In vitro activity: Indoximod significantly reverses the suppression of T cells created by IDO-expressing dendritic cells, using both human monocyte-derived dendritic cells and murine dendritic cells isolated directly from tumor-draining lymph nodes. Indoximod activates a Trp sufficiency signal that stimulates mTOR, and relieves IDO-induced Trp deprivation as well as an autophagic response.

Kinase Assay: 1MT enantiomers are solubilized in DMSO containing 0.1N HCl and added at concentrations of 100, 50, and 0 μM to wells containing the reaction mixture with tryptophan (0-200 μM) followed by addition of IDO enzyme. Plates are sealed and incubated 1 hr in a humidified 37°C incubator, after which the reactions are terminated by addition of 12.5 μl 30% TCA per well. Plates are then resealed in plastic wrap, incubated 30 min at 50°C to hydrolyze the reaction product N-formylkynurenine to kynurenine, and centrifuged. Supernatants are transferred to a flat-bottom 96-well plate, mixed with 100 μl Ehrlich reagent and incubated 10 min at room temperature. Absorbance at 490 nm is read.

Cell Assay: The immunosuppressive activity of IDO leads to an increase in the number of T-regulatory cells, as measured by their Foxp3+/CD4+/CD25+ phenotype. Indoximod has also been shown to reduce the number of T-regulatory cells.