Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: a B220 + cells and Th17 cells in the BM niche in WT mice and BCR-ABL tTA mice ( n = 3 mice per group) were subjected to immunofluorescence staining. Representative images were shown. b The numbers of Th17 cells migrating to culture medium or to leukemia cells treated with or without 50 ng/ml rhIL-17A were determined by cell counting and FCS analysis. c Real-time PCR analysis of the relative mRNA levels of chemokine genes in SupB15 (left) and BV173 cells (right) treated with rhIL-17A (100 ng/ml) or PBS control. d The human IL-5, CXCL5, CCL5 and CXCL16 levels in the culture supernatant of SupB15 cells treated with or without rhIL-17A (100 ng/ml) were measured by ELISA. e The CXCL16 concentrations in the serum of BCR-ABL tTA mice and WT mice ( n = 5 mice per group) were quantified with a Mouse Chemokine Assay Q1 (Raybiotech). f The CXCL16 concentrations in the serum of HDs ( n = 7 samples) and primary Ph + B-ALL patients ( n = 14 samples) were measured by ELISA. g Schematic strategy for studying the differentiation of Th17 cells with or without rmCXCL16 stimulation in vitro. h Representative FACS plots and quantification of the percentage of Th17 cells in naïve CD4 + T cells with or without rmCXCL16 treatment. i Schematic strategy for studying the migration of Th17 cells cocultured with leukemia cells with or without anti-CXCL16 mAb treatment in vitro. j , k Flow cytometric analysis of the percentage of Th17 cells in the lower chambers of the inserts ( j ) and proliferation activity of leukemia cells ( k ) in a coculture system with or without anti-CXCL16 treatment. The gating strategy for isolating Th17 cells is shown in Supplementary Fig. . (b–d , h , j , k) n = 3 independent experiments. Statistical significance was calculated by ( c – f , j , k ) two-tailed Student’s t test; ( b , h ) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Immunofluorescence, Staining, Cell Counting, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, In Vitro, Migration, Activity Assay, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: Primary Ph + B-ALL cells were treated with or without rhIL-17A for 24 h, and relative CXCL16 mRNA level ( a ) and CXCL16 secretion ( b ) were evaluated by real-time PCR and ELISA, respectively. c Representative immunofluorescence images of B220 (red) and CXCL16 (green) staining in spleen tissue sections of WT mice and BCR-ABL tTA mice treated with or without 50 µg/kg rmIL-17A for a duration of 3 weeks (twice a week) ( n = 3 mice per group). d Flow cytometric analysis and quantification of CXCL16 expression in primary mouse B-ALL cells treated with or without rmIL-17A. e The protein levels of p65 and p-p65 in the cytoplasm and p-p65 in the nucleus of primary mouse leukemia cells treated with or without rmIL-17A were measured by Western blotting. f Immunofluorescence of p-P65 in primary B-ALL cells treated with or without rhIL-17A. g The effect of rhIL-17A treatment on NF-kB transcriptional activity. HEK 293T cells were transfected with a synthetic NF-kB luciferase reporter construct (pNF-kB-Luc) for 12 h and then treated with different concentrations of rhIL-17A for 24 h. NF-kB transcriptional activity was detected by a luciferase assay. h ChIP‒qPCR analyses of the binding of NF-kB to the CXCL16 promoter region in SupB15 cells treated with or without rhIL-17A. i – k The effect of BAY11-7082, rIL-17A or BAY11-7082 in combination with rIL-17A on the cytoplasmic and nuclear protein levels of p65, p-p65, and CXCL16 in primary Ph + B-ALL cells. i The indicated protein band intensities were quantified using ImageJ software. The relative CXCL16 mRNA level ( j ) in primary Ph + B-ALL cells and CXCL16 protein level ( k ) in the supernatant of primary Ph + B-ALL cells were measured by RT‒PCR and ELISA, respectively. (a, b , d – k ) n = 3 independent experiments. Statistical significance was calculated by ( a , b , d ) two-tailed Student’s t-test; ( e , g – k ) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Expressing, Western Blot, Activity Assay, Transfection, Luciferase, Construct, Binding Assay, Software, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: a Flow cytometric analysis of the percentages of B220 dim CD19 + cells in BM, spleens, LNs and PB of mice with secondary transplantation treated with or without rmCXCL16 ( n = 6 mice per group). b – d Representative images of Wright-Giemsa-stained PB smears ( b , top), H&E staining of spleens ( b , bottom), spleens ( c ) and spleen weights ( d ) from the indicated mice ( n = 6 mice per group). e Representative images of Ki67 staining in the spleen tissues from the indicated mice. n = 4 fields, two different mice per group. f Flow cytometric analysis of the percentages of Th17 cells in the PB, LNs, spleens and BM from the indicated mice ( n = 6 mice per group). g Spleen tissues from the indicated mice were subjected to immunofluorescence staining for IL-17A (red), CD4 (green), and B220 (rose red). Representative images of Th17 cells were shown. n = 4 fields, two different mice per group. h Kaplan–Meier survival curves for the indicated mice ( n = 8 mice per group). i Schematic strategy for investigating the effects of anti-CXCL16 mAb alone or combined with imatinib on Ph + B-ALL progression. j – l Representative images of spleens ( j ), spleen weights ( k ), Wright-Giemsa-stained PB smears ( l, top ), and H&E staining of the spleen ( l, bottom ) from leukemia mice treated with the indicated agents ( n = 3 mice per group). m Flow cytometric analysis of the percentages of B220 dim CD19 + cells in the PB, BM, LNs and spleens from leukemia mice treated with the indicated agents ( n = 3 mice per group). n The percentage of Ki-67 + cells in the spleen was detected by immunofluorescence staining in the indicated mice. Data are presented as means ± S.E.M of eight random fields of view from three different mice per group. o Flow cytometric analysis of the percentages of Th17 cells in the PBMCs, LNs, spleens and BM of leukemia mice treated with the indicated agents ( n = 3 mice per group). (a , m ) The gating strategy for B220 dim CD19 + cells was shown in Supplementary Fig. . ( f , o ) The gating strategy for Th17 cells in the CD4 + T cells was shown in Supplementary Fig. . Statistical significance was calculated by ( a, d – g ) two-tailed Student’s t-test; ( k , m – o) one-way ANOVA with Tukey’s multiple comparison tests ; Data are presented as means ± S.E.M. Source data are provided as a Source Data file.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Transplantation Assay, Staining, Immunofluorescence, Two Tailed Test, Comparison

Journal: Nature Communications

Article Title: Targeting IL-17A enhances imatinib efficacy in Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia

doi: 10.1038/s41467-023-44270-3

Figure Lengend Snippet: The Th17 cell population and IL-17A expression are distinctively increased in Ph + B-ALL patients, and high expression of IL-17A promotes the progression of Ph + B-ALL. IL-17A promotes the proliferation and survival of Ph + B-ALL cells by activating the BCR-ABL and IL6/JAK/STAT3 signaling pathways. Moreover, IL-17A can increase the secretion of the chemokine CXCL16 from leukemia cells by activating NF-kB, which in turn mediates the differentiation and recruitment of Th17 cells to the leukemia niche microenvironment. Targeting IL-17A or CXCL16 in the leukemia niche microenvironment attenuates the progression of Ph + B-ALL.

Article Snippet: The following fluorescently labeled antibodies against surface proteins were used for human/mouse cell staining: mouse anti-CXCL16 antibody (R&D, AF503, 1:100), followed by staining with secondary antibodies labeled with Alexa Fluor 555; PE-conjugated anti-mouse/human CD45R/B220 antibody (BioLegend, 103208, 1:100); APC/Cyanine7-conjugated anti-mouse CD19 antibody (BioLegend, 152412, 1:100); PerCP-conjugated anti-mouse CD19 antibody (BioLegend, 115531, 1:100), Alexa Fluor 647-conjugated anti-mouse Ly-51 (BP-1) antibody (BioLegend, 108311, 1:100), PE/Cyanine7-conjugated anti-mouse CD43 antibody (BioLegend, 143209, 1:100), APC-conjugated anti-mouse CD4 antibody (BioLegend, 116014, 1:100), FITC-conjugated anti-mouse IL-17A antibody (BioLegend, 506907, 1:100), PE/Cyanine7-conjugated anti-mouse Ki-67 antibody (BioLegend, 652426, 1:100), PerCP anti-mouse CD19 Antibody (BioLegend, 115531, 1:100), PE-conjugated anti-human CD19 antibody (BioLegend, 302254, 1:100), FITC-conjugated anti-human CD4 antibody (BioLegend, 357405, 1:100), APC-conjugated anti-human CD4 antibody (BioLegend, 317416, 1:100), FITC-conjugated anti-mouse/human Ki-67 antibody (BioLegend, 151212, 1:100), PE/Cyanine7-conjugated anti-human IL-17A antibody (BioLegend, 512315, 1:100), Alexa Fluor 647-conjugated anti-human IL-17A antibody (BioLegend, 512310, 1:100), APC-conjugated human IL-17RA/IL-17R antibody (R & D, FAB177A, 1:100), and Alexa Fluor® 488-conjugated human IL-17RC antibody (R & D, FAB22691,1:100).

Techniques: Expressing

Journal: Life Science Alliance

Article Title: Dendritic cells maintain anti-tumor immunity by positioning CD8 skin-resident memory T cells

doi: 10.26508/lsa.202101056

Figure Lengend Snippet: (A) Heat map showing gene expression of chemokines and receptors in CD11c + CD11b neg , CD11c + CD11b + , and CD8 + T cells sorted from skin of MAV-affected mice. Data presented as log2-normalized expression. (B) Surface expression of CXCL16 on CD11c + cells isolated from skin of naïve (n = 3) or MAV-affected (n = 5) mice, skin-draining LNs, or spleen 7, 14, and 35 d after surgery. Representative of three independent experiments. (C) Surface expression of CXCL16 on CD45 + CD11c + , CD45 + CD11c neg , and CD45 neg cells isolated from skin of MAV-affected mice, 35 d after surgery. Representative of two independent experiments with skin collected between 35- and 60-d post-surgery. Dotted horizontal line indicates background CXCL16 expression in unstained cell sample. (D) Expression of CXCL16 and CD11c in unaffected and MAV-affected mice; CD11c (green), CXCL16 (red), and nuclei (blue). Scale bar, 50 μm. (E) CXCL16 expression in IF images from skin of unaffected and MAV-affected mice; n = 5–8 mice. (F) CXCL16 expression in hair follicles with or without CD11c + cell clusters in MAV-affected skin; n = 8 mice. (G) CD11c + and CXCL16 + cells in skin from MAV-affected patients. Stains identify CXCL16 (green) and CD11c (red); dark red identifies colocalization of CD11c and CXCL16. Scale bar, 25 μm. (H) Percent of CD11c + cells expressing CXCL16 in MAV-affected patient skin. (B, C, H) Symbols represent individual mice (B, C) or patients (H); horizontal lines indicate mean. MFI, mean fluorescence intensity. Significance was determined by one-way ANOVA; * P ≤ 0.03, ***< P = 0.001, **** P ≤ 0.0001.

Article Snippet: Antibodies from BioLegend: anti-mouse CD45-APC/Fire 750 (clone 30-F11; #147713), anti-mouse CD8α-PerCP/Cynine5.5, -PE/Cy7 (clone 53-6.7; #100733, #100721), anti-mouse Thy1.1-PerCP/Cynine5.5 (clone OX-7; #202515), anti-mouse CD103-Alexa Fluor 647, -FITC (clone 2E7; #121409, #121419), anti-mouse CD44-APC/Fire 750 (clone IM7; #103061), anti-mouse CD62L-Brilliant Violet 510 (clone MEL-14; 104441), anti-mouse CXCR6-Brilliant Violet 421 (clone SA051D1; #151109), anti-mouse CD11c Alex Fluor 647 (clone N418; #117314), anti-mouse CD11b-Alexa Fluor 488 (clone M1/70; #101219); Bioss: anti-mouse CXCL16 Alexa Fluor 488 (polyclonal; #bs-1441R-A488).

Techniques: Expressing, Isolation, Fluorescence

Journal: Life Science Alliance

Article Title: Dendritic cells maintain anti-tumor immunity by positioning CD8 skin-resident memory T cells

doi: 10.26508/lsa.202101056

Figure Lengend Snippet: (A, B, C, D) Melanoma-associated vitiligo (MAV) was induced in CD11c.DTR + and CD11c.DTR neg mice. 30 d after surgery, mice received three doses of 50 ng diphtheria toxin (DT) i.d. over 7 d at the surgical site before the skin was analyzed. (B) Expression of CD8 T cells and CD11c + cells in skin from CD11c.DTR neg and CD11c.DTR + MAV-affected mice; CD11c (green), CD8β (red), and nuclei (blue). Scale bar, 50 μm. (C) Number of clusters found per cm 2 of skin from CD11c.DTR + and CD11c.DTR neg MAV-affected mice treated with DT. (D) Expression of CXCL16 and CD11c in skin from CD11c.DTR neg and CD11c.DTR + MAV-affected mice treated with DT; CD11c (green), CXCL16 (red), and nuclei (blue). Scale bar, 50 μm. (E) Relative CXCL16 expression in skin from CD11c.DTR + and CD11c.DTR neg MAV-affected mice treated with DT. (F, G) 6 wk after lethally irradiated WT congenic CD45.1 mice received CD11c.DTR bone marrow, MAV was induced and beginning d30 after surgery mice received 250 ng DT or PBS i.p. every 3 d until skin was harvested d60 post-surgery. (G) Percent of CD8 T cells or CD11c + cells isolated from skin of MAV-affected mice treated with either DT or PBS. (C, G) Symbols represent individual clusters (C), individual mice (G); horizontal lines indicate mean. (B, C, D, E, G) n = 4–6 mice pooled from two independent experiments (B, C, D, E) n = 10–14 mice pooled from two independent experiments (G) Significance was determined by Mann–Whitney test; * P ≤ 0.04, *** P ≤ 0.0001.

Article Snippet: Antibodies from BioLegend: anti-mouse CD45-APC/Fire 750 (clone 30-F11; #147713), anti-mouse CD8α-PerCP/Cynine5.5, -PE/Cy7 (clone 53-6.7; #100733, #100721), anti-mouse Thy1.1-PerCP/Cynine5.5 (clone OX-7; #202515), anti-mouse CD103-Alexa Fluor 647, -FITC (clone 2E7; #121409, #121419), anti-mouse CD44-APC/Fire 750 (clone IM7; #103061), anti-mouse CD62L-Brilliant Violet 510 (clone MEL-14; 104441), anti-mouse CXCR6-Brilliant Violet 421 (clone SA051D1; #151109), anti-mouse CD11c Alex Fluor 647 (clone N418; #117314), anti-mouse CD11b-Alexa Fluor 488 (clone M1/70; #101219); Bioss: anti-mouse CXCL16 Alexa Fluor 488 (polyclonal; #bs-1441R-A488).

Techniques: Expressing, Irradiation, Isolation, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Expression and function of CXCL16 in cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and effect of CXCL16 neutralizing antibody on neutrophil and mononuclear leukocyte adhesion. HUAEC were stimulated with 1% CSE, INF-γ, or TNF-α (20 ng/ml) for 1, 4, or 24 h. Relative quantification of mRNA levels for CXCL16 and GAPDH after (A) 1 h and (B) 4 h ( n = 5–8 independent experiments). Columns show fold increase in CXCL16 mRNA expression relative to control GAPDH. Values are expressed as mean ± SEM of the 2 −ΔΔCt values. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (C) Protein expression was determined by flow cytometry. Results are expressed as mean of fluorescence intensity (MFI) ( n = 7–8 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group. (D) Following a similar protocol, CXCL16 was visualized in non-permeabilized HUAEC by immunofluorescence (green). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI) ( n = 4–5 independent experiments). (E,F) Endothelial cells were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, human neutrophils (E) or mononuclear cells (F) (1 × 10 6 cells/ml) incubated with or without EDTA were perfused over the monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte accumulation quantified ( n = 5–7 independent experiments). Values are expressed as the mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to the stimulus MOPC-21-treated group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Flow Cytometry, Fluorescence, Immunofluorescence, Incubation

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Inhibition of cigarette smoke extract (CSE)-induced CXCL16 expression in arterial endothelial cells. (A) CXCL16 expression was determined by flow cytometry in human umbilical arterial endothelial cells preincubated or not with apocynin (30 µM) or allopurinol (100 µM) for 1 h and then stimulated with 1% CSE for 24 h ( n = 7 independent experiments). Results are expressed as mean of fluorescence intensity (MFI). Values are expressed as the mean ± SEM. * P < 0.05 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group. Endothelial cells were transfected with (B) Nox2, (C) Nox4, or (D) Nox5 siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 5–11 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the medium group; ++ P < 0.01 relative to 1% CSE group in control siRNA transfected cells.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Inhibition, Expressing, Flow Cytometry, Fluorescence, Transfection

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Cigarette smoke extract (CSE)-induced CXCL16 overexpression is decreased by RhoA, p38 mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB inhibition in human umbilical arterial endothelial cells (HUAEC). (A) CXCL16 expression was determined by flow cytometey in endothelial cells preincubated or not with a RhoA inhibitor (C3 transferase, 2 µg/ml) for 4 h and then stimulated with 1% CSE for 24 h. Results are expressed as mean of fluorescence intensity (MFI) ( n = 5 independent experiments). Values are expressed as mean ± SEM. * P < 0.05 relative to values in the medium group; + P < 0.05 relative to 1% CSE group. (B) HUAEC were transfected with RhoA siRNA or control siRNA. At 48 h post-transfection, cells were stimulated with 1% CSE for 24 h. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to their respective group in control siRNA-transfected cells. (C) HUAEC were stimulated with 1% CSE for 24 h. Some cells were pretreated with PD098059 (20 µM), SB202130 (20 µM), or MOL-294 (2.5 µM) for 1 h before CSE stimulation. CXCL16 expression was determined by flow cytometry. Results are expressed as MFI ( n = 4–7 independent experiments). Values are expressed as mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 relative to values in the 1% CSE group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Over Expression, Inhibition, Expressing, Fluorescence, Transfection, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Percentage of circulating platelets expressing PAC-1, P-selectin, CXCL16, and CXCR6 from active and not active smoking chronic obstructive pulmonary disease (COPD) patients and aged-matched controls by flow cytometry. Platelets were stained with conjugated antibodies against (A) CD41 and PAC-1, (B) CD41 and P-selectin, (C) CD41 and CXCL16, and (D) CD41 and CXCR6. Results are expressed as percentage of positive cells ( n = 15 aged-matched controls, n = 17 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as mean ± SEM. * P < 0.05 or ** P < 0.01 relative to values in the control group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Leukocyte recruitment by cigarette smoke extract (CSE)-stimulated human umbilical arterial endothelial cells (HUAEC) and CXCL16 plasma levels from whole blood of active and not active smokers patients with chronic obstructive pulmonary disease (COPD) and aged-matched controls. HUAEC were stimulated with 1% CSE for 24 h. Some cells were incubated with a CXCL16 neutralizing antibody (2 µg/ml) or an irrelevant isotype-matched monoclonal antibody (MOPC-21, 2 µg/ml). Subsequently, whole blood from patients with COPD active or not active smokers and healthy aged-matched controls incubated (A) without, or (B) with EDTA, was perfused over endothelial monolayers for 5 min at 0.5 dyn/cm 2 and leukocyte adhesion quantified ( n = 13 aged-matched controls, n = 14 active smokers COPD patients, n = 16 ex-smokers COPD patients). Values are expressed as the mean ± SEM. ** P < 0.01 relative to values in the medium group; + P < 0.05 or ++ P < 0.01 relative to 1% CSE group; Δ P < 0.05 or ΔΔ P < 0.01 relative to the values in the aged-matched control group. (C) CXCL16 plasmatic levels were measured by ELISA ( n = 17 aged-matched controls, n = 17 active smokers COPD patients, n = 18 ex-smokers COPD patients). Values are expressed as the mean ± SEM.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo . Potential Consequences in Chronic Obstructive Pulmonary Disease

doi: 10.3389/fimmu.2017.01766

Figure Lengend Snippet: Effect of cigarette smoke (CS) exposure in CXCR6-expressing and CXCRC6 knockout mice. Heterozygous (CXCR6 −/+ ) and homozygous (CXCR6 −/– ) mice were exposed or not to CS for 3 days and responses were examined 16 h later. (A) Leukocyte–arteriolar endothelium interactions was measured by intravital microscopy. Results are expressed as mean ± SEM ( n = 5–8 animals per group). * P < 0.05 or ** P < 0.01 relative to non-exposed animals; + P < 0.05 relative to CXCR6 −/+ mice. (B) Relative quantification of CXCL16 and β-actin mRNA was determined by RT-PCR. Columns show fold increase in expression of CXCL16 mRNA relative to control GAPDH values ( n = 5 independent experiments). Values are represented as mean ± SEM of the 2 −ΔΔCt values. ** P < 0.01 relative to non-exposed animals. (C) Cremaster muscle was fixed for CXCL16 and endothelium (CD31) staining. CXCL16 expression is shown in green (stained with an Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody) and vessel endothelium (red) was stained with a PE-conjugated anti-mouse CD31 monoclonal antibody. Overlapping expression of CXCL16 and CD31 is shown in yellow. Results are representative of five to six animals per group.

Article Snippet: The APC-conjugated rat monoclonal anti-human CXCL16, the recombinant human CXCL16, the rat polyclonal anti-human CXCL16, and the biotinylated goat polyclonal anti-human CXCL16 antibodies were purchased from R&D Systems (Abingdon, UK).

Techniques: Expressing, Knock-Out, Intravital Microscopy, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: Cancers

Article Title: Phosphodiesterase 5 (PDE5) Is Highly Expressed in Cancer-Associated Fibroblasts and Enhances Breast Tumor Progression

doi: 10.3390/cancers11111740

Figure Lengend Snippet: Role for C-X-C motif chemokine 16 (CXCL16) in mediating tumor-promoting features of PDE5-overexpressing fibroblasts. ( A ) Mouse cytokine arrays for the detection of secreted proteins in conditioned medium derived from vector (V) and PDE5-overexpressing (PDE5 2) MEFs. Left panels: membranes, 62 targets detected. Right panels: raw numerical densitometry data were extracted, the background subtracted, and the data normalized to the positive control signals. Results are shown as fold change of PDE5 2 versus V stable clones. ( B ) Real-time RT-PCR assay for CXCL16 mRNA expression in V, PDE5 1, and PDE5 2 stable clones. ( C ) ELISA for CXCL16 protein secretion in V, PDE5 1, and PDE5 2 stable clones. ( D ) Immunofluorescent staining of CXCL16 protein expression in V, PDE5 1, and PDE5 2 stable clones. DAPI staining was used for nuclei detection (inset). Scale bar = 5 µm. ( E ) Trypan blue cell count assays in V, PDE5 1, and PDE5 2 stable clones treated with vehicle (-) or CXCL16 blocking antibody (Ab CXCL16, 1.5 µg/mL) for 72 h. ( F ) Boyden chamber transmigration and ( G ) invasion assays in cells treated with vehicle (-) or Ab CXCL16. ( H ) Soft agar growth (upper panel) and wound healing (lower panel) assays in MCF-7 breast cancer cells exposed with conditioned medium (CM) derived from V, PDE5 1, and PDE5 2 stable MEFs treated with vehicle (-) or Ab CXCL16. Inset, time 0. Pictures are representative of three independent experiments. The values represent the mean ±SEM of three different experiments, each performed in triplicate. * p < 0.05; ** p < 0.005; *** p < 0.0005.

Article Snippet: Neutralizing antibody to CXCL16 (MBA503) was from R&D Systems (Minneapolis, MN, USA).

Techniques: Derivative Assay, Plasmid Preparation, Positive Control, Clone Assay, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Cell Counting, Blocking Assay, Transmigration Assay

Journal: Cancers

Article Title: Phosphodiesterase 5 (PDE5) Is Highly Expressed in Cancer-Associated Fibroblasts and Enhances Breast Tumor Progression

doi: 10.3390/cancers11111740

Figure Lengend Snippet: CXCL16 and PDE5 correlation in the stroma of breast cancer patients. ( A ) Real-time RT-PCR assay for CXCL16 mRNA expression in CAFs treated with vehicle (−) or the PDE5 inhibitors: sildenafil (S, 10 µM), tadalafil, (T, 10 µM), and vardenafil (V, 10 µM) for 24 h. ( B ) ELISA and ( C ) immunofluorescent staining for CXCL16 protein levels in CAFs treated as indicated for 24 h. DAPI staining was used for nuclei detection (inset). Scale bar = 5 µm. The values represent the mean ± SEM of three different experiments, each performed in triplicate. * p < 0.05; *** p < 0.0005. ( D ) Left panel: human CXCL16 immunohistochemical staining of MCF-7/CAF xenograft tumor sections. Scale bar = 25 µm. Right panel: immunohistochemistry scores. Cases were scored according to Allred immunohistochemistry (IHC) score which includes both the proportion and intensity scores (range from 0 to 8). * p < 0.05. ( E ) Gene expression levels of CXCL16 in normal (N) and breast cancer (T) stroma samples. ( F ) Kaplan–Meier survival analysis relating stromal CXCL16 levels and overall survival (OS) in breast cancer patients. ( G ) Kaplan–Meier survival analysis relating high stromal PDE5-CXCL16 levels and OS in breast cancer patients.

Article Snippet: Neutralizing antibody to CXCL16 (MBA503) was from R&D Systems (Minneapolis, MN, USA).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunohistochemical staining, Immunohistochemistry, Gene Expression