Anti 1 a 1 e m5 114

Thymic epithelial cells were acquired as described above. Cells were incubated with 2.4G2 before staining with other antibodies. The antibodies used included anti-CD45 (30-F11, eBioscience), anti-EpCAM (G8.8, Biolegend), anti-Ly51 (6C3, Biolegend), FITC labelled Ulex europaeus agglutinin-1 (UEA-1; Vector Laboratory), anti-IA/IE (M5/114.15.2, Biolegend), anti-SSEA-1 (MC-480, Biolegend), anti-LTβR (eBio3C8, eBioscience). C-CPE (C. perfringens enterotoxin) was produced as described47 (link) and biotin-conjugated; Streptavidin APC-eFluor 780 (eBioscience) was used for visualization of C-CPE. For intracellular staining of Ki-67 (B56, BD) and active caspase 3 (C92-605, BD), cells were fixed and permeabilzated with BD Cytofix/Cytoperm™ Fixation/Permeabilization Solution Kit (554714) and stained according to the manufacture’s protocols. Fixable viability dye (L-34967, ThermoFisher) was used to exclude dead cells. For thymocyte and splenocyte analysis, cells were stained with anti-CD4 (RM4-5, eBioscience), anti-CD8 (53-6.7, eBioscience), anti-Va2 (B20.1, eBioscience), anti-Vb5 (MR9-4, eBioscience), and anti-CD24 (M1/69, Biolegend) before flow cytometry analysis. The samples were analyzed on BD LSRFortessa and FlowJo software (Tree Star Inc).

Tissues were excised from mice, minced and digested in Collagenase for 30 min. The samples were filtered through a 40-μm filter. Erythrocytes were removed with Lysing buffer (BD Biosciences). The samples were then incubated for 10 min with anti-CD16/CD32 blocking antibodies (BD Biosciences). The cells were stained with the following antibodies: anti-CD206 (C068C2, BioLegend), anti-CD11c (HL3, BD Biosciences), anti-CD11b (M1/70, BioLegend), anti-CD45 (30-F11, eBioscience), anti-F4/80 (CI: A3-1, BD Bioscience), anti-I-A/I-E (M5/114.15.2, BioLegend), anti-CD8a (53-6.7, BioLegend), anti-CD4 (GK1.5, BioLegend), anti-TCRβ (H57-597, BioLegend), anti-PD-1 (29F.1A12, BioLegend), and anti-CD44 (IM7, BioLegend). The samples were washed, incubated with 7-amino-actinomycin D (BD Biosciences) and then analysed on a FACSAria II (BD Biosciences).

Antibodies used for flow cytometry analysis of surface marker expression on BMDCs are the following: anti-CD11c (N418); anti-CD40 (1C10); anti-CD80 (16-10A1); anti-CD86 (GL1); and anti-I-A/I-E (M5/114.15.2) (all eBioscience). Antibodies used for flow cytometry analysis of cells from mediastinal LNs are the following: anti-CD19 (eBio1D3); anti-F4/80 (BM8) (eBioscience); anti-CD3ε (145-2C11); anti-CD45 (30-F11); anti-CD11c (N418); and anti-I-A/I-E (M5/114.15.2) (BioLegend). For staining of intracellular protein iNOS (C-11) (Santa Cruz Biotechnology), samples were first fixed with IC Fixation Buffer (eBioscience) and stained in Permeabilization Buffer (eBioscience). Samples were acquired on the BD LSR Fortessa and data analyzed using FlowJo software.