Fig. 4

AS-derived M-MDSCs suppress T cell responses in an arginase-I dependent manner. a ROS levels in M-MDSCs from patients with ankylosing spondylitis or healthy controls (n = 6) were measured by flow cytometric analysis (left). HLA-DR^–/lowCD11b^intCD33^int cells were first gated, and the percentage of CM-H₂DCFDA⁺ cells is shown. Both representative results (left) and means ± SEM from three independent experiments (right) are included. b NO content in plasma in sorted M-MDSCs from ankylosing spondylitis patients and healthy controls. c Arginase activity in M-MDSCs from healthy controls (n = 6) and from patients at ankylosing spondylitis. d Effects of different inhibitors on the suppressive function of M-MDSCs from ankylosing spondylitis patients were evaluated by allogeneic mixed lymphocytes reaction. T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with anti-CD3/anti-CD28 (5 ng/ml). These cells were then co-cultured with M-MDSCs from the same donor at a 2:1 ratio with treatments as indicated for 3 days, and T cell proliferation was evaluated by a flow cytometry. N-hydroxy-L-arginine (NOHA) (an arginase inhibitor, 100 mM); L-NG-monomethyl-L-arginine [L-NMMA, an inducible nitric oxide synthase (iNOS) inhibitor, 100 mM]; N-acetylcysteine (NAC, a ROS inhibitor, 1 mM). e Production of interferon (IFN)-γ by T cell supernatants from (d) was measured by enzyme-linked immunosorbent assay (ELISA). f p-STAT3 levels in M-MDSCs from ankylosing spondylitis patients (n = 11) and the control population from healthy donors (n = 11) were measured by flow cytometric analysis. AS ankylosing spondylitis, CFSEcarboxyfluorescein succinimidyl ester, IFN interferon, L-NMMA L-NG-monomethyl-L-arginine, M-MDSCs monocytic myeloid-derived suppressor cells, NAC N-acetylcysteine, NOHA N-hydroxy-nor-L-arginine