Uptake Of Neutrophil Elastase Research Articles

Cathepsin G Is Expressed By B Cell Acute Lymphoblastic Leukemia and Is an Effective Immunotherapeutic Target

The azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3) have been established as effective immunotherapeutic targets in hematological malignancies. Expression of PR1, an HLA-A2 restricted nonameric peptide derived from NE and P3, has been demonstrated in acute myeloid leukemia (AML). We have successfully targeted PR1 in AML patients using PR1-peptide vaccine, anti-PR1/HLA-A2 antibody and PR1-specific cytotoxic T-lymphocytes (PR1-CTL). We have also identified the uptake and cross-presentation of NE and P3 by breast cancer, lung cancer and melanoma, which underlines their potential as immunotherapeutic targets in non-myeloid malignancies.Cathepsin G is also an azurophil granule serine protease that is highly expressed by AML blasts and leukemic stem cells. Our previous studies showed a high level of efficacy in targeting AML blasts that express CG1 (FLLPTGAEA) using CG1-specific CTL (CG1-CTL), while sparing normal hematopoietic cells. However, two previous reports (Fujiwara et al., Clinical Cancer Research, 2005 and Gorodkiewicz et al., Analytical Biochemistry, 2012) and our published data (Alatrash G, Leukemia, 2017) have reported CG expression in lymphoid leukemia. We therefore hypothesized that CG is an effective immunotherapeutic target in lymphoid malignancies, specifically acute lymphoid leukemia (ALL).We first analyzed CG expression in ALL patient samples. Using reverse phase protein array (RPPA), we analyzed CG expression in ALL blasts from 345 adult ALL patient samples. We detected CG in 345 patient samples at levels comparable to G-CSF stimulated myeloid progenitors. The differential expression of CG in sorted ALL blasts ranged from -1.8030 to 5.4390, with a median value of 0.3506. In estimating CG levels, we found that more than 15% of our ALL samples expressed Log2 concentrations of CG between 0.3 and 0.4 (Figure 1). This was comparable to what we have previously reported in AML. We then wanted to determine whether the ALL-associated CG was due to uptake or endogenous expression. We performed PCR and western blotting and confirmed CG expression in 4 of 9 and 9 of 9 patient ALL samples, respectively (Figure 2). To assess whether CG is also taken up by ALL, we examined CG uptake by the B-ALL cell lines SB, RS4, Nalm6 and HMY (B-ALL). ALL cell lines were incubated with CG protein at increasing doses and durations. Intracellular staining of CG followed by flow cytometry analysis was used to determine uptake of CG by ALL cell lines. Our data show that all of the ALL cell lines take up CG; uptake correlated directly with increasing doses and time of incubation up to 24 hours.Having characterized the expression of CG by ALL, we sought to determine its potential as an immunotherapeutic target, as we have previously shown for AML. In these experiments, CG1-specific CTLs were generated using standard dendritic cells methodology, interleukin-7 (IL-7), IL-2 and CG1-peptide pulsing. Expanded CG1-CTL were confirmed by staining cells with CG1 tetramer followed by flow cytometry analysis. We employed standard calcein-AM cytotoxicity assays to determine specific lysis. In these experiments, patient ALL sample UPN8 was pulsed with calcein-AM and then co-cultured with CG1-CTL for 4 hours. Our results indicated that UPN8 was effectively lysed by CG1-CTL, reaching a maximum specific lysis of ~30% at the highest effector:target ratio (Figure 3).Overall, our analysis confirms CG endogenous expression and uptake by ALL. Furthermore, we conclude that CG is an effective target for immunotherapy in B-ALL. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 3984: Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells

Abstract We previously showed that soluble neutrophil elastase (NE) is rapidly taken up by tumor cells that lack endogenous NE expression, including breast cancer (BrCa). NE uptake led to cross-presentation of PR1, an NE-derived HLA-A2-restricted peptide that is an immunotherapeutic target in hematologic malignancies. The mechanism of NE uptake, however, remains unknown. Here, we demonstrated that NE internalization was temperature- and time-sensitive, dose-saturation was observed, and uptake was partially blocked by chlorpromazine (CPZ) and wortmannin, supporting a receptor-mediated process and suggesting a role for clathrin- and PI3K-dependent mechanisms. To identify a candidate NE receptor, we performed mass spectrometry (MS) of proteins after anti-NE co-immunoprecipitation of NE-pulsed MDA-MB-231 BrCa cells and we identified neuropilin-1 (NRP1) as an NE co-associated protein. Binding of NE to NRP1 was confirmed by ELISA, and peptide epitope mapping studies indicated NE bound via it consensus sequence RRXR, which is also known to bind the b1b2 domain of NRP1. NE bound to NRP1 with high affinity (Kd=38.7 nM) as measured with biolayer interferometry. To confirm the role of NRP1 in cellular uptake of NE, we transfected MDA-MB-231 BrCa cells with siRNA or shRNA against NRP1 and showed that NRP1 knockdown resulted in a 2-fold decrease in NE uptake as determined with flow cytometry. Similarly, blocking with an NRP1-neutralizing antibody decreased NE uptake by 60% vs. isotype control antibody, an effect that was also observed in other breast cancer cell lines that expressed NRP1. Conversely, transient expression of NRP1 in the NRP1-deficient T47D BrCa cell line was sufficient to induce uptake of NE. Importantly, knockdown of NRP1 expression in MDA-231 cells also prevented PR1 cross-presentation as determined with the anti-PR1/HLA-A2 monoclonal antibody 8F4. This was confirmed by the loss of susceptibility of NE-pulsed MDA-MB-231 cells to lysis by PR1-specific cytotoxic T cells following knockdown of NRP1 expression. Our data support a novel function of NRP1 in the uptake and cross-presentation of neutrophil-derived proteins by non-hematopoietic cancer cells. Because 8F4 mediates killing of PR1/HLA-A2+ leukemia and NRP1 is broadly expressed on many tumors, our results suggest a role for immunotherapy strategies that target NE-derived peptides on NRP1+ tumors. Citation Format: Celine Kerros, Satyendra C. Tripathi, Dongxing Zha, Sergeeva Anna, Haley L. Peters, Hiroyuki Katayama, Pariya Sukhumalchandra, Kathryn R. Cox, Alexander A. Perakis, Lisa S. St John, Gheath Alatrash, Elizabeth A. Mittendorf, Karen C. Dwyer, Samir M. Hanash, Jeffrey J. Molldrem. Neuropilin-1 mediates neutrophil elastase uptake and antigen cross-presentation in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3984. doi:10.1158/1538-7445.AM2017-3984

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

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.

Multiple Myeloma Cross-Presents Neutrophil Elastase and Proteinase 3 and Becomes Susceptible to PR1-Targeting Immunotherapy

Abstract 4973PR1 (VLQELNVTV) is a human leukocyte antigen (HLA)-A2 restricted nonameric peptide that has been effectively targeted in myeloid leukemia using PR1-peptide vaccine and PR1-specific cytotoxic T cells (CTL). A phase I/II clinical trial has been initiated with PR1 peptide vaccine, which demonstrated complete remission and immunologic responses in patients with acute (AML) and chronic (CML) myeloid leukemia, as well as myelodysplastic syndrome. We recently developed an anti-PR1/HLA-A2 antibody (8F4) and have demonstrated its activity against myeloid leukemia blasts in vitro and in a human leukemia xenograft mouse model.PR1 is derived from the serine proteases proteinase-3 (P3) and neutrophil elastase (NE), which are normally found within neutrophil azurophil granules and are released into the inflammatory milieu. We have shown that through cross-presentation, a process whereby exogenous antigen is taken up, processed and expressed on cell surface in association with human leukocyte antigen class I molecules, PR1 can be expressed on the surface of cells that do not endogenously express NE and P3. Specifically, we showed that P3 and NE are taken up and cross-presented by antigen presenting cells (APC), including B cells, as well as breast cancer and melanoma cells. Furthermore, cross-presentation by these non-myeloid cells led to their killing by PR1-CTL and 8F4 antibody.Since multiple myeloma is derived from B cells, which are deficient in NE and P3 but have intrinsic APC functions including cross-presentation, we hypothesized that P3 and/or NE may be taken up and cross-presented by multiple myeloma cells, hence rendering them targets for PR1-CTL and 8F4 antibody. Using RT-PCR and western blotting, we first show that NE and P3 are absent in myeloma cell lines including ARK, ARP-1, OMP-2, LP-1, U-266, IM9, and RPMI 8226. Intracellular staining for P3 and NE after culturing myeloma cell lines with P3 (10 ug/mL) or NE (10 ug/mL) confirmed uptake of P3 and NE by multiple myeloma cell lines; uptake is time dependent with variable plateauing between the cell lines over a 30-hour time course. Using 8F4, the novel PR1-HLA-A2 monoclonal antibody, we demonstrate that P3 and NE are cross-presented by the HLA-A2 positive U-266 myeloma cell line. Peak P3 and NE cross-presentation occurs at 24 hours and 6 hours, respectively. Next, we studied whether PR1 cross-presentation causes cells to be susceptible to PR1-specific killing by PR1-CTL and 8F4 monoclonal antibody. We show that cross-presentation increases susceptibility of U-266 myeloma cell line to killing by PR1-CTL. Specifically, following incubation with P3 (10 ug/mL) and NE (10 ug/mL), we show 30% and 25% killing, respectively, of U-266 cells by PR1-CTL versus unpulsed U-266 cells (10% killing). Furthermore, we show dose-dependent killing of the U-266 myeloma cell line by 8F4 antibody in a complement dependent cytotoxicity assay, with maximum 30% and 20% killing of U-266 cells after incubation with P3 (10 ug/mL) or NE (10 ug/mL), respectively, versus no killing in the unpulsed group. Finally, using PR1/HLA-A2 dextramer staining, we show PR1-CTL in peripheral blood from patients with multiple myeloma following stem cell transplantation (Median, 0. 06%; range, 0. 03%-0. 1%). Our data therefore show that if the uptake of P3 or NE, present in the inflammatory milieu of multiple myeloma bone marrow and extramedullary tumors, leads to PR1 cross-presentation, then PR1-based immunotherapy may be useful to treat patients with multiple myeloma. These results also support a new paradigm linking inflammation and innate immunity to adaptive immune responses to cancer. Disclosures:No relevant conflicts of interest to declare.

Breast Cancer Cell Uptake of the Inflammatory Mediator Neutrophil Elastase Triggers an Anticancer Adaptive Immune Response

There is little understanding of the impact of tumor-associated neutrophils (TAN) on adaptive immunity to tumors. In this study, we report the results of an investigation of the pathobiologic basis for the prognostic significance of neutrophil elastase, a serine protease found in neutrophil granules, in a model of cyclin E (CCNE)-overexpressing breast cancer. We established that neutrophil elastase was expressed by TAN within breast cancer tissues but not by breast cancer cells. Neutrophil elastase modulated killing of breast cancer cells by CTLs specific for CCNE-derived HLA-A2-restricted peptide (ILLDWLMEV). Breast cancer cells exhibited striking antigen-specific uptake of neutrophil elastase from the microenvironment that was independent of neutrophil elastase enzymatic activity. Furthermore, neutrophil elastase uptake increased expression of low molecular weight forms of CCNE and enhanced susceptibility to peptide-specific CTL lysis, suggesting that CCNE peptides are naturally presented on breast cancer cells. Taken together, our findings reveal a previously unknown mechanism of antitumor adaptive immunity that links cancer cell uptake of an inflammatory mediator to an effective cytolytic response against an important breast cancer antigen.

The Role of Antigen Cross-presentation From Leukemia Blasts on Immunity to the Leukemia-associated Antigen PR1

Cross-presentation is an important mechanism by which exogenous tumor antigens are presented to elicit immunity. Because neutrophil elastase (NE) and proteinase-3 (P3) expression is increased in myeloid leukemia, we investigated whether NE and P3 are cross-presented by dendritic cells (DC) and B cells, and whether the NE and P3 source determines immune outcomes. We show that NE and P3 are elevated in leukemia patient serum and that levels correlate with remission status. We demonstrate cellular uptake of NE and P3 into lysosomes, ubiquitination, and proteasome processing for cross-presentation. Using anti-PR1/human leukocyte antigen-A2 monoclonal antibody, we provide direct evidence that B-cells cross-present soluble and leukemia-associated NE and P3, whereas DCs cross-present only leukemia-associated NE and P3. Cross-presentation occurred at early time points but was not associated with DC or B-cell activation, suggesting that NE and P3 cross-presentation may favor tolerance. Furthermore, we show aberrant subcellular localization of NE and P3 in leukemia blasts to compartments that share common elements of the classic major histocompatibility class I antigen-presenting pathway, which may facilitate cross-presentation. Our data demonstrate distinct mechanisms for cross-presentation of soluble and cell-associated NE and P3, which may be valuable in understanding immunity to PR1 in leukemia.

Abstract 788: Uptake and cross-presentation of the leukemia associated antigens neutrophil elastase (NE) and proteinase-3 (P3) increases susceptibility of solid tumors to PR1-targeted immunotherapy

Abstract We have shown that the HLA-A2-restricted nonapeptide PR1 (VLQELNVTV) is a leukemia-associated peptide derived from P3 and NE. Furthermore, immunologic and clinical responses to PR1 peptide vaccination occur in patients with acute (AML), chronic (CML) myeloid leukemia and myelodysplastic syndrome. We have also shown that PR1 expression results from NE and P3 cross-presentation by B-cells and dendritic cells. Because NE and P3 are present in lung and breast cancer tumor tissue, we investigated whether uptake of NE and P3 might result in PR1 expression on solid tumors, which could induce susceptibility of non-hematopoietic tumors to PR1-targeted immunotherapy. We studied melanoma, breast, ovarian and lung cancer cell lines to determine whether NE and P3 are cross-presented. Using flow cytometry, we show that numerous malignant cell lines take up 5 to 10 °g/ml of soluble NE and P3 and was dose- and time-dependent, consistent with a receptor-mediated mechanism. In addition to soluble NE and P3, uptake of granulocyte-associated NE and P3 was also demonstrated; however, uptake of soluble NE and P3 were 3- and 2-fold higher, when compared to granulocyte-associated proteins. Within 4 hours of uptake, confocal microscopy showed that soluble P3 localized to endosomes and lysosomes, suggesting the possibility of lysosomal degradation of P3, a common pathway for MHC-I processing of cross-presented proteins. With the use of 8F4, a monoclonal antibody that is specific for a PR1/HLA-A2 conformational epitope, we show that PR1/HLA-A2 is expressed by 12-hours on P3- and NE-pulsed HLA-A2+ melanoma and breast cancer cell lines. Because 8F4 mediates complement-dependent cytotoxicity (CDC) of AML, we studied whether PR1 expression induced susceptibility of P3- and NE-pulsed tumor cells to killing by 8F4 and PR1-CTL. Twelve hours after pulsing the HLA-A2+ breast cancer cell lines MDA-MB-453 and MDA-MB-231 with P3 or NE, up to 40% and 60% of pulsed breast cancer cells were lysed by PR1-CTL and 8F4, respectively. We conclude that PR1 is cross-presented by solid tumors following receptor-mediated uptake of soluble NE and P3 at concentrations observed in tumors and inflammatory sites. Importantly, this cross-presentation increased susceptibility of embryological distinct tumors to PR1-specific immunotherapy. Together, these data suggest that cross-presentation of extracellular inflammatory proteins that are normally not expressed in the tumor may be a mechanism shared by many tumors, which could be exploited by immunotherapy strategies targeting these proteins. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 788. doi:10.1158/1538-7445.AM2011-788

Abstract 801: Uptake of exogenous neutrophil elastase by breast cancer cells: A novel link between innate immunity, inflammation and breast cancer immunity

Abstract Introduction: High level of neutrophil elastase (NE) in tumor tissue from breast cancer (BrCa) patients is associated with poor metastasis-free survival. Modest endogenous expression of NE in some BrCa cell lines has also been observed. However, because the major source of NE is from azurophil granules of activated neutrophils, we hypothesized that breast cancer cells may take up soluble NE released by abundant inflammatory cells in the tumor microenvironment. Methods: Endogenous NE expression was evaluated in MCF7, HER18, MDA-MB-231 (231) and MDA-MB-453 BrCa cell lines by western blot analysis and RT-PCR. Uptake of soluble NE was quantified by flow cytometry by intracellular staining of NE-pulsed BrCa and compared to non-specific uptake of OVA. Dose and time course experiments were performed. Confocal immunoflourescence microscopy was used to confirm uptake and demonstrate subcellular localization of NE. BrCa cells were also co-incubated with activated neutrophils to determine if they were able to take up cell-associated NE. Results: Western blot analysis of BrCa cell lysates showed no NE protein expression in all 4 cell lines. Furthermore, RT-PCR with NE-specific primers demonstrated no NE mRNA expression in the 4 BrCa cell lines. However, BrCa cells pulsed with 5 to 25 μg/ml of soluble NE took up NE in a time and dose dependent manner, consistent with a receptor-mediated mechanism. Maximal intracellular uptake of NE by 231 cells occurred by 24 hours when NE accumulated at comparable levels to that of HL60 leukemia cells that express high levels of NE (987 vs 1300 MFI, p=ns). Uptake was enzyme-independent because it was not affected by alpha-1-antitrypsin, a protease inhibitor. Confocal microscopy demonstrated that NE accumulates in membrane bound compartments by 20 minutes after uptake, whereas there was no significant uptake of OVA by BrCa cells. Similarly, after co-incubation of 231 cells with activated neutrophils, significant uptake of NE was detected (p<.001). Conclusions: These data show that BrCa cells do not express NE but the cells take up extracellular NE by a receptor-mediated enzyme-independent mechanism. This establishes a novel link between NE, a naturally secreted mediator of inflammation and cell growth, and BrCa. Because neutrophils are the major source of NE and chronic inflammation by tumor-infiltrating neutrophils is associated with positive tumor growth, this suggests NE could mediate growth control of BrCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 801. doi:10.1158/1538-7445.AM2011-801

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

AbstractAbstract 4301The 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. Disclosures:No relevant conflicts of interest to declare.

Soluble Inflammatory Mediators Proteinase-3 and Neutrophil Elastase Are Targeted by PR1-Specific Immunotherapy After Cellular Uptake and Cross Presentation of PR1 Peptide by Breast Cancer Cells

AbstractAbstract 2089The human leukocyte antigen (HLA)-A2 restricted nonapeptide PR1 (VLQELNVTV) was shown to be immunogenic in leukemia. A phase I/II clinical trial has been initiated with PR1 peptide vaccine and to date has demonstrated clinical efficacy, including complete remission and immunologic responses in patients with acute (AML) and chronic (CML) myeloid leukemia, as well as myelodysplastic syndrome. PR1 is derived from the serine proteases proteinase-3 (P3) and neutrophil elastase (NE), which are normally found within neutrophil azurophil granules and are released into the inflammatory milieu. We have shown that P3 and NE are taken up and cross presented by antigen presenting cells and that their cross presentation elicits PR1 immunity. Because P3 and NE are present in breast cancer biopsies, we hypothesized that P3/NE may be taken up by breast cancer cells and cross presented to PR1-CTL. We recently demonstrated that the breast cancer cell lines MDA-MB-231, MDA-MB-453, MCF-7 and HER18 do not endogenously express NE and that NE is taken up by these cell lines. In this report, using PCR, western blot and flow cytometry, we show that P3 also is NOT endogenously expressed by the breast cancer cell lines MDA-MB-231, MDA-MB-453, MCF-7 or HER18. Using confocal microscopy, we demonstrate that P3 is taken up by these breast cancer cell lines within 10 minutes of pulsing and localizes to LAMP-2 containing lysosomal vesicles by 4 hours, suggesting its processing for presentation by (HLA)-I (i.e. HLA-A2). Using 8F4, the novel PR1-HLA-A2 monoclonal antibody, we show that PR1 is cross presented from P3 by 3 of 4 HLA-A2+ breast cancer cell lines (MDA-MB-231, MDA-MB-453-A2+, MCF-7), and from NE by 1 of 4 breast cancer cell lines (MDA-MB-231). Next, we studied whether PR1 presentation made cells susceptible to PR1-specific killing by PR1-CTL and the 8F4 monoclonal antibody. We show that following 12-hour pulsing of the MDA-MB-231 cell line with NE or P3, PR1 CTLs killed up to 31% and 38% of the NE- or P3-pulsed breast cancer cells respectively, vs. <1% of ovalbumin (ova)-pulsed MDA-MB-231cells. Additionally, in a complement mediated cytotoxicity assay using 8F4 antibody, pulsing of MDA-MB-231 cells with P3 led to 60% cytotoxicity (vs. 40% in ova-pulsed cells). In conclusion, this study shows that 1) PR1 is cross presented by breast cancer cells following uptake of soluble P3 and NE and 2) PR1 expression makes breast cancer a target of PR1-specific immunotherapy. If uptake of P3 or NE, present in the inflammatory milieu of other solid tumors, also leads to PR1 cross presentation, then PR1-based immunotherapy may be useful to treat other non-hematopoietic tumors. These results support a new paradigm linking inflammation and innate immunity to adaptive immune responses to cancer. Disclosures:No relevant conflicts of interest to declare.

Surface Expression of PR1 Peptide on Thymic Dendritic Cells Does Not Eliminate PR1-Specific CD8+ Cells In Umbilical Cord Blood

AbstractAbstract 959Positive and negative selection of developing thymocytes is mediated primarily by cortical and medullary epithelial cells (CEC and MEC), respectively, in the thymus. Tolerance to peripheral tissue antigens (PTA) not normally expressed in the thymus can result when PTA expression is induced by AIRE in MEC or when corticomedullary dendritic cells (DC) present endogenous antigens, both mechanisms contributing to deletion of potentially auto-reactive T cells. However, in some instances, negative selection may be incomplete, leading to the release of autoreactive cells into the periphery. Indeed, T cells specific for PR1, the HLA-A2-restricted self-peptide derived from proteinase 3 (P3) and neutrophil elastase (NE), are present in peripheral blood of healthy adults, albeit at extremely low frequency of fewer than 0.0005% of CD8+ T cells. This suggests there is strong thymic central tolerance to PR1. However, PR1-CTL can increase from 0.1 to 2% of peripheral blood CD8 T cells in CML patients treated with interferon or stem cell transplant, and similar levels can be achieved after PR1 peptide vaccination, which suggests that under some circumstances this central tolerance can be reversed. Because of the increasing use of umbilical cord blood (UCB) as an alternative donor source and because incomplete graft-versus-leukemia (GVL) immunity after UCB transplant contributes to relapse, we sought to determine whether PR1-CTL can be expanded from UCB. We hypothesized that PR1-CTL frequency should be low in UCB due to central tolerance. Surprisingly, we found PR1-CTL at a frequency ranging from 0.007 to 0.345% (mean 0.117%) of CD8+ cells in 57 HLA-A2+ cord blood units, similar to what is observed in immunologically responsive vaccine patients and 100- to 1000-fold higher than in healthy adults. The PR1-CTL were predominantly CCR7+CD45+CD28+ and did not efficiently expand ex vivo following peptide stimulation and low dose IL-2, which was consistent with a naive T cell subset. Therefore, this data suggests that central tolerance to PR1 is incomplete. To study whether PR1 is expressed in human thymus, we used the PR1/HLA-A2-specific antibody 8F4 to study PR1 expression. Thymic CEC and MEC expressed no P3, NE, or PR1 by flow cytometry or immunofluorescence imaging of sectioned fetal thymus, which is consistent with previous reports showing absence of P3 or NE induction by AIRE in MEC. Interestingly, by flow cytometric analysis, we found that PR1 is expressed on the surface of thymic dendritic cells (DC) exclusively. This selective expression of PR1 was further confirmed via immunoflourescent staining of sectioned fetal thymus, and the PR1-expressing DC were localized to the corticomedullary junction and medulla. Thus it appears that PR1 expression by DC in the thymus is insufficient for complete central tolerance to PR1. Furthermore, because we have previously shown that PR1-overexpressing CML cells can induce apoptosis of high affinity PR1-CTL, it is possible that peripheral tolerance mechanisms are most critical for preventing autoreactivity to PR1 in humans. These observations suggest possible strategies to overcome tolerance to PR1 by modifying DC uptake and cross-presentation of soluble P3 and NE, when selective autoimmunity may be desirable for leukemia patients or detrimental for patients with vasculitis such as Wegener's granulomatosis. Disclosures:No relevant conflicts of interest to declare.