Abstract
Introduction
PR1 is a nonameric HLA-A2 restricted leukemia-associated antigen derived from serine proteases neutrophil elastase (NE) and proteinase 3 (P3). NE and P3 are primarily expressed in cells of myeloid lineage including granulocytes, bone marrow progenitors and myeloid leukemia. However, NE and P3 are also cross-presented by antigen presenting cells (APCs) such as B-cells and some non-hematopoietic solid tumors, a mechanism whereby exogenous antigens are endocytosed and presented on HLA class I molecules. Therefore, identifying non-myeloid tumors capable of PR1 cross-presentation broadens the application of PR1-targeted immunotherapies, which to date include PR1 peptide vaccine, PR1 cellular therapy, and 8F4, a T cell receptor (TCR)-like monoclonal antibody (mAb).
One possible source of NE and P3 in the microenvironment is neutrophil extracellular traps (NETs). NETs are composed of deoxyribonucleic acid (DNA)/histones and the antimicrobial proteases, NE and P3, which are extruded from activated polymorphonuclear neutrophils (PMNs) during inflammation. Although a main function of NETs is elimination of pathogens, studies have shown immune-regulatory effects of NETs. Thus there is a great interest in understanding the role of NETs in modulating adaptive immune system in the setting of anti-tumor immunity. We hypothesized that NETs facilitate NE and P3 cross-presentation in malignant B-cell acute lymphoblastic leukemia (ALL) due to their high abundance in the bone marrow, which could lead to susceptibility to PR1-targeted immunotherapies.
Methods
B-ALL cell lines SB, Nalm6, RS4 and Nalm6 were co-cultured with soluble NE and P3 (10 ug/mL) and PMNs at a ratio of 3:1 (PMN: ALL) in low serum media and analyzed for NE and P3 cross-presentation by staining for HLA-A2/PR1 complex using 8F4 mAb. T2 cells pulsed with PR1 and HIV peptide were used as a positive and negative control, respectively. To determine susceptibility to killing my PR1-CTLs, SB cell line was co-cultured with 10 μg/mL soluble NE and P3 for 24 hours and incubated with healthy PR1-CTLs for 4 hours following a protocol for a standard calcein-AM cytotoxicity assay. T2 cells were pulsed with PR1 and CG1 peptide as a positive and negative control, respectively.
To identify NETs as a source of NE and P3, healthy PMNs were treated with ionomycin to induce NETosis, co-cultured with B-ALL cell lines at a ratio of 3:1 (PMN: ALL) overnight and analyzed for NE and P3 uptake. NETs were also treated with DNAse to determine the effect on uptake.
Results
The B-ALL cell line, SB, cross-presented PR1 from soluble P3 and NE, as well as supernatants derived from quiescent PMNs. There was a 1.3 fold significant increase in PR1/HLA-A2 surface expression when co-cultured with soluble and PMN-associated protease (Figure 1A). We then investigated whether PR1 cross-presentation by SB cell line rendered it susceptible to killing by PR1-CTLs. Dose-dependent killing of SB cells was observed following PR1 cross-presentation compared to nonpulsed cells (Figure 1B). The percent specific lysis was evident even at a very low ratio of 0.625:1 Effector: Target cells (E:T). As controls, T2 cells pulsed with PR1 showed dose-dependent killing by PR1-CTLs, whereas T2 cells pulsed with CG1 and nonpulsed did not, as expected (Figure 1B).
To evaluate whether NET-associated NE and P3 facilitate uptake in B-ALL, healthy PMNs were co-cultured with B-ALL cell lines overnight. The co-culture resulted in both NE (Figure 2A-B) and P3 (Figure 2C-D) uptake in RS4 and Nalm6 cell lines with an average MFI of 350 and 426, respectably, compared to nonpulsed MFI of 145. Measurement of intracellular P3 and NE by flow cytometry showed that although both NET-associated and PMN-associated NE and P3 are taken up, uptake of supernatants from resting PMNs was significantly greater than NET-associated (Figure 2). Similar to uptake of soluble and PMN-associated proteases, uptake between B-ALL cell lines was heterogeneous.
Conclusion
Collectively, our data demonstrate that B-ALL cell lines are capable of PR1 cross-presentation rendering them susceptible to killing by PR1-CTLs. Further, NET-associated NE and P3 are a source of protease uptake. Because NET-associated antigens are less efficient at uptake, our data suggest that NETs produced during systemic inflammation might be less immunogenic in B-cell malignancies.
No relevant conflicts of interest to declare.
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