recombinant murine ccl21  (PeproTech)

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: A soluble proteoform of CCL21 (CCL21-ΔC) with chemotactic activity is present in murine skin and increased in CHS-inflamed skin. (A) Representative western blot of CCL21 performed on steady-state (CTR) and CHS-inflamed (CHS) murine ear skin protein extracts. Recombinant CCL21 was loaded as a CTR. (B) Western blot analysis of recombinant human full-length CCL21 and CCL21-ΔC protein. One out of two experiments is shown. (C) Quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentages. Pooled data from n = 4 independent biological replicates. (D) ELISA-based quantification of total CCL21 in tissue protein extracts, performed with antibody clone AF457, which detects full-length CCL21 and CCL21-ΔC. Pooled data from n = 5 mice/condition. Statistics: unpaired Student’s t test. (E–J) Skin elution assay and analyses were performed on the supernatants. (E) Schematic depiction of the assay and representative western blot analysis. (F) Image-based quantification of the CCL21-ΔC band intensity from western blots as in E. A.U., arbitrary units, as produced by the western blot imager (G) ELISA-based quantification of total CCL21, performed with antibody clone AF457. Pooled data from n = 6–7 mice/condition, with one CHS-inflamed and a contralateral CTR ear, are shown in E and F. Data from the same mouse are connected by a line. The mean is shown in red, paired Student’s t test. (H–J) Transwell chemotaxis assays were performed on elution assay supernatants (see E) with 1:1 mixtures of LPS-matured labelled WT and CCR7 −/− DCs in presence/absence of a CCL21-blocking antibody. Flow cytometry–based quantification of the total numbers of transmigrated (H) WT DCs and (I) CCR7 −/− DCs, as well as of (J) the ratio of transmigrated WT to CCR7 −/− DCs. Data points from 3–7 experiments per condition are shown. Mixed effects statistical analysis. (K) Ratio of WT: CCR7 −/− DCs measured in seven paired experiments performed for CTR and CHS-inflamed condition. Statistical analysis: paired Student's t test. (L and M) Western blot analysis of protein extracts of (L) steady-state human skin (CTR) and (M) donor-matched steady-state (CTR) and inflamed (INF) human skin from a psoriasis patient. Data from one out of two experiments in L and one experiment in M are shown. Recombinant human full-length CCL21 was loaded as a CTR. Source data are available for this figure: .

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: Activity Assay, Western Blot, Recombinant, Enzyme-linked Immunosorbent Assay, Produced, Chemotaxis Assay, Blocking Assay, Flow Cytometry

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: In vitro assays demonstrating CCL21 cleavage by plasmin. (A and B) Western blot analysis of (A) human and (B) murine CCL21 cleavage after incubation with recombinant plasmin with a fixed molar ratio of 1:0.08 for increasing times at 37°C, as indicated in the figure. (C) Dose titration of the plasmin inhibitor C3 to a fixed molar ratio of murine CCL21:plasmin (1:0.08) and incubation for up to 4 h, as indicated in the figure. Representative western blots of n = 2 (A) or n = 3 (B and C) experiments are shown. (D) Fluorometric plasmin activation assay, with indicated plasmin (12 μM) and inhibitor concentrations. The % increase from T 0 (0 min) is depicted. PIC: broad spectrum protease inhibitor. Representative results of n = 3 independent experiments are shown. Source data are available for this figure: .

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: In Vitro, Western Blot, Incubation, Recombinant, Titration, Activation Assay, Protease Inhibitor

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: LECs activate plasminogen to plasmin, thereby generating CCL21-ΔC with enhanced chemotactic activity. (A and B) Quantification of (A) plasminogen and (B) plasmin activity in tissue protein extracts generated from CTR or CHS-inflamed ear skin. n = 6–7 mice per condition. (C) CTR experiment with CHS-inflamed ears documenting that the plasmin activity observed in C can be completely blocked in presence of the plasmin inhibitor C3. (D) Schematic depiction of the experimental hypothesis: Inflammation leads to enhanced extravasation of plasminogen. uPA bound to uPAR on CCL21-secreting LECs converts plasminogen to plasmin, thereby inducing CCL21 cleavage into CCL21-ΔC. (E–G) In vitro CCL21 cleavage experiment: (E) Schematic depiction of the experiment: immortalized LECs were incubated with recombinant CCL21 (100 nM) and plasminogen (20 nM) for 4 h or 24 h at 37°C in absence or presence of the plasmin inhibitor C3, mU1, or PIC. Supernatants were analyzed by western blot for CCL21. (F) Representative western blot of the cell culture supernatant at indicated time points and conditions and (G) quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentage. Pooled data from n = 4 independent experiments. Mean ± SEM, one-way ANOVA, and P values are relative to the “plg only” condition. (H and I) Cell culture supernatants generated as in E were evaluated in a 3D collagen migration assay. Recombinant human CCL21 and CCL21-ΔC were used as positive CTRs (H) Cell trajectory plots of migrating BMDCs’ migratory tracks in response to the stimuli applied on either side of the collagen channel. (I) Quantification of DC directionality, displacement, and velocity in response to the stimuli applied. Pooled data from n = 2 independent experiments with a total of n = 40–50 tracks analyzed per condition. Mean ± SEM, unpaired Student's t test for each comparison. (J–L) Analysis of the CCL21 cleavage activity of LECs isolated from uPA −/− mice or mice with defective uPA binding to uPAR (uPA mut ) (J) Schematic illustration of the three genotypes investigated. (K and L) Representative western blot of the cell culture supernatants after (K) 4 h and (L) 24 h of incubation (top) and quantification of the full-length CCL21 (gray) and CCL21-ΔC (white) relative band percentage (bottom). Pooled data from n = 5 independent experiments. Mean ± SEM, one-way ANOVA, and Source data are available for this figure: . plg, plasminogen.

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: Activity Assay, Generated, In Vitro, Incubation, Recombinant, Western Blot, Cell Culture, Migration, Comparison, Isolation, Binding Assay

Journal: The Journal of Cell Biology

Article Title: uPA-mediated remodeling of CCL21 gradients regulates lymphatic migration of dendritic cells

doi: 10.1083/jcb.202412190

Figure Lengend Snippet: Video showing fluorescently labeled bone marrow–derived DCs (green) moving in 3D collagen toward recombinant human CCL21 (up) and CCL21+plg (down) provided in reservoirs on left and right, respectively, of the chamber. WT DCs display enhanced migration toward CCL21-ΔC provided on the left. Video specifications: 5-min intervals; 5 frames/s (1500-fold accelerated). The original length of the recording: 200 min. Video length: 8 s. plg, plasminogen.

Article Snippet: Murine recombinant CCL21 (#250-13; PeproTech) and recombinant murine plasmin (Molecular Innovations) at molar ratios of CCL21 to plasmin of 1:0.02–1:0.16 (starting with 100 nM CCL21 and 2 nM plasmin) were incubated in PBS or serum-free ProCHO medium (Lonza) for 4 h at 37°C.

Techniques: Labeling, Derivative Assay, Recombinant, Migration

Journal: Cancer Immunology, Immunotherapy

Article Title: Chemokine CCL21 determines immunotherapy response in hepatocellular carcinoma by affecting neutrophil polarization

doi: 10.1007/s00262-024-03650-4

Figure Lengend Snippet: Association between CCL21 levels and immunotherapy response in HCC. A Transcriptome analysis showed that CCL21 was upregulated in responders. B Serum CCL21 levels of responders and non-responders in the training cohort. C Using the median serum CCL21 concentration (1736 pg/ml) as the cutoff value, the proportion of responders and non-responders in different serum CCL21 levels. D Serum CCL21 levels of responders and non-responders in the validation cohort. E Using the median serum CCL21 concentration of training cohort as the cutoff value, the proportion of responders and non-responders in different serum CCL21 levels. F ROC curve analysis of predictive performance of high serum CCL21 level for predicting immunotherapy response in training and validation cohorts. One-way ANOVA with a post hoc LSD test. Categorical variables were presented as n (%) and constituent ratio between groups was compared using Pearson χ 2 and Fisher’s exact test. HCC, hepatocellular carcinoma; CCL21, Chemokine C–C motif ligand 21; ROC curve analysis, receiver operating characteristic curve analysis

Article Snippet: Once palpable, the following treatment commenced and mice were randomly divided into four groups (five mice per group): for CCL21 therapy, mice were injected intraperitoneally with two times per week doses of 0.25 μg recombinant murine CCL21 (Cat. 250-13, PeproTech) or vehicle control; for anti-PD-1 therapy, mice were treated intraperitoneally with two times per week doses of 100 μg anti-mouse PD-1 antibody (Cat. BP0273, Bio X Cell) or IgG isotype control.

Techniques: Concentration Assay, Biomarker Discovery

Journal: Cancer Immunology, Immunotherapy

Article Title: Chemokine CCL21 determines immunotherapy response in hepatocellular carcinoma by affecting neutrophil polarization

doi: 10.1007/s00262-024-03650-4

Figure Lengend Snippet: CCL21 inhibits neutrophil N2 polarization through regulating NF-κB signaling pathway. A Schematic diagram of neutrophil isolation and culture in vitro. B qPCR analysis of the transcription of N1 and N2 markers in neutrophils treated with or without recombinant human CCL21. C Flow cytometry analysis of CD206 expression of neutrophils in indicated groups. D Heatmap of the differential genes in negative control and CCL21 treatment groups. E Differentially enriched pathways in negative control and CCL21 treatment groups identified by GSEA. F Western blot of NF-κB pathway in neutrophils treated with or without recombinant human CCL21. G qPCR analysis of the transcription of N1 and N2 markers in neutrophils treated with or without recombinant human CCL21 and/or NF-κB activator 1. H Flow cytometry analysis of CD206 expression of neutrophils in indicated groups. One-way ANOVA with a post hoc LSD test. CCL21, Chemokine C–C motif ligand 21; qPCR, quantitative real- time PCR; GSEA, gene set enrichment analysis; NF-κB, nuclear factor kappa B

Article Snippet: Once palpable, the following treatment commenced and mice were randomly divided into four groups (five mice per group): for CCL21 therapy, mice were injected intraperitoneally with two times per week doses of 0.25 μg recombinant murine CCL21 (Cat. 250-13, PeproTech) or vehicle control; for anti-PD-1 therapy, mice were treated intraperitoneally with two times per week doses of 100 μg anti-mouse PD-1 antibody (Cat. BP0273, Bio X Cell) or IgG isotype control.

Techniques: Isolation, In Vitro, Recombinant, Flow Cytometry, Expressing, Negative Control, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancer Immunology, Immunotherapy

Article Title: Chemokine CCL21 determines immunotherapy response in hepatocellular carcinoma by affecting neutrophil polarization

doi: 10.1007/s00262-024-03650-4

Figure Lengend Snippet: CCL21 enhances the efficacy of anti-PD-1 antibody in HCC in vivo. A Schematic diagram of anti-PD-1 antibody and recombinant murine CCL21 treatment in the mice model of HCC subcutaneous tumors. B The volume of subcutaneous tumors in each group during treatment. C Gross appearance of the subcutaneous HCC tumors from the indicated treatment groups. D The weight of subcutaneous tumors in each group at the endpoint. E – G Flow cytometry analyses of CD206 expression of neutrophils and the tumor infiltration percentage of CD3 + CD8 + T cells in each group. H Schematic diagram depicting that CCL21 determines immunotherapy response in HCC by affecting neutrophil polarization. One-way ANOVA with a post hoc LSD test. CCL21, Chemokine C–C motif ligand 21; PD-1, programmed death receptor 1; HCC, hepatocellular carcinoma

Article Snippet: Once palpable, the following treatment commenced and mice were randomly divided into four groups (five mice per group): for CCL21 therapy, mice were injected intraperitoneally with two times per week doses of 0.25 μg recombinant murine CCL21 (Cat. 250-13, PeproTech) or vehicle control; for anti-PD-1 therapy, mice were treated intraperitoneally with two times per week doses of 100 μg anti-mouse PD-1 antibody (Cat. BP0273, Bio X Cell) or IgG isotype control.

Techniques: In Vivo, Recombinant, Flow Cytometry, Expressing

Journal: International Journal of Biological Sciences

Article Title: IL-38 Aggravates Atopic Dermatitis via Facilitating Migration of Langerhans cells

doi: 10.7150/ijbs.93843

Figure Lengend Snippet: IL-38 has non-chemotactic properties of LCs, but can upregulate CCR7 expression to promote migration of LCs, aggravating the progression of AD. (A) Bone marrow cells were isolated in vitro and induced into LCs (BM-LCs). 100 ng/ml rmIL-38 or equal volume of vehicle was added to the lower chamber of the Transwell and incubated at 37°C in a 5% CO 2 incubator for 3 h. The number of BM-LCs migrating into the lower chamber was collected and analyzed by flow cytometry (n=3). (B and C) Bone marrow cells were isolated in vitro and induced into LCs (BM-LCs). BM-LCs were inserted in the Transwell upper chamber with being pretreated with rmIL-38 or vehicle for 24 hours. The lower chamber of Transwell was loaded recombinant mouse CCL21 protein or vehicle. After incubation, the number of cells migrating to the bottom chamber was determined by flow cytometry (n=3). (D) Relative expression of CCR7L on AD-lesion skin in K14 Cre/+ -IL-38 f/f (n=5) and IL-38 f/f (n=5) mice were quantified by RT-qPCR after DNFB-induced AD. (E) Relative expression of CCR7L on lymph nodes in K14 Cre/+ -IL-38 f/f (n=5) and IL-38 f/f (n=5) mice were quantified by RT-qPCR after DNFB-induced AD. (F) Flow cytometry measurement of CCR7 expression in LCs migrating to lymph nodes in K14 Cre/+ -IL-38 f/f (n=5) and IL-38 f/f (n=5) after DNFB-induced AD. (G) Flow cytometry measurement of CCR7 expression in LCs on AD-lesion skin in K14 Cre/+ -IL-38 f/f (n=5) and IL-38 f/f (n=5) after DNFB-induced AD. Error bars represent the mean ± SD. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; p values were calculated using Student's t test or One-way ANOVA and Two-way ANOVA.

Article Snippet: Depending on the purpose of the experiment, 100ng/mL of recombinant murine CCL21 (Peprotech, 250-13), rmIL-38 (Adipogen, AG-40B-0101-C010) or vehicle was added to the lower chamber, which was then incubated at 37°C in a 5% CO2 incubator for 3h.

Techniques: Expressing, Migration, Isolation, In Vitro, Incubation, Flow Cytometry, Recombinant, Quantitative RT-PCR