Samples were split up in 3 groups: (1) non-preprocessed samples, (2) T cell-depleted samples and (3) Thymus epithelial cells (TECs)-enriched sample. We isolated 20 neonatal thymuses per condition were pooled together per condition/treatment: [1] (P0.5), [2] NTOC lobes day 1.5 and [3] NTOC lobes day 3 treated with either vehicleor TL1A+IL-18. For the non-processed sample, single-cell suspensions were obtained as described previously. T cell-depleted sample was obtained  by negative beads enrichment using biotinylated antibodies as follows: (1) Biotin-anti-CD4 [1:100], (2) Biotin-anti-CD8α [1:100], (3) Biotin-anti-CD19 [1:200] and (4) Biotin-anti-Ter-119 [1:200]. Samples were incubated for 15 min at RT. After 15 min incubation, the cells were centrifuged at 400g, 4ºC for 5 min to remove the excess of non-bound antibodies. 150µL of MagniSort™ Streptavidin Negative Selection Beads (eBioscience, #MSNB-6002) were added  per sample and incubated for 10 min at RT. The samples were placed in a MagniSort™ Magnet (eBioscience, #MAG-4902-10) for 5 min at RT and purified-enriched cells were recovered in clean round bottom polystyrene tubes ready for sorting. Thymus epithelial cells (TECs)-enriched samples were obtained by enzymatic digestion with the following digestion mix:  RPMI, 2%FCS, 2mM EDTA (Invitrogen, #15575020), 0.125 mg/mL Liberase™TM (Roche, #5401119001), 1mg/mlDispase II (Roche, #04942078001) and 10ng/mlDNase I (Roche, #10104159001). Thymic lobes were incubated for 30–40min in a 37 °C water bath in the enzymatic solution. After digestion, the remaining pieces were filtered through a 70-μm mesh filter and washed with 5–10ml MACS buffer (1× PBS with 5mM EDTA and 2% FCS). Cells were centrifuged at 400g, 4ºC for 5 min. Lastly, cells were subjected to negative bead enrichment with the following biotinylated antibodies: (1) Biotin-anti-CD4 [1:100], (2) Biotin-anti-CD8β [1:100], (3) Biotin-anti-CD19 [1:200], (4) Biotin-anti-CD11b [1:300], and (4) Biotin-anti-Ter-119 [1:200] and FACS sorted based on CD45- EpCAM+. Sorted cells were finally poled in a (1)20:(2)60:(3)20 ratio. Sequencing was done using the following parameters: Novaseq6000 flowcell 100 cycles kit v1.5 paired reads (28-8-0-91), 100 pM + 1 % PhiX. Thymic NTOC lobes were processed to single-cell suspension by pressing the organ through a 70 μm cell strainer. NTOC supernatant cells were harvested by repeated pipetting of the NTOC well and filter after the lobes were removed from the well. Samples were spun down at 400g, 4ºC for 7 min. Subsequently, samples were washed once more with 12.5 mL of cold PBS and spin down again at 400g, 4ºC for 7 min. Finally, we added 2mL of ACK lysis buffer (Westburg b.v., #10-548E) to each sample for 2 min at RT. Once incubated, we added 10mL of cold PBS to stop the lysis reaction and samples were centrifuged at 400g, 4ºC during 7 min. If needed, lysis step can be repeated again for better removal of red blood cells. Samples were incubated at 37ºC water bath for 45 min and resuspended with a glass pipette each 15 min. Once incubated, we added 5mL of FACS-EDTA buffer and the cells were transferred to a new 15mL falcon tube through a 70µm cell strainer. Subsequent flow cytometry or FACS preparation of all samples was performed on ice. Antibody staining was performed 30min in the dark. For intracellular staining of transcription factors cells were fixed after extracellular staining using eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher, #00-5523-00). For intracellular cytokine measurements, cells were first extracted from each tissue as mentioned above, and incubated with Brefeldin A (Enzo Life Sciences, #BML-G405-0025) and Monensin (Biolegend, #420701) for 4 hours, following extracellular staining and fixation and permeabilisation using BD Cytofix/Cytoperm™ (BD Biosciences, #554714). Fixable Viability Dye eFluor™ 780 (ThermoFisher, #65- 0865-14) was used to exclude dead cells in all flow cytometry and FACS experiments. For counting purposes, we added 10 µL/sample of e123count eBeads (ThermoFisher, #01-1234-42) or samples were counted in a  BD FACSVerse™ Cell Analyzer. Cell suspensions were analyzed with a BD FACSymphony™ A5, BD FACSymphony™ A3 or BD LSRFortessa™ or purified using a BD Symphony S6, BD FACSAria II or III.