Introduction: Neutrophil elastase (NE) is an inflammatory mediator that is taken up by breast cancer cells. We have previously shown that NE uptake increases susceptibility to lysis by cytotoxic T lymphocytes (CTL) targeting the tumor antigens PR1 and cyclin E. We investigated whether NE uptake alters adaptive immunity by affecting MHC class I antigen presentation and T cell inhibitory molecules.

Methods: MDA-MB-231 breast cancer cells were maintained in standard media ± NE. Healthy donor HLA-A2+ peripheral blood mononuclear cells were used to generate antigen specific CTL by pulsing dendritic cells with E75, a HER2-derived epitope we are currently investigating as a vaccine in clinical trials. Calcein-AM cytotoxicity assays evaluated E75-specific lysis. Flow cytometry was used to show NE uptake, HLA-A2, pan-HLA class I (HLA-ABC) and PD-L1 surface expression. Western blot analysis was used to show total MHC class I heavy chain, B2M, LMP2, TAP1, calnexin and tapasin expression. Relative PD-L1 mRNA levels were determined using qPCR.

Results: NE uptake enhanced tumor cell lysis by E75-specific CTL at all effector to target (E:T) ratios (lysis for 20 to 1 E:T ratio was 50% ± 7 for NE treated vs 28% ± 7 for untreated, p < 0.05), and led to an increase in HLA-A2 and HLA-ABC surface expression (p < 0.05; Table). However, NE uptake did not change total expression of MHC class I or components of the MHC class I pathway. Additionally, NE uptake led to a decrease in PD-L1 surface expression and transcript levels (p < 0.05; Table).

Conclusion: NE increases tumor antigen presentation as well as MHC class I on the cell surface. However, NE uptake does not change total MHC class I expression or expression of MHC class I antigen processing components, suggesting that NE increases peptide availability as a mechanism of increased MHC class I antigen presentation. NE also leads to the downregulation of PD-L1. Together, our data indicate that NE uptake enhances adaptive immune responses.

 HLA-A2 Surface Expression (MFI) HLA-ABC Surface Expression (MFI) PD-L1 Surface Expression (MFI) PD-L1 mRNA Expression (RQ) 
NE Treated (Mean ± SD) 7341 ± 867 1317 ± 120 1723 ± 55 0.274 ± 0.02 
Untreated (Mean ± SD) 5144 ± 272 802 ± 133 6966 ± 451 
 HLA-A2 Surface Expression (MFI) HLA-ABC Surface Expression (MFI) PD-L1 Surface Expression (MFI) PD-L1 mRNA Expression (RQ) 
NE Treated (Mean ± SD) 7341 ± 867 1317 ± 120 1723 ± 55 0.274 ± 0.02 
Untreated (Mean ± SD) 5144 ± 272 802 ± 133 6966 ± 451 

Citation Format: Akhil Chawla, Anne V. Philips, Na Qiao, Pariya Sukhumalchandra, Celine Kerros, Gheath Alatrash, Jeffrey J. Molldrem, Elizabeth A. Mittendorf. Neutrophil elastase enhances adaptive immunity via increase of peptide presentation and downregulation of T cell inhibitory molecules. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4843. doi:10.1158/1538-7445.AM2014-4843