Generating Stem-like and Exhausted T Cells

For cell-tracing experiments, OTI cells from both WT or IFNγRKO were in vitro-treated to induce stem-like T cells or exhausted T cells. To obtain stem-like T cells, 2 × 10⁶ OTI/mL were incubated 24 h at 37 °C in RPMI 1640 media (Gibco) containing 10% fetal bovine serum (FBS) (Sigma), 0.5 μM beta-mercaptoethanol (Gibco) and Penicillin/Streptomycin (Gibco) (called R10 thereafter) supplemented with 10IU/ml of IL-2, 100 ng/mL of N4 peptide (SIINFEKL) and 7uM of TWS119 (Merk)^{48 (link)}. To obtain in vitro induced exhausted T cells, 500 000 OTI/mL were plated in R10 containing 1% HEPES (Gibco), 5 ng/mL IL-15 (Peprotech), 5 ng/mL IL-7 (Peprotech), and 10 ng/mL of N4 peptide (SIINFEKL). Cells were incubated at 37 C and 10 ng/mL of N4 peptide (SIINFEKL) were added daily for 5 days. Cells were split with fresh media when confluent and underwent Ficoll separation (Ficoll-paque Premium, Cytiva) prior to in vivo injection^{47 (link)}. Mice were adoptively transferred with 2 × 10⁶ OTI WT and IFNγRKO at 1:1 ratio by intravenous injections 6 days after B16-OVA cell engraftment.