Fig. 1

Mass spectrometry analysis of HLA Class I epitopes in PDAC tumor cell lines and tissues. MaxQuant was used to identify the peptide sequences with a false discovery rate (FDR) of 1%. A Histograms show the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC cell lines, Panc10.05 and Panc06.03. These peptides correspond to 363 and 1238 unique proteins, respectively. B Representative histograms show the numbers of different lengths of peptides affinity purified by anti-HLA Class I antibody from human PDAC tissues. C The numbers of HLA Class I epitopes and their associated proteins identified from each individual PDAC tissues. From the 10 PDACs, a total of 14,632 peptides and 11,849 unique peptides, corresponding to 6086 non-redundant proteins, were identified. Note that the numbers of eluted peptides from different PDAC specimens varied between 296 and 3270 (1331 on average). These peptides correspond to 123–2041 proteins (782 on average), respectively. D Predicted HLA Class I binding affinity of eluted peptides from representative PDAC tissues, Pan12 and Pan11, using the NetMHC4.0 algorithm. The black dot lines represent the 500 nM threshold of high binding affinity. Note that 339 eluted peptides and 219 eluted peptides from the Pan12 PDAC specimen (81.7% and 52.8% of the total of 415 9-mer peptides, respectively) showed a low predicted binding affinity to the patient’s class I HLA types, HLA-A*2902 and HLA-A*3301, respectively. Similarly, 343 eluted peptides and 319 eluted peptides from the Pan11 PDAC specimen (73.3% and 68.2% of the total of 468 9-mer peptides, respectively) showed a low predicted binding affinity to HLA-A*0101 and HLA-A*2902, respectively. A similar degree of netMHC in missing the peptides with high binding affinity was reported in the prior study [11]. E Criteria of selecting peptides for validation: (1) those that are shared by multiple patients; (2) those whose predicted HLA-binding affinity ranks among the top 0.5% of all peptides, which is the recommended threshold for the selection of peptides by NetMHC); (3) those whose corresponding proteins are overexpressed in tumor epithelia of PDAC compared to normal pancreas according to the Human Protein Atlas (https://www.proteinatlas.org/). These criteria were used with a consideration of developing therapeutic agents in the future. Eight peptides that met the selection criteria include four HLA-A2 peptides (COL6A3, ELOVL1, LAMC2, RASAL2) and four HLA-A3 peptides (DYNLRB1, ICE1, LAMB3, MYH9) (Additional file 1: Fig. S4). F Numbers of HLA class I peptides from representative PDAC samples including Pan04, Pan06, and Pan07 and those of completely overlapped peptides among all three or any two of three PDAC samples were indicated (left). Numbers of peptides considered as strong binders (ranks among the top 0.5%) for HLA-A0201 (upper right) and HLA-A0301 (lower right) in Pan06 and Pan07, respectively, and those of overlapped peptides between Pan06 and Pan07 were also indicated. The sources of eight selected peptides were indicated. G–I T2 cell binding assays of selected HLA-A2 and A29 peptides binding to HLA-A2 expressing T2 cells (G), HLA-A3 expressing T2 cells (H), and HLA-A1 expressing T2 cells (I). Twelve peptides that consisted of five peptides (ORMDL3, MYL12A, LAMC2, WDR82, TRRAP) shared by multiple HLA-A2 PDACs and seven peptides (TFIP11, ACBD3, CKS2, IGF1, TRAPPC11, ZMYND11, CTNNBIP1) shared by multiple HLA-A29 PDACs were selected according to the criteria listed in (E) (Additional file 1: Fig. S8) and their sequences were shown. Controls indicate negative control peptides. MFI: mean fluorescent intensity. Unpaired t test and 1-way ANOVA was used for comparing between samples. *p < 0.05, **p < 0.01