(A) Heatmap representing mean MFI of H-2Kb from RMA-S assay. Green, immunogenic controls. (B) Immunization schema. (C) Representative IFNγ ELISpot from CD8+ T cells upon stimulation with syngeneic peptide-loaded splenocytes. Each row is a peptide used for in vivo immunization. Columns, T cells reacted with the indicated stimuli. PMA: Phorbol 12-myristate 13-acetate; Iono: ionomycin. (D) Spots per 105 CD8+ T cells from IFNγ ELISpot for peptides identified as immunogenic. Bar indicates median. SIINFEKL, positive control. Each dot is one technical replicate. (E) Representative IFNγ ELISpot of CD8+ T cells from immunized mice, following stimulation with syngeneic peptide-loaded splenocytes. Each row is one dose. Columns, T cells reacted with the indicated stimuli. Plots on right quantify numbers of dots per well; each dot is one technical replicate. (F) Comparisons of predicted MHC I binding for immunogenic (IFNγ ELISpot-positive) versus nonimmunogenic peptides. (G) As (F), but with RMA-S MFIs. For (F-G), p from two-sided Wilcoxon rank-sum test. See also Figure S7 and Table S7.