Fig. 4

CD40L expressed on aAPC enhances the proliferation of CD8⁺ T cells stimulated with CMV pp65 antigen. Sorted CD8⁺ T cells were weekly stimulated with irradiated aAPC or aAPC-CD40L loaded with HLA-A*02:01 restricted peptides of pp65 in the presence of IL-2(10U/mL). After two rounds of stimulation, fold expansion, flow cytometry analyses and ELISPOT assay was done on day 12. (A) Fold expansion of CD8⁺ T cells stimulated with antigen-loaded aAPC was compared to that of CD8⁺ T cells stimulated with antigen-loaded aAPC-CD40L. (B, C) Tetramer positive CD8⁺ T cell proportion and tetramer positive CD8⁺ T cell number was compared between aAPC stimulated group and aAPC-CD40L stimulated group. Tetramer positive CD8⁺ T cell number was calculated as (% of tetramer positive CD8⁺ T cell)×(total CD8⁺ T cell number on day 12). (D, E) For ELISPOT assay, CD8⁺ T cells were co-cultured with antigen-loaded T2 cells. After 24 h of incubation, ELISPOT was done to analyzed the IFN-γ secretion from CD8⁺ T cells. IFN-γ spots were compared between aAPC stimulated CD8⁺ T cells and aAPC-CD40L stimulated CD8⁺ T cells. IFN-γ secreting total cell number was analyzed by multiplying the IFN-γ spot number to the number of CD8⁺ T cells on day 12 (n = 8). Wilcoxon paired rank test one tailed, **p < 0.005