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1.
Figure 2

Figure 2. FITC-dextran uptake and responses to chemokines are not affected in Plxna1−/− DCs. From: Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

(a) FITC-dextran uptake by wild-type and Plxna1−/− BMDCs at 37°C for 30 min. Cells cultured with FITC-dextran on ice were used as controls. (b) Chemotaxis of wild-type and Plxna1−/− DCs toward CCL19, CCL21 or CXCL12 gradients in transwell migration assays (pore size: 5 μm). (c) Directional sensing in wild-type or Plxna1−/− DCs in response to a CCL21 gradient in a Zigmond chamber. Scatter plots show the position of wild-type and Plxna1−/− DCs relative to their original positions after 60 min of chemokine gradient. The percentages of cells that ended up within a 30° arc facing the CCL21 source are shown. (d) CCR7 and CXCR4 expression in wild-type and Plxna1−/− DCs.

Hyota Takamatsu, et al. Nat Immunol. ;11(7):594-600.
2.
Figure 4

Figure 4. Sema3A-Nrp1-plexin-A1 interactions are responsible for DC trafficking. From: Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

(a) Wild-type DC trafficking in the lymphatics after adoptive transferred into wild-type and Sema3A−/−, Sema6C−/− or Sema6D−/− mice. Data are pooled from three independent experiments. Standard error ± 95% confidence interval, **p<0.01, by Mann-Whitney’s U test. (b) Left: DCs trafficking into the lymphatics following adoptive transferred of wild-type and Nrp1sema--knock-in (KI) DCs into wild-type recipients. Mean ± SE, *p<0.05, by Mann-Whitney’s U test. Right: Chemotaxis of wild-type and Nrp1sema--knock-in (KI) DCs through transwells (pore size: 5 μm) layered with lymphatic ECs in response to a CCL21 gradient. Mean ± SD, **p<0.01, by Student’s t test. (d) In vitro CD4+ T cell proliferation in response to KLH following KLH in CFA immunization of Sema3A−/−, Nrp1sema--knock-in (KI), Sema6C−/−, Sema6D−/− and wild-type mice. Mean ± SD. **p<0.01, ***p<0.001, by Student’s t test.

Hyota Takamatsu, et al. Nat Immunol. ;11(7):594-600.
3.
Figure 1

Figure 1. Plxna1−/− mice show impaired T-cell responses due to defects in DC migration into the LNs. From: Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

(a) CFSE dilution by CD4+ OT-II T cells intravenously transferred into wild-type or Plxna1−/− mice, subcutaneously injected in the footpads with OVA peptides in CFA. Ag-specific T cell responses were assayed in the draining LNs (black lines) or non-draining LNs (red lines). The data are representative of three independent experiments. (b) Two-photon microscopy imaging of wild-type or Plxna1−/− CMTMR-labeled BMDCs (orange) injected into the footpads of wild-type recipient mice that also received CMFDA-labeled CD4+ OT-II T-cells (green). DC trafficking into the popliteal LNs was observed 24 hours post injection (c) Numbers of wild-type or Plxna1−/− DCs trafficking into the popliteal LNs of wild-type recipient mice following foodpad injections. Donor BMDCs were CFSE-labeled and the following calculation was used: (% of input cells) = (total cell number) x (% of CFSE+ cells)/(input cell number). Mean ± SD, *p<0.01, **p<0.001, by Mann-Whitney’s U test. (d) Absolute numbers of endogenous DCs isolated at the indicated time points the brachial LNs of wild-type and Plxna1−/− mice after epicutanous administration of FITC-isomer to the shoulder skin.

Hyota Takamatsu, et al. Nat Immunol. ;11(7):594-600.
4.
Figure 5

Figure 5. Sema3A acts on the rear side of DCs. From: Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

(a) Chemotaxis of DCs in the presence of human IgG or recombinant Sema3A proteins in the lower (left) or upper (right) chambers of transwells, while CCL21 was absent or present in the lower chambers. Mean ± SD. *p<0.01, by Student’s t test. (b) DC velocities in two-dimensional DC chemotaxis assays using EZ-TAXIScan in which Sema3A or human IgG was added to the opposite side of the CCL21. The frequency distribution (bar chart) and cumulative frequency distribution (line chart) of the instantaneous speed were determined. p<0.001, by Mann-Whitney’s U test. Data are representative of three independent experiments. (c) Confocal time-lapse video-microscopy of plexin-A1-EGFP expressing BMDCs treated with LPS, suspended in type I collagen gels and placed into a Zigmond chamber with chemokine gradients. DC locomotion was examined at 1-min intervals. (d) Left: Confocal Z-stack imaging showing localization and intensity of plexin-A1 (anti-plexin-A1 polyclonal antibody plus anti-rabbit IgG-Cy3-red) and F-actin (Alexa 488-conjugated phalloidin-green) in DC. Scale bars, 10 μm. Right: Percentage of cells with no co-localization of signals. Data are representative of three independent experiments.

Hyota Takamatsu, et al. Nat Immunol. ;11(7):594-600.
5.
Figure 3

Figure 3. Plxna1−/− DCs exhibit impaired transmigration across the lymphatics. From: Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

(a) Upper: Confocal Z-stack imaging of wild-type or Plxna1−/− CFSE-labeled BMDCs (green) intradermally injected into the ear tissues of oxazolone-sensitized mice. Whole-mount staining was performed 24 h post transfer using biotinylated anti-LYVE-1 with streptavidin-Cy3 (red). Scale bars, 50 μm (upper). Lower: Quantification of the number of retained DCs in the fields. Red circles indicate the mean number of cells. *p<0.001, by Mann-Whitney’s U test. (b) Transmigration of wild-type and Plxna1−/− BMDCs across a lymphatic EC monolayer. Interactions between DCs and the lymphatic ECs were recorded every 30 sec by a time-lapse video microscope. The yellow dotted lines show the cellular junctions of the ECs. White arrows indicate DCs that were contacting the lymphatic ECs. Red arrows indicate the transmigration process that was observed in wild-type DCs. Scale bars, 50 μm. (c) Left: Confocal microscopy of wild-type and Plxna1−/− CFSE-labeled DCs added to EC monolayers, incubated for 45 min, fixed, and then stained with Alexa 546-conjugated phalloidin. Images were obtained with an optical section separation (Z-interval) of 0.22 μm. Right: Quantification of DC transmigration determined by confocal microscopy and displayed as percentage of transmigrated DCs relative to the total number of DCs. Mean ± SD. *p<0.001, by Student’s t-test. (d) Chemotaxis of wild-type or Plxna1−/− DC across transwells (pore size: 5 μm) layered with lymphatic EC in response to a CCL21 gradient. Mean ± SD. *p<0.001, by Student’s t test.

Hyota Takamatsu, et al. Nat Immunol. ;11(7):594-600.
6.
Figure 6

Figure 6. Sema3A induces phosphorylation of MLC and promotes actomyosin contraction. From: Semaphorins guide the entry of dendritic cells into the lymphatics by activating myosin II.

(a) Left: Confocal Z-stack imaging of DCs on fibronectin-coated coverslips treated with human IgG or Sema3A-Fc and stained with an anti-pMLC antibody plus an anti-rabbit-IgG-Cy3 (lower). Eight Z-stack images with an optical section separation (Z-interval) of 0.36 μm were were projected onto one single image. Scale bars, 10 μm (left). Right: Relative frequency distribution (bar chart) and cumulative frequency distribution (line chart) of the average intensities of dendrite regions in DCs that were stimulated with human IgG or Sema3A-Fc. p<0.001, by Mann-Whitney’s U test. Data are representative of three independent experiments. (b) Mean velocities (left) and relative frequency distribution (bar chart) or cumulative frequency distribution (line chart) of the instantaneous speed (right) of a single DC in response to chemokines in the presence of human IgG or Sema3A-Fc in type I collagen matrices analyzed by time-lapse microscopy imaging and MetaMorph software. ***p<0.001, by Mann-Whitney’s U test. Data are representative of three independent experiments. (c) Chemotaxis of wild-type or Plxna1−/− DCs in response to CCL21 in the presence of human IgG or Sema3A-Fc. DCs were added to the upper chambers of transwell assays with type I collagen. An overall difference between the groups was determined by one-way ANOVA. Post hoc multiple comparisons were made using Tukey’s test. *p<0.05. (d) Chemotaxis of DCs treated with 50 μM blebbistatin or 30 μM Y-27632 for 30 min at 37°C and added to the upper chambers of transwells (pore size: 5 μm) layered with 3 mg/ml type I collagen (left) and a HMVEC-dLy monolayer (right) in response to CCL21 in the presence of human IgG or Sema3A-Fc in the upper chambers. An overall difference between the groups was determined by one-way ANOVA. Post hoc multiple comparisons were made using Tukey’s test. *p<0.05, **p<0.01.

Hyota Takamatsu, et al. Nat Immunol. ;11(7):594-600.

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