Robust Production of Cytomegalovirus pp65-Specific T Cells By a Fully Automated IFN-γ Cytokine Capture System

Cytomegalovirus (CMV) related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). As an alternative to antiviral drugs, CMV-specific cytotoxic lymphocytes (CMV-CTLs) can provide long-term CMV-specific immunity without major side effects. In this study, we apply the IFN-γ cytokine capture system (CCS) using the fully automated CliniMACS Prodigy device to rapidly produce CMV-CTLs. Five validation runs were performed using apheresis samples from randomly selected CMV-seropositive healthy blood donors. CliniMACS Prodigy automatically performed successive processes including antigen stimulation, anti-IFN-γ labelling, magnetic enrichment, and elution which took approximately 13 hours. The original apheresis samples consisted of 0.3% CD3+IFN-γ+ T cells which were mainly CD45RA+CD62L+ naïve T cells. Following IFN-γ enrichment, the target fraction contained 51.3% CD3+IFN-γ+ cells with reduction in naïve T cells and the selection of CD45RA-CD62L- and CD45RA+CD62L- memory T cells. Interestingly, the frequency of CMV pp65 specific CD3+ T cells was also increased by 1.7-fold. Furthermore, extended culture of isolated cells revealed efficient proliferation with sustained antigen-specific IFN-γ secretion and cytotoxicity against pp65 pulsed target cells. Therefore, we suggest IFN-γ CCS by CliniMACS Prodigy as a simple and robust approach to produce CMV-CTLs, which may be highly feasible and applicable for clinical use.