Fig. 1. mTECs express a set of TLRs and their signaling adaptors independently of Aire.

a Gating strategy used for the analysis of TEC populations and general thymic conventional DCs. MACS enriched CD45^– and EpCAM⁺CD11c^– pre-gated cells were further divided into cTECs (Ly51⁺), mTECs^low (MHCII^lowCD80^low), and mTECs^high (MHCII^highCD80^high). Thymic conventional DCs were gated as CD11c⁺MHCII⁺ from the CD45⁺ fraction. A more detailed gating strategy is found in Supplementary Fig. 1a, b. b Representative flow cytometry histograms of TLR expression on mTECs and DCs isolated from the thymus (n = 3 independent experiments). c MyD88 and Trif mRNA expression is determined by qRT-PCR from FACS sorted mTECs and DCs. The expression is calculated relative to Casc3 and normalized to the highest value within each experiment=1 (mean ± SEM, n = 3 samples). d Representative flow cytometry histograms of TLR expression on mTECs from Aire^+/+ and Aire^–/– mice, (n = 3 independent experiments). e Representative comparative flow cytometry plots of different TEC subpopulations in MyD88^fl/fl and MyD88^ΔTECs mice. f Quantification of TEC frequencies from plots in e (mean ± SEM, n = 6 mice). Statistical analysis was performed by unpaired, two-tailed Student’s t-test, p-values are shown. ns = not significant.