Concomitant signaling through CD40 and TLR7 drives expansion of self-antigen–specific CD8⁺ T cells with enhanced cytolytic activity. C57BL/6 mice were immunized intravenously with 100 μg of the tumor-associated antigen ΔV, 100 μg αCD40 FGK45, and 100 μg S-27609 in combinations as indicated. Seven days later, mice were bled and cells were restimulated in vitro with TRP2_(180-188) to assess the ability to produce IFNγ and translocate CD107a as described in “Methods.” Lymphocytes were identified by forward and side scatter and subsequently gated on all CD8⁺ events. (A) Representative dot plots from vaccinated mice. The numbers in the upper right corners indicate the frequency of CD8⁺ T cells that are positive for IFNγ and CD44 (top row) or IFNγ and CD107a (bottom row). (B) Percentage of peripheral blood lymphocytes expressing the CD8 antigen. P ≤ .001 by one-tailed ANOVA (C) Quantification of the percentages of CD8⁺ cells that degranulated in response to peptide restimulation. In all cases, data presented are representative of at least 3 independent experiments. Data are plotted as means plus or minus SEM (n = 8 in each group). P ≤ .001 by one-tailed ANOVA.