Abstract 1257: Investigating the protective role of HLA-B35 in pancreatic cancer.
Authors: Yanagisawa Ryu , Keirnan Jacqueline M. , Katari Usha L. , Anurathapan Usanarat , Brenner Malcolm K. , Rooney Cliona M. , Vera Juan F. , Leen Ann M. 
Published: August 2014

HLA-B35 has recently been reported as a protective marker for pancreatic cancer development. The goal of the current study was to investigate whether this link between B35 and cancer protection was related to cellular immunity and correlated with the presence of HLA-B35-restricted cytotoxic T cell activity. As a model antigen we chose mesothelin (MSLN), a tumor antigen expressed by &gt;90% of pancreatic tumors and known to be immunogenic to T cells. To activate MSLN-specific T cells we generated autologous DCs from 15 HLA-B35 positive and 12 HLA-B35 negative donors and loaded them with a pepmix (15mers overlapping by 11aas) spanning MSLN in order to use an HLA unbiased approach for antigen stimulation. These were used to stimulate PBMCs in the presence of a cytokine cocktail (IL7, 12, 15, 6), which we have optimized for activating tumor-directed T cells in vitro. We were successfully able to amplify an immune response against MSLN in 12 of 15 (80%) B35+ donors based on IFNg ELIspot analysis (range 23-10108 SFC/2x10e5). In contrast we were successful in only 1 of 12 (8%) B35-ve donors. Since the CTL lines were polyclonal (CD8+, median 69%, range 38-97%; CD4+, median 30%, range 2-55%) we next investigated, using ICS, whether this anti-tumor activity was in the helper or cytotoxic T cell compartments. Eight of 13 MSLN responders had activity exclusively in CD8+ T cells (range 3-64% IFNg+ cells) and 2 had activity in both CD8 and CD4 T cells, while in the only non-B35 responder activity was detected exclusively in the CD4+ T cell compartment. We next performed epitope mapping studies using peptide minipools and detected activity against 3 published CD8+ epitopes [HLA-24 FYP (aa435-443) and LYP (aa475-483); HLA-A2 VLP (aa530-538)] as well as 5 novel B35-restricted epitopes, one of which was detected in 7 of our 12 B35 responders. Finally, to assess whether these novel epitopes were recognized in the context of HLA-B35 we loaded autologous and partially HLA-matched APCs with the individual peptides and assessed cytolytic activity. Autologous peptide-loaded targets were killed (range 52-98% killing, E:T 40:1) as were allogeneic targets exclusively matched at HLA-B35 (range 32-92% killing), confirming that all epitopes were presented by the HLA-B35 allele. These B35-restricted CTLs were also able to kill HLA-matched tumors expressing MSLN - we saw specific lysis of CFPAC-1 (B35+MSLN+) (36% killing E:T 40:1) with no recognition of CAPAN-1 (B35-MSLN+) (3% killing). Thus, our results support a contribution of HLA-B35-restricted T cells and anti-tumor activity in pancreatic cancer and highlight the importance of cellular immunity in protection against cancer.

Citation Format: Ryu Yanagisawa, Jacqueline M. Keirnan, Usha L. Katari, Usanarat Anurathapan, Malcolm K. Brenner, Cliona M. Rooney, Juan F. Vera, Ann M. Leen. Investigating the protective role of HLA-B35 in pancreatic cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1257. doi:10.1158/1538-7445.AM2013-1257