Peripheral Tolerance to PR1 Is Maintained After Uptake of Soluble Proteinas-3 and Neutrophil Elastase

Abstract 4301

The leukemia associated antigens (LAA) proteinase-3 (P3) and neutrophil elastase (NE) are serine proteases found within neutrophil azurophil granules and are aberrantly expressed by leukemia. The human leukocyte antigen (HLA)-A2 restricted nonomeric peptide PR1, derived from P3 and NE, has been detected on leukemia cell surface in association with HLA-A2. PR1-specific cytotoxic T lymphocytes (CTL) were detected in patients following hematopoietic stem cell transplantation and were associated with clinical remission. Furthermore, in a phase I/II clinical trial, PR1-vaccine showed immunologic and clinical responses in patients with MDS and AML. Antigen cross presentation is a mechanism by which exogenous antigens are taken up by antigen presenting cells (APC) and presented in association with major histocompatibility (MHC)-I molecule. Cross presentation is thought to be the primary mechanism by which tumor antigens are presented to elicit tumor specific immune responses. Cross presentation by dendritic cells (DC), which express co-stimulatory molecules, leads to immune priming, while antigen presentation by B cells is associated with immune tolerance. Since cross presentation of leukemia antigens has been associated with anti-leukemia immune responses, we investigated whether P3 and NE are cross presented by DCs and B cells, and whether the source of P3 and NE (soluble vs. leukemia-cell associated) determines priming of the anti-PR1 immune response. We have previously reported that serum P3 levels are significantly higher in leukemia patients, and we now also report higher serum NE levels in patients with AML (median±standard deviation (SD)= 1236 ±572 ng/ml; n=15) and CML (median ± SD = 941 ± 494 ng/ml; n=15), in comparison with healthy donor blood (median ± SD = 65 ± 177 ng/ml; n=5) (p<0.05). Using flow cytometry and confocal microscopy, we demonstrate uptake of soluble P3 and NE into lysosomes by B cells as well as normal and leukemia-derived DCs. Using western blots (WB), we show that leukemia-cell associated P3 and NE are found in nuclear, membrane, cytosolic and skeletal compartments, unlike neutrophils where they are primarily limited to the granule-containing compartment. Moreover, immunoprecipitation and WB assays showed that intracellular P3 and NE are ubiquitinated, suggesting that they are processed by proteasomes for subsequent PR1 presentation. Using BrDU uptake assay, we show that cross presentation of PR1 by HMY.CIR-A2 APC cell line leads to a 2-fold increase in proliferation of in vitro generated PR1-specific CTLs through a proteasomal dependent pathway. Using the monoclonal antibody 8F4 (anti-PR1-HLA-A2), we show that PR1 is cross presented by normal B cells from soluble P3 and NE (1.6 fold increase in PR1 expression vs. unpulsed B cells). Furthermore, we show that B cells cross present PR1 from cell-associated NE and P3 from irradiated HLA-A2+ and HLA-A2- primary leukemia and leukemia cell lines (1.7 fold increase in PR1 expression vs. unpulsed B cells). In contrast to B cells, normal DCs cross presented cell-associated, but not soluble, P3 and NE (1.6-2.0 fold increase in PR1 expression vs. unpulsed or soluble NE/P3 pulsed DCs). Furthermore, cell-associated NE and P3 increased the co-stimulatory receptors CD83, CD86, and HLA-DR in normal DCs, while soluble NE and P3 did not affect, and in some cases decreased, expression of co-stimulatory DC receptors. In conclusion, this study shows that 1) soluble and cell-associated P3 and NE are cross presented by B cells, 2) only cell-associated P3 and NE are cross presented by DCs, and 3) cell-associated, but not soluble, P3 and NE increased co-stimulatory receptors in DCs, all suggesting that cross presentation of soluble P3 and NE leads to tolerance to PR1. Since serum P3 and NE are increased in leukemia, this may be a mechanism by which leukemia evades the anti-PR1 immune response. Our findings may also be applicable not only to the anti-leukemia immune response, but also to autoimmune diseases where serum P3 and NE were shown to be increased and to inflammatory states that are common to many cancers.