Tetramer analyses 7 days (Fig. 5B, i-HEK-LyUV) revealed low responses dominated by NP396 and GP33. With regard to NP205 and GP276, the values obtained were barely higher than the background staining of naïve mice (Fig. 5B, 0.3%). We also compared these data with APC pulsed with each peptide separately, then pooled at equal ratios and injection i.v. Tetramer analysis on day 7 revealed Ipilimumab manufacturer that CTL were dominated by NP396 and GP33 epitopes (Fig. 5B, DC2.4-peptide) similar to the cross-priming data (Fig. 5B). To confirm these data, we expanded all four epitope-specific CTL obtained 7 days after cross-priming, for further

8 days with peptide-pulsed APC in separate wells. When we tested these CTL in a peptide restimulation assays, we found that the response was again dominated by NP396- and GP33-specific CTL, with few detectable NP205 and GP276-specific CTL (Fig. 5C). These experiments indicated that cross-priming after LCMV infections favors the CTL response toward GP33 and NP396. To address which

pAPC subsets are required to cross-present LCMV antigens in vivo, we harvested peritoneal exudates cells 8 h post-i.p. injection and separated the cells based on CD11c expression. As shown in Fig. 6A, the sorted CD11c+ population was of high purity (80–90%). We examined the AZD1208 mouse cross-presentation capacity of CD11c+versus CD11c− cells by incubating them with epitope-specific CTL. The data obtained in Fig. 6B show that CD11c+ were more efficient than CD11c− cells at cross-presenting the various LCMV epitopes. Although the values obtained were low, ID-8 it was still clear that cross-presentation was most efficient with NP396 compared with NP205, GP33, and GP276 and that the CD11c− cells cross-presented GP33 but with low efficiency. We also examined cross-presentation capacities of spleen resident pAPC in similar experimental protocols but could not detect any significant CTL activation probably due the limited antigen threshold (data not shown). Additionally, we asked whether

inducing cross-priming of different epitopes could affect the immunodominance during subsequent viral infection. Control WT HEK cells did not impact the immunodominance hierarchy when compared with PBS, with GP33>NP396>GP276=NP205 (Fig. 7A). If infected HEK-LyUV were introduced first, it caused GP276>NP205, but GP33 remained>NP396 (Fig. 7A, i-HEK-LyUV). We compared these data with LyUV-treated HEK-NP where NP396 was the main epitope being cross-presented (Fig. 7A, HEK-NP). In the latter condition, NP396 was the only immunodominant epitope possibly due to the prior expansion of NP396-specfic CTL, which competed out the naïve GP33-specific T cells. This did not occur when infected-ADC were tested since GP33 was also cross-presented (Fig. 7A, i-HEK-LyUV).