a Treatment schedule used in this study. b Flow cytometry analysis of splenocytes and blood cells from tumor-free mice primed with Ad-Ova and boosted or not with MRB co-administered with Ova peptide 7 days later. Blood samples were collected on days 14, 28 and 35 and splenocytes were also analyzed on day 35. The bar charts show the percentage of Ova-tetramer+ cells (within the CD8+ live cell population) (top panel) or of live cells that are CD8+, Ova-tetramer+ (bottom panel) (n = 2 (no prime, no boost) and 3 (Ad-Ova, no boost and Ad-Ova, MRB + Ova)). c Contour plots showing CD44 expression and IFNγ or TNFα production by CD8+ splenocytes (day 35) re-stimulated ex-vivo with Ova peptides. d Quantification of the percentage of CD8+ cells that are CD44+, IFNγ+ or CD44+, TNFα+ with and without ex-vivo re-stimulation n = 2 (no prime, no boost) and 3 (Ad-Ova, no boost and Ad-Ova, MRB + Ova). e Contour plots showing the expression of KLRG1 and CD127 by Ova-tetramer+ CD8 T cells. The graphs also delineate different T cell subpopulations: memory precursor effector cells (MPECs), double-positive effector cells (DPECs), short-lived effector cells (SLECs) and early effector cells (EECs) (n = 2 (no prime, no boost) and 3 (Ad-Ova, no boost and Ad-Ova, MRB + Ova)). f Quantification of the populations identified in (e) for all mice (n = 3). Data are presented as mean values ± SEM. g Absolute number of MPECs, DPECs, SLECs and EECs from the spleen of the mice at day 35 (n = 3). Data are presented as mean values ± SEM. h Representative contour plots showing different T cell populations based on CD44 and CD62L expression of splenocytes from untreated animals (left panel) or of CD8+, Ova-tetramer+ cells from the blood and the spleen of both treatment groups at day 35 (right panels). The graphs also show the gating strategy used to distinguish between naïve, central memory (CM), effector memory (EM) and double negative (DN) CD8+ T cells (n = 2 (no prime, no boost) and 3 (Ad-Ova, no boost and Ad-Ova, MRB + Ova)). i Quantification of the populations identified in (g) for all mice at different time points (top panel) and absolute number of naive, CM, EM and DN CD8+, Ova-tetramer+ cells from the spleen of mice at day 35 (bottom panel) (n = 3). Data are presented as mean values ± SEM. Unless indicated otherwise, the statistical analyses refer to the comparison between the corresponding ex-vivo “No restim” and “restim” conditions. NS: p > 0.05, *: p < 0.05, **: p < 0.01, ***: p < 0.001 (unpaired two-tailed t-test). Source data are provided as a Source Data file. Exact p values can be found in the Source Data.