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 spatially confined expression of CCRL1 was necessary and sufficient for the creation of functional chemokine gradients.
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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.).
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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.
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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.
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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
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DOI: https://doi.org/10.1038/ni.2889
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