Generation of Antigen-Specific T Cell Retrogenic Mice

SP33rg mice were generated as described previously (Leonard et al., 2017 (link); McDonald et al., 2015 (link)). In brief, the SP33 TCRα was cloned into a retroviral construct modified from Turner and colleagues (McDonald et al., 2015 (link); Turner et al., 2010 (link)). Plat-E cells, also previously described (Morita et al., 2000 (link)), were used to generate retrovirus. TCRα^−/− CD4-Cre⁺ TCRβtg⁺ mice on a B6 background were injected with 5-fluorouracil (APP Pharmaceuticals) 3 d before bone marrow harvest. Bone marrow cells were cultured for 2 d in X-VIVO 10 (Lonza) containing 15% FCS, 1% pen/strep, 100 ng/ml mouse stem cell factor, 10 ng/ml mouse IL-3, and 20 ng/ml mouse IL-6 (BioLegend). Cells were infected with retrovirus by spinfection in the presence of 6 µg/ml polybrene (EMD Millipore) and cultured for an additional 24 h. All spinfected cells were then mixed with 5 × 10⁶ freshly harvested bone marrow ‘‘filler’’ cells from Rag1^−/− mice and injected into lethally irradiated (900 rads) CD45^.1/.1B6.SJL recipient mice to generate SP33rg mice. SP33rg cells were isolated from retrogenic mice 6–8 wk after bone marrow reconstitution. CD4⁺ T cells were FACS–purified from SP33rg mice following CD4 MACS (Miltenyi Biotec) enrichment and staining with the following antibodies: anti-CD8b (Ly-3), anti-CD45.1 (A20), anti-CD45.2 (104), and anti-Thy1.1 (OX-7).