γδTreg诱导免疫耐受性树突状细胞在移植物抗宿主病中的作用及机制研究

发布时间：2018-01-12 21:33

本文关键词：γδTreg诱导免疫耐受性树突状细胞在移植物抗宿主病中的作用及机制研究　出处：《浙江大学》2017年博士论文　论文类型：学位论文

【摘要】：背景:异基因造血干细胞移植(allo-HSCT)是治疗恶性血液系统疾病的有效手段,急性移植物抗宿主病(aGVHD)成为阻碍移植成功的关键因素之一。目前aGVHD的最主要预防和治疗手段为免疫抑制药物,但容易导致移植后感染风险增加;体内外去除T细胞是另一种常用的策略,但也伴随着植入失败、移植后复发率增高等风险。近年来越来越多的免疫细胞亚群被发现在aGVHD调控中起重要作用,过继免疫细胞输注调控aGVHD具有较好的临床应用前景。调节性γδT细胞(γδTreg)是新发现的γδT细胞亚群,该细胞亚群具有与Foxp3+αβTreg细胞相似的生物学特性,发挥负向免疫调控功能。我们前期建立了高效的γδTreg体外诱导扩增富集体系,并发现该群细胞能负向调控小鼠allo-HSCT后aGVHD但不影响GVL效应,但机制尚未完全明确。通过细胞间接触的方式抑制T细胞的增殖活化是其中的机制之一。进一步阐明γδTreg负向调控aGVHD的完整机制将为其临床应用奠定基础。抗原提呈细胞树突状细胞(DC)在aGVHD发生发展的病理生理过程中起重要作用。DC在预处理后活化,活化后的DC处理并递呈宿主抗原给供者来源的T细胞,并进一步通过共刺激分子信号使得T细胞活化和增殖,这是aGVHD起始阶段的关键事件。近年来通过诱导免疫耐受性DC的生成是aGVHD治疗中重要的研究方向。αβTreg与免疫耐受性DC之间有互相诱导生成的作用,两者共同参与维持移植后的免疫耐受。因此,我们推测γδTreg诱导免疫耐受性DC的生成是其发挥负向调控aGVHD的机制之一。在本研究的前两部分,我们首先在体外研究了γδTreg诱导免疫耐受性DC的生成,包括对DC成熟、吞噬、刺激活化T细胞、黏附、迁移等功能影响,并探讨了可能的机制;进一步在人源化小鼠aGVHD模型体内环境中验证了 γδTreg诱导免疫耐受性DC是其发挥负向调控aGVHD作用的机制之一。另一方面,目前关于Foxp3+γδTreg的研究都是采用富集了 γδTreg的γδT混合细胞,但这不能完整反映Foxp3+γδTreg的真实生物学功能。目前尚未发现Foxp3+γδTreg与Foxp3-γδT之间具有差异表达的膜蛋白可供分选纯化,这也大大限制了对Foxp3+γδTreg的生物学功能的进一步研究和相应的临床应用。因此,本研究的第三部分通过单细胞转录组测序技术探索了 Foxp3+γδTreg与Foxp3-γδT的转录组差异,为寻找两者之间差异表达的膜蛋白从而进一步能纯化分离Foxp3+γδTreg提供新的思路。具体研究内容分为3部分:1)γδTreg细胞体外诱导免疫耐受性DC生成的作用及机制研究;2)γδTreg细胞体内诱导免疫耐受性DC的生成发挥负向免疫调控aGVHD的作用;3)γδTreg细胞表面特异性分子标记的筛选和纯化。第一章γδTreg细胞体外诱导免疫耐受性DC生成的作用及机制研究目的:研究γδTreg体外诱导免疫耐受性DC生成的作用,包括对DC成熟、吞噬、迁移、黏附、活化T细胞等功能的负向免疫调控作用,并阐明相应的机制。方法:从健康成年人外周血中分离单个核细胞(PBMCs),分别进行DC和yδTreg的诱导培养。γδTreg与iDC或mDC共培养48h后磁珠分选去除γδTreg,通过流式细胞术检测共刺激分子荧光强度,采用FITC-Dextran的胞吞实验,混合淋巴细胞反应,与内皮细胞的黏附实验,Transwell下趋化因子的迁移实验等检测DC吞噬、黏附、迁移等功能,并在共培养过程中加用Transwell或阻断抗体来进一步阐明γδTreg诱导免疫耐受性DC生成的机制。结果:γδTreg与iDC共培养后能显著抑制LPS促进的iDC成熟,CD80、CD86、CD40、HLA-DR表达水平显著下调,该抑制作用依赖于ICOS/ICOSL的结合,同时还能显著抑制iDC的吞噬功能。yδTreg与mDC共培养后能显著抑制mDC的黏附功能,以及向CXCL12的迁移功能,相应的CXCR4表达下调,而对CCR7无影响,对黏附功能的抑制同样依赖于ICOS/ICOSL的结合。与yδTreg共培养后iDC和mDC刺激活化T细胞的能力均显著降低。结论:γδTreg体外可通过抑制DC成熟、吞噬、迁移、黏附、活化T细胞等功能诱导免疫耐受性DC的作用,γδTreg对DC的负向免疫调控功能部分依赖于ICOS/ICOSL 的结合。第二章 γδTreg细胞体内诱导免疫耐受性DC的生成发挥负向免疫调控aGVHD的作用目的:在第一章中我们在体外已经明确了 γδTreg可诱导免疫耐受性DC的生成,本章通过构建人源化小鼠aGVHD模型验证γδTreg在aGVHD体内环境下对DC的调控作用。方法:采用NOG小鼠辐照后回输PHA活化的hPBMCs与体外培养的iDC构建人源化小鼠aGVHD模型,其中一组同时回输γδTreg细胞,比较两组小鼠的aGVHD症状、生存时间、aGVHD靶器官损伤;移植后第7天检测比较两组小鼠人细胞的嵌合程度,以及脾脏、外周血中DC共刺激分子表达水平、DC的黏附迁移能力。结果:1)小鼠异体移植模型中,aGVHD组的DC共刺激分子水平显著高于无aGVHD组;2)γδTreg对人源化小鼠aGVHD模型具有保护作用,小鼠aGVHD症状减轻,生存时间延长,靶器官损伤小;3)γδTreg对小鼠aGVHD体内环境中外周血、脾脏的人DC免疫调控功能不同。γδTreg显著下调外周血人DC的共刺激分子表达水平,但对脾脏中人DC的共刺激分子抑制作用不明显;相反,γδTreg能显著抑制脾脏中人DC的迁移黏附功能,但对外周血中人DC的黏附迁移功能无显著影响。结论:γδTreg对人源化小鼠aGVHD模型具有保护作用。γδTreg在aGVHD体内环境下可诱导免疫耐受性DC的生成,但对外周血和脾脏DC的调控功能不同。γδTreg诱导免疫耐受性DC是其发挥负向调控aGVHD作用的机制之一。第三章 γδTreg细胞表面特异性分子标记的筛选和纯化目的:探索比较Foxp3+γδTreg与Foxp3-γδT之间的转录组差异,筛选Foxp3+γδTreg表面特异性分子标记。方法:采用流式细胞术检测常见的可能分子标记在Foxp3+γδTreg与Foxp3-γδT两群细胞中的表达;采用单细胞转录组测序在单细胞水平比较Foxp3+γδTreg与Foxp3-γδT的转录组差异,多种策略对比筛选出在两群细胞间差异表达基因,并通过蛋白定位数据库筛选出其中定位于膜的蛋白基因,进一步通过高通量单细胞qPCR验证筛选。结果:常见的可能分子标记CD127、CD45RA、CD49d、CTLA-4、GITR、ICOS、CD27、CD69在Foxp3+γδTreg与Foxp3-γδT两群细胞中均未见显著差异表达,不能用于Foxp3+γδTreg的纯化分选;Foxp3在诱导培养体系中是不稳定表达的,诱导培养早期迅速增高,高峰期维持约15天,40天左右小于5%,但Foxp3+比例小于5%时细胞状态差,难以用于转录组测序比较分析;单细胞水平Foxp3 mRNA转录水平呈现连续分布,单细胞转录组测序对比筛选出278差异表达基因,通过蛋白定位数据库筛出膜蛋白差异表达基因35个,进一步通过高通量单细胞qPCR验证筛选出8个可能在两群细胞间差异表达的分子标记。结论:我们首次通过单细胞转录组测序在单细胞水平揭示了 Foxp3+γδTreg与Foxp3-γδT细胞的转录组差异,并通过筛选验证获得了 8个潜在的Foxp3+γδTreg特异性分子标记。
[Abstract]:Background: allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an effective method for treatment of malignant hematological disorders, acute graft-versus-host disease (aGVHD) has become one of the key factors hindering the successful transplantation. At present the most important means of prevention and treatment of aGVHD as immunosuppressive drugs, but easily lead to increased risk of infection after transplantation in vivo; the removal of T cells is another common strategy, but also accompanied by increased risk of graft failure, relapse rate after transplantation. In recent years, more and more immune cell subsets were found to play an important role in aGVHD regulation, after following the immune cell infusion regulation of aGVHD has good clinical application prospect. Regulatory gamma delta T cells (gamma delta Treg) is a newly discovered gamma delta T cell subsets, the cells have similar biological characteristics with Foxp3+ alpha beta Treg cells, play a negative immune regulation function. We previously established an efficient gamma delta T The enrichment of reg in vitro amplification system, and found that the cells can negatively regulate allo-HSCT in mice after aGVHD but does not affect the GVL effect, but the mechanism is not clear yet. The cell contact inhibits the proliferation of T cell activation is one of the mechanisms. The complete negative regulation of aGVHD will lay the foundation for its clinical application to further clarify the gamma delta Treg. Antigen presenting cells, dendritic cells (DC) play an important role in the activation of.DC after pretreatment in the pathophysiological process of the occurrence and development of aGVHD, DC activation and presenting antigens to host donor derived T cells, and further through the costimulatory signal makes the activation of T cells and this proliferation is a key event in aGVHD initial stages. In recent years through the formation of immune tolerance induced by DC is an important research direction in the treatment of aGVHD. With each other between alpha beta Treg and induce immune tolerance of DC The role of both involved in the maintenance of immune tolerance after transplantation. Therefore, we speculate that the generation of gamma delta Treg induced immune tolerance of DC is its mechanism of negative regulation of aGVHD. In this study, the first two parts, we first studied in vitro gamma delta Treg induced immune tolerance of DC generation DC, including phagocytosis, mature, stimulate the activation of T cells, adhesion, migration effects and other functions, and discuss the possible mechanism; further in the humanized aGVHD mice model in vivo environment to verify the gamma delta Treg induced immune tolerance of DC is the mechanism of regulation of aGVHD play a negative role. On the other hand, the current research on Foxp3+ gamma delta Treg are used in the enrichment of gamma delta T cells mixed gamma delta Treg, but it can not completely reflect the true biological function of gamma delta Treg. Foxp3+ has not yet been found with differential expression between Foxp3+ Treg and Foxp3- gamma delta gamma delta T Membrane protein for separation and purification, which greatly limits the further study on the biological function of Foxp3+ gamma delta Treg and corresponding clinical applications. Therefore, the third part of the study explored the differences of the transcriptome Foxp3+ gamma delta Treg and Foxp3- gamma delta T by single-cell sequencing, for membrane protein expression the differences between the two so as to further separation and purification of Foxp3+ gamma delta Treg can provide new ideas. The concrete research content is divided into 3 parts: 1) the function and mechanism of immune tolerance induced by DC generation of gamma delta Treg cells in vitro; 2) generation of immune tolerance induced by DC gamma delta Treg cells play a negative immune to aGVHD the role of regulation and control; 3) gamma delta Treg cell surface specific molecular marker screening and purification. In the first chapter, the function and mechanism of immune tolerance induced by DC generation of gamma delta Treg cells in vitro Objective: To study the in vitro immune tolerance induced by gamma delta Treg DC formation, including DC maturation, migration, adhesion, phagocytosis, the negative immune regulation of activated T cell function, and clarify the corresponding mechanism. Methods: mononuclear cells were isolated from healthy adult peripheral blood (PBMCs), DC and y were cultured. Gamma delta Treg 8 Treg and iDC or mDC were cultured after 48h multisort removal of gamma delta Treg cells by flow cytometry detection of costimulatory molecule fluorescence intensity, the endocytosis of FITC-Dextran experiments, mixed lymphocyte reaction and endothelial cell adhesion test, migration test. DC phagocytosis, chemokine Transwell adhesion, migration and other functions, and in the process of co cultured with Transwell or blocking antibodies to further clarify the mechanism of gamma delta Treg induced immune tolerance of DC generation. Results: gamma delta Treg co cultured with iDC significantly inhibited LPS promote iDC maturation, CD80, CD86, CD40, HLA-DR expression Flat were significantly reduced, combined with the inhibition of ICOS/ICOSL dependent, while iDC significantly inhibited the adhesion function of the phagocytic function of.Y Delta Treg were co cultured with mDC significantly inhibited mDC, as well as to the transfer function of the CXCL12, the corresponding CXCR4 expression, but had no effect on CCR7, with the inhibition of adhesion function also depends on the ICOS/ICOSL. Y co cultured with iDC and mDC Delta Treg ability to stimulate the activation of T cells decreased significantly. Conclusion: gamma delta Treg in vitro can inhibit DC maturation, migration, adhesion, phagocytosis, activation of T cell function of immune tolerance induced by DC, combined with gamma delta Treg to DC negative the immune regulation function is partially dependent on ICOS/ICOSL. Formation of immune tolerance induced by DC in the second chapter of gamma delta Treg cells play a negative role to the immune regulation of aGVHD Objective: in the first chapter we have identified in vitro gamma delta Treg can induce immune The tolerance of DC generation, this chapter through the construction of humanized mouse aGVHD model validation of gamma delta Treg in aGVHD in vivo environment's effects on DC. Methods: NOG mice after irradiation to cultured hPBMCs and in vitro transport PHA activated iDC build humanized mouse model of aGVHD, one group with transfusion gamma delta Treg cells, compared with two groups of mice aGVHD symptoms, survival time, aGVHD of target organ damage; chimeric degree, seventh days compared with two groups of mice were detected after transplantation, spleen, peripheral blood DC expression level of costimulatory molecules, adhesion and migration ability of DC. Results: 1) mouse xenograft model in group aGVHD, the DC costimulatory molecules was significantly higher than that of non aGVHD group; 2) gamma delta Treg has a protective effect on the humanized mouse model of aGVHD mice, aGVHD symptoms, survival time prolonged, the damage of target organs; 3) gamma delta Treg on mouse aGVHD in peripheral blood, spleen Dirty DC immune regulation function. Gamma delta Treg significantly decreased peripheral blood DC expression level of costimulatory molecules, but on spleen DC costimulatory molecule inhibition is not obvious; on the contrary, migration and adhesion function of gamma delta Treg can significantly inhibit the spleen in DC, but the adhesion and migration of peripheral function in the blood of DC had no significant effect. Conclusion: gamma delta Treg has a protective effect on the humanized mouse model of aGVHD. Generation of gamma delta Treg can induce immune tolerance in vivo DC aGVHD environment, but the regulation function of peripheral blood and spleen of DC. Gamma delta Treg induced immune tolerance is DC play a negative role. One of the mechanisms of regulation of aGVHD to the third chapter screening and purification of gamma delta Treg cell surface specific molecular markers: To explore the differences of transcriptome comparison between Foxp3+ Treg and Foxp3- gamma delta gamma delta T, screening of Foxp3+ gamma delta Treg surface specific molecular markers. Methods: Using May the expression of molecular markers in Foxp3+ gamma delta Treg and Foxp3- gamma delta T two cells in common flow cytometry; single cell transcriptome sequencing comparison at the single cell level Foxp3+ gamma delta Treg and Foxp3- gamma delta T transcriptome differences, comparison of various strategies to screen differentially expressed genes in two cells between, and through the protein localization database screened protein gene located in the membrane, further through high-throughput screening of single cell qPCR test. Results: the possible molecular marker CD127, common CD45RA, CD49d, CTLA-4, GITR, ICOS, CD27, showed no significant difference in the expression of CD69 Foxp3+ and Foxp3- Treg gamma delta gamma delta T two groups of cells, can not be used for the purification of sorting Foxp3+ gamma delta Treg; Foxp3 in the induction is not a stable expression system, inducing early cultivation increased rapidly and the peak last for about 15 days, 40 days less than 5%, but the Foxp3+ ratio is less than 5% cellular The state is poor, which is difficult for the comparative analysis of transcriptome sequencing; single cell level Foxp3 mRNA transcription level showed a continuous distribution, single cell transcriptome sequencing comparison screened 278 differentially expressed genes, the protein localization database screened 35 genes differentially expressed membrane proteins, further through high-throughput single-cell qPCR validation screened 8 possible molecular markers differential expression in two cells. Conclusion: we first through single-cell sequencing revealed differences in transcriptome Foxp3+ gamma delta Treg and Foxp3- gamma delta T cells at the single cell level, and obtained 8 potential Foxp3+ gamma delta Treg specific molecular markers by screening test.

【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R457.7

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