DataSheet_1_Robust Production of Merkel Cell Polyomavirus Oncogene Specific T Cells From Healthy Donors for Adoptive Transfer.docx

Virus positive Merkel cell carcinoma (VP-MCC) is an aggressive but immunogenic skin malignancy driven by Merkel cell polyomavirus (MCPyV) T antigen (TAg). Since adoptive T cell transfer (ACT) can be effective against virus-driven malignancies, we set out to develop a methodology for generating MCPyV TAg specific T cells. MCPyV is a common, asymptomatic infection and virus-exposed healthy donors represent a potential source of MCPyV TAg specific T cells for ACT. Virus specific T cells were generated using monocyte-derived dendritic cells (moDCs) pulsed with MCPyV TAg peptide libraries and co-cultured with autologous T cells in supplemented with pro-inflammatory and homeostatic cytokines for 14 days. Specific reactivity was observed predominantly within the CD4+ T cell compartment in the cultures generated from 21/46 random healthy donors. Notably, responses were more often seen in donors aged 50 years and older. TAg specific CD4+ T cells specifically secreted Th1 cytokines and upregulated CD137 upon challenge with MCPyV TAg peptide libraries and autologous transduced antigen presenting cells. Expanded T cells from healthy donors recognized epitopes of both TAg splice variants found in VP-MCC tumors, and minimally expressed exhaustion markers. Our data show that MCPyV specific T cells can be expanded from healthy donors using methods appropriate for the manufacture of clinical grade ACT products.

病毒阳性默克尔细胞癌（Virus positive Merkel cell carcinoma, VP-MCC）是一种由默克尔细胞多瘤病毒（Merkel cell polyomavirus, MCPyV）T抗原（TAg）驱动的侵袭性且具有免疫原性的皮肤恶性肿瘤。鉴于过继性T细胞移植（adoptive T cell transfer, ACT）对病毒驱动的恶性肿瘤具有治疗效果，本研究旨在开发一种制备MCPyV T抗原特异性T细胞的方法。默克尔细胞多瘤病毒是一种常见的无症状感染病毒，病毒暴露的健康供体可作为过继性T细胞移植所需的MCPyV T抗原特异性T细胞的潜在来源。本研究采用负载MCPyV T抗原肽库的单核细胞衍生树突状细胞（monocyte-derived dendritic cells, moDCs），将其与自体T细胞在添加促炎细胞因子与稳态细胞因子的培养基中共培养14天，以制备病毒特异性T细胞。在21/46例随机选取的健康供体的培养体系中，特异性免疫反应主要存在于CD4+ T细胞亚群中。值得注意的是，50岁及以上的供体更易检测到此类特异性反应。MCPyV T抗原特异性CD4+ T细胞在受到MCPyV T抗原肽库以及自体转导抗原呈递细胞刺激时，可特异性分泌Th1型细胞因子，并上调CD137的表达。从健康供体中扩增得到的T细胞可识别VP-MCC肿瘤中两种T抗原剪接变体的表位，且仅低表达耗竭标志物。本研究数据表明，采用适配临床级过继性T细胞移植产品制备的方法，可从健康供体中扩增得到MCPyV特异性T细胞。