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有很高的风险重新激活病毒，并因免疫抑制而发生危及生命的并发症。我们假设NKG2D配体(NKG2D-L)的病毒表达可以增强巨细胞病毒特异性免疫反应(NK和CD8T细胞)。目前在感染细胞中过表达ULBP2的候选主要病毒TB40-ULBP2将与现有的表达ULBP1、MICA和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志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激活时表达荧光，以鉴定由NKG2D-L诱导的各种巨细胞病毒特异性T细胞应答所介导的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共同培养，NKG2D介导的信号转导对不同dNaM-1、TIGIT和CD96表达的T细胞亚群的表型特征及其对其激活和扩增的影响将被检测。单细胞信使核糖核酸(scRNA-seq)、CITE和T细胞受体测序(TCR-seq)的组合将用于对激活的HCMV特异性CTL(例如，人类白细胞抗原A*02/NLV特异性CTL)进行精确的分子表征。基于我们目前在肺内组织驻留记忆(TRM)T细胞方面的工作，我们将从终末性肺衰竭患者的移植肺组织中分离出HCMV特异性T细胞。TRM-CTL(针对HLAA*02/NLV或其他多肽)将通过多聚体分离，并进行表型鉴定和scRNA-seq、TCR-seq和CITE-seq的组合。将这些TRM-CTL与表达NKG2D-L的突变体共同培养，分析其dNaM-1、TIGIT和PD-1亚群，并与人巨细胞病毒特异性循环T细胞进行比较。在这些比较的基础上，我们的目标是确定与TRM相关的长寿命CTL的最佳开发条件。

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.