DLI逆转T细胞功能耗竭有效治疗移植后B-ALL复发的实验研究

Donor lymphocyte infusion (DLI) is a highly effective immunotherapy for relapsed acute lymphoid leukemia (ALL) after allogeneic hematopoietic stem cell transplant (HSCT), but the precise basis of its effectiveness remains elusive. Recently, increasing evidence suggests that T-cell exhaustion contributes to T cell dysfunction in tumor specific response. Program death-1 (PD-1) signaling and regulatory T cells (Tregs) collaborate to resist the function of adoptively transferred cytotoxic T lymphocytes PD-1/PD-L1interaction and BTLA/HVEM contribute to functional T-cell impairment. DLI can effectively treat post-transplant relapse in B-ALL probably via reversal of T cell exhaustion. In this project, using prospective cohort, we will study the function of T cells and subsets in bone marrow and peripheral blood before and after DLI, as well as the expression varieties of co-stimulating molecules related to T cell exhaustion. We will elucidate the possible mechanism that reversal of T-cell exhaustion after DLI can effectively attack leukemia from the aspect of tumor immunology. We will explore whether blocking the co-stimulating molecules can offer an appealing immunotherapeutic strategy by in vitro functional tests or animal models. The results of this project will provide experimental basis for the mechanism of DLI for prevention and treatment of B-ALL relapse after transplantation and provide further experimental basis for a reasonable application of DLI.

供者淋巴细胞输注（DLI）防治造血干细胞移植（HSCT ）后急性淋巴细胞白血病（ALL）复发疗效肯定，但机制不明。近年来的新观点，T细胞的耗竭使之失去了肿瘤的杀伤能力，PD-1/PD-L1和BTLA/HVEM途径是导致T细胞耗竭的主要信号途径，且血液肿瘤细胞上可高表达PD-L1及HVEM。DLI可能通过逆转T细胞功能耗竭有效治疗HSCT后B细胞ALL复发，其机制涉及IL-2途径、PD-1/PD-L1和BTLA/HVEM信号途径。本课题拟利用前瞻性、临床队列研究探讨DLI通过逆转骨髓微环境中T细胞功能耗竭有效治疗HSCT后B-ALL复发：利用体外实验探讨DLI逆转T细胞功能耗竭的机制；利用小鼠模型阐明PD-1/PD-L1和BTLA/HVEM信号途径在DLI逆转T细胞功能耗竭中的作用，揭示其分子机制。本课题研究的结果将为DLI防治移植后B-ALL复发机制提供实验依据及进一步完善的实验基础。

供者淋巴细胞输注（DLI）防治造血干细胞移植后急性淋巴细胞白血病（ALL）复发疗效肯定，既往研究表明DLI可能通过逆转T细胞功能耗竭有效治疗移植后B-ALL复发，然而具体机制仍不清楚。.本课题收集B-ALL移植后复发+/-患者骨髓验证T细胞耗竭与复发的相关性，收集复发患者DLI后完全缓解及未缓解患者骨髓探讨DLI有效性与T细胞耗竭之间的相关性，研究髓样来源免疫抑制细胞（MDSC）可能影响DLI逆转T细胞功能耗竭有效治疗移植后B-ALL复发的疗效，检测复发+/-患者MDSC细胞百分比及MDSC与NK细胞、T细胞及GFP+Nalm-6共培养对NK、T细胞表型及功能、GFP+Nalm-6增殖影响。流式分选B-ALL复发患者和健康供者骨髓MDSC及T细胞进行RNA-seq，探讨MDSC抑制T细胞功能的具体机制。.移植后复发患者PD-1+Tim+CD8+T及PD-1+Tim+CD4+T细胞增加且CD4+T及CD8+T细胞IFN-γ和TNF-α分泌能力降低；复发患者DLI后持续完全缓解骨髓中PD-1+Tim+CD8+T细胞较DLI前比例降低且IFN-γ分泌能力显著增加，DLI后未达到持续缓解骨髓中PD-1+Tim+CD8+T及PD-1+Tim+CD4+T细胞无明显变化；B-ALL移植后复发患者骨髓中MDSC细胞百分比较非复发患者或健康供者显著升高且骨髓单核细胞与MDSC细胞共培养时GFP+Nalm-6细胞数量显著高于无MDSC共培养细胞数量。B-ALL移植后复发患者骨髓中 T细胞与NK细胞分别与MDSC细胞共培养+/-，MDSC抑制T细胞增殖呈细胞剂量依赖性：随着MDSC比例的增加CD4+和CD8+T细胞百分比显著降低，CD4+和CD8+T细胞中PD-1+细胞百分比显著升高；MDSC显著抑制NK细胞细胞毒（CD107a+、Granzyme B+及IFN-γ+）功能。流式分选B-ALL移植后复发患者和健康供者骨髓MDSC及T细胞进行RNA-seq分析MDSC差异基因及T细胞差异基因显示复发患者MDSC脱颗粒和活性氧（ROS）通路激活，T细胞受体信号通路和氧化磷酸化通路下调。.DLI可能通过逆转T细胞衰竭而有效预防和治疗B-ALL移植后复发。移植后B-ALL复发的机制之一是MDSC促进白血病细胞增殖，抑制T细胞和NK细胞的功能；DLI逆转耗竭T细胞功能和抵抗B-ALL复发的治疗机制