Cytomegalovirus (CMV) infection remains a life-threatening condition in individuals with a suppressed immune system. CMV may also represent a clinically relevant target for immune responses in CMV-positive malignancies. We established a protocol to expand CMV-specific T cells (CMV-T) using peripheral blood mononuclear cells (PBMCs). PBMCs from 16 HLA-A*0201 donors were cultured with a cytokine cocktail comprising IL-2/IL-15/IL-21 along with overlapping peptides from CMV-pp65. Ten days later, T cells were stimulated with anti-CD3 (OKT3) and irradiated autologous PBMCs. CMV-T were detected by HLA-A*0201 CMV-pp65_NLVPMVATV wild type and q226a mutant tetramers (for high-affinity T cells), intracellular cytokine staining, a CD107a mobilization assays as well as IFN-γ and TNF-α production in cell culture supernatants. We reliably obtained 50.25 ± 27.27% of CD8+ and 22.08 ± 21.83% of CD4+ T cells post-CMV-pp65 stimulation of PBMCs with a Th1-polarized phenotype and decreased Th2/Th17 responses. Most CD3 + CD8 + tetramer+ T cells were effector-memory cells, particularly among high-affinity CMV-T (q226a CMV-tetramer+). High-affinity CMV-T cells, compared to WT-tetramer+ cells, expressed higher IL-21R and lower FasL post-stimulation with CMV-pp65. The IL-2/IL-15/IL-21 cocktail also promoted CCR6 and CXCR3 expression necessary for T-cell migration into tissues. We have optimized methods for generating high-affinity CMV-specific T cells that can be used for adoptive cellular therapy in clinical practice.

巨细胞病毒（CMV）感染在免疫系统受到抑制的个体中仍然威胁着生命。CMV也可能代表CMV阳性恶性肿瘤免疫应答的临床相关靶标。我们建立了使用外周血单核细胞（PBMC）扩展CMV特异性T细胞（CMV-T）的协议。将来自16个HLA-A * 0201供体的PBMC与包含IL-2 / IL-15 / IL-21以及来自CMV-pp65的重叠肽的细胞因子混合物一起培养。十天后，用抗CD3（OKT3）和放射自体PBMC刺激T细胞。通过HLA-A * 0201 CMV-pp65 _NLVPMVATV检测到CMV-T野生型和q226a突变四聚体（用于高亲和力T细胞），细胞内细胞因子染色，CD107a动员测定以及细胞培养上清液中的IFN-γ和TNF-α产生。我们可靠地获得了CMV-pp65刺激具有Th1极化表型和降低的Th2 / Th17反应的PBMC后50.25±27.27％的CD8 +和22.08±21.83％的CD4 + T细胞。大多数CD3 + CD8 +四聚体+ T细胞是效应记忆细胞，特别是在高亲和力CMV-T（q226a CMV-tetramer +）中。与WT-tetramer +细胞相比，高亲和力的CMV-T细胞在用CMV-pp65刺激后表达更高的IL-21R和更低的FasL。IL-2 / IL-15 / IL-21混合物还促进了T细胞向组织迁移所必需的CCR6和CXCR3表达。