Yalin Emre, Magali Irla, Isabelle Dunand- Sauthier, Romain Ballet, Mehdi Meguenani, Stephane Jemelin, Christian Vesin, Walter Reith and Beat A.

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1 Supplementary information Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T- cell output Yalin Emre, Magali Irla, Isabelle Dunand- Sauthier, Romain Ballet, Mehdi Meguenani, Stephane Jemelin, Christian Vesin, Walter Reith and Beat A. Imhof Supplementary Figure S1 Supplementary Figure S2 Supplementary Figure S3 Supplementary Figure S4 Supplementary Figure S5 Supplementary Figure S6 Supplementary Figure S7 Supplementary Figure S8 Supplementary Table S1 Supplementary Table S2

2 Supplementary Figure S1! a Gated on mtec (EpCAM +, CD45 neg, Ly51 neg ) Gated on ctec (EpCAM +, CD45 neg, Ly51 + ) control Ig anti-cyr61 Ab CYR61 surface staining b K8 K5 control antibody for CYR61 Supplementary Figure S1: Control staining for CYR61, related to Figure 1. (a) Cell surface staining for CYR61. In that case, thymic cell suspensions were not permeabilized before CYR61 staining. No surface expression was detected for CYR61. (b) Thymic sections from WT adult mice were stained for CYR61, K5 (mtec marker) and K8 (ctec marker) expression. Scale bar represents 5 µm.

3 Supplementary Figure S2! 3 a 5 b Relative CYR61 binding Number of thymocytes / field rabbit Ig 2 1 IT76 anti-integrin α6 anti-icam1 anti-integrin αm Number of IT76 / field rabbit Ig primary TEC anti-integrin α6 anti-icam1 anti-integrin αm Number of primary TEC / field Fc CYR61-Fc thymocytes rabbit Ig anti-integrin α6 anti-icam1 anti-integrin αl anti-integrin αm c Number of thymocytes / field Supplementary Figure S2: CYR61 binding enhances TEC interactions with thymocytes, related to Figure 2. (a) Quantification of the adhesion of thymocytes and TEC to recombinant CYR61-Fc or Fc molecule ±sem from experiments presented in Fig 2a., p.1;, p.1 (two-tailed unpaired Student's t-test). Three experiments for thymocytes and IT76. Four preparations for primary TEC. (b) Quantification of the binding of CYR61 assessed by flow cytometry and presented in Fig 2b. Binding to CYR61-Fc of each of three experiment was normalized to 1 ±sem., p.5; (one-way ANOVA, Dunnet test). (c) Quantification of the adhesion of CFSE-labeled thymocytes on IT76M1 monolayers pretreated CYR61-Fc (5 µg/ml) or Fc molecules at C for 6 min from three experiments ±sem presented in Fig 2c., p.1 (two-tailed unpaired Student's t- test).

4 Supplementary Figure S3! 9.1±1.3! mtec! ctec! control! 8.9±1.2! % gated! 87.9±.4! CYR61! Ly51! UEA1! 11.1±.4! % gated! I-Ab! Supplementary Figure S3: CYR61 does not affect TEC maturation and mtec to ctec ratio, related to Figure 3. 2DG-FTOC were cultured in presence or absence of CYR61 for 4 days and analyzed by flow cytometry gated on TEC (CD45neg, EpCAM+). Percentages of mtec (Ly51 -/low, UEA-1 + ) and ctec (Ly51 +, UEA-1 - ) are shown ±sem as well as MHC class II (I-Ab) expression levels. Data are representative of three experiments.

5 Supplementary Figure S4! time (min)!! 2! 4! 6! 12! p-erk! t-erk! p-jnk! t-jnk! Quantification! (phospho/total)! ! p-p38! t-p38! Quantification! (phospho/total)! 1.!.5!.! 1.5! I-κB! actin! Quantification! (I-κB/actin)! 1.!.5!.!! 2! 4! 6!12! time (min)! Supplementary Figure S4: CYR61 treatment does not affect JNK, p38 and NF-κB signaling in IT76M1, related to Figure 4. Immunoblot analysis of phospho- and total-erk1/2, phospho- and total-jnk, phospho- and total-p38, I-κB and actin in lysates of IT76M1 stimulated with CYR61-Fc for the indicated times. Data are representative of 2-4 experiments. Quantifications are provided on the right. Data ±sem of 3 experiments for ERK. Data ±sem of 4 experiments for p38. Data ±sd of 2 experiments for JNK., p.5;, p.1 (one-way ANOVA, Dunnet test)

6 Supplementary Figure S5! a control CYR61 b C57BL6 CYR61 actin embryo c control-lobes control-nude CYR61-lobes CD45+ TEC C57BL6 nude GFP 8 weeks d blood sampling h 3! 2! 1! control-nude g control-nude 1! CYR61-nude 8! 6! 4! 2!! control-nude.8 CYR61-nude 6.4± ! 12! 6.5±.7 CD4 4! lung CYR61-nude 1 f 5! Serum Ig (AU) Spleen/body weight (x1-3) sacrifice FACS analyses of grafts and spleen e GFP CYR61-nude Spleen weight (mg) control-nude CYR61-nude % gated 2DG-FTOC. control vector transduction with. CYR61 vector 2.9±.4 2.1±.3 CD8 liver pancreas kidney

7 Supplementary Figure S5: CYR61 overexpression in thymic stroma does not induce autoimmunity, related to Figure 5. (a) Strategy for CYR61 overexpression in thymic stroma. 2DG-FTOC were transduced with a lentiviral CYR61 expression vector or a control vector. Then lobes were grafted under renal capsules of C57BL6 nude mice. Control-nude mice and CYR61-nude mice were followed for 8 weeks. Once a week, blood was collected to assess T-cell count. (b) Immunoblot analysis of CYR61 overexpression in 2DG-FTOC 3 days after transduction. (c) Transduction efficiency. Representative FACS histograms for GFP expression in TEC 8 weeks after engraftment of nude mice. Over 9% of TEC were transduced with control vector or CYR61 expression vector. Spleen weight (d), spleen/body weight ratio (e), serum Ig levels (f) of control-nude and CYR61-nude mice. Mean±sem of control-nude (n=6) and CYR61-nude mice (n=5). (g) CD4/CD8 plots of spleen from control-nude and CYR61-nude mice. Percentages of cells ±sem. (h) Hematoxylin-eosin staining of formalin-fixed sections of lung, liver, pancreas, and kidney of control-nude and CYR61-nude mice 8 weeks after engraftment. Scale bar represents 2 µm.

8 Supplementary Figure S6! a! 74.±5.6! mtec! ctec! control! 81.3±.8! 2.1±1.3! % gated! CYR61! Ly51! UEA1! 18.1±.7! % gated! I-Ab! b! 8.4±1.2! mtec! ctec! control! 81±1.3! CYR61! Ly51! UEA1! 17.8±1! 17.5±1! % gated! % gated! I-Ab! Supplementary Figure S6: CYR61 induces TEC proliferation but does not affect their maturation in FTOC, related to Figure 6. FTOC were cultured in presence or absence of CYR61 for 2 (a) or 6 (b) days and analyzed by flow cytometry gated on TEC (CD45 neg, EpCAM + ). Percentages of ctec and mtec ±sem and MHC class II expression levels are shown. Data are representative of three experiments.

9 Supplementary Figure S7! control-lobes! a! CYR61-lobes! WT C57BL6! 3 weeks! 3x1 6 CFSE + BM cells! from WT C57BL6! i.v. 48 hours! b! relative mrna levels! normalized to cyclophilin (A.U.)! ! sacrifice! FACS analyses of grafts! Supplementary Figure S7: CYR61 increased TEC number in thymic lobes, related to Figure 7 (a) Strategy for experiments of progenitor hosting capacity of thymic lobes. The hosting capacity of control-lobes and CYR61-lobes for circulating progenitors was assessed with short-term homing assays. WT-C57BL6 mice were transplanted under the renal capsule of both kidneys. Control-lobes in one kidney and CYR61-lobes in the other kidney. After three weeks, 3x1 6 CFSE-labeled WT-bone marrow cells were injected i.v. After 48h, thymic receptivity of grafted lobes was assessed by counting the number of recruited Lin neg CFSE + cells and Lin + CFSE + cells. (b) FTOC were cultured in the presence of CYR61-Fc (5 µg/ml) for 6 days. mrna levels of EpCAM, PDGFRα and VE-cadherin were analyzed by qpcr. Data ±sem are representative of 2 independent experiments., p.5; (two-tailed unpaired Student's t-test).

10 Supplementary Figure S8! p-akt t-akt p-erk t-erk p-jnk t-jnk p-p38 t-p38 I-kB actin Supplementary Figure S8: Full immunoblot scans

11 Supplementary Table S1. List of antibodies used Gene Clone Supplier PercP-anti-CD4 GK1.5 AF488-,, PE- AC7-anti-CD anti-cd11a M17/4 and 2D7 AC7-anti-CD11c N418 biotin-anti-cd19 MB19-1 APC-anti-CD25 PC61 BioLegend BV421-CD45 3-F11 AC7-anti-EpCAM G8.8 PC7-anti-I-Ab AF biotin-anti-ly6g 1A8 PE-anti-Ly51 6C3 streptavidin-ac7 anti-rat IgG2a RMG2a-62 alexa7-, PerCP-Cy5.5-, biotin-anti-cd4 RM4-5 biotin-, efluor45-anti-cd biotin-anti-cd11b M1/7 biotin-anti-cd11c N418 PE-anti-CD44 IM7 efluor45-anti-cd45 3-F11 biotin-anti-cd117 2B8 biotin-anti-nk1.1 NKR-P1C biotin-anti-pdca1 ebio927 ebioscience APC-anti-PDGFRα APA5 FITC-anti-PECAM-1 39 APC-anti-Vβ 6 TCR RR4-7 biotin-anti-vβ 1b TCR B21.5 PerCP-eFluor71-anti-Vβ 5.1/5.2 TCR MR9-4 PerCP-eFluor71-anti-Vβ 13 TCR MR12-3 PerCP-eFluor71-anti-Vβ 11 TCR RR3-15 PerCP-eFluor71-anti-Vβ 12 TCR MR11-1 PerCP-eFluor71-anti-Vβ 8.1/ 8.2 TCR KJ anti-rat IgG2a ebr2a anti-armenian hamster IgG ebio299arm streptavidin-apc anti-cd16/32 2.4G2 BDPharmingen FITC-anti-Ki67 B56 alexa488-, APC-donkey anti-sheep IgG Jackson Immuno Research anti-pecam-1 was coupled to Alexa6 with a kit from Thermo Scientific biotin-anti-ter119 TER-119 Caltag biotin-uea1 Vector anti-cd45 MicroBeads Miltenyi Biotec anti-biotin MicroBeads anti phospho- and total- ERK, JNK, p38, Cell Signaling Technology Akt and anti-ikb Anti-mouse actin Millipore Cy3- anti-rabbit IgG Invitrogen Cy5-streptavidin

12 biotin-anti-keratin 8 rabbit anti-cyr61 antibody, rabbit anti-integrin-α6, rabbit anti-integrin-αl, rabbit anti-integrin-αm rabbit anti-icam1 ProGen Biotechnik Santa Cruz Biotechnology

13 Supplementary Table S2. List of primer sequences used Gene primer sense primer sequence CYR61 forward TGGAAAAGGCAGCTCACTGAA CYR61 reverse GCGTGCAGAGGGTTGAAAA GAPDH forward CCCGTAGACAAAATGGTGAAG GAPDH reverse AGGTCAATGAAGGGGTCGTTG cyclophilin forward ATGGCAAATGCTGGACCAA cyclophilin reverse GCCATCCAGCCATTCAGTCT VE-cadherin forward GTGGCCAAAGACCCTGACAA VE-cadherin reverse TTCGGAAGAATTGGCCTCTGT CCL25 forward GCCTGGTTGCCTGTTTTGTT CCL25 reverse ACCCAGGCAGCAGTCTTCAA FGF7 forward GGATTGACAAACGAGGCAAAG FGF7 reverse ACTGCCACGGTCCTGATTTC IL7 forward GGGTCCTGGGAGTGATTATGG IL7 reverse CGGGAGGTGGGTGTAGTCAT P-selectin forward CCTAGGAACATACGGAGTCTTCACTA P-selectin reverse CCTGAGAAATCGAATGAGACATTG EpCAM forward AAAGCCCCTACGACCATCAGA EpCAM reverse TCTGATTCAGCTTATATCGAGATGTGA