, Tissue sections were co-stained with a-CD90.2 and a-B220 Abs Scale bars (white) represent 100 mm. (B) Number of CPs and ILFs in the small intestine and colon of Rorc(gt) GFP Gpr183 +/+ and Rorc(gt) GFP Gpr183 À/À mice (n = 3?5) The upper panel shows representative images of a CP and an ILF from Rorc(gt) GFP Gpr183 +/+ mice. Scale bars (red) represent 100 mm. (C) Number of peripheral and mesenteric lymph node cells (n = 6?8), Peyer's patches (n = 10), and colonic patches (n = 3) from Gpr183 +/+ and Gpr183 À/À mice. (D) Number of RORgt + clusters in the colon of bone marrow chimeras (n = 5?9), Bone marrow cells from Gpr183 +/+ or Gpr183 À/À mice were injected into either Gpr183 +/+ or Gpr183 À/À irradiated recipient mice. (E) Number of CPs in the colon of Rag1-deficient Gpr183 +/+ and Gpr183 À/À mice
, The biology of innate lymphoid cells, Nature, vol.69, issue.7534, pp.293-301, 2015.
DOI : 10.1038/ni.1947
, Identification and distribution of developing innate lymphoid cells in the fetal mouse intestine, Nature Immunology, vol.160, issue.2, pp.153-160, 2015.
DOI : 10.1002/eji.200636745
, Development and Function of Secondary and Tertiary Lymphoid Organs in the Small Intestine and the Colon, Frontiers in Immunology, vol.211, issue.Suppl 10, p.342, 2016.
DOI : 10.1084/jem.20131737
, Innate lymphoid cells drive interleukin-23-dependent innate intestinal pathology, Nature, vol.148, issue.7293, pp.1371-1375, 2010.
DOI : 10.1038/nature08949
URL : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3796764/pdf
, 25-Hydroxycholesterols in innate and adaptive immunity, Nature Reviews Immunology, vol.37, issue.11, pp.731-743, 2014.
DOI : 10.1038/ni.2570
, Development, Differentiation, and Diversity of Innate Lymphoid Cells, Immunity, vol.41, issue.3, pp.354-365, 2014.
DOI : 10.1016/j.immuni.2014.09.005
, Thymic Origin of Intestinal ???? T Cells Revealed by Fate Mapping of ROR??t+ Cells, Science, vol.305, issue.5681, pp.248-251, 2004.
DOI : 10.1126/science.1096472
, An essential function for the nuclear receptor ROR??t in the generation of fetal lymphoid tissue inducer cells, Nature Immunology, vol.5, issue.1, pp.64-73, 2004.
DOI : 10.1038/ni1022
, Innate lymphoid cells: A new paradigm in immunology, Science, vol.15, issue.5, p.6566, 2015.
DOI : 10.1038/ni.2830
URL : https://hal.archives-ouvertes.fr/pasteur-01402704
, Guidance of B Cells by the Orphan G Protein-Coupled Receptor EBI2 Shapes Humoral Immune Responses, Immunity, vol.31, issue.2, pp.259-269, 2009.
DOI : 10.1016/j.immuni.2009.06.016
, The chemotactic receptor EBI2 regulates the homeostasis, localization and immunological function of splenic dendritic cells, Nature Immunology, vol.174, issue.5, pp.446-453, 2013.
DOI : 10.1038/ni1100
, IL-23???responsive innate lymphoid cells are increased in inflammatory bowel disease, The Journal of Experimental Medicine, vol.208, issue.6, pp.1127-1133, 2011.
DOI : 10.1172/JCI21404
, Identification of Multiple Isolated Lymphoid Follicles on the Antimesenteric Wall of the Mouse Small Intestine, The Journal of Immunology, vol.168, issue.1, pp.57-64, 2002.
DOI : 10.4049/jimmunol.168.1.57
, Oxysterols direct immune cell migration via EBI2, Nature, vol.65, issue.7357, pp.524-527, 2011.
DOI : 10.1016/S0039-128X(00)00131-8
URL : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4297623/pdf
, Cutting Edge: TGF-?? Signaling Is Required for the In Vivo Expansion and Immunosuppressive Capacity of Regulatory CD4+CD25+ T Cells, The Journal of Immunology, vol.173, issue.11, pp.6526-6531, 2004.
DOI : 10.4049/jimmunol.173.11.6526
, Host???microbe interactions have shaped the genetic architecture of inflammatory bowel disease, Nature, vol.286, issue.7422, pp.119-124, 2012.
DOI : 10.1074/jbc.M111.291492
, Identification of novel lymphoid tissues in murine intestinal mucosa where clusters of c-kit+ IL-7R+ Thy1+ lympho-hemopoietic progenitors develop, Journal of Experimental Medicine, vol.184, issue.4, pp.1449-1459, 1996.
DOI : 10.1084/jem.184.4.1449
, EBI2 Guides Serial Movements of Activated B Cells and Ligand Activity Is Detectable in Lymphoid and Nonlymphoid Tissues, The Journal of Immunology, vol.187, issue.6, pp.3026-3032, 2011.
DOI : 10.4049/jimmunol.1101262
, Natural Aryl Hydrocarbon Receptor Ligands Control Organogenesis of Intestinal Lymphoid Follicles, Science, vol.187, issue.6, pp.1561-1565, 2011.
DOI : 10.4049/jimmunol.1100912
, Nonredundant Function of Soluble LT??3 Produced by Innate Lymphoid Cells in Intestinal Homeostasis, Science, vol.120, issue.12, pp.1243-1246, 2013.
DOI : 10.1172/JCI43918
, Tissue-infiltrating neutrophils represent the main source of IL-23 in the colon of patients with IBD, Gut, vol.8, issue.10, pp.1632-1641, 2016.
DOI : 10.1038/mi.2014.78
, AHR drives the development of gut ILC22 cells and postnatal lymphoid tissues via pathways dependent on and independent of Notch, Nature Immunology, vol.181, issue.2, pp.144-151, 2011.
DOI : 10.4049/jimmunol.181.6.4052
, EBI2 augments Tfh cell fate by promoting interaction with IL-2-quenching dendritic cells, Nature, vol.1291, issue.7601, pp.110-114, 2016.
DOI : 10.1007/978-1-4939-2498-1_1
, Oxysterols direct B-cell migration through EBI2, Nature, vol.408, issue.7357, pp.519-523, 2011.
DOI : 10.1016/j.ab.2010.09.031
, T cells, The Journal of Experimental Medicine, vol.205, issue.6, pp.1381-1393, 2008.
DOI : 10.1084/jem.20071451
URL : https://hal.archives-ouvertes.fr/pasteur-01402753
, Distinct oxysterol requirements for positioning na?¨vena?¨ve and activated dendritic cells in the spleen, 2017.
, Innate Lymphoid Cells in Inflammation and Immunity, Immunity, vol.41, issue.3, pp.366-374, 2014.
DOI : 10.1016/j.immuni.2014.09.006
URL : https://hal.archives-ouvertes.fr/pasteur-01402714
, Developing Lymph Nodes Collect CD4 + CD3 ??? LT?? + Cells That Can Differentiate to APC, NK Cells, and Follicular Cells but Not T or B Cells, Immunity, vol.7, issue.4, pp.493-504, 1997.
DOI : 10.1016/S1074-7613(00)80371-4
, Lymphatic Endothelial Cells Control Initiation of Lymph Node Organogenesis, Immunity, vol.47, issue.1, pp.80-92, 2017.
DOI : 10.1016/j.immuni.2017.05.008
, EBI2 mediates B cell segregation between the outer and centre follicle, Nature, vol.316, issue.7259, pp.1122-1126, 2009.
DOI : 10.1038/nature08226
, The Transcription Factor T-bet Regulates Intestinal Inflammation Mediated by Interleukin-7 Receptor+ Innate Lymphoid Cells, Immunity, vol.37, issue.4, pp.674-684, 2012.
DOI : 10.1016/j.immuni.2012.09.008
, The development and function of mucosal lymphoid tissues: a balancing act with micro-organisms, Mucosal Immunology, vol.7, issue.3, pp.455-466, 2014.
DOI : 10.1200/JCO.2007.15.0284
, RORgammat and commensal microflora are required for the differentiation of mucosal interleukin 22-producing NKp46+ cells, 2009.
, Microbial Flora Drives Interleukin 22 Production in Intestinal NKp46+ Cells that Provide Innate Mucosal Immune Defense, Immunity, vol.29, issue.6, pp.958-970, 2008.
DOI : 10.1016/j.immuni.2008.11.001
URL : https://hal.archives-ouvertes.fr/pasteur-01402754
, The Chemokine Receptor CXCR6 Controls the Functional Topography of Interleukin-22 Producing Intestinal Innate Lymphoid Cells, Immunity, vol.41, issue.5, pp.776-788, 2014.
DOI : 10.1016/j.immuni.2014.10.007
URL : https://hal.archives-ouvertes.fr/pasteur-01090414
, Innate Lymphoid Cells, Science, vol.330, issue.6004, pp.665-669, 2010.
DOI : 10.1016/j.immuni.2004.07.007
URL : https://hal.archives-ouvertes.fr/pasteur-01402753
, Transcriptional regulation of innate lymphoid cell fate, Nature Reviews Immunology, vol.157, issue.7, pp.415-428, 2015.
DOI : 10.1016/j.cell.2014.08.026
URL : https://hal.archives-ouvertes.fr/pasteur-01168713
, Innate lymphoid cells in the initiation, regulation and resolution of inflammation, Nature Medicine, vol.62, issue.7, pp.698-708, 2015.
DOI : 10.1136/gutjnl-2012-304154
, Oxysterols are agonist ligands of RORgt and drive Th17 cell differentiation, Proc. Natl. Acad. Sci. USA 111, pp.12163-12168, 2014.
, mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury, Proc. Natl, 2017.
DOI : 10.1016/j.cell.2010.09.016
URL : https://hal.archives-ouvertes.fr/pasteur-01449402
, Requirement for Lymphoid Tissue-Inducer Cells in Isolated Follicle Formation and T Cell-Independent Immunoglobulin A Generation in the Gut, Immunity, vol.29, issue.2, pp.261-271, 2008.
DOI : 10.1016/j.immuni.2008.05.014
, Differential Activity of IL-12 and IL-23 in Mucosal and Systemic Innate Immune Pathology, Immunity, vol.25, issue.2, pp.309-318, 2006.
DOI : 10.1016/j.immuni.2006.05.017
, Regulated Expression of Nuclear Receptor ROR??t Confers Distinct Functional Fates to NK Cell Receptor-Expressing ROR??t+ Innate Lymphocytes, Immunity, vol.33, issue.5, pp.736-751, 2010.
DOI : 10.1016/j.immuni.2010.10.017
, Author response image 1 Author response, eLife, vol.37, issue.2, p.757, 2013.
DOI : 10.7554/eLife.00757.016
, blocking steps, sections were stained with primary rat a-RORgt (AFKJS-9) Ab, followed by signal amplification with secondary (FITCconjugated donkey a-rat secondary Ab), tertiary (Alexa Fluor 488-conjugated rabbit a-fluorescein Ab), and quaternary (Alexa Fluor 488-conjugated donkey a-rabbit Ab) Abs. Finally, sections were co-stained with APC-conjugated a-B220 Ab. GATA3 immunofluorescence was determined using the same protocol with rat a-GATA3 (TWAJ) Ab and rat IgG2a isotype was used as a control, KLRG1 was detected with hamster a-KLRG1 (2F1) Ab and visualized with AF647-conjugated goat a-hamster IgG (ThermoFisher). Hamster IgG2a isotype was used as a control for KLRG1 staining. Images were acquired on a Nikon A1R confocal microscope and processed with NIS-Elements Imaging software
, Briefly, cells were blocked with 40 mg/ml donkey a-mouse Fab fragments (Jackson ImmunoResearch) for 20 min on ice before staining with 1 mg/ml LTbR-Fc chimera for 30 min on ice. After washing, cells were incubated for 20 min on ice with 6.5 mg/ml biotinylated donkey a-mouse IgG, LTa 1 b 2 Cell Surface Expression Surface LTa 1 b 2 was detected using a LTbR-Fc chimera (R&D Systems) as described Cells were then washed, blocked with 2% mouse/rat serum, and stained with surface Ab mix including PE-conjugated streptavidin, 2015.
, Colon and small intestine sections were washed in PBS, cut into 1cm-pieces, and incubated with 20mL of HBSS containing 5% FCS, 5mM EDTA, 1mM DTT, and 15mM HEPES at 37 C for 30 min on a shaking incubator The supernatant, i.e., the intestinal epithelial cell (IEC) fraction, was saved and the pieces were subsequently washed and incubated with 10mL of serum-free HBSS containing 0.15mg/mL Liberase TL (Roche) and 0.1mg/mL DNase I at 37 C for 45 min at 600rpm. The resulting lamina propria (LP) cell suspension was then passed through a 100 mm nylon mesh and centrifuged. For isolation of epithelial (CD45 -Epcam + ), CD45 -Epcam -, and CD45 + cells (Figure 6B, upper panel), the IEC and LP fractions were mixed, centrifuged and stained. For isolation of ILC3s, the LP fraction was further processed for Percoll gradient separation (44%/67%, Sigma) at 600 3 g for 20 min, washed and stained, Cell Sorting Cells were isolated from the colon and small intestine as previously described Figure 4D and Figure S6E, ILC3s were sorted from Rag1-deficient Rorc(gt) GFP Gpr183 +/+ and Gpr183 À/À mice and were defined as DAPI -CD45 + CD11c À CD90.2 + KLRG1 À RORgt-GFP + cells. In Figure 6B (middle panel), cells were sorted from B6 mice as B cells (CD45 + B220 + MHC class II + ), LTi-like ILC3s (CD45 + Lin(CD3/CD5/B220/CD11c/NK1.1) À CD127 + CD90 + KLRG1 À CCR6 + ), DCs (CD45 + CD11c + Class II + ), and CD45 À Epcam -FSC hi cells. Cell populations were sorted using BD FACSAriaI (BD Biosciences), 2011.
, CXCL13 + stromal cells (Figures S7F and G) were isolated from Cxcl13-EYFP reporter mice 2017) as follows: Colonic tissue was harvested and incubated three times for 15 min at room temperature under constant agitation with PBS containing 5% FCS (Lonza), 5mM EDTA (Sigma), 10mM HEPES (Sigma) and 1mM DTT (PD buffer) in order to dissociate the epithelial layer. The tissue was subsequently washed with HBSS containing 10mM HEPES and digested three times for 20 min at 37 C under constant agitation with 120mg/ml collagenase P (Roche), 25mg/ml DNase I (Applichem) and 5mg/ml Dispase I (Roche) in RPMI 1640. To enrich the fraction of stromal cells, suspensions were first purified with a 30% Percoll gradient for 20 min at 1,800rpm and 4 C. Then hematopoietic cells and erythrocytes were depleted by incubating the cell suspension with MACS a-CD45 and a-Ter119 microbeads and passing through a MACS LS column (Miltenyi Biotec) Unbound single cell suspensions were used for further flow cytometric analysis with a, Isolation of Stromal Cells Colonic stromal cell subsets (Figure 6B, lower panel) were isolated by cell sorting as describedPerCP/Cy5.5 and a-Podoplanin (8.1.1)-PE Abs (Biolegend) and cell sorting was performed via Bio-Rad S3 cell sorter, p.31, 2017.
, For B cell (TCRb À CD4 À CD11b À CD11c À Ly6G À B220 + ) chemotaxis B6 mice were used The chemotactic response was measured either as frequency of cells compared to input (Figure 1D) or as relative migration, i.e., number of cells migrated toward 7a,25-OHC compared to medium (Figures 1E and 1F), Figure 1F, NK cells (B220 À CD11c À CD127 À NK1.1 + ), ILC2s (B220 À CD11c À CD127 + CD90.2 + NK1.1 À KLRG1 + ), and ILC3s (B220 À CD11c À CD127 + CD90.2 + NK1.1 À KLRG1 À CD45 mid ) were identified in Rag1 À/À mice, 2011.
, RNA was extracted from colon tissue ($1cm piece) or purified cells with either TRIzol reagent (Invitrogen) or RNeasy Micro Kit (QIAGEN) and used for cDNA synthesis with the SuperScript First-Strand Synthesis System, iScript cDNA Synthesis Kit (BioRad), or High-Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems). Quantitative RT-PCR was performed on a, p.7500
, 2003) were flushed with cold PBS (GIBCO) and opened longitudinally. Mucus and epithelium were scraped mechanically using 1.5 mm coverslips (Thermo Scientific) Under a fluorescence Stereo Lumar Zeiss V12 microscope, intestines were imaged with a NeoLumar S 0.8x objective using the GFP filter. CPs, ILFs, colonic patches, and Peyer's patches were dissected using a surgical blade (Swann-Morton). Tissues were collected in cold RPMI supplemented with 1% HEPES, sodium pyruvate, glutamine, streptomycin and penicillin and 0.1% b-mercaptoethanol (GIBCO) After tissue dissection , supplemented media was removed and tissues were snap-frozen using liquid nitrogen and dry ice for later RNA extraction, Gene Expression in Colonic Lymphoid Structures Intestines from human CD2 GFP transgenic mice
, Ab (Bio X Cell) into mice on a Rag1-deficient background as described At the indicated time points, proximal colons were harvested for RNA extraction, chemotaxis assay, immunofluorescence microscopy, or fixed in 3.7% formalin for histological analysis. Gut inflammation was assessed in paraffin-embedded colon sections stained with H&E. Inflammatory foci were identified as clusters of leukocytes near/at the tip of colonic folds in the proximal colon as described They were distinguished from CPs by their distinct location because the latter are found at the base of the colonic folds near the crypts. Colitis score was calculated using a semiquantitative criterion-based method Colon sections were scored in a blinded manner, Colitis Model Colitis was induced by i.p. injection of 100 mg CD40 (FGK45), 2004.
, Statistical parameters including number of biological replicates and repeat experiments, data dispersion and precision measures (mean and SEM) and p values for statistical significance (a = 0.05) are reported in Figures and Figure Legends. Student's t test was used to determine statistical significance between two groups. For multigroup comparisons, we applied one-way ANOVA with post hoc testing using Tukey's Multiple Comparison Test