soluble intercellular adhesion molecule 1 sicam 1  (R&D Systems)

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Design of different ICAM1 isoforms carrying eGFP-plasmids. ( B ) Representative pictures of HCT 116 or ICAM1 KO cells transfected with ICAM-1-eGFP-plasmids. Pictures were obtained 20 hr after transfection with a 10 x objective using phase contrast channel as well as the green fluorescent channel (n=3). Scale bars, 400 μm. ( C ) eGFP + cells one day post transfection (dpt) measured by flow cytometry. Bar graphs show mean frequency ± s.e.m. (n=3). ( D ) Flag-tag level on the cell surface after 1 day of transfected cells measured by flow cytometry. Bar graphs show mean fluorescent intensity ± s.e.m. (n=3). ( E ) Fold change of sICAM-1 in the supernatant of transfected cells compared to WT (left) or KO (right) measured by IQELISA. Bar graphs show mean ± s.e.m. (n=3.). Two-tailed t tests with adjustments for multiple comparisons were performed (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Transfection, Flow Cytometry, FLAG-tag, Two Tailed Test

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) sICAM-1 levels of conditioned medium harvested from stimulated Panc-1 WT or ICAM1 KO cells. Bar graphs show normalized mean ± SD of three independent experiments. ( B ) Cell survival of antigen loaded HCT 116 cells against CTL killing either cultured in conditioned medium harvest from Panc-1 WT or ICAM1 KO cells after 3 days with different ratios of PBMCs containing antigen specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative for or three independent experiments (n=3).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Cell Culture

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Cell survival of antigen loaded or untreated HCT 116 cells against CTL killing cultured in sICAM-1 (Invitrogen or Preprotech) conditioned medium as indicated after 3 days with a target:effector (T:E) ratio of 1:2. ( B ) Cell survival of antigen loaded or untreated HCT 116 cells against CTL killing in presence of different dosages given once or continuously over time of sICAM-1 (Invitrogen) after 3 days with different ratios of PBMCs containing antigen-specific CTLs. Bar graphs show normalized mean ± SD of triplicates.

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Cell Culture

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Membrane-bound ICAM-1 (mICAM-1) on the cell surface and ( B ) soluble ICAM-1 in the supernatant of untreated or stimulated cells with 100 ng/mL IFN-γ, 20 ng/mL TNF-α or both. Bar graphs show normalized mean ± SD of triplicate for each condition. Two-tailed t tests with adjustments for multiple comparisons were performed (*p<0.05, **p<0.01, ***p<0.001, **** p<0.0001). ( C ) ICAM1 expression in normal (N) or tumor tissue (T) of 22 different human cancers. Number of samples used for analysis as indicated. ( D ) Heatmaps showing expression of ICAM1 and ICAM-1 cleavage related proteases MMP9, ELANE, CTSG, ADAM10, and ADAM17 in normal or tumor tissue of 22 different cancer types. Expression data were obtained using GEPIA. ( E ) Kaplan–Meier survival plots of patient overall survival with the expression of ICAM1 and MMP9 (left), ICAM1 and ADAM10 (middle) , ICAM1 and ADAM17 (right). Patients were categorized into ‘high’ and ‘low’ groups according to the highest and the lowest quartiles of each individual gene expression. Data were obtained from TCGA and GTEx. ( E ) Schematic describing the effect on tumor killing by mICAM-1 and sICAM-1. More details see text.

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Membrane, Two Tailed Test, Expressing

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Cell survival of antigen loaded and untreated HCT 116 WT or ILKAP KO cells using 3 sgRNAs after 3 days of co-culturing with different ratios of PBMCs containing antigen specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative of three independent experiments. ( B ) Cell survival of antigen loaded and untreated Panc-1 WT or ILKAP KO cells using 2 sgRNAs after 3 days of co-culturing with different ratios PBMCs containing antigen-specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative of three independent experiments ( C ) ILKAP protein levels normalized to β-actin determined by Simple Western system. Bar graphs show normalized mean ± SD (n=3). (n.d. – not detectable). ( D ) Cell death of HCT 116 WT or ILKAP KO cells untreated or treated with 100 ng/mL IFN-γ or 40 ng/mL TNF-α determined with live/dead staining (FVS780) using flow cytometry. Bar graphs show mean ± s.e.m (n=3). ( E ) Fold change of HLA-A2, ICAM-1 and PD-L-1 cell surface levels after treatment with 100 ng/mL IFN-γ or 40 ng/mL TNF-α of WT or HCT 116 ILKAP KO cells. Bar graphs show mean ± s.e.m (n=3). ( F ) Mean fluorescence intensities (MFI) of HLA-A2, ICAM-1 and PD-L-1 of HCT 116 WT or HCT 116 ILKAP KO cells. Bar graphs show mean ± s.e.m (n=3). ( G ) ILKAP protein levels of Panc-1 cells transiently transfected with ILKAP or ILKAP H154D assessed by western blot. ( H ) Level of mICAM-1 measured with flow cytometry and ( I ) secreted sICAM-1 levels determined by ELISA of transiently transfected Panc-1 cells. For ( A ) and ( B ), two-way ANOVA corrected for multiple comparison according to Dunnett was used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.001****, p<0.0001). Two-tailed t tests with adjustments for multiple comparisons were performed ( D and E ). For ( C and F ) unpaired two-tailed t test was used to determine statistical significance (*p<0.05, **p<0.01).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Western Blot, Staining, Flow Cytometry, Fluorescence, Transfection, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) LFA-1 cell surface expression of CMV-specific CD8 + T cells measured by flow cytometry displayed as histogram (n=2). ( B ) Histograms showing ICAM-1 levels of HCT 116, Panc-1 and UACC-257 cell lines and respective KO pools after fluorescence activated cell sorting (n=3). ( C ) Cell survival of antigen loaded and untreated HCT 116, Panc-1, UACC-257 cells and ICAM1 KO pools using CRISPR KO and 2 sgRNAs cells against CTL killing after 3 days of co-culturing with different ratios of PBMCs containing antigen specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative for two (Panc-1, UACC-257) or three (HCT-116) independent experiments. Two-way ANOVA corrected for multiple comparison according to Dunnett was used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). ( D ) Mean fluorescence intensities of HLA-A2 and PD-L-1 on the cell surface of HCT 116 WT or HCT 116 ICAM1 KO cells. Bar graphs show mean ± s.e.m (n=2). Unpaired two-tailed t test was used to determine statistical significance (*p<0.05). ( E ) Cell survival of untreated or antigen loaded HCT 116 and HCT 116 PD-L1 cells in the presence of Nivolumab or isotype with different ratios of PBMCs containing antigen-specific CTLs. Bar graphs show normalized mean ± s.e.m. in triplicate representative for two independent experiments. ( F ) Representative histogram of CRISPRa-induced PDL1 expression in HCT 116 cells (n=2). NTC = non-targeting control. ( G ) Representative histogram of PD-1 expression of stimulated CMV-specific CTLs (n=3).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Expressing, Flow Cytometry, Fluorescence, FACS, CRISPR, Comparison, Two Tailed Test

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Design of different ICAM1 isoforms carrying eGFP-plasmids. ( B ) Representative pictures of HCT 116 or ICAM1 KO cells transfected with ICAM-1-eGFP-plasmids. Pictures were obtained 20 hr after transfection with a 10 x objective using phase contrast channel as well as the green fluorescent channel (n=3). Scale bars, 400 μm. ( C ) eGFP + cells one day post transfection (dpt) measured by flow cytometry. Bar graphs show mean frequency ± s.e.m. (n=3). ( D ) Flag-tag level on the cell surface after 1 day of transfected cells measured by flow cytometry. Bar graphs show mean fluorescent intensity ± s.e.m. (n=3). ( E ) Fold change of sICAM-1 in the supernatant of transfected cells compared to WT (left) or KO (right) measured by IQELISA. Bar graphs show mean ± s.e.m. (n=3.). Two-tailed t tests with adjustments for multiple comparisons were performed (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Transfection, Flow Cytometry, FLAG-tag, Two Tailed Test

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Real time kinetic of tumor cell killing by PBMCs with T:E ratio of 1:4. HCT 116 ICAM1 KO cells were transfected with empty vector (gray), ICAM1 (green), ICAM1 Y474A+Y485 A (black) or ICAM1 P404E (red). ( B ) Real-time kinetic of tumor cell killing by PBMCs with T:E ratio of 1:4. WT or HCT 116 ICAM1 KO cells were transfected with empty vector (WT – black; KO – gray) or sICAM1 (WT – orange; KO – blue). ( C ) Real-time kinetic of tumor cell killing by PBMCs with T:E ratio of 1:4. HCT 116 ICAM1 KO cells were transfected with empty vector (gray), ICAM1 - ΔC (purple), ICAM1 -ΔTM-ΔC-GPI (light blue). Cell survival was determined counting green objects every 6 hours by using the IncuCyte system and normalized to timepoint zero. Conditions were performed in triplicate and four pictures of each triplicate were used for analysis (in total 12). Line graphs show mean ± SD for each timepoint representative for at least two independent experiments. Two-way ANOVA with Geisser-Greenhouse correction was used to determine statistical significance of each timepoint. Depicted stars represent statistical significance for t=42 hr (*p<0.05, **p<0.01, ***p<0.001, **** p<0.0001). Figure 6—source data 1. Sequences of ICAM-1 isoform eGFP-plasmids.

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Transfection, Plasmid Preparation

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) sICAM-1 levels of conditioned medium harvested from stimulated Panc-1 WT or ICAM1 KO cells. Bar graphs show normalized mean ± SD of three independent experiments. ( B ) Cell survival of antigen loaded HCT 116 cells against CTL killing either cultured in conditioned medium harvest from Panc-1 WT or ICAM1 KO cells after 3 days with different ratios of PBMCs containing antigen specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative for or three independent experiments (n=3).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Cell Culture

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Cell survival of antigen loaded or untreated HCT 116 cells against CTL killing cultured in sICAM-1 (Invitrogen or Preprotech) conditioned medium as indicated after 3 days with a target:effector (T:E) ratio of 1:2. ( B ) Cell survival of antigen loaded or untreated HCT 116 cells against CTL killing in presence of different dosages given once or continuously over time of sICAM-1 (Invitrogen) after 3 days with different ratios of PBMCs containing antigen-specific CTLs. Bar graphs show normalized mean ± SD of triplicates.

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Cell Culture

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Membrane-bound ICAM-1 (mICAM-1) on the cell surface and ( B ) soluble ICAM-1 in the supernatant of untreated or stimulated cells with 100 ng/mL IFN-γ, 20 ng/mL TNF-α or both. Bar graphs show normalized mean ± SD of triplicate for each condition. Two-tailed t tests with adjustments for multiple comparisons were performed (*p<0.05, **p<0.01, ***p<0.001, **** p<0.0001). ( C ) ICAM1 expression in normal (N) or tumor tissue (T) of 22 different human cancers. Number of samples used for analysis as indicated. ( D ) Heatmaps showing expression of ICAM1 and ICAM-1 cleavage related proteases MMP9, ELANE, CTSG, ADAM10, and ADAM17 in normal or tumor tissue of 22 different cancer types. Expression data were obtained using GEPIA. ( E ) Kaplan–Meier survival plots of patient overall survival with the expression of ICAM1 and MMP9 (left), ICAM1 and ADAM10 (middle) , ICAM1 and ADAM17 (right). Patients were categorized into ‘high’ and ‘low’ groups according to the highest and the lowest quartiles of each individual gene expression. Data were obtained from TCGA and GTEx. ( E ) Schematic describing the effect on tumor killing by mICAM-1 and sICAM-1. More details see text.

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Membrane, Two Tailed Test, Expressing

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: ( A ) Cell survival of antigen loaded and untreated HCT 116 WT or ILKAP KO cells using 3 sgRNAs after 3 days of co-culturing with different ratios of PBMCs containing antigen specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative of three independent experiments. ( B ) Cell survival of antigen loaded and untreated Panc-1 WT or ILKAP KO cells using 2 sgRNAs after 3 days of co-culturing with different ratios PBMCs containing antigen-specific CTLs. Bar graphs show normalized mean ± SD of triplicate representative of three independent experiments ( C ) ILKAP protein levels normalized to β-actin determined by Simple Western system. Bar graphs show normalized mean ± SD (n=3). (n.d. – not detectable). ( D ) Cell death of HCT 116 WT or ILKAP KO cells untreated or treated with 100 ng/mL IFN-γ or 40 ng/mL TNF-α determined with live/dead staining (FVS780) using flow cytometry. Bar graphs show mean ± s.e.m (n=3). ( E ) Fold change of HLA-A2, ICAM-1 and PD-L-1 cell surface levels after treatment with 100 ng/mL IFN-γ or 40 ng/mL TNF-α of WT or HCT 116 ILKAP KO cells. Bar graphs show mean ± s.e.m (n=3). ( F ) Mean fluorescence intensities (MFI) of HLA-A2, ICAM-1 and PD-L-1 of HCT 116 WT or HCT 116 ILKAP KO cells. Bar graphs show mean ± s.e.m (n=3). ( G ) ILKAP protein levels of Panc-1 cells transiently transfected with ILKAP or ILKAP H154D assessed by western blot. ( H ) Level of mICAM-1 measured with flow cytometry and ( I ) secreted sICAM-1 levels determined by ELISA of transiently transfected Panc-1 cells. For ( A ) and ( B ), two-way ANOVA corrected for multiple comparison according to Dunnett was used to determine statistical significance (*p<0.05, **p<0.01, ***p<0.001****, p<0.0001). Two-tailed t tests with adjustments for multiple comparisons were performed ( D and E ). For ( C and F ) unpaired two-tailed t test was used to determine statistical significance (*p<0.05, **p<0.01).

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Western Blot, Staining, Flow Cytometry, Fluorescence, Transfection, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: eLife

Article Title: Complementary CRISPR screen highlights the contrasting role of membrane-bound and soluble ICAM-1 in regulating antigen-specific tumor cell killing by cytotoxic T cells

doi: 10.7554/eLife.84314

Figure Lengend Snippet: sgRNA sequences used to knockout ILKAP and ICAM1 for validation experiments.

Article Snippet: The amount of sICAM-1 in harvested cell culture supernatants was measured by using either RayBio human sICAM-1 IQELISA kit (RayBiotech, IQH-ICAM1) or human sICAM-1 ELISA kit (RayBiotech, ELH-ICAM-1) according to manufacturer’s instructions in duplicates for each sample.

Techniques: Knock-Out, Sequencing