NKG2D、DNAM-1信号通路结合基因筛选在双阴性T细胞治疗UCBT后复发AML患者的作用机制研究

The primary disease relapse after transplantation is still the main cause of death after transplantation. Donor lymphocyte infusion (DLI) is an effective method in prevention and treatment of relapse after transplantation. Since umbilical cord blood transplantation (UCBT) cannot use the traditional DLI treatment due to the lack of donor, once the UCBT patients relapse, the treatment is very limited. Our preliminary results showed that peripheral blood from normal person or acute myeloid leukemia (AML) patients could both culture double negative T cells (DNT). DNT cells not only had the selective anti-leukemia effect, but also do not cause xenogeneic GVHD. The same DNT cells had different killing effect to different leukemia cells. Clinical study found that patients had different sensitivity to DNT treatment . NKG2D and DNAM-1 were highly expressed on DNT cells, and blocking of these interactions significantly, but not completely, inhibited DNT-mediated cytotoxicity. Based on the clinic research on DNT treatment, this project will select samples from AML patients relapse after UCBT which are highly susceptible or resistant to DNT cells-mediated killing, detect the level of NKG2D,DNAM-1, related ligands and signal regulation pathway to reveal the mechanisms that are important for effective DNT cells therapy; Further send for RNA-seq/micro array to identify genes that are associated with highly susceptible or resistant to DNT cells, look for the biomarkers for selection of patients responding to DNT therapy;. The completion of this project will help to make up the defect of no traditional DLI treatment for relapse after UCBT, provide new ideas and ways for the treatment of relapse after UCBT.

复发是恶性血液病患者造血干细胞移植后死亡主要原因之一，供者淋巴细胞输注（DLI）是预防或治疗移植后复发有效方法。非血缘脐血移植（UCBT）因缺乏供体而不能采用传统DLI治疗，一旦复发，治疗方法非常有限。我们前期结果表明，正常人外周血能培养双阴性T（DNT）细胞，这群细胞不仅具有选择性抗白血病效应，且不引起异源性GVHD；同一DNT细胞对不同白血病细胞杀伤效应不同；临床研究中发现不同患者对DNT治疗敏感性不同；DNT细胞高表达NKG2D和DNAM-1，应用相应抗体可部分降低其杀伤效应。本项目立足于临床研究，筛选出对DNT高度敏感/耐受的UCBT后复发AML患者样本，检测NKG2D、DNAM-1相关信号通路，揭示DNT细胞的作用机制；进一步高通量RNA测序技术筛选对DNT高度敏感/耐受的相关基因，寻找可作为选择DNT有效治疗的生物学标志。项目的完成弥补了UCBT后复发无传统DLI治疗的缺陷。

复发是恶性血液病患者造血干细胞移植后死亡主要原因之一，供者淋巴细胞输注（DLI）是预防或治疗移植后复发的有效方法。非血缘脐血移植（UCBT）因缺乏供体而不能采用传统DLI治疗，一旦复发，治疗方法非常有限。我们前期结果表明，正常人外周血能培养双阴性T（DNT）细胞，这群细胞不仅具有选择性抗白血病效应，且不引起异源性GVHD；同一DNT细胞对不同白血病细胞杀伤效应不同；临床研究中发现不同患者对DNT治疗敏感性不同。本研究通过加拿大多伦多大学Dr. Li Zhang及其团队研发的DNT细胞培养专利技术，成功分离并体外扩增DNT细胞，纯度＞90%，满足临床治疗剂量；通过对DNT敏感及耐受标本的单细胞测序，首次发现CD96可能为导致对DNT治疗不敏感的重要分子，并构建CD96过表达慢病毒载体，成功转染THP-1细胞株，为进一步探究DNT有效杀伤白血病细胞提供基础。本研究通过体外培养DNT细胞验证其抗白血病效应以筛选供者，并首次发起并完成了第三方健康供者DNT细胞治疗allo-HSCT后复发AML患者的first-in-human I期临床试验（ChiCTR1900022795），通过临床研究进一步验证了DNT细胞在体内应用的安全性和有效性，为寻找新型有效防治AML患者移植后复发提供新的思路和途径。此外，通过对362例健康人群的外周血免疫细胞亚群尤其是DNT细胞亚群的分布进行检测和分析，确定了合适的参考范围，并基于此标准，回顾性分析UCBT后免疫重建的规律，为UCBT后动态免疫监测及相关并发症的抢先治疗提供方向，具有重要的临床应用价值。