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Dendritic cells control fibroblastic reticular network tension and lymph node expansion


After immunogenic challenge, infiltrating and dividing lymphocytes markedly increase lymph node cellularity, leading to organ expansion1,2. Here we report that the physical elasticity of lymph nodes is maintained in part by podoplanin (PDPN) signalling in stromal fibroblastic reticular cells (FRCs) and its modulation by CLEC-2 expressed on dendritic cells. We show in mouse cells that PDPN induces actomyosin contractility in FRCs via activation of RhoA/C and downstream Rho-associated protein kinase (ROCK). Engagement by CLEC-2 causes PDPN clustering and rapidly uncouples PDPN from RhoA/C activation, relaxing the actomyosin cytoskeleton and permitting FRC stretching. Notably, administration of CLEC-2 protein to immunized mice augments lymph node expansion. In contrast, lymph node expansion is significantly constrained in mice selectively lacking CLEC-2 expression in dendritic cells. Thus, the same dendritic cells that initiate immunity by presenting antigens to T lymphocytes3 also initiate remodelling of lymph nodes by delivering CLEC-2 to FRCs. CLEC-2 modulation of PDPN signalling permits FRC network stretching and allows for the rapid lymph node expansion—driven by lymphocyte influx and proliferation—that is the critical hallmark of adaptive immunity.

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Figure 1: CLEC-2 binding uncouples PDPN from RhoA/C- and actomyosin-driven fibroblast contractility.
Figure 2: CLEC-2 binding causes redistribution of PDPN within the plasma membrane.
Figure 3: The FRC network stretches to accommodate acute increases in lymph node cellularity.
Figure 4: CLEC-2+ dendritic cells are required for lymph node swelling during adaptive immune responses.


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We thank B. Reizis for CD11c-Cre mice, the LRI Biological Resources staff for animal care and assistance with experiments and R. Horton-Harpin for production of CLEC-2–Fc. We are grateful to M. Y. Gerner and R. N. Germain for critical reading of the manuscript and to all the members of the Immunobiology Laboratory and Tumour Cell Biology Laboratory for helpful discussions and comments. This work was supported by a Henry Wellcome Postdoctoral fellowship (S.E.A.) and core funding from Cancer Research UK (I.R., G.S., E.S. and C.R.S.).

Author information

Authors and Affiliations



S.E.A., E.S. and C.R.S. designed the study, analysed data and wrote the manuscript. S.E.A. conducted experiments with assistance from A.J.F., J.v.B., R.P.J., K.J.S. and S.H. Preliminary results were generated with S.J.T. and J.L.A. E.N. and G.S. carried out immunohistochemistry and morphometric analysis. D.M.-S., N.C.R. and I.R. generated CD11cΔCLEC-2 mice. N.C.R. managed breeding and genotyping of mouse strains. L.F.M. provided key reagents.

Corresponding authors

Correspondence to Sophie E. Acton or Caetano Reis e Sousa.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Screen for inhibitors of PDPN-mediated cell contractility.

a, Quantification of proportion of contracted NIH/3T3 fibroblasts expressing enhanced (e)GFP control or PDPN–CFP and treated with the indicated inhibitors or vehicles. Statistically significant inhibition: ****P < 0.00001 and *P = 0.01, Fisher’s exact test. Data represent mean ± s.d. of three independent experiments. b, Chemical inhibitors used in a and their targets. c, Contraction score of PDPN-expressing NIH/3T3 fibroblasts transfected with siRNA smartpools targeting the indicated Rho GEFs (MU-046870-01-0002, MU-040120-00-0002, MU-047092-01-0002, MU-041056-01-0002, Dharmacon, GE Healthcare). **P < 0.05, one-way ANOVA. d, Maximum length of FRCs in collagen gels measured in three dimensions from 100-μm-deep confocal z-stacks. Each point represents one FRC. **P < 0.05, one-way ANOVA. e, PCR analysis of Rho GEF mRNA expression in FRC cell lines after siRNA knockdown in comparison to expression of GAPDH.

Extended Data Figure 2 Generation of FRC lines.

Comparison of an FRC cell line generated by immortalization of primary FRCs (bottom, blue) with primary FRCs in lymph node (LN) cell suspensions cultured for 7 days (top, red). Grey histograms indicate isotype-matched control antibody staining. Histograms of primary lymph node cultures are gated on CD45 CD140a+ cells to exclude haematopoietic cells and other stromal subsets. Histograms of the FRC cell line are gated only on live cells.

Extended Data Figure 3 Loss of PDPN results in FRC spreading and actin polymerization.

a, Single optical slice (1 μm) showing morphology and pMLC organization of control and PDPN knockdown (KD) FRCs. pMLC (S19) (green), F-actin (red). Scale bar, 50 μm. b, Quantification of the number of ARP2/3-positive protrusions per FRC comparing control (green) and PDPN knockdown (red) cell lines. Data represent mean ± s.d., each point represents an individual FRC. P < 0.0001, Mann–Whitney U-test. c, Quantification of tail retraction defects comparing control and PDPN knockdown FRCs. Data are collated from >100 cells; P < 0.0001, Fisher’s exact test. ‘Present’ means that tail retraction defects were deemed present by the observer.

Extended Data Figure 4 Loss of pMLC and F-actin filaments after treatment of FRCs with CLEC-2.

Single optical slices (1 μm) of FRC cell lines treated for 30 min with 10 μg ml−1 soluble CLEC-2–Fc protein, fixed and stained for pMLC (S19) (green) and F-actin (red). Scale bars, 50 μm. Higher magnification shown to the right. Scale bars, 5 μm.

Extended Data Figure 5 Elongated morphology of CLEC-2-treated FRCs in three-dimensional cultures.

Quantification of maximum cell length for 100-μm-deep z-stacks. FRCs cultured in three-dimensional collagen/matrigel matrix for 3 days treated with CLEC-2–Fc, ROCK inhibitor 10 μM (Y27632), or stably knocked down (KD) for PDPN expression. Left, projection of the three-dimensional stack; staining F-actin (red), DNA (cell nuclei, blue). Scale bar, 200 μm. Centre, x,y,z coordinates and length of each vector for each end of each cell in three dimensions as quantified using Imaris image analysis software. Right, example of cell morphology in each treatment group; staining F-actin (red), DNA (cell nuclei, blue). Scale bar, 50 μm.

Extended Data Figure 6 Time course of lymph node expansion after OVA/CFA immunization.

Total cellularity, number of T cells (CD45+, CD3+), B cells (CD45+, CD19+) and biliary epithelial cells (BECs; CD45 PDPN CD31+) in draining lymph nodes (LNs) at different times after OVA/CFA immunization. Each point represents one lymph node and data show mean ± s.d. of two independent experiments scoring 8–12 mice. Day 0 represents lymph nodes from non-immunized mice. *P < 0.05, **P > 0.001, ***P > 0.0001, ****P < 0.00001, differences between non-immunized and immunized mice as calculated using one-way ANOVA test followed by Tukey’s multiple comparisons test.

Extended Data Figure 7 FRCs are selectively labelled in PDGFRαKI-H2B-GFP mice.

a, Analysis of skin draining lymph nodes from PDGFRαKI-H2B-GFP mice showing lymph node stromal cells co-expressing CD140a (PDGFRα) and GFP. Left, gate for CD45 stroma; right, GFP and CD140a expression of CD45 gate. b, Flow cytometry analysis showing that GFP+ cells are CD140a+. Left, gating for GFP+ lymph node cells; right, CD140a expression of GFP+ gate (green) compared with CD45 cells (grey). c, z-Stack of PDGFRαKI-H2B-GFP lymph node imaged ex vivo using two-photon microscopy. FRC nuclei (green), second harmonic signal (collagen) (blue). Wheat germ agglutinin AF647 (red) was injected subcutaneously 5 min before lymph node extraction to label conduits. Scale bar, 200 μm. d, Immunohistochemical staining of paraffin-embedded sections of lymph nodes from PDGFRαKI-H2B-GFP mice. Staining GFP (brown) and PDPN (pink), counterstained with haematoxylin (blue). Scale bar, 200 μm.

Extended Data Figure 8 Generation and characterization of CD11cΔCLEC-2 mice.

a, Scheme of targeting approach to allow conditional deletion of Clec1b exons 2, 3 and 4. loxP sites are shown in yellow. b, Clec1b mRNA in lipopolysaccharide (LPS)-treated bone marrow dendritic cells (BMDCs) or freshly isolated CD11c+ splenocytes from CD11cΔCLEC-2 mice and Creneg littermates. Data are represented as relative expression compared to control and depict mean ± s.d. from six replicates from two independent experiments. P values were calculated using Students’s t-test. c, Quantification of bone-marrow-derived dendritic cell (DC) morphology cultured in contact with FRCs. Data indicate score of perimeter2/(4π × area), with area and perimeter calculated from immunofluorescence imaging using ImageJ software. Higher scores indicate increased elongation and/or protrusions. P = 0.0007, Mann–Whitney U-test. d, Representative images from c showing dendritic cells spreading over FRCs. F-Actin (red), cell nuclei (blue). Scale bar, 20 μm. e, f, Total dendritic cell numbers (e) and total FRC numbers (f) in steady-state skin draining lymph nodes of control versus CD11cΔCLEC-2 mice. Each data point represents one lymph node. g, PDPN surface expression by FRCs from control and CD11cΔCLEC-2 mice as measured by flow cytometry and represented relative to the control group. MFI, mean fluorescence intensity.

Supplementary information

Confocal (20x objective) timelapse imaging showing the expression of PDPN-CFP (green) and ezrin-cherry (red) in NIH/3T3 fibroblasts

Imaging starts 16 hours after transfection. Acquisition rate 1 frame per 2 minutes. (MOV 1172 kb)

Confocal (63x objective) timelapse imaging of FRCs stably expressing a RhoA biosensor

FRET ratio shown, red indicates highest and blue indicates lowest RhoA activity. Acquisition rate 1 frame per minute. CLEC-2-Fc beads were added after 10 minutes. (AVI 1450 kb)

Confocal (63x objective) timelapse imaging of FRCs stably expressing a Rac1 biosensor

FRET ratio shown, red indicates highest and blue indicates lowest Rac1 activity. Acquisition rate 1 frame per minute. CLEC-2-Fc beads were added after 10 minutes. (AVI 5150 kb)

Confocal (40x objective, 0.6 zoom) timelapse imaging of FRCs stably expressing GFP-MLC and treated with CLEC-2-Fc beads

Acquisition rate 1 frame/3 min. CLEC-2-Fc beads were added from the beginning of acquisition. (MOV 4185 kb)

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Acton, S., Farrugia, A., Astarita, J. et al. Dendritic cells control fibroblastic reticular network tension and lymph node expansion. Nature 514, 498–502 (2014).

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