Role of NKG2D ligands for activation of the human cytomegalovirus-specific immune response

HCMV记忆t细胞反应不足的实体器官移植受者，由于免疫抑制，病毒重新激活和发生危及生命的并发症的风险很高。我们假设NKG2D配体（NKG2D- l）的病毒表达可以促进hcmv特异性免疫反应（NK和CD8+ T细胞）。目前的主要候选病毒TB40-ULBP2在感染细胞中过表达ULBP2，将与已有的表达ULBP1-、MICA-、micb的病毒在NK细胞和细胞毒性T细胞（CTL）的激活以及这些免疫细胞最有效地限制病毒传播的能力方面进行比较。潜在的机制将通过测量激活和脱颗粒标记物以及细胞因子的分泌来确定。此外，我们将使用TCR转基因Jurkat三重报告细胞系，在转录因子NFAT、NF-kB或AP-1的激活下表达荧光色素，以鉴定由各种nkg2d - l诱导的hcmv特异性t细胞反应介导的TCR和共刺激信号。通过将感染的成纤维细胞与hcmv血清阳性个体的PBMC共培养，nkg2d介导的信号传导对DNAM-1、TIGIT和CD96表达不同的t细胞亚群的影响将通过表型表征及其激活和扩增进行检测。单细胞mRNA （scRNA-seq）、CITE和t细胞受体测序（TCR-seq）的组合将用于对活化的hcmv特异性CTL（例如HLA-A*02/ nlv特异性CTL）进行精确的分子表征。基于我们目前对肺组织驻留记忆（TRM） T细胞的研究，我们将对从终末期肺衰竭患者的外植肺组织中分离的hcmv特异性T细胞进行表征。TRM-CTL（特异性针对HLA-A*02/NLV或其他多肽）将通过多聚体分离，并通过表型分析和scRNA-seq、TCR-seq和CITE-seq组合进行表征。这些TRM-CTL将与表达nkg2d - l的突变体共培养，分析其DNAM-1、TIGIT和PD-1亚群，并与hcmv特异性循环T细胞进行比较。基于这些比较，我们的目标是确定与trm相关的长寿命CTL发展的最佳条件。

Solid organ transplant recipients with an insufficient HCMV memory T-cell response are at high risk of reactivating the virus and developing life-threatening complications due to immunosuppression. We hypothesize that HCMV-specific immune responses (NK and CD8+ T cells) can be boosted by viral expression of NKG2D ligands (NKG2D-L). The current lead virus candidate, TB40-ULBP2, overexpressing ULBP2 in infected cells, will be compared with the already available ULBP1-, MICA-, MICB-expressing viruses in terms of activation of NK cells and cytotoxic T cells (CTL) and the ability of these immune cells to most effectively limit virus spread. Underlying mechanisms will be determined by measuring activation and degranulation markers and secretion of cytokines. Additionally, we will use TCR transgenic Jurkat triple reporter cell lines expressing fluorochromes upon activation of the transcription factors NFAT, NF-kB, or AP-1 to identify TCR and costimulatory signals mediated by the various NKG2D-L-induced HCMV-specific T-cell responses. By coculturing infected fibroblasts with PBMC from HCMV-seropositive individuals, effects of NKG2D-mediated signaling on T-cell subpopulations differing in DNAM-1, TIGIT, and CD96 expression will be examined by phenotypic characterization, and on their activation and expansion. A combination of single-cell mRNA (scRNA-seq), CITE, and T-cell receptor sequencing (TCR-seq) will be used for precise molecular characterization of activated HCMV-specific CTL (e.g., HLA-A*02/NLV-specific CTL). Based on our current work on tissue-resident memory (TRM) T cells in the lung, we will characterize HCMV-specific T cells isolated from explanted lung tissue from patients with terminal lung failure. TRM-CTL (specific for HLA-A*02/NLV or other peptides) will be isolated via multimers and characterized by phenotyping and combined scRNA-seq, TCR-seq and CITE-seq. These TRM-CTL will be cocultured with NKG2D-L-expressing mutants, their DNAM-1, TIGIT, and PD-1 subsets analyzed, and compared with HCMV-specific circulating T cells. Based on these comparisons, we aim to define the optimal conditions for the development of TRM-related long-lived CTL.