Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Nectin-2 is a functional receptor for VSIG2 on T cells. a SDS-PAGE and immunoblot analysis of T cell lysates following pull-down with hVSIG2-His protein (n =3 independent experiments). b SDS-PAGE and immunoblot analysis of CD11b + monocyte lysates following pull-down with hNectin-2-Fc protein (n =3 independent experiments). c Molecular docking model of the VSIG2-Nectin-2 interaction interface. d Key amino acid residues involved in hydrogen bond and ionic interaction formation between VSIG2 and Nectin-2, as predicted by molecular docking. e Flow cytometry analysis of Nectin-2 expression on the surface of T cells in both resting and activated states, and ( f ) statistical analysis of the results (n =3 independent experiments). g SDS-PAGE and immunoblot analysis of hVSIG2-His protein pull-down with hNectin-2-Fc protein (n =3 independent experiments). h SDS-PAGE and immunoblot analysis of hNectin-2-Fc protein pull-down with hVSIG2-His protein (n =3 independent experiments). i Isothermal titration calorimetry (ITC) curve and thermodynamic parameters for the hVSIG2-hNectin-2 binding interaction at 25°C. Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA with Tukey’s test. Source data are provided as a Source Data file

Article Snippet: VSIG2 (Human, HEK293, His, purchased from MCE, HY- P70503 ) was dissolved in PBS buffer (pH 7.4) to a final concentration of 20 μM, and Nectin-2 (Human, HEK293, Fc, purchased from MCE, HY- P78372 ) was dissolved in the same buffer to a concentration of 200 μM.

Techniques: Functional Assay, SDS Page, Western Blot, Flow Cytometry, Expressing, Isothermal Titration Calorimetry, Binding Assay

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Anti-Nectin-2 Ab blocks the inhibitory effect of hVSIG2-Ig on T cells. CD3⁺ T cells were isolated by magnetic bead sorting and pre-incubated with 5 µg/ml anti-Nectin-2 antibody. Cells were then plated in 96-well plates pre-coated with anti-CD3/CD28 (1 µg/ml/0.5 µg/ml) and 6400 ng/ml hVSIG2-Ig, followed by culture for 24–72 h. a Schematic illustration of Nectin-2 receptor blockade on T cells using anti-Nectin-2 antibody. b Representative flow cytometry plots of CD69, CD25, and CD40L expression in CD4⁺ and CD8⁺ T cells, with ( c ) quantitative analysis of T cell activation ( n = 3 independent experiments). d Representative flow cytometry plots of Ki67 expression in CD4⁺ and CD8⁺ T cells, with e quantitative analysis of T cell proliferation ( n = 3 independent experiments). f Cytokine microsphere array (CBA) analysis of TNF-α, IFN-γ, IL-17 A, IL-2, and IL-6 in culture supernatants ( n = 3 independent experiments). g Immunoblot analysis of p-STAT1, GBP2, and IRF1 protein expression in T cells, with ( h ) densitometric quantification ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by one-way ANOVA with Tukey’s test. Source data are provided as a Source Data file

Article Snippet: VSIG2 (Human, HEK293, His, purchased from MCE, HY- P70503 ) was dissolved in PBS buffer (pH 7.4) to a final concentration of 20 μM, and Nectin-2 (Human, HEK293, Fc, purchased from MCE, HY- P78372 ) was dissolved in the same buffer to a concentration of 200 μM.

Techniques: Isolation, Incubation, Flow Cytometry, Expressing, Activation Assay, Western Blot

Journal: Journal of Neuroinflammation

Article Title: VSIG2 as a novel immunosuppressive ligand interacts with Nectin-2 to regulate T cell responses

doi: 10.1186/s12974-025-03645-7

Figure Lengend Snippet: Knockdown of Nectin-2 blocks the inhibitory effect of hVSIG2-Ig on Jurkat cells. a Schematic illustration of hVSIG2-Ig blockade in Jurkat cells with lentiviral-mediated Nectin-2 knockdown ( n = 3 independent experiments). b Flow cytometry analysis of Nectin-2 surface expression in resting and activated Jurkat cells after lentiviral transfection ( n = 3 independent experiments). c Western blot validation of Nectin-2 knockdown efficiency in Jurkat cells ( n = 3 independent experiments). d Representative flow cytometry plots of CD69, CD25, and IL-2 expression in Jurkat cells, with quantitative analysis of T cell activation markers ( n = 3 independent experiments). e Representative flow cytometry plots of Ki67 expression in Jurkat cells, with statistical analysis of proliferation rates ( n = 3 independent experiments). f Western blot analysis of p-STAT1, GBP2, and IRF1 protein levels in Jurkat cells, with g densitometric quantification of band intensities ( n = 3 independent experiments). Data are presented as mean ± SD. Statistical significance was assessed by two-way ANOVA with Tukey’s test

Article Snippet: VSIG2 (Human, HEK293, His, purchased from MCE, HY- P70503 ) was dissolved in PBS buffer (pH 7.4) to a final concentration of 20 μM, and Nectin-2 (Human, HEK293, Fc, purchased from MCE, HY- P78372 ) was dissolved in the same buffer to a concentration of 200 μM.

Techniques: Knockdown, Flow Cytometry, Expressing, Transfection, Western Blot, Biomarker Discovery, Activation Assay

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: Journal of Hematology & Oncology

Article Title: Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors

doi: 10.1186/s13045-021-01112-3

Figure Lengend Snippet: Both early and late blockades of PVRIG inhibit tumor growth in MC38 tumor-bearing mice. a Experimental protocol for murine colon cancer model used in b – e . Mice were injected with PBS, anti-PVRIG mAb or isotype-matched control mAb (rat IgG) intraperitoneally (i.p.) at various time points after injection of 5 × 10 4 MC38 tumor cells subcutaneously (s.c.) on day 0. b Median tumor size ( n = 10 (PBS), n = 8 (rat IgG), n = 12 (anti-PVRIG)) measured at each time point. c Overall survival of mice treated with PBS ( n = 33), Rat IgG ( n = 31) or anti-PVRIG mAb ( n = 38). d Representative photograph and e weight of tumor ( n = 8 per group) on day 28 after challenge. f C57BL/6 mice were inoculated subcutaneously (s.c.) with 5 × 10 4 MC38 colon cancer cells on day 0. Mice were grouped randomly on day 3. And then mice were injected with isotype-matched control mAb (Rat IgG), anti-PVRIG mAb, anti-PD-L1 mAb or anti-PVRIG + anti-PD-L1 mAb intraperitoneally (i.p.) starting on day 3 for six times ( n = 8 per group). And median tumor size was shown. g , h C57BL/6 mice were inoculated subcutaneously (s.c.) with 2 × 10 5 MC38 colon cancer cells. Mice were grouped randomly when tumor size reaches around 100–150 mm 3 and treated with isotype-matched control mAb (Rat IgG) or anti-PVRIG mAb intraperitoneally (i.p.) for six times ( n = 8 per group). g Median (left) and individual (right) tumor size was shown. h Overall survival of tumor-bearing mice was shown. Each symbol represents an individual mouse. Data were representative of at least two independent experiments. Error bars represent means ± s.e.m. Statistical significance was determined using two-way ANNOVA ( b , f , g ), Mantel–Cox test ( c , h ) or unpaired two-tailed t test ( e ). ns, p > 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001

Article Snippet: Paraffin sections were de-waxed, rehydrated, subjected to heat-induced epitope retrieval (HIER) and followed by incubation with primary antibodies to human PVRIG (Clone 2, generated in house) and PVRL2 (AF2229, R&D Systems, Minneapolis, USA) respectively.

Techniques: Injection, Two Tailed Test

Journal: Journal of Hematology & Oncology

Article Title: Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors

doi: 10.1186/s13045-021-01112-3

Figure Lengend Snippet: Blockade of PVRIG reverses exhaustion of tumor-infiltrating NK cells in tumor-bearing mice. Mice were injected with PBS, anti-PVRIG mAb or isotype-matched control mAb (rat IgG) intraperitoneally (i.p.) at various time points after injection of 5 × 10 MC38 tumor cells subcutaneously (s.c.) on day 0 and killed on day 28 after challenge as described in Fig. a ( n = 8 per group). a Representative histograms (top) and quantification (bottom) of PD-1 and CD96 expression in tumor-infiltrating NK cells. b Representative histograms (top) and quantification (bottom) of NKG2D and TRAIL expression in tumor-infiltrating NK cells. c Representative histograms (top) and quantification (bottom) of CD107a and Granzyme B (GzmB) expression in tumor-infiltrating NK cells. d Representative histograms (top) and quantification (bottom) of IFN-γ synthesis in tumor-infiltrating NK cells. e Absolute number of tumor-infiltrating NK cells in mice treated with rat IgG or anti-PVRIG mAb. Each symbol represents an individual mouse ( n = 8 ( a – d ) or n = 5 ( e ) per group). Data were representative of two independent experiments. Error bars represent means ± s.e.m. Statistical significance was determined using unpaired two-tailed t test. * p < 0.05; ** p < 0.01; *** p < 0.001 and **** p < 0.0001

Article Snippet: Paraffin sections were de-waxed, rehydrated, subjected to heat-induced epitope retrieval (HIER) and followed by incubation with primary antibodies to human PVRIG (Clone 2, generated in house) and PVRL2 (AF2229, R&D Systems, Minneapolis, USA) respectively.

Techniques: Injection, Expressing, Two Tailed Test

Journal: Journal of Hematology & Oncology

Article Title: Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors

doi: 10.1186/s13045-021-01112-3

Figure Lengend Snippet: Both NK cells and CD8 + T cells contribute to the anti-tumor efficacy of PVRIG blockade. a Experimental protocol for murine colon cancer model used in d – g . Mice were injected with isotype-matched control mAb (rat IgG), anti-PVRIG mAb, anti-PVRIG mAb combined with PK136 (anti-NK1.1), anti-PVRIG mAb combined with 53-5.8 (anti-CD8β) or anti-PVRIG mAb combined with PK136 and 53-5.8 intraperitoneally (i.p.) at various time points after injection of 5 × 10 4 MC38 tumor cells subcutaneously (s.c.) on day 0. b Representative flow plots of splenic NK cells in mice treated with PK136 (anti-NK1.1) antibody or control antibody. c Representative flow plots of splenic CD8 + T cells in mice treated with 53-5.8 (anti-CD8β) antibody or control antibody. d Median tumor size measured at various time points and e overall survival of mice with different treatments ( n = 5 (Rat IgG), n = 7 (anti-PVRIG), n = 10 (anti-PVRIG + NK depletion), n = 10 (anti-PVRIG + NK depletion + CD8 + T depletion)). f Median tumor size measured at various time points and g overall survival of mice with different treatments ( n = 5 (Rat IgG), n = 7 (anti-PVRIG), n = 10 (anti-PVRIG + CD8 + T depletion), n = 10 (anti-PVRIG + NK depletion + CD8 + T depletion)). h Rag1 −/− mice were injected with isotype-matched control mAb (rat IgG) or anti-PVRIG mAb intraperitoneally (i.p.) at various time points after injection of 5 × 10 4 MC38 tumor cells subcutaneously (s.c.) on day 0. And median tumor size was shown on the right. Data are representative of two independent experiments. Error bars represent means ± s.e.m. Statistical significance was determined using two-way ANNOVA ( d , f , h ) or Mantel–Cox test ( e , g ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Paraffin sections were de-waxed, rehydrated, subjected to heat-induced epitope retrieval (HIER) and followed by incubation with primary antibodies to human PVRIG (Clone 2, generated in house) and PVRL2 (AF2229, R&D Systems, Minneapolis, USA) respectively.

Techniques: Injection

Journal: Journal of Hematology & Oncology

Article Title: Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors

doi: 10.1186/s13045-021-01112-3

Figure Lengend Snippet: Blockade of PVRIG enhances human NK cell cytotoxicity and inhibits tumor growth in NK cell- or PBMC-reconstituted xenograft mice. a Representative histogram of PVRIG expression in human NKG cell line. b NKG cells were co-cultured with SW620 cells at 2.5:1 ratio for 24 h in the presence of anti-hPVRIG antibody or mIgG1 control antibody. The expression of NKG2D, CD107a and perforin in NKG cells was analyzed by flow cytometry. c Cytotoxicity of NKG cells against human colon cancer cell line SW620 in the presence of anti-hPVRIG antibody (red) or mIgG1 control antibody (blue) at E/T ratios of 1:1 and 5:1. d Flow cytometric analysis of PVRIG expression in CD56 + NK cells, CD8 + T cells and CD4 + T cells from healthy human PBMCs ( n = 21). e Human PBMCs were co-cultured with SW620 cells at an E/T ratio of 25:1 for 24 h in the presence of anti-hPVRIG antibody or mIgG1 control antibody. The expression of IFN-γ ( n = 47), TNF-α ( n = 47) and CD107a ( n = 26) in NK cells was analyzed by flow cytometry. f Cytotoxicity of purified human NK cells against SW620 cells in the presence of anti-hPVRIG antibody or mIgG1 control antibody at indicated E/T ratios. g Cytotoxicity of human PBMCs against SW620, A375 and SK-OV-3 tumor cells in the presence of anti-hPVRIG antibody or mIgG1 control antibody at various E/T ratios was analyzed, respectively. h Representative histogram of PVRIG expression in expanded human NK cells. i B-NDG mice were inoculated subcutaneously with SW620 colon cancer cells on day 0. Mice were grouped randomly and expanded NK cells were injected intravenously (i.v.) on days 7, 12 and 17. Mice were then treated with anti-hPVRIG mAb ( n = 9) or isotype-matched control mAb (mouse IgG) ( n = 9) intraperitoneally (i.p.) every three days starting on day 7 for five times. All mice were injected intraperitoneally with 50,000 IU recombinant human IL-2 every two days starting on day 7. And median tumor size was shown on the right. j B-NDG mice were inoculated subcutaneously with SW620 colon cancer cells on day 0. Mice were grouped randomly and human PBMCs were injected intravenously (i.v.) on day 7. Mice were then treated with PBS ( n = 9), anti-hPVRIG mAb ( n = 9) or isotype-matched control mAb (mouse IgG) ( n = 7) intraperitoneally (i.p.) every three days starting on day 8 for five times. And median tumor size was shown on the right. Each symbol represents an individual health donor ( d , e , g ) or B-NDG mouse ( i , j ). Data are representative of at least two independent experiments. Error bars represent means ± s.e.m. Statistical significance was determined using unpaired two-tailed t test ( b , c , f , g ), paired two-tailed t test ( e ) or two-way ANNOVA ( i , j ). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Paraffin sections were de-waxed, rehydrated, subjected to heat-induced epitope retrieval (HIER) and followed by incubation with primary antibodies to human PVRIG (Clone 2, generated in house) and PVRL2 (AF2229, R&D Systems, Minneapolis, USA) respectively.

Techniques: Expressing, Cell Culture, Flow Cytometry, Purification, Injection, Recombinant, Two Tailed Test

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: 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: 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: 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: 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: 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