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Presentation of Telomerase Reverse Transcriptase, a Self-Tumor Antigen, Is Down-Regulated by Histone Deacetylase Inhibition

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Presentation of Telomerase Reverse Transcriptase, a Self-Tumor Antigen, Is Down-Regulated by Histone Deacetylase Inhibition Research Article Presentation of Telomerase Reverse Transcriptase, a Self-Tumor Antigen, is Down-regulated by Histone Deacetylase Inhibition Ilenia Pellicciotta,1 Xochitl Cortez-Gonzalez,1 Roman Sasik,2 Yoram Reiter,3 Gary Hardiman,2 Pierre Langlade-Demoyen,4 and Maurizio Zanetti1 1The Laboratory of Immunology, Department of Medicine and Moores Cancer Center and 2Department of Medicine and BIOGEM, University of California, San Diego, La Jolla, California; 3Department of Biology, Technion-Israel Institute of Technology, Haifa, Israel; and 4Unite’ de Retrovirologie Moleculaire, Institut Pasteur, Paris, France Abstract chromatin structure influence antigen presentation in human Histone deacetylases (HDAC) modify the architecture of tumor cells. chromatin, leading to decreased gene expression, an effect Here,westudiedtheeffectofHDACinhibitononthe that is reversed by HDAC inhibition. The balance between presentation of telomerase reverse transcriptase (TRT), a bona fide deacetylation and acetylation is central to many biological universal antigen in human cancer cells (10, 11) using trichostatin events including the regulation of cell proliferation and cancer A (TSA), a natural product hydroximate with high affinity for the but also the differentiation of immune T cells. The effects of catalytic site of class I and II HDAC (12). We monitored the surface HDAC inhibition on the interaction between antitumor density of complexes formed between the major histocompatibil- effector T cells and tumor cells are not known. Here, we ity/Human Leukocyte Antigen (HLA)-A2 molecule and two high- affinity 9mer hTRT peptides: 540ILAKFLHWL548 (p540) and studied presentation of a universal self-tumor antigen, 865 873 telomerase reverse transcriptase, in human tumor cells during RLVDDFLLV (p865; ref. 11). Because telomerase is expressed HDAC inhibition. We found that HDAC inhibition with in >85% of human cancers (13) and CD8 T cells specific for p540 trichostatin A was associated with a decreased presentation are found in the blood of cancer patients at high frequency (14), and diminished killing of tumor cells by CTLs. Using gene array the model system studied herein is relevant to better understand- analysis, we found that HDAC inhibition resulted in a decrease ing the relation between cancer cells and the antitumor immune of genes coding for proteasome catalytic proteins and for response in humans. Our findings show a reduction of HLA-A2/ tapasin, an endoplasmic reticulum resident protein involved hTRT peptide complexes at the cell surface of TSA-treated human in the MHC class I pathway of endogenous antigen presenta- tumor cells. More importantly, using a highly specific human tion. Our findings indicate that epigenetic changes in cytotoxic T-lymphocyte (CTL) clone, we found that TSA-treated tumor cells decrease self-tumor antigen presentation and cancer cell killing was reduced compared with untreated cells. contribute to reduced recognition and killing of tumor cells This finding may be relevant to understand how immunosurvel- by cytotoxic T lymphocytes. This mechanism could contribute liance of cancer cells can be affected in vivo. to tumor escape from immune surveillance. [Cancer Res 2008;68(19):8085–93] Materials and Methods Cell Lines and Inhibitors Introduction The human B-lymphoblastoid JY cell line and prostate cancer cells LnCap Epigenetic modifications influence gene activity without altering (the kind gift of Dr. A. Vitiello, The R.W. Johnson Pharmaceutical Research DNA sequences from the transcriptional activity of selected genes Institute, San Diego, CA), melanoma cells 629.38 (the kind gift of Dr. S. to the individual’s susceptibility to disease (1), and include both Rosenberg, Surgery Branch, National Cancer Institute, Bethesda, MD), and transient modifications and stable heritable changes (2). Acetyla- TAP-deficient T2 cells (the kind gift of Dr. P. Creswell, Yale University, New Haven, CT) were cultured in complete RPMI 1640 supplemented with 10% tion and deacetylation of histones impart modification on the fetal bovine serum (FBS). TSA, Lactacystin, Sodium butyrate, and BFA were architecture of chromatin (3), which reflect on increased or purchased from Sigma. decreased gene expression through the opposing activities of histone acetyltransferases and histone deacetylases (HDAC). Immunochemical Detection of hTRT, HLA-A2/hTRT Acetylation plays a critical role in the regulation of cell proliferation Complexes, and HLA-A2 (4) and transcriptional silencing associated with a generalized loss Detection of HLA-A2/hTRT complexes at the cell surface. HLA-A2/ of acetylation has been found in cancer (5). Levels of acetylation p540 and HLA-A2/p865 complexes were detected with the human Fab 4A9 A also play a role in the differentiation of T cells (6, 7) and in the and 3H2 (15), respectively. Briefly, cells were treated with TSA (1 g/mL), BFA (10 Ag/mL), or lactacystin (10 Amol/mL). After washing, cells were expression of ligands for natural killer (NK) cells (8). Although incubated with human Fab 4A9 or 3H2 (2 Ag per reaction) for 1.5 h at +4jC. mouse tumor cells treated with high dose HDAC inhibitors are After washing, cells were counterstained with FITC-conjugated goat immunogenic and confer partial protection from tumor growth antibody to human Fab (Jackson Laboratories) for 45 min. Surface staining in vivo (9), little is known on whether epigenetic changes of for HLA-A2 was performed using monoclonal antibody BB7.2 (ATTC) followed by a FITC-conjugated goat antibody to mouse IgG (eBioscience). Cells were analyzed in a FACSCalibur (Becton Dickinson) using the BD Note: I. Pellicciotta and X. Cortez-Gonzalez contributed equally to this work. CellQuest data acquisition and analysis software. Requests for reprints: Maurizio Zanetti, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0815. Phone: 858-822-5412; Fax: 858-822-5421; E-mail: [email protected]. Cytotoxicity Assays I2008 American Association for Cancer Research. Buffy coats from HLA-A2.1 healthy donors were purchased from the doi:10.1158/0008-5472.CAN-08-1014 Blood bank of Hopital Henry Mondor in accordance with an approved www.aacrjournals.org 8085 Cancer Res 2008; 68: (19). October 1, 2008 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 2008 American Association for Cancer Research. Cancer Research Institutional Review Board protocol. HTERT-specific human CTL lines were DNA Microarray generated according to published procedure (11). Briefly, peripheral blood Biotinylated cRNA was prepared using the Illumina RNA Amplification mononuclear cells were stimulated with hTERT peptides in vitro in 24-well kit (Ambion, Inc.) according to the manufacturer’s directions starting with plates using autologous irradiated adherents cells as antigen-presenting cells 250 ng total RNA. The labeling approach uses a modified Eberwine and interleukin (IL)-2 and IL-7. After three to four rounds of culture, CTLs protocol (17) by which mRNA is converted to cDNA, followed by an were screened in a standard 51Cr-release assay. The cold target inhibition amplification/labeling step mediated by T7 DNA polymerase. The cDNA assay (11) was performed to ensure the specificity of the CTL clone. T2 cells and cRNA filter cartridges (Ambion) were used according to the were pulsed with either p540 or the HIV p17 gag 77-85 peptide as a control. manufacturer’s instructions for RT and IVT cleanup, respectively. For An adaptation of this assay was used to investigate killing inhibition where microarray analysis, the Illumina Human Expression BeadChip was used 51Cr-labeled targets treated with TSA were used (Fig. 6C and D). In these (Illumina). Hybridization of labeled cRNA to the BeadChip, and washing experiments, the E:T ratio was kept constant at 20:1, whereas the cold/hot and scanning were performed according to the Illumina BeadStation 500 inhibitor ratio varied as indicated in the legend to the figure. manual. Essentially, the amplified, biotin-labeled human cRNA samples Proteasome Activity Assay were resuspended in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) A A JY cells were treated with TSA (1 Ag/mL), Sodium Butyrate (20 Ag/mL), formamide at a final concentration of 25 ng/ L. cRNA (1.5 g of each) Lactacystin (3.8 Ag/mL), or 5 AL of DMSO (untreated) in RPMI supplemented were hybridized. Hybridization was allowed to proceed at 55 C, for 18 h after which, the bead array matrix was washed for 10 min with with 5% FBS for 18 h. Sodium Butyrate was used as a control based on the  fact that to the best of our knowledge it is the only HDAC inhibitor reported 1 High temperature buffer (Illumina), followed by a subsequent 10-min wash in Wash E1BC buffer. The arrays were then washed with 100% to reduce proteasome activity beside TSA (16). Lactacystin served as a ethanol for 10 min to strip off any remaining adhesive on the chip. A generic inhibitor of proteasome function. Cells were harvested, washed, and 2-min E1BC wash was performed to remove residual ethanol. The arrays resuspended in complete medium. Proteasome activity assay was performed were blocked for 5 min with 1% (w/v) casein-PBS (Pierce). The array using the cell-based luminescent assay Proteasome-Glo (Promega) in white signal was developed via 10-min incubation with Streptavidin-Cy3 at a flat-bottomed 96-well plates (Costar) with 20,000 cells per well. Lumines- final concentration of 1 Ag/mL solution of (GEHealthcare) in 1% casein- cence was measured in a plate reader Infinite M200 (TECAN). Assays were PBS blocking solution. The Human Expression BeadChip was washed a done in triplicate and repeated twice with similar results. final time in Wash E1BC buffer for 5 min and subsequently dried via Deconvolution and Fluorescence Microscopy centrifugation for 4 min at a setting of 275 rcf. The arrays were scanned Cells treated with TSA and immunostained as described above were also on the Illumina BeadArray Reader, a confocal-type imaging system with used for deconvolution microscopy studies. Briefly, after staining with 532 (Cye3) nm laser illumination.
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