Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.
DZIF-National Centre for Infection Research, Munich, Germany.
PLoS One. 2018 Feb 28;13(2):e0193554. doi: 10.1371/journal.pone.0193554. eCollection 2018.
Human Cytomegalovirus (CMV) reactivation remains a major source of morbidity in patients after solid organ and hematopoietic stem cell transplantation (HSCT). Adoptive T cell therapy (ACT) with CMV-specific T cells is a promising therapeutic approach for HSCT recipients, but might be counteracted by CMV's immune evasion strategies. HLA-C07:02 is less susceptible to viral immune evasion suggesting HLA-C07:02-restricted viral epitopes as promising targets for ACT. For a better understanding of HLA-C07:02-restricted CMV-specific T cells we used recently generated reversible HLA-C07:02/IE-1 multimers (Streptamers) recognizing a CMV-derived Immediate-Early-1 (IE-1) epitope and analyzed phenotypic and functional T cell characteristics. Initially, we detected very high frequencies of HLA-C07:02/IE-1 multimer+ T cells (median = 11.35%), as well as robust functional responses after stimulation with IE-1 peptide (IFNγ+; median = 5.02%) in healthy individuals. However, MHC-multimer+ and IFNγ-secreting T cell frequencies showed a relatively weak correlation (r2 = 0.77), which could be attributed to an unexpected contribution of CMV-epitope-independent KIR2DL2/3-binding of HLA-C07:02/IE-1 multimers. Therefore, we developed a MHC-multimer double-staining approach against a cancer epitope-specific HLA-C07:02 multimer to identify truly HLA-C07:02/IE-1 epitope-specific T cells. The frequencies of these truly HLA-C07:02/IE-1 multimer+ T cells were still high (median = 6.86%) and correlated now strongly (r2 = 0.96) with IFNγ-secretion. Interestingly, HLA-C07:02/IE-1-restricted T cells contain substantial numbers with a central memory T cell phenotype, indicating high expansion potential e.g. for ACT. In line with that, we developed a clinical enrichment protocol avoiding epitope-independent KIR-binding to make HLA-C07:02/IE-1-restricted T cells available for ACT. Initial depletion of KIR-expressing CD8+ T cells followed by HLA-C07:02/IE-1 Streptamer positive selection using paramagnetic labeling techniques allowed to enrich successfully HLA-C07:02/IE-1-restricted T cells. Such specifically enriched populations of functional HLA-C07:02/IE-1-restricted T cells with significant central memory T cell content could become a potent source for ACT.
人巨细胞病毒 (CMV) 再激活仍然是实体器官和造血干细胞移植 (HSCT) 后患者发病的主要原因。使用 CMV 特异性 T 细胞的过继性 T 细胞疗法 (ACT) 是 HSCT 受者有前途的治疗方法，但可能会受到 CMV 免疫逃避策略的影响。HLA-C07:02 对病毒免疫逃避的敏感性较低，这表明 HLA-C07:02 限制性病毒表位是 ACT 的有希望的靶标。为了更好地了解 HLA-C07:02 限制性 CMV 特异性 T 细胞，我们使用了最近生成的可逆 HLA-C07:02/IE-1 多聚体（Streptamers）来识别源自 CMV 的即时早期-1（IE-1）表位，并分析了表型和功能 T 细胞特征。最初，我们在健康个体中检测到非常高的 HLA-C07:02/IE-1 多聚体+ T 细胞频率（中位数=11.35%），以及在用 IE-1 肽刺激后产生的强大功能反应（IFNγ+；中位数=5.02%）。然而，MHC-多聚体+和 IFNγ 分泌 T 细胞频率显示出相对较弱的相关性（r2=0.77），这可能归因于 HLA-C07:02/IE-1 多聚体对 CMV-表位非依赖性 KIR2DL2/3 结合的意外贡献。因此，我们开发了一种针对癌症表位特异性 HLA-C07:02 多聚体的 MHC-多聚体双重染色方法，以鉴定真正的 HLA-C07:02/IE-1 表位特异性 T 细胞。这些真正的 HLA-C07:02/IE-1 多聚体+ T 细胞的频率仍然很高（中位数=6.86%），并且现在与 IFNγ 分泌强烈相关（r2=0.96）。有趣的是，HLA-C07:02/IE-1 限制性 T 细胞含有大量中央记忆 T 细胞表型的细胞，表明其具有很高的扩增潜力，例如用于 ACT。与此一致，我们开发了一种临床富集方案，避免了对非表位依赖性 KIR 结合，以使 HLA-C07:02/IE-1 限制性 T 细胞可用于 ACT。最初的 KIR 表达 CD8+T 细胞耗竭，然后使用顺磁标记技术对 HLA-C07:02/IE-1 Streptamer 进行阳性选择，成功富集了 HLA-C07:02/IE-1 限制性 T 细胞。具有显著中央记忆 T 细胞含量的这种特异性富集的功能性 HLA-C07:02/IE-1 限制性 T 细胞群体可能成为 ACT 的有效来源。
