Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD112 , REA1195 , 50 , 130-122-770 , PE , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Cellular neighborhood analysis of PD1 high/low T cells in the tumor margin and core. (A–D) Topology of PD1 high (left) and PD1 low (right) T cells and their cellular neighborhood within a 5 µm range. (A, B) represent tumor margin and (C, D) show tumor core areas. Cell types showing different distribution patterns around PD1 high and PD1 low T cells (mDCs, M1-like M, M2-like M, MDSCs, Fibroblasts, vessels, tumor cells) are highlighted by arrowheads. (E, F) Quantification of cells in a 5 µm range around of PD1 high/low T cells for tumor margin and tumor core, (E) represents immune cells and (F) stroma/tumor cells. (G) Violin plots for expression levels of eight immune-modulating markers (CD112, CD155, CD276, CD39, CD73, IDO, PD-L1, and VISTA) for the most important immune and tumor cells around PD1 high/low T cells in the tumor core area. Violin plots for tumor margin are shown in Supplementary Figure S5E . Depicted markers and annotated cell types as indicated by the color code. ROI sizes: Tumor margin (ROI15) and tumor core (ROI16): 975 x 769 µm.

Article Snippet: CD112 , REA1195 , 50 , 130-122-770 , PE , Miltenyi Biotec.

Techniques: Expressing

Journal: Cancers

Article Title: Targeting High-Risk Neuroblastoma Patient-Derived Xenografts with Oncolytic Virotherapy

doi: 10.3390/cancers14030762

Figure Lengend Snippet: Neuroblastoma PDXs express viral entry receptors. ( A ) Flow cytometry was used to detect the cell surface expression of the four main HSV receptors: CD111, CD112, syndecan, and HVEM in COA3, COA6, and COA129 human neuroblastoma PDX cells. All PDXs expressed all four receptors. ( B ) Representative histograms of CD111, CD112, syndecan, and HVEM cell surface expression and negative controls of COA6 are shown. Data reported as mean ± SEM and represent at least three biologic replicates.

Article Snippet: After 15 min, 10 μL of phycoerythrin (PE) conjugated anti-human CD111 antibody (Miltenyi Biotec), PE anti-human CD112 antibody (Miltenyi Biotec), allophycocyanin (APC) conjugated anti-human syndecan (Miltenyi Biotec), or APC anti-human HVEM (Miltenyi Biotec), were added for 20 min on ice in the dark.

Techniques: Flow Cytometry, Expressing

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: AML cells exhibit no, or heterogeneous, expression of DNAM-1 ligands. (A) The indicated AML cell lines were analyzed for expression of CD112, CD155 and CD33 by flow cytometry. Iso refers to immunoglobulin isotype matched control antibody while stain indicates specific antibody staining. (B) The percentage of cells expressing the indicated ligands within populations of the indicated cell type, as analyzed from (A). (C) CD155 expression on the indicated AML cell lines was visualized by confocal microscopy. Scale bar represents 20 µm.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Flow Cytometry, Control, Staining, Confocal Microscopy

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands are required for NK cell activity against AML targets. (A) K562 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (B) K562 cells were FACS sorted as in (A), then used as targets in an NK cell degranulation assay. (C) K562 cells were FACS sorted as in (A), then used as targets in an NK cell chromium release assay. (D) MV4-11 cells were stained with anti-CD112 and anti-CD155 antibodies, then FACS sorted into high expressing (both CD112 and CD155) and low expressing (both CD112 and CD155) populations. (E) MV4-11 cells were FACS sorted as in (D), then used as targets in an NK cell degranulation assay. (F) MV-411 cells were FACS sorted as in (D), then used as targets in an NK cell chromium release assay. (G) NK cell chromium release assay against the indicated AML targets (5:1 E:T ratio) in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). (H–I) NK cell chromium release assay at the indicated E:T ratios using FACS sorted high and low CD112/CD155 expressing K562 and MV-411 cells, in the presence or absence of anti-DNAM-1-neutralizing antibody (5 μg/mL). Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Activity Assay, Staining, Expressing, Degranulation Assay, Release Assay

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: DNAM-1 ligands increase the frequency of ‘normal’ NK-target cell synapses. (A–B) FACS sorted (CD112/155 high and low) K562 and MV-411 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later, followed by fixing. The percentage of targets that had conjugated with an NK cell was then quantitated by confocal microscopy. A minimum of 20 fields of view was analyzed and is representative of two independent experiments. (C) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells 30 min later. After 1 h, cells were fixed, stained with the antibody combinations indicated, then analyzed by confocal microscopy. Representative images of NK-target cell synapses are presented. Scale bar represents 10 µm. (D) The percentage of NK cells that had polarized LFA-1 and perforin to the synapse was quantified from (C). A minimum of 20 NK-target cell synapses was analyzed and data from two independent experiments was pooled. Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Confocal Microscopy, Staining

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: Live imaging reveals that AML cells lacking DNAM-1 ligand expression drive NK cell failed killing. (A) FACS sorted (CD112/155 high and low) MV4-11 cells were seeded in chamber slides using serum free media, then overlaid with NK cells labeled with fluo-4 acetoxymethyl AM (green) to indicate calcium signaling, and analyzed by time-lapse microscopy. PtdIns (red) (100 μg/mL) was added to the medium to indicate perforin-induced target membrane puncture. Representative still images at the indicated time-points are depicted (hr:min). (B–C) Individual NK-MV-411 CD112/CD155 high and low contacts were monitored for events that did (successful kill) or did not (failed kill) result in target killing, as indicated by PI influx and apoptotic morphology. (D) The time interval between initial NK-target cell contact and target cell death (PtdIns influx) was analyzed. (E–G) K562 cells were used as targets in the assays described in (B–D) above. All quantification data is pooled from individual movies (n = 3). Error bars represent the mean ± SEM *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Imaging, Expressing, Labeling, Time-lapse Microscopy, Membrane

Journal: Oncoimmunology

Article Title: Loss of DNAM-1 ligand expression by acute myeloid leukemia cells renders them resistant to NK cell killing

doi: 10.1080/2162402X.2016.1196308

Figure Lengend Snippet: NK cells preferentially target DNAM-1 ligand-expressing cells and drive clonal selection of DNAM-1 ligand negativity. (A) FACS-sorted K562 CD112/CD155 high and low cells were labeled with CFSE and CTV, respectively, then exposed to NK cells at the indicated E:T ratios. After 4 h, cells were analyzed by flow cytometry and loss of dye was monitored from viable populations. (B) Extended E:T ratio titration for the assay described in (A), using FACS-sorted K562 and MV-411 CD112/CD155 high and low cells as targets. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 2). *p < 0.05 by unpaired Student's t test. (C–D) K562 and MV-411 cells were either exposed to NK cells (1:1 E:T Ratio), or not, for 5 d. Viable AML cells (fixable yellow negative, CD33 positive) were then analyzed for CD112 and CD155 expression by flow cytometry, and compared to parental cells (no NK cell exposure). Bar charts represent the number of CD112/CD155 double positive cells after 5 d in the presence or absence of NK cell exposure. Error bars represent the mean ± SEM of triplicate determinations from a representative experiment (n = 3). *p < 0.05 by unpaired Student's t test.

Article Snippet: Antibodies Directly conjugated antibodies were used for of the analysis of NK cell ligands and consisted of anti-human CD155-PE (FAB25301, R&D systems), CD112-FITC (FAB2229G, R&D systems), CD33 PECy7 (333946, BD) and anti-mouse CD112-FITC (690912 R&D systems), CD155-PE (690912, R&D systems).

Techniques: Expressing, Selection, Labeling, Flow Cytometry, Titration

Journal: Scientific Reports

Article Title: Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

doi: 10.1038/s41598-017-10403-0

Figure Lengend Snippet: PVR and Nectin2 are mainly found as intracellular pool in MM cells. ( a ) CD38 + CD138 + malignant PCs derived from BM aspirates of MM patients (n = 34) were analysed for PVR and Nectin2 surface and total (surface plus intracellular) expression before and after fixation and permeabilization, respectively. Cells were acquired using FACSCanto flow cytometer (BD Biosciences). Each dot represents a single patient, ****p < 0.001, Wilcoxon matched pairs test. ( b ) PVR (left panel) and Nectin2 (right panel) surface and total (surface plus intracellular) expression was analysed on MM cell lines before and after fixation and permeabilization, respectively. Cells were acquired using FACSCalibur flow cytometer (BD Biosciences). Data represent the means ± SD of PVR and Nectin2 from three independent experiments. *p < 0.05, Student T test. MFI: mean fluorescence intensity.

Article Snippet: Surface ligands expression on patient-derived PCs was evaluated by means of PE-conjugated anti-PVR mAb (Biolegend, SKII.4), and APC-conjugated anti-Nectin2 mAb (R&D Systems, FAB2229A) after gating on the CD38 + CD138 + PC population.

Techniques: Derivative Assay, Expressing, Flow Cytometry, Fluorescence

Journal: Scientific Reports

Article Title: Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

doi: 10.1038/s41598-017-10403-0

Figure Lengend Snippet: SUMOylation controls PVR but not Nectin2 surface expression in MM cell lines. ( a–c ) Inhibition of the SUMO pathway was achieved by means of overnight treatment with 25μg/mL Ginkgolic Acid (GA). The efficacy of treatment was verified by means of western blot analysis on total cell lysates ( a ). ( b , c ) PVR and Nectin2 surface expression was evaluated on ARK and OPM2 cell lines by immunofluorescence and FACS analysis using FACSCalibur flow cytometer (BD Biosciences). One out of three independent experiments ( b ) and means ± SD of PVR and Nectin2 MFI from three independent experiments ( c ) are shown. **p < 0.01, ***p < 0.001, Two-way ANOVA. ( d–f ) Inhibition of the SUMO pathway was achieved by means of UBC9 gene silencing. Silencing efficiency was verified by means of western blot analysis on total cell lysates ( d ). ( e , f ) PVR and Nectin2 surface expression was evaluated on ARK and OPM2 cell lines by immunofluorescence and FACS analysis using FACSCanto flow cytometer (BD Biosciences). One out of three independent experiments ( e ) and means ± SD of PVR and Nectin2 MFI from three independent experiments ( f ) are shown. **p < 0.01, ***p < 0.001, Two-way ANOVA.

Article Snippet: Surface ligands expression on patient-derived PCs was evaluated by means of PE-conjugated anti-PVR mAb (Biolegend, SKII.4), and APC-conjugated anti-Nectin2 mAb (R&D Systems, FAB2229A) after gating on the CD38 + CD138 + PC population.

Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Flow Cytometry

Journal: Scientific Reports

Article Title: Innate immune activating ligand SUMOylation affects tumor cell recognition by NK cells

doi: 10.1038/s41598-017-10403-0

Figure Lengend Snippet: Ginkgolic Acid treatment up-regulates PVR but not Nectin2 surface expression in malignant PCs. Malignant PCs were treated overnight with 25 μg/mL of GA or vehicle alone (DMSO), and PVR ( a ) and Nectin2 ( b ) surface expression was evaluated on cells gated as in Supplementary Fig. . Data from two representative patients are shown in left panels. Data from 9 patients analysed are shown in right panels. Each dot represents a single patient. ***p < 0.001 Wilcoxon matched pairs test.

Article Snippet: Surface ligands expression on patient-derived PCs was evaluated by means of PE-conjugated anti-PVR mAb (Biolegend, SKII.4), and APC-conjugated anti-Nectin2 mAb (R&D Systems, FAB2229A) after gating on the CD38 + CD138 + PC population.

Techniques: Expressing

Journal: eLife

Article Title: MPDZ promotes DLL4-induced Notch signaling during angiogenesis

doi: 10.7554/eLife.32860

Figure Lengend Snippet:

Article Snippet: For Nectin-2 silencing, three different siRNAs were used (SR321541, Origene, Rockville, MD).

Techniques: shRNA, Western Blot, Recombinant, Clone Assay, Plasmid Preparation, Mutagenesis