【作者】 张晓龙；

【导师】 魏海明；

【作者基本信息】 中国科学技术大学 ， 细胞生物学， 2013， 博士

【摘要】 调节性T细胞是不同于Thl, Th2和Th17的具有调节功能的T细胞群体,因其具有免疫抑制作用,在自身免疫耐受、获得性免疫耐受以及维持体内的免疫平衡状态中发挥着重要作用。调节性T细胞的种类很多,包括Tr1细胞,natural killer T cells,一小群CD8+T细胞以及CD4+CD25+Foxp3+T细胞,其中CD4+CD25+Foxp3+T细胞是最重要的一群调节性T细胞,常被叫做Treg细胞,这群细胞的缺失或者功能缺陷将会导致机体自发多种自身免疫性疾病。Treg细胞在体内根据产生部位不同可以分为胸腺中发育成熟输送到外周的natural Treg细胞(nTreg)和在外周组织中特定的环境下经过抗原刺激产生的adaptive Treg细胞(iTreg),两者在体内都起着重要的免疫调节作用。对于nTreg细胞在体内的起源和产生目前已经有了比较清楚的认识,认为nTreg细胞是在胸腺内产生的,然而对于iTreg细胞在体内的产生和来源却还没有完全明确的定义。早期的研究表明,在体内很多的情况下胸腺外周能够诱导形成iTreg细胞,并且证明这群iTreg细胞来源于外周的CD4+CD25-T细胞活化后产生的效应T细胞,还发现在体外通过anti-CD3抗体刺激CD4+CD25-T细胞的时候加入TGF-(3能够诱导形成iTreg细胞。但是CD4+CD25-T细胞中的初始型CD4T细胞或者记忆性CD4T细胞这两群细胞在经刺激活化后都可以形成效应T细胞,所以对于形成iTreg细胞的效应T细胞是来源于两者中的哪一群细胞并不是十分明确。最近有些小鼠和人的研究工作发现分离纯化得到的初始型CD4T细胞在体外很容易通过TGF-β的诱导方法得到iTreg细胞,而分离纯化得到的记忆性CD4T细胞在体外却很难诱导形成iTreg细胞。因此,目前研究者都普遍认为,在体内,iTreg细胞来源于初始型CD4T细胞经刺激活化后形成的效应T细胞这一观点。但是也存在和此观点相矛盾的一些证据,有研究者通过人体内同位素追踪实验表明iTreg细胞可能来源于外周的记忆性CD4T细胞,还有研究者发现小鼠的Th2型记忆性T细胞能够在体外通过改善后的刺激诱导方法诱导形成iTreg细胞,所以并不能排除体内产生的iTreg细胞来源于由记忆性T细胞形成的效应T细胞。由于iTreg细胞在机体针对外来抗原和体内肿瘤来源的抗原的耐受形成当中发挥着重要的作用,清楚的了解它们的来源和形成将为Treg细胞的临床应用提供更多有价值的信息。本研究主要目的在于研究形成iTreg细胞的前体T细胞的来源,通过流式细胞仪对HBV病人的外周血标本进行检测,利用流式细胞分选仪分选得到不同的CD4T细胞亚群进行体外刺激诱导iTreg细胞实验,发现初始型CD4T细胞可能并不是体内形成iTreg细胞的T细胞来源,进一步对细胞的表型分析找出了特定的一群能够诱导形成iTreg细胞的记忆性CD4T细胞,然后利用CFSE染色的方法检测了诱导形成的iTreg细胞是否具有抑制功能,并且通过亚硫酸氢盐修饰后测序法(BSP)比较了由记忆性CD4T细胞和初始型CD4T细胞诱导形成的iTreg细胞的TSDR区域的去甲基化情况。通过上述的实验方法我们获得了以下结果以及结论：1. CD45RO区分出体内和体外形成的iTreg细胞的不同首先分选得到的初始型CD4T细胞在体外通过anti-CD3和anti-CD28抗体刺激时加入TGF-β能够诱导形成高比例的Foxp3+iTreg细胞,但是Foxp3+细胞表面却几乎不表达CD45RO记忆性T细胞的标志。接下来检测分析了38例感染HBV的孕妇和22例未感染孕妇的外周血标本,发现HBV感染孕妇外周血中CD4+Foxp3+的Treg细胞比例较正常人显著增加,而进一步统计分析发现只有CD45RO+CD4+Foxp3+Treg细胞的比例升高,CD45RO-CD4+Foxp3+的Treg细胞比例基本没有变化。综上所述,初始型CD4T细胞体外诱导形成的iTreg细胞不表达CD45RO记忆性细胞标志,体内病毒持续性感染形成的iTreg细胞却基本都表达CD45RO,表明体内形成的iTreg细胞可能不是直接来源于初始型CD4T细胞。2. CD4+CD62L+CCR7+中枢记忆性T细胞能够诱导形成iTreg细胞分选得到的人的记忆性CD4T细胞体外刺激诱导虽然很难得到高比例的iTreg细胞,但是却发现有一小部分记忆性CD4T细胞能诱导出Foxp3的表达,通过分选得到记忆性CD4T细胞中的不同亚群细胞,分别体外进行诱导iTreg细胞实验,发现具有CD62L+CCR7+中枢记忆性CD4T细胞表型的细胞亚群能够在体外诱导形成高比例Foxp3+iTreg细胞,而其他表型的记忆性CD4T细胞则很难诱导形成Foxp3+iTreg细胞。上述结果说明记忆性CD4T细胞虽然总体诱导形成iTreg细胞的能力很弱,但是其中的CD4+CD62L+CCR7+中枢记忆性T细胞却具有较强的诱导形成iTreg细胞的能力。3. CD4+CD62L+CCR7+中枢记忆性T细胞诱导得到的iTreg细胞在体外具有抑制功能将CD4+CD62L+CCR7+中枢记忆性T细胞和初始型CD4T细胞体外诱导得到iTreg细胞分别和CFSE标记的CD4+CD25-T细胞体外混合刺激培养,观察CFSE标记细胞的增殖情况,结果显示两种细胞诱导得到的iTreg细胞在体外具有相同的抑制CD4+CD25-T细胞增殖的能力,而CD62L-CCR7-记忆性CD4T细胞经过诱导处理后的细胞则不具有这种抑制功能。以上结果说明CD4+CD62L+CCR7+中枢记忆性T细胞可以诱导形成有功能的iTreg细胞。4. CD4+CD62L+CCR7+中枢记忆性T细胞诱导得到的iTreg细胞在TSDR区域的去甲基化程度提高分别用亚硫酸氢盐修饰后测序法分析初始型CD4T细胞和CD4+CD62L+CCR7+中枢记忆性T细胞体外诱导形成的iTreg细胞,以及分选得到的Treg细胞中FOXP3基因TSDR区域中CpG岛位点的去甲基化情况,结果发现CD4+CD62L+CCR7+中枢记忆性CD4T细胞体外诱导得到iTreg细胞,其TSDR区域去甲基化程度明显比初始型CD4T细胞诱导形成的iTreg细胞高,但还是比直接分选得到Treg细胞的TSDR区域去甲基化程度低。结果表明CD62L+CCR7+中枢记忆性CD4T细胞诱导得到的iTreg细胞比初始型CD4T细胞诱导得到iTreg细胞具有更稳定的性能。综上所述,我们首次发现CD4+CD62L+CCR7+中枢记忆性T细胞亚群具有分化形成iTreg细胞的能力,说明体内的iTregs可能是由CD4+CD62L+CCR7+中枢记忆性T细胞活化后产生,我们的研究为了解体内iTreg细胞来源提供了新的信息,对Treg细胞的临床治疗应用有价值。更多还原

【Abstract】 Regulatory T cells is a population of T cells with regulatory function that differ-ent from Thl, Th2and Th17cells, they play an important role in self-tolerance and acquired tolerance as well as immunological homeostasis. There are multiple types of immune regulatory T cells, including Trl cells, natural killer T cells, a subset of CD8+T cells, and CD4+CD25+Foxp3+cells. CD4+CD25+Foxp3+cells (commonly known as Tregs) are the predominant regulatory cells. Deficiency or dysfunction of Tregs leads to the development of various autoimmune diseases. Tregs can be divided into natural arising Treg cells (nTregs) generated in the thymus and adaptive Treg cells (iTregs) generated in the peripheral through antigen stimulation, both of which are implicated in immune regulation. It is well known that nTregs are derived from the thymus, but the exact origin of iTregs remains incompletely defined.Previous studies suggested that iTregs may be derived from various conditions outside the thymus. It is clear that iTregs are converted from T effector cells in the periphery. And further in vitro studies found that iTregs can be efficiently differen-tiated from purified CD4+CD25-T cells stimulation with TGF-β.Naive CD4+T cells and memory CD4+T cells both can formation effect T cells after stimulation, thus which effect T cells will differentiation into iTregs is unknow. Recently, other studies reported that of CD4+CD25-T cells, only the naive CD4+T cells (Tn) but not the memory CD4+T cells (Tm) were able to differentiate into iTregs in vitro both in mouse and human models. So it is generally accepted that iTregs are converted from T effector cells that originate from activated naive CD4+CD25-T cells in vivo. However, there is controversial evidence regarding this view. One report showed that human iTregs were derived from peripheral memory CD4+Foxp3-T cells in vivo by isotopes tracking experiments and another found that mouse Th2memory T cells could also be efficiently converted into Foxp3+Tregs with a differentiation protocol in vitro. Thus, the possibility that iTregs are converted from T effector cells that are derived from activated memory CD4+CD25-T cells in vivo cannot be excluded.Because iTregs play essential roles in developing immune tolerance to foreign antigens and tumor derived neo-antigens, a better understanding of their origin and differentiation would provide important information regarding their therapeutic poten-tial. Our study wanted to define the origin of effect T cells that could differentiation into Tregs in vivo. Through flow cytometer analyzing the peripheral blood of HBV infected patients and inducing iTregs from flow cytometer sorted naive CD4+T cells, we found that naive CD4+T cells might not be the source of iTregs de-rived from effect T cells. Through phenotype analysis we found a subset of memoy CD4+T cells which could be induced into iTregs, and determined the suppressive function of these cells by CFSE labeling assay. And we analyzed the TSDR sequence of iTregs that derived from memory CD4+T cells and naive CD4+T cells by bisulfite sequencing PCR. The results and conclusions are shown as follow:1. CD45RO distinguishes the different between iTregs in vivo an in vitroPurified naive CD4+T cells from human PBMCs were activation in vitro by an-ti-CD3/anti-CD28plus TGF-β to generate Foxp3+iTregs, these iTregs were rarely co-expressed memory cell phenotype CD45RO with Foxp3. PBMCs from HBV-infected pregnant (n=38) and healthy pregnant (n=22) women were analyzed by flow cytometry, we found that the proportion of CD4+Foxp3+Tregs was significantly higher in HBV-infected pregnant women than the healthy controls, further statistic analysis found that only the CD45RO+CD4+Foxp3+Treg proportion was increased, and the CD45RO-CD4+Foxp3+Treg proportion had no change.The above results collectively showed that iTregs generated in vitro had different surface CD45RO expression from in vivo chronic virus infection-induced iTregs, which suggested that iTregs induced in vivo might not be directly derived from naive CD4+T cells.2. CD4+CD62L+CCR7+central memory T cells can be induced to differentiate into iTregsPurified memory CD4+T cells are difficult to be induced into iTregs in vitro. However, a small undefined population of memory CD4+T cells could be induced to express Foxp3. Through analysis the capability of different subsets of memory CD4+t cells to be induced to differentiate into iTregs, we found that CD62L+CCR7+central memory T cells can be induced to differentiate into iTregs efficaciously.The above results suggested that although overall memory CD4+T cells were re-sistant to differentiate into iTregs, a subset of memory CD4+T cells defined as CD62L+CCR7+central memory T cells had the capability to be induced to differen-tiate into iTregs. 3. Induced iTregs from CD4+CD62L+CCR7+memory T cells have potent suppressive function in vitroInduced iTregs from CD62L+CCR7+memory CD4+T cells or naive CD4+T cells were stimulated with CFSE labeled CD4+CD25" T cells, the proliferation of CFSE labeled cells showed that both iTregs had suppressive function, and cells de-rived from CD62L-CCR7-memory CD4+T cells through induction process did not exhibit any suppressive function.These results showed that CD62L+CCR7+central memory CD4+T cells could be induced to differentiate into functional iTregs.4. iTregs derived from CD4+CD62L+CCR7+central memory T cells showed signifi-cantly more FOXP3TSDR demethylationBisulfite sequencing PCR analysis of the status of the CpG sites in FOXP3TSDR sequence among iTregs derived from CD62L+CCR7+central memory CD4+T cells and naive CD4+T cells and purified Tregs. The results showed that CD62L+CCR7+central memory CD4+T cells derived iTregs displayed more deme-thylation of TSDR sequence than naive CD4+T cells derived iTregs, but less than pu-rified Tregs. These results indicated that CD62L+Tcm cells derived iTregs might have a more stable Treg phenotype than those from naive CD4+T cells.In conclusion, we proposed for the first time that CD62L+CCR7+central memory CD4+T cells can be induced into iTregs, and iTregs might be converted from T effec-tor cells that are derived from activated CD62L+CCR7+central memory CD4+T cells in vivo. Our study provided novel information on the origin of human iTregs and might have important clinical value in adoptive Tregs therapy.更多还原