Tws119

Sorted T cells were washed once with CM supplemented with 100 mM Imidazole and once with CM, before being plated in pre-warmed CM supplemented with 100 ng/mL IL15. Cells were plated in wells of either 96- or 48-well-plates, and plates were incubated at 37 °C, 5% CO₂ for 5 days (tilt for the first 12 h). T cells were activated at day 5 by adding peptide-pulsed DCs (1:5 to 1:1 DCs-to-T cells) suspended in CM supplemented with IL15. DCs were thawed the night before activation, let rest in R10 for 12–14 h and activated with 1 μg/mL LPS or poly I:C for 6 h. During the last hour, DCs were pulsed with 1–5 μM AH1 peptide. Before adding them to T cells, DCs were washed in CM. One-half of the medium was replaced every 3–4 days. T cells were re-activated at day 15, and 10 ng/mL IL7 was added to culture medium from day 15 onwards. One-half of the medium was replaced every 3–4 days, and cells were split in additional wells whenever confluent. T cells were harvested and used for analysis and therapy between days 21 and 24. In some experiments, T cells were expanded in the presence of either 0.5 μM Doramapimod (Cayman Chemical), 7 μM TWS119 (Sigma) or 2.5 μg/mL mDLL1-Fc (R&D Systems).

Obeticholic acid (OCA, SML3096, Sigma-Aldrich, St Louis, MO, USA), CHIR-99021 (Selleck Chemicals, Houston, TX, USA), CHIR-98014 (Selleck Chemicals), GW4064 (G5172, Sigma-Aldrich), SB-216763 (S3442, Sigma-Aldrich), TWS-119 (SML1271, Sigma-Aldrich), Tideglusib (SML0339, Sigma-Aldrich), SB-415286 (S3567, Sigma-Aldrich), and nanaomycin A(sc-396527, Selleckchem) were used in the study.

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.

TILs were isolated from tumor-bearing WT mice, enriched using Ficoll, and labeled with 2uM of CFSE. Between 2 × 10⁵ to 2 × 10⁶ cells were resuspended in complete RPMI supplemented with 2 ug/mL of anti-CD28 (Biolegend-37.51) and with or without blocking IFNγ antibody (BioXcell XMG1.2 at 10 ug/mL) or recombinant mouse IFNγ (Biolegend at 100 ng/mL). Cells were then plated at a similar density in 96-well or 12-well plates previously coated with 2 ug/mL of anti-CD3 (Biolegend – 145-2C11) in PBS for 2 h at 37 °C. Cells were incubated at 37 °C and were fed every 2 days with fresh media according to the different conditions. At the desired time points cells were harvested and processed for flow cytometry.
In some experiments, naïve T cells were stimulated by 2 µg/ml coated anti-CD3 and 2 µg/ml soluble anti-CD28 with or without blocking IFNγ antibody as above and treated with 3.5 µM of the inhibitor TWS119 (Merk) when indicated. IL-2 at 10IU/ml was added to the culture two days after priming.