Human cytomegalovirus-mediated modulation of leukemia and its immune control by donor T cell alloreactivity to HLA-DPB1 after hematopoietic stem cell transplantation

人巨细胞病毒（HCMV）是一种具有重要临床意义的典型β-疱疹病毒，特别是对于免疫抑制患者，如接受异基因造血干细胞移植（HSCT）作为白血病治愈性治疗的个体。在这种情况下，HCMV再活化已被证明会影响免疫重建和移植后发生移植物抗宿主病（GvHD）或复发的风险。在拟定项目期间，将通过两名PI的协同努力探索解释这些临床观察结果的三种新的、互不排斥的假设，这两名PI将分别在HSCT和巨细胞病毒的免疫生物学方面贡献其互补的专业知识：1）HCMV特异性CD 4 + T细胞受体（TCR）交叉识别HLA-DPB 1同种抗原（主要是PI Fleischhauer）的异源免疫。HCMV特异性和HLA-DPB 1同种异体反应性CD 4 + T细胞培养物将从UK-Essen的HLA-DPB 1不匹配无关HSCT供体中独立获得。其TCR CDR 3序列将通过下一代测序确定。在两种培养物中鉴定的具有相同CDR 3序列的TCR指示异源免疫，并且将在移植后具有或不具有急性GvHD或白血病复发的患者中纵向追踪。2)调节同种异体HLA-DPB 1免疫原性的HCMV编码蛋白（PI Fleischhauer和Trilling）。将覆盖典型HCMV蛋白的可用表达载体文库单独引入单个HLA-DP表达模型细胞系中，以测试基因特异性对HLA-DPB 1特异性CD 4 + T细胞的同种异体识别的影响。鉴定的候选基因将表征其对HLA-DPB 1表达和异位表达后加工以及HCMV感染背景下的作用。3)HCMV诱导的白血病细胞增殖受损的细胞和病毒决定因素（主要是PI Trilling）。为了鉴定和表征先前观察到的灭活HCMV颗粒对急性髓样白血病（AML）细胞引起的抗增殖作用的介质，将进行一系列生化和免疫学测定以及抑制研究，以检验HCMV与AML细胞接触后引发自杀性先天免疫应答的假设，并鉴定负责因素。总之，对T细胞同种异体反应性、HCMV和白血病的免疫生物学的新见解将在细胞治疗中具有潜在的转化意义。

Human cytomegalovirus (HCMV) is a prototypical β-herpesvirus with important clinical relevance, especially for immunosuppressed patients like individuals receiving allogeneic hematopoietic stem cell transplantation (HSCT) as curative therapy for leukemia. In this setting, HCMV reactivation has been shown to impact both immune reconstitution and the risk of developing graft versus host disease (GvHD) or relapse after transplantation. Three novel, mutually non-exclusive hypotheses to explain these clinical observations will be explored during the proposed project, through the synergistic effort of the two PIs who will contribute their complementary expertise in the immunobiology of HSCT and cytomegaloviruses, respectively: 1) Heterologous immunity by HCMV-specific CD4+ T cell receptors (TCR) cross-recognizing HLA-DPB1 alloantigens (mainly PI Fleischhauer). HCMV-specific and HLA-DPB1 alloreactive CD4+ T cell cultures will be independently obtained from donors of HLA-DPB1-mismatched unrelated HSCT at UK-Essen. Their TCR CDR3 sequences will be determined by next generation sequencing. TCR with identical CDR3 sequences identified in both cultures are indicative of heterologous immunity and will be longitudinally traced in the patient with or without acute GvHD or leukemia relapse after transplantation. 2) HCMV-encoded proteins modulating the immunogenicity of allogeneic HLA-DPB1 (PI Fleischhauer and Trilling). An available library of expression vectors covering the canonical HCMV proteins will be introduced individually into single HLA-DP expressing model cell lines, to test the gene-specific impact on allorecognition by HLA-DPB1-specific CD4+ T cells. Identified candidate genes will be characterized concerning their effects on HLA-DPB1 expression and processing upon ectopic expression as well as in the context of HCMV infection. 3) Cellular and viral determinants of the HCMV-induced impairment of leukemic cell proliferation (mainly PI Trilling). To identify and characterize the mediators of previously observed anti-proliferative effects elicited by inactivated HCMV particles on acute myeoloid leukemia (AML) cells, a series of biochemical and immunological assays as well as inhibition studies will be conducted to test the hypothesis that HCMV triggers a suicidal innate immune response after contact with AML cells, and to identify the responsible factors. Together, the new insights into the immunobiology of T cell alloreactivity, HCMV and leukemia expected from this project will have potential translational implications in cellular therapy.