DCs matured with TLR4 and TLR7/8 agonists together prime an antigen-specific response containing an increased number of CD28⁺ CD8⁺ T cells. Monocyte-derived DCs were matured with LPS, R848, LPS + R848, or the cytokine cocktail together with Melan-A_26-35 peptide, and subsequently cultured with purified naive (CD45RO^neg) autologous CD8⁺ T cells. Quantitation and phenotyping of Melan-A specific CD8⁺ T cells was performed 9 days after priming. (A) Representative data from 2 donors showing the mean frequency and absolute number of CD8⁺/Melan-A pentamer⁺ cells after priming by the indicated DC population (± SEM). (B) Representative data from 3 donors showing the percentage of CD27⁺/CD28⁺ of CD8⁺/Melan-A pentamer⁺ cells primed by Cytokine Mix, LPS, R848, or LPS/R848 matured DCs. Data points indicate individual priming wells measured from 3 (donor 1) or 2 (donors 2 and 3) independent experiments, and lines indicate means ± SEM. (C) Representative flow cytometric analysis of CD27/CD28 surface expression on Melan-A pentamer⁺ CD8⁺ cells superimposed on CD8⁺ pentamer-negative cells within the same priming well. Percentages represent the proportion of each subset within CD8⁺/Melan-A⁺ cells. (D) Representative CD45RO surface expression on Melan-A pentamer⁺ CD8⁺ cells overlaid on CD8⁺ pentamer-negative cells with the percentage of CD45RO⁺/CD8⁺/Melan-A⁺ cells indicated. (E) Mean percentage of CD45RO⁺ of CD8⁺/Melan-A⁺ cells from 3 donors primed by the indicated DCs ± SEM. **Statistically significant differences with P values shown.