1 methyl d tryptophan 1 mt

To assess the effect of drugs on CAR T cell cytotoxicity, cancer cells were plated at 5,000/well concentration in 96 well plates in triplicate. Next day, cells were treated with drugs (1-Methyl-d-tryptophan (1-MT, #452483, Sigma), indomethacin (#17378, Sigma), gemcitabine hydrochloride (#G6423, Sigma), paclitaxel (Taxol equivalent, #P3456, Invitrogen) and 5-Fluorouracil (#F6627, Sigma)) at indicated concentrations for 24 h. After 24 h, the drug was removed, cells were washed and further incubated with mock or CAR T cells at T:E ratio of 1:10 for 72 h. Thereafter, the percentage of surviving cancer cells were measured using the MTT assay. For Gal-9 and PD-1 blocking experiments, cancer cells were plated at 10,000 cells/well concentration in 96 well plates in triplicate. After 24 h, mock or CAR T cells were added at T:E ratio of 1:10. 0.1, 1, and 10 μg/mL of Gal-9 (clone 9M1-3, BioLegend, San Diego, CA, USA) or PD-1 blocking Ab (clone EH12.2H7, BioLegend) was added to the media and incubated at 37 °C for 72 h. Survival was measured using the MTT assay.

Fluoxetine hydrochloride (FLX) was obtained from Sequoia Research Products Ltd (UK). The IDO inhibitor 1-methyl-D-tryptophan (1-MT) was purchased from Sigma-Aldrich and dissolved in saline (NaCl 0.9%) containing 10% dimethyl sulfoxide. Vehicle, FLX (15mg/kg/day) and 1-MT (70mg/kg/day) were administered intraperitoneally (i.p.), based on previous experiments [35 (link)] and literature data [36 (link)]. Both drugs were injected every day (1pm) from the second week of UCMS till the end of the experiment.

Mice were irradiated by 2.5 cGy TBI using a ¹³⁷Cs source 2–4 h before PBMC injection. CD25-depleted PBMC (20 × 10⁶) were injected intravenously into mice via tail vein. Four hours later, PBS, GMSCs, human Fibroblasts, or nTregs were intravenously co-transfused. Mice were monitored every 2–3 days for weight loss and assessed for symptoms of GVHD. Blood samples were taken weekly to detect the percentages of human CD3⁺ T cells and cytokine production and Foxp3 expression among CD4⁺ cells. Serum samples were used to measure cytokines and antibodies by ELISA. At the terminal point of observation, mice were sacrificed for cytokine measurements and histologic analysis. To determine the underlying mechanisms, a tryptophan analog 1-Methyl-d-tryptophan (1-MT) (Sigma) was administered i.p. at 10 mg/mouse/day for 14 consecutive days. Additionally, GMSCs cells were pretreated with POM-1 (Tocris Bioscience; 100 µM) overnight before being injected into mice.