All viable cells (DAPI-) from Day 6 NTOC supernatant treated with: TL1A and TL1A+IL-18 were FACS-purified (Aria III, BD Biosciences) and pelleted by centrifugation at 400g for 5 mins. Cells were incubated with CD16/CD32 mAb (BD Bioscience, #553142) for 20mins at 4°C to avoid non-specific Fc receptor binding of CITE-seq antibodies, prior CITE-seq performance. Cells were washed twice with  PBS with 2% FCS used directly in downstream CITE-seq analysis. We FACS-purified 15.000 cells per sample (treatment) were loaded on a Chromium GemCode Single Cell Instrument (10x Genomics) to create single-cell gel beads-in-emulsion (GEM). The scRNA-Seq libraries were prepared using GemCode Single Cell 3' Gel Bead and Library kit, version 3 (NTOC supernatant) and version NextGEM 3.1 (Thymus lobes & NTOC lobes) (10x Genomics) according to the manufacturer's instructions with the addition of amplification primer (3 nM, 5'CCTTGGCACCCGAGAATT*C*C) during cDNA amplification to enrich the TotalSeq-A cell surface protein oligos. 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.