MUC1/sec and its unique IEP block induction of arginase in MDSCs. (A) Splenic MDSCs from DA-3/TM tumor-bearing mice were purified and cultured in vitro in the presence of 20% CM from DA-3/TM and/or DA-3/sec tumor cell cultures for 24 hours. MDSC lysates were then analyzed by Western blot for the enzyme arginase I and β-actin. Relative density of arginase to β-actin is presented. (B) Splenic MDSCs from DA-3/TM tumor-bearing mice were cultured as in panel A, and after 48 hours MDSC lysates were obtained and an arginase assay was performed to detect levels of arginase. Urea is measured as a by-product of exogenous arginine hydrolysis. IL-4 was used (100 ng/mL) as a positive control for induction of arginase in MDSCs. (C) MDSCs were cultured and assayed for the presence of arginase as in panel B, with the addition to the cultures of 10 μg/mL of the unique MUC1/sec peptide (MAP-IEP), or as a control a scrambled peptide sequence (MAP-SCR). (D) Splenic MDSCs were cultured as in panel B with IL-4 and DA-3/sec CM, or DA-3/sec CM separated using a 100-kDa filter into a MUC1/sec-containing fraction (sec > 100 K), and a fraction lacking MUC1/sec (sec < 100 K). Antibody specific for IEP (aIEP) or a control IgY was also added to MDSC cultures, and an arginase assay was performed. Error bars representing SEM are provided, and data are representative of at least 3 experiments.