Measurement of CD8⁺ and CD4⁺ T cell frequencies specific for EBV LMP1 and LMP2a using mRNA-transfected DCs

An EBV-specific cellular immune response is associated with the control of EBV-associated malignancies and lymphoproliferative diseases, some of which have been successfully treated by adoptive T cell therapy. Therefore, many methods have been used to measure EBV-specific cellular immune responses. Previous studies have mainly used autologous EBV-transformed B-lymphoblastoid cell lines (B-LCLs), recombinant viral vectors transfected or peptide pulsed dendritic cells (DCs) as stimulators of CD8⁺ and CD4⁺ T lymphocytes. In the present study, we used an interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay by using isolated CD8⁺ and CD4⁺ T cells stimulated with mRNA-transfected DCs. The frequency of latent membrane protein 1 (LMP1)-specific IFN-γ producing CD4⁺ T cells was significantly higher than that of LMP2a. The frequency of IFN-γ producing CD4⁺ T cells was significantly correlated with that of CD8⁺ T cells in LMP1-specific immune responses (r = 0.7187, Pc < 0.0001). To determine whether there were changes in LMP1- or LMP2a-specific immune responses, subsequent peripheral blood mononuclear cells (PBMCs) samples were analyzed. Significant changes were observed in 5 of the 10 donors examined, and CD4⁺ T cell responses showed more significant changes than CD8⁺ T cell responses. CD8⁺ and CD4⁺ T cells from EBV-seropositive donors secreted only the Th1 cytokines IFN-γ, TNF-α, and IL-2, while Th2 (IL-4) and Th17 (IL-17a) cytokines were not detected. CD4⁺ T cells secreted significantly higher cytokine levels than did CD8⁺ T cells. Analysis of EBV-specific T cell responses using autologous DCs transfected with mRNA might provide a comprehensive tool for monitoring EBV infection and new insights into the pathogenesis of EBV-associated diseases.