2 μg S1-conjugated anti–DNGR-1 (7H11) or rat IgG1 isotype-matched control mAbs were injected s.c. with or without anti-CD40 (25 μg) as indicated. Target cells were injected 5 days later, and mice were analyzed on day 6. (A) In vivo CTL activity as measured by target cell elimination. Histograms show target cell frequency in representative mice from each group (CFSE^lo, 20 nM peptide; CFSE^int, 200 nM peptide; CFSE^hi, no peptide). Graph shows mean ± SEM of percentage of specific lysis in 1 experiment of 3 (n = 6 mice/group). All groups are shown, but the only one in which killing was detectable was that receiving anti–DNGR-1–S1 plus anti-CD40. (B) H-2K^b–SIINFEKL tetramer staining of splenocytes. Left panel shows representative dot plots of tetramer staining versus CD8 in gated CD8⁺ Thy1⁺ T cells. Right panel shows frequency of tetramer-positive CD8⁺ T cells in 1 experiment of 3 (n = 6 mice/group). (C) In vitro restimulation with 1 μM SIINFEKL (PEPTIDE) or medium alone (CTR). Left panel IFN-γ content in supernatants at the end of the 5-day culture. Right panel shows specific CTL activity of in vitro–restimulated cells against EL4 targets loaded with 2 μM of SIINFEKL. Data are the average + SEM of all cultures (n = 6 mice/group, restimulated individually). P values were calculated using Student’s t test. (D) OVA protein conjugated to anti–DNGR-1 induces CTL priming in vivo. Mice were immunized s.c. in the paw with 2 μg anti–DNGR-1 or isotype control antibodies conjugated to OVA protein in the presence of 25 μg of anti-CD40. In vivo killing activity was analyzed as in A using targets loaded with 200 nM SIINFEKL peptide. Results represent individual mice and the mean for 1 representative experiment out of 3. n = 5; P < 0.01, t test.