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因免疫抑制而重新激活病毒并出现危及生命的并发症的风险很高。我们假设 NKG2D 配体 (NKG2D-L) 的病毒表达可以增强 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 在受感染细胞中过度表达 ULBP2，将与现有的表达 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限制病毒传播的能力方面进行比较。通过测量激活和脱粒标记物以及细胞因子的分泌来确定潜在机制。此外，我们将使用在转录因子 NFAT、NF-kB 或 AP-1 激活后表达荧光染料的 TCR 转基因 Jurkat 三重报告细胞系来识别由各种 NKG2D-L 诱导的 HCMV 特异性 T 细胞反应介导的 TCR 和共刺激信号。通过将受感染的成纤维细胞与来自 HCMV 血清阳性个体的 PBMC 共培养，将通过表型特征及其激活和扩增来检查 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。单细胞 mRNA (scRNA-seq)、CITE 和 T 细胞受体测序 (TCR-seq) 的组合将用于激活 HCMV 特异性 CTL（例如 HLA-A*02/NLV 特异性 CTL）的精确分子表征。基于我们目前对肺部组织驻留记忆 (TRM) T 细胞的研究，我们将表征从终末肺衰竭患者的外植肺组织中分离出的 HCMV 特异性 T 细胞。 TRM-CTL（针对 HLA-A*02/NLV 或其他肽）将通过多聚体进行分离，并通过表型分析和组合 scRNA-seq、TCR-seq 和 CITE-seq 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，我们的目标是确定开发 TRM 相关长寿命 CTL 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.