Magnetic negative selection

Total CD4⁺ T cells were isolated from single-cell spleen suspensions using magnetic negative selection (eBioscience). Cells were stimulated at 37 °C, 5% CO₂ for 16 h in RPMI culture media supplemented with 10% fetal calf serum (Sigma), 1% penicillin and streptomycin (pen/strep; Gibco), and 50μM β-mercaptoethanol, non-essential amino acids (Gibco) and Glutamax (Gibco). Cells were plated at density of 1×10⁵ cells/well on 96-well plates in the presence or absence of 10ng/mL recombinant human IL-2 (Peprotech) with or without 300nM Tofacitinib (Sigma) or 10μM BI 605906 (Tocris). Where indicated, plates were coated overnight with 5 μg/mL anti-CD3 (clone 145.2C11; BioXcell Cat #BE0001-1) and 5 μg/mL anti-CD28 (clone 37.51 BioXcell Cat #BE0015-1) monoclonal antibodies in PBS before washing and plating of cells for stimulation. Cells were harvested and analysed by flow cytometry. To detect LPS-induced GARP expression on B cells, cells from erythrocyte-lysed blood were stimulated in RPMI complete media (RPMI, 10% FCS, 1% pen/strep, and 50μM β-mercaptoethanol) containing 0.5 mg/ml lipopolysaccharide (Sigma) at 37 °C, 5% CO₂ for 48 hr. At the end of the stimulation period, cells were collected and analysed using flow cytometry.

CD4⁺Foxp3^GFP+ T_reg and CD4⁺Foxp3^GFP– T_conv cells were sorted by FACS from WT and Enh-KO Foxp3^GFP reporter animals. Briefly, total CD4⁺ T cells were pre-enriched from single cell spleen suspensions using magnetic negative selection (eBioscience) prior to FACS-sorting of GFP⁺ and GFP^– CD4⁺ T cells using a BD Influx instrument (Becton Dickinson Biosciences). Cells were sorted into solutions of RPMI 1640 medium supplemented with 20% FBS and pellets were stored in RNAlater (Ambion) at -80°C. Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen) according to manufacturer instructions. Barcoded libraries were prepared using the SmartSeq2 protocol as previously described⁴ using a Hamilton NGS-STAR library preparation automation system at the BI Sequencing Facility and sequenced using a HiSeq 2500 (Illumina).
RNA-seq reads were trimmed using Trim Galore v0.4.4 using default parameters to remove the Nextera adapter sequence. Mapping was performed using HISAT2 v2.1.0 against the mouse NCBIM37 genome, guided by gene models from Ensembl annotation release 68. Aligned fragments were imported into SeqMonk (v1.44.0) and filtered to remove mappings with MAPQ scores of <20. Differential gene expression analysis was performed using the DESeq2 algorithm within SeqMonk.