Immunosuppressed patients are susceptible to virus reactivation or de novo infection. Adoptive immunotherapy, based on virus-specific T lymphocytes (VST), can prevent or treat viral diseases. However, donor availability, HLA-compatibility restrictions, high costs, and time required for the production of personalized medicines constitute considerable limitations to this treatment. Ex vivo rapid and large-scale expansion of VST, compliant with current good manufacturing practice (cGMP) standards, with an associated cell donor registry would overcome these limitations. This study aimed to characterize a VST product obtained through an expansion protocol transferable to cGMP standards. Antigenic stimulus consisted of cytomegalovirus (CMV) pp65 peptide pool-pulsed autologous dendritic cells (DCs) derived from monocytes. G-Rex technology, cytokines IL-2, IL-7, and IL-15, and anti-CD3 and anti-CD28 antibodies were used for culture. At day 14 of cell culture, the final product was characterized regarding T cell subsets, specificity, and functionality. The final product, comprised mainly CD4⁺ and CD8⁺ T lymphocytes (49.2 ± 24.7 and 42.3 ± 25.2, respectively). The culture conditions made it possible to achieve at least a 98.89-fold increase in pp65-specific CD3⁺ IFN-γ⁺ cells. These cells were specific, as pp65-specific cytotoxicity was demonstrated. Additionally, in complete HLA mismatch and without the presence of pp65, alloreactivity resulted in <5% cell lysis. In conclusion, a cGMP scalable process for the generation of a large number of doses of CMV-specific cytotoxic T cells was successfully performed.

免疫抑制的患者易感染病毒或 从头感染。基于病毒特异性T淋巴细胞（VST）的过继免疫疗法可以预防或治疗病毒性疾病。但是，供体的可获得性，HLA相容性限制，高成本以及生产个性化药物所需的时间构成了这种治疗的很大局限性。离体VST的快速大规模扩展，符合当前的良好生产规范（cGMP）标准，并具有相关的细胞供体注册表，将克服这些限制。这项研究旨在表征通过可扩展至cGMP标准的扩展协议获得的VST产品。抗原刺激由巨细胞病毒（CMV）pp65肽池脉冲自体单核树突状细胞（DC）组成。使用G-Rex技术，细胞因子IL-2，IL-7和IL-15以及抗CD3和抗CD28抗体进行培养。在细胞培养的第14天，就T细胞亚群，特异性和功能性对最终产品进行了表征。最终产品主要包括CD4 ⁺和CD8 ⁺T淋巴细胞（分别为49.2±24.7和42.3±25.2）。培养条件使得有可能在pp65特异性CD3 ⁺ IFN-γ ⁺细胞中获得至少98.89倍的增长。这些细胞具有pp65特异性的细胞毒性，具有特异性。此外，在完全HLA错配且没有pp65的情况下，同种异体反应导致细胞裂解率低于5％。总之，成功地进行了cGMP可扩展过程，用于生成大量剂量的CMV特异性细胞毒性T细胞。