[Pentamer guided HLA-restricted epitope identification for mucoprotein 4 antigen of pancreatic ductal adenocarcinoma].

Objectives To identify HLA-restricted epitope of mucoprotein 4 (MUC4) antigen as a tumor associated antigen of pancreatic ductal adenocarcinoma (PDAC), and to validate its natural presentation in PDAC patient peripheral blood. Methods Two epitope prediction databases (SYFPEITHI and ProPred- Ⅰ)were used to predict HLA-A * 0201 restricted MUC4 epitope, T2 cell assay was used to determine the peptide binding affinity with HLA-A * 0201 molecule. Dendritic cells(DCs) were induced from the HLA-A * 0201-positive healthy individuals' peripheral blood mononuclear cells (PBMC). Mature DCs were pulsed with synthesized peptides. Autologous CD8 + T cells from the HLA-A * 0201 healthy donor were stimulated with the peptide-pulsed DCs as CTL. CTL activity was assessed by lactate dehydrogenase release assay and IFN-γreleased by enzyme-linked immunospot assay. Pentamer was synthesized for HLA-A * 0201 restricted epitope P1126,then was used to detect specific CTL in PBMC of PDAC patients. Results Five candidate HLA-A * 0201 epitopes were predicted, LLLGVGTFV ( P1125 ) and LLGVGTFVV ( P1126 ) were determined as the two with more HLA-A * 0201 affinity. Mature DCs could be induced from PBMCs. CTL induced by peptide P1126 could lyses T2 cells pulsed with peptide P1126 and HCT-116 cells[MUC4+,HLA-A2+]. The number of CTL induced by peptide P1126 which could secret IFN-γ( 130.3 ± 6.6 ) was obviously higher than that in the negative group. By Pentamer assay, P1126-pentamer and CD8 double positive CTL could be detected in PBMC of PDAC patients with MUC4( + )than patients with MUC4( - ),but no significant difference of CTL frequency between patients with HLA-A2( + )and with HLA-A2( - )in MUC4( + ) PDAC patients. Conclusions Tumor associated antigen MUC4-derived HLA-A * 0201-restrictive cytotoxic T lymphocyte (CTL)epitope P1126 can induce CTL reaction. The CTL can secret immunologic active material to induce the specific target cells lysis. P1126 epitope can be naturally presented in PBMC of PDAC patients,but its HLA-restriction may not be perfect. Key words: Pancreatic neoplasms; HLA-A antigens; Monocytes