Treatment with 5-Aza-2'-Deoxycytidine Induces Expression of NY-ESO-1 and Facilitates Cytotoxic T Lymphocyte-Mediated Tumor Cell Killing

<div><p>Background</p><p>NY-ESO-1 belongs to the cancer/testis antigen (CTA) family and represents an attractive target for cancer immunotherapy. Its expression is induced in a variety of solid tumors via DNA demethylation of the promoter of CpG islands. However, NY-ESO-1 expression is usually very low or absent in some tumors such as breast cancer or multiple myeloma. Therefore, we established an optimized <i>in vitro</i> treatment protocol for up-regulation of NY-ESO-1 expression by tumor cells using the hypomethylating agent 5-aza-2'-deoxycytidine (DAC).</p><p>Methodology/Principal Findings</p><p>We demonstrated <i>de novo</i> induction of NY-ESO-1 in MCF7 breast cancer cells and significantly increased expression in U266 multiple myeloma cells. This effect was time- and dose-dependent with the highest expression of NY-ESO-1 mRNA achieved by the incubation of 10 μM DAC for 72 hours. NY-ESO-1 activation was also confirmed at the protein level as shown by Western blot, flow cytometry, and immunofluorescence staining. The detection and quantification of single NY-ESO-1 peptides presented at the tumor cell surface in the context of HLA-A*0201 molecules revealed an increase of 100% and 50% for MCF7 and U266 cells, respectively. Moreover, the enhanced expression of NY-ESO-1 derived peptides at the cell surface was accompanied by an increased specific lysis of MCF7 and U266 cells by HLA-A*0201/NY-ESO-1(_157–165) peptide specific chimeric antigen receptor (CAR) CD8⁺ T cells. In addition, the killing activity of CAR T cells correlated with the secretion of higher IFN-gamma levels.</p><p>Conclusions/Significance</p><p>These results indicate that NY-ESO-1 directed immunotherapy with specific CAR T cells might benefit from concomitant DAC treatment.</p></div

Effects of DAC treatment on NY-ESO-1 mRNA and protein expression in MCF7, U266, and ARK cell lines.

<p>A. NY-ESO-1 specific mRNA, quantified as copy numbers/μg RNA using qRT-PCR. All data are representative of three independent experiments performed in triplicate. B. NY-ESO-1 protein expression analyzed by Western blotting (n = 3). The first and second line show total cell lysate of the respective cell line +/- DAC treatment and detection via a NY-ESO-1 specific or α-Tubulin specific (loading control) antibody. The third line shows a Western blot of recombinant NY-ESO-1 protein as control.</p

Optimization of DAC treatment for MCF7, U266, and ARK tumor cell lines.

<p>A. Variation of DAC concentration in cell culture medium as indicated (0–15 μM); white bars: isotype control, grey bars: detection with HLA-A2/NY-ESO-1p_157-165 specific Fab-tetramers. B. Variation of DAC-treatment intensity (0–4 times per day). C. Variation of DAC-treatment duration (1–3 days). All data are representative of at least three independent experiments performed in triplicate.</p

Increased specific lysis of MCF7 and U266 tumor cells by CAR redirected CD8⁺ T cells after DAC treatment.

<p>Retrovirally transduced NY-ESO-1-specific and CEA-specific CAR redirected CD8⁺ T cells were cocultivated with U266 or MCF7 cells. NY-ESO-1 expression after DAC treatment statistically significantly enhanced the antigen specific killing of anti-NY-ESO-1 CAR redirected T cells in U266 (A), whereas the increased lysis of MCF7 was only detectable after DAC treatment (B). Antigen specific activation of anti-NY-ESO-1 CAR redirected CD8⁺ T cells was determined by IFN-gamma (C and D). All data are representative of three independent experiments performed in triplicate.</p

Quantification of HLA-A2/NY-ESO1p_157-165 complexes at the cell surface of MCF7, U266, and ARK tumor cell lines.

<p>A. Flow cytometry histogram after staining of the indicated tumor cell lines with HLA-A2/NY-ESO-1p_157-165 peptide specific Fab-T1 tetramer (blue/red) and isotype control (black/green). Cells were either untreated (black/blue) or DAC- treated (green/red). B. Quantification of the NY-ESO-1 peptides at the surface of the untreated (white bars) or DAC-treated (grey bars) tumor cell lines. Peptide numbers were calculated as described in materials and methods. C. Relative change in HLA-A2/NY-ESO-1p_157-165 peptide presentation on the tumor cell lines. Increase of NY-ESO-1p_157-165 peptide presentation is shown in relation to the total number of HLA-A2 molecules on the tumor cell lines following DAC treatment. ARK cells were used as a negative control. All data are representative of at least five independent experiments performed in triplicate.</p

Cadherin expression in RP1-mutants.

<p><b>4A</b> N-cadherin levels of HEK293 cells containing empty vector (c), wt, ALA and ASP were determined by immunoblotting with an N-terminal N-cadherin antibody. The middle panel shows a processed N-cadherin fragment (named CTF1) detected by a fragment specific antibody in respective lysates. The lower panel shows the β-tubulin loading control. <b>4B</b> The Western blot signals of <b>4A</b> were quantified using the Image-J software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, <a href="http://rsb.info.nih.gov/ij/" target="_blank">http://rsb.info.nih.gov/ij/</a>, 1997–2008). Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001) comparing control versus wt and mutants regarding complete N-cadherin and comparing wt versus mutants regarding N-cadherin cleavage fragment (CTF1). In the right panel, the empty c lane indicates no detectable CTF in control cells. <b>4C</b> N-cadherin levels were measured by incubation with a monoclonal antibody directed against the cytoplasmic tail and subsequent FACS analysis. The negative control (yellow line) was incubated with secondary antibody only. The positive control (red) was empty vector containing HEK293 cells. The results for HEK293 expressing RP1-wt are depicted in black, RP1-ALA²³⁶ in green and RP1-ASP²³⁶ in blue. <b>4D</b> Quantification of N-cadherin levels from FACS analysis<b>.</b> Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p<0.001).</p

Binding and Phosphorylation of RP1 by CK2.

<p><b>1A</b> Identification of CK2 phosphorylation site - RP1-sequence (amino acid), three potential CK2 kinase sites S59, S72, S236 (underlined) were identified by prosite scan (<a href="http://www.expasy.ch" target="_blank">www.expasy.ch</a>). The peptides used for in vitro experiments (1C) are marked in bold. S236 the actual CK2 phosphorylation site is shown in red. <b>1B</b> Interaction–assay RP1/CK2 - Endogenous RP1 (first panel) was co-precipitated with its potential binding partners. RP1/CK2 kinase interaction could be detected by specific α/ß CK2 subunit antibodies. The black wedges in this panel indicate increasing stringency of washing procedure (% Tween20/PBS). In a reverse experiment (right side panel), endogenous RP1 was verified as genuine CK2 binding substrate. By using CK2 subunits as baits, RP1 could be detected in the pulldowns by its specific RP1 antibody (right panel). No signal was seen when an insignificant IgG antibody was used. On the far right 1/10 of cell lysate of the foregoing experiments is depicted as an input control. The black wedges in this panel indicate stringency of the washing procedure (0.01% and 0.3% Tween/PBS). <b>1C</b> Biotinylated peptides (A: aa54–65, B: aa70–80, C: aa229–240) containing the potential CK2 phosphorylation sites S<i>⁵⁹</i>, S<i>⁷²</i>, S<i>²³⁶ were</i> synthesized and tested as CK2 phosphorylation substrates (A, B, C, 3 µg each) in an <i>in vitro</i> phosphorylation assay. A known positive CK2 kinase site peptide (DDDDSDDDDD, 3 µg) served as a control. The black wedge indicates incubation times (minutes). <b>1D</b> CK2 kinase assay - Recombinant CK2 and ³³P-gamma-ATP were incubated in vitro with different amounts of RP1-wt protein (first panel shows a coomassie stain of his-tagged purified RP1 protein used for the assay) and phosphorylation was measured by autoradiography (middle panel). The amounts of RP1 protein used are indicated above the middle panel. Autophosphorylation of CK2 at its subunit ß served as positive control RP1-ALA236 mutated protein (ALA) was almost non-phosphorylated in comparison to the wild type protein (right side upper panel). The lower panel on the right side shows a coomassie stain representing the amount of RP1 used for this experiment.</p

G- and F-Actin in RP1-expressing cells.

<p><b>3A</b> G- and F-actin content of 10×10⁵ constitutively RP1 expressing cells were measured by FACS analysis. G-Actin (green) was measured in the FL-1 channel (Fluor 488, green) and F-Actin in the FL-3 channel (phalloidin rodamine staining, red). The upper left quadrant of each panel represents the F-actin pool, the upper right quadrant the G-Actin pool. The top three panels are the controls: Upper left panel, unstained control cells. Upper middle panel: Boiled fluoresceine conjugated DNAse I unable to bind G-Actin serving as a negative control. Upper right panel: Double staining of HEK293 cells with Fluor 488 conjugated DNAse I and Phalloidin-Rhodamine carrying the empty pEAK8 vector to determine the general content of G-actin and F-actin pools as reference. The bottom panels show the respective degree of G-actin decrease seen in the RP1-wt, ALA, ASP containing cell lines. <b>3B</b> Quantification of G-Actin content in RP1 expressing HEK293 cells compared to mock transfected cells from <b>3A</b>. Differences marked by asterisks were statistically significant using the two-tailed Fisher’s exact test (**p = 0.0001;*p = 0.0003).</p

Shear Stress experiments.

<p>2A Analysis of shear stress-dependent adhesion of RP1 mutants on endothelial cells under flow 1×10⁵ HEK293 cells stably transfected with different RP1 mutants were allowed to settle for 3 min on parallel plate flow chambers with pre grown confluent HUVECs. Subsequently, preheated HBSS/0.1% BSA was flushed through the chambers at the indicated calculated shear stress, and shear stress levels were increased every 30 s. Photographs were taken and adherent cells were counted in four fields for every condition. Cell line with empty vector (black squares), RP1 wild type (wt) (black circles), RP1-ALA²³⁶ (ALA) mutant (white circles), RP1-ASP²³⁶ (ASP) mutant (white squares). Values are means of n = 5–6+/− SEM. Asterisks denote statistically significant differences *p<0.05 or **p<0.01 between parental cell line and ASP mutant as determined by a two-tailed t-test. <b>2B Analysis of RP1 expression under fluid shear stress</b> Native HEK293 cells were exposed to fluid shear stress or simply cultured (control). Thereafter, cells were lysed and total protein from the lysates was employed in immunoprecipitation of RP1. Endogenous RP1 was detected by an RP1 specific antibody. α-tubulin served as a loading control. RP-1protein detected by Western blot was quantified using the ImageJ software. Asterisks mark statistically significant differences **p<0.01 between sheared and non-sheared cells as determined by a two-tailed t-test. <b>2C </b><b>Analysis of RP1 phosphorylation under fluid shear stress</b> HEK293 cells overexpressing RP1-wt were exposed to 1dynes/cm² shear stress. From cell lysate RP1 was immunoprecipitated and subjected to Western blotting. In parallel the phosphorylation status of RP1 was detected with an anti phospho-serine antibody (anti PS). As control HEK293 cells overexpressing RP1-wt were cultured without shear stress and otherwise processed alike. Total RP1 (RP1) expression served as loading control. The difference between phosphorylation intensity in sheared versus control cells was statistically significant (**, p<0.05). <b>2D Analysis of RP1 shRNA regulated cells and RP1 phosphor-mutants under fluid shear stress</b> HEK293 containing empty vector or various mutants were exposed to increasing shear stress. The curves show the percentage of adhering cells under different shear stress intensity (0, 1.5, 4.5 and 8.5 dynes/cm2) on control cells transfected with irrelevant shRNA served as a reference (black boxes) and were compared to RP1 specific shRNA bearing cells and the mutant cell lines RP1-ALA236, RP1-ASP236. A significant gain of cell adhesion is seen for RP1 specific shRNAs (white boxes). The statistical differences of adherent cells between the depicted cell lines and the control cells are marked with Asterisks *p<0.05 or **p<0.01.</p

Reduced expansion of pro-inflammatory CD4+ T-cells from spleen and MLN in Tg-HD5 rats.

<p><i>In vitro</i> stimulated cells obtained from Tg-HD5, Tg-ctrl and WT-littermates were assessed by ICS for the presence of pro-inflammatory cells expressing TNF <b>(A)</b>, IL-17 <b>(B)</b> and IFN-γ <b>(C)</b>. MLN and spleens cells were obtained at week 15 (n = 5) and at week 23 (n = 5). Results are plotted as the percentage of CD4+ T-cells gated positive for TNF, IL-17 and IFN-γ. Values are expressed as mean±SEM. *p<0.05, **p<0.01, ***p<0.005 as determined by one-way ANOVA followed by Bonferroni post-hoc analysis.</p