影响及其机制初探

目的:以链脲佐菌素(STZ)腹腔注射小鼠,建立Ⅰ型糖尿病动物模型,探讨TNFSF14在Ⅰ型糖尿病病理发生的作用。方法:以55 mg/kg的剂量,腹腔注射STZ于野生型(WT)和TNFSF14缺陷(TNFSF14 KO)小鼠体内,连续注射5 d,并在最后一次注射的当天开始测量血糖,根据情况每周测量2～3次;在指定的时间点处死小鼠,收集胰腺组织、脾脏和胰腺淋巴结。HE染色观察胰腺组织的病理情况;流式细胞术检测脾脏和胰腺淋巴结的淋巴细胞中T细胞亚群(CD3~+T细胞、CD4~+T细胞以及CD4~+FoxP3~+调节性T细胞)的频率。结果:连续5 d注射STZ后,WT组小鼠的血糖随即急剧上升,23 d后所有小鼠均超过了正常水平,而TNFSF14 KO小鼠的血糖值相对稳定,23 d后仍有80%的小鼠维持在正常血糖水平。HE染色显示,与WT小鼠相比,TNFSF14 KO小鼠的胰岛中淋巴细胞浸润明显减少,胰岛相对完整。流式细胞术结果表明,与WT组小鼠相比,TNFSF14 KO小鼠脾脏和胰腺淋巴结淋巴细胞中的CD3~+T细胞百分比都明显下调(脾脏:P0.05;胰腺淋巴结:P0.01),而CD4~+T细胞百分比差异不显著(P0.05),但CD4~+FoxP3~+调节性T细胞百分比显著升高(脾脏:P0.01;胰腺淋巴结:P0.05)。结论:TNFSF14在STZ诱导的Ⅰ型糖尿病中发挥重要作用,其可能通过下调CD4~+FoxP3~+调节性T细胞的分化来介导Ⅰ型糖尿病的发生。     

microRNA-7对小鼠肠系膜淋巴结αβT淋巴细胞组成的影响

目的:研究miR-7(MicroRNA-7,miRNA-7)对小鼠肠系膜淋巴结αβT淋巴细胞比例的影响并初步探讨其意义。方法:利用miR-7基因敲减(Knockdown,KD)小鼠模型,观察其肠系膜淋巴结大小、重量和淋巴结细胞总数,以及HE染色的病理学变化;流式细胞术(FACS)检测淋巴结中αβT淋巴细胞亚群(CD3+T细胞、CD4+T细胞和CD8+T细胞)以及CD4+T细胞和CD8+T细胞的活化相关分子CD69、CD62L和功能相关因子IFN-γ表达的情况。结果:与野生型(Wild type,WT)小鼠相比,miR-7KD小鼠肠系膜淋巴结大小、重量指数和淋巴结细胞总数均显著增高(P0.05),且HE染色显示MLNs有病理性改变;肠系膜淋巴结中αβCD3+T细胞比例增加显著,其中以CD4+T细胞比例变化最为明显(P0.05),而CD19+B淋巴细胞比例明显下调(P0.05),但各亚群细胞总数均明显增加(P0.05);最后,CD4+T细胞和CD8+T细胞中的CD62L+细胞的比例均显著下调(P0.05),而CD69+细胞和IFN-γ+细胞比例均显著上调(P0.05)。结论:miR-7敲减后可显著影响肠系膜淋巴结中αβT淋巴细胞的组成比例,提示其对肠系膜淋巴结中淋巴细胞的组成和功能具有重要的调控作用。     

敲除CD226通过调节小鼠脾脏和肠道免疫细胞比例缓解小鼠的慢性束缚应激引起的抑郁样行为

目的 探讨CD226在小鼠慢性束缚应激(CRS)诱导的抑郁样行为中发挥的免疫调控作用及其可能机制。方法 选取4～6周龄野生型(WT)雄性C57/BL6J和同品系CD226基因敲除(CD226KO)小鼠建立CRS模型，通过强迫游泳测试、蔗糖偏好测试等行为学检测方法对小鼠进行应激抑郁评分，利用流式细胞术分析脾脏、Peyer淋巴结及肠上皮内淋巴细胞亚群的差异。结果 与WT CRS组相比，CD226KO CRS组强迫游泳测试静止时间显著降低，蔗糖偏好率显著上升；脾脏CD4⁺T细胞和CD8⁺ T细胞之比显著降低；小肠上皮内淋巴细胞中T细胞受体αβ(TCRαβ)和TCRαβ CD8αβ细胞亚群比例显著升高。结论敲除CD226能够缓解小鼠CRS模型诱导的抑郁样行为，影响CRS状态下小鼠脾脏及肠道免疫细胞比例，改善小鼠应激状态下的整体免疫状态。     

The susceptibility of Aire(-/-) mice to experimental myasthenia gravis involves alterations in regulatory T cells

The autoimmune regulator (Aire) is involved in the prevention of autoimmunity by promoting thymic expression of tissue restricted antigens which leads to elimination of self-reactive T cells. We found that Aire knockout (KO) mice as well as mouse strains that are susceptible to experimental autoimmune myasthenia gravis (EAMG) have lower thymic expression of acetylcholine receptor (AChR- the main autoantigen in MG), compared to wild type (WT) mice and EAMG-resistant mouse strains, respectively. We demonstrated that Aire KO mice have a significant and reproducible lower frequency of CD4+Foxp3+ cells and a higher expression of Th17 markers in their thymus, compared to wild type (WT) mice. These findings led us to expect that Aire KO mice would display increased susceptibility to EAMG. Surprisingly, when EAMG was induced in young (2 month-old) mice, EAMG was milder in Aire KO than in WT mice for several weeks until the age of about 5 months. However, when EAMG was induced in relatively aged (6 month-old) mice, Aire KO mice presented higher disease severity than WT controls. This age-related change in susceptibility to EAMG correlated with an elevated proportion of Treg cells in the spleens of young but not old KO, compared to WT mice, suggesting a role for peripheral Treg cells in the course of disease. Our observations point to a possible link between Aire and Treg cells and suggest an involvement for both in the pathogenesis of myasthenia.     

Impaired regulatory T cell function in germ-free mice

Regulatory T cells (Treg) are crucial for the maintenance of tolerance to auto-antigens and harmless exogenous antigens. Here, we studied the role of the commensal microbiota for the development and function of Treg. CD4+CD25+ T cells were obtained from peripheral lymph nodes (PLN) and mesenteric lymph nodes (MLN) of germ-free (GF) and conventional (conv) NMRI mice and tested for phenotype and functional suppressive capacity. CD4+CD25+ T cells from GF mice showed a lower relative gene expression of fork head box p3 gene (Foxp3) and were not as potent suppressors in vitro as CD4+CD25+ T cells from conv animals. Intracellular staining for Foxp3 and CTLA-4 revealed proportional and regional differences in putative Treg subsets between conv and GF mice. Fewer of the CD4+CD25+ T cells in GF MLN expressed Foxp3 and CTLA-4, while the expression of these markers was similar amongst the CD4+CD25+ T cells in PLN of conv and GF mice. The largest difference between conv and GF Treg was observed in the liver draining celiac lymph node, where GF mice had fewer putative Treg as compared to conv mice. We propose that the presence of a microbial flora favors the development of a fully functional Treg population.     

Lymphocyte subpopulations and function in chronic murine toxoplasmosis.

A long-lasting, chronic infection with Toxoplasma gondii was established in C57Bl/6J mice as a model system of slow parasite infection. We quantified all nucleated cells and Thy-1+ and Thy-1- cell subpopulations in thymus, spleen and peripheral and mesenteric lymph nodes throughout the first 8 months of life of mice infected at 8 weeks of age. We found a physiological pattern of change with age in the lymphocyte subpopulations examined; the pattern was distinctive for each lymphoid organ. These normal patterns were altered in infected mice from the beginning of the observation period. The most prominent findings were (a) a relatively early atrophy of the thymus and (b) no increase in Thy-1+ lymphocyte numbers in either spleen or lymph nodes during the first 3 months post-infection, followed by decreasing numbers afterwards. Other findings were a rapid increase in the number of Thy-1- cells in the thymus before the onset of generalized atrophy; accumulation of erythrocytes in the spleen and splenomegaly during the first 3 months post-infection, despite a steady decrease in the number of nucleated cells; and an increase in Thy-1- cells in the peripheral but not in the mesenteric nodes, thereby diluting the Thy-1+ lymphocyte subpopulation.     

The effect of peripheral immunization with Mls-1a on the emigration of antigen-specific cells from the thymus.

Mature T cells found in the lymph nodes and spleen have the capacity to become activated and to proliferate in response to foreign antigens. The response of the thymus to such immunization is less well understood. We have examined one aspect of the thymic response by determining the effect of peripheral immunization upon cell emigration from the thymus. BALB/c (Mls-1b) mice were injected with spleen cells from DBA/2 (Mls-1a) mice, and V beta 6+ (Mls-1a-reactive) thymic emigrants were identified 3-30 days after immunization. Neither the rate of total cell migration from the thymus nor the proportion of V beta 6+ cells was altered, even though the immunizing spleen cells elicited an immune response in the draining (parathymic) lymph nodes. The same immunogen caused deletion of V beta 6+ cells in both the thymus and lymph nodes after intraperitoneal injection into the neonate. The inability of DBA/2 splenocytes to modify the development of adult thymocytes after intrathymic injection of the cells precluded the lack of entry into the thymus as the reason for the lack of any observed effect in the adult. Our results, therefore, indicate that the development of adult thymocytes is not modified by immunization, and suggest that the differing thymic response of mice injected as adults or neonates is related to changes in the intrathymic antigen presentation capacity associated with age.     

Peripheral tolerance through clonal deletion of mature CD4-CD8+ T cells.

Transgenic mice bearing the alpha beta transgenes encoding a defined T cell receptor specific for the male (H-Y) antigen presented by the H-2Db class I MHC molecule were used to study mechanisms of peripheral tolerance. Female transgenic mice produce large numbers of functionally homogeneous CD8+ male antigen-reactive T cells in the thymus that subsequently accumulate in the peripheral lymphoid organs. We have used three experimental approaches to show that male reactive CD8+ T cells can be eliminated from peripheral lymphoid organs after exposure to male antigen. (i) In female transgenic mice that were neonatally tolerized with male spleen cells, male reactive CD8+ T cells continued to be produced in large numbers in the thymus but were virtually absent in the lymph nodes. (ii) Injection of thymocytes from female transgenic mice into female mice neonatally tolerized with the male antigen, or into normal male mice, led to the specific elimination of male-reactive CD8+ T cells in the lymph nodes. (iii) Four days after male lymphoid cells were injected intravenously into female transgenic mice, male antigen-reactive CD8+ T cells recovered from the lymph nodes of recipient mice were highly apoptotic when compared to CD4+ (non-male reactive) T cells. These data indicate that tolerance to extrathymic antigen can be achieved through elimination of mature T cells in the peripheral lymphoid organs.     

Vitamin D receptor is required to control gastrointestinal immunity in IL-10 knockout mice

The vitamin D receptor (VDR) is a nuclear receptor expressed in a number of different cells of the immune system. This study was performed to determine the effect of VDR deficiency on immune function and inflammation of the gastrointestinal tract in a model of inflammatory bowel disease, namely interleukin-10 (IL-10) knockout mice. IL-10 knockout mice were generated which either could or could not respond to vitamin D (double IL-10/VDR knockout; DKO). The distribution and function of lymphocytes in both the primary and secondary lymphoid organs were compared and determined as a function of the severity of intestinal inflammation. DKO mice had normal thymic development and peripheral T-cell numbers at 3 weeks of age, but a week after intestinal disease was detected the thymus was dysplastic with a reduction in cellularity. The atrophy was coupled with increased apoptosis. The spleen weight of DKO mice increased as a result of the accumulation of red blood cells; however, there was a 50% reduction in the numbers of T and B cells. Conversely, the mesenteric lymph nodes were enlarged and contained increased numbers of lymphocytes. The T cells from DKO mice were of a memory phenotype and were hyporesponsive to T-cell receptor stimulation. Colitis in the DKO mice was associated with local and high expression of IL-2, interferon-gamma, IL-1beta, tumour necrosis factor-alpha and IL-12. The primary and secondary lymphoid organs in DKO mice are profoundly altered as a consequence of the fulminating inflammation in the gastrointestinal tract. VDR expression is required for the T cells and other immune cells to control inflammation in the IL-10 KO mice.     

Development and activation of regulatory T cells in the human fetus

There is an increasing amount of knowledge on the functional properties of regulatory T cells (Treg) in the adult immune system, but data on the generation and function of these cells during human embryonic development are scarce. In this study, we show that in the fetal thymus, double-positive cells initiate expression of CD25, GITR, CTLA4 and CD122 during their transition from the CD27- to the CD27+ stage. Moreover, CD4+CD25+ fetal thymocytes already have the potential to suppress proliferation of CD25- cells. After leaving the thymus, FoxP3+CD4+CD25+ Treg enter the fetal lymph nodes and spleen, where they acquire a primed/memory phenotype. A model is proposed for the development of human fetal Treg that encompasses two sequential maturation steps: initiation of a regulatory phenotype and suppressive activity in the thymus; and subsequent activation within the peripheral lymphoid organs. Upon activation, FoxP3+CD4+CD25+ Treg suppress potentially deleterious responses by autoreactive lymphocytes and maintain homeostasis within the developing fetus.     

Influence of dietary protein restriction on immune competence. II. Effect on lymphoid tissue.

Weanling mice fed a 4 percent diet showed a generalized loss of lymphoid tissue which was greater than the loss of total body weight. This effect was greatest in the thymus greater than spleen greater than mesenteric lymph node. Cell loss was most pronounced during the 1st week on diets, then remained at stable levels for 3 weeks and showed a gradual rise thereafter. The effect was shown to be mediated partly by a cessation of growth in lymphoid organs due to the low protein intake and, secondly, an adrenal corticosteroid induced lympholysis which actually reduced cell numbers. Recirculating T cells and resident B cells were amongst the least affected cells whereas stem cells, non-migratory T cells and other reticuloendothelial cells were most depressed in numbers. At no stage was the germinal centre forming capacity of the mesenteric node lost although cell recruitment to antigenically stimulated nodes was diminished. During nutritional repletion the spleen, thymus and mesenteric node all showed different and characteristic regrowth. The spleen was most active initially and rapidly reconstituted haemopoietic cells and B cells. This was followed by the thymus which showed a delayed reinitiation of its normal growth kinetics which had been interrupted by the diet. The evidence suggested that full rehabilitation of the immune apparatus took place even after 2 months of nutritional deprivation.