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

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

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

Hole Transport in Exfoliated Monolayer MoS₂

Ideal monolayers of common semiconducting transition-metal dichalcogenides (TMDCs) such as MoS₂, WS₂, MoSe₂, and WSe₂ possess many similar electronic properties. As it is the case for all semiconductors, however, the physical response of these systems is strongly determined by defects in a way specific to each individual compound. Here we investigate the ability of exfoliated monolayers of these TMDCs to support high-quality, well-balanced ambipolar conduction, which has been demonstrated for WS₂, MoSe₂, and WSe₂, but not for MoS₂. Using ionic-liquid gated transistors, we show that, contrary to WS₂, MoSe₂, and WSe₂, hole transport in exfoliated MoS₂ monolayers is systematically anomalous, exhibiting a maximum in conductivity at negative gate voltage (<i>V</i>_<i>G</i>) followed by a suppression of up to 100 times upon further increasing <i>V</i>_<i>G</i>. To understand the origin of this difference, we have performed a series of experiments including the comparison of hole transport in MoS₂ monolayers and thicker multilayers, in exfoliated and CVD-grown monolayers, as well as gate-dependent optical measurements (Raman and photoluminescence) and scanning tunneling imaging and spectroscopy. In agreement with existing <i>ab initio</i> calculations, the results of all these experiments are consistently explained in terms of defects associated with chalcogen vacancies that only in MoS₂ monolayers, but not in thicker MoS₂ multilayers nor in monolayers of the other common semiconducting TMDCs, create in-gap states near the top of the valence band that act as strong hole traps. Our results demonstrate the importance of studying systematically how defects determine the properties of 2D semiconducting materials and of developing methods to control them

Scalable Patterning of One-Dimensional Dangling Bond Rows on Hydrogenated Si(001)

Silicon dangling bonds exposed on the monohydride silicon (001) (Si(001):H) surface are highly reactive, thus enabling site-selective absorption of atoms and single molecules into custom patterns designed through the controlled removal of hydrogen atoms. Current implementations of high-resolution hydrogen lithography on the Si(001):H surface rely on sequential removal of hydrogen atoms using the tip of a scanning probe microscope. Here, we present a scalable thermal process that yields very long rows of single dimer wide silicon dangling bonds suitable for self-assembly of atoms and molecules into one-dimensional structures of unprecedented length on Si(001):H. The row consists of the standard buckled Si dimer and an unexpected flat dimer configuration

Induced miRNAs showing no or only weak expression in EBV-negative T-cell lymphoma.

<p>Induced miRNAs showing no or only weak expression in EBV-negative T-cell lymphoma.</p

Validation of miRNA expression by quantitative Real-Time PCR.

<p>Total RNA out of the different samples used for sequencing was reverse transcribed and the expression of miRNAs was analysed in a light cycler by quantitative Real-Time PCR. The relative quantification was carried out by the 2^−ΔΔct-method after measurement the amount of 5.8sRNA in each sample. Represented are the comparisons between (A) EBV-positive lymphomas and thymus, (B) EBV-negative lymphomas and thymus as well as (C) EBV-associated and EBV-negative lymphomas. The shaded bars represent the sequencing results and the filled bars the PCR-results. Bars above the x-axis show induced miRNAs and the bars below repressed miRNAs.</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