Fig 2: Stable expression of HLA molecules and binding to various antigen peptides in murine 3T3 cells. (A) Western blot analysis of HLA-DR expression levels in various HLA-expressing 3T3 cells. (B,C) FACS analysis of cell-surface expression of HLA-DR molecules in 3T3 cells. The grey peak and pink peak represent untransfected control cells and respective DRB1 transfected cells, respectively. The graph shown is a summary of the FACS results of two independent measurements and expressed as mean ± range (C). Western blotting and peptide binding assay were performed, and one representative result is shown. (D) Summary of the relative binding affinity of various HLA-DR and antigen peptides. Peptide binding assay was carried out in various HLA-expressing 3T3 cells (Supplementary Fig. 2). For each peptide group, peptide binding/HLA was calculated by subtracting the MFI value for the parental 3T3 cells from individual MFI. Then, the net MFI values were divided by the respective MFI value for HLA expression levels (Fig. 2C). The values are mean ± inter-assay deviation expressed as SEM of three independent experiments. Statistical significance is as follows: *P < 0.05 and **P < 0.01.

Fig 3: Immunopeptidomic profiling of HLA-DR and HLA-DP-bound peptides presented by S-pulsed MDDCs(A and B) Number of unique peptide sequences identified in each HLA-DR (A) or HLA-DP (B) immunoprecipitation sample.(C and D) Number of unique peptide sequences that map to S identified in the HLA-DR (C) or HLA-DP (D) samples.(E) Total number of MDDCs harvested from each donor.(F and G) Length distribution of peptide sequences identified as mapping to human proteins (blue) or S protein (orange).(H) Proportion of peptide sequences predicted to bind to each donor’s HLA-DR and -DP alleles by NetMHCIIpan 4.0.(I) Proportion of total predicted HLA-DR binders stratified by allele for each donor.(J) All 12–20 mers in each sample were clustered using the online (unsupervised) GibbsCluster algorithm. Each cluster is represented by a sequence logo, which corresponds to at least one of the HLA-DRB alleles expressed by the donor MDDCs. Amino acids are represented by their single letter code; the more frequently an amino acid occurs a position within peptides, the larger the letter is displayed. The number (n) of peptides within each cluster is indicated along with the number of outlier peptides removed, and clusters are presented with the specific sequence motifs for donor DR alleles as reported by NetMHCIIpan 4.0.See also Figures S4 and S5 and Table S1.