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）是一种典型的β-疱疹病毒，具有重要的临床意义，特别是对于接受同种异体造血干细胞移植（HSCT）治疗白血病的免疫抑制患者。在这种情况下，HCMV再激活已被证明影响免疫重建和发生移植物抗宿主病（GvHD）或移植后复发的风险。在拟建的项目中，将通过两位在HSCT和巨细胞病毒免疫生物学方面的互补专业知识的PI的协同努力，探索三个新的，相互不排斥的假设来解释这些临床观察结果：1)hcmv特异性CD4+ T细胞受体（TCR）交叉识别HLA-DPB1异体抗原的异源免疫（主要是PI Fleischhauer）。hcmv特异性和HLA-DPB1同种反应性CD4+ T细胞培养将分别从UK-Essen的HLA-DPB1不匹配的非相关HSCT供体中获得。它们的TCR CDR3序列将通过下一代测序确定。在两种培养中鉴定出具有相同CDR3序列的TCR，表明异种免疫，并将在移植后患有或不患有急性GvHD或白血病复发的患者中进行纵向追踪。2)调节同种异体HLA-DPB1免疫原性的hcmv编码蛋白（PI Fleischhauer and Trilling）。将覆盖典型HCMV蛋白的可用表达载体库单独导入单个表达HLA-DP的模型细胞系中，以测试hla - dp1特异性CD4+ T细胞对同种异体识别的基因特异性影响。确定的候选基因将被表征其对HLA-DPB1表达和异位表达加工的影响，以及在HCMV感染的背景下。3) hcmv诱导白血病细胞增殖损伤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颗粒对急性髓细胞白血病（AML）细胞的抗增殖作用的介质，我们将进行一系列生化和免疫学试验以及抑制研究，以验证HCMV与AML细胞接触后引发自杀性先天免疫反应的假设，并确定相关因素。总之，该项目对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.