human recombinant 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: Scientific Reports

Article Title: Immune regulation and lymphangiogenesis by lymphatic endothelial cells in the decidua in severe preeclampsia

doi: 10.1038/s41598-026-35667-3

Figure Lengend Snippet: Reduced CCL21 expression and impaired dendritic cell (DC) migration in PE-dLECs. ( A ) PE-dLECs showed significantly reduced expression of CCL21 at both the mRNA and protein levels, as assessed using RT-qPCR and western blot, respectively. ( B ) DC trafficking through LECs was evaluated in three steps: migration, adhesion, and transmigration. PE-dLECs exhibited reduced activity in all three steps. ( C ) Decreased DC migration observed with conditioned media from PE-dLECs was restored by the addition of recombinant CCL21. Data are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA followed by Tukey’s test. All experiments represent three independent experiments, each performed in duplicate. **, p < 0.01 vs. N-dLEC. N-dLECs, dLECs from normal pregnancies; PE-dLECs, dLECs from preeclamptic pregnancies; DC, dendritic cell; dLECs, decidua lymphatic endothelial cells; PE, preeclampsia; CCL 21, chemokine (C-C motif) ligand 21; RT-qPCR, quantitative reverse-transcription PCR.

Article Snippet: Conditioned media from PE or normal pregnancies collected after 24 h of culture in EBM with 1% FBS were added to the lower chamber, with or without recombinant human CCL21 (250 ng/mL; R&D Systems, Minneapolis, MN, USA).

Techniques: Expressing, Migration, Quantitative RT-PCR, Western Blot, Transmigration Assay, Activity Assay, Recombinant, Reverse Transcription

Journal: Scientific Reports

Article Title: Immune regulation and lymphangiogenesis by lymphatic endothelial cells in the decidua in severe preeclampsia

doi: 10.1038/s41598-026-35667-3

Figure Lengend Snippet: Proposed model of immune and lymphatic dysfunction at the maternal-fetal interface in preeclampsia. This schematic illustrates the functional and genetic differences observed in PE-dLECs. In PE-dLECs, reduced expression of lymphangiogenesis-related genes leads to impaired lymphatic vessel formation and functional deficits. Decreased CCL21 expression disrupts DC trafficking, while reduced NO levels impair the suppression of cytotoxic T cells. Finally, these changes may contribute to immune imbalance and the pathogenesis of preeclampsia. LV, lymphatic vessel; BV, blood vessel; M, amnion chorion; D, decidua; dLECs, decidual lymphatic endothelial cells; PE, pre-eclampsia; NO, nitric oxide; CCL 21, chemokine (C-C motif) ligand 21; DC, dendritic cell.

Article Snippet: Conditioned media from PE or normal pregnancies collected after 24 h of culture in EBM with 1% FBS were added to the lower chamber, with or without recombinant human CCL21 (250 ng/mL; R&D Systems, Minneapolis, MN, USA).

Techniques: Functional Assay, Expressing

Journal: bioRxiv

Article Title: Initiation of primary T cell—B cell interactions and extrafollicular antibody responses in an organized microphysiological model of the human lymph node

doi: 10.1101/2025.01.12.632545

Figure Lengend Snippet: (a-c) Naïve T cells were cultured on chip with IL-7. Representative image (b) and quantification (c) of T cell viability after 4 days, labeled with (Calcein AM, green, and Dapi, blue) for 3 donors. (d-g) A CCL21 gradient was established on chip, with CCL21 added to the left-hand media lane. Representative images (e) and quantification (f) of naive CD4+ T cells after migrating toward CCL21 for 1 hr and staining with Calcein AM (green). (g) Quantification of cell velocity 30 min after gradient set up (cells were unlabeled). (h-k) Naïve T cells were cultured without (i) and with (ii) a-CD3/CD28 (StemCell). Images of CD69+ signal (FITC-anti-CD69, green) for (i) naïve and (ii) activated T cells on-chip, and quantification (j) of CD69 signal after 48 hours (unpaired T test, **:p<0.005). (k) Quantification of IFN-γ secretion by activated or naïve CD4+T cells on-chip measured by ELISA of supernatants collected at day 5. Panels f, g, k analyzed with ordinary two-way ANOVA with Sidak’s multiple comparisons test w/single pooled variance, ns: p>0.05, *:p<0.05, ****:p<0.00005.

Article Snippet: For assessing chemotactic activity of naïve CD4+ T cells on chip, a solution of 0.1 μM CCL21 (recombinant human, Peprotech, NJ, USA, Cat# 300–35A) in AIMV media was added to the left media reservoir.

Techniques: Cell Culture, Labeling, Staining, Enzyme-linked Immunosorbent Assay

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: Monocyte-derived DCs (Mo-DCs) were treated (or not) with HIF1A inhibitor PX-478 (PX) or LDH inhibitor oxamate (OX) and stimulated with iMtb for 24 hr. ( A ) Lactate release as measured in supernatants in DCs stimulated or not with iMtb in the presence of OX (N = 5). ( B ) Percentage of migrated cells toward CCL21 relative to the number of initial cells per condition (N = 6). ( C, D ) Three-dimensional amoeboid migration of DCs through a collagen matrix after 24 hr. Cells within the matrix were fixed and stained with DAPI. Images of the membrane of each insert were taken and the percentage of cells per field were counted. ( C ) Mo-DCs stimulated with iMtb for 24 hr (N = 5). ( D ) Mo-DCs infected with Mtb for 24 hr (N = 4). The data are represented as scatter plots, where each circle represents a microphotograph sourced from either five ( C ) or four ( D ) independent donors, with each experiment typically including between 5 to 10 microphotographs. ( E ) Representative schematic of the experimental setup for in vivo migration assays. ( F ) Percentages of migrating bone marrow-derived DCs (BMDCs) (CFSE-labeled among CD11c + ) recovered from inguinal lymph nodes (N = 3). Statistical significance assessed by ( A, B ) ANOVA followed by Dunnett’s multiple-comparisons test (*p<0.05; **p<0.01); ( C, D ) Nested ANOVA followed by Dunnett’s multiple-comparisons test (*p<0.05; **p<0.01); ( F ) ANOVA followed by Holm–Sidak’s multiple-comparisons test (*p<0.05).

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Derivative Assay, Migration, Staining, Membrane, Infection, In Vivo, Labeling

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: ( A, B ) Monocyte-derived DCs (Mo-DCs) were treated or not with the glycolysis inhibitor GSK2837808A and stimulated with iMtb. ( A ) Lactate release (N = 5). ( B ) Chemotactic activity toward CCL21 (N = 5). ( C, D ) Murine bone marrow-derived DCs (BMDCs) were treated or not with PX-478 or oxamate and stimulated with iMtb for 24 hr. ( C ) Lactate release (N = 3). ( D ) Representative dot blots showing the percentages of migrating BMDCs (CD11c + , CFSE-labeled) determined from inguinal lymph nodes. ( E ) Mo-DCs were stimulated with iMtb in the presence of either PX-478 or oxamate and CCR7 expression was measured by FACS (N = 6). Two-way ANOVA followed by Tukey’s multiple-comparisons test (*p<0.05; **p<0.01; ***p<0.001), as depicted by lines. The data are represented as scatter plots, with each circle representing a single individual, means ± SEM are shown.

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Derivative Assay, Activity Assay, Labeling, Expressing

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: Tolerogenic Mo-DCs were generated by dexamethasone (Dx) treatment and were stimulated (or not) with irradiated Mycobacterium tuberculosis (iMtb) in the presence or absence of HIF1A activator dimethyloxalylglycine (DMOG). ( A ) Lactate release and glucose uptake as measured in supernatant (N = 8). ( B ) Mean fluorescence intensity (MFI) of HIF1A. Representative histograms and quantification are shown (N = 6). ( C ) Three-dimensional amoeboid migration of DCs through a collagen matrix. After 24 hr of migration, images of stacks within the matrix were taken every 30 µm. Percentage of migrating cells was defined as cells in the stacks within the matrix relative to total number of cells (N = 6). ( D ) Chemotactic activity toward CCL21 in vitro (N = 6). ( E–H ) Mo-DCs were generated from healthy subjects (HS) or TB patients, and DCs were stimulated (or not) with iMtb. ( E ) Chemotaxis index toward CCL21 (relative to unstimulated DCs) (N = 6). ( F ) Lactate production ratio relative to unstimulated DCs (N = 6). ( G ) Glycolytic capacity assessed by SCENITH (N = 4). ( H ) Chemotactic activity toward CCL21 of Mo-DCs from TB patients stimulated with iMtb and treated or not with DMOG (N = 6). Statistical significance assessed by ( A–D ) two-way ANOVA followed by Tukey’s multiple-comparisons test (*p<0.05; **p<0.01); ( E–G ) unpaired t -test (*p<0.05); ( H ) paired t -test (*p<0.05). The data are represented as scatter plots, with each circle representing a single individual, means ± SEM are shown.

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Generated, Irradiation, Fluorescence, Migration, Activity Assay, In Vitro, Chemotaxis Assay

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet: ( A ) Ex vivo determination of HIF1A expression by monocytes from healthy subjects (HS) or TB patients (TB) for each monocyte subset (CD14 + CD16 - , CD14 + CD16 + , and CD14 dim CD16 + ) (N = 6). ( B, C ) Monocytes from HS were treated with dimethyloxalylglycine (DMOG) during the first 24 hr of differentiation with IL-4/GM-CSF (earlyDMOG) and removed afterward. On day 6 of differentiation, cells were stimulated (or not) with irradiated Mycobacterium tuberculosis (iMtb). ( B ) Monocyte lactate release after 24 hr of DMOG addition (N = 4). ( C ) Chemotactic activity toward CCL21 of DCs (N = 5). Statistical significance was assessed by ( B ) paired t -test (p<0.01); ( C ) two-way ANOVA followed by Tukey’s multiple-comparisons test (*p<0.05). The data are represented as scatter plots, with each circle representing a single individual, means ± SEM are shown.

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Ex Vivo, Expressing, Irradiation, Activity Assay

Journal: eLife

Article Title: Elevated glycolytic metabolism of monocytes limits the generation of HIF1A-driven migratory dendritic cells in tuberculosis

doi: 10.7554/eLife.89319

Figure Lengend Snippet:

Article Snippet: Each DC population (4 × 10 5 cells in 75 μl) was placed on the upper chamber of a transwell insert (5 μm pore size, 96-well plate; Corning), and 230 μl of media (RPMI with 0.5% FCS) with human recombinant CCL21 (200 ng/ml) (Peprotech) was placed in the lower chamber.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Sequencing, Software