發(fā)布時間：2018-01-12 21:33

  本文關(guān)鍵詞：γδTreg誘導(dǎo)免疫耐受性樹突狀細(xì)胞在移植物抗宿主病中的作用及機(jī)制研究　出處：《浙江大學(xué)》2017年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 調(diào)節(jié)性γδT細(xì)胞 樹突狀細(xì)胞 免疫耐受 移植物抗宿主病 單細(xì)胞轉(zhuǎn)錄組測序

【摘要】：背景:異基因造血干細(xì)胞移植(allo-HSCT)是治療惡性血液系統(tǒng)疾病的有效手段,急性移植物抗宿主病(aGVHD)成為阻礙移植成功的關(guān)鍵因素之一。目前aGVHD的最主要預(yù)防和治療手段為免疫抑制藥物,但容易導(dǎo)致移植后感染風(fēng)險增加;體內(nèi)外去除T細(xì)胞是另一種常用的策略,但也伴隨著植入失敗、移植后復(fù)發(fā)率增高等風(fēng)險。近年來越來越多的免疫細(xì)胞亞群被發(fā)現(xiàn)在aGVHD調(diào)控中起重要作用,過繼免疫細(xì)胞輸注調(diào)控aGVHD具有較好的臨床應(yīng)用前景。調(diào)節(jié)性γδT細(xì)胞(γδTreg)是新發(fā)現(xiàn)的γδT細(xì)胞亞群,該細(xì)胞亞群具有與Foxp3+αβTreg細(xì)胞相似的生物學(xué)特性,發(fā)揮負(fù)向免疫調(diào)控功能。我們前期建立了高效的γδTreg體外誘導(dǎo)擴(kuò)增富集體系,并發(fā)現(xiàn)該群細(xì)胞能負(fù)向調(diào)控小鼠allo-HSCT后aGVHD但不影響GVL效應(yīng),但機(jī)制尚未完全明確。通過細(xì)胞間接觸的方式抑制T細(xì)胞的增殖活化是其中的機(jī)制之一。進(jìn)一步闡明γδTreg負(fù)向調(diào)控aGVHD的完整機(jī)制將為其臨床應(yīng)用奠定基礎(chǔ)�？乖�提呈細(xì)胞樹突狀細(xì)胞(DC)在aGVHD發(fā)生發(fā)展的病理生理過程中起重要作用。DC在預(yù)處理后活化,活化后的DC處理并遞呈宿主抗原給供者來源的T細(xì)胞,并進(jìn)一步通過共刺激分子信號使得T細(xì)胞活化和增殖,這是aGVHD起始階段的關(guān)鍵事件。近年來通過誘導(dǎo)免疫耐受性DC的生成是aGVHD治療中重要的研究方向。αβTreg與免疫耐受性DC之間有互相誘導(dǎo)生成的作用,兩者共同參與維持移植后的免疫耐受。因此,我們推測γδTreg誘導(dǎo)免疫耐受性DC的生成是其發(fā)揮負(fù)向調(diào)控aGVHD的機(jī)制之一。在本研究的前兩部分,我們首先在體外研究了γδTreg誘導(dǎo)免疫耐受性DC的生成,包括對DC成熟、吞噬、刺激活化T細(xì)胞、黏附、遷移等功能影響,并探討了可能的機(jī)制;進(jìn)一步在人源化小鼠aGVHD模型體內(nèi)環(huán)境中驗(yàn)證了 γδTreg誘導(dǎo)免疫耐受性DC是其發(fā)揮負(fù)向調(diào)控aGVHD作用的機(jī)制之一。另一方面,目前關(guān)于Foxp3+γδTreg的研究都是采用富集了 γδTreg的γδT混合細(xì)胞,但這不能完整反映Foxp3+γδTreg的真實(shí)生物學(xué)功能。目前尚未發(fā)現(xiàn)Foxp3+γδTreg與Foxp3-γδT之間具有差異表達(dá)的膜蛋白可供分選純化,這也大大限制了對Foxp3+γδTreg的生物學(xué)功能的進(jìn)一步研究和相應(yīng)的臨床應(yīng)用。因此,本研究的第三部分通過單細(xì)胞轉(zhuǎn)錄組測序技術(shù)探索了 Foxp3+γδTreg與Foxp3-γδT的轉(zhuǎn)錄組差異,為尋找兩者之間差異表達(dá)的膜蛋白從而進(jìn)一步能純化分離Foxp3+γδTreg提供新的思路。具體研究內(nèi)容分為3部分:1)γδTreg細(xì)胞體外誘導(dǎo)免疫耐受性DC生成的作用及機(jī)制研究;2)γδTreg細(xì)胞體內(nèi)誘導(dǎo)免疫耐受性DC的生成發(fā)揮負(fù)向免疫調(diào)控aGVHD的作用;3)γδTreg細(xì)胞表面特異性分子標(biāo)記的篩選和純化。第一章γδTreg細(xì)胞體外誘導(dǎo)免疫耐受性DC生成的作用及機(jī)制研究目的:研究γδTreg體外誘導(dǎo)免疫耐受性DC生成的作用,包括對DC成熟、吞噬、遷移、黏附、活化T細(xì)胞等功能的負(fù)向免疫調(diào)控作用,并闡明相應(yīng)的機(jī)制。方法:從健康成年人外周血中分離單個核細(xì)胞(PBMCs),分別進(jìn)行DC和yδTreg的誘導(dǎo)培養(yǎng)。γδTreg與iDC或mDC共培養(yǎng)48h后磁珠分選去除γδTreg,通過流式細(xì)胞術(shù)檢測共刺激分子熒光強(qiáng)度,采用FITC-Dextran的胞吞實(shí)驗(yàn),混合淋巴細(xì)胞反應(yīng),與內(nèi)皮細(xì)胞的黏附實(shí)驗(yàn),Transwell下趨化因子的遷移實(shí)驗(yàn)等檢測DC吞噬、黏附、遷移等功能,并在共培養(yǎng)過程中加用Transwell或阻斷抗體來進(jìn)一步闡明γδTreg誘導(dǎo)免疫耐受性DC生成的機(jī)制。結(jié)果:γδTreg與iDC共培養(yǎng)后能顯著抑制LPS促進(jìn)的iDC成熟,CD80、CD86、CD40、HLA-DR表達(dá)水平顯著下調(diào),該抑制作用依賴于ICOS/ICOSL的結(jié)合,同時還能顯著抑制iDC的吞噬功能。yδTreg與mDC共培養(yǎng)后能顯著抑制mDC的黏附功能,以及向CXCL12的遷移功能,相應(yīng)的CXCR4表達(dá)下調(diào),而對CCR7無影響,對黏附功能的抑制同樣依賴于ICOS/ICOSL的結(jié)合。與yδTreg共培養(yǎng)后iDC和mDC刺激活化T細(xì)胞的能力均顯著降低。結(jié)論:γδTreg體外可通過抑制DC成熟、吞噬、遷移、黏附、活化T細(xì)胞等功能誘導(dǎo)免疫耐受性DC的作用,γδTreg對DC的負(fù)向免疫調(diào)控功能部分依賴于ICOS/ICOSL 的結(jié)合。第二章 γδTreg細(xì)胞體內(nèi)誘導(dǎo)免疫耐受性DC的生成發(fā)揮負(fù)向免疫調(diào)控aGVHD的作用目的:在第一章中我們在體外已經(jīng)明確了 γδTreg可誘導(dǎo)免疫耐受性DC的生成,本章通過構(gòu)建人源化小鼠aGVHD模型驗(yàn)證γδTreg在aGVHD體內(nèi)環(huán)境下對DC的調(diào)控作用。方法:采用NOG小鼠輻照后回輸PHA活化的hPBMCs與體外培養(yǎng)的iDC構(gòu)建人源化小鼠aGVHD模型,其中一組同時回輸γδTreg細(xì)胞,比較兩組小鼠的aGVHD癥狀、生存時間、aGVHD靶器官損傷;移植后第7天檢測比較兩組小鼠人細(xì)胞的嵌合程度,以及脾臟、外周血中DC共刺激分子表達(dá)水平、DC的黏附遷移能力。結(jié)果:1)小鼠異體移植模型中,aGVHD組的DC共刺激分子水平顯著高于無aGVHD組;2)γδTreg對人源化小鼠aGVHD模型具有保護(hù)作用,小鼠aGVHD癥狀減輕,生存時間延長,靶器官損傷小;3)γδTreg對小鼠aGVHD體內(nèi)環(huán)境中外周血、脾臟的人DC免疫調(diào)控功能不同。γδTreg顯著下調(diào)外周血人DC的共刺激分子表達(dá)水平,但對脾臟中人DC的共刺激分子抑制作用不明顯;相反,γδTreg能顯著抑制脾臟中人DC的遷移黏附功能,但對外周血中人DC的黏附遷移功能無顯著影響。結(jié)論:γδTreg對人源化小鼠aGVHD模型具有保護(hù)作用。γδTreg在aGVHD體內(nèi)環(huán)境下可誘導(dǎo)免疫耐受性DC的生成,但對外周血和脾臟DC的調(diào)控功能不同。γδTreg誘導(dǎo)免疫耐受性DC是其發(fā)揮負(fù)向調(diào)控aGVHD作用的機(jī)制之一。第三章 γδTreg細(xì)胞表面特異性分子標(biāo)記的篩選和純化目的:探索比較Foxp3+γδTreg與Foxp3-γδT之間的轉(zhuǎn)錄組差異,篩選Foxp3+γδTreg表面特異性分子標(biāo)記。方法:采用流式細(xì)胞術(shù)檢測常見的可能分子標(biāo)記在Foxp3+γδTreg與Foxp3-γδT兩群細(xì)胞中的表達(dá);采用單細(xì)胞轉(zhuǎn)錄組測序在單細(xì)胞水平比較Foxp3+γδTreg與Foxp3-γδT的轉(zhuǎn)錄組差異,多種策略對比篩選出在兩群細(xì)胞間差異表達(dá)基因,并通過蛋白定位數(shù)據(jù)庫篩選出其中定位于膜的蛋白基因,進(jìn)一步通過高通量單細(xì)胞qPCR驗(yàn)證篩選。結(jié)果:常見的可能分子標(biāo)記CD127、CD45RA、CD49d、CTLA-4、GITR、ICOS、CD27、CD69在Foxp3+γδTreg與Foxp3-γδT兩群細(xì)胞中均未見顯著差異表達(dá),不能用于Foxp3+γδTreg的純化分選;Foxp3在誘導(dǎo)培養(yǎng)體系中是不穩(wěn)定表達(dá)的,誘導(dǎo)培養(yǎng)早期迅速增高,高峰期維持約15天,40天左右小于5%,但Foxp3+比例小于5%時細(xì)胞狀態(tài)差,難以用于轉(zhuǎn)錄組測序比較分析;單細(xì)胞水平Foxp3 mRNA轉(zhuǎn)錄水平呈現(xiàn)連續(xù)分布,單細(xì)胞轉(zhuǎn)錄組測序?qū)Ρ群Y選出278差異表達(dá)基因,通過蛋白定位數(shù)據(jù)庫篩出膜蛋白差異表達(dá)基因35個,進(jìn)一步通過高通量單細(xì)胞qPCR驗(yàn)證篩選出8個可能在兩群細(xì)胞間差異表達(dá)的分子標(biāo)記。結(jié)論:我們首次通過單細(xì)胞轉(zhuǎn)錄組測序在單細(xì)胞水平揭示了 Foxp3+γδTreg與Foxp3-γδT細(xì)胞的轉(zhuǎn)錄組差異,并通過篩選驗(yàn)證獲得了 8個潛在的Foxp3+γδTreg特異性分子標(biāo)記。
[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.

【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R457.7

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6 周幽心,張學(xué)光,鮑耀東,周岱,朱鳳清,陳忠平;去腦垂體大鼠誘導(dǎo)免疫耐受性的實(shí)驗(yàn)研究[J];上海免疫學(xué)雜志;1998年03期

7 陳政良，，謝佩蓉，鄭萍;多聚Clq誘導(dǎo)免疫細(xì)胞的Ca~（2＋）動員[J];上海免疫學(xué)雜志;1996年04期

8 孫早喜;孫誠誼;王福生;;樹突狀細(xì)胞疫苗誘導(dǎo)免疫效應(yīng)細(xì)胞對PC3的凋亡和抑制[J];海南醫(yī)學(xué)院學(xué)報;2006年03期

9 陳紅梅;白雪帆;任廣立;;重組質(zhì)粒pcDNA3.1S2S/Fc在小鼠誘導(dǎo)免疫反應(yīng)的觀察[J];實(shí)用肝臟病雜志;2009年03期

10 沈佰華,周洪,謝晉,陳福祥,李寧麗,周光炎;混合表皮淋巴細(xì)胞培養(yǎng)體系中IL-10表達(dá)在誘導(dǎo)免疫抑制中的作用[J];基礎(chǔ)醫(yī)學(xué)與臨床;2001年02期

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