Tritiated thymidine

For the proliferation assay with conditioned medium, DCs were isolated by FACS sorting and 2.5‐3 × 10⁴ DCs were cultured for 48 hours in 100 μL complete RPMI medium with 10 μM L‐tryptophan, indicated stimuli and with or without 10 μM epacadostat. DCs from three different donors were pooled at the same ratio for each subset to obtain a sufficient number of cells and to even out donor variation. After 48 h, 75 μL of this conditioned medium was added to 75 μL complete RPMI medium containing 1.5 × 10⁵ PBLs and 3 × 10⁴ anti‐CD3/CD28 beads (Gibco). After 3 days, proliferation was assessed by adding 1 μCi [0.037 MBq]/well of tritiated thymidine (MP Biomedicals, Irvine, CA) to the cells. Tritium incorporation over 16 h was measured with a scintillation counter. All experiments were performed in triplicate for cDC2s and pDCs and in duplicate for cDC1s.
For classical MLR assays, DCs were isolated by FACS sorting and 2.5‐4 × 10⁴ DCs (always the same numbers across subsets) were cultured for 16 h in 100 μL complete RMPI medium with 10 μM L‐tryptophan, indicated stimuli and with or without 10 μM epacadostat. Then, 1.5 × 10⁵ PBLs were added and incubated with the DCs for 3 days. Proliferation was assessed by tritiated thymidine incorporation as above. All experiments were performed in triplicate for cDC2s and pDCs and in duplicate for cDC1s.