Fig. 2

IDO1-mediated KYN production from CR cells suppressed immunomodulatory profile. A Immunoblot of IDO1, IDO2, and TDO2. Resistant cells were treated with either IDO1 inhibitor or shRNA targeting IDO1 (shCTRL represents scramble sequence, shIDO A, B, and C represent 3 unique shRNA sequences). B Detection of amino acid KYN and TRP concentrations in culture supernatants using Amino Acid Analyzer Biochrome30 + . C Immune profile of CS vs. CR co-cultured with hPBMC. Using the same experimental condition as Fig. 1E above, IDO1 inhibitions significantly enhanced the percent of NKG2D on NK cells (CD3-CD56 + NKG2D +) and the percent of NKG2D on CD8 + (CD3 + CD8 + NKG2D +) cells, but significantly suppressed Treg (CD4 + CD25 + FoxP3 +) and MDSC (HLA-DR.^loCD14-CD11b + CD33 +) populations (see the gating strategy in Supplemental Figure S1). D The indicated cytokines are quantified in culture supernatants by LEGENDplex™ bead-based immunoassay. The panel below indicated that anti-NKG2D blocking antibodies blunt the effect of IDO1 inhibition. Note: To detect the MDSC population, cells were activated by PHA + IL2 instead of antiCD2/28 + IL2. Cell lines A and FA are cisplatin-sensitive, and ALC and FC are cisplatin-resistant counterparts. In all experiments, data presented as mean ± SEM of 3 independent experiments and were analyzed using one-way ANOVA followed by Tukey’s multiple comparison analysis with *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001