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Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues

Nature Immunology volume 22, pages 571–585 (2021)Cite this article

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Abstract

Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell–zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T–B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.

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Fig. 1: ScRNA-seq identifies Grem1 as a marker for T cell zone FRCs in LNs.
Fig. 2: Grem1-expressing FRCs are localized at the interface of B and T cell zones in SLOs.
Fig. 3: Multi-omics analyses define immune hallmarks of Grem1+ FRCs.
Fig. 4: Selective ablation of Grem1+ FRCs has minimal impact on LN architecture.
Fig. 5: Grem1+ FRC ablation leads to a loss of lymphoid tissue-resident cDCs.
Fig. 6: Grem1+ FRCs cohabitate with pre-cDCs and cDCs in the parafollicular region of LNs and regulate cDC homeostasis.
Fig. 7: Characterization of Grem1+ FRC paracrine signaling events with DCs.
Fig. 8: Decreased T cell response following depletion of Grem1+ FRCs.

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Data availability

ScRNA-seq and bulk RNA-seq data are available in the ArrayExpress database under accession nos. E-MTAB-10197 (single-cell mouse FRCs), E-MTAB-10196 (single-cell mouse DCs), E-MTAB-10205 (bulk mouse FRCs) and E-MTAB-10206 (single-cell human DCs + FRCs). Source data are provided with this paper.

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Acknowledgements

We thank G. Ortiz-Munoz at Genentech for technical assistance with in vivo studies. We thank S. Carlisle at Genentech for assistance with bulk RNA-seq data analysis. We thank members of the Turley lab for helpful discussions. This work was supported by Genentech.

Author information

Author notes

  1. Viviana Cremasco

    Present address: Novartis Institutes for BioMedical Research, Cambridge, MA, USA

  2. These authors contributed equally: Varun N. Kapoor, Sören Müller.

Authors and Affiliations

  1. Department of Cancer Immunology, Genentech Inc., San Francisco, CA, USA

    Varun N. Kapoor, Shilpa Keerthivasan, Markus Brown, Alessandra Castiglioni, Ryan Lane, Claudia X. Dominguez, Jillian L. Astarita, Akshay T. Krishnamurty, Catherine B. Carbone, Ira Mellman, Christine Moussion & Shannon J. Turley

  2. Department of Oncology Bioinformatics, Genentech Inc., San Francisco, CA, USA

    Sören Müller, Yasin Senbabaoglu, Amber W. Wang & Richard Bourgon

  3. Center for Advanced Light Microscopy, Genentech Inc., San Francisco, CA, USA

    Cecile Chalouni

  4. Department of Molecular Oncology, Genentech Inc., San Francisco, CA, USA

    Elaine E. Storm & Frederic J. de Sauvage

  5. Research Biology, Genentech Inc., San Francisco, CA, USA

    Maximilian Nitschke, Merone Roose-Girma, Lucinda Tam & Andrey S. Shaw

  6. Department of Molecular Biology, Genentech Inc., San Francisco, CA, USA

    Xiumin Wu & Jonas Doerr

  7. Department of Cancer Immunology and AIDS, Dana–Farber Cancer Institute, Boston, MA, USA

    Viviana Cremasco

  8. Immunology and Infectious Diseases, Genentech Inc., San Francisco, CA, USA

    Mark Z. Chen & Wyne P. Lee

  9. Department of Microchemistry, Proteomics and Lipidomics, Genentech Inc., San Francisco, CA, USA

    Zora Modrusan, Yeqing Angela Yang & Wendy Sandoval

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Contributions

V.N.K. designed and performed experiments, analyzed and interpreted the data, and wrote the manuscript. S.M. analyzed and interpreted the bioinformatics data and wrote the manuscript. C.C. performed imaging analysis. M.B., S.K. and E.E.S. performed experiments and analyzed the data. Y.S. and A.W.W. analyzed bioinformatics data. C.B.C. and R.L. analyzed imaging data. A.T.K, C.X.D, J.L.A., A.C., M.N., X.W., J.D., M.Z.C., Z.M. and Y.A.Y. performed a subset of experiments. M.R.G. and L.T. generated a mouse model. V.C., W.P.L., R.B., W.S., A.S.S., F.J.D.S. and I.M. provided input for the experimental design. C.M. designed and performed experiments and interpreted the data. S.J.T. oversaw the project, generated the mouse model, interpreted the results and wrote the manuscript.

Corresponding author

Correspondence to Shannon J. Turley.

Ethics declarations

Competing interests

All of the authors on the manuscript are employees at Genentech and are stock holders of Genentech/Roche. V.C. worked on this project at Dana–Farber Cancer Institute and declares no competing interest.

Additional information

Peer review information Nature Immunology thanks Antonio P. Baptista, Alison Simmons and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. L. A. Dempsey was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 scRNA-seq analysis of CD45− cells in lymph nodes.

a, UMAP plot of 5,470 stromal cells (dots) from lymph nodes of 10 mice colored by cluster (top) and split by replicate (bottom). b, Left: Projection as in a) colored by expression of indicated genes. Right: Heatmap of the 50 most strongly upregulated genes (rows) in cells from each of the 3 main cell populations (columns): Blood endothelial cells [BECs], lymph node endothelial cells [LECs] and fibroblasts. c, Left: Heatmap of the 10 most strongly upregulated genes (rows) for each LEC subpopulation compared to all other LEC cells (columns). Right: Heatmap of the 10 most cluster-specific genes for each BEC subpopulation compared to all other BECs. d, UMAP plot of fibroblastic cells from Fig. 1a colored by the expression of indicated marker genes. e, Heatmap as described in (c), here of the 10 most cluster-specific genes for each Cd34+ fibroblast subpopulation from Fig. 1a compared to all other Cd34+ fibroblasts. f, Heatmap as described in (c), here of the most strongly enriched genes for fibroblast subpopulations from Fig. 1a. For all heatmaps most strongly enriched genes were defined as the genes with largest LogFC compared to all other cells in the dataset; at most 10 genes are shown per cluster, but less if one or more genes were not uniquely enriched in just the cluster under consideration.

Extended Data Fig. 2 Gremlin1 expressing FRCs are localized outside the B-cell follicle in Peyer’s patches.

a, Fraction of SLN Grem1+ cells in FRCs from Fig. 1a for each replicate separately. b, Left: Gating strategy for identification of FDC (follicular dendritic), MRC (marginal reticular) and FRC (fibroblastic reticular) cells within the CD45−Ter119−CD31−PDPN+ gate; Middle: relative proportion; Right: absolute numbers of EYFP+ (Grem1+) in FRC, MRC and FDC subsets from Grem1 Cre.YFP mice (n = 4) post-tamoxifen induction. c, Top Left: UMAP dimensionality reduction of stromal cells isolated from mouse spleen, colored by cluster membership. Right/bottom left: UMAP as on top left, colored by indicated marker gene expression. Bottom right: Fraction of Grem1+ fibroblasts shown on the top left. d, EYFP in CD45− stromal cells in bone marrow and bones of Grem1 Cre.YFP mice (n = 3) post-tamoxifen induction. e, Plots showing in-vitro EYFP expression by lymph node FRCs in control and 4-hydroxytamoxifen treated cultures (4-OHT) from Grem1 Cre.YFP mice. (f, g) Imaging of lymph node sections from Grem1 Cre.YFP mice post-tamoxifen induction. f, Anti-YFP (Grem1+ FRCs), anti-PDPN (stromal cells) staining in SLN. Scale bars, 150μm. g, Anti-YFP (Grem1+ FRCs), anti-PDPN (stromal cells), anti-PNAd (HEV), anti-LYVE-1 (lymphatic, LEC) staining in SLN. Scale bars, 150μm. h, Imaging of spleen sections from Grem1 Cre.YFP mice post-tamoxifen induction; stained for anti-B220 and anti-YFP (left). Scale bars, 150μm; (right) enlargement of sections on the left. Scale bars, 75μm. i, Imaging of Peyer’s patches from Grem1 Cre.YFP mice post-tamoxifen induction, stained for anti-YFP (Grem1+ FRCs), anti-PDPN (stromal cells), anti-B220 (B cells). Scale bars, 150μm; zoomed image on right, scale bar, 75μm. Data representative of two independent experiments (b, d); data pooled from three independent experiments (e) [control n = 3; OHT treated n = 6]; data representative of images from 13 SLN, three independent experiments (f, g); representative image from 5 spleens, two independent experiments (h); representative images from 4 Peyer’s patches, two independent experiments (i). Data shown as mean ± SEM.

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Extended Data Fig. 3 RNA-seq reveals differences in chemokine activity and integrin mediated signaling between Grem1+ and Grem1− FRCs.

a, Top: UMAP as in Fig. 1a), here colored by Cd200 expression. Bottom: Violin plot showing the expression of Cd200 in Grem1+ clusters 1 and 3 from Fig. 1a. b, Representative plots showing CD200 expression on EYFP+ (Grem1+) FRCs (FRC: CD45−Ter119−CD31−Madcam1−CD21/35− PDPN+) in skin draining lymph nodes (SLN) of Grem1-Cre-ERT2 Rosa26 EYFP mice (n = 4) post-tamoxifen induction c, Left: Representative plots showing CD90 and CD26 expression on EYFP− (Grem1−) CD34+ FRCs in SLN of Grem1-Cre-ERT2 Rosa26 EYFP mice (n = 4) at day 25 post-tamoxifen induction. Right: UMAP as in Fig. 1a, here colored by Cd34, Dpp4, and Thy1 expression. d, Relative average expression of 25 of the most up – and downregulated genes in bulk RNA-seq samples of Grem1+ and Grem1− FRCs. e, Heatmap of expression of marker genes (columns) determined via scRNA-seq [MAST, adj. p-val <0.05 in both replicates comparing Grem1+ FRCs to all other fibroblastic cells] in bulk RNA-seq samples of Grem1+ and Grem1− fibroblasts (rows). f, Gene set enrichment analysis comparing the distribution of the log2FC of Grem1+ vs Grem1− cells across all genes (blue) to the distribution of genes in indicated categories (orange); fry with p-value adjustment. g, Heatmap of genes (rows) from indicated gene ontology (GO) categories with significant (voom/limma, adj. p-val <0.05) differences in gene expression between Grem1+ and Grem1− bulk RNA-seq libraries (columns). h, Enrichment of transcription factor binding sites in genes with significant (MAST, adj. p-val <0.05) upregulation in Grem1+ FRCs compared to Grem1− FRCs in scRNA-seq. i, Volcano plot visualizing the fold-change (x-axis, log2 transformed) and adjusted p-value (y-axis, Spectronaut adjusted p-val <0.001) for proteins (dots) between Grem1+ and Grem1− cells. j, Comparison of indicated proteins from proteomic analysis between Grem1+ and Grem1− FRCs. Data are representative of two independent experiments (b, c); data are compilation of proteomic analysis from three independent sorts (j). ***P < 0.001, **P < 0.01, *P < 0.05 (two-tailed unpaired Student’s t-test). Exact P-values in extended source data. Data are shown as mean ± SEM.

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Extended Data Fig. 4 Normal lymph node HEV morphology and numbers upon Grem1+ FRC ablation.

a, Representative plots with relative proportion (top) and absolute numbers (bottom) of CD45− EYFP+ stromal cells in Grem1-Cre-ERT2 Rosa26 EYFP (n = 5) and Rosa26 EYFP/iDTR (n = 5) mice upon DTxn mediated ablation. Plots are gated on CD45− stromal cells. b, Spleen weight (left) and cellularity (right) in Grem1-Cre-ERT2 Rosa26 EYFP (n = 12) and Rosa26 EYFP/iDTR (n = 14) mice upon DTxn mediated ablation. c, Immuno-fluorescence imaging of lymph node sections from Grem1-Cre-ERT2 Rosa26 EYFP (top) and Rosa26 EYFP/iDTR mice (bottom) upon DTxn mediated ablation, sections were stained for anti-LYVE-1(lymphatics, LECs) and anti-PNAd (HEVs). Scale bars, 150μm. d, Number of HEVs per lymph node as identified by PNAd staining as in (c), here comparing Grem1-Cre-ERT2 Rosa26 EYFP and Rosa26 EYFP/iDTR mice upon DTxn mediated ablation. e, Images of lymph node sections from Grem1-Cre-ERT2 Rosa26 EYFP (left) and Rosa26 eYFP/iDTR mice (right) upon DTxn mediated ablation, sections were stained for anti-PDPN (stromal cells) and anti-PNAd (HEVs). Scale bars, 25μm. f, Immuno-fluorescence imaging of lymph node sections from Grem1-Cre-ERT2 Rosa26 EYFP (left) and Rosa26 EYFP/iDTR mice (right) upon DTxn mediated ablation, sections were stained for anti-PDPN (stromal cells). Scale bars, 75μm. g, Relative proportion of ITGA2b + LEC in Grem1-Cre-ERT2 Rosa26 EYFP (n = 10) and Rosa26 EYFP/iDTR (n = 9) mice upon DTxn mediated ablation. Plots are gated on LEC: CD45− Ter119− CD31+ PDPN+. Data are representative of three independent experiments (a); pooled data from three independent experiments (b); Representative image and quantitation of ten lymph nodes from Grem1-Cre-ERT2 Rosa26 EYFP and fourteen lymph nodes from Rosa26 EYFP/iDTR mice from three independent experiments (c, d, f); representative image of seven lymph nodes from three independent experiments (e); pooled data from two independent experiments (g). ****P < 0.0001, **P < 0.01 (two-tailed unpaired Student’s t-test). Exact P-values in extended source data. Data are shown as mean ± SEM.

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Extended Data Fig. 5 Comparison of lymphoid cellularity upon Grem1+ FRC ablation.

a, Relative proportion (top row) and absolute numbers (bottom row) of indicated cell subsets in lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP (n = 12) and Rosa26 EYFP/iDTR (n = 14) mice upon DTxn mediated ablation. b, Representative plot with relative proportion of EYFP (Grem1) expression on CD11chi MHCIIinter cDCs in lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP (n = 5) mice. c, Relative proportion (top row) and absolute numbers (bottom row) of CD8a+ (left column) or CD11b+ CD4+ (right column) cDCs in spleens of Grem1-Cre-ERT2 Rosa26 EYFP (n = 12) and Rosa26 EYFP/iDTR mice (n = 14) upon DTxn mediated ablation d, Total cell numbers (top, left) and relative proportions of B cells (top, right), CD4+ T (bottom, left) and CD8+ T (bottom, right) in lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP (n = 7) and Rosa26 EYFP/iDTR (n = 9) mice at day 5 post-DTxn mediated ablation. Data are pooled from three independent experiments (a, c); representative of three independent experiments (b); data are pooled from two independent experiments (d). ****P < 0.0001, ***P < 0.001, *P < 0.05 (two-tailed unpaired Student’s t-test). Exact P-values in extended source data. Data are shown as mean ± SEM.

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Extended Data Fig. 6 T cell and pre-cDC trafficking to secondary lymphoid organs is not affected upon Grem1+ FRC ablation.

a, High resolution image of lymph nodes from Grem1-Cre-ERT2 Rosa26 EYFP mice, panels were stained for anti-YFP, anti-CD11c and anti-IgD to detect Grem1+ FRC, CD11c+ and B cells respectively at the B cell follicle boundary. Scale bars, 25μm. b, Confocal imaging of lymph nodes from Grem1-Cre-ERT2 Rosa26 EYFP (left) and Rosa26 EYFP/iDTR (right) mice upon DTxn mediated ablation, panels were stained for anti-YFP, anti-DEC205 and anti-CD11b to detect Grem1+ FRC, cDC1 and cDC2 cells respectively. Scale bars, 125μm. c, Top: schematic of T cell transfer and trafficking experiment; relative proportion of donor T cells two hours after adoptive transfer in lymph nodes (bottom, left) and spleen (bottom, right) of Grem1-Cre-ERT2 Rosa26 EYFP (n = 9) and Rosa26 EYFP/iDTR mice (n = 10 for LN, n = 9 for SPL). d, Top: schematic of pre-cDC transfer and trafficking experiment; relative proportion of donor pre-cDCs twenty hours after adoptive transfer in lymph nodes (bottom, left) and spleen (bottom, right) of Grem1-Cre-ERT2 Rosa26 EYFP (n = 8) and Rosa26 EYFP/iDTR mice (n = 8). e, Relative proportion of BrdU incorporation (19 hours pulse) by CD8a + cDC1s (top) and CD4 + CD11b + cDC2s (bottom) in lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP (n = 12) and Rosa26 EYFP/iDTR (n = 13) at day 7 after DTxn mediated ablation. Data are representative of images of six lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP mice from two independent experiments (a); representative of image from thirteen lymph nodes from Grem1-Cre-ERT2 Rosa26 EYFP and eight lymph nodes from Rosa26 EYFP/iDTR mice from three independent experiments (b); data are pooled from two independent experiments (c, d); data are pooled from three independent experiments (e). ****P < 0.0001, *P < 0.05 (two-tailed unpaired Student’s t-test). Exact P-values in extended source data. Data are shown as mean ± SEM.

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Extended Data Fig. 7 Interactions between Grem1+ FRCs and DCs.

a, Left: Uniform Manifold Approximation and Projection (UMAP) plot visualization of 12,620 murine dendritic cell sorts (dots) colored by cluster identity. Right: UMAP as on the left, here colored by expression of indicated marker genes. b, Average relative expression of the 10 most strongly upregulated genes (by LogFC, rows) across clusters from (a). Two representative genes per cluster are highlighted. c, Fold change in expression of Flt3l mRNA in indicated cell types from skin draining lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP/iDTR mice with respect to Grem1-Cre-ERT2 Rosa26 EYFP mice upon DTxn mediated ablation, results are normalized to those of the gene encoding Rpl19 (ribosomal protein L19). d, Quantification of FLT3L by ELISA of total lymph node protein lysates from skin draining lymph nodes of Grem1-Cre-ERT2 Rosa26 EYFP and Rosa26 EYFP/iDTR mice upon DTxn mediated ablation. e, Left: UMAP plot visualizing 16,226 human CD11c + cells form lymph nodes of three patients colored by cluster identity. Middle: UMAP as on the left, here colored by expression of marker genes. Right: Average relative expression of the 10 most strongly upregulated genes (by LogFC, rows) in clusters from the left. f, Percentage of cells from each patient in each of the clusters from Fig. 7f. g, Enrichment analysis of ligands expressed by GREM1+ FRCs predicted to be involved in paracrine signaling events with DC1. (c) data are pooled from three independent experiments; (d) data are pooled from three independent experiments (YFP n = 14; YFP/iDTR n = 13). **P < 0.01 (two-tailed unpaired Student’s t-test). Exact P-values in extended source data. Data are shown as mean ± SEM.

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Extended Data Fig. 8 Decreased CD4/CD8 T cell responses upon Grem1+ FRC depletion.

a, schematic of anti-DEC205−OVA immunization protocol. b, relative proportion (top row) and absolute numbers (bottom row) of IFNg+ OT-I T cells in lymph nodes (left column) and spleen (right column) of Grem1-Cre-ERT2 Rosa26 EYFP (n = 5) and Rosa26 EYFP/iDTR (n = 5) at day 8 after DTxn mediated ablation. c, schematic of CFA/OVA immunization protocol. d, relative proportion (top) and absolute numbers (bottom) of transferred Thy1.1 OT-I T cells (left) or transferred labeled OT-II T cells (right) in spleen of Grem1-Cre-ERT2 Rosa26 EYFP (n = 5) and Rosa26 EYFP/iDTR (n = 5) at day 7 after DTxn mediated ablation. Data are representative of two independent experiments (b, d). **P < 0.01, *P < 0.05 (two-tailed unpaired Student’s t-test). Exact P-values in extended source data. Data are shown as mean ± SEM.

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Supplementary information

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Kapoor, V.N., Müller, S., Keerthivasan, S. et al. Gremlin 1+ fibroblastic niche maintains dendritic cell homeostasis in lymphoid tissues. Nat Immunol 22, 571–585 (2021). https://doi.org/10.1038/s41590-021-00920-6

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