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: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 1 | TWEAK (tumor necrosis factor–like weak inducer of apoptosis) induces renal CXCL16 expression in vivo. (a) Quantification of CXCL16 mRNA by real-time quantitative reverse transcription-PCR in kidneys from mice 4 h after the administration of TWEAK and/or parthenolide. *Po0.003 vs. control. #Po0.05 vs. TWEAK. Data were normalized with murine glyceraldehyde-3-phosphate dehydrogenase mRNA. (b) Quantification of CD3-positive cells 4 h after TWEAK or/and parthenolide injection. *Po0.009 vs. control. #Po0.01 vs. TWEAK. Mean±s.e.m. of six animals per group. h.p.f, high power field.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Expressing, In Vivo, Reverse Transcription, Control, Injection

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 2 | Localization of TWEAK (tumor necrosis factor–like weak inducer of apoptosis)-induced renal CXCL16 expression in vivo. In control kidneys CXCL16 located mainly to tubules. In TWEAK-injected animals, an increased number of CXCL16- expressing tubules is noted. CXCL16 immunohistochemistry. Original magnification 400. Pictures are representative of six animals per group.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Expressing, In Vivo, Control, Injection, Immunohistochemistry

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 3 | TWEAK (tumor necrosis factor–like weak inducer of apoptosis) neutralization decreases CXCL16 and CXCR6 mRNA expression in experimental tubulointerstitial inflammation (TII). (a) During experimental TII, CXCL16 mRNA is increased. TWEAK neutralization decreased kidney CXCL16 mRNA (real-time quantitative reverse transcription-PCR (qRT-PCR)). *Po0.001 vs. control, **Po0.01 vs. TII, #Po0.05 vs. TII þ IgG. Data were normalized with 18s eukaryotic ribosomal RNA. (b) During experimental TII, CXCR6 mRNA is increased. TWEAK neutralization decreased kidney CXCR6 mRNA (real-time qRT-PCR). *Po0.01 vs. control, **Po0.03 vs. TII. Data were normalized with 18s eukaryotic ribosomal RNA. (c) Histological assessment confirmed that cellular tubulointerstitial injury was decreased by anti-TWEAK antibodies. *Po 0.001 vs. control, **Po0.003 vs. TII, #Po0.003 vs. TII þ IgG. Mean±s.e.m. of eight animals per group.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Neutralization, Expressing, Reverse Transcription, Quantitative RT-PCR, Control

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 4 | Localization of CXCL16 expression in experimental tubulointerstitial inflammation (TII). Note tubular cell localization of increased CXCL16 expression in mice with TII, when compared with healthy control or mice with TII treated with anti- TWEAK (tumor necrosis factor–like weak inducer of apoptosis). Immunohistochemistry. Original magnification 400. Pictures representative of eight animals per group.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Expressing, Control, Immunohistochemistry

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 6 | Increased tubular fibroblast growth factor– inducible 14 (Fn14) and CXCL16 expression is associated with interstitial inflammatory infiltrates in human tubulointerstitial inflammation (TII). (a) CXCL16 immunohistochemistry. CXCL16 is observed in renal tubules (arrows) and in surrounding inflammatory infiltrates (arrowheads) in a case of TII secondary to glomerular injury (rapidly progressive glomerulonephritis), but not in minimal change disease (MCD). (b) Fn14 immunohistochemistry. Fn14 is observed in the same renal tubules (arrows) that were stained for CXCL16 in serial sections of the same case of TII, but not in MCD. Original magnifications 200 and 400. Control for the technique is shown in Supplementary Figure S3 online. Representative images of two patients with MCD, and two with rapidly progressive glomerulonephritis.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Expressing, Immunohistochemistry, Staining, Control

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 7 | Increased tubular fibroblast growth factor–inducible 14 (Fn14) and CXCL16 expression is associated with interstitial inflammatory infiltrates in human acute tubulointerstitial nephritis. (a) CXCL16 immunohistochemistry. CXCL16 is observed in renal tubules (arrows) and in surrounding inflammatory infiltrates (arrowheads). (b) Fn14 immunohistochemistry. Fn14 is observed in renal tubules (arrows) of the same case. Original magnification 400.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Expressing, Immunohistochemistry

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 8 | TWEAK (tumor necrosis factor–like weak inducer of apoptosis) modulates CXCL16 expression in cultured renal tubular cells. (a) MCT cells were stimulated with 100 ng/ml TWEAK. Quantification of CXCL16 mRNA expression (real-time quantitative reverse transcription-PCR). Expression level at 6 h control was considered to be 100%. Mean±s.e.m. of three independent experiments. *Po0.04 vs. control 6 h; #Po0.05 vs. control 24 h. (b) MCT cells were pretreated for 1 h with 10 mmol/l parthenolide and stimulated with TWEAK for 3 h. The nuclear factor-kB inhibitor parthenolide prevented TWEAK-induced CXCL16 upregulation. Mean±s.e.m. of three independent experiments. *Po0.05 vs. vehicle; #Po0.05 vs. vehicle þ TWEAK. Data were normalized with murine glyceraldehyde-3-phosphate dehydrogenase mRNA. (c) MCT cells stimulated with TWEAK for 6 h or 24 h were analyzed for CXCL16 surface expression by flow cytometry. Mean±s.e.m. of three independent experiments. *Po0.03 vs. control. (d) NP-1 cells were treated with 100 ng/ml TWEAK for 24 h. Soluble and cellular CXCL16 were quantified by ELISA. Mean±s.e.m. of three independent experiments. *Po0.05 vs. control.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Expressing, Cell Culture, Reverse Transcription, Control, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 9 | Confocal microscopy detection of CXCL16 and CXCR6 expression in cultured tubular cells. Cells were stimulated with 100 ng/ml TWEAK (tumor necrosis factor–like weak inducer of apoptosis) or vehicle (control) for 24 h. (a) Constitutive CXCR6 (green) and inducible CXCL16 (red) expression in proximal tubular MCT cells. Original magnification 40. (b) Constitutive CXCR6 and inducible CXCL16 expression in proximal tubular MCT cells. Note the increased peripheral location of CXCL16 following TWEAK with areas of overlap (yellow). Original magnification 40, zoom 7. (c) Constitutive CXCR6 and inducible CXCL16 expression in distal tubular NP1 cells. Original magnification 40, zoom 7. Indirect immunofluorescence using anti-CXCR6 with secondary Alexa Fluor 488–conjugated antibody (green) and anti-CXCL16 antibodies with secondary Alexa Fluor 633–conjugated antibody (red). Nuclei were stained with 40-6-diamidino-2-phenylindole (blue). Images are representative of three independent experiments.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Confocal Microscopy, Expressing, Cell Culture, Control, Immunofluorescence, Staining

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 10 | CXCL16 does not modulate tubular cell survival or proliferation. (a) Tubular MCT cell death and (b) proliferation were assessed by flow cytometry of DNA content after culture for 24 h in the presence of 100 ng/ml TWEAK and different concentrations of CXCL16. Hypodiploid cells were considered apoptotic and cells in S/G2/M phase were considered proliferating. Mean±s.e.m. of four independent experiments. *Po0.02 vs. control.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Flow Cytometry, Control

Journal: Kidney international

Article Title: TWEAK (tumor necrosis factor-like weak inducer of apoptosis) activates CXCL16 expression during renal tubulointerstitial inflammation.

doi: 10.1038/ki.2011.475

Figure Lengend Snippet: Figure 11 | CXCL16 modulates TWEAK (tumor necrosis factor–like weak inducer of apoptosis)-induced tubular inflammation. MCT cells were pretreated with 50 ng/ml CXCL16 for 1 h, and then stimulated with TWEAK for 6 h or 24 h. (a) ICAM-1 mRNA, *Po0.02 vs. control 6 h, **Po0.03 vs. control 24 h, #Po0.05 vs. TWEAK 6 h, ##Po0.05 vs. TWEAK 24 h. (b) MCP-1 mRNA, *Po0.01 vs. control 6 h, **Po0.03 vs. control 24 h, #Po0.05 vs. TWEAK 6 h. (c) RANTES mRNA, *Po0.01 vs. control 6 h, **Po0.03 vs. control 24 h, ##Po0.05 vs. TWEAK 24 h. Real-time quantitative reverse transcription-PCR. Values for mRNA were normalized to glyceraldehyde-3-phosphate dehydrogenase expression. Expression level at 6 h control was considered to be 100%. Mean±s.e.m. of four independent experiments.

Article Snippet: The primary antibody was CXCL16 goat antibody (R&D Systems) and anti-Fn14 mouse antibody (eBiosciences, San Diego, CA) with the buffer containing 1% immunoglobulin-free bovine serum albumin overnight at 4 1C.

Techniques: Control, Reverse Transcription, Expressing

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 1. CXCL16 and CXCR6 are expressed by astrocytes, microglia, and neurons. A, RT-PCR analysis for CXCL16 and CXCR6 mRNAinadultbraintissuesofwtandcxcr6 gfp/gfpmiceandwtprimarycellcultures as indicated (left), Western blot analysis for

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 3. CXCL16 protects hippocampal neurons from excitotoxic cell death. A, CXCL16 concentration–survival response rela- tionship. Primary hippocampal neurons were exposed to Glu (100 M, 30 min) in the presence or in the absence of CXCL16 (at indicatedconcentrations),andafter18hanalyzedforcellsurvival(seeMaterialsandMethods).ResultsrepresentthemeanSEM (n5–9).B,Exampleofimmunohistochemicalanalysisofcleavedcaspase-3(green),NeuNorGFAP(red)expressionincontrol, Glu-challenged and Glu/CXCL16-treated mixed hippocampal cultures. Upon Glu challenge there is an increase in the number of caspase-3 positive cells that are also positive for NeuN with respect to control or Glu/CXCL16-treated cultures. Nuclei are stained withHoechst(blue);whitearrowsindicatecaspase-3-positiveneurons;whitestarsindicatecaspase-3-negativeneurons;images acquiredwith40objective.C,HistogrambarofcellsurvivaluponGluinsultinwtandcxcr6 gfp/gfpmice.Incxcr6 gfp/gfphippocam- palcultures,CXCL16(100nM)isnotabletoincreaseneuronalsurvivalupontoxicinsult.ResultsrepresentthemeanSEM(n 4). Data are expressed as the percentage of viable cells in treated cultures taking as 100% the number of viable cells in wt control condition.Statisticalanalysis:one-wayANOVAfollowedbyHolm–Sidakposthoctest*p0.05(A);two-wayANOVAfollowedby Holm–Sidak post hoc test *p 0.05 (C).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Concentration Assay, Control, Staining

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 4. CXCL16 acts on astrocytes to promote neuroprotection. A, Minocycline (MC) pre- vented CX3CL1- but not CXCL16-induced neuroprotection upon Glu insult. Hippocampal cul- tures were preincubated or not with MC (200 nM, 30 min) and then stimulated for 30 min with Glu, Glu/CXCL16 (100 nM), or Glu/CX3CL1 (100 nM) in the presence or in the absence of MC, as indicated. In normal condition (nil) both CXCL16 and CX3CL1 increased neuronal survival upon Glu insult; in the presence of MC only CX3CL1 failed to protect neurons. Results represent the mean SEM (n 4–5). Data are expressed as percentage of viable cells in treated cultures takingas100%thenumberofviablecellsincontrolnilcondition.B,AstrocytesmediateCXCL16 neuroprotection.Left,Whenhippocampalcellsfromcxcr6 gfp/gfpmicewerecoculturedwithwt microgliaandstimulatedwithGlu,CXCL16wasineffectiveoncellsurvival.Resultsrepresentthe mean SEM (n 4). Data are expressed as the percentage of viable cells in treated cultures taking as 100% the number of viable cells in microglia control condition. Right, When hip- pocampal cells from cxcr6 gfp/gfp mice were cocultured with wt astrocytes, in presence or ab- sence of MC, CXCL16 determined increased neuronal survival. Results represent the mean SEM (n 3–7). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in astrocytes control condition. Statistical analysis: one-way ANOVA followed by Holm–Sidak post hoc test *p 0.05 (A, B).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Control

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure5. CXCL16neuroprotectionrequiresA3Ractivity.A,CXCL16neuroprotectionrequiresADOactivity.Toremoveextracel- lularADO,primaryrathippocampalcultureswereincubatedwithADA(1U/ml,1hbefore,during,andfollowingGluinsult).While inthepresenceofADO(niltreatment)CXCL16preventedneuronalcelldeath;inabsenceofADO(ADAtreatment)CXCL16wasnot able to promote neuronal survival. To prevent ADO activity hippocampal cultures were incubated with the generic AR antagonist CGS15943(100nM).UponthistreatmenttheprotectiveeffectofCXCL16wassignificantlyreducedcomparedwithcontrolcondition (niltreatment).ResultsrepresentthemeanSEM(n4–11).Dataareexpressedaspercentageofviablecellsintreatedcultures takingas100%thenumberofviablecellsincontrolnilcondition.B,EffectsofpharmacologicalinhibitionofARs.Rathippocampal neuronsweretreatedwithspecificantagonists(DPCPX,50nM;SCH58261,5nM;MRS1706,20nM;MRS1523,100nM)andusedfor Glu-excitotoxic experiments, as indicated. Only in the presence of MRS1523 was CXCL16 neuroprotection significantly reduced compared with the control condition (nil). Results represent the mean SEM (n 5–9). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in control nil condition. C, Genetic deletion of A3R preventedCXCL16effect.HippocampalneuronsobtainedfromwtorA3R /micewerestimulatedwithGluinthepresenceorin theabsenceofCXCL16.InA3R /cultures,CXCL16failedtoincreasecellsurvivaluponGluinsult.Resultsrepresentthemean SEM (n 4). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in wt controlcondition.Statisticalanalysis:one-wayANOVAfollowedbyHolm–Sidakposthoctest;*p0.05(A–B);two-wayANOVA followed by Holm–Sidak post hoc test *p 0.05 (C).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Activity Assay, Incubation, Control

Journal: Journal of Neuroscience

Article Title: CXCL16 Orchestrates Adenosine A3 Receptor and MCP-1/CCL2 Activity to Protect Neurons from Excitotoxic Cell Death in the CNS

doi: 10.1523/jneurosci.4046-11.2012

Figure Lengend Snippet: Figure 6. The release of CCL2 by astrocytes upon CXCL16 stimulation is determinant for CXCL16 neuroprotection. A, HEK cells thatexpressCCR2migrateinresponsetoconditionedmedium(c.m.)derivedfromCXCL16stimulatedastrocytes.Thec.m.derived fromastrocytestreatedwithvehicle,CXCL16(100nM)or2-CL-IB-MECA(100nM),wascollectedafter18h.Thec.m.ofCXCL16-and 2-CL-IB-MECA-treatedastrocytesspecificallyincreasedthechemotaxisofHEK-expressingCCR2(CCR2-HEK).Resultsrepresentthe mean SEM (n 8–10). B, CXCL16 stimulation induces CCL2 release from astrocytes. Astrocytes were incubated with CXCL16 (100nM,30min)orvehicleandthemediawerereplacedandcollectedafter6h.CCL2levelsinthemediaweremeasuredbyELISA. ResultsrepresentthemeanSEM(n7).C,NeutralizationofCCL2activitysignificantlypreventedCXCL16neuroprotection.In primary hippocampal cultures treated with neutralizing -CCL2 Ab (3 g/ml; 30 min before, during, and after Glu challenge), neuronalsurvivalfollowingCXCL16stimulationwasreducedcomparedwithsurvivalobtainedincontrolcondition(IgGtreatment; 3 g/ml). Results represent the mean SEM (n 5–8). Data are expressed as percentage of viable cells in treated cultures taking as 100% the number of viable cells in IgG control condition. D, CCL2 is able to reduce Glu-excitotoxic cell death. Primary hippocampal cultures were exposed to Glu (100 M; 30 min) in the presence or in the absence of CCL2 (100 nM) as indicated, and analyzed for cell survival. Results represent the mean SEM (n 5–6). Statistical analysis: one-way ANOVA followed by Holm–Sidak post hoc test, *p 0.05 (A, C, D); Student’s t test *p 0.05 (B).

Article Snippet: Primary antibodies were as follows: polyclonal mouse CXCL16 (1: 100 AF503; R&D Systems), monoclonal mouse -tubulin III (1:1000 MCA2047; Serotec), monoclonal mouse NeuN (1:100 MAB377; Millipore Bioscience Research Reagents), and monoclonal mouse GFAP (1: 1000 MAB360; Millipore Bioscience Research Reagents).

Techniques: Derivative Assay, Incubation, Control

Journal: Nature Immunology

Article Title: Inflammation triggers ILC3 patrolling of the intestinal barrier

doi: 10.1038/s41590-022-01284-1

Figure Lengend Snippet: a , Heatmap of relative expression of chemokine receptors in sorted NKp46 + ILC3s ( n = 6) and CCR6 + ILC3s ( n = 6) from WT mice detected by bulk multiplex Biomark assay. b , Representative flow cytometry analysis of chemokine receptors profiles of intestinal NKp46 + ILC3s, CCR6 + ILC3s and T cells from one of three independent experiments in WT mice. A negative or positive population is shown as a control for each chemokine receptor. c , Heatmap of relative expression of chemokines in whole ileum of WT ( n = 10), Rag2 −/− ( n = 10) and Rag2 −/− adoptively transferred with T cells ( n = 8) detected by bulk multiplex Biomark assay. d – g , Intravital imaging of intestinal ILC3s in RR22 mice with combination of isotype controls (mouse IgG1, rat IgG2a and rat IgG2b) or blocking monoclonal antibodies (anti-CXCL12, anti-CXCL16, anti-CCL21 and anti-CCL25). Data are representative of three independent experiments. Representative image (left; scale bar, 50 µm), time-lapse images ( d ) (right; scale bar, 15 µm), speed over time ( e ) ( n = 216 and 248), individual tracks ( f ), mean speed, arrest coefficient and straightness ratio ( g ) of intestinal ILC3s as in d at the indicated time points. Results in f , g are from 3 movies per condition obtained in 3 independent experiments ( n = 364, 0 h; n = 617, 1 h; n = 310, 2 h). Each bar corresponds to the mean ± s.e.m. of the values obtained (*** P < 0.001; one-way ANOVA; exact P values are provided in the source data).

Article Snippet: For chemokine neutralization, mice were injected intravenously during imaging with a mixture of Hoechst 33342 (see above) to control intravenous injection and the following monoclonal blocking antibodies (50 µg): anti-CXCL12 (catalog no. MAB310); anti-CXCL16 (catalog no. MAB503); anti-CCL21 (catalog no. MAB4572) and anti-CCL25 (catalog no. MAB481), all from R&D Systems.

Techniques: Expressing, Multiplex Assay, Flow Cytometry, Control, Imaging, Blocking Assay

Journal: Nature Immunology

Article Title: Inflammation triggers ILC3 patrolling of the intestinal barrier

doi: 10.1038/s41590-022-01284-1

Figure Lengend Snippet: a , Intestinal ILC3s were imaged in RR22 mice using intravital imaging for one hour. Then, mice were injected i.v . with a combination of blocking monoclonal antibodies (anti-CXCL12, anti-CXCL16, and anti-CCL21) and subsequently imaged for 2 hours. Individual tracks of intestinal ILC3s before (−60–0 min), 1 h (0–60 min) and 2 h (60–120 min) after blocking antibodies injection. Mean speed, arrest coefficient and straightness ratio of intestinal ILC3s at indicated timepoints after blocking antibodies injection. Results in ( a ) are from at least five movies per condition obtained in two independent experiments (n = 129, n = 100, and n = 184 ILC3s for 0 h, 1 h and 2 h, respectively). b , Individual tracks of intestinal NKp46 + Il22 + ILC3s before (−60–0 min), 1 h (0–60 min) and 2 h (60–120 min) after CCL25 Ab injection and 5 h after Flag injection. Mean speed, arrest coefficient and straightness ratio of intestinal NKp46 + Il22 + ILC3s at indicated timepoints after CCL25 Ab injection. Results in ( b ) are from at least five movies per condition obtained in two independent experiments (n = 78, 0 h; n = 50, 1 h; n = 49, 2 h). Each bar corresponds to the mean ± s.e.m of the values obtained (ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA; exact P values are provided in the source data). RR22, Rag2 −/− Rorc GFP Il22 TdT; CCL25 Ab, CCL25 blocking antibody; Flag, Flagellin.

Article Snippet: For chemokine neutralization, mice were injected intravenously during imaging with a mixture of Hoechst 33342 (see above) to control intravenous injection and the following monoclonal blocking antibodies (50 µg): anti-CXCL12 (catalog no. MAB310); anti-CXCL16 (catalog no. MAB503); anti-CCL21 (catalog no. MAB4572) and anti-CCL25 (catalog no. MAB481), all from R&D Systems.

Techniques: Imaging, Injection, Blocking Assay