Fig. 9: The LTα₁β₂-LTβR axis provided by mTEC limits Foxp3^lo T_reg development in an IL-4 dependent manner.

a Representative flow cytometry profiles, frequencies, and numbers of thymic CCR6⁻CD4⁺ CD25⁺ T_regP, Foxp3^lo T_regP and mature T_reg from OTIIxRag2^−/− and RipmOVAxOTIIxRag2^−/− mice (n = 5 from two independent experiments). b T_reg conversion assay: OVA_323-339-loaded mTEC were co-cultured with CD69⁺ semi-mature CD4⁺ thymocytes from OTII or OTIIxLta^−/− mice. c LTα₁β₂ expression in CD69⁺ OTII CD4⁺ thymocytes compared to control (II ab). d Flow cytometry profiles and fold change in the frequencies of CD25⁺ and Foxp3^lo T_regP derived from CD69⁺ OTII or OTIIxLta^−/− CD4⁺ thymocytes after 18 h of co-culture (n = 16 from two independent experiments). e T_reg conversion assay: OVA_323-339-loaded mTEC were co-cultured with CD69⁺ semi-mature CD4⁺ thymocytes pretreated or not with the blocking LTβR-Fc receptor. f Flow cytometry profiles and fold change in the frequencies of CD25⁺ and Foxp3^lo T_regP derived from CD69⁺ OTII CD4⁺ thymocytes pretreated or not with the blocking LTβR-Fc receptor after 18 h of co-culture (n = 9 from two independent experiments). g, h Flow cytometry profiles and fold change in the frequencies of CD25⁺Foxp3⁺ mature T_reg derived from CD69⁺ OTII (n = 10) or OTIIxLta^−/− (n = 9) CD4⁺ thymocytes co-cultured with OVA_323-339-loaded mTEC (g), mTEC^lo or mTEC^hi (n = 5 for OTII and OTIIxLta^−/−) (h) after 5 days. Data are pooled from three (g) and two (h) independent experiments. i Flow cytometry profiles and fold change in the frequencies of CD25⁺Foxp3⁺ mature T_reg derived from CD69⁺ OTII CD4⁺ thymocytes pretreated (n = 7) or not (n = 8) with the blocking LTβR-Fc receptor after 5 days of co-culture. Data are pooled from two independent experiments. j T_reg conversion assay: OVA_323-339-loaded mTEC were co-cultured with CD25⁺ T_regP or Foxp3^lo T_regP from RipmOVAxOTIIxFoxp3^eGFP mice. T_regP were pretreated or not with the blocking LTβR-Fc receptor. k, l Flow cytometry profiles (k) and fold change in the frequencies of mature T_reg (l) derived from the conversion of pretreated or not CD25⁺ T_regP or Foxp3^lo T_regP after 72 h or 24 h of co-culture, respectively (n = 7 from two independent experiments). m Relative expression of Il4 and Rela in co-cultures of OVA_323-339-loaded mTEC and CD69⁺ semi-mature OTII or OTIIxLta^−/− CD4⁺ thymocytes (n = 9 from two independent experiments). n Fold change in the frequencies of CD25⁺Foxp3⁺ mature T_reg derived from CD69⁺ OTII or OTIIxLta^−/− CD4⁺ thymocytes co-cultured with OVA_323-339-loaded mTEC with (n = 6) or without (n = 10) a neutralizing anti-IL4 antibody. Data are pooled from two independent experiments. Error bars show mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 using unpaired two-tailed Mann–Whitney U test for (a), (g–i) and (l), unpaired two-tailed Student’s t test for (d), (f) and (m) and Kruskal–Wallis test for (n). Source data are provided as a Source Data File.