CD4+CD25+Foxp3+调节T细胞介导的急性髓系白血病免疫逃逸机制研究

Immune escape contributes to the pathogenesis of acute myeloid leukemia (AML). CD4+CD25+Foxp3+ regulatory T cells (Tregs), a type of immunosuppressive T cells, have been found higher in solid tumor. Recently, increased Tregs in AML has also been shown. We hypothesized that frequencies of marrow and peripheral blood Tregs might be greater in patients with de novo AML than in control subjects, Tregs in AML might have enhanced homing potiential to bone marrow and increased suppression of the immune cells, which contribute to the immune escape of AML. We also hypothesized that regulatory B cells (Bregs) might be the upstream factor of the abnormalities of the Tregs in AML. To test these hypotheses, we would first detect the frequencies of marrow and blood Tregs in patients with AML by flow cytometry, then examine the migratory capacity of Tregs towards stromal-derived factor-1α and bone marrow fluid and explore the expression levels of CXCR4 and CXCR7 on Tregs from AML patients. Moreover, we would design coculture assays and criss-cross experiments by Tregs and several lymphoid/immune cells, such as CD4+, CD8+ T cells, natural killer cells, dendritic cells in vitro to explore whether Tregs from AML over suppress the function of immune cells. We will perform coculture of Bregs and CD4+CD25- T cells to address the efficiency of Bregs-mediated conversion/expansion for Tregs. Our study would be of great helpful to further understand the immune escape in the pathogenesis of AML with an intention to probe novel targets of immunotherapy for AML.

免疫逃逸是急性髓系白血病（AML）的发病机制之一。近来研究发现，与实体瘤一样，AML中具有免疫抑制作用的CD4+CD25+Foxp3+调节T细胞(Tregs)也增高。我们的假说是AML患者Tregs数量增多，从外周血向骨髓趋化的能力及对免疫细胞的抑制功能增强，参与AML的免疫逃逸；而可以扩增Tregs的调节B细胞（Bregs）在AML中增多，是导致CD4+ T细胞向Tregs转化增多的上游因素。为此，拟研究AML中：① Tregs数量异常；② Tregs生物学特性，迁移至骨髓的趋化功能，明确其趋化异常机制；③ Tregs与CD4+和CD8+ T细胞、自然杀伤细胞、树突状细胞共培养比较AML与正常Tregs对免疫细胞的抑制能力，交叉培养比较AML和正常免疫细胞抵抗Tregs抑制的能力；④ Bregs促进Tregs转化、扩增的能力。对AML免疫逃逸的深入研究将为免疫治疗靶点的选择提供理论依据。

调节性T细胞（regulatory T cells，Tregs）是一种免疫应答负调节细胞，主要通过细胞-细胞接触依赖和可溶性细胞因子介导的方式抑制许多免疫细胞的活性、增殖及功能。肿瘤细胞可以通过Tregs逃避免疫识别和免疫监视，导致肿瘤免疫耐受。急性髓系白血病（Acute Myeloid Leukemia，AML）是白血病的一个主要类型，其异常主要表现在增殖失控，凋亡障碍和分化受阻。近年研究发现，Tregs 除了在实体肿瘤中增高外，在AML患者中也发现增高，本研究进一步研究AML患者是否存在Tregs的功能异常和可能的上游机制。.目的：研究AML患者Tregs 的数量、趋化及其对免疫细胞的抑制功能，并探讨导致AML患者Tregs异常可能的上游因素，阐明其在AML免疫逃逸机制中的作用，为提高AML临床疗效寻找新的治疗方向。.方法：①流式细胞术检测初诊AML患者和正常对照骨髓液单个核细胞中Tregs比例及Tregs表面PD1、CTLA4、GITR的表达；②transwell实验检测Tregs迁移至骨髓的趋化功能；③流式细胞术检测Tregs细胞表面CXCR4、CXCR7表达，ELISA方法检测骨髓和血浆中SDF-1α水平；④Tregs分别与CD4+T细胞和CD8+T细胞共培养、交叉共培养比较AML与正常Tregs对免疫细胞的抑制能力；⑤Bregs与CD4+CD25-T细胞共培养、交叉共培养比较AML与正常Bregs诱导CD4+CD25-T细胞向Tregs转化的能力。.结果：①初诊急性髓系白血病患者外周血和骨髓液中Tregs比例增多；②AML患者外周血Tregs趋化至骨髓的能力增强；③AML患者外周血Tregs趋化至骨髓的能力增强源于CXCR4表达升高；④AML患者Tregs抑制CD4+ T细胞增殖促进CD4+ T细胞凋亡的能力增强并影响其INF-γ分泌；⑤AML患者Bregs诱导CD4+CD25-T 细胞向CD4+CD25+Foxp3+ Tregs 转化。.结论：AML患者Tregs数量增多，迁移至骨髓能力增强，并影响CD4+T细胞增殖、凋亡、细胞因子分泌等功能，而AML患者Bregs促进CD4+ CD25- T细胞向CD4+ CD25+Foxp3+ Tregs 的转化，从而放大Tregs的免疫抑制效应，为AML基于Tregs的免疫抑制治疗提供理论依据。