Figure 7. IDO-activated Tregs in TDLNs.

(A) Tumors were grown in wild-type or IDO-KO hosts. Tregs from day 7 TDLNs were sorted and added to readout assays (A1 T cells + CBA DCs) with and without PD-1/PD-L blocking antibodies. Mean ± SD of 4 pooled experiments with B78H1–GM-CSF, 4 experiments with B16-OVA, and 3 experiments with IDO-KO hosts (2 with B78H1–GM-CSF and 1 with B16-OVA). (B) Wild-type mice were treated throughout tumor growth with vehicle control or sustained-release 1MT. Tregs from day 7 tumors were tested in readout assays as described above with added isotype, PD-1/PD-L–blocking antibodies, or a combination of anti–PD-1/PD-L plus IL-2 plus anti–IL-10/TGF-β antibodies. One of 3 experiments using B78H1–GM-CSF and B16-OVA. (C) Upper panels: CFSE-labeled OT-I were injected into mice with B16-OVA tumors (days 7–8) with and without oral 1MT administration after transfer. After 4 days, TDLNs and contralateral LNs (CLN) were stained for the 1B11 activation marker. Percentages show the CFSE⁺ OT-I in total LN cells. Histogram shows 1B11 on OT-I in TDLNs. Representative of 4 transfers each. Lower panels: Similar experiments as described above using OT-I^GCN2-KO cells transferred into WT or GCN2-KO hosts bearing B16-OVA tumors. One of 3 similar experiments. (D) B78H1–GM-CSF tumors were treated on day 11 with vehicle (control), cyclophosphamide (CY; 150 mg/kg), or cyclophosphamide plus 1MT pellets. Seven days later cells from TDLNs were harvested and added to readout assays (allospecific BM3 T cells plus B6 splenocytes, as described in ref. 1). One group in each readout assay also received 1MT added during the assay, as shown on the last bar of each graph. One of 3 experiments.