Fig. 3

Analysis of HAdV-specific T-cell responses to the immunodominant epitopes A02Hexon_TLLY and A01Penton_STDV in healthy donors. HAdV-specific T cells induced by A02Hexon_TLLY and A01Penton_STDV were characterized in relation to a proliferative capacity of peptide-specific T cells within CD3⁺ and CD3⁺CD8⁺ T-cell populations b phenotype and frequency of HAdV-specific CD8⁺ T cells, (C) cytotoxicity of CD8⁺ T cells by multicolor flow cytometry. a PBMCs from healthy donors were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated over 7 days with A02Hexon_TLLY or A01Penton_STDV, respectively and analyzed for the frequency of viable proliferative CD3⁺ and CD3⁺CD8⁺ T cells (CFSE low). The proliferative capacity of unstimulated CSFE-labeled PBMCs (negative control) was subtracted from the values for peptide-stimulated PBMCs. Results of independent experiments (n = 5) are expressed as mean ± SD. b Frequency of A02Hexon_TLLY- and A01Penton_STDV-specific CD8⁺ T cells and their four subsets—naïve (T_N), central (T_CM), effector memory (T_EM), and terminally differentiated effector memory (T_EMRA) T cells—from healthy donors before (day 0) and after two restimulation cycles (day 14), including dot plots for phenotype and multimer⁺CD8⁺ T cells of one representative donor (upper right). Results of independent experiments (n = 3) are expressed as mean ± SD. c Cytotoxic activity of expanded A02Hexon_TLLY- and A01Penton_STDV-specific T cells from healthy donors (day 14, effector cells, E) was analyzed in five-hour cytotoxicity assays using CFSE-labeled peptide-loaded/unloaded PBMCs as target cells (T). Effector T cells were co-cultured with target cells at an E:T ratio of 10:1, 30:10, or 60:1, respectively. Basal cytotoxic activity of effector T cells induced by A02Hexon_TLLY or A01Penton_STDV against the unloaded target cells was subtracted from the cytotoxic T-cell values against peptide-loaded PBMCs. Results (n = 2) are expressed as the mean percentage of target cell lysis ± SD. Asterisks shown in the figure indicate statistically significant differences between T-cell proliferation, phenotype and cytotoxicity in response to A02Hexon_TLLY- and A01Penton_STDV (*p < 0.05, **p < 0.01, ***p < 0.001, SD, standard deviation)