LAUSR

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-pp65NLVPMVATV 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 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.