Thymocyte

Animal work was performed according the Animals (Scientific Procedures) Act, UK. Dicer^lox/lox mice were crossed with LckCre transgenic mice (17 (link)) to generate lckCre Dicer^Δ/Δ mice. Thymocytes were stained, analyzed, and sorted by flow cytometry as described previously (24 (link)). Where indicated, thymocytes were incubated with 40 nM DiOC6 (Molecular Probes) for 10 min at 37°C as described previously (20 (link)). To down-regulate Tdt expression, DP thymocytes were cultured with 7.5 ng/ml PMA (Sigma-Aldrich) and 180 ng/ml ionomycin (Sigma-Aldrich) as described previously (28 (link)).

Processed thymic B cells were stained with biotinylated anti-CD4 and anti-CD8a antibodies, and thymocytes were depleted using anti-Biotin microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. CD4/CD8a⁻ thymocytes were surface stained with indicated antibodies, and CD19⁺B220⁺ IgM⁻IgD⁻ were isolated using a FACSAria (BD Biosciences). The RNeasy Micro Kit (Qiagen) was used to isolate RNA obtained from each sample per the manufacturer’s instructions.

DN, DP, CD8SP, CD4SP, CD25⁺ Treg precursor, Foxp3^lo Treg precursor, thymic Treg cells were isolated by FACS sorting from a thymocyte suspension. Tcon and Treg cells were isolated from the spleen by pre-enrichment with EasySep Mouse CD4+ T cell Isolation Kit (STEMCELL Technologies, Cat# 19852), and FACS sorting. The individual cell population was sorted by FACS using the following markers. DN: CD45⁺CD4⁻CD8⁻; DP:CD45⁺CD4⁺CD8⁺; CD8SP: CD45⁺CD4⁻CD8⁺; CD4SP: CD45⁺CD4⁺CD8⁻CD25⁻ Foxp3-reporter⁻; CD25⁺ Treg precursor: CD45.2⁺CD4⁺CD8⁻CD25⁺Foxp3-reporter⁻; Foxp3^lo Treg precursor: CD45⁺CD4⁺CD8⁻CD25⁻Foxp3-reporter^low; mature thymic Treg: CD45⁺CD4⁺CD8⁻CD25⁺Foxp3-reporter⁺; Splenic Tcon cells: CD45⁺TCRb⁺CD4⁺CD8⁻ Foxp3-reporter⁻ ; splenic Treg cells: CD45⁺ TCRb⁺CD4⁺CD8⁻Foxp3-reporter (Thy1.1 or GFP)⁺.
To analyze immune cell compositions in Foxp3 DSM mice, a single cell suspension was prepared from the spleen or lymph nodes, treated with red cell lysis buffer, and filtered through 70 μm cell strainer. For transcription factor staining, cells were first stained for surface markers, followed by fixation and permeabilization with reagents from the Foxp3/Transcription Factor Staining Buffer Set (eBioscience, 00-5521-00) and incubated with antibodies according to the manufacturer’s protocol. For cytokine analysis, cells were stimulated with phorbol 12-myristate 13-acetate (PMA) (50 ng ml⁻¹; Sigma), ionomycin (500 ng ml⁻¹; Sigma) and GolgiStop (BD) for 5 hours. Cells were incubated with cell surface antibodies on ice for 30 min, and then subjected to intracellular staining using Foxp3/Transcription Factor Staining Buffer Set as described above. Samples were run on a BD FACSAria II Flow Cytometer (Becton Dickinson) and data were analyzed by FlowJo software (Tree Star).