Figure 3.

Experimental validation of in silico predictions of the MHC-I binding affinity. RMA-S cells were pre-incubated for 1 h at 4°C. Then, 4 × 10⁶ cells were incubated for 2 h with one of the indicated peptides at two different concentrations (A) 1 µg /mL or (B) 0.1 µg/mL in a volume of 1 mL. The presence of H-2Kb molecules on the membrane was measured by flow cytometry and normalized against cells incubated with no peptide (negative control). (C) OT-I splenocytes were labeled with CFSE dye. Then, 3 × 10⁴ cells were incubated with different stimuli for 72 h in a volume of 200 µL of complete media. Then the percentage of proliferating (i.e., CFSE diluted) CD3⁺CD8⁺ T-cells was determined by flow cytometry; data was normalized against positive control Concanavalin A (defined as 100% of proliferation). All the data are represented as mean ± SD; Significance was assessed by One-way ANOVA with Tukey's post hoc test; *p < 0.05, ** p <0.01.