Journal: Journal for Immunotherapy of Cancer

Article Title: Interactions between LAMP3+ dendritic cells and T-cell subpopulations promote immune evasion in papillary thyroid carcinoma

doi: 10.1136/jitc-2024-008983

Figure Lengend Snippet: The recruitment of LAMP3 + DCs by tumor cells promotes PTC clinical progression. Heatmaps (A) and circle (B) plots show the comparison of interaction quantity and interaction strength between thyrocytes and cDCs between non-progressive PTC and progressive PTC. Red indicated that the increase of communication in the latter. (C) The interaction between thyrocytes and cDCs is more numerous and stronger in Group B. (D) Summary of selected ligand-receptor interactions between thyrocytes and cDC-C3 cells in the two groups. (E) Circle plots showing the interaction between NECTIN3-NECTIN2 ligand-receptor pairs in the cDCs and thyrocytes. (F) The expression level of S100A2 is positively correlated with LAMP3. (G) Representative immunofluorescence images illustrating the interaction between thyrocytes and cDC_C3 in two groups (A1 and B9). The small panels show the magnification of the selected region highlighted in red. Scale bars correspond to 50 µm in the large panel. (H) Schematic showing the crosstalk among thyrocytes, LAMP3 + DCs, CD8 + T cells, and Tregs involved in the recruitment of immune cells and the formation of an immunosuppressive microenvironment in the progressive PTC. cDC. conventional DC; DC, dendritic cell; CTLA-4, cytotoxic T-lymphocyte associated protein 4; ICAM, intercellular adhesion molecule 1; LAMP3, lysosomal associated membrane protein 3; PTC, papillary thyroid cancer; SPN, sialophorin; TME, tumor microenvironment; Treg, regulatory T cell.

Article Snippet: The following antibodies were used in the current study: primary antibodies against DC_LAMP/CD208 (1:400, CST, Cat#47778), CD8A (1:100 dilution; Abcam, Cat#ab217344), TIGIT (1:1,000, Cell Signaling Technology, Cat#99567T), NECTIN2 (1:400, Cell Signaling Technology, Cat#95333T), NECTIN3 (1:500, R&D, Cat#AF3064-SP), CCL17 (1:1,000, Abcam, Cat#ab195044), KRT19 (1:2,000, Abcam, Cat#ab76539), FOXP3 (1:2,000, Abcam, Cat#ab215206), or CCR4 (1:2,000, Novus Biologicals, Cat#NBP1-86584).

Techniques: Comparison, Expressing, Immunofluorescence, Membrane

Journal: Nature Communications

Article Title: Cancer cell plasticity defines response to immunotherapy in cutaneous squamous cell carcinoma

doi: 10.1038/s41467-024-49718-8

Figure Lengend Snippet: a Percentage of PD-L1 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 23), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 27). b Percentage of CD112 + cells within the indicated cancer cells ( n = 12 tumors per group). c Percentage of Gal9 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 27), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 17), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). d Percentage of CD80 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 24), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 18), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 21). e Percentage of CD155 + cells within full epithelial cancer cells from epithelial cSCCs ( n = 10), EpCAM high , EpCAM low , and EpCAM − cancer cells from mixed cSCCs ( n = 9), and full mesenchymal cancer cells from mesenchymal cSCCs ( n = 12). f , h Representative immunofluorescence images of Ecad + (green), f CD80 + or h CD155 + (red), and DAPI nuclear (blue) staining in the indicated patient cSCCs. Scale bar, 100 µm. g Percentage of CD80 + cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). i Percentage of CD155 + cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Each dot indicates the average quantification of at least five fields from different tumor regions. j Percentage of CD80 − Ecad + , CD80 + Ecad + , and CD80 + Ecad − cancer cells relative to total cancer cells in the indicated patient cSCCs ( n = 4 per group). k Percentage of CD155 − Ecad + , CD155 + Ecad + , and CD155 + Ecad − cancer cells relative to total cancer cells in epithelial ( n = 4), mixed ( n = 5), and mesenchymal ( n = 4) patient cSCCs. Data are represented as the mean ± SD ( a – e ) or ± SEM ( g , i , j , k ), and n values indicate independent tumors ( a – e , g , i ). P values are determined by one-way ANOVA with Dunnett’s ( a – e ) or Tukey’s ( g , i ) multiple comparison tests. See Supplementary Fig. for the gating strategy ( a – e ). Source data are provided as a Source Data file.

Article Snippet: For cell-surface staining, cells were blocked with 1 mg/ml IgG (Sigma, I5381) and stained with a cocktail of cell-surface antibodies in staining buffer (5% FBS in PBS) for 30 min at 4 °C: from Biolegend, CD11b-APC 1:250 (M1/70, 101211), CD11b-PE/Cy7 1:250 (M1/70, 101215), CD152 (CTLA-4)-PE/Cy7 1:250 (UC10-4B9, 106313), CD155-PE/Cy7 1:200 (TX56, 131511), CD223 (LAG-3)-PE/Cy7 1:250 (C9B7W, 125225), CD226 (DNAM-1)-PE/Cy7 1:250 (10E5, 128811), CD25-PE/Cy7 1:200 (PC61, 102015), CD274 (PD-L1)-PE/Cy7 1:200 (10F.9G2, 124313), CD279 (PD-1)-APC/Cy7 1:250 (29F.1A12, 135223), CD28-PE/Cy7 1:250 (37.51, 102125), CD3ε-APC 1:200 (145-2C11, 100311), CD366 (TIM-3)-PE/Cy7 1:250 (B8.2C12, 134009), CD4-PE/Cy7 1:200 (RM4-5, 100528), CD49f (α6-integrin)-FITC 1:10 (GoH3, 313605), CD69-PE/Cy7 1:200 (H1.2F3, 104511), CD8a-PE 1:200 (53-6.7, 100707), CD80-PE/Cy7 1:250 (16-10A1, 104733), F4/80-APC/Cy7 1:200 (BM8, 123118), Galectin9-PE/Cy7 1:250 (108A2, 137913), Ly-6G/Ly-6C (Gr-1)-PE/Cy7 1:250 (RB6-8C5, 108415), Ly-6C-PE/Cy7 1:250 (HK1.4, 128017), Ly-6G-APC 1:250 (1A8, 127613), NK-1.1-PE 1:200 (PK136, 108707), TIGIT (Vstm3)-PE/Cy7 1:250 (1G9, 142107); from BD Bioscience, CD11b-PE 1:250 (M1/70, 557397); from eBioscience, CD206-APC 1:200 (MR6F3, 17-2061-80), CD326 (EpCAM)-APC-eF780 1:400 (G8.8, 47-5791-82); from TONBO, CD45-PE 1:350 (30-F11, 50-0451); from R&D Systems, CD112 (Nectin-2)-APC 1:200 (829038, FAB3869A).

Techniques: Immunofluorescence, Staining, Comparison

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: Nature Communications

Article Title: Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

doi: 10.1038/s41467-021-24010-1

Figure Lengend Snippet: a We examined the immune checkpoint interactions between lymphocytes and APCs (tumor cells and TAMs) and identified the prominent interaction via the TIGIT – NECTIN2 axis (circle size indicates the statistical significance and circle color indicates the level of interaction). The empirical P value was estimated by 1000 imputations. b The expression of TIGIT and NECTIN2 was respectively enriched in T cells and APCs. c Upregulation of NECTIN2 was detected in HCC tumors, as compared to non-tumorous livers in both in-house and TCGA datasets. Student’s t test (2-sided). Source data are provided as a Source Data file.

Article Snippet: In the coculturing experiment of T and parental Hepa1–6 cells, 15 μg/mL anti-Nectin2 neutralizing antibody (MAB3869, R&D Systems, MN, USA) (Supplementary Table ) was added.

Techniques: Expressing

Journal: Nature Communications

Article Title: Single-cell RNA sequencing shows the immunosuppressive landscape and tumor heterogeneity of HBV-associated hepatocellular carcinoma

doi: 10.1038/s41467-021-24010-1

Figure Lengend Snippet: a CellTrace Violet (CTV)-labeled mouse splenic T cells were isolated and co-cultured with Hepa1–6 cells in the presence or absence of anti-Nectin2 neutralizing antibody (15 µg/mL). Mean ± SD is presented. b CTV-labeled T cells were cocultured with Hepa1–6 (WT), - Nectin2 -KO1, - Nectin2 -KO2, - Nectin2 -KO3 cells. Mean ± SD is presented. c Representative picture and weight of Nectin2 WT ( Nectin2 WT: Tp53 KO: c-Myc OE), and Nectin2 KO ( Nectin2 KO: Tp53 KO: c-Myc OE) HCC tumors. Scale bar = 1 cm. d – f Numbers of tumor-infiltrating lymphocytes were analyzed by flow cytometry. g , h Representative pictures, and quantification of CD4 + T cells and CD8 + T cells in HCC tumors by IHC staining. Scale bar = 100μm in IHC representative pictures. a – h Student’s t test. The experiment was performed with a variable number of biologically independent samples ( n number) ( a n = 6, n = 3, and n = 3 for T cells only Ctrl and anti-Nectin2 respectively in CD4+ cells, and n = 3 for all groups in CD8+ cells; b n = 10, n = 6, n = 4, and n = 4 for WT, Nectin2 -KO1, Nectin2 -KO2, and Nectin2 -KO3, respectively in both CD4+ and CD8+ cells; c – e , g : n = 7 per group; f , h : n = 21 per group). Source data are provided as a Source Data file.

Article Snippet: In the coculturing experiment of T and parental Hepa1–6 cells, 15 μg/mL anti-Nectin2 neutralizing antibody (MAB3869, R&D Systems, MN, USA) (Supplementary Table ) was added.

Techniques: Labeling, Isolation, Cell Culture, Flow Cytometry, Immunohistochemistry