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

NKG2D 配体在激活人巨细胞病毒特异性免疫反应中的作用

• 批准号: 421451057
• 负责人: Professorin Dr. Christine Falk
• 金额: --
• 依托单位: Institut für Virologie
• 依托单位国家: 德国
• 项目类别: Research Units
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/421451057?language=en
• 关键词: Role; NKG2D; ligands; activation; human

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.

HCMV记忆T细胞应答不足的实体器官移植受者由于免疫抑制而处于病毒再激活和发展危及生命的并发症的高风险中。我们假设HCMV特异性免疫应答（NK和CD 8 + T细胞）可以通过NKG 2D配体（NKG 2D-L）的病毒表达来增强。将在NK细胞和细胞毒性T细胞（CTL）的活化以及这些免疫细胞最有效地限制病毒传播的能力方面，将在感染细胞中过表达ULBP 2的当前先导候选病毒TB 40-ULBP 2与已经可用的表达ULBP 1、云母、MICB的病毒进行比较。通过测量活化和脱粒标志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，以鉴定由各种NKG 2D-L诱导的HCMV特异性T细胞应答介导的TCR和共刺激信号。通过将感染的成纤维细胞与来自HCMV血清阳性个体的PBMC共培养，NKG 2D介导的信号传导对在DNAM-1、TIGIT和CD 96表达方面不同的T细胞亚群的影响将通过表型表征以及对它们的活化和扩增进行检查。单细胞mRNA（scRNA-seq）、CITE和T细胞受体测序（TCR-seq）的组合将用于活化的HCMV特异性CTL（例如，HLA-A*02/NLV特异性CTL）。基于我们目前对肺中组织驻留记忆（TRM）T细胞的研究，我们将表征从终末期肺衰竭患者的肺组织中分离的HCMV特异性T细胞。TRM-CTL（对HLA-A*02/NLV或其他肽具有特异性）将通过多聚体分离，并通过表型分析和组合的scRNA-seq、TCR-seq和CITE-seq表征。将这些TRM-CTL与表达NKG 2D-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TRM相关的长寿命CTL的发展的最佳条件。