il17rb  (R&D Systems)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Distinct immune signatures for predicting the immunotherapy efficacy of esophageal squamous cell carcinoma or adenocarcinoma

doi: 10.1007/s00262-024-03904-1

Figure Lengend Snippet: Detecting unique cytokines linked to clinical outcomes in esophageal squamous cell carcinoma and adenocarcinoma separately. a Venn diagram shows genes in the cytokine-cytokine receptor pathway for ESCC and EAC from TCGA, annotated with gene counts in distinct and overlapping categories. b Overall survival of patients with TCGA-ESCC and different expression levels of TNFSF10 and CXCL14. c TNFSF10 and CXCL14 expression comparison between early and advanced ESCC tissues (TCGA). d Survival probabilities of patients with EAC classified by CXCL8 and IL17RB expression levels (TCGA). e CXCL8 and IL17RB expression during EAC progression from early to advanced stages. f TNFSF10 and CXCL14 expression in early and advanced ESCC tissues of Chinese patients. g, h The left panel presents immunohistochemical images illustrating TNFSF10 or CXCL14 staining in tissue microarrays obtained from patients diagnosed with ESCC, arranged sequentially from left to right based on ascending expression levels quantified by the H-score. On the far left, the lowest expression level is shown, followed by a sample in the lowest 33.33 percentile, then a moderate expression sample in the next 33.33 percentile, and finally, the highest expression level on the far right. The right panel displays survival curves stratified by the expression of TNFSF10 and CXCL14, categorized into low and high expression groups based on their H-scores. i, j The left panel displays immunohistochemical staining for CXCL8 or IL17RB in tissue microarrays from patients with EAC, with samples ordered from left to right by increasing H-scores. The sequence starts with the lowest expression level, followed by a sample in the first 33.33 percentile, then a sample with moderate expression in the next percentile, and ends with the highest expression level on the far right. P values were calculated using the Mann–Whitney (log-rank) test. Early stage: Stage 0-II; Advanced stage: Stage III-IV; ns: no significance; * P < 0.05, Mann–Whitney test

Article Snippet: The tissue sections were incubated with primary antibodies against TNFSF10 (Catalog No. ab2056; Abcam), CXCL14 (catalog no. ab137541; Abcam), CXCL8 (catalog no. ab106350; Abcam), IL17RB (Catalog No. AF1207; R&D Systems), CXCL10 (Catalog No. ab306587; Abcam, Cambridge, UK), HIF1α (catalog no. ab51608; Abcam), CSF2 (catalog no. ab316862; Abcam), and NOS2 (catalog no. ab283655; Abcam) (diluted at 1:500 in phosphate-buffered saline) overnight at 4℃.

Techniques: Expressing, Comparison, Immunohistochemical staining, Staining, Sequencing, MANN-WHITNEY

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB positivity in tumor tissues is a poor prognostic factor in breast cancer. Tumor tissues obtained from stage I-III breast cancer patients (n = 115) were immunohistochemically analyzed for IL17RB expression, and the expression patterns were classified into 3 levels: Level 0, no expression; Level 1, weak expression; and Level 2, strong expression. Diseases-free survival of the patients was statistically compared between two groups divided by the positivity (Level 0 versus Level 1+2).

Article Snippet: After deparaffinization, formalin-fixed and paraffin-embedded tumor tissue sections were stained with HRP-conjugated anti-IL17RB mAb (Clone 938923; Novus) or the isotype control, and were treated with DAB and hematoxylin for visualization.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: Patient characteristics categorized by IL17RB expression levels in tumors

Article Snippet: After deparaffinization, formalin-fixed and paraffin-embedded tumor tissue sections were stained with HRP-conjugated anti-IL17RB mAb (Clone 938923; Novus) or the isotype control, and were treated with DAB and hematoxylin for visualization.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB transduction confers cancer stem-like properties to murine mammary cancer 4T1 cells. A. IL17RB transduction simultaneously upregulates IL17RA expression, and confers anoikis resistance. Murine mammary cancer 4T1 cells were transfected with a plasmid vector encoding murine il17rb (TR1, TR2, and TR3) or the empty vector as mock control (mock). The immunofluorescence intensity was measured as pixel counts at 400× magnification (3 fields per slide × 2 slides/clone, n = 6). Scale = 100 µm. B. IL17RB transduction confers high self-renewal and invasive on tumor cells (n = 3). Photos show the appearance of sphere colony formation and invaded cells (scale = 100 µm). C. IL17RB transduction simultaneously upregulates gene expression related to cancer stemness. RT-PCR was conducted to analyze gene expression in the tumor cells. D. The IL17RB+ tumor cells are resistant to cyclin-dependent kinase 4/6 inhibitors. The TR cells (closed circles) or mock cells (open circles) were cultured with abemaciclib/LY2835219 (CDKI-LY) or palbociclib/PD0332991 (CDKI-PD) for 2 days, and the cell proliferation was assessed by WST1 assay (n = 3). Graphs were depicted as the percentage of the control without agents (100%). E. CDKI therapy rather promotes IL17RB+ tumor growth in the implanted mice. BALB/c mice were subcutaneously (s.c.) implanted with tumor cells (5 × 105), and were orally administered with CDKI-LY (5 mg/kg) or PBS as a control daily on days 3-7 after tumor implantation (n = 5). Mock + PBS, open circles. Mock + CDKI-LY, closed circles. TR + PBS, open triangles. TR + CDKI-LY, closed triangles. All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock control. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After deparaffinization, formalin-fixed and paraffin-embedded tumor tissue sections were stained with HRP-conjugated anti-IL17RB mAb (Clone 938923; Novus) or the isotype control, and were treated with DAB and hematoxylin for visualization.

Techniques: Transduction, Expressing, Transfection, Plasmid Preparation, Control, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Tumor Implantation

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB+ tumor cells systemically expand IL25+ macrophages in the implanted mice. A. IL17RB+ tumor growth is retarded after implantation in mice. The TR (closed triangles and squares) or mock cells (open circles) were s.c. implanted in mice (5 × 105, n = 5). B. F4/80+ macrophages (Møs) and possibly exhausted CD3+PD1+Tim3+ T cells increase in the TR tumors (day 15 after implantation, n = 3). C. F4/80+ Møs also systemically increase in spleen of the TR-implanted mice (day 15; n = 3). D. Cytotoxic activities of anti-tumor effector cells are extremely reduced in the TR-implanted mice. CD8+ T cells were isolated from spleen cells (SPCs) stimulated with a tumor antigen AH1 peptide (1 µg/ml) for 6 days, and the sorted CD8+ T cells as CTLs were co-cultured with 4T1 cells as a target at ET ratio = 40:1 for 4 hours (n = 3). Splenic DX5+ NK cells were also co-cultured with Yac1 cells as a target at ET ratio = 20:1 for 4 hours (n = 3). E. The IL17RB+ tumor-induced Møs highly produce IL25. F4/80+ Møs were isolated from tumor-infiltrating cells (TILs) and SPCs of mice on day 15 after implantation with mock tumors (mock-Mø) or TR tumors (TR-Mø), and the adherent Møs after 2-hour-culture were stained with anti-IL25 mAb or the isotype control (n = 10, pooled). Scale = 100 µm. The 6-day-cultured splenic Mø supernatant was also tested for IL25 or IL17B by ELISA (n = 3). F. The IL17RB+ tumor-induced Møs suppress CTL induction partly by the secreted IL25. The sorted F4/80+ Møs were cocultured with the AH1-prestimulated CD8+ T cells in the presence of the AH1 peptide (10 µg/ml), IL2 (100 IU/ml), and anti-IL25 mAb or mIgG as a control (10 µg/ml) for 6 days, and the sorted CD8+ T cells were tested for cytotoxic activity (ET ratio = 20:1; n = 3). All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock control. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After deparaffinization, formalin-fixed and paraffin-embedded tumor tissue sections were stained with HRP-conjugated anti-IL17RB mAb (Clone 938923; Novus) or the isotype control, and were treated with DAB and hematoxylin for visualization.

Techniques: Isolation, Cell Culture, Staining, Control, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: The IL17RB+ tumor-induced Møs intensify tumor functions. A. The TR-Møs enhance tumor invasive and self-renewal abilities partly utilizing the secreted IL25. Tumor cells were stimulated with IL25 (10 ng/ml) or the 6-day-cultured F4/80+ Mø supernatants (6 days) in the presence of anti-IL25 mAb or mIgG as a control (10 µg/ml) for 3 days before assays (n = 3). B. The TR-Møs promote in vivo tumor progression utilizing the secreted IL25. 4T1 cells were s.c. coinjected (1:1) with splenic Møs in mice, and the subcutaneous tumors were injected with anti-IL25 mAb or mIgG as a control (100 µg) on days 3 and 10 after coinjection (n = 5). Open circles, tumor only. Closed circles, tumor + mock-Møs + mIgG. Gray circles, tumor + mock-Møs + anti-IL25 mAb. Closed triangles, tumor + TR-Møs + mIgG. Gray triangles, tumor + TR-Møs + anti-IL25 mAb. All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After deparaffinization, formalin-fixed and paraffin-embedded tumor tissue sections were stained with HRP-conjugated anti-IL17RB mAb (Clone 938923; Novus) or the isotype control, and were treated with DAB and hematoxylin for visualization.

Techniques: Cell Culture, Control, In Vivo, Injection

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: Significance of the IL17RB-IL25 axis in human breast cancer system. A. IL17RB transduction confers high self-renewal and invasive properties on human breast cancer (n = 3). Human breast cancer MCF7 cells were transfected with a plasmid vector encoding human il17rb (2 clones: TR1 and TR2) or the empty vector as mock control (mock). Photos show IL17RB expression and anoikis-resistant appearance (scale = 100 µm). B. The IL17RB+ tumor cells are resistant to CDKI treatment. The TR cells (closed triangles and squares) or mock cells (open circles) were cultured with CDKI-LY for 2 days, and the cell proliferation was assessed by WST1 assay (n = 3). Graphs were depicted as the percentage of the control without agents (100%). C. IL17RB+ tumor growth is retarded after implantation in mice. The TR (closed triangles and squares) or mock cells (open circles) were s.c. implanted in immunodeficient nude mice (1 × 106, n = 5). D. The IL17RB+ tumor-expanded Møs highly produce IL25. F4/80+ Møs were isolated from SPCs of the tumor-implanted mice (day 30), and the 6-day-cultured Mø supernatants were tested for IL25 by ELISA (n = 3). E. Combination therapy with CDKI and anti-IL25 mAb significantly suppresses tumor progression in the xenograft models (n = 5). Nude mice were s.c. implanted with parental MCF7 cells (1 × 106), and received two cycles of the treatments: oral administration with CDKI-LY (5 mg/kg) or PBS as a control daily on days 5-9 and days 16-20 after tumor implantation, and/or i.p. injection with anti-IL25 mAb or mIgG as a control (10 mg/kg) on days 5, 9, 16 and 20. Open circles, PBS + mIgG. Closed circles, CDKI-LY + mIgG. Gray triangles, PBS + anti-IL25 mAb. Closed triangles, CDKI-LY + anti-IL25 mAb. All graphs show means ± SDs. *P < 0.01 versus mock. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After deparaffinization, formalin-fixed and paraffin-embedded tumor tissue sections were stained with HRP-conjugated anti-IL17RB mAb (Clone 938923; Novus) or the isotype control, and were treated with DAB and hematoxylin for visualization.

Techniques: Transduction, Transfection, Plasmid Preparation, Clone Assay, Control, Expressing, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Tumor Implantation, Injection

Journal: Cancer Gene Therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: A Representative IHC images of PanCK (red signal), IL-17RB (green signal), and Hoechst 33342 (blue signal) expression in FFPE samples of PDAC patients. PanCK (Pan-Cytokeratin) is an epithelial marker to indicate tumor cells. Scale bar, 25 μm. B H score of IL-17RB expression levels in regions with 0–20 μm and 20–40 μm distance from the tumor–stromal interface in PDAC patients (n = 29). **, P < 0.01 (paired t-test). C , D Representative IHC images (G) and H scores (H) of IL-17RB expression in regions of invasive and noninvasive tumors. The brown signal denotes IL-17RB expression. Scale bar, 50 μm. Each dot represents the datum from one high-power field (n = 50). E , F Kaplan–Meier analysis of progression-free survival (I) and overall survival (J) in regions with 0–20 μm distance from the tumor ( P = 0.046 and P = 0.053, separately, Log-rank test). IL-17RB expression levels were scored by 0 (no staining), 1+ ( < 10% among cancer cells), 2+ (10 to 50% among cancer cells, and 3+ ( ≥ 50% among cancer cells). For statistical analyses, the score of 0 and 1+ was combined as the low expression group, and the score of 2+ and 3+ was combined as the high expression group. Blue line, IL-17RB low in 0–20 μm, n = 29; Red line, IL-17RB high in 0–20 μm, n = 15. Values are expressed as mean ± SD. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (two-tailed Student’s t-test).

Article Snippet: For co-staining of SNAI2 or VIM with IL-17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Expressing, Marker, Staining, Two Tailed Test

Journal: Cancer Gene Therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: Matrigel invasion assays of HPAF-II ( A ) and MIA PaCa-2 ( B ) cells from parental or IL-17RB Del clones. Each dot represents the datum from one independent experiment (n = 4). Values are expressed as mean ± SD. ***, P < 0.001 (two-tailed Student’s t-test). C Western blotting analysis of proteins harvested from parental or IL-17RB Del tumor cells (HPAF-II or MIA PaCa-2). D Western blotting analysis of proteins harvested from HPAF-II or MIA PaCa-2 cells transduced with lentiviral pLAS5w (vector) or pLAS5w-IL17RB-Flag (IL17RB-Flag). E Representative IHC images of IL-17RB (green signal), SNAI2 (red signal) and Hoechst 33342 (blue signal) expression in FFPE samples of PDAC patients. Scale bar, 100 μm. F Relative intensity of SNAI2 in IL-17RB low and high expressing regions. IL-17RB low expressing regions were defined as IL-17RB staining expressed in <10% tumor cells per high power field, and IL-17RB high expressing regions were defined as IL-17RB staining expressed in ≥50% tumor cells per high power field. Each dot represents the datum from one high-power fields (n = 8). Values are expressed as mean ± SD. **, P < 0.01 (two-tailed Student’s t-test). G Representative IHC images of IL-17RB (green signal), VIM (red signal), Hoechst 33342 (blue signal) expression in FFPE samples of PDAC patients. Scale bar, 100 μm. H Relative intensity of VIM in IL-17RB low and high expressing regions. IL-17RB low expressing regions were defined as IL-17RB staining expressed in <10% tumor cells per high power field, and IL-17RB high expressing regions were defined as IL-17RB staining expressed in ≥50% tumor cells per high power field. Each dot represents the datum from one high-power field n = 10). Values are expressed as mean ± SD. *, P < 0.05 (two-tailed Student’s t-test). I , J Western blotting analysis (I) and Matrigel invasion assays (J) of cells from parental, IL-17RB Del (none), IL-17RB Del transduced with pLAS5w (vector), and IL-17RB Del transduced with pLAS5w-SNAI2 (SNAI2). Each dot represents the datum from one independent experiment (n = 4). Values are expressed as mean ± SD. ****, P < 0.0001 (one-way ANOVA).

Article Snippet: For co-staining of SNAI2 or VIM with IL-17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Clone Assay, Two Tailed Test, Western Blot, Transduction, Plasmid Preparation, Expressing, Staining

Journal: Cancer Gene Therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: A Representative IHC images of ɑSMA (red signal), IL-17RB (green signal), DAPI (blue signal) expression in FFPE samples of PDAC patients. Scale bar, 50 μm. B H score of ɑSMA expression levels in stromal regions adjacent to IL-17RB + , IL-17RB ++ , or IL-17RB +++ cancer regions in PDAC patients (n = 5 high-power fields per group). IL-17RB expression levels were scored by 0 (no staining), 1+ ( < 10% among cancer cells), 2+ (10 to 50% among cancer cells, and 3+ ( ≥ 50% among cancer cells). Values are expressed as mean ± SD. *, P < 0.05 (one-way ANOVA). C , D Representative IHC images of ɑSMA (red signal), IL-17RB (green signal), DAPI (blue signal) (C) and H Score of IL-17RB expression (D) in xenograft tumors collected from orthotopic injection of HPAF-II cells and mixed HPAF-II cells/fibroblasts (PSC, HSC, CAF-1, and CAF-2) in NOD/SCID/IL2Rγ null mice at day 14 after inoculation (n = 3 mice per group). Scale bar, 100 μm. Values are expressed as mean ± SD. *, P < 0.05; ***, P < 0.001 (one-way ANOVA). E , F Representative IHC images (E) and H Score (F) of IL-17RB expression in xenograft tumors collected from subcutaneous injection of HPAF-II cells and mixed HPAF-II/PSC cells in NOD/SCID/IL2Rγ null mice treated with DMSO or 2 μM all-trans retinoid acid (ATRA, R2625, Sigma) every 3 days and collected tumor samples at day 14 after inoculation (n = 6 mice per group). Scale bar, 100 μm. Values are expressed as mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA).

Article Snippet: For co-staining of SNAI2 or VIM with IL-17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Expressing, Staining, Injection

Journal: Cancer Gene Therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: A , C Immunofluorescence staining of IGFBP-3 (green signal) and Hoechst 33342 (blue signal) was conducted on PSC (A) or BxPC-3-mcherry (red signal) (C) under monocultured, transwell-cocultured, or direct-cocultured conditions with the other cell type, and images were captured using a confocal microscope. A protein transport inhibitor (containing monensin) was used for IGFBP-3 accumulation in the Golgi complex before staining. Scale bar, 20 μm. B , D Quantification results of IGFBP-3 intensity in perinuclear regions of PSC (B) or BxPC-3 (D) analyzed by Image J. Each dot represents the datum from one cell (n = 20). Values are expressed as mean ± SD. **, P < 0.01 (one-way ANOVA). E RNAi knockdown screening of candidate plasma membrane proteins (n = 15) that induced IL-17RB expression during direct tumor-fibroblast contact. Luciferase activity of the IL-17RB reporter in cocultured PSCs/BxPC-3 cells was normalized to monocultured BxPC-3 cells. F , G Western blotting analysis of proteins harvested from BxPC-3 cells stably expressing lentiviral-based LacZ shRNA , ITGB3 shRNA (F), or SEMA7A shRNA (G). H IL-17RB mRNA levels were measured by quantitative real-time PCR in BxPC-3 cells (shLacZ, shITGB3, shSEMA7A) following 24-hrs mono-culture or direct coculture with PSCs with three technical repeats. I IGFBP-3 protein levels were measured by ELISA analysis in supernatant of BxPC-3 cells (shLacZ, shITGB3, shSEMA7A) following 48-hrs transwell (T) or direct (D)-cocultured with PSCs with three technical repeats. Values were presented as mean ± SD. *, P < 0.05 (two-tailed Student’s t-test). J , K Immunofluorescence staining of IGFBP-3 (green signal) in PSCs under different conditions. In these conditions, PSCs are grown alone or cocultured with BxPC-3 cells and treated with IgG (control), anti-ITGB3 (sc-52685, Santa Cruz Biotechnology), or anti-SEMA7A (sc-374432, Santa Cruz Biotechnology) neutralizing antibodies (all at 1 μg/mL). Representative images (J) and quantification results (K) of IGFBP-3. Scale bar, 20 μm. Each dot represents the datum from one cell (n = 8). Values are expressed as mean ± SD. ****, P < 0.0001 (one-way ANOVA). L , M Representative images (L) and quantification results (M) of Matrigel invasion assays of BxPC3 cells under different conditions. These conditions include BxPC-3 cells alone or those cocultured with PSCs treated with IgG, anti-SEMA7A (sc-374432, Santa Cruz Biotechnology), or anti-IGFBP-3 (AF675, R&D Systems) neutralizing antibodies (all at 1 μg/mL). Each dot represents the datum from one independent experiment (n = 5). Values are expressed as mean ± SD. *, P < 0.05; **, P < 0.01; ****, P < 0.0001 (one-way ANOVA). N , O HPAF-II cells and mixed HPAF-II/PSC cells were injected subcutaneously in NOD/SCID/IL2Rγ null mice. Mice in the mixed HPAF-II/PSC group received treatment every 3 days with IgG (control), anti-SEMA7A (sc-374432, Santa Cruz Biotechnology), or anti-IGFBP (AF675, R&D Systems) neutralizing antibodies (all at 1 μg/mL). The neutralizing antibodies were subcutaneously injected adjacent to subcutaneous tumors every 2 days. After 14 days, all mice were sacrificed, and tumor samples were collected for analysis. Representative IHC images (N) and H scores (O) of IL-17RB expression. Scale bar, 50 μm. Each dot represents the datum from a single high-power field (n = 10). Values were presented as mean ± SD. *, P < 0.05; ****, P < 0.0001 (one-way ANOVA).

Article Snippet: For co-staining of SNAI2 or VIM with IL-17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Immunofluorescence, Staining, Microscopy, Knockdown, Clinical Proteomics, Membrane, Expressing, Luciferase, Activity Assay, Western Blot, Stable Transfection, shRNA, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Control, Injection

Journal: Cancer Gene Therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: A , B Correlation of SEMA7A and IGFBP-3 protein expression levels in tumor specimens from PDAC patients. (A) Representative images of SEMA7A protein expression (brown signal, IHC) and IGFBP-3 mRNA expression (pink signal, in situ hybridization RNAscope). Scale bar, 25 μm. B Pearson’s correlation coefficient analysis of SEMA7A and IGFBP-3 according to H score of SEMA7A and tumor-adjacent IGFBP-3 + cells (%) (n = 30; r = 0.42; P = 0.021). Each dot represents the datum from one high-power field. C Representative images of in situ hybridization (RNAscope) using a probe for IGFBP-3 mRNA in tumor specimen from a PDAC patient. mRNAs of IGFBP-3 (pink signal dots) were located in peri-tumoral regions. The nucleus was stained with hematoxylin. Scale bar, 25 μm. D Percentage of IGFBP-3 + adjacent stromal cells in normal pancreatic duct, PanIN (Pancreatic intraepithelial neoplasia), and PDAC (pancreatic ductal adenocarcinoma) in patient specimen. Each dot represents the datum from one high-power field. Values are expressed as mean ± SD (n = 10). ****, P < 0.0001 (one-way ANOVA). E Representative images of in situ hybridization (RNAscope) using probes for IGFBP-3 (pink) and INHBA (blue, gene name of activin A) mRNA in tumor specimen froma PDAC patient. The pink signal dots (IGFBP-3) were co-localized with blue signal dots (INHBA) in peri-tumoral regions. The nucleus was stained with hematoxylin. Scale bar, 25 μm. F Percentage of IGFBP-3 + /INHBA - , IGFBP-3 - /INHBA + , and IGFBP-3 + /INHBA + tumor-adjacent stromal cells. Each dot represents the datum from one high-power field. Values are expressed as mean ± SD (n = 30). ****, P < 0.0001 (one-way ANOVA). G , H Representative images (G) and Pearson’s correlation analysis (H) of IGFBP-3 + tumor-adjacent fibroblasts and IL-17RB + tumor cells in PDAC patient specimen (n = 30). Scale bar, 25 μm. Each dot represents the datum from one high-power field. I , J Representative images (I) and Pearson’s correlation analysis (J) of INHBA + tumor-adjacent fibroblasts and IL-17RB + tumor cells in IGFBP-3 low expression regions in tumor specimen from PDAC patients (n = 30). Scale bar, 25 μm. Each dot represents the datum from one high-power field. K , L Representative images (K) and Pearson’s correlation analysis (L) of INHBA + tumor-adjacent fibroblasts and IL-17RB + tumor cells in IGFBP-3 high expression regions in tumor specimen from PDAC patients (n = 30). Scale bar, 25 μm. Each dot represents the datum from one high-power field.

Article Snippet: For co-staining of SNAI2 or VIM with IL-17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Expressing, In Situ Hybridization, RNAscope, Staining

Journal: Cancer Gene Therapy

Article Title: SEMA7A-mediated juxtacrine stimulation of IGFBP-3 upregulates IL-17RB at pancreatic cancer invasive front

doi: 10.1038/s41417-024-00849-6

Figure Lengend Snippet: A Immunofluorescence staining of SEMA7A (green signal), ATP1A1 (magenta signal) and Hoechst 33342 (blue signal) in BxPC-3 cells (red signal). Plasma membrane proteins were stained without cell permeabilization. Scale bar, 5 μm. B Western blotting analysis of protein immunoprecipitated using IgG and anti-ATP1A1 antibodies in BxPC-3 cells. The left panels were input; the right panels were immunoprecipitated fractions. C Western blotting analysis of proteins harvested from BxPC-3 cells stably expressing lentiviral-based LacZ shRNA and ATP1A1 shRNA . D – F Immunofluorescence staining (D) and quantification results of SEMA7A (green signal) (E) and ATP1A1 (magenta signal) (F) in mcherry + BxPC-3 shLacZ , BxPC-3 shATP1A1 , and BxPC-3 shSEMA7A cells. Scale bar, 10 μm. Each dot represents the datum from one cell (n = 12). Values are expressed as mean ± SD. ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). G , H Immunofluorescence staining (G) and quantification results (H) of IGFBP-3 in PSC alone, cocultured with BxPC-3 shLacZ -mcherry, or cocultured with BxPC-3 shATP1A1 -mcherry. Cocultured cells were treated with IgG (control) or anti-SEMA7A (sc-374432, Santa Cruz Biotechnology) neutralizing antibodies (all at 1 μg/mL). Scale bar, 20 μm. Each dot represents the datum from one cell (n = 10). Values are expressed as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA). I Western blotting analysis of proteins harvested from BxPC-3 cells stably expressing lentiviral-based LacZ shRNA and ATP1A1 shRNA . J , K HPAF-II shLacZ and HPAF-II shATP1A1 cells, either alone or mixed with PSCs, were injected subcutaneously in NOD/SCID/IL2Rγ null mice. Mice in the mixed HPAF-II/PSC groups received treatment every 3 days with IgG (control) or anti-SEMA7A SEMA7A (sc-374432, Santa Cruz Biotechnology) neutralizing antibodies (all at 1 μg/mL). After 14 days, all mice were sacrificed, and tumor samples were collected for analysis. Representative IHC images (J), H scores (K), and IL-17RB expression. Scale bar, 50 μm. Each dot represents the datum from one high-power field (n = 10). Values are expressed as mean ± SD. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA).

Article Snippet: For co-staining of SNAI2 or VIM with IL-17RB in human PDAC specimens, anti-SNAI2 antibody (1:30; 9585, Cell Signaling) or anti-vimentin antibody (1:500; 10366-1-AP, Proteintech) and anti-IL-17RB antibody (1:500; A81, homemade) were used.

Techniques: Immunofluorescence, Staining, Clinical Proteomics, Membrane, Western Blot, Immunoprecipitation, Stable Transfection, Expressing, shRNA, Control, Injection

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Flow diagram of the i.p. HDM sensitization model. Challenge phase: D24, Remission phase: D30. B Airway resistance of mice was determined on day 1 (D1), D8, D15, D24 and D30 under methacholine (0, 6.25, 12.5, 25 and 50 mg/mL in PBS) stimulation. C Serum IgE ( n = 8), D lung IL-4, IL-5 and IL-13 ( n = 8), E eosinophils in peripheral blood ( n = 6), and F cytology of bronchoalveolar lavage fluid (BALF) ( n = 10) were determined at indicated time points. G Gating strategy. In live CD45 + lymphocytes, Th2s are defined as CD4 + GATA3 + , and total ILC2s are defined as lineage – CD90.2 + NK1.1 – NKp46 – Rorγt – GATA3 + . H Kinetics of the frequency and number of ST2 – KLRG1 + IL-17RB + ILC2 subset in the lamina propria of small intestines (siLP) ( n = 10). I , J IL-13 + percentage and MFI I and IL-5 + percentage and MFI J of ST2 – KLRG1 + IL-17RB + ILC2s in siLP after stimulation with PMA, ionomycin, brefeldin A and monensin. K , L Pie charts of IL-13 + K and IL-5 + L cell populations in siLP in remission under stimulation ( n = 10). Mice in control group (Ctrl) were treated with corresponding volume of sterile normal saline. Data points are individual mice pooled from three independent experiments for all panels. Data are shown as the mean ± SD. Statistical comparisons were performed using unpaired one-way ANOVA with Dunnett’s test except in B and F using unpaired two-way ANOVA. p values are shown on the graphs. Source data are provided as a Source Data file.

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Control, Sterility, Saline

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Donor CD45.1 + mice were sensitized and challenged with HDM follow the i.p. HDM sensitization model protocol. On day 30 (remission), lineage-negative cells were isolated from bone marrow (BM), spleen, lungs and siLP, respectively and cultured in vitro for expansion. Subsequently, 5 × 10 5 CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ILC2s were sorted and injected into the irradiated CD45.2 + mice via tail vein. Recipient CD45.2 + mice were then treated with 25 μg HDM intranasally for 3 times once daily. 24 hours later, single cells in lungs were prepared for flow cytometry analysis. B – E Representative plots and statistical results (bar graph) showing the percentages and the numbers of ST2 – KLRG1 + IL-17RB + ILC2s at lungs in mice that received cells derived from B BM, C spleen, D lungs, or E siLP, respectively. F , G IgE levels in serum F and IL-13 and IL-5 protein levels in lungs ( G ) of recipient mice in each group were detected by ELISA. H Histological scores and representative images of lungs of recipient mice by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. Results are representative of three independent experiments. Means ± SD from 8 mice each group. Statistical comparisons were performed using unpaired, two-sided Welch’s t-test except in F – H using unpaired two-way ANOVA. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 2A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Isolation, Cell Culture, In Vitro, Injection, Irradiation, Flow Cytometry, Derivative Assay, Enzyme-linked Immunosorbent Assay, Staining

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A CD45.1 + and irradiated CD45.2 + mice were surgically jointed from olecranon to knee joint and recovered for 4 weeks to establish blood chimerism. Subsequently, CD45.1 + mice were treated with HDM as Fig. . B The percentages and the numbers of ST2 – KLRG1 + IL-17RB + ml-ILC2s in siLP of CD45.1 + and CD45.2 + were analyzed on day 30. Cells were gated on CD45.1 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + . For in vivo imaging experiments, mice were treated with HDM to establish the asthma relapse model as portrayed in Fig. . C On day 30 (remission), ST2 – KLRG1 + IL-17RB + ILC2s were sorted from siLP and stained with 5 mM Xenolight DiR, and then injected intravenously (5 × 10 5 cells/each) into various groups of mice. Specifically, mice in DiR- group were injected with unstained cells. For the remission + CCX282-B group, recipient mice resting from asthma symptoms were treated with the CCR9 antagonist CCX282-B at 50 mg/kg (daily injection subcutaneously) from day 24 to day 30. For the remission + Etrolizumab group, recipient mice resting from asthma symptoms were treated with the anti-α4β7 monoclonal antibody Etrolizumab at 5 mg/kg (injection intravenously) on day 24, 26, 28 and 30. For the relapse group, mice in remission were injected with DiR-stained cells, followed with treatment of HDM at 50 μg intranasally for 2 times once per hour. Mice in the relapse + FTY720 group received intravenous injection of FTY720 along with 2-time intranasal administration of HDM. Mice in all groups were imaged 2 hours post cell injection, and organs including lungs, spleen, liver, SI and large intestine were then rapidly harvested and imaged. D S1P expression in plasma collected from mice post asthma relapse (D34, D37 and D40) was measured by ELISA. The S1P signaling antagonist FTY720 was administered intraperitoneally to asthmatic mice on days 31 to 33 for 3 times. E The percentages and the numbers of ST2 – KLRG1 + IL-17RB + ILC2s in lungs were detected on D34. F Histological examination of lungs by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. G Protein levels of IL-13 in lungs was detected by ELISA. Results are representative of three independent experiments. Means ± SD from 6 mice for each group. Statistical comparison was conducted using unpaired, two-sided Welch’s t test for B and unpaired one-way ANOVA with Dunnett’s test for the rest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 3A Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Irradiation, In Vivo Imaging, Staining, Injection, Expressing, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A WT or Rag1 – / – mice were stimulated with HDM, and anti-KLRG-1 mAb or isotype IgG were administered intraperitoneally as indicated. B The percentage and the number of CD45 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in lungs were analyzed on day 24. C Pulmonary IL-13 was detected by ELISA. D Airway resistance was measured under stimulation of methacholine. E Pulmonary histological changes were observed by hematoxylin-eosin staining. Magnification: ×200, scale bar = 100 μm. F Rag1 – / – mice were stimulated with HDM, and CD45 + lineage – CD90.2 + ST2 – KLRG1 + IL-17RB + ILC2s were sorted from siLP on D30. In vitro culturing cells were treated with anti-KLRG1 mAb or isotype control, and then adoptively transferred into untreated ILC2 depleted (anti-CD90.2 mAb or isotype control) Rag1 – / – mice via tail vein (5 × 10 5 cells/mouse). Recipient mice were intranasally treated (i.n.) with 25 μg HDM or 500 ng recombinant IL-25 for 3 consecutive days. G The percentage and the number of CD45 + lineage – CD90.2 + NK1.1 – NKp46 – GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in lungs. H , I IL-13 level in lungs homogenates H and airway resistance I was measured. J Pulmonary histological changes were analyzed. Magnification: ×200, scale bar = 100 μm. Results are representative of three independent experiments. Data was shown as Means ± SD. n = 6 in all panels except for n = 3 in D and I . Statistical comparison was conducted using unpaired, two-sided Welch’s t test, except in ( D ) and ( I ) using unpaired two-way ANOVA with the Bonferroni posttest to analyze dose response to methacholine. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 4F Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, In Vitro, Control, Recombinant, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A Flow chart of asthma mice model establishment and sample collection. B , C On days 21 to 23, BrdU (0.8 mg/mice) was given intranasally. At indicated time points, single-cell suspension was prepared and subjected to flow cytometry analysis; Dynamic changes of the percentage B and numbers C of CD45 + lineage – CD90.2 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in siLP. D The proportions of BrdU + ST2 – KLRG1 + IL-17RB + ILC2s in siLP of HDM-challenged mice were analyzed by flow cytometry. A control histogram (shaded in blue) was generated using cells from ctrl mice stained with BrdU. E During asthma remission (AR) (day 30) of the i.p. HDM sensitization model, three ILC2 subsets including KLRG1 – , KLRG1 + IL-17RB + and KLRG1 + IL-17RB + (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB + and AR-KLRG1 + IL-17RB + ) were purified from the total CD45 + lineage – CD90.2 + ST2 – cells from siLP. Besides, ST2 – KLRG1 + IL-17RB + ILC2s (marked as naive-KLRG1 + IL-17RB + ILC2s) were sorted from siLP of untreated WT mice. Cells were then cultured (5 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins + ) or without (alarmins-) 200 ng/mL IL-25 and 200 ng/mL IL-33 every other day for 14 days. Subsequently, cells were collected for further analysis. (F-H) Ki-67 + percentage F , number ( G ) and fold changes H of expansion of cells were determined. I Representative plots and statistical results (bar graph) showing the percentages of IL-13 + and IL-5 + of these four ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. Data are representative of four independent experiments. Means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm \,$$\end{document} ± SD; n = 5 for the naive-KLRG1 + IL-17RB + group, and n = 9 for the other groups. B , C statistical comparisons were performed using unpaired, two-sided Welch’s t test. F – I unpaired two-way ANOVA with the Bonferroni posttest analysis was performed. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 5E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Suspension, Flow Cytometry, Control, Generated, Staining, Purification, Cell Culture

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: A In asthma remission, KLRG1 – , KLRG1 + IL-17RB – , KLRG1 + IL-17RB + subsets (marked as AR-KLRG1 – , AR-KLRG1 + IL-17RB – and AR-KLRG1 + IL-17RB + ) in total CD45 + lineage – CD90.2 + ST2 – ILC2s in siLP were sorted. KLRG1 + IL-17RB + ILC2 (marked as naive-KLRG1 + IL-17RB + ) were isolated from siLP of naive WT mice (5 ~ 7 × 10 5 for every subset, purity > 98%). Total RNA was isolated, and qRT-PCR was performed. Heat map of mRNA expression values of selected genes (see Fig. ). n = 6 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and AR-KLRG1 + IL-17RB + ILC2, n = 3 for naive-KLRG1 + IL-17RB + ILC2. B – F Sorted CD45 + lineage – CD90.2 + NK1.1 – NKp46 – ST2 – KLRG1 + IL-17RB + ml-ILC2s were transfected with siRNAs targeting Tcf7 or Tox (si Tcf7 or si Tox ) or negative control (Negative Ctrl). Together with AR-KLRG1 – , AR-KLRG1 + IL-17RB – and naive-KLRG1 + IL-17RB + ILC2s (5 ± 10 3 per well), a combination of 200 ng/mL IL-25 and 200 ng/mL IL-33 were added every two days with IL-2 and IL-7 for 8 days. Knockdown efficiency of Tcf7 or Tox was confirmed (see Fig. ). B Cell expansion was calculated relative to day 0. C Protein levels of IL-13 in culture medium were detected by ELISA. n = 8 for ml-ILC2 Ctrl, ml-ILC2 Negative Ctrl, ml-ILC2 si Tcf7 and ml-ILC2 si Tox , n = 5 for AR-KLRG1 – ILC2, AR-KLRG1 + IL-17RB – ILC2 and naive-KLRG1 + IL-17RB + ILC2. D Heat map of mRNA expression values of selected genes (see Fig. ). n = 3. Expression of E CCR9 and F S1PR1 of all cells in each group were detected with flow cytometry. n = 3. G Flow chart of asthma mice model, BrdU administration, RNA interference and sample collection. On days 7–11, BrdU (0.8 mg/mice) was given intranasally daily. Rag1 – / – mice ( n = 5) were injected with Ambion in vivo si-negative control or indicated siRNAs on days 12–18, and were intranasally treated with 25 μg HDM on days 72-74. The percentage of BrdU + in ST2 – KLRG1 + IL-17RB + ILC2s in H siLP on day 71 and in ( I ) lungs on day 75 were detected with flow cytometry. J Flow chart of asthma mice model, adoptive transfer, RNA interference, and sample collection. Rag1 – / – mice were exposed with HDM and rested till AHR vanished as described in Fig. . Mice were injected with Ambion in vivo si-negative control, si Tcf7 or si Tox on days 16-18, and were intranasally treated with 25 μg HDM on days 19–21. For the groups of in vivo si Tcf7 +ml-ILC2 AT + HDM and in vivo si Tox +ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of si-negative control on day 19 before HDM treatment; For the groups of in vivo si Tcf7 +si Tcf7 -ml-ILC2 AT + HDM and in vivo si Tox +si Tox -ml-ILC2 AT + HDM, mice were injected (i.v.) with 5 × 10 5 ml-ILC2s precultured with transfection of siRNAs on day 19 before HDM treatment. Twenty-four hours after the last HDM treatment, K airway resistance ( n = 3) and L IL-13 level in lung homogenates were measured ( n = 6). M Pulmonary histological changes were analyzed ( n = 6). Magnification: ×200, scale bar = 100 μm. Data was shown as Means ± SD. Results are representative of three independent experiments. Statistical comparison was conducted using unpaired one-way ANOVA with Dunnett’s test, except in ( B ) and ( K ) using unpaired two-way ANOVA with the Bonferroni posttest. p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 6G, J Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Isolation, Quantitative RT-PCR, Expressing, Transfection, Negative Control, Knockdown, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Injection, In Vivo, Adoptive Transfer Assay, Comparison

Journal: Nature Communications

Article Title: TCF-1 and TOX regulate the memory formation of intestinal group 2 innate lymphoid cells in asthma

doi: 10.1038/s41467-024-52252-2

Figure Lengend Snippet: Flow cytometry was performed to analyze CD45 + lineage – CRTH2 + CD127 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s in PBMCs purified from asthmatic patients during remission phase (all rested for 50 days or longer) and healthy donors. A Gating strategy, B statistical plots, C IL-13 + percentage and MFI and D IL-5 + percentage and MFI of CD45 + lineage – CRTH2 + CD127 + GATA3 + ST2 – KLRG1 + IL-17RB + ILC2s. E KLRG1 – , KLRG1 + IL-17RB – and KLRG1 + IL-17RB + subsets were purified from the total CD45 + lineage – CRTH2 + CD127 + ST2 – ILC2s in PBMC of 6 asymptomatic asthmatic patients (marked as A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ). KLRG1 + IL-17RB + subsets were sorted from total ILC2s in PBMC of 3 healthy donors (marked as H-KLRG1 + IL-17RB + ). Cells were then cultured (5 × 10 3 /well) with 10 ng/mL IL-2 and 20 ng/mL IL-7, and treated with (alarmins+) or without (alarmins−) 200 ng/mL recombinant human IL-17E/IL-25 and 200 ng/mL recombinant human IL-33 every other day for 21 days. Subsequently, cells were collected for further analysis. F Ki-67 + percentage, G absolute number and H fold changes of expansion of cells were determined. Representative plots and statistical results (bar graph) showing the percentages of I IL-13 + and J IL-5 + of three ILC2 subsets after stimulation with PMA, ionomycin, brefeldin A and monensin. KLRG1 – , KLRG1 + IL-17RB – and KLRG1 + IL-17RB + subsets in total CD45 + lineage – CRTH2 + CD127 + ST2 – ILC2s in PBMC of asymptomatic asthmatic patients (marked as A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ) and KLRG1 + IL-17RB + subsets in total ILC2s in PBMC of healthy donors (marked as H-KLRG1 + IL-17RB + ) were sorted (4.5 ~ 6.8 × 10 5 for every subset, purity > 98%). K , L Total RNA was isolated, and qRT-PCR was performed. K Heat map of mRNA expression values of selected genes. L Bar plots of mRNA expression values (relative to GAPDH ) for genes significantly changed. Data was shown as Means ± SD. n = 8 H and 15 A for ( A – D ). n = 11 ( H ) and 18 A for E – J . n = 6 (A-KLRG1 – , A-KLRG1 + IL-17RB – and A-KLRG1 + IL-17RB + ) and 3 (H-KLRG1 + IL-17RB + ) for ( K, L ). Statistical comparisons were performed using unpaired, two-sided Welch’s t test for ( B – D ), and unpaired one-way ANOVA with Dunnett’s test for ( L ). p values are shown on the graphs. Source data are provided as a Source Data file. Fig. 7E Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license https://creativecommons.org/licenses/by-nc-nd/4.0/deed.en .

Article Snippet: To analyze human ILC2s, antibodies against CD45 (clone HI30; #MHCD4512; Invitrogen; 1:500), lineage markers (CD2, clone RPA-2.10, CD3, clone OKT3, CD14, clone 61D3, CD16, clone CB16, CD19, clone HIB19, CD56, clone TULY56, and CD235a, clone HIR2; #22-7778-72; eBioscience; 1:250), CRTH2 (clone BM16; #12-2949-41; eBioscience; 1:500), CD127 (clone eBioRDR5; #15-1278-42; Invitrogen; 1:500), GATA3 (clone TWAJ; #48-9966-42; Invitrogen; 1:200), ST2 (clone hIL33Rcap; #46-9338-42; Invitrogen; 1:200), KLRG1 (clone 47-9488-41; #47-9488-41; eBioscience; 1:200), IL-17RB (clone 170220; #FAB1207A; R&D systems; 1:200), IL-13 (clone 85BRD; #51-7136-42; Invitrogen; 1:200), IL-5 (clone TRFK5; #48-7052-82; Invitrogen; 1:200), Ki-67 (clone SolA15; #53-5698-82; Invitrogen; 1:500) and corresponding isotype controls were applied.

Techniques: Flow Cytometry, Purification, Cell Culture, Recombinant, Isolation, Quantitative RT-PCR, Expressing

Journal: JCI Insight

Article Title: IL-17B alleviates the pathogenesis of systemic lupus erythematosus by inhibiting FASN-mediated differentiation of B cells

doi: 10.1172/jci.insight.181906

Figure Lengend Snippet: ( A ) Serum IL-17B levels and ( B ) IL-17RB expression on CD19 + B cells in healthy controls (HC, n = 37), inactive-SLE patients ( n = 16), and active-SLE patients ( n = 36). Correlation of IL-17RB with ( C ) CD86 and ( D ) CD40 on CD19 + B cells in SLE ( n = 52) and HC ( n = 37). Correlation of IL-17RB with levels of ( E ) anti-dsDNA antibody, ( F ) SLEDAI, ( G ) C3 complement, and ( H ) anti-nucleosome antibodies on CD19 + B cells with SLE ( n = 52). ( I ) Serum FASN levels and ( J ) FASN expression on CD19 + B cells in HC ( n = 37), inactive-SLE patients ( n = 16), and active-SLE patients ( n = 36). Correlation of FASN with ( K ) CD86 and ( L ) CD40 on CD19 + B cells in SLE ( n = 52) and HC ( n = 37). Correlation of FASN with levels of ( M ) anti-dsDNA antibodies, ( N ) SLEDAI, ( O ) C3 complement, and ( P ) anti-nucleosome antibodies on CD19 + B cells in SLE ( n = 52). ( Q ) PCA dot plot, ( R ) volcano plot, ( S ) heatmap, and ( T and U ) GSEA plots were generated based on the lipid profiles of peripheral blood serum from SLE patients ( n = 40) and HC ( n = 40). The data are shown as the mean ± SEM and are representative of 3 independent experiments. * P < 0.05, *** P < 0.001, **** P < 0.0001 by 1-way ANOVA with Tukey’s multiple-comparison test ( A , B , I , and J ). Correlation coefficients were calculated using linear regression analysis. NS, P > 0.05.

Article Snippet: APC-labeled anti–human IL-17RB (catalog FAB1207A) was purchased from R&D Systems, and APC-labeled anti–human FASN (catalog ab223965) was purchased from Abcam.

Techniques: Expressing, Generated, Comparison

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB positivity in tumor tissues is a poor prognostic factor in breast cancer. Tumor tissues obtained from stage I-III breast cancer patients (n = 115) were immunohistochemically analyzed for IL17RB expression, and the expression patterns were classified into 3 levels: Level 0, no expression; Level 1, weak expression; and Level 2, strong expression. Diseases-free survival of the patients was statistically compared between two groups divided by the positivity (Level 0 versus Level 1+2).

Article Snippet: After limiting dilution, the transduction efficacy was evaluated by flow cytometry and immunostaining using anti-mouse IL17RB-PE (BioLegend), anti-human IL17RB-AF488 (R&D), or each isotype control, and several clones with IL17RB overexpression were chosen for assays.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: Patient characteristics categorized by IL17RB expression levels in tumors

Article Snippet: After limiting dilution, the transduction efficacy was evaluated by flow cytometry and immunostaining using anti-mouse IL17RB-PE (BioLegend), anti-human IL17RB-AF488 (R&D), or each isotype control, and several clones with IL17RB overexpression were chosen for assays.

Techniques: Expressing

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB transduction confers cancer stem-like properties to murine mammary cancer 4T1 cells. A. IL17RB transduction simultaneously upregulates IL17RA expression, and confers anoikis resistance. Murine mammary cancer 4T1 cells were transfected with a plasmid vector encoding murine il17rb (TR1, TR2, and TR3) or the empty vector as mock control (mock). The immunofluorescence intensity was measured as pixel counts at 400× magnification (3 fields per slide × 2 slides/clone, n = 6). Scale = 100 µm. B. IL17RB transduction confers high self-renewal and invasive on tumor cells (n = 3). Photos show the appearance of sphere colony formation and invaded cells (scale = 100 µm). C. IL17RB transduction simultaneously upregulates gene expression related to cancer stemness. RT-PCR was conducted to analyze gene expression in the tumor cells. D. The IL17RB+ tumor cells are resistant to cyclin-dependent kinase 4/6 inhibitors. The TR cells (closed circles) or mock cells (open circles) were cultured with abemaciclib/LY2835219 (CDKI-LY) or palbociclib/PD0332991 (CDKI-PD) for 2 days, and the cell proliferation was assessed by WST1 assay (n = 3). Graphs were depicted as the percentage of the control without agents (100%). E. CDKI therapy rather promotes IL17RB+ tumor growth in the implanted mice. BALB/c mice were subcutaneously (s.c.) implanted with tumor cells (5 × 105), and were orally administered with CDKI-LY (5 mg/kg) or PBS as a control daily on days 3-7 after tumor implantation (n = 5). Mock + PBS, open circles. Mock + CDKI-LY, closed circles. TR + PBS, open triangles. TR + CDKI-LY, closed triangles. All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock control. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After limiting dilution, the transduction efficacy was evaluated by flow cytometry and immunostaining using anti-mouse IL17RB-PE (BioLegend), anti-human IL17RB-AF488 (R&D), or each isotype control, and several clones with IL17RB overexpression were chosen for assays.

Techniques: Transduction, Expressing, Transfection, Plasmid Preparation, Immunofluorescence, Reverse Transcription Polymerase Chain Reaction, Cell Culture, Tumor Implantation

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: IL17RB+ tumor cells systemically expand IL25+ macrophages in the implanted mice. A. IL17RB+ tumor growth is retarded after implantation in mice. The TR (closed triangles and squares) or mock cells (open circles) were s.c. implanted in mice (5 × 105, n = 5). B. F4/80+ macrophages (Møs) and possibly exhausted CD3+PD1+Tim3+ T cells increase in the TR tumors (day 15 after implantation, n = 3). C. F4/80+ Møs also systemically increase in spleen of the TR-implanted mice (day 15; n = 3). D. Cytotoxic activities of anti-tumor effector cells are extremely reduced in the TR-implanted mice. CD8+ T cells were isolated from spleen cells (SPCs) stimulated with a tumor antigen AH1 peptide (1 µg/ml) for 6 days, and the sorted CD8+ T cells as CTLs were co-cultured with 4T1 cells as a target at ET ratio = 40:1 for 4 hours (n = 3). Splenic DX5+ NK cells were also co-cultured with Yac1 cells as a target at ET ratio = 20:1 for 4 hours (n = 3). E. The IL17RB+ tumor-induced Møs highly produce IL25. F4/80+ Møs were isolated from tumor-infiltrating cells (TILs) and SPCs of mice on day 15 after implantation with mock tumors (mock-Mø) or TR tumors (TR-Mø), and the adherent Møs after 2-hour-culture were stained with anti-IL25 mAb or the isotype control (n = 10, pooled). Scale = 100 µm. The 6-day-cultured splenic Mø supernatant was also tested for IL25 or IL17B by ELISA (n = 3). F. The IL17RB+ tumor-induced Møs suppress CTL induction partly by the secreted IL25. The sorted F4/80+ Møs were cocultured with the AH1-prestimulated CD8+ T cells in the presence of the AH1 peptide (10 µg/ml), IL2 (100 IU/ml), and anti-IL25 mAb or mIgG as a control (10 µg/ml) for 6 days, and the sorted CD8+ T cells were tested for cytotoxic activity (ET ratio = 20:1; n = 3). All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock control. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After limiting dilution, the transduction efficacy was evaluated by flow cytometry and immunostaining using anti-mouse IL17RB-PE (BioLegend), anti-human IL17RB-AF488 (R&D), or each isotype control, and several clones with IL17RB overexpression were chosen for assays.

Techniques: Isolation, Cell Culture, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: The IL17RB+ tumor-induced Møs intensify tumor functions. A. The TR-Møs enhance tumor invasive and self-renewal abilities partly utilizing the secreted IL25. Tumor cells were stimulated with IL25 (10 ng/ml) or the 6-day-cultured F4/80+ Mø supernatants (6 days) in the presence of anti-IL25 mAb or mIgG as a control (10 µg/ml) for 3 days before assays (n = 3). B. The TR-Møs promote in vivo tumor progression utilizing the secreted IL25. 4T1 cells were s.c. coinjected (1:1) with splenic Møs in mice, and the subcutaneous tumors were injected with anti-IL25 mAb or mIgG as a control (100 µg) on days 3 and 10 after coinjection (n = 5). Open circles, tumor only. Closed circles, tumor + mock-Møs + mIgG. Gray circles, tumor + mock-Møs + anti-IL25 mAb. Closed triangles, tumor + TR-Møs + mIgG. Gray triangles, tumor + TR-Møs + anti-IL25 mAb. All graphs show means ± SDs. *P < 0.01, **P < 0.05 versus mock. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After limiting dilution, the transduction efficacy was evaluated by flow cytometry and immunostaining using anti-mouse IL17RB-PE (BioLegend), anti-human IL17RB-AF488 (R&D), or each isotype control, and several clones with IL17RB overexpression were chosen for assays.

Techniques: Cell Culture, In Vivo, Injection

Journal: American Journal of Cancer Research

Article Title: IL25 + macrophages are a key determinant of treatment resistance of IL17RB + breast cancer

doi:

Figure Lengend Snippet: Significance of the IL17RB-IL25 axis in human breast cancer system. A. IL17RB transduction confers high self-renewal and invasive properties on human breast cancer (n = 3). Human breast cancer MCF7 cells were transfected with a plasmid vector encoding human il17rb (2 clones: TR1 and TR2) or the empty vector as mock control (mock). Photos show IL17RB expression and anoikis-resistant appearance (scale = 100 µm). B. The IL17RB+ tumor cells are resistant to CDKI treatment. The TR cells (closed triangles and squares) or mock cells (open circles) were cultured with CDKI-LY for 2 days, and the cell proliferation was assessed by WST1 assay (n = 3). Graphs were depicted as the percentage of the control without agents (100%). C. IL17RB+ tumor growth is retarded after implantation in mice. The TR (closed triangles and squares) or mock cells (open circles) were s.c. implanted in immunodeficient nude mice (1 × 106, n = 5). D. The IL17RB+ tumor-expanded Møs highly produce IL25. F4/80+ Møs were isolated from SPCs of the tumor-implanted mice (day 30), and the 6-day-cultured Mø supernatants were tested for IL25 by ELISA (n = 3). E. Combination therapy with CDKI and anti-IL25 mAb significantly suppresses tumor progression in the xenograft models (n = 5). Nude mice were s.c. implanted with parental MCF7 cells (1 × 106), and received two cycles of the treatments: oral administration with CDKI-LY (5 mg/kg) or PBS as a control daily on days 5-9 and days 16-20 after tumor implantation, and/or i.p. injection with anti-IL25 mAb or mIgG as a control (10 mg/kg) on days 5, 9, 16 and 20. Open circles, PBS + mIgG. Closed circles, CDKI-LY + mIgG. Gray triangles, PBS + anti-IL25 mAb. Closed triangles, CDKI-LY + anti-IL25 mAb. All graphs show means ± SDs. *P < 0.01 versus mock. Representative data of an experiment out of three independent experiments with consistent results.

Article Snippet: After limiting dilution, the transduction efficacy was evaluated by flow cytometry and immunostaining using anti-mouse IL17RB-PE (BioLegend), anti-human IL17RB-AF488 (R&D), or each isotype control, and several clones with IL17RB overexpression were chosen for assays.

Techniques: Transduction, Transfection, Plasmid Preparation, Clone Assay, Expressing, Cell Culture, Isolation, Enzyme-linked Immunosorbent Assay, Tumor Implantation, Injection