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Tolerance is also induced by the cross-presentation of tissue antigens by non-hematopoietic cells, which occurs in the thymus and is facilitated by medullary thymic epithelial cells Table I Pathologic characteristics of breast and ldy tumor tissues used for laser capture microdissection and confocal microscopy.
Data demonstrated that breast cancer cells can take up both soluble P3 as well as cell-associated P3. Spatial and mechanistic separation of cross-presentation and endogenous antigen presentation.
Together, our data demonstrate the ability of solid tumors to cross-present antigen and suggest PR1 as a broadly expressed tumor antigen. This process is necessary for immunity to most tumors.
le Human tonsil tissue sections Origene were used as positive staining control for CD Our study provides evidence of a novel mechanism whereby hematopoietic antigens can be taken up and cross-presented on major histocompatibility MHC class I by non-hematopoietic tumors, and it suggests that in addition to leukemia, exogenous P3 and NE may also be tumor antigens in non-hematopoietic tumors.
Antibodies to neutrophil elastase: Discussion P3 and NE are serine proteases that are normally expressed in hematopoietic cells and are abundant in leukemia and the microenvironment of non-hematopoietic tumors 161719 Hematoxylin was used to stain nuclei after tissue hydration. Cytotoxic T lymphocytes specific for a nonpolymorphic proteinase 3 peptide preferentially inhibit chronic myeloid leukemia colony-forming units.
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A recent report by Francois et al. Journal of Clinical Oncology. Dapi-blue was used to stain for cell nuclei. Our data also highlight the role of cross-presentation in expanding the number of tumor types that could be targeted by existing immunotherapeutic modalities.
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. Healthy donor and patient peripheral blood mononuclear cells PBMC and polymorphonuclear neutrophils PMN were enriched using standard Histopaque and Sigma gradient centrifugation, respectively.
N Engl J Med. It is therefore important to understand whether normal tissues can also cross-present P3 and NE and express PR1 and whether this mechanism plays a role in maintaining tolerance to these tissue antigens, which is being currently investigated in our laboratory.
Efficient major histocompatibility complex class I presentation of exogenous antigen upon phagocytosis by macrophages. Leica LCS software version 2.
The publisher’s final edited version of this article is available free at J Immunol. Immunohistochemistry staining of primary breast cancer detected P3 in leh cancer tissue, but the P3 was limited to the inflammatory component within the breast tumor, and not in the breast tumor cells Fig.
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The areas used for microdissection were identified using hematoxylin and eosin staining. These data are ldy with a study by Houghton et al. B, MDA-MB cells were incubated with increasing lwy 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. Mechanisms of MHC class I-restricted antigen processing and cross- presentation.
Both images are taken from the same patient and are representative of 5 tissues. Dapi-blue was used to stain cell nuclei. 819-11 cytotoxicity assay To determine whether cross-presentation increases breast cancer susceptibility to 8F4, we performed complement mediated cytotoxicity assay, as previously described 22 Author manuscript; available in PMC Dec 1. For melanoma, tissue sections were fixed with cold acetone, permeabilized with 0.
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Introduction Proteinase 3 P3 and neutrophil elastase NE are proteases leyy stored in neutrophil primary azurophil granules. Fluorescence was measured and specific killing was calculated as key above. There was no lej increase in HLA-A2 expression on the cell surface data not shown. U leukemia cell line was used as a positive control for NE and P3. Following denaturation for 5 minutes at 95 C, samples were amplified for 35 cycles using an iCycler Bio-Rad.
Impact of cyclins E, neutrophil elastase and proteinase 3 expression levels on clinical outcome in primary breast cancer patients. 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 leyy common malignancy in women, we investigated cross-presentation of NE and P3 in breast cancer. Fold increase in MFI vs. Cytotoxicity was determined by measuring released calcein-AM.
Our data demonstrate the localization of P3 and NE 15 to lysosomal and endosomal compartments, respectively, which are both known to play a role in antigen cross-presentation 314647thus providing further support to NE and P3 cross-presentation by non-APCs.
Nuclei appear blue using DAPI.
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.