Lcmv gp33 41

Spleens were harvested from B6.Cg-Tcra^tm1MomTg(TcrLCMV)327Sdz/TacMmjax (P14), B6.Cg-Ptprc^aPepc^bTg(TcrLCMV)1Aox/PpmJ (SMARTA), or C57BL/6-Tg(TcraTcrb)1100Mjb/J (OT-1) transgenic mice (Jackson Labs) and mechanically dissociated into a single cell suspension using a 70μm nylon mesh strainer (Fisherbrand). All mouse experiments were conducted under institutional care and use committee approval. T cells were stimulated with 1μg/mL of appropriate viral peptide (LCMV gp_33-41, LCMV gp_61-80, or OVA_257-264, respectively) (GenScript; Anaspec). Cells were cultured in a 96-well plate for 8 days using RPMI complete medium (10% FBS, 1% PSG 100X) supplemented with 2.5x10^-5 μg/μL IL-2 (Gibco) and 100nM of indicated rexinoid treatment or ATRA in a final volume of 200μL. 8 day treatment timeframe was determined using a time course assay (Supplementary Figure 1). Fresh culture medium with IL-2 and rexinoid or ATRA was replaced every 48 hours. Vitamin A deficient media was made using charcoal-stripped FBS (ThermoFisher).

Cells culture was set up in DMEM containing 10% FBS and 5 × 10⁻⁵ M 2-Mercaptoethanol. To activate CD8⁺ T cells, naïve T cells (CD62L^hiCD44^loCD8⁺) were stimulated with anti-CD3 or anti-CD3/28 antibodies for 72 h and harvested. CD8⁺ T_eff used in this study were from naïve CD28WTCD8⁺ T cells stimulated by plate-bound anti-CD3/28 antibodies. To generate P14 CD8⁺ T_eff cells, activate P14 CD8⁺ T cells (1 × 10⁵ cell/mL) were activated by M2 antigenic peptide (LCMV gp33-41, KAVYNFATM, 0.08 nM, AnaSpec, Inc., San Jose, CA) presented by LPS-stimulated B6 B blast cells^{37 (link)} (mitomycin C-treated^{38 (link)}, 5 × 10⁵ cell/mL). At the end of the 3-day activation culture, the activated cells were washed and cultured in the presence of rhIL-2 (5 ng/mL) for 3–4 more days under 5% CO2.