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

Background: Cytomegalovirus(CMV)-related diseases are a serious cause of morbidity and mortality following hematopoietic stem cell transplantation (HSCT). CMV-specific cytotoxic T lymphocytes (CMV-CTLs) have been reported as an alternative to antiviral drugs that provide long-term CMV-specific immunity without major side effects. However, their application has been limited by the prolonged manufacturing process required. Methods: In this study, we applied the IFN-γ cytokine capture system (CCS) using the fully automated CliniMACS Prodigy device for rapid production of CMV-CTLs, which may be applicable in clinically urgent CMV-related diseases. Five validation runs were performed using apheresis samples from randomly selected CMV-seropositive healthy blood donors. Successive processes, including antigen stimulation, anti-IFN-_γ labeling, magnetic enrichment and elution, were then performed automatically using the CliniMACS Prodigy, which took approximately 13 h. Results: The original apheresis samples consisted mainly of CD45RA+ CD62L+ naïve T cells as well as 0.3% IFN-_γ-secreting CD3+ T cells that showed a response to the CMV pp65 antigen (CD3+ IFN-γ+ cells). Following IFN-_γ enrichment, the target fraction contained 51.3% CD3+ IFN-γ+ cells with a reduction in naïve T cells and selection of CD45RA- CD62L- and CD45RA+ CD62L- memory T cells. Furthermore, extended culture of these isolated cells revealed functional activity, including efficient proliferation, sustained antigen-specific IFN-_γ secretion, and cytotoxicity against pp65-pulsed target cells. Conclusion: The findings reported here suggest that the IFN-_γ CCS by the CliniMACS Prodigy is a simple and robust approach to produce CMV-CTLs, which may be applicable for the treatment of clinically urgent CMV-related diseases.