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 DCs crawling around or inside LVs in uninflamed murine ear skin. Intravital microscopy was performed on uninflamed ear skin of anesthetized Prox1-mOrange YFP-CD11c mouse. DCs (green) can be observed crawling around or inside LVs (red). The video was generated from 3D reconstructions of Z-stacks. Video specifications: 20× objective, 30-s intervals, and 10 frames/s (300-fold accelerated). The original length of the recording: 60 min. Scale bar, 50 μm. Video length: 16 s.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Intravital Microscopy, Generated

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 DCs crawling around or inside LVs in inflamed murine ear skin. Intravital microscopy was performed on CHS-inflamed ear skin of anesthetized Prox1-mOrange YFP-CD11c mouse. DCs (green) can be observed crawling around or inside LVs (red). The video was generated from 3D reconstructions of Z-stacks. Video specifications: 10× objective, 30-s intervals, and 10 frames/s (300-fold accelerated). The original length of the recording: 60 min. Scale bar, 100 μm. Video length: 16 s.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Intravital Microscopy, Generated

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: Flow cytometry–based analysis of uPAR, uPA, and plasminogen protein levels on dermal cell subsets in vivo . Mice were sensitized with 2% oxazolone on the belly on day 0 and challenged on day 5 by applying 1% oxazolone to the skin of one ear. Flow cytometry was performed on both ears, i.e., the CTR and the CHS-inflamed ear, 1 day later. (A) Depiction of the gating strategy used for the identification of LECs (CD45 − CD31 + podoplanin + ), blood endothelial cells (CD45 − CD31 + podoplanin - ), leukocytes (CD45 + CD31 − ), and other nonvascular stromal cells (CD45 − CD31 − ). (B–E) Representative FACS plots (top) and summary of the delta mean fluorescent intensity (ΔMFI; specific - isotype staining) values obtained (bottom) when analyzing the expression of uPAR, uPA, and plasminogen in (B) LECs, (C) blood endothelial cells (BECs), (D) leukocytes, and (E) other nonvascular stromal cells of CTR or CHS-inflamed skin. Data points from the same animal (i.e., with one CTR and one CHS-inflamed ear, n = 4–7 mice in total) are connected by a line. Red lines indicate the mean. Paired Student’s t test.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Flow Cytometry, In Vivo, Staining, Expressing

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: Loss of uPA or of its cell surface localization alters DC entry into lymphatics. Quantitative whole-mount analysis of endogenous DC positioning in the steady-state ear skin of WT, uPA mut , uPA −/− , and CCR7 −/− mice. (A) Top row: Representative confocal images from the four genotypes. DCs are identified as CD45 + CD11c + cells (yellow). Bottom row: Confocal images with orthogonal views provided for one selected DC in the upper row image. Scale bars: 50 μm. The number in each image indicates the DC tissue position with respect to the LV, as defined in B. (B) Schematic depiction of the three different types of DC tissue positionings: (1) interstitial space, (2) adherent to the outer surface of the LV, and (3) within the LV lumen. (C) Quantification of the percentage DCs colocalized with lymphatics (i.e., percentage of (2+3)/(1+2+3), as defined in B). (D) Quantification of the percentage DCs localized inside the LV lumen (i.e., percentage of (3)/(1+2+3), as defined in B). Data points from the same experiment (involving one mouse per genotype) are connected by a line. n = 4–10 mice per condition. Paired Student’s t test.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques:

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: Supplemental data to FITC painting experiments and analysis of LNs draining CHS-inflamed skin. (A) Schematic depiction of the experiment: FITC was applied to the ear skin in WT and uPA mut mice, and ear skin or ear-draining auricular LNs were collected for analysis after 24 h. (B and C) Analysis of dermal DC numbers. Ear skin was enzymatically digested, and single-cell suspensions were generated for flow cytometry–based analysis. (D and E) Gatings used to identify migratory DCs (CD11c + MHCII hi ), and, amongst those, FITC + DCs in single-cell suspensions generated from (D) enzymatically digested LNs and (E) undigested LNs. (F–I) Analysis of immobilized CCL21 and plasmin(ogen) in CHS-dLNs. A CHS response was induced in the ear skin of WT mice, and ear-draining auricular LNs were collected 24 h later. (F and G) Analysis of CCL21 levels in LNs draining CTR or CHS-inflamed ear skin. Freshly cut LN sections were immediately (i.e., without fixation/permeabilization) stained for B220 (B cell follicles), LYVE-1, and CCL21. (F) Representative images of the immunofluorescent staining and of the AI-based tissue segmentation used for differentiating between the T cell zone and B cell follicles/SCS. Scale bar: 100 μm. (G) Quantification of CCL21 staining intensity of observed in the T cell zone. Each dot represents data from a stained auricular LN of one mouse (average of 4–6 images per LN). Student’s t test. (H) ELISA-based quantification of (H) plasminogen and (I) plasmin activity in LN protein extracts, generated after perfusing the mice with PBS. Pooled data from n = 6 mice per group are shown in H and I, Student’s t test. (J) Representative gating strategy used for the quantification of FITC + CD11c + cells in LN sections from WT and uPA mut FITC-painted auricular LNs in . CD11c + cells were identified based on CD11c-AF647 positivity. From the CD11c + population, FITC + cells were identified and subsequently quantified. Marker-negative cells (red color) served as negative control to check background staining and to set the fluorescence thresholds. Please note that the data points of the WT CTR group in C are identical to those shown in , as these extracts were prepared and measured simultaneously.

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Generated, Flow Cytometry, Staining, Enzyme-linked Immunosorbent Assay, Activity Assay, Marker, Negative Control, Fluorescence

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: DC migration from skin into the LN parenchyma is enhanced in uPA mut mice. FITC was applied to the ear skin in WT and uPA mut mice, and ear-draining auricular LNs were collected for analysis after 24 h. (A–D) Flow cytometry–based quantification of DCs in single-cell suspensions of enzymatically digested LNs. Percentage (A and C) and absolute numbers (B and D) of all migratory DCs (A and B; CD11c + MHCII + ) and FITC + migratory DCs (C and D). Pooled data from two similar experiments are shown ( n = 16–19 mice per group). (E–G) . Immunofluorescence-based analysis of sections prepared from auricular LNs after FITC painting. (E) Representative whole-slide multiplex immunofluorescence images of WT and uPA mut LNs. Images on the far left: overall LN architecture and examples of regions of interest (yellow boxes) used for high-resolution analysis of the SCS and parenchymal compartments. Subsequent images (left to right): higher-magnification images showing a merge of FITC signal, CD11c (DCs) and LYVE-1 (LVs) staining, followed by single-channel views of FITC and CD11c. Images on the far right: AI-based segmentation maps of FITC⁺CD11c⁺ DCs in the SCS or parenchyma. Scale bar.: 100 μm. (F) Percentage of FITC + CD11c + DCs localized in the SCS or in the LN parenchyma, and (G) ratio of FITC + CD11c + DCs in the SCS vs. LN parenchyma in WT and uPA mut LNs. Pooled data from the analysis of 6–7 mice per genotype are shown. Each dot presents the average of 3–6 images analyzed per one mouse. ( H ) Comparison of the LN cellularity retrieved from enzymatically digested (“D”) or nondigested (“ND”) LNs. Data points belong to the experiments described in A–D; “D” and I–L, “ND”. (I–L) Flow cytometry–based quantification of DCs in single-cell suspensions generated not digested LNs. Percentage and absolute numbers of (I and J) all migratory DCs (CD11c + MHCII + ) and (K and L) FITC + migratory DCs. Pooled data from three similar experiments are shown ( n = 31–34 mice per group). Unpaired Student’s t test (all graphs).

Article Snippet: Localization and quantification of CD11c + CD45 + cells were performed with the Imaris software (Bit Plane) in a blinded manner.

Techniques: Migration, Flow Cytometry, Immunofluorescence, Multiplex Assay, Staining, Comparison, Generated