Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence imaging of immune synapses between H1299 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. H1299 lung cancer cells are pretreated with phosphate- buffered saline (PBS) or DAC prior to coculture with γδ T cells. Quantifications of immune synapses per cancer cell on eight randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). Scale bar: 100 μm. p value is calculated by the Mann-Whitney test. ( B ) A scatter plot of DAC-induced surface proteomes in H1299 (y-axis) and A549 (x-axis) human lung cancer cells following daily treatment of 100 nM DAC for 72 hours and culture in drug-free medium for 3 days (D3R3). ICAM-1 is among the top upregulated surface proteins by DAC in both cells. ( C ) Western blot analyses of ICAM-1 protein expression in mock-treated vs. DAC-treated human lung cancer cells. D3: daily treatment of 100 nM decitabine for 72 hours. D3R3: daily treatment for 72 hours, followed by a 3-day rest period in drug-free medium. β-actin: loading control. ( D ) Immunofluorescence staining of ICAM-1 and immune synapse molecules (e.g., LFA-1, LAT) at immune synapses formed between γδ T cells and DAC-treated H1299 lung cancer cells. Scale bar: 10 μm. ( E ) Representative flow cytometric dot plot showing H1299 lung cancer cells with CRISPR-knockout of ICAM1 (KO-ICAM1) subject to γδ T cell killing for 2 hours. The effector to target (E: T) ratio is 3:1. Lung cancer cells are pre-treated with mock, DAC alone, γδ T cells alone or a combination of DAC and γδ T cells. The X-axis denotes surface ICAM1 levels. Y-axis represents signal intensities of propidium iodide. ( F ) Bar graphs showing percent cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with CRISPR-knockout of ICAM-1 subject to γδ T cell killing for 2 hours. Cell death is measured by Annexin V and propidium iodide apoptosis assays (mean ± SEM, n = 3). Statistical significance is determined by one-way ANOVA test. ( G ) Representative flow cytometric dot plot showing H1299 lung cancer cells with a Tet-on expression system of ICAM1 (OV-ICAM1) subject to γδ T cell killing for 2 hours. Doxycycline (1 μg/mL) is added 24 hours prior to coculture to induce ICAM-1 protein expression. Cell death is measured by Annexin V (x-axis) and propidium iodide (y-axis) apoptosis assays. ( H ) Bar graphs showing cell death of human lung cancer cell lines (i.e., H1299, CL1-0, and A549) with ICAM-1 over-expression subject to γδ T cell killing for 2 hours. E:T ratio is 3:1. Cell death is measured by Annexin V and propidium iodide apoptosis assays. Statistical significance is determined by one-way ANOVA test (* p < 0.05, ** p < 0.01, ***, p < 0.001). ( I ) Immunofluorescence imaging of immune synapses between H1299 KO-ICAM1 lung cancer cells and γδ T cells by phosphotyrosine (pTyr) staining. Scale bar: 100 μm. Quantifications of immune synapses per cancer cell on six randomly taken high power fields for each treatment are shown in the dot plots (mean ± SD). p value is calculated by the Mann-Whitney test.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Imaging, Staining, Saline, MANN-WHITNEY, Western Blot, Expressing, Control, CRISPR, Knock-Out, Over Expression

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Sequencing results of the KO-ICAM1 lung cancer cells are aligned against the reference sequence of the ICAM1 genome locus. Alignment gaps are denoted as hyphens (-) to mark the lost (knockout) regions of the edited ICAM1 genome locus.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Sequencing, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Immunofluorescence staining of F-actin (red), ICAM-1 (green), pTyr (phosphotyrosine, white) at immune synapses between γδ T cells and DAC-pretreated H1299 lung cancer cells at D3R3. Accumulation of F-actin beneath the cell membrane is noted in DAC-pretreated lung cancer cells. DAPI: 4′,6-diamidino-2-phenylindole, as a nuclear counterstain. Scale bar: 10 μm. ( B ) Representative immunofluorescence images of the interfaces between γδ T cells and H1299 lung cancer cells (parental vs. ICAM-1 knockout (KO-ICAM1)). Signals of F-actin (red) in the periphery of H1299 cancer cells are shown in two- and-a-half-dimensional (2.5D) images in the lower panels. Scale bar: 10 μm. ( C ) Dot plots of signal intensities of F-actin (left panel) and ICAM-1 (right panel) from five pTry-positive immune synapses between γδ T cells and H1299 lung cancer cells (parental or KO-ICAM1). p value is calculated by two-way ANOVA test. ( D ) Immunofluorescence images of immune synapses between γδ T cells (marked with T) and H1299 lung cancer cells (marked with C) stained for ICAM-1 (green), F-actin (Red) and phosphotyrosine (pTyr, white). Lung cancer cells (parental or KO-ICAM1) are pretreated with PBS (Mock) or 100 nM DAC and cocultured with γδ T cells at D3R3. ( E ) Dot plots of F-actin signal intensities at immune synapses between γδ T cells and H1299 cells. H1299 cells are pretreated with PBS (Mock), DAC alone or combination of DAC pretreatment (D3R3) and 1 μg/mL Cyto B (cytochalasin B, an inhibitor of actin filament polymerization) for 1.5 hours prior to coculture with γδ T cells (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (***, p < 0.001; ****, p < 0.0001). ( F ) Representative immunofluorescence images of immune synapses (pTyr staining) between γδ T and H1299 cells pretreated with PBS (Mock), DAC alone, and combination of DAC and Cyto B. Blow-up images of the square areas for each treatment are shown in the lower panels. Arrows denote immune synapses between γδ T and H1299 cells. Scale bar: 100 μm (upper) and 20 μm (lower panels). ( G ) Dot plots showing numbers of immune synapses per cancer cell on eight randomly taken high power fields for H1299 cells pretreated with PBS (Mock), DAC, and combination of DAC and Cyto B (mean ± SD). p value is calculated by one-way ANOVA with Tukey’s multiple comparisons test (*, statistical significance).

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Immunofluorescence, Staining, Membrane, Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: Parental or ICAM-1 knockout (KO-ICAM1) H1299 cells are pretreated daily with PBS (Mock) or 100 nM DAC for 72 hours followed by 3-day drug-free culture before coculture with γδ T cells. Signal intensities of each protein (F-actin, red; ICAM-1, green; phosphotyrosine, pTyr, white) along the immune synapse area are graphed on the right. DAPI: 4’,6-diamidino-2-phenylindole, as nuclear counterstain. Scale bar: 10 μm.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Knock-Out

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Visualization of multi-omics data (i.e., mRNA-seq, Omni-ATAC-seq, and MethylationEPIC arrays) for DAPK3 , EVPLL, and TUBE1 in H1299 lung cancer cells. ( B ) Promoter methylation status and mRNA expression levels of the ICAM1 gene measured by Infinium MethylationEPIC arrays (left panels) and mRNA-seq (right panels) in human lung cancer cells treated without and with DAC 100 nM DAC for 3 days followed by a 3-day drug-free culture. ( C ) Open chromatin regions in the promoter areas of the ICAM1 gene in human lung cancer cells upon 100 nM DAC treatment analyzed by Omni-ATAC-seq. The green bar represents a CpG island. ( D ) Validation of Omni-ATAC-seq by quantitative real-time PCR on transposase-accessible chromatin at the ICAM1 promoter of human lung cancer cells subject to daily treatment of 100 nM DAC treatment for 3 days, followed by a 3-day drug-free culture. Experiments are performed in triplicates, and data are presented as mean ± SD. p value was calculated by unpaired t test (*, p < 0.05). ( E ) IPA Network analysis of mRNA expression changes in human lung cancer cells treated by DAC reveals coordinated changes of the immune-related surface molecules and the cytoskeleton-associated genes. ( F ) IPA upstream regulator analysis of mRNA expression changes in human lung cancer cells treated by DAC. T cell effector cytokines such as TNF-α and IFN-γ may enhance DAC-induced expression changes of immune-related molecules and ICAM-1 in lung cancer cells. TP53 is a potential master regulator for cancer cytoskeleton reorganization essential for DAC-potentiated γδ T cell killing.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Biomarker Discovery, Methylation, Expressing, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Epigenetic therapy remodels the immune synaptic cytoskeleton to potentiate cancer susceptibility to γδ T cells

doi: 10.1101/2020.04.30.069955

Figure Lengend Snippet: ( A ) Diagram of transcription factor binding sites at the ICAM1 promoter derived from the ENCODE ChIP-seq data ( https://www.encodeproject.org ). Visualizations of ATAC-seq peaks at the ICAM1 promoter in PC9 and CL1-5 lung cancer cell lines subject to DAC treatment are shown above. ( B ) Promoter methylation status and mRNA expression levels of putative transcription factors (i.e., RELB, NFKB2, STATS, and RUNX3) at the ICAM1 promoter in A549, H1299, PC9, and CL1-5 lung cancer cells. Dot and line plots represent methylation levels (β values) of promoter probes measured by Infinium MethylationEPIC arrays. The promoter probes with β values greater or equal to 0.5 at baseline (Mock) are shown. Bar graphs represent relative mRNA expression levels based on normalized FPKM measured by mRNA-seq.

Article Snippet: The overexpression and loss of ICAM-1 protein were validated by flow cytometry with an anti-ICAM1 antibody (BBA20, R&D Systems).

Techniques: Binding Assay, Derivative Assay, ChIP-sequencing, Methylation, Expressing

Journal: Nature Communications

Article Title: Severe COVID-19 patients have impaired plasmacytoid dendritic cell-mediated control of SARS-CoV-2

doi: 10.1038/s41467-023-36140-9

Figure Lengend Snippet: Cells were infected by SARS-CoV-2 (indicated as +) or not (indicated as −) 2 days prior to coculture with PBMCs, pDC-depleted PBMCs, and isolated pDCs (16 h-coculture). Infected cell types included the human alveolar basal epithelial cell lines, i.e., Calu-3 and A549-ACE2. Statistical analyses of the results were performed using Wilcoxon rank-sum test and p values were calculated with Tukey and Kramer test. For the different panels, the significant contrasts are indicated when p-values are: ≤0.05 as *; ≤0.005 as **; ≤0.0005 as ***; and ≤0.00005 as ****. a , b Quantification of IFN-α in the supernatants of total PBMCs [tPBMC], pDC-depleted PBMCs [PBMC/no pDC], and isolated pDCs [iso pDC] cocultured with either SARS-CoV-2-infected or uninfected Calu-3 cells ( a ) or A549-ACE2 cells ( b ), or treated with 100 µl of cell-free supernatant (SN) collected from SARS-CoV-2-infected cells. Of note, viral titers of SARS-CoV-2: SN ≈ 2.5 × 10 5 foci forming units (ffu)/ml, MOI ≈ 1 per pDCs and no detection of IFN-α by SARS-CoV-2-infected cells themselves [no PBMC/pDC]. Arrows indicate results below the detection threshold of the IFN-α ELISA (i.e., 12.5 pg/ml). Each dot represents one independent experiment performed with distinct healthy donors (ELISA results are similarly presented in all other Figures). Error bars represent the means ± standard deviation (SD); from independent experiments ( n = 5, 8, and 9 for the conditions of cocultures, respectively, with PBMCs, pDCs/Calu-3 cells, and pDCs/A549-ACE2 cells); exact p-values are indicated to Supplementary Fig. . c , d Total PBMCs were cocultured with SARS-CoV-2-infected or uninfected A549-ACE2 cells or treated with TLR agonists [31.8 µM R848 and 42.22 µM polyI:C] for 14–16 h. Cell populations gated as pDCs, non-pDC PBMCs, non-pDC enriched mDCs, mDC1, and mDC2 subsets, see gating strategies in Supplementary Fig. . Representative dot blots of flow cytometry analyses ( c ) and frequencies of cells ( d ) positive for IFN-α + but IFN-λ1 − or double positive for IFN-α + /IFN-λ1 + in gated pDCs versus non-pDC PBMCs (upper panels) and in gated pDCs versus non-pDC enriched mDCs, mDC1 and mDC2 subsets (lower panels). Means ± SD; Bars represent n = 10–11 independent experiments/distinct healthy donors. e – h Quantification of IFN-α in SNs of pDCs cocultured with the indicated cell types infected or not by SARS-CoV-2. e , f pDCs were cocultured with infected cells, either in direct contact [coculture] or physically separated by the semi-permeable membrane of transwell [TW], or treated with SN from the corresponding SARS-CoV-2-infected cells. IFN-α concentration was also determined in the SN of SARS-CoV-2-infected cells cultured without pDC (no pDC). Means ± SD; n = 5 independent experiments for pDC cocultured with infected cells or SN and n = 4 for TW and no pDC. g Dots represent IFN levels for pDCs purified from distinct donors in independent experiments; including n = 11 and n = 15 for A549-ACE2 and Calu-3 cells, respectively. Means ± SD. h Quantification of IFN-α in SNs of pDCs cocultured with SARS-CoV-2-infected cells A549-ACE2 treated or not with blocking antibodies against α L -integrin and ICAM-1 at 10 µg/mL; means ± SD; each dots represent n = 4 independent experiments. Source data are provided as a Source Data file.

Article Snippet: Other reagents included LPS, TLR3 agonist (Poly(I:C); LMW) and TLR7 agonist (R848 and Imiquimod) (Invivogen); TLR7 antagonist (IRS661, 5’-TGCTT GCAAGCTTGCAAGCA-3’ synthesized on a phosphorothionate backbone; MWG Biotech); mouse anti-α L integrin (clone 38; Antibodies Online); mouse anti-ICAM-1 (Clone LB-2; BD Bioscience): Arp2/3 complex inhibitor I (CK-666; Merck Millipore); Fc Blocking solution (MACS Miltenyi Biotec); Golgi-Plug, cytoperm/cytofix and permeabilization-wash solutions (BD Bioscience); IFN-α and IFN-λ 1/2/3 ELISA kit (PBL Interferon Source); IL-6 and TNF ELISA kit (Affymetrix, eBioscience); 96-well format transwell chambers (Corning); IL-6 and IFN-λ by U-PLEX Custom Human Cytokine assay (Meso Scale Diagnostics, Rockville, MD); 96-Well Optical-Bottom Plates (Thermo Fisher Scientific); cell-labeling solution using CellTrace Violet Cell Proliferation Kit (Life Technologies ref # C34557, C34571), Live/Dead Fixable Dead Cell Stain Near-IR (Life Technologies ref #10119); Fixable Viability Dye eFluor 450 (Life Technologies); Zombie Aqua and Zombie Green Fixable Viability Kits (Biolegend); FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend); cDNA synthesis and qPCR kit (Life Technologies); poly-L-lysin (P6282, Sigma-Aldrich).

Techniques: Infection, Isolation, Enzyme-linked Immunosorbent Assay, Standard Deviation, Flow Cytometry, Concentration Assay, Cell Culture, Purification, Blocking Assay

Journal: Nature Communications

Article Title: Severe COVID-19 patients have impaired plasmacytoid dendritic cell-mediated control of SARS-CoV-2

doi: 10.1038/s41467-023-36140-9

Figure Lengend Snippet: Our longitudinal study of the innate responses by ex-vivo stimulation of PBMCs from COVID-19 patients, and across distinct disease severities (i.e., mild/asymptomatic versus severe COVID-19 and healthy donors, as reference) highlighted the following proposed model. pDCs from mild/asymptomatic patients and healthy donors (in purple) robustly produce IFN-I/λ upon cell contact with SARS-CoV-2-infected cells (upper panel). As opposed, pDCs from severe patients (in red) produce IFN-I/λ in absence of ex vivo stimulation, but fail to be activated by contact with SARS-CoV-2-infected cells (lower panel). This non-responsive/exhausted state of pDCs in severe COVID-19 patients is associated with an elevated level of pro-inflammatory cytokines (here represented by IL-6, red round symbols) that are most likely produced by the HLA-DR + CD14 + monocytes. As shown on the zoomed view of the contact site (right panel, at the top), the short-range sensing of SARS-CoV-2-infected cells by pDCs requires cell contact mediated by adhesion complexes, identified as α L β 2 integrin and ICAM-1. This triggers TLR7-induced signaling via IRF7 leading to an IFN-I/λ-prioritized response while leaving inactive the NF-κB-mediated signaling.

Article Snippet: Other reagents included LPS, TLR3 agonist (Poly(I:C); LMW) and TLR7 agonist (R848 and Imiquimod) (Invivogen); TLR7 antagonist (IRS661, 5’-TGCTT GCAAGCTTGCAAGCA-3’ synthesized on a phosphorothionate backbone; MWG Biotech); mouse anti-α L integrin (clone 38; Antibodies Online); mouse anti-ICAM-1 (Clone LB-2; BD Bioscience): Arp2/3 complex inhibitor I (CK-666; Merck Millipore); Fc Blocking solution (MACS Miltenyi Biotec); Golgi-Plug, cytoperm/cytofix and permeabilization-wash solutions (BD Bioscience); IFN-α and IFN-λ 1/2/3 ELISA kit (PBL Interferon Source); IL-6 and TNF ELISA kit (Affymetrix, eBioscience); 96-well format transwell chambers (Corning); IL-6 and IFN-λ by U-PLEX Custom Human Cytokine assay (Meso Scale Diagnostics, Rockville, MD); 96-Well Optical-Bottom Plates (Thermo Fisher Scientific); cell-labeling solution using CellTrace Violet Cell Proliferation Kit (Life Technologies ref # C34557, C34571), Live/Dead Fixable Dead Cell Stain Near-IR (Life Technologies ref #10119); Fixable Viability Dye eFluor 450 (Life Technologies); Zombie Aqua and Zombie Green Fixable Viability Kits (Biolegend); FITC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend); cDNA synthesis and qPCR kit (Life Technologies); poly-L-lysin (P6282, Sigma-Aldrich).

Techniques: Ex Vivo, Infection, Produced