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biotinylated anti ccl21 (R&D Systems)

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R&D Systems biotinylated anti ccl21
Biotinylated Anti Ccl21, supplied by R&D Systems, used in various techniques. Bioz Stars score: 93/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/anti+human+ccl21/Human+CCL21%2F6Ckine+Biotinylated+Antibody/pm40010560-185-12-15
Average 93 stars, based on 5 article reviews

biotinylated anti ccl21 - by Bioz Stars, 2026-09

93/100 stars

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Recombinant:

Article Title: IL-21 promotes survival and maintains a naive phenotype in human CD4+ T lymphocytes.
Article Snippet: .. The chemoattractant recombinant human CCL21, anti-human CCL21 (both obtained from R&D Systems) or control medium was added at indicated concentrations to the 24-well tissue culture plates. .. A polyvinylpyrrolidone-free polycarbonate membrane with 8-lm pores for T lymphocytes (Nunc A/S) was layered onto the wells.

Control:

Hybridization:

Article Title: Disparate lymphoid chemokine expression in mice and men: no evidence of CCL21 synthesis by human high endothelial venules.
Article Snippet: T-cell homing to secondary lymphoid tissues generally depends on chemokineinduced firm adhesion in high endothelial venules (HEVs) and is primarily mediated through the CC chemokine receptor 7 (CCR7) on lymphocytes.. The CCR7 ligand designated CCL21 is considered the most important trigger because it appears constitutively expressed by murine HEVs.. Surprisingly, when we analyzed human tissues, no CCL21 mRNA could be detected in HEVs.

Immunofluorescence:

Staining:

Immunostaining:

Chromatin Immunoprecipitation:

Article Title: Immune-privileged tissues formed from immunologically cloaked mouse embryonic stem cells survive long term in allogeneic hosts.
Article Snippet: .. 3 nature portfolio | reporting sum m ary April 2023 Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Clinical data Dual use research of concern Plants Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used Anti-mouse anti-mouse CD3ε (BD Biosciences 553058) anti-mouse CD28 (BD Biosciences 553295) anti-mouse PD-L1 (alexa488, Novus NBP1-43262AF488) anti-mouse CD47, (BV421, BD Biosciences 740055) anti-mouse CD200 (alexa647, BD Biosciences 565544) anti-mouse FASL (Novus NBP1-97519) anti-mouse H2-M3 (BD Biosciences 551769) anti-mouse SERPINB9 (HycultBiotech HP8035) anti-mouse CCL21 (R&D AF457) anti-mouse MFGE8 (Biolegend 518603) anti-mouse CD31 (Biolegend 102417) anti-mouse Neurofilament (Fisher Scientific MS359R7) anti-mouse SSEA1 (Sigma MAB4301) anti-mouse OCT4 (Santa Cruz sc-8628) anti-mouse Smooth Muscle Actin (Abcam ab5694) anti-mouse FOXA2 (Abcam ab108422) anti-mouse SOX17 (R&D AF1924) anti-mouse Troponin (cTNT, abcam, ab8295) anti-mouse H-2Kb (Biolegend 116511) anti-mouse H-2Kq (Biolegend 115106) Anti-human anti-human PDL1 (APC, Biolegend 329708) anti-human FALSG (PE, Biolegend 306406) anti-human CD200 (APC/Cy7, Biolegend 329220) anti-human CD47 (PerCP/Cy5, Biolegend 323109) anti-human SERPINB9 (ThermoFisher MA5-17648) anti-human CCL21 (R&D AF366-SP) anti-human MFGE8 (R&D IC27671A) anti-human CD45 (APC, BD Biosciences 560973) anti-human CD3 (Pe-Cy5, BD Biosciences 561006) anti-human HLA-A,B,C (APC, Biolegend 311409) anti-human CD31 (Pe-Cy7, Biolegend 303118) anti-human CD144 (PE, Biolegend 138009) anti-human CD16/CD32 Fc blocker (BD Biosciences 553141) anti-human Human TruStain FxC Fc blocker (Biolegend 422301) Secondaries and Others anti mouse IgG (alexa488, ThermoFisher A10680) anti goat IgG (alexa488 ThermoFisher A27012) anti-Rat Fc (alexa568 secondary antibody (ThermoFisher A21112). anti-hamster (Dylight 488 Biolegend 405503) anti-rat IgG (DyLight 549 Novus NBP1-72975) anti-mouse (alexa647 ThermoFisher 21244) Annexin V (BV421, Biolegend 640923) PI (Biolegend, 421301) Validation Antibody performance and validations are available from the vendor and/or manufacturer. ..

Flow Cytometry:

Magnetic Resonance Imaging:

Biomarker Discovery:

Microscopy:

Article Title: CCL21 overexpressed on lymphatic vessels drives thymic hyperplasia in myasthenia.
Article Snippet: Objective: Myasthenia gravis (MG), a neuromuscular disease mediated by anti-acetylcholine receptor (AChR) autoantibodies, is associated with thymic hyperplasia characterized by ectopic germinal centers that contain pathogenic antibody-producing B cells.. Our thymic transcriptome study demonstrated increased expression of CCL21, a recruiter of immune cells.. Accordingly, we are investigating its implication in MG pathogenesis.

Immunohistochemical staining:

Article Title: Activation of Arterial Wall Dendritic Cells and Breakdown of Self-tolerance in Giant Cell Arteritis
Article Snippet: .. The following Abs were used for immunohistochemical procedures: mouse anti–human CD83 (1:1,000; Research Diagnostics), mouse anti–human CCL19 (1:300), goat anti–human CCL21 (1:300; both obtained from R&D Systems), rabbit anti–S-100 (1:500), mouse anti–human CD3 (1:300), mouse anti–human fascin (1:100), mouse anti–human CD11c (1:100), goat anti–rabbit Ig, rabbit anti–mouse Ig, and goat anti–mouse Ig (all obtained from DakoCytomation). ..

Image Search Results

Primary antibodies used for immunohistochemistry

Journal: Respiratory Research

Article Title: Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

doi: 10.1186/1465-9921-14-65

Figure Lengend Snippet: Primary antibodies used for immunohistochemistry

Article Snippet: After incubation with antibodies against goat anti-human CCL21 or mouse anti-human D6, sections were incubated with biotinylated rabbit anti-goat (1:200, BA-1000, Vector Laboratories, Inc., Burlingame, CA, USA) or biotinylated horse anti-mouse IgG secondary antibodies (1:200, BA-2000, Vector Laboratories).

Techniques:

Increased number of alveolar CCL21- and D6-positive lymphatic vessels in patients with advanced COPD. (A) Percentage of CCL21-positive lymphatic vessels among all Prox1-positive lymphatics and (B) number of CCL21-positive lymphatic vessels in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma in never smokers and patients with GOLD stage IV COPD. (C) Percentage of D6-positive lymphatic vessels among all Prox1-positive lymphatics and (D) number of D6-positive lymphatic vessels in similar lung compartments as for (A and B) in never smokers and patients with GOLD stage IV COPD. Mann–Whitney rank sum test was used for comparison between two groups. Horizontal lines indicate median value for each cohort. ** p < 0.01. (E and F) Immunohistochemical staining for CCL21 (red) and Prox1 (brown nuclei) and (G) immunohistochemical staining for D6 (red) and Prox1 (brown nuclei) in sections of patients with GOLD stage IV COPD. LA, lymphoid aggregate. Sections were counterstained with Mayer’s haematoxylin (blue stain). Scale bars: (E , G) 20 μm; (F) 30 μm.

Journal: Respiratory Research

Article Title: Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

doi: 10.1186/1465-9921-14-65

Figure Lengend Snippet: Increased number of alveolar CCL21- and D6-positive lymphatic vessels in patients with advanced COPD. (A) Percentage of CCL21-positive lymphatic vessels among all Prox1-positive lymphatics and (B) number of CCL21-positive lymphatic vessels in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma in never smokers and patients with GOLD stage IV COPD. (C) Percentage of D6-positive lymphatic vessels among all Prox1-positive lymphatics and (D) number of D6-positive lymphatic vessels in similar lung compartments as for (A and B) in never smokers and patients with GOLD stage IV COPD. Mann–Whitney rank sum test was used for comparison between two groups. Horizontal lines indicate median value for each cohort. ** p < 0.01. (E and F) Immunohistochemical staining for CCL21 (red) and Prox1 (brown nuclei) and (G) immunohistochemical staining for D6 (red) and Prox1 (brown nuclei) in sections of patients with GOLD stage IV COPD. LA, lymphoid aggregate. Sections were counterstained with Mayer’s haematoxylin (blue stain). Scale bars: (E , G) 20 μm; (F) 30 μm.

Techniques: MANN-WHITNEY, Immunohistochemical staining, Staining

Increased immunoreactivity for CCL21 and D6 in lymphatic endothelium in patients with advanced COPD. (A) Accumulated lymphatic immunoreactivity of CCL21 and (B) D6 in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma in never smokers and patients with GOLD stage IV COPD. Each point indicates the total amount of lymphatic immunoreactivity per unit area. (C) Immunoreactivity of CCL21 and (D) D6 per lymphatic vessel endothelial length in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma. (E and F) Scatter graphs representing the immunoreactivity for (E) CCL21 and (F) D6 in each analysed lymphatic vessel in (C and D) . Mann–Whitney rank sum test was used for comparison between two groups. Horizontal lines indicate median value for each cohort. * p < 0.05; ** p < 0.01; *** p < 0.001.

Journal: Respiratory Research

Article Title: Increased number and altered phenotype of lymphatic vessels in peripheral lung compartments of patients with COPD

doi: 10.1186/1465-9921-14-65

Figure Lengend Snippet: Increased immunoreactivity for CCL21 and D6 in lymphatic endothelium in patients with advanced COPD. (A) Accumulated lymphatic immunoreactivity of CCL21 and (B) D6 in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma in never smokers and patients with GOLD stage IV COPD. Each point indicates the total amount of lymphatic immunoreactivity per unit area. (C) Immunoreactivity of CCL21 and (D) D6 per lymphatic vessel endothelial length in bronchiolar subepithelial tissue, adventitia of bronchiole-associated arteries and alveolar parenchyma. (E and F) Scatter graphs representing the immunoreactivity for (E) CCL21 and (F) D6 in each analysed lymphatic vessel in (C and D) . Mann–Whitney rank sum test was used for comparison between two groups. Horizontal lines indicate median value for each cohort. * p < 0.05; ** p < 0.01; *** p < 0.001.

Techniques: MANN-WHITNEY

Figure 2 Tobacco smoke exposure promoted CCL21 secretion by regulating the CCL21-CCR7 axis. (A) Analysis of 12 TLS- related chemokine expression by qRT-PCR in tissues from 136 patients with LUAD at Jiangsu Cancer Hospital. (B) CCL21 expression in TLS− and TLS+ tumor tissues from TCGA-LUAD database. (C) Correlation of CCL21 expression levels with germinal center B cells in tobacco-exposed patients using TCGA-LUAD database. (D) LLC cells were injected into the lungs of C57BL/6J mice for 1 week, then exposed to tobacco smoke for 3 weeks, and finally sacrificed. The expression of CCL21 in serum was detected by ELISA, and (E) the expression of CCR7 in peripheral blood lymphocytes was detected by flow cytometry. (F) Tissues of 136 patients with LUAD from Jiangsu Cancer Hospital. H&E staining low-power field of view (scale bar=2000 µm) and high-power field of view (scale bar=100 µm) and IHC of lung tissue sections using anti-CD20, anti-CD23, and anti-CCR7 antibodies. (G) Histograms showing the percentage of CCR7+TLSs (identified by H&E stain and CCR7 IHC) in all TLSs from current smokers with LUAD compared with life-long non-smokers with LUAD. (H) Orthotopic implantation models of LLC-Luciferase cells. One week after orthotopic implantation, intravenous tail injections with or without CCL21 neutralizing antibody were administered, and then the mice were exposed to tobacco smoke for 3 weeks before being sacrificed. (I) LLC cells were injected into the lungs of C57BL/6J mice for 1 week, then exposed to tobacco smoke for 3 weeks, and finally sacrificed, the representative images of mouse bioluminescence imaging at Week 1 and Week 4 (left panel) and the corresponding quantification analysis (right panel). (J) Representative image of IHC assay of lung tissue sections using anti- CD20 and anti-CCL21 antibodies, and (K) quantified results. Data are presented as the mean ± SD. ns, not significant. **p<0.01, ***p<0.001, two-tailed Student’s t-test. IHC, immunohistochemistry; LUAD, lung adenocarcinoma; qRT-PCR, quantitative real- time PCR; TCGA, The Cancer Genome Atlas; TLSs, tertiary lymphoid structures; TLS(+), TLS-present tissues; TLS(−); TLS- absent tissues.

Journal: Journal for immunotherapy of cancer

Article Title: Tobacco exposure primes the secretion of CCL21 positively associated with tertiary lymphoid structure and response to immunotherapy.

doi: 10.1136/jitc-2023-006939

Figure Lengend Snippet: Figure 2 Tobacco smoke exposure promoted CCL21 secretion by regulating the CCL21-CCR7 axis. (A) Analysis of 12 TLS- related chemokine expression by qRT-PCR in tissues from 136 patients with LUAD at Jiangsu Cancer Hospital. (B) CCL21 expression in TLS− and TLS+ tumor tissues from TCGA-LUAD database. (C) Correlation of CCL21 expression levels with germinal center B cells in tobacco-exposed patients using TCGA-LUAD database. (D) LLC cells were injected into the lungs of C57BL/6J mice for 1 week, then exposed to tobacco smoke for 3 weeks, and finally sacrificed. The expression of CCL21 in serum was detected by ELISA, and (E) the expression of CCR7 in peripheral blood lymphocytes was detected by flow cytometry. (F) Tissues of 136 patients with LUAD from Jiangsu Cancer Hospital. H&E staining low-power field of view (scale bar=2000 µm) and high-power field of view (scale bar=100 µm) and IHC of lung tissue sections using anti-CD20, anti-CD23, and anti-CCR7 antibodies. (G) Histograms showing the percentage of CCR7+TLSs (identified by H&E stain and CCR7 IHC) in all TLSs from current smokers with LUAD compared with life-long non-smokers with LUAD. (H) Orthotopic implantation models of LLC-Luciferase cells. One week after orthotopic implantation, intravenous tail injections with or without CCL21 neutralizing antibody were administered, and then the mice were exposed to tobacco smoke for 3 weeks before being sacrificed. (I) LLC cells were injected into the lungs of C57BL/6J mice for 1 week, then exposed to tobacco smoke for 3 weeks, and finally sacrificed, the representative images of mouse bioluminescence imaging at Week 1 and Week 4 (left panel) and the corresponding quantification analysis (right panel). (J) Representative image of IHC assay of lung tissue sections using anti- CD20 and anti-CCL21 antibodies, and (K) quantified results. Data are presented as the mean ± SD. ns, not significant. **p<0.01, ***p<0.001, two-tailed Student’s t-test. IHC, immunohistochemistry; LUAD, lung adenocarcinoma; qRT-PCR, quantitative real- time PCR; TCGA, The Cancer Genome Atlas; TLSs, tertiary lymphoid structures; TLS(+), TLS-present tissues; TLS(−); TLS- absent tissues.

Article Snippet: The primary antibodies applied included anti- CCL21 (#AF366, R&D), ICAM1 (#Ab222736, Abcam), and CD11α (#26703, CST) overnight.

Techniques: Expressing, Quantitative RT-PCR, Injection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Luciferase, Imaging, Two Tailed Test, Immunohistochemistry, Real-time Polymerase Chain Reaction

Figure 3 CCL21 activated the CD11a-ICAM1 axis. (A) Five major cell subsets (CD45+ cells, CD31+ cells, EpCAM+ cells, NG2+ cells, and SPARC+ cells) were sorted and cultured from fresh LUAD tissues using flow cytometry. (B) Determination of CCL21 secretion by CD45+, CD31+, EpCAM+, NG2+, and SPARC+ cells cultured in CSE medium. The supernatant from cultured cells was collected and assayed using ELISA. (C) The t-SNE plots showed epithelial cell subpopulations in subcluster analysis, according to current smoker (above) and non-smoker (below) single-cell RNA sequencing. (D) Correlation between CCL21 and CD11a in the TCGA-lung database. (E) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated either with or without CCL21 for 48 hours. The expression of CD11a in peripheral blood lymphocytes was assayed by flow cytometry. (F) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated with or without CSE or CSE+ αCCL21 for 48 hours. The expression of CD11a in peripheral blood lymphocytes was assayed by flow cytometry. (G) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated either with or without CCL21 for 48 hours. B cell and T cell interactions were detected by the T cells static adhesion assay (scale bar=100 µm). (H) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated with or without CSE or CSE+ αCCL21 for 48 hours. B cell and T cell interactions were detected by the T cells static adhesion assay (scale bar=100 µm). (I) B cells co-cultured with CD4+ T cells treated with CCL21 recombinant protein schematic. (J) Immunofluorescence staining of B cells co-cultured with CD4+ T cells using anti-CD4 anti-B, anti-CD11a, and anti-ICAM-1 antibodies (scale bar=100 µm). (K) The co-immunoprecipitation assay demonstrated the interaction between CD11a and ICAM1. (L) Representative immunofluorescence images of stained lung tissue derived from smoking and non-smoking patients using an anti-CD20, anti-CD4, anti-CD11a, and anti-ICAM-1 antibodies (scale bar=50 µm). Data are presented as the mean ± SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. CSE, cigarette smoke extract; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; t-SNE, t-distributed stochastic neighbor embedding.

Journal: Journal for immunotherapy of cancer

Article Title: Tobacco exposure primes the secretion of CCL21 positively associated with tertiary lymphoid structure and response to immunotherapy.

doi: 10.1136/jitc-2023-006939

Figure Lengend Snippet: Figure 3 CCL21 activated the CD11a-ICAM1 axis. (A) Five major cell subsets (CD45+ cells, CD31+ cells, EpCAM+ cells, NG2+ cells, and SPARC+ cells) were sorted and cultured from fresh LUAD tissues using flow cytometry. (B) Determination of CCL21 secretion by CD45+, CD31+, EpCAM+, NG2+, and SPARC+ cells cultured in CSE medium. The supernatant from cultured cells was collected and assayed using ELISA. (C) The t-SNE plots showed epithelial cell subpopulations in subcluster analysis, according to current smoker (above) and non-smoker (below) single-cell RNA sequencing. (D) Correlation between CCL21 and CD11a in the TCGA-lung database. (E) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated either with or without CCL21 for 48 hours. The expression of CD11a in peripheral blood lymphocytes was assayed by flow cytometry. (F) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated with or without CSE or CSE+ αCCL21 for 48 hours. The expression of CD11a in peripheral blood lymphocytes was assayed by flow cytometry. (G) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated either with or without CCL21 for 48 hours. B cell and T cell interactions were detected by the T cells static adhesion assay (scale bar=100 µm). (H) CD4+ T cells were extracted by magnetic beads and co-cultured with A549 cells treated with or without CSE or CSE+ αCCL21 for 48 hours. B cell and T cell interactions were detected by the T cells static adhesion assay (scale bar=100 µm). (I) B cells co-cultured with CD4+ T cells treated with CCL21 recombinant protein schematic. (J) Immunofluorescence staining of B cells co-cultured with CD4+ T cells using anti-CD4 anti-B, anti-CD11a, and anti-ICAM-1 antibodies (scale bar=100 µm). (K) The co-immunoprecipitation assay demonstrated the interaction between CD11a and ICAM1. (L) Representative immunofluorescence images of stained lung tissue derived from smoking and non-smoking patients using an anti-CD20, anti-CD4, anti-CD11a, and anti-ICAM-1 antibodies (scale bar=50 µm). Data are presented as the mean ± SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. CSE, cigarette smoke extract; LUAD, lung adenocarcinoma; TCGA, The Cancer Genome Atlas; t-SNE, t-distributed stochastic neighbor embedding.

Article Snippet: The primary antibodies applied included anti- CCL21 (#AF366, R&D), ICAM1 (#Ab222736, Abcam), and CD11α (#26703, CST) overnight.

Techniques: Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, RNA Sequencing, Magnetic Beads, Expressing, Cell Adhesion Assay, Recombinant, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Derivative Assay, Two Tailed Test

Figure 4 BaP promoted CCL21 expression both in vitro and in vivo. (A) Cigarette smoke extract (CSE) medium was prepared by the modified Carp and Janoff method.43 (B) CSE medium was used to treat HBE (normal lung epithelial cells), A549, PC9, H1299, H1975 (NSCLC), and LLC cells; the expression of CCL21 was assessed by qRT-PCR. (C) A549 and H1975 cells were treated with CSE medium at indicated concentrations for 48 hours; the expression of CCL21 was assessed by qRT-PCR (D) and ELISA. (E) A549 and H1975 cells were treated with BaP at indicated concentrations (0, 2.5, 5, or 10 µM) for 48 hours; and the expression of CCL21 was assessed by qRT-PCR and (F) ELISA assay. (G) LLC cells were injected into the lungs of C57BL/6J mice for 1 week, then treated with BaP for 3 weeks, and finally sacrificed. (H) Representative images of mouse bioluminescence imaging at Week 1 and Week 4 (left panel) and the corresponding quantification analysis (right panel). (I)The expression of CCL21 was assessed by ELISA. (J) The number of TLSs was assessed by H&E staining (scale bar=50 µm). (K) The expression of CCR7 in peripheral blood lymphocytes was detected by flow cytometry. (L) The expression of CD11a in peripheral blood lymphocytes was detected by flow cytometry. (M) The expression of CD11a was detected by the T cell static adhesion assay (scale bar=100 µm). Data are presented as the mean ± SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test. NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time PCR; TLSs, tertiary lymphoid structures.

Journal: Journal for immunotherapy of cancer

Article Title: Tobacco exposure primes the secretion of CCL21 positively associated with tertiary lymphoid structure and response to immunotherapy.

doi: 10.1136/jitc-2023-006939

Figure Lengend Snippet: Figure 4 BaP promoted CCL21 expression both in vitro and in vivo. (A) Cigarette smoke extract (CSE) medium was prepared by the modified Carp and Janoff method.43 (B) CSE medium was used to treat HBE (normal lung epithelial cells), A549, PC9, H1299, H1975 (NSCLC), and LLC cells; the expression of CCL21 was assessed by qRT-PCR. (C) A549 and H1975 cells were treated with CSE medium at indicated concentrations for 48 hours; the expression of CCL21 was assessed by qRT-PCR (D) and ELISA. (E) A549 and H1975 cells were treated with BaP at indicated concentrations (0, 2.5, 5, or 10 µM) for 48 hours; and the expression of CCL21 was assessed by qRT-PCR and (F) ELISA assay. (G) LLC cells were injected into the lungs of C57BL/6J mice for 1 week, then treated with BaP for 3 weeks, and finally sacrificed. (H) Representative images of mouse bioluminescence imaging at Week 1 and Week 4 (left panel) and the corresponding quantification analysis (right panel). (I)The expression of CCL21 was assessed by ELISA. (J) The number of TLSs was assessed by H&E staining (scale bar=50 µm). (K) The expression of CCR7 in peripheral blood lymphocytes was detected by flow cytometry. (L) The expression of CD11a in peripheral blood lymphocytes was detected by flow cytometry. (M) The expression of CD11a was detected by the T cell static adhesion assay (scale bar=100 µm). Data are presented as the mean ± SD. ns, not significant. *p<0.05, **p<0.01, ***p<0.001, two-tailed Student’s t-test. NSCLC, non-small cell lung cancer; qRT-PCR, quantitative real-time PCR; TLSs, tertiary lymphoid structures.

Article Snippet: The primary antibodies applied included anti- CCL21 (#AF366, R&D), ICAM1 (#Ab222736, Abcam), and CD11α (#26703, CST) overnight.

Techniques: Expressing, In Vitro, In Vivo, Modification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Injection, Imaging, Staining, Flow Cytometry, Cell Adhesion Assay, Two Tailed Test, Real-time Polymerase Chain Reaction

Figure 5 Upstream mechanism of BaP-mediated TLS generation. (A) A549 cells were stably transfected with sh-Ctrl+DMSO, sh-AhR+DMSO, sh-Ctrl+BaP, or sh-AhR+BaP, (B) the expression of CCL21 was detected by qRT-PCR, western blot, and (C) ELISA assay. (D) Correlation between AhR and CCL21 in the GTEx-lung database. (E) AhR expression in TLS− and TLS+ tumor tissues in TCGA-LUAD database. (F) Representative images of mouse bioluminescence imaging at Week 1 and Week 4 (left panel) and the corresponding quantification analysis (right panel). (G) LLC cells were transfected with sh-AhR or sh- Ctrl, injected into the lungs of C57BL/6J mice for 1 week, then treated with BaP for 3 weeks, and finally sacrificed. The number of TLSs was detected by H&E staining. (H)The expression of CCR7 in peripheral blood lymphocytes was detected by flow cytometry. (I) The expression of CCL21 in serum was detected by ELISA. (J) A549 cells were treated with either DMSO or BaP for 48 hours, then detected by the nuclear-cytoplasmic isolations assay and western blot and (K) assessed by immunofluorescence assay (scale bar=20 µm). (L) Design of the positive control group pGL3-CCL21 wild-type (WT) double luciferase reporter gene plasmid and the two motif mutant group double luciferase reporter gene plasmids pGL3-CCL21 MT# 1 and pGL3-CCL21 MT#2. (M) A549 cells were transfected with the WT or mutant CCL21 promoter-luciferase reporter constructs MT#1 or MT#2, treated with BaP for 48 hours, and assessed by ChIP-qPCR. (N) A549 cells were transfected with the WT, MT#1, or MT#2 plasmids and treated with BaP using a co-transfection strategy. The si-Ctrl, si-AhR, pcDNA3.1, and overexpressed- AhR groups were assessed by the luciferase assay. (O) A549 cells were transfected with the WT, MT#1, or MT#2 plasmids and then treated with BaP using a co-transfection strategy. The DMSO-administered and BaP-administered groups were assessed by the luciferase assay. (P) Mechanism diagram. BaP activates and induces AhR into the nucleus, binds to the CCL21 promoter region, and promotes the transcription of CCL21. Data are presented as the mean ± SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AhR, aryl hydrocarbon receptor; ChIP, chromatin immunoprecipitation; LUAD, lung adenocarcinoma; MFI, mean fluorescence intensity; qRT-PCR, quantitative real-time PCR; TCGA, The Cancer Genome Atlas; TLS, tertiary lymphoid structure; TLS(+), TLS-present tissues; TLS(−); TLS-absent tissues.

Journal: Journal for immunotherapy of cancer

Article Title: Tobacco exposure primes the secretion of CCL21 positively associated with tertiary lymphoid structure and response to immunotherapy.

doi: 10.1136/jitc-2023-006939

Figure Lengend Snippet: Figure 5 Upstream mechanism of BaP-mediated TLS generation. (A) A549 cells were stably transfected with sh-Ctrl+DMSO, sh-AhR+DMSO, sh-Ctrl+BaP, or sh-AhR+BaP, (B) the expression of CCL21 was detected by qRT-PCR, western blot, and (C) ELISA assay. (D) Correlation between AhR and CCL21 in the GTEx-lung database. (E) AhR expression in TLS− and TLS+ tumor tissues in TCGA-LUAD database. (F) Representative images of mouse bioluminescence imaging at Week 1 and Week 4 (left panel) and the corresponding quantification analysis (right panel). (G) LLC cells were transfected with sh-AhR or sh- Ctrl, injected into the lungs of C57BL/6J mice for 1 week, then treated with BaP for 3 weeks, and finally sacrificed. The number of TLSs was detected by H&E staining. (H)The expression of CCR7 in peripheral blood lymphocytes was detected by flow cytometry. (I) The expression of CCL21 in serum was detected by ELISA. (J) A549 cells were treated with either DMSO or BaP for 48 hours, then detected by the nuclear-cytoplasmic isolations assay and western blot and (K) assessed by immunofluorescence assay (scale bar=20 µm). (L) Design of the positive control group pGL3-CCL21 wild-type (WT) double luciferase reporter gene plasmid and the two motif mutant group double luciferase reporter gene plasmids pGL3-CCL21 MT# 1 and pGL3-CCL21 MT#2. (M) A549 cells were transfected with the WT or mutant CCL21 promoter-luciferase reporter constructs MT#1 or MT#2, treated with BaP for 48 hours, and assessed by ChIP-qPCR. (N) A549 cells were transfected with the WT, MT#1, or MT#2 plasmids and treated with BaP using a co-transfection strategy. The si-Ctrl, si-AhR, pcDNA3.1, and overexpressed- AhR groups were assessed by the luciferase assay. (O) A549 cells were transfected with the WT, MT#1, or MT#2 plasmids and then treated with BaP using a co-transfection strategy. The DMSO-administered and BaP-administered groups were assessed by the luciferase assay. (P) Mechanism diagram. BaP activates and induces AhR into the nucleus, binds to the CCL21 promoter region, and promotes the transcription of CCL21. Data are presented as the mean ± SD. ns, not significant. *p<0.05, ***p<0.001, two-tailed Student’s t-test. AhR, aryl hydrocarbon receptor; ChIP, chromatin immunoprecipitation; LUAD, lung adenocarcinoma; MFI, mean fluorescence intensity; qRT-PCR, quantitative real-time PCR; TCGA, The Cancer Genome Atlas; TLS, tertiary lymphoid structure; TLS(+), TLS-present tissues; TLS(−); TLS-absent tissues.

Article Snippet: The primary antibodies applied included anti- CCL21 (#AF366, R&D), ICAM1 (#Ab222736, Abcam), and CD11α (#26703, CST) overnight.

Techniques: Stable Transfection, Transfection, Expressing, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Imaging, Injection, Staining, Flow Cytometry, Immunofluorescence, Positive Control, Luciferase, Plasmid Preparation, Mutagenesis, Construct, ChIP-qPCR, Cotransfection, Two Tailed Test, Chromatin Immunoprecipitation, Fluorescence, Real-time Polymerase Chain Reaction

Figure 6 bCCL21 was associated with the clinical benefit of tislelizumab combined with chemotherapy. (A) Schematic diagram of the treatment and evaluation process of immunotherapy combined with chemotherapy. (B)Clinical characteristics, bCCL21 in peripheral blood, and the response of patients to tislelizumab. Objective response: PR, partial response; SD, stable disease; PD, progression of the disease. Gender: F, female; M, male. (C) Association between the therapeutic efficacy and clinical characteristics of 29 patients treated with tislelizumab analyzed by multivariate logistic analyses. (D) Random forests method to determine the predictive weight of the efficacy of immunotherapy combined with chemotherapy. (E) ROC curve analysis of serum CCL21 in predicting the curative effect. (F) ROC curve analysis of PD-L1 TPS in predicting the curative effect. AUC, area under the curve; LUAD, lung adenocarcinoma; PD-L1, programmed death-ligand; ROC, receiver operating characteristic; TPC, tislelizumab+pemetrexed+carboplatin; TPS, tumor proportion score.

Journal: Journal for immunotherapy of cancer

Article Title: Tobacco exposure primes the secretion of CCL21 positively associated with tertiary lymphoid structure and response to immunotherapy.

doi: 10.1136/jitc-2023-006939

Figure Lengend Snippet: Figure 6 bCCL21 was associated with the clinical benefit of tislelizumab combined with chemotherapy. (A) Schematic diagram of the treatment and evaluation process of immunotherapy combined with chemotherapy. (B)Clinical characteristics, bCCL21 in peripheral blood, and the response of patients to tislelizumab. Objective response: PR, partial response; SD, stable disease; PD, progression of the disease. Gender: F, female; M, male. (C) Association between the therapeutic efficacy and clinical characteristics of 29 patients treated with tislelizumab analyzed by multivariate logistic analyses. (D) Random forests method to determine the predictive weight of the efficacy of immunotherapy combined with chemotherapy. (E) ROC curve analysis of serum CCL21 in predicting the curative effect. (F) ROC curve analysis of PD-L1 TPS in predicting the curative effect. AUC, area under the curve; LUAD, lung adenocarcinoma; PD-L1, programmed death-ligand; ROC, receiver operating characteristic; TPC, tislelizumab+pemetrexed+carboplatin; TPS, tumor proportion score.

Article Snippet: The primary antibodies applied included anti- CCL21 (#AF366, R&D), ICAM1 (#Ab222736, Abcam), and CD11α (#26703, CST) overnight.

Techniques: Drug discovery

Chemokines and antibodies used in SDS/PAGE and Western blot analysis.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: Chemokines and antibodies used in SDS/PAGE and Western blot analysis.

Article Snippet: The Vectastain ABC Elite kits (Vector Laboratories, Inc., Burlingame, CA, USA) were used as described , with 1:500 dilution of 100µg/ml rat polyclonal anti-human CCL21 (GTX31167, GeneTex, Irvine, CA, USA).

Techniques: Western Blot, Concentration Assay, Recombinant

CCL21 expression in systemic sclerosis with associated pulmonary arterial hypertension from explanted lung tissue. CCL21 immunohistochemistry demonstrated protein expression (black arrows) from (A-C) airway epithelial cells (20x) and (D) alveolar macrophages (20x), which was confirmed by (E) CD68 macrophage marker in a serial cut of the same histopathology block.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: CCL21 expression in systemic sclerosis with associated pulmonary arterial hypertension from explanted lung tissue. CCL21 immunohistochemistry demonstrated protein expression (black arrows) from (A-C) airway epithelial cells (20x) and (D) alveolar macrophages (20x), which was confirmed by (E) CD68 macrophage marker in a serial cut of the same histopathology block.

Techniques: Expressing, Immunohistochemistry, Marker, Histopathology, Blocking Assay

CCL21 serum levels and correlation analysis in ELISA 1, Luminex and ELISA 2. (A) The mean serum levels of CCL21 in ELISA 1 were higher in SSc patients compared to healthy controls (i) in Luminex (ii) and ELISA 2 (iii). (B) In ELISA 1, CCL21 serum levels were higher in patients diagnosed with PAH compared to patients with no PH (i) while similar in PAH patient and no PH (ii) in Luminex. The mean serum levels of CCL21 in ELISA 2 however were, like in ELISA 1, higher in PAH patients compared to those without PH (iii). (C) The correlation of CCL21 serum levels between ELISA 1 and Luminex (r=0.515, p<0.001) (i) and the correlation of CCL21 serum levels between ELISA 2 and Luminex (r=0.52, p<0.001) (ii) were poor. However, the correlation of CCL21 serum levels between ELISA 2 and ELISA 1 (r=0.788, p<0.001) (iii) were good.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: CCL21 serum levels and correlation analysis in ELISA 1, Luminex and ELISA 2. (A) The mean serum levels of CCL21 in ELISA 1 were higher in SSc patients compared to healthy controls (i) in Luminex (ii) and ELISA 2 (iii). (B) In ELISA 1, CCL21 serum levels were higher in patients diagnosed with PAH compared to patients with no PH (i) while similar in PAH patient and no PH (ii) in Luminex. The mean serum levels of CCL21 in ELISA 2 however were, like in ELISA 1, higher in PAH patients compared to those without PH (iii). (C) The correlation of CCL21 serum levels between ELISA 1 and Luminex (r=0.515, p<0.001) (i) and the correlation of CCL21 serum levels between ELISA 2 and Luminex (r=0.52, p<0.001) (ii) were poor. However, the correlation of CCL21 serum levels between ELISA 2 and ELISA 1 (r=0.788, p<0.001) (iii) were good.

Techniques: Enzyme-linked Immunosorbent Assay, Luminex

ROC analysis of CCl21 levels from ELISA 1, Luminex and ELISA 2. (A) ROC analysis of CCL21 levels from ELISA 1 with AUC=0.843, 95% CI = 0.084-0.883 and p<0.001, (B) ROC analysis of CCL21 levels from Luminex with AUC=0.772, 95% CI = 0.711-0.833 and p<0.001, and (C) ROC analysis of CCL21 levels from ELISA 2 with AUC=0.770, 95% CI = 0.711-0.830 and p<0.001.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: ROC analysis of CCl21 levels from ELISA 1, Luminex and ELISA 2. (A) ROC analysis of CCL21 levels from ELISA 1 with AUC=0.843, 95% CI = 0.084-0.883 and p<0.001, (B) ROC analysis of CCL21 levels from Luminex with AUC=0.772, 95% CI = 0.711-0.833 and p<0.001, and (C) ROC analysis of CCL21 levels from ELISA 2 with AUC=0.770, 95% CI = 0.711-0.830 and p<0.001.

Techniques: Enzyme-linked Immunosorbent Assay, Luminex

Peptides detected by MS of CCL21 immunoprecipitated from SSc patient serum.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: Peptides detected by MS of CCL21 immunoprecipitated from SSc patient serum.

Techniques: Immunoprecipitation

Multivariable logistic and cox regression analysis showing CCL21 and other variables associated with PAH and predictive of PAH.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: Multivariable logistic and cox regression analysis showing CCL21 and other variables associated with PAH and predictive of PAH.

Techniques:

Serum anti-CCL21 antibody level in healthy controls and patients with different systemic connective tissue diseases. The dots in healthy controls (green), Systemic sclerosis (SSc; pink), primary Sjøgren syndrome (SS; purple), Systemic lupus erythematosus (SLE; blue) and Mixed connective tissue disease (MCTD; orange) shows the level of anti-CCL21 in the different patients. The dotted line represents the cut off value for considering the patients positive or negative for anti-CCL21, in which the patients above the dotted line are positive for anti-CCL21 antibodies. Levels of anti-CCL21 antibodies were significantly higher in patients with SSc, SS and SLE than in healthy controls (p-value).

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: Serum anti-CCL21 antibody level in healthy controls and patients with different systemic connective tissue diseases. The dots in healthy controls (green), Systemic sclerosis (SSc; pink), primary Sjøgren syndrome (SS; purple), Systemic lupus erythematosus (SLE; blue) and Mixed connective tissue disease (MCTD; orange) shows the level of anti-CCL21 in the different patients. The dotted line represents the cut off value for considering the patients positive or negative for anti-CCL21, in which the patients above the dotted line are positive for anti-CCL21 antibodies. Levels of anti-CCL21 antibodies were significantly higher in patients with SSc, SS and SLE than in healthy controls (p-value).

Techniques:

CCL21 levels in SSc, SS and MCTD of anti-CCL21 antibody positive and negative patients. (A) Mean serum levels of CCL21 in anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc, SS and MCTD. (B) Mean serum levels of CCL21 of anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc patients diagnosed with PAH.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: CCL21 levels in SSc, SS and MCTD of anti-CCL21 antibody positive and negative patients. (A) Mean serum levels of CCL21 in anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc, SS and MCTD. (B) Mean serum levels of CCL21 of anti-CCL21 antibody positive (green) and anti-CCL21 antibody negative (red) patients in SSc patients diagnosed with PAH.

Techniques:

Test of CCL21-antibodies using Western blot analysis. The Western blot analysis showed that three of the CCL21 antibodies detected both full length and tailless CCL21 (four left lanes). The custom-made anti-C-terminal tail of CCL21 antibody from Sicgen only detected full length CCL21 (right lane).

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: Test of CCL21-antibodies using Western blot analysis. The Western blot analysis showed that three of the CCL21 antibodies detected both full length and tailless CCL21 (four left lanes). The custom-made anti-C-terminal tail of CCL21 antibody from Sicgen only detected full length CCL21 (right lane).

Techniques: Western Blot

Western blot of SSc serum samples. The four first lanes show the IP product of 1: costume made CCL21, 2: Tailless CCL21, 3: Patient serum 1, and 4: patient serum 2. The last two lanes CCL21 and Tailless CCL21 directly loaded on the gel.

Journal: Frontiers in Immunology

Article Title: Target organ expression and biomarker characterization of chemokine CCL21 in systemic sclerosis associated pulmonary arterial hypertension

doi: 10.3389/fimmu.2022.991743

Figure Lengend Snippet: Western blot of SSc serum samples. The four first lanes show the IP product of 1: costume made CCL21, 2: Tailless CCL21, 3: Patient serum 1, and 4: patient serum 2. The last two lanes CCL21 and Tailless CCL21 directly loaded on the gel.

Techniques: Western Blot

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biotinylated anti ccl21 (R&D Systems)

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