LEY 189-11 PDF

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PR1 is a let leukocyte antigen HLA -A2 restricted peptide that has been targeted successfully in myeloid leukemia with immunotherapy. PR1 is derived from the neutrophil granule proteases proteinase 3 P3 and neutrophil elastase NEwhich are both found in the tumor microenvironment. We recently showed that P3 and NE are taken up and cross-presented by normal and leukemia-derived antigen presenting cells, and that NE is taken up by breast cancer cells.

We now extend our findings to show that P3 and NE are taken up and cross-presented by human solid tumors. Together, our data identify cross-presentation as a novel mechanism through which cells that lack endogenous expression of an antigen become susceptible to leh that target cross-presented antigens and suggest PR1 as a broadly expressed tumor antigen.

Proteinase 3 P3 and neutrophil elastase NE are proteases normally stored in neutrophil primary azurophil granules.

They play a role in 189-1, leukemogenesis and autoimmune disease e. PR1 has shown 189-1 in the therapy of myeloid leukemia 78. We have shown that P3 and NE are cross-presented by normal donor antigen presenting cells APC and leukemia, and that cross-presentation by leukemia renders cells susceptible to killing by PR1 targeting therapy Furthermore, cross-presentation is thought 18911 be the primary mechanism through which tumor antigens are presented to the immune system, and is believed to be restricted to subpopulations of APCs 11 A recent report by Francois et al.

P3 and NE are both endogenously expressed in myeloid hematopoietic cells and therefore provide a source for PR1 in myeloid malignancies. Since NE was shown to be taken up by lung cancer 14 and as we have shown that breast cancer cells take up NE ldywe hypothesized that NE and P3 uptake by solid tumors may lead to PR1 cross-presentation, thereby rendering non-myeloid malignancies susceptible to killing by PR1-targeting therapy.

We first show NE and P3 uptake by a number of solid tumors. Since breast cancer was shown to contain an inflammatory component that may be the source for NE and P3 1617is susceptible to immunotherapy 18and is the most common malignancy in women, we investigated cross-presentation of NE and P3 in breast cancer. We show that P3, like NE 15 is absent in breast cancer cell lines and primary breast cancer tumors, and is taken up by breast cancer cells.

We then extend our findings to melanoma, which like breast cancer also contains an inflammatory component 19 and has demonstrated susceptibility to immunotherapy 20 Together, our data demonstrate the ability of solid tumors to cross-present antigen and suggest PR1 as a broadly expressed tumor antigen.

Patient breast cancer frozen tissue blocks were purchased from Origene. 18-911 and healthy donor Lfy samples were collected after informed consent was obtained to participate in a study approved by the institutional review board at MD Anderson Cancer Center. Healthy donor and patient peripheral blood mononuclear cells PBMC and polymorphonuclear neutrophils PMN were enriched using standard Histopaque and Sigma gradient centrifugation, respectively.

The following primers were used: Following denaturation for 5 minutes at 95 C, samples were amplified for 35 cycles using an iCycler Bio-Rad.

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Samples were run on 1. Chemiluminescence was captured on Kodak film. To determine protein uptake, cells were pulsed in reduced serum medium 0.

To determine cross-presentation, ly were surface-stained with fluorescently-conjugated 8F4 as previously described For all flow cytometry experiments, light scatter was used to establish the initial gating followed by aqua live dead stain. To inhibit cross-presentation, cells were co-incubated with the endoplasmic reticulum ER to Golgi antegrade inhibitor brefeldin A BFA Sigma or the proteasome inhibitor lactacystin Sigma 1323 Flow cytometry was performed using the Cytomation CyAn flow cytometer Dako.


Cryopreserved breast and melanoma tumor tissues Origene were formalin fixed and then paraffin-embedded for immunohistochemistry.

Prior to staining, tissue sections were de-paraffinized in xylene, re-hydrated and quenched for endogenous peroxidase activity. Melanoma slides were co-stained with anti-Microphthalmia-associated transcription factor MITF antibody Thermoscientific.

Slides were then washed and incubated with secondary anti-mouse IgG-biotin antibody 1: All slides were counterstained with hematoxylin. PMN staining of normal tonsil tissue was used as a positive control. Negative controls were stained as above after deletion of primary antibodies. A standard cytotoxicity assay was used to determine specific lysis as described previously 5 Percent specific cytotoxicity was calculated as follows: To determine whether cross-presentation increases breast cancer susceptibility to 8F4, we performed complement mediated cytotoxicity assay, as previously described 22 One million cells were mixed with increasing doses of 8F4 antibody 0.

Fluorescence was measured and specific killing was calculated as described above. Laser capture microdissection LCM was preformed to isolate breast cancer cells from breast tumor biopsy tissue with an Arcturus PixCell laser capture microscope with an IR diode laser Life Technologies, Applied Biosystems. Hematoxylin was used to stain nuclei after tissue hydration. Samples were stored in xylene after graded alcohol dehydration until ready for LCM. The areas used for microdissection were identified using hematoxylin and eosin staining.

PBMC from patients were stained with the following fluorescent antibodies: Aqua live dead stain Invitrogen was used to exclude dead cells. Cryopreserved tissue sections were fixed with cold acetone. Breast cancer tissues were stained with the breast cancer marker Alexa conjugated mouse anti-Cytokeratin-7 CK7 antibody Abcam and Alexa conjugated 8F4 antibody Human tonsil tissue sections Origene were used as positive staining control for CD For melanoma, tissue sections were fixed with cold acetone, permeabilized with 0.

ProLong Gold antifade reagent with dapi Invitrogen was added. Leica LCS software version 2. We show that not all tumor types take up NE and P3, and furthermore the degree of uptake varies among different tumor types Fig. In addition, NE uptake appears to plateau over time and is much lower than P3 uptake, indicating different uptake mechanisms and suggesting a receptor-mediated process that may be involved in NE uptake.

Solid tumor cell lines take up NE and P3. Since we have previously shown that NE is absent in breast cancer and is taken up by breast cancer cells 15 and to differentiate P3 uptake from endogenous expression, we analyzed breast cancer cell lines and primary tumor tissues for P3 expression at the mRNA and protein levels.

Similarly, breast cancer cells extracted from 3 different breast tumors Fig. Immunoblots of whole cell lysates WCL from cell lines confirmed the absence of P3 protein in breast cancer cells Fig. Immunohistochemistry staining of primary breast cancer detected P3 in breast cancer tissue, but the P3 was limited to the inflammatory component within the breast tumor, and not in the breast tumor cells Fig.

These data are consistent with previous reports showing P3 in breast cancer 29although our data suggest that the source of P3 is inflammatory cells within the tumor, and not the breast cancer cells. Breast cancer does not endogenously express P3.

Jurkat and HL leukemia cell lines were used as negative and positive controls, respectively. Primary breast cancer cells from patient tissues, samples Breast 1—3, were obtained by laser capture microdissection LCM performed on tumor obtained from patients at the time of surgical resection.

Mammaglobin MGB -1 was used to confirm analysis of breast cancer cells. C, Immunoblots demonstrate lack of P3 protein in whole cell lysates from 5 different breast cancer cell lines.

D, Immunohistochemistry showing absence of P3 in patient breast cancer tissue Breast 3. Right panel shows positive staining of P3 in the admixed neutrophils but not in the breast cancer cells.

The inset X shows a rare tumor cell engulfing a neutrophil. Both images are taken from the same patient and are representative of 5 tissues.


Pathologic characteristics of breast and melanoma tumor tissues used for laser capture microdissection and confocal microscopy. Since we showed that P3 is not expressed endogenously by breast cancer cells, we hypothesized that P3 may be taken up by breast cancer cells, as we have previously shown for NE We detected a time-dependent increase in P3 uptake in all three cell lines.

We also demonstrated a dose dependent uptake of P3 that does not appear to plateau, suggesting a non-receptor mediated process for P3 uptake Fig. To further characterize P3 uptake as it relates to antigen cross-presentation, which occurs in distinct lfy compartments 30we performed laser confocal microscopy and showed that following uptake, P3 localizes within lysosomes, as shown by P3 co-staining with lysosomal-associated membrane protein 2 LAMP-2 Fig.

Uptake into lysosomal compartments occurred at early time points 1—4 hours and may be the initial step in antigen degradation as it is being processed for cross-presentation on HLA class-I molecules P3 is taken up by breast cancer cell lines and localizes to lysosomal compartments.

Median fluorescence intensity MFI was measured for triplicate experimental groups and was normalized to the MFI of unpulsed cells. Fold increase in MFI vs.

B, MDA-MB cells were incubated with increasing doses of soluble P3 or ovalbumin ova and analyzed by flow cytometry for intracellular uptake of P3 or ova using anti-P3 or anti-ova antibodies, respectively. Confocal microscopy images demonstrate localization of P3 in lysosomal compartments 4 hours following uptake as shown by overlay images yellow. Nuclei appear blue using DAPI. Because different cellular lwy are involved in uptake and processing of soluble and cell-associated proteins, which can determine whether or not they are cross-presented 32and since neutrophils were reported in tumor tissues including breast cancer 1617we evaluated if there was difference in the uptake of soluble and 189-111 P3 by breast cancer cells.

Data demonstrated that breast cancer cells can take up both soluble P3 as well as cell-associated P3. Cells were permeabilized, stained with anti-P3 antibody and analyzed by flow cytometry. Cytotoxicity was 1189-11 by measuring released calcein-AM. NE- or P3-pulsed cells show higher killing vs.

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PR1-pulsed and unpulsed T2 cells were used as positive and negative controls, respectively. Unpaired t test was performed using Prism 5. Since we have shown that NE is also taken up by breast cancer 15 and since PR1 is derived from both of the neutrophil azurophil granule proteases NE and P3, we investigated whether NE and P3 are cross-presented by ,ey cancer cells following uptake.

Significant PR1 cross-presentation was primarily seen at 24 hours Fig. There was no significant increase in HLA-A2 expression on the cell surface data not shown. Similarly, using 8F4 antibody in a complement dependent cytotoxicity assay Fig. Since we showed that cultured breast 189–11 cell lines and tumor tissues lack endogenous NE and P3, and because we observed in vitro evidence of NE and P3 cross-presentation by breast cancer cells and subsequent susceptibility to PR1-targeting therapies, we investigated whether PR1 could be detected in primary beast cancer patient tissues and whether PR1-CTLs could be detected in peripheral blood from patients with breast cancer.

Dapi-blue was used to stain cell nuclei. Mann-Whitney U test was performed using Prism 5. Dapi-blue was used to stain for cell nuclei. Taken together, these in vivo data suggest 189–11 the 18-911 proteases NE and P3 present in the tumor microenvironment can be taken up and cross-presented by breast cancer cells, which may contribute to an key immune response against the NE and P3-derived epitope PR1.