【作者】 吕凌；

【导师】 张峰；

【作者基本信息】 南京医科大学 ， 普通外科， 2010， 博士

【摘要】 目的:了解TGF-β家族成员BMP-2/BMP-4是否能够替代TGF-β,或者促进TGF-β体外诱导调节性T细胞（Treg）的能力,及其可能的机制。方法:利用免疫磁珠分选出不同基因型小鼠脾脏的Na?ve CD4+细胞,经过TCR刺激,加入IL-2 （20 U/ml）,按照不同的培养要求加TGF-β（2 ng/ml）, BMP-2 （10 ng/ml）, BMP-4 （10 ng/ml）。在体外诱导3天后收集细胞,用细胞内染色检测Foxp3的变化。部分培养条件中加入Noggin,JNK和ERK的阻断剂。Western Blot检测BMP对Smad1磷酸化的影响。另外,将BMP体内注射,观察外周血,淋巴和脾脏内CD4⁺CD25⁺Foxp3⁺T细胞占CD4⁺T细胞的比例。分离出脾脏的CD4⁺CD25⁺T细胞通过混合淋巴细胞培养分析其抑制功能。结果:体外TCR刺激的情况下,BMP-2,BMP-4本身并不能够诱导产生Foxp3⁺T细胞。Noggin阻断BMP受体,但并不影响TGF-β体外诱导Treg的能力。BMP-4能够增加TGF-β体外诱导Treg的能力。体内注射BMP不能增加Foxp3⁺细胞的比例,但能够促进TsA增加Foxp3的能力。BMP本身不能够扩增自然Treg,另外BMP联合TGF-β诱导后的T细胞更能抵抗凋亡。BMP-2,BMP-4除了增加Smad1的磷酸化,主要能够增加ERK,JNK磷酸化来促进Foxp3⁺ T细胞的诱导。结论:BMP-2,BMP-4单独并不能够诱导Treg;无论在体内还是体外,BMP都能促进TGF-β诱导Treg的能力;BMP这种能力主要依赖于MAKP的ERK和JN K信号。目的:探讨TGF-β诱导调节性T细胞和Th17生成中TGF-β下游信号机制,了解这两群细胞体外诱导分化中TGF-β作用的不同信号机制。方法:利用Auto MACS来分选出不同基因型小鼠脾脏内的Na?ve CD4⁺T细胞。按照调节性T细胞和Th17细胞不同的诱导条件进行体外诱导。根据实验要求分别加入MAKP, P38,JNK或ERK阻断剂。体内实验通过向小鼠腹腔注射TsA,由FACS分析不同基因型小鼠体内Foxp3⁺T细胞的变化。利用不同基因型小鼠来免疫诱导EAE小鼠模型,分离出脾脏淋巴细胞分析Th17含量的变化,一组EAE模型在免疫诱导后注射P38阻断剂,在相同时间点来分析Th17的含量的变化。结果:在体外实验中,敲除Smad2/Smad3基因后,TGF-β仍然能够诱导出Treg,但是能力稍弱。当加入MAKP,JNK或ERK阻断剂后能够明显削弱TGF-β体外诱导Treg的能力。体内实验中,缺乏Smad3信号时atRA仍然能够促进iTreg的生成和抑制Th17的生成。在EAE模型中,P38抑制剂能明显降低EAE的发病程度。结论: TGF-β信号是体外TGF-β诱导调节性T细胞和Th17细胞生成所必须的信号通路;Smad信号在TGF-β诱导调节性T细胞中只有部分作用;TGF-β的MAKP信号通路在TGF-β诱导调节性T细胞和Th17细胞生成中也起着关键性的作用。TGF-β下游的MAKP中,ERK和JNK信号通路参与了Treg的分化,而JNK和P38参与了Th17的分化。方法:体外分离出人外周血PBMC中的Naive CD4⁺ T细胞,在体外经过anti-CD3CD28-beads的TCR刺激后,伴有IL-2,和/或TGF-β,和/或Rapamycin（RAPA）诱导7天后,检测细胞表面CTLA-4和细胞内FOXP3等的表达。通过混合淋巴细胞培养分析诱导后细胞体外抑制活性。将体外诱导的细胞和PBMC同时注射到免疫缺陷鼠体内观察其对异种移植物抗宿主反应的抑制情况。同时分析人细胞在小鼠组织内的浸润情况。结果:RAPA能够诱导TCR刺激后的Na?ve CD4⁺细胞成为FOXP3⁺T细胞,该功能在加入ALK5阻断剂后消失。RAPA联合TGF-β能够诱导出具有免疫抑制功能的CD4RAPA,该细胞表达膜表面TGF-β,其体外抑制功能在加入ALK5阻断剂后丧失。将PBMC（20×10⁶）注射给RAG2^-/-γc^-/-小鼠会诱发急性异种移植物抗宿主病,同时给予CD4_RAPA则能够明显延长小鼠的存活时间,减少人细胞的组织浸润。结论:RAPA能够增加CD4⁺ T细胞FOXP3的表达,依赖于TGF-β信号;RAPA能够联合TGF-β在体外诱导出类似于nTreg表型的FOXP3⁺ T细胞。这群细胞高表达趋化因子的受体,该细胞本身在TCR刺激时并不在增殖。RAPA联合TGF-β诱导的Treg表达膜表面的TGF-β,其抑制活性依赖于mTGF-β。将RAPA联合TGF-β诱导的Treg输注给RAG2^-/-γc^-/-小鼠,能够明显减轻异种移植物抗宿主反应,延长小鼠的存活。更多还原

【Abstract】 AIM: To clarify the possibility and mechanism of TGF-βsuperfamily members BMPs affect the development of Foxp3⁺ Treg cells in vitro and in vivo.METHODS: Na?ve CD4⁺ T cells were separated from the spleen cells from wild type and transgenic mice. These cells were underwent TCR stimulation, with IL-2 （20 U/ml） for 3days. TGF-β（2 ng/ml）, BMP-2 （10 ng/ml）, or BMP-4 （10 ng/ml） was added in some culture. Cell was intracellular stained with Foxp3. Some conditions were added Noggin, P38 inhibitor, JNK inhibitor, or ERK inhibitor. The effect of phosphorylation Smad by BMP was analyzed by Weston Blot. In vivo, BMP was i.p. injected into the mouse. The percentage of CD4⁺CD25⁺Foxp3⁺T cells in the blood, lymph nodes and spleen was analyzed by FACS at day 12. Spleen CD4⁺CD25⁺ T cells was separated and assayed for assessment of the mixed lymphocyte reaction RESULT: BMP-2 and BMP-4, typical representatives of the BMPs, are unable to induce non-regulatory T cells to become Foxp3⁺ regulatory T cells. Neutralization studies with Noggin have revealed that BMP-2/4 and the BMP receptor signaling pathway is not required for TGF-βto induce naive CD4⁺CD25- cells to express Foxp3. However, BMP-2/4 and TGF-βhave a synergistic effect on the induction of Foxp3⁺ regulatory T cells. BMP-2/4 affects non-Smad signaling molecules including phosphorylated ERK and JNK, which could subsequently promote the differentiation of Foxp3⁺ regulatory T cells induced by TGF-β.CONCLUTION: TGF-βis a key signaling factor for Foxp3⁺ regulatory T cell development. BMP-2 and BMP-4 significantly increased the ability of TGF-βto promote the generation of Foxp3⁺ iTreg cells, and this synergistic effect was also dependent upon TGF-βreceptor signaling, as well as ERK and/or JNK MAPK pathways.AIM: To observation that Smad and non-Smad signaling pathways differentially affect the Th17 and Foxp3⁺ Treg cell differentiation.METHODS: Na?ve CD4⁺ T cells were separated from the spleen cells from wild type and transgenic mice. Under induction of Treg, cells were underwent TCR stimulation, with IL-2 （20 U/ml） for 3days. TGF-β（2 ng/ml）, P38 inhibitor, JNK inhibitor, or ERK inhibitor, or atRA （10 nM） was added in some culture. The percentage of Foxp3⁺ in T cells was analyzed by FACS following the injection of TsA. T cells were isolated from spleen, LN and blood in EAE mice at day 18 after immunization, intracellular IL-17 expression was stained and analyzed by FACS.RESULT: TGF-βcan induce regulatory cells from Smad2 or Smad gene knockout mice. The MAKP JNK, and ERK inhibitor can down regulate the ability of Treg induction by TGF-β. AtRA also can favorate the Treg and inhibitor the Th17 cells in Smad3 knockout mice.CONCLUTION: Neither Smad2 nor Smad3 deficiency abrogates TGF-β-dependent iTreg induction by a deacetylase inhibitor Trichostatin A （TsA） in vivo although loss of Smad2 or Smad3 partially reduces iTreg induction in vitro. Similarly, Smad2 and Smad3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis （EAE）. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTregs, while the p38 pathway predominately modulates Th17 and EAE induction.AIM: The possibility to induce human regulatory cells with stable suppressor function both in vitro and in vivo.METHODS: Na?ve CD4⁺ T cells were separated from health donor PBMC. These cells were stimulated with anti-CD3CD28-beads. IL-2, TGF-β, and RAPA were added to some culture. The cells were harvested and stained the phenotypes of nature regulatory cells. Membran bound TGF-βwas stained after restimulation for 72 hours. The suppress function of different condition cells was analyzed by the dilution of CFSE of T cells. Human peripheral blood mononuclear cells （huPBMCs） with or whithout different condition cells were injected into RAG2^-/-γc^-/- mice. The weight and engraft human T cells of these mice were monitored.RESULT: Combine RAPA with TGF-βnot only enhanced FOXP3 expression by CD4⁺ cells, but also appeared more resistant to apoptosis compared to others. The combination of RAPA and TGF-βenabled CD4⁺ cells to express a phenotype and trafficking receptors similar to natural Tregs, and no significant cytotoxicity was observed. CD4RAPA were anergic and had potent in vitro suppressive activity. When transfer human peripheral blood mononuclear cells （huPBMCs） with CD4_RAPA into RAG2^-/-γc^-/- mice, CD4RAPA can decrease human cell engraftment and extend survival.CONCLUTION: Stimulation of human CD4+T cells in the presence of RAPA results in a highly increased suppressor function is TGF-βdependent, these cells also has stable in vivo suppressor function as compared with that of CD4+ T cells stimulated in the absence of the drug.更多还原