mouse nectin 4 proteins  (R&D Systems)

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: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Staining, Control, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, Control, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: Then the following antibodies or corresponding isotype controls were added for 30 min at 4 °C: APC/Cy7 rat anti-mouse CD45 (BioLegend), BV421 rat anti-mouse CD31 (BioLegend), APC Syrian hamster anti-mouse Podoplanin (BioLegend), PE/Cy7 or APC Armenian hamster anti-mouse CD11c (BioLegend), BV421 rat anti-mouse MHC class II (BioLegend), Alexa Fluor 488 rat anti-mouse CD112 (clone:829038, R&D system) and Zombie Aqua fixable viability dye (dilution as recommended by the manufacturer, BioLegend).

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed in BM-DCs and LECs and supports DC transmigration. ( A ) Flow cytometry analysis of immature (−LPS) and LPS-matured (+LPS) BM-DCs (gated on live/single cells). ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression of 11 independent experiments. ( C – F ) FACS analysis of CD112 expression in ( C ) LPS-matured BM-DCs and ( E ) primary LN-LECs, derived from WT and CD112 KO mice. ( D , F ) Summary of the ∆MFI values of CD112 expression of 4–6 independent experiments. Data points of the same experiment in ( B , D , F ) are connected by a line, and the mean ΔMFI values are indicated by horizontal lines. ( G ) Set up of the transmigration experiments to investigate the transmigration of BM-DCs (WT or KO) across an LEC monolayer (WT or KO). ( H ) Impact of ICAM-1 blockade on transmigration of WT BM-DCs. ( I,J ) Impact of loss of CD112 in either ( I ) LECs or ( J ) BM-DCs on transmigration. ( K ) Impact of simultaneous loss of CD112 in LECs and BM-DCs on transmigration. For each condition in ( H – K ), one representative experiment with n = 3 technical replicates is shown on the left, and a summary of the averages of 4 independent experiments (biological replicates, each experiment in a different color) is shown on the right. Data points of the same experiment are connected by a line. ( L ) Adhesion assay of WT and KO BM-DCs to WT or KO lymphatic endothelium. The pool of two independent experiments with three replicates per condition is shown (each dot represents a sample). # BM-DCs: number of BM-DCs. Data in all graphs show mean ± standard error of the mean (SEM). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns: not significant.

Article Snippet: Nectin-2 expression on matured BM-DCs was analyzed by using AF488 rat anti-mouse Nectin-2/CD112 and corresponding isotype control from R&D Systems, Minneapolis, MN, USA.

Techniques: Transmigration Assay, Flow Cytometry, Staining, Expressing, Derivative Assay, Cell Adhesion Assay

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 expression is high in LECs but low in DCs present in murine skin. ( A , B ) FACS analysis was performed to detect CD112 expression in dermal LECs and BECs. ( A ) Depiction of the gating strategy in one representative experiment. ( B ) Summary of the delta mean fluorescent intensity (∆MFI; specific-isotype staining) values of CD112 expression observed in 5 independent experiments. ( C – G ) Impact of TPA-induced skin inflammation on the expression of CD112 in LECs. ( C ) Schematic depiction of the experiment: Inflammation was induced in the murine ear skin by topical application of TPA and the ear skin and draining auricular LNs analyzed 24 h later. ( D – G ) FACS analyses were performed to quantify CD112 expression levels in LECs present in control or inflamed tissues. ( D , E ) Analysis of murine ear skin and ( F , G ) auricular LN single-cell suspensions. ( E , G ) The summary of ∆ MFI values was recorded in 5–6 different experiments performed in one control (CTL) and one TPA-inflamed (TPA) ear skin. ( H , I ) FACS gating and quantification of CD112 expression in DCs present in CTL and TPA-inflamed ear skin. ( H ) Gating strategy and ( I ) summary of ∆MFI values recorded in 3 different experiments. ( J – P ) Crawl-out experiments. ( J ) Schematic depiction of the experiment performed to evaluate CD112 expression in ( K – M ) DCs that had emigrated from murine ear skin into the culture medium or in ( N – P ) DCs that had remained in the cultured ear skin at the end of the experiment. Representative ( K , N ) FACS dot plots (gating on single/live cells), identifying DCs as MHCII + CD11c + cells. ( L , O ) Representative histogram plots showing CD112 expression in WT and KO DCs as well as the corresponding fluorescence minus one (FMO) control. ( M , P ) Summary of ∆MFI values (defined as specific staining—FMO) recorded in 4 different experiments performed with one WT and one KO mouse each. Data points in ( B , E , G , I , M , P ) of the same experiment are connected by a line.

Article Snippet: Nectin-2 expression on matured BM-DCs was analyzed by using AF488 rat anti-mouse Nectin-2/CD112 and corresponding isotype control from R&D Systems, Minneapolis, MN, USA.

Techniques: Expressing, Staining, Cell Culture, Fluorescence

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Loss of CD112 does not impact the in vivo migration of adoptively transferred or endogenous DCs to dLNs. ( A – D ) Adoptive transfer experiment. ( A ) Scheme of the experiment. ( B ) Gating strategy to identify fluorescently labeled adoptively transferred BM-DCs in popliteal LNs. ( C ) The ratio of KO–WT DCs recovered from popliteal LNs draining control (CTL) or CHS-inflamed (CHS) footpads of WT or KO mice. ( D – J ) FITC painting experiment. ( D ) Scheme of the experiment. ( E ) ΔEar thickness, defined as the difference between the ear thickness measured at the start and at the end of the experiment. ( F ) Cellularity and ( G ) weight of the ear-draining auricular LN at the end of the experiment. ( H ) Gating strategy to identify and quantify the number (#) of ( I ) all CD11c + MHCII hi migratory DCs (mDCs) and ( J ) FITC + mDCs. Summaries of three ( A – D ) and two ( D – J ) independent experiments, each with 2–7 mice per condition, are shown. Each dot represents one mouse. Mann–Whitney t -test was used. Red bars in all graphs show the mean. ns: not significant.

Article Snippet: Nectin-2 expression on matured BM-DCs was analyzed by using AF488 rat anti-mouse Nectin-2/CD112 and corresponding isotype control from R&D Systems, Minneapolis, MN, USA.

Techniques: In Vivo, Migration, Adoptive Transfer Assay, Labeling, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: Nectin-2 expression on matured BM-DCs was analyzed by using AF488 rat anti-mouse Nectin-2/CD112 and corresponding isotype control from R&D Systems, Minneapolis, MN, USA.

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: Nectin-2 expression on matured BM-DCs was analyzed by using AF488 rat anti-mouse Nectin-2/CD112 and corresponding isotype control from R&D Systems, Minneapolis, MN, USA.

Techniques: Expressing, Derivative Assay, Blocking Assay, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: Blockade of CD112 decreases in vitro transmigration of human moDCs across human dermal LEC monolayers. ( A – C ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in in vitro-differentiated ( A ) immature (−LPS) and ( B ) LPS-matured (+LPS) human moDCs. LPS was added 24 h prior to FACS analysis. Representative FACS plots are shown in ( A , B ). ( C ) Summary of the delta mean fluorescent intensity (∆MFI; defined as specific-isotype staining) values recorded for each corresponding marker in 3–6 independent experiments (biological replicates). Data points of the same experiment are connected by a line, and the means of the ΔMFI values are indicated by horizontal red lines. ( D , E ) Analysis of CD112, DNAM-1, TIGIT and CD113 expression in primary human dermal LECs. ( D ) Representative FACS histograms recorded upon gating on CD31 + podoplanin + cells, and ( E ) summary of the MFI values recorded for all markers and corresponding isotype controls in 4–5 independent experiments performed on LECs from two different donors. Data points of the same experiment are connected by a line, and the means of the MFI values are indicated by horizontal red lines. ( F – I ) Transmigration experiments involving human moDCs and human dermal LECs, performed in the presence/absence of ( F , G ) αICAM-1 or of ( H , I ) αCD112 or the corresponding isotype controls; ( F – I ) The number of transmigrated DCs (# DCs) was assessed. ( F , H ) show representative results from one representative experiment with n = 6 technical replicates per condition. ( G , I ) show the summaries of four independent experiments (i.e., different biological replicates, shown with different colors) with 3–6 replicates per condition. The averages from each experiment are connected by a line. The standard error of the mean (SEM) is shown; the Mann–Whitney t -test was used. * p < 0.05; ** p < 0.01.

Article Snippet: CD112-binding partners’ expression on in vitro matured BM-DCs and in vitro cultured primary LN-LECs was analyzed by using the following antibodies and corresponding isotype controls: PE/Cy7 rat anti-mouse CD226 (BioLegend), BV421 mouse anti-mouse TIGIT (BioLegend) and goat anti-mouse CD113 (R&D system).

Techniques: In Vitro, Transmigration Assay, Expressing, Staining, Marker, MANN-WHITNEY

Journal: Cells

Article Title: CD112 Supports Lymphatic Migration of Human Dermal Dendritic Cells

doi: 10.3390/cells13050424

Figure Lengend Snippet: CD112 is expressed by DCs and LECs in human skin. ( A – D ) FACS-based analysis of CD112 expression in endothelial cells and DCs present in human skin. ( A , C ) Gating strategy used to detect CD112 expression in ( A ) BECs and LECs and ( C ) DCs. ( B , D ) Summary of mean fluorescent intensity (MFI) values of CD112 expression in ( B ) LEC and BECs or ( D ) HLA-DR + CD86 + DCs in 2 independent experiments (i.e., different biological replicates) was analyzed. Data points of the same experiment are connected by a line. ( E , F ) Confocal images of human skin sections depicting ( E ) CD112 expression (white) by dendritic cells (examples indicated by white arrows), identified as HLA-DR + (green) and CD11c + (red). Scale bar = 100 μm ( F ) CD112 expression (white) by lymphatic vessels, LYVE-1 (green) and PLVAP (red). Scale bar = 100 μm. ( G ) Top: Gating strategy and Bottom: representative histogram plot showing CD112 expression on DCs that had emigrated from a human breast skin punch biopsy. ( H ) Crawl-out experiments from punch biopsies derived from either breast or abdominal skin were performed in the presence of a CD112-blocking antibody or media/isotype control (CTL) in the culture medium. Top: Representative FACS gating plot from abdominal skin. Bottom: Quantification of emigrated HLA-DR+CD86 + DCs. Pooled data from 5 independent experiments with 4–10 punches per condition are shown. ( I ) Crawl-out experiment from abdominal skin punch biopsies to verify the expression of CD112-binding partners DNAM-1, TIGIT and CD113 on human DCs, identified as live, HLA-DR + cells. Representative stainings from one out of three independent experiments are shown. The mean and standard deviation (SD) are shown in (H). Mann–Whitney t -test was used. ** p < 0.01.

Article Snippet: CD112-binding partners’ expression on in vitro matured BM-DCs and in vitro cultured primary LN-LECs was analyzed by using the following antibodies and corresponding isotype controls: PE/Cy7 rat anti-mouse CD226 (BioLegend), BV421 mouse anti-mouse TIGIT (BioLegend) and goat anti-mouse CD113 (R&D system).

Techniques: Expressing, Derivative Assay, Blocking Assay, Control, Binding Assay, Standard Deviation, MANN-WHITNEY

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity

doi: 10.1101/2023.11.23.568355

Figure Lengend Snippet: (A-B) NK cells from BALB/c or C57BL/6 mice were activated for 24 hours, and then incubated with NMFs, pCAFs, or sCAFs at a 5:1 NK:fibroblast ratio to determine cytotoxicity. NK cells were treated with either 50μg/ml IgG control or combinations of 50μg/ml α-NKG2D/DNAM1 antibodies to determine dependency on either receptor for cytotoxicity of fibroblast targets. The relative cytotoxicity (normalized to cytotoxicity of NK cells activated with IgG control) is presented (N=3 biologically independent experiments, data is presented as mean ± SEM). (C) NK cells from BALB/c mice were activated for 24 hours alone or in the presence of pCAFs that were pre-incubated with either 50μg/ml IgG control or combinations of 50μg/ml α-CD155, Nectin2, and Rae-1 antibodies. Subsequently, NK cells were subjected to FACS staining for NKG2D and DNAM-1. Quantification of the FACS experiments is shown in the top panel. N=5 biologically independent experiments, data is presented as mean ± SEM. The representative histograms (bottom) were normalized to the modal value. (D) NK cells from BALB/c mice were activated for 24 hours alone or as in (C), and then incubated with 4T1 cancer cells at a 5:1 NK:cancer ratio to determine cytotoxicity. Cytotoxicity (normalized to cytotoxicity of NK cells activated alone) is presented. N=6 biologically independent experiments, data is presented as mean ± SEM.

Article Snippet: For CAF ligand blockade experiments, CD155, Nectin2, and Pan-Rae-1 expressed on CAFs were blocked utilizing 25 μg/ml of anti-mouse pan Rae-1 (R&D,AF1136), Ultra-LEAF™ anti-mouse CD155 (Biolegend, 942103), anti-mouse Nectin2 (R&D, MAB3869), or Mouse IgG1 Isotype Control control (R&D, MAB002) for 15 minutes at room temperature prior to culture with NK cells.

Techniques: Incubation, Staining

Journal: bioRxiv

Article Title: Cancer-associated fibroblasts serve as decoys to suppress NK cell anti-cancer cytotoxicity

doi: 10.1101/2023.11.23.568355

Figure Lengend Snippet: (A) Representative IHC staining of NK cells (NKp46) in TMAs of TNBC patient cohorts. (B) Quantification of IHC images was conducted on the proportion of NK cells in stromal areas compared to cancer specified regions (Exact Wilcoxon rank sum test p−value = 0.00005) (C-D) Representative immunofluorescence images of 2 patient TMAs. Staining was conducted on DAPI, cytokeratin (cancer cells), CD45 (immune cells), and NK cell ligands NECTIN2, PVR, and MICA/B. (E) Kaplan-Meir survival analysis of patients stained in (C-D). Analysis was conducted on the ratio of CAFs expressing NECTIN2 out of the total CAFs, compared to the ratio of positive cancer cells out of total cancer cells, with groups stratified above (high) or below (low) the median value. Statistical analysis was conducted using two-sided log-rank test. (F) Scheme of proposed model. Reduction of NK cell receptors following engagement with CAFs may result in impaired cytolysis of cancer cells by NK cells in the TME

Article Snippet: For CAF ligand blockade experiments, CD155, Nectin2, and Pan-Rae-1 expressed on CAFs were blocked utilizing 25 μg/ml of anti-mouse pan Rae-1 (R&D,AF1136), Ultra-LEAF™ anti-mouse CD155 (Biolegend, 942103), anti-mouse Nectin2 (R&D, MAB3869), or Mouse IgG1 Isotype Control control (R&D, MAB002) for 15 minutes at room temperature prior to culture with NK cells.

Techniques: Immunohistochemistry, Immunofluorescence, Staining, Expressing