, Charles River), CD45.2 WT (Stock n°000664, Charles River), CD45.2 Lt? ?/?58 , CD45.2 Ccr2-red fluorescent protein (RFP)/RFP (CCR2 RFP/RFP ) 59 , CD45.2 Ccr1 ?/?60, vol.45

, RipmOVA mice were backcrossed on a Rag2 ?/? background and OTII-Rag2 specific pathogen-free condition at the Centre d'Immunologie de Marseille-Luminy, France. Standard food and water were given ad libitum. For all experiments, males and females were used at the age of 6 weeks. Mice were killed using CO 2 and terminated via cervical dislocation. All experiments were done in accordance with national and European laws for laboratory animal welfare (EEC Council Directive 2010/63/UE), and were approved by the Marseille Ethical Committee for Animal Experimentation, Ccr5 ?/?61 , OTII 62 and RipmOVA 27 mice were on a C57BL/6J background. OTII and

, After flushing with RPMI medium, 5 × 10 6 BM cells from tibia and femurs of OTII-Rag2 ?/? mice were injected i.v. into lethally ?-irradiated OTII-Rag2 ?/? and RipmOVA-Rag2 ?/? recipients

X. ,

, Total mTECs were purified by sorting CD45 -Ep-CAM + BP-1 lo UEA-1 + cells after depletion of CD45 haematopoietic cells, using anti-CD45 magnetic beads by autoMACS with the DepleteS program (Miltenyi Biotec). mTEC lo and mTEC hi cells were sorted as CD80 lo and CD80 hi mTECs, respectively. Flow cytometry gating strategies are shown in Fig. 5a. For co-culture experiments, resident cDCs, migratory cDCs and pDCs were purified from the thymus of WT mice, by sorting of SHPS-1 -CD11c hi BST-2 lo , SHPS-1 + CD11c hi BST-2 lo and CD11 int BST-2 hi , respectively. Flow cytometry gating strategies are shown in Supplementary Fig. 1. CD4 + thymocytes from OTII or OTIIxLt? ?/? mice were purified by sorting CD4 + CD8 ? cells after depletion of CD8 + cells, using biotinylated anti-CD8 antibody with anti-biotin microbeads by AutoMACS via the DepleteS program, The same experiment was performed by transplanting 5 × 10 6 BM cells from CD45.2 WT or Lt? ?/? mice into lethally ?-irradiated CD45.1 WT recipients

B. +-cd11c-hi and . Lo, Flow cytometry gating strategies are shown in Supplementary Fig. 14a,b. BM-derived cDCs, pDCs and macrophages, defined as CD11c hi MHCII + , CD11c + BST-2 hi and F4/80 + CD11b + , respectively were cell-sorted. Flow cytometry gating strategies are shown in Supplementary Fig. 14c-e. All purified cell types were cell-sorted, CD11 int BST-2 hi and F4/80 + CD11b + cells, respectively

, BP-1), anti-CD45.1 (A20), anti-CD11b (M1/70), anti-CD45 (30-F11) and anti-TCR V?5.1,5.2 (MR9-4) antibodies were purchased from BD Biosciences. Anti-CCR2 (475301) was purchased from RnD systems. FITC-conjugated UEA-1 lectin was purchased from Vector Laboratories. Anti-Ep-CAM (G8.8), anti-Foxp3 (FJK-16s), anti-Ki-67 (SolA15) and anti-LT?R (ebio3C8) antibodies were purchased from eBioscience. For intracellular staining of anti-Foxp3, anti-Helios, anti-Ki-67, anti-NF-?B p65, anti-phospho NF-?B p65, anti-RelB and anti-MCP1-4, cells were fixed, permeabilized and stained with the Foxp3 staining kit, according to the manufacturer's instructions (eBioscience). Cells were incubated 1 h at 37°C with anti-CCR1 and anti-CCR5 antibodies. For MCP1-4 detection, cells were incubated for 3 h with brefeldin A (BioLegend), Flow cytometry. DCs, macrophages, mTECs and thymocytes were analysed by flow cytometry with standard procedures. Cells were incubated for 15 min at 4°C with Fcblock (anti-CD16/CD32, 2.4G2, BD Biosciences) before staining with the following antibodies: anti-CD11c (N418)

, All 2 × 10 3 cell-sorted CD45 ? Ep-CAM + BP-1 lo UEA-1 + mTECs, SHPS-1 + CD11c hi BST-2 lo cDCs, SHPS-1 -CD11c hi BST-2 lo cDCs and CD11 int BST-2 hi pDCs from WT thymi were loaded or not for 1 h with OVA 323-339 peptide (10 ?g per ml, Polypeptide group) at 37°C. Cells were then cultured for 24 h with 1 × 10 4 purified CD4 + thymocytes, from OTII-Rag2 ?/? or OTII-Rag2 ?/? xLt? ?/? mice, p.10

, Sigma Aldrich) supplemented with L-glutamine (2 mM, ThermoFisher), sodium pyruvate (1 mM, ThermoFisher), 2-mercaptoethanol (2 × 10 ?5 M, Ther-moFisher), penicillin (100 IU per ml, ThermoFisher) and streptomycin (100 ?g per ml, ThermoFisher). When indicated, recombinant LT?R-Fc chimera (2 ?g per ml, FBS

, Adoptive transfer of blood and splenic CD45.1 donor cells. Red blood cells from CD45.1 WT congenic mice or Ccr2 RFP/RFP homozygous mice were lysed with RBC lysis buffer (eBioscience)

X. ,

. Rs-, , 2000.

, For co-adoptive transfer experiments, 6 × 10 6 nucleated blood cells from CD45.1 WT congenic mice and Ccr2 RFP/RFP homozygous mice were co-injected i.v. into sublethally ?-irradiated CD45.2 WT or Lt? ?/? recipients

. Rs-, For AT of DC and macrophage-enriched cells, spleens and lymph nodes from CD45.1 WT congenic mice or Ccr2 RFP/RFP homozygous mice were depleted in T and B cells, using biotinylated anti-CD3 and anti-CD19 antibodies with anti-biotin microbeads by AutoMACS via the deplete program (Miltenyi Biotec), 2000.

, BM cells from WT mice were cultured for 7-10 days in complete RPMI medium containing murine M-CSF (1 ng per ml, PeproTech), murine Flt3-ligand (100 ng per ml, PeproTech) and GM-CSF (20 ng per ml, PeproTech) for the generation of cDCs, pDCs and macrophages, respectively, Bone marrow-derived cDCs/pDCs/macrophages. After red blood cell lysis with RBC lysis buffer (eBioscience)

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