Neutrophil elastase enhances antigen presentation by upregulating human leukocyte antigen class I expression on tumor cells

Abstract

Neutrophil elastase (NE) is an innate immune cell-derived inflammatory mediator that we have shown increases the presentation of tumor-associated peptide antigens in breast cancer. In this study, we extend these observations to show that NE uptake has a broad effect on enhancing antigen presentation by breast cancer cells. We show that NE increases human leukocyte antigen (HLA) class I expression on the surface of breast cancer cells in a concentration and time-dependent manner. HLA class I upregulation requires internalization of enzymatically active NE. Western blots of NE-treated breast cancer cells confirm that the expression of total HLA class I as well as the antigen-processing machinery proteins TAP1, LMP2, and calnexin does not change following NE treatment. This suggests that NE does not increase the efficiency of antigen processing; rather, it mediates the upregulation of HLA class I by stabilizing and reducing membrane recycling of HLA class I molecules. Furthermore, the effects of NE extend beyond breast cancer since the uptake of NE by EBV-LCL increases the presentation of HLA class I-restricted viral peptides, as shown by their increased sensitivity to lysis by EBV-specific CD8+ T cells. Together, our results show that NE uptake increases the responsiveness of breast cancer cells to adaptive immunity by broad upregulation of membrane HLA class I and support the conclusion that the innate inflammatory mediator NE enhances tumor cell recognition and increases tumor sensitivity to the host adaptive immune response.

Keywords: Adaptive immunity; Breast cancer; HLA class I; Neutrophil elastase; Tumor-associated neutrophils.

Fig. 1

NE uptake enhances the susceptibility of breast cancer cells to lysis by E75-CTL. E75-CTL was incubated with the HLA-A2⁺ breast cancer cell lines MDA-MB-231, HER18, and SKBR3-A2 that were maintained in standard media ± NE (10 µg/mL) in calcein-AM cytotoxicity assays at various E (effector): T (target) ratios. NE uptake by all three cell lines resulted in their increased lysis by E75-CTL. Assays were performed in triplicate; results are representative of 5 separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 comparing NE treated to untreated

Fig. 2

NE uptake leads to increased surface expression of HLA class I in breast cancer. MDA-MB-231, MDA-MB-468, MCF-7, HER18, and SKBR3 breast cancer cells were maintained in standard media ± NE (10 µg/mL). a–c NE uptake corresponded with an increase in HLA-ABC and HLA-A2 on the cell surface. d, e NE uptake in MDA-MB-231 cells led to concentration-dependent and time-dependent increases in surface expression of HLA-A2 and HLA-ABC. f IFN-γ treatment of MDA-MB-231 cells led to an increase in HLA-A2 compared to untreated cells. The addition of NE further increased HLA-A2 surface expression compared to IFN-γ treatment alone. Experiments were performed in triplicate; results are representative of 3 separate experiments. *p < 0.05, **p < 0.01, ***p < 0.001 comparing NE treated to untreated, unless designated; MFI, median fluorescence intensity

Fig. 3

Increased cell surface HLA class I expression is not due to an increase in total HLA class I protein expression. MEL526, OVCAR3, H2023 and MDA-MB-231 cells were maintained in media supplemented with either NE or IFN-γ. a Western blot analysis showed no change in HLA class expression in any of the cell lines after NE uptake. b In contrast, Western blot analysis showed increased HLA class I expression in all cells lines maintained in media supplemented with IFN-γ. c RT-qPCR evaluating HLA-A, B, or C mRNA was carried out using RNA extracted from MEL526, OVCAR3, H2023 and MDA-MB-231 cells maintained in media supplemented with either NE or IFN-γ. For cells maintained in NE-supplemented media, there was no change, suggesting that HLA class I expression is post-transcriptionally regulated. In contrast, for cells maintained in IFN-γ-supplemented media, there was an increase in HLA-A, B and C transcripts suggesting transcriptional regulation. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

Fig. 4

Uptake of enzymatically active NE is required for the regulation of HLA class I by NE. a–c CPZ treatment abrogated the effects of NE on HLA-A2 and HLA-ABC surface expression by inhibiting NE uptake. d, e Inhibition of NE enzymatic activity by co-culture of NE with either elafin or PMSF abrogated the increase in HLA-A2 and HLA-ABC expression in MDA-MB-231 cells. f Enzymatic inhibition of NE following the incubation of MDA-MB-231 cells with PMSF or elafin resulted in increased NE uptake compared with enzymatically active NE. Experiments were performed in triplicate; results are representative of 3 separate experiments. *p < 0.05; **p < 0.01; ***p < 0.001; MFI median fluorescence intensity, PMSF phenylmethanesulfonyl fluoride, CPZ chlorpromazine

Fig. 5

NE uptake does not affect the HLA class I antigen-processing machinery. a Western blot analysis showed no change in expression of HLA class I heavy chain or beta-2-microglobulin (β2M) in MDA-MB-231 cells after NE uptake. b NE did not affect APM components, including LMP2 (23 kDa), TAP1 (81 kDa), calnexin (75 kDa), and tapasin (50 kDa). Actin or vinculin was used as loading controls. Results are representative of 3 separate experiments. APM, antigen-processing machinery