Fig. 6.

Aire⁺ mTEC control the activation and suppressive signature of recirculating T_reg through antigen-dependent contact. A The expression level of Tgfb1, Gzmb, Fasl, Entpd1 and Nt5e was measured by qPCR in splenic T_reg from Aire^WT or Aire^KO mice co-cultured with Aire^WT or Aire^KO mTEC. Data are derived from 2 independent experiments. B Experimental setup: purified splenic T_reg from CD45.1 Foxp3^eGFP mice were adoptively transferred i.v. into sublethally irradiated CD45.2 Aire^WT or Aire^KO recipients. Adoptively transferred CD45.1 Foxp3^eGFP T_reg were cell-sorted from the thymus of recipient mice one week later and their suppressive signature was analyzed by qPCR. C Flow cytometry profiles, frequencies and numbers of CD45.1 Foxp3^eGFP donor T_reg observed in the thymus of CD45.2 Aire^WT or Aire^KO recipients. D The expression level of Il10, Tgfb1, Gzmb, Fasl, Lag3 and Nt5e was measured by qPCR in CD45.1 Foxp3^eGFP donor T_reg transferred into Aire^WT (n = 4) or Aire^KO (n = 4) recipients. E Representative images of WT thymic sections stained for Aire (green), Foxp3 (red) and CD73 (white). Yellow and white arrowheads denote developing CD73⁻ and recirculating CD73⁺ T_reg, respectively. Scale bar, 30 µm. F Flow cytometry profiles of CD69 activation of splenic OTII T_reg from Rip-mOVA x OTII x Rag2^KO mice co-cultured with OVA_323-339-loaded Aire^het (Aire^eGFP/WT) or Aire^KO (Aire^eGFP/eGFP) mTEC^hi after 24 h later. The histogram shows the frequencies of CD69⁺ T_reg normalized to the activation of OTII T_reg co-cultured with Aire^het mTEC^hi. G The expression level of Il10, Tgfb1, Gzmb, Fasl, Lag3, Entpd1 and Nt5e was measured by qPCR in splenic OTII T_reg from Rip-mOVA x OTII x Rag2^KO mice co-cultured with OVA_323-339-loaded Aire^het (Aire^eGFP/WT; n = 6) or Aire^KO (Aire^eGFP/eGFP; n = 6) mTEC^hi. Data are derived from 2 independent experiments. Bar graphs show mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 using two-tailed Mann–Whitney test for (A, C, D, F, G)