去甲基化治疗联合HLA半相合T细胞输注诱导白血病特异性CTL生成的机制

Leukemia remains a serious threat to the health of the people. Immune escape is the major cause for leukaemia occurrence and recurrence after treatment.The most important method to cure leukemia is to reverse the immune escape, and to reactivate immune killing to leukemia. Demethylating agents can induce reexpression of some silent genes by inhibiting the DNA methyltransferase. Our previous studies demonstrated that demethylating treatment can induce the expression of CD80 in malignant cells, increasing its immunogenicity.As a result, tumor-specific CTL responses were enhanced to kill tumor cells in vivo. In a registered a clinical trial (NCT01690507), demethylating agent combined with CAG followed by HLA haploidentical T cells infusion was given to elderly patients with acute myeloid leukemia.The remission rate after the first two cycle induction is higher than 80%. In this project, samples collected from clinical trial will be tested to validate the augmentation of immunogenicity of leukemic cells after demethylating treatment at molecules, protein and cell levels. The generation of leukemia specific CTL will be confirmed after in fusion of HLA haploidentical T cells infusion. In addition, a special mouse model will be employed to explore the source of leukemia specific CTL and identify the role of each subgroup of infused cells. This study will provide theoretical basis for establishing a new combination therapy pattern, meanwhile, we might find some clues for optimizing treatment strategies.

白血病是严重威胁人民健康的重大疾病，免疫逃逸是白血病发生与治疗后复发的重要原因，逆转免疫逃逸，重激活免疫杀伤是治愈白血病的关键。去甲基化药物可通过抑制DNA甲基转移酶,激活多种已沉默基因的表达。我们前期的研究发现去甲基化治疗可诱导恶性细胞共刺激分子CD80的表达，增加其免疫原性，强化体内肿瘤特异性CTL的杀伤。据此设计并注册临床试验（NCT01690507），应用去甲基化药物联合CAG化疗后继HLA半相合T细胞输注治疗老年急性髓系白血病，结果两周期诱导缓解率高于80%。本课题拟对临床试验中留取的标本进行检测，从分子、蛋白与细胞水平验证去甲基化治疗后体内白血病细胞免疫原性增加，确认HLA半相合T细胞输注后白血病特异性CTL生成；并利用特异的小鼠模型，复制临床治疗经过，阐明白血病特异性CTL的来源，明确输注各T细胞亚群的作用，为建立新的治疗模式提供理论依据，为进一步优化治疗策略提供思路。

免疫逃逸是白血病发生与治疗后复发的重要原因，逆转免疫逃逸，重激活免疫杀伤是治愈白血病的关键。本研究旨在验证核心假说：表观调控化疗与细胞免疫治疗结合，前者强化白血病细胞免疫原性，后者强化白血病细胞特异性免疫反应。我们应用去甲基化药物地西他滨联合CAG方案化疗后继HLA 半相合T细胞输注治疗急性髓系白血病，对临床试验中留取的标本进行相关检测。成果如下：（1）完成8例患者去甲基化治疗前后骨髓标本real-time PCR 检测一组CTA 的表达（包括HAGE、MAGE、SSX-1、LAGE、PASD1、SPANXB、WT1等），确认治疗后CTA总体呈上调趋势。（2）完成8例患者去甲基化治疗前后骨髓标本流式细胞仪检测，分析白血病细胞表面CD80、CD86、MHC 分子、细胞间粘附分子-1（ICAM-1）表达变化，确认CD80治疗后呈上调趋势。（3）完成6例患者去甲基化治疗前后NY-ESO-1特异性T细胞在CD8+ T 细胞中的比例的检测，发现2例患者治疗后白血病特异性CTL上升。所获结果初步证明核心假说成立，并在具体操作过程中积累了很多经验，对标本冻存及检测过程的具体细节进行了优化，为进一步扩大样本深入研究做好了准备。