ABSTRACT Afferent lymph–borne dendritic cells essentially rely on the chemokine receptor CCR7 for their transition from the subcapsular lymph node sinus into the parenchyma, a migratory step

driven by putative gradients of CCR7 ligands. We found that lymph node fringes indeed contained physiological gradients of the chemokine CCL21, which depended on the expression of CCRL1,

the atypical receptor for the CCR7 ligands CCL19 and CCL21. Lymphatic endothelial cells lining the ceiling of the subcapsular sinus, but not those lining the floor, expressed CCRL1, which

scavenged chemokines from the sinus lumen. This created chemokine gradients across the sinus floor and enabled the emigration of dendritic cells. _In vitro_ live imaging revealed that

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LYMPHANGIOGENESIS INDEPENDENT OF YAP/TAZ ACTIVITY IN LYMPHATIC ENDOTHELIAL CELLS Article Open access 12 September 2024 ATYPICAL CHEMOKINE RECEPTORS IN THE IMMUNE SYSTEM Article 07 May 2024 B

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cells. _J. Exp. Med._ 198, 569–580 (2003). Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS We thank J. Caamano for reading the manuscript and for

suggestions; C. Bleul (Novartis) for CCRL1-eGFP mice; M. Gunn (Duke University) for plt mice; and D. Kioussis (Medical Research Council, National Institute for Medical Research) for the TEP

cell line. Supported by the Medical Research Council (G0802838 to A.R. and G9818340 to the Medical Research Council Centre), The European Union Marie Curie Actions (CRITICS to M.H.U. and

A.R.), the Wellcome Trust (WT090962MA to I.N.-B. and A.R.), the European Research Council (322645 to R.F.), Deutsche Forschungsgemeinschaft (SFB738-B5 and EXC62, 'Rebirth', to

R.F.) and Boehringer Ingelheim Fonds (A.B.). AUTHOR INFORMATION Author notes * Maria H Ulvmar & Asolina Braun Present address: Present addresses: Department of Immunology, Genetics and

Pathology, Rudbeck Laboratory, Uppsala University, Uppsala, Sweden (M.H.U.), and Institute of Microbiology and Immunology, University of Melbourne, Australia (A.B.)., * Maria H Ulvmar and

Kathrin Werth: These authors contributed equally to this work. AUTHORS AND AFFILIATIONS * MRC Centre for Immune Regulation, School of Immunity and Infection, University of Birmingham,

Birmingham, UK Maria H Ulvmar, Poonam Kelay, Elin Hub, Kathrin Eller, Li Chan, Beth Lucas, Igor Novitzky-Basso, Kyoko Nakamura & Antal Rot * Institute of Immunology, Hannover Medical

School, Hannover, Germany Kathrin Werth, Asolina Braun, Tim Worbs & Reinhold Förster * Division of Nephrology, Medical University of Graz, Graz, Austria Kathrin Eller * Institute of

Laboratory Animal Science, University of Veterinary Medicine, Vienna, Austria Thomas Rülicke * Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, UK Robert J

B Nibbs Authors * Maria H Ulvmar View author publications You can also search for this author inPubMed Google Scholar * Kathrin Werth View author publications You can also search for this

author inPubMed Google Scholar * Asolina Braun View author publications You can also search for this author inPubMed Google Scholar * Poonam Kelay View author publications You can also

search for this author inPubMed Google Scholar * Elin Hub View author publications You can also search for this author inPubMed Google Scholar * Kathrin Eller View author publications You

can also search for this author inPubMed Google Scholar * Li Chan View author publications You can also search for this author inPubMed Google Scholar * Beth Lucas View author publications

You can also search for this author inPubMed Google Scholar * Igor Novitzky-Basso View author publications You can also search for this author inPubMed Google Scholar * Kyoko Nakamura View

author publications You can also search for this author inPubMed Google Scholar * Thomas Rülicke View author publications You can also search for this author inPubMed Google Scholar * Robert

J B Nibbs View author publications You can also search for this author inPubMed Google Scholar * Tim Worbs View author publications You can also search for this author inPubMed Google

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inPubMed Google Scholar CONTRIBUTIONS M.H.U., K.W., A.B., E.H., K.E., T.W., R.F. and A.R. designed the experiments and evaluated the data; M.H.U., K.W., A.B., P.K., E.H., L.C. and I.N.-B.

did the experiments; T.W. wrote evaluation software; M.H.U., K.W., A.B., P.K., E.H., R.F. and A.R. prepared the figures; M.H.U., K.W., A.B. and E.H. contributed to the preparation of the

manuscript; B.L. and K.N. produced cell lines; R.J.B.N. provided a mouse strain; T.R. rederived mouse strains; and R.F. and A.R. conceived of the project, directed the research and wrote the

manuscript. CORRESPONDING AUTHORS Correspondence to Reinhold Förster or Antal Rot. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. INTEGRATED

SUPPLEMENTARY INFORMATION SUPPLEMENTARY FIGURE 1 SPECIFICITY CONTROLS FOR STAINING WITH ANTIBODY TO CCRL1. Acetone-fixed frozen sections of WT LN stained (A) with anti-CCRL1 antibody (red)

and (B) with control isotype IgG. Acetone-fixed cryo sections of WT LN (C) and CCRL1 ko LN (D) stained with anti-CCRL1 (red), anti-LYVE-1 (blue) and anti-gp38 (green) antibodies. Scale bars

50 μm. Representative of more than 3 studies. SUPPLEMENTARY FIGURE 2 CCRL1 EXPRESSION IN THE LNS IS CONFINED TO THE GP38HILYVE-1LO LEC SUBSET. Flow cytometry analysis of LECs in lymph nodes

from CCRL1-eGFP mice. (A-B) Stromal cells in the live cell gate (A), gated from the CD45neg, Ter119neg cells (B). (C) LECs are defined as gp38high CD31high cells, BECs as gp38low CD31high,

FRC as gp38high CD31neg, and DN stromal cells are defined as gp38neg CD31neg. (D) gp38high CD31high LECs analysed for expression of LYVE-1 and eGFP. (E-F) Analysis of the eGFPneg and eGFPpos

gated LEC subsets for expression of gp38 (E) and Lyve-1 (F). Geometric mean (MFI) and LYVE-1 positive cells in gate L1 are shown. eGFP positive cells in the gp38high CD31low, FRCs (G), the

gp38low CD31high, BECs, (H) the gp38low CD31low DN stromal cells (I) and CD45pos gated cells (J). Representative graphs from inguinal LN from 3 CCRL1-eGFP mice. SUPPLEMENTARY FIGURE 3

TEP-CCRL1 CELLS BIND AND SCAVENGE CCR7 LIGANDS. (A) CCRL1 expressed in TEP cells scavenges CCL19. TEP-CCRL1 or TEP-mock cells were incubated at 37°C with 10nM CCL19-Alexa647 for the time

indicated. Cells were washed with cold PBS 3% FBS and analyzed by flow cytometry revealing accumulation of CCL19 over time in TEP-CCRL1 cells but not TEP-mock cells (B,C). The expression of

CCRL1 in monolayers inhibits CCL19- (B) and CCL21-induced (C) _in vitro_ transmigration of BM-DCs. Monolayers of TEP-CCRL1 and empty vector-transfected control TEP-mock cells were grown to

confluence on the lower side of Transwell insert filters with 5μm pores. BM-DCs were added to triplicate inserts and allowed to migrate in response to 0.8nM murine recombinant CCL19 or CCL21

or RPMI in the lower well. After incubation at 37o C for 3 h, BM-DCs were collected from the bottom well, stained by an anti-CD11c Ab and analyzed by FACS using counting beads. Data shown

are mean and STDEV for triplicate wells from one representative experiment each from three and four performed using CCL19 and CCL21, respectively. Significance indicated: *: P < 0.05. In

all experiments migration indices (ratios of BM-DCs migrated to chemokine and to RPMI) were 1.6±0.2 and 3.5±0.3 for CCL19 and 1.7±0.9 and 6.6±4.2 for CCL21, across TEP-CCRL1 and TEP-mock

monolayers, respectively (median±STDEV; for both chemokines significant difference between TEP-CCRL1 and TEP-mock, p<0.05, Mann-Whitney, U-test). SUPPLEMENTARY FIGURE 4 LOWER PROPORTIONS

OF CCR7+CD86+ DCS IN SKIN-DRAINING LNS OF CCRL1-DEFICIENT MICE. (A) Gating strategy to identify “migratory” CD86pos CCR7pos DC in inguinal LNs of wild type (WT)and CCRL1 deficient (KO) mice.

After single cell gate and exclusion of dead cells, total CD45+ cells were analysed for CD11c and CD11b expression as displayed for representative wild type and CCRL1 deficient LNs.

Populations of CD11bhigh (blue gate) and CD11blow (green gate) DCs were further analysed for CCR7 and CD86 expression to identify CCR7+CD86+ “migratory” DCs. To identify LDCs total DCs (red

gate) were analysed for langerin against CCR7. This gate encompasses both epidermal-derived Langerhans cells and langerinpos dermal DCs. (B) Proportions of both CD11bhighCD11c+CD86highCCR7+

and CD11blowCD11c+CD86highCCR7+ DCs are significantly lower in CCRL1 deficient mice (KO, white box) compared to wild-type (WT grey box), Significance indicated: *, _P_ < 0.00. Data are

from four litters of 8 week old F2 female mice. n=9 per group. (C) Representative histogram of CCR7 expression in WT (blue) versus CCRL1 ko (red) gated on all DCs, representative of 3

independent experiments. (E) Geometric mean fluorescent intensity of CCR7 in gated CD86high cells from WT (grey box) and KO (white box). F2 littermates, n=4 and 5 for CCRL1 ko and WT mice,

respectively. Box-plot graphs show median values with quartiles and minimum/maximum. SUPPLEMENTARY FIGURE 5 CELL POPULATIONS IN LNS OF WILD-TYPE AND CCRL1-DEFICIENT MICE. (A) LN cellularity

in WT (grey box) and CCRL1 ko mice (white box) determined by FACS using counting beads after LN digestion. (B-E) Absolute cell numbers (left) and relative proportions (right) of main immune

cell populations. (B) T cells (CD3+), (C) B cells (B220+CD19+) (D) Myeloid cells (CD11c-CD11b+) and (E) DC (CD11c+). Data are from F2 generation 8 week old females; n=6-7 in A-C and n=5 in

D-E across three and two litters, respectively. Significance indicated: *, _P_ < 0.05, **, _P_ < 0.01. Box-plot graphs show median values with quartiles and minimum/maximum.

SUPPLEMENTARY FIGURE 6 DC-BORNE ANTIGEN–INDUCED T CELL PROLIFERATION IN WILD-TYPE AND CCRL1-DEFICIENT LNS (A-D) Representative proliferation curves of CellTrace violet labelled pmel (Thy1.1+

Vbeta13 TCR+) T-cells after transfer of 5x104 cognate antigen loaded, LPS matured WT BM-DCs into the footpad of WT or CCRL1 deficient mice. Proliferating T cells in draining LNs of WT (A)

and CCRL1 deficient (B), and non-draining LNs of WT (C) and CCRL1 deficient (D) mice. Data expressed as % of pmel cells showing dilution of the CellTrace dye. (E-F) Cumulative data from

draining (E) and non-draining (F) LNs of WT and CCRL1 deficient mice; n=18 and 19 (E) and 9 and 8 (F), respectively. (G) Homing of pmel T-cells into resting WT and CCRL1 ko LNs after i.v.

transfer. Cumulative data from 10 WT and 8 CCRL1 deficient mice. Significance indicated,*, _P_ < 0.001. Box-plot graphs show median values with quartiles and minimum/maximum.

SUPPLEMENTARY INFORMATION SUPPLEMENTARY TEXT AND FIGURES Supplementary Figures 1–6 (PDF 2021 kb) CCRL1-EXPRESSING TEP CELLS CREATE GRADIENTS OF CCR7 LIGANDS. (MOV 4545 KB) CCRL1

'PATTERNS' FUNCTIONAL _IN VITRO_ GRADIENTS OF CCL19. (MOV 8923 KB) SOURCE DATA SOURCE DATA TO FIG. 1 SOURCE DATA TO FIG. 2 SOURCE DATA TO FIG. 3 SOURCE DATA TO FIG. 4 RIGHTS AND

PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Ulvmar, M., Werth, K., Braun, A. _et al._ The atypical chemokine receptor CCRL1 shapes functional CCL21 gradients in

lymph nodes. _Nat Immunol_ 15, 623–630 (2014). https://doi.org/10.1038/ni.2889 Download citation * Received: 03 December 2013 * Accepted: 04 April 2014 * Published: 11 May 2014 * Issue

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