MDSCs inhibit T-cell alloresponses through the expression of arginase-1. (A) RT-PCR showing that MDSCs express arginase-1 and iNOS and up-regulate arginase-1 on stimulation with IL-13. Hypoxanthine phosphoribosyl transferase shown as endogenous control. M indicates marker. (B) Real-time RT-PCR showing quantitatively the extent of arginase-1 and iNOS up-regulation by IL-13-stimulated MDSCs. Standardized to glyceraldehyde-3-phosphate dehydrogenase endogenous control and relative expression compared with untreated BM. (C) Western blot showing arginase-1 and actin control at the protein level in untreated BM, MDSCs, and MDSC IL-13. (D) Arginase activity was determined by measuring the production of urea over time. MLR was performed by mixing B6 purified T cells with irradiated BALB/c stimulators (1:1 ratio) and MDSCs (1:10 ratio). Cultures were pulsed with ³H-thymidine on the indicated days and harvested after a 16-hour incubation. (E) MLR/MDSC cocultures were left untreated or were treated with arginase inhibitor, nor-NOHA (300μM), nitric oxide inhibitor, L-NMMA (300μM), or both. (F) Arginase inhibitor (nor-NOHA) was added to MLR/MDSC cocultures at increasing concentrations (30, 100, and 300μM). (G) B6 WT T cells or GCN2 KO T cells were used as responder cells, and MDSCs were added at 1:10 ratio. (H) Addition of excess L-arginine (5mM) or tryptophan (5mM) was added back to MLR/MDSC cocultures and assessed for T-cell proliferation.