Fig. 1

Foxp1 is required to maintain stable expression of Foxp3 in iTreg cells. a Intracellular staining of Foxp1 to confirm T-cell-specific deletion of Foxp1 in Foxp1^f/fCD4-Cre mice. b Expression of CD44 or CD62L in the indicated cell populations derived from lymph nodes of young and aged Foxp1^+/−CD4-Cre (red line) and Foxp1^f/fCD4-Cre (black line) mice. c Intracellular staining to confirm Treg-specific deletion of Foxp1 in the lymph nodes (top), thymus (middle), and thymic CD24^hiFoxp3⁺ and CD24^loFoxp3⁺ cells (bottom) of Foxp1^f/fFoxp3^IRES-YFP-Cre (KO) mice compared to littermate control Foxp1^+/+Foxp3^IRES-YFP-Cre mice (WT). d Representative FACS plots and quantification of percentage of in vivo iTreg conversion within CD45.2⁺ compartment, 2 months after adoptive transfer of Tnv cells isolated from WT or KO mice into RAG1^−/− host mice. e Foxp1 expression status within live CD4⁺Foxp3⁺ in vitro-generated iTreg cells originating from WT- or KO-derived Tnv cells after TGF-β treatment for the indicated number of days. IgG staining is shown as isotype control. f Status of Foxp3 expression within CD4⁺ live cell population in the same iTreg assay shown in e. g Mean fluorescent intensity (MFI) changes for Foxp3 and YFP-Cre in KO-derived iTreg cells relative to WT at the indicated time points. *P < 0.05, ***P < 0.0001 (Student’s t test, error bars, s.e.m). h WT- or KO-derived iTreg cells were sorted at high purity after 3 days of TGF-β treatment and co-transferred in RAG1^−/− hosts along with allelically marked CD4⁺ T cells derived from CD45.1⁺Foxp3^GFPKO mice. Representative FACS plots of Foxp3 expression within CD4⁺CD45.2⁺ compartment from lymph nodes of recipient mice at the indicated time points are shown. i Percentage of cells retaining Foxp3 (left) and its expression per cell depicted by MFI (right) after 3 days post-transfer. j Percentage of CD45.2⁺ cells recovered (left) and retaining Foxp3 (right), 7 days post-transfer. Each filled circle represents a single RAG1^−/− recipient mouse analyzed. Data are representative of 2–5 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Student’s t test, error bars, s.d.)