Biomag

Lymph nodes were harvested and gently tweezed to remove lymphocytes. CD8 lymph node T cells were enriched for via antibody-mediated depletion of B cells using anti-mouse IgG magnetic beads (BioMag, Qiagen). CD4 T cells were removed via anti-rat IgG magnetic beads (BioMag, Qiagen) following incubation with anti-mouse CD4 antibodies conjugated with rat IgG (GK1.5). Lymphocytes were electronically sorted for the further purification of naive CD8 T cells (CD44^lo CD25^loCD8⁺CD4^-) (Figure 3—figure supplement 1A).
Cells were stimulated either with irradiated splenocytes loaded with anti-CD3 mAbs (10 μg/mL), or plate-bound anti-TCRβ mAbs (10 μg/mL) and anti-CD28 mAbs (5 μg/mL), then differentiated for 5 days in RPMI supplemented with 10% fetal bovine serum, 1% HEPES, 1% sodium pyruvate, 1% penicillin/streptomycin, 1% L-glutamine, 1% non-essential amino acids, 0.3% β-mercaptoethanol, 100 U/mL IL-2, 100 mg/mL gentamicin, and 2 μg/mL doxycycline when necessary.

Different T cell populations were identified by flow cytometry using LSRII or LSRFortessa (BD Biosciences). Specifically, the double negative (DN) thymocytes were first stained with biotinylated antibodies against γδTCR, CD45R/B220, NK1.1, and TCRβ, washed with MACS buffer (PBS with 0.5% BSA, 1 mM EDTA) and then incubated with Strepatavidin Microbeads, CD4 (L3T4) Microbeads and CD8 (Ly-2) Microbeads (Miltenyi Biotec). Negatively selected thymocytes were then stained with anti-CD4, CD8, and CD69 monoclonal antibodies and cell sorted using FACSAria II (BD Biosciences). Pre-selection unsignaled DP thymocytes were sorted based on CD4⁺, CD8α⁺, and CD69⁻ gates from B6 or MHC-KO animals. For purification of naïve T cells, lymph node (LN) T cells were first depleted of MHC-II⁺ and Ig⁺ B cells by antibody-mediated magnetic beads depletion (Biomag, Qiagen) and then FACS sorted for γδTCR⁻/TCRβ⁺, and CD44⁻ /CD62L⁺ populations.

Single-cell suspensions were generated from spleens, and CD8 or CD4 T cells purified by negative selection using magnetic beads (Dynabeads, Invitrogen or BioMag, Qiagen). Enrichment of cells was verified by flow cytometry. Purified T cells were labelled with Cell Trace Violet (CTV) (Molecular Probes, OR, USA) according to the manufacturer's instructions. In vitro stimulation assays were performed by plating cells at 10⁴ cells per well in RPMI 1640 medium containing 10% (v/v) heat-inactivated FCS (Sigma-Aldrich, MO, USA), 5×10⁻⁵ M 2-ME (Sigma-Aldrich), 100 µg/mL streptomycin and 100 U/mL penicillin (Invitrogen Life Technologies) into anti-CD3-coated (10 µg/mL, clone KT3-1-1) 96-well plates. Recombinant mouse IL-2 (20 ng/mL) was also added to wells. Cells were harvested at different timepoints, propidium iodide (2 µg/mL) and 5000 of Sphero Nile Red Fluorescent Particles (BD Biosciences) added per well and cell division analysed by flow cytometry. Each sample was analysed in duplicate.