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Supplementary Materials and methods Primary cell cultures Primary human thymic epithelial cell (TEC) cultures were established following the protocol previously described in several published manuscripts (40, 41, 86). Briefly, after removal of the thymic capsule, fresh human thymic tissue was mechanically minced with scissors in Hanks buffer to obtain 0.5 mm explants. Explants were washed twice with hanks buffer to remove as much as possible thymocytes. Explants were set down onto 75 cm2 flasks for 20 min to allow proper adhesion on the flask surface, and grown in RPMI 1640 medium (Invitrogen) supplemented with L-glutamine, Ultroser-g and 20% of horse sera. Minor changes to the published model are provided, the replacement of horse serum by charcoal-treated SVF and the RPMI medium used was a phenol free RPMI medium, in order to avoid phenol hormone-like effects. The percentage of medullary epithelial cells was assessed throughout the culture. Preliminary experiments indicated that the optimal time for high mTEC percentage was between days 6 and 8. As an example of a representative experiment the percentage of mTEC was estimated by immunofluorescence using an anti-Claudin 4 and anti-Keratin 5/14 antibodies, both known as specific markers of mTEC (19, 87, 88) and was over 85% (Supplemental Figure S6). The other cells were either contaminating fibroblasts or residual thymocytes. Thus, all experiments were performed at day 7, in order to achieve experiments only with high enriched mTECs. Under a percentage of 80% cells being Keratin 14 positive, the cell culture was discarded. After 7 days of culture, mTECs were trypsinized and transferred in 24-well plates at 105 cells/well. The cells were let to attach overnight and were then treated with several doses of sex hormones (estrogen, testosterone, DHT or progesterone) for 24 hours. 42 Isolation of murine thymic epithelial cells Murine thymuses were prepared from 6 to 9 weeks old C57BL/6 mice (Janvier Laboratory, Saint Berthevin, France), as previously described by Gray et al. (24). Connective and fat tissue were removed from each individual thymus and the capsule was incised with fine scissors. Thymuses were then cut in RPMI-1640 (Life Technologies, Carlsbad, USA), to slowly remove as many thymocytes as possible. The supernatant was collected and replaced as it became visibly cloudy and kept on ice. Thymuses were then incubated at 37 °C for 5 minutes in 5 ml of RPMI-1640 containing 0.15% (w/v) liberase DH and 0.1% (w/v) DNAse I (Roche, Meylan, France). A regular and gentle agitation was performed. Fragments were allowed to settle. The supernatant was collected, kept on ice, and the digestion repeated with fresh enzyme mixture using the remaining settled thymic fragments. After 3 digestions, the remaining aggregates were incubated for 10 min with 5 ml of RPMI-1640, containing 0.125% (w/v) Collagenase/Dispase (Roche, Meylan France) and 300 μl of 0.1%(w/v) DNAse I. Cells from all supernatant fractions were then centrifuged at 472 ×g for 5 min, pooled, and resuspended in cold EDTA (5mM EDA/PBS with 5% FCS). Cells were filtered through 70 μm mesh and then stained. mTECs were sorted on a FACSAria II (Becton Dickinson, Le Pont de Claix, France) according to the phenotype CD45- MHC+Ly51-UEA+ as described by Gray et al (24). The staining was performed with fluorescein coupled ulex europaeus agglutinin I (UEA I) (Vector Laboratories, Burlingame, USA), RPE coupled-anti-mouse Ly-51 (clone 6C3/BP-1; Biolegend, San Diego, USA), RPE-Cy7 coupled anti-mouse I-A/I-E (clone M5/114.15.2, Biolegend, San Diego, USA), APC coupled anti-mouse CD45 (clone 30-F11, Becton Dickinson, Le Pont de Claix, France), and Live/Dead fixable IR dead markers (Becton Dickinson, Le Pont de Claix, France). 43 Fetal Thymus Organ Culture (FTOC) Fetal mouse thymuses were extracted from wild type C57BL6 fetal mice at gestational day 12 (GD12). Thymic lobes were placed on membrane filters (Millipore) in petri dishes with 3 ml complete medium (89) containing complete RPMI (Invitrogen, Carlsbad, CA) containing 12% FCS (HyClone, Logan, UT), 100 U/ml penicillin and streptomycin (Invitrogen, Carlsbad, CA), and 50 μ Logan, UT), 100 U/ml penicillin and streptomycin. After 7 days of culture, thymuses were treated with several doses of estrogen for 24 to 72 hours. NSG mouse experiments To generate xenografts, 4 fragments of human thymuses from immunologically normal patients (less than 1 year old) were grafted s.c. into both flanks of 6 week-old female severe combined immunodeficient (NOD Scid gamma C-/- (NSG)) mice provided by Janvier Laboratory (Saint Berthevin, France). The treatment began the day of the graft. To assess estrogen effects, we injected daily in intra-peritoneal, 200 μg of E2 (Sigma-Aldrich, Lyon, France). At day 4, mRNA expression level in two grafted human thymic explants was analyzed. Control mice were injected with vehicle daily. Flow cytometry For cytometry analysis of the Aire protein in mTECs, we used the protocol described by Monte et al. (90) for optimal staining in the nucleus. Nuclei were isolated from cultured mTECs in cold nuclei extraction buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES, 1% Triton X-100 at pH 7.4). Cells were gently vortexed for 10 seconds and allowed to incubate at 4°C for 10 minutes. Nuclei were then pelleted by centrifugation at 2000 g and washed twice with nuclei wash buffer (320 mM sucrose, 5 mM MgCl2, 10 mM HEPES at pH 7.4). Nuclei quality and integrity were confirmed by microscopic examination with trypan blue staining. Nuclei were 44 incubated overnight with uncoupled goat anti-AIRE (clone D17, sc-17986, Santa Cruz Biotechnologies, Heidelberg, Germany) and washed with nuclei wash buffer. Aire staining was detected by PeCy7 coupled rabbit anti goat IgG (Dako, Trappes, France). The labeling was performed in nuclei wash buffer supplemented with 1% BSA and 0.1% sodium azide, at 4°C. Nuclei were analyzed on FACSVerse using the Facs suite software (Becton Dickinson, Le Pont de Claix, France). Immunofluorescence microscopy Cortical and medullary thymic surface analyses Cryostat serial sections (7 µm) of frozen mouse and human thymic tissues were affixed to glass superfrost slides, dried overnight, and stained by hematoxylin. The whole slide pictures were done using Axio Vision software (Carl Zeiss, Le Pecq, France). Quantification of medulla and cortex surfaces was obtained with Image J software. Quantification of Aire expressing cells To quantify Aire-positive cells in mouse and human thymuses, frozen thymic sections were incubated with a goat anti Aire antibody (clone D-17, sc-17986, Santa Cruz Biotechnologies, Heidelberg, Germany) and a rabbit anti-K14 antibody (Eurogentec, Seraing, Belgium) for 2 hours at room temperature. The labeled cells were revealed with Alexa 488 coupled chicken anti goat IgG and Alexa 594 coupled donkey anti rabbit IgG. For each thymus, AIRE+ cells were counted in the medullary area on the whole section (about 20-25 mm2) as shown in supplemental Figure S2. Since the number of K14+ cells was generally higher than 3000 in the thymic entire section, these cells were not counted in the whole section but in four different representative medullary areas and then normalized to the whole section. The surface of these areas, as well as the whole medullary area, were estimated by image 45 j analysis. The number of K14+ cells in the whole medullary area of the section was calculated as follows: number of K14+ cells/surface of the zones counted multiplied by the total medullary surface. The number of AIRE+ cells was then normalized to the number of K14+ cells in the whole section. Alternatively, the number of AIRE+ cells was calculated per mm2 of thymic section or per mm2 of thymic medulla. All countings of AIRE+ cells were done in blind by the same experimenter directly on the microscope in order to be able to change the focus, when required. The individual number of AIRE+ cells from males and females is detailed for humans and mice in supplemental Tables S2 and S4, respectively. Of note, due to ageing process, in adult human thymuses, the thymic medulla was dispersed and its surface could not be correctly evaluated. In this case, the normalization was done to the thymic section total surface only. Quantification of cell infiltrates in the thyroid To quantify cell infiltration in mouse thyroids, we analyzed CD8 T cell infiltrates in mouse thyroids (54). Frozen thyroid sections were incubated with a rat anti-mouse CD8 antibody (Clone: 53-6.7, Becton Dickinson, Le Pont de Claix, France) for 2 hours at room temperature. The labeled cells were revealed with a donkey anti-rat IgG coupled to an Alexa 594. Frozen thyroid sections were also incubated with a rat anti mouse F4/80 (glycoprotein express in mouse macrophage) antibody (Clone: BM8, eBiosciences, San Diego, USA) for 2 hours at room temperature. The labeled cells were revealed with Alexa 488 coupled donkey anti rat IgG. In the thyroid, F4/80 positive cells were counted and normalized per thyroid surface unit. Images were acquired with a Zeiss Axio Observer Z1 Inverted Microscope using 20X magnification. IHC analyses in the mouse thyroids were done in blind. 46 Demethylation and deacetylation experiments Primary human TECs were seeded at 2.105 cells per well in a 12 well-plate, incubated for 24h in RPMI 1640 medium with 5% charcoal-treated serum, and then supplemented with fresh medium containing 5-aza-2’-deoxycytidine (10 μM) (Sigma-Aldrich, Lyon, France) or trichostatin A (50 nM) (Sigma-Aldrich, Lyon, France) for 24h. DNA isolation and bisulfite genomic DNA sequencing The methylation status of CpG sites of AIRE human promoters was performed on bisulfite treated genomic DNA.