(A) Loss of ARG1 and NOS2 activity in α-GalCer–pulsed MDSCs incubated with BM-derived iNKT cells. MDSCs were treated with α-GalCer in the presence of iNKT cells and collected at different time points. ARG1 and NOS2 activities were measured using a colorimetric assay to evaluate urea and nitrate/nitrite release, respectively (17, 18). An ELISA was used to evaluate IL-12p40 production. Values are shown as a percentage relative to time point 0. The maximum amount of urea released by untreated MDSCs (1 × 10⁶) was 91.7 μg. The maximum amount of NO₂^– and NO₃^– measured in the supernatant of untreated MDSCs was 120 μM. The maximum amount of IL-12p40 was 200 ng/ml. (B) Incubating α-GalCer–pulsed MDSCs with BM-derived iNKT cells abolishes their suppressive function. CFSE-labeled OT-I cell proliferation was analyzed in the presence (red) or absence (green) of MDSCs derived from WT, Jα18^–/–, or CD1d^–/– mice. MDSCs were left untreated or pulsed with α-GalCer. Proliferation of OT-I cells without SIINFEKL peptide is superimposed in all panels (black). (C) The effect of iNKT cells on α-GalCer–pulsed MDSCs is CD40 dependent. CFSE-labeled OT-I proliferation was analyzed in the presence (red) or absence (green) of MDSCs derived from WT, CD40^–/–, or CD40L^–/– mice. MDSCs were left untreated or pulsed with α-GalCer. To assess the role of the CD40 molecules, BM-derived MDSCs were treated with anti–CD40 agonist Ab for 48 hours. Proliferation of CFSE-labeled OT-I cells without SIINFEKL peptide is superimposed in all panels (black).