L nmma

Recombinant murine and human IFN-γ and TNF-α were purchased from PeproTech (Rocky Hill, USA). Antibodies against β-actin, GAPDH, UCHL1, iNOS, NF-κB p65, Phospho-NF-κB p65, IκBα, histone H3, STAT1, Phospho-STAT1 (Tyr701), IDO, PARP, cleaved PARP, p53, Bcl-2, caspase 3, and cleaved caspase 3 were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA). LDN57444, L-NMMA, Fludarabine, PDTC were purchased from Selleckchem (Houston, USA). ConA, Griess reagents and 1-MT were from Sigma (St Louis, MO, USA). Annexin V/prodium iodide (PI) staining kit were purchased from Life Technologies GmbH (Darmstadt, Germany). C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and maintained under specific pathogen-free conditions in the vivarium of Shanghai Jiao Tong University School of Medicine. All animal procedures were approved by the Animal Welfare and Ethics Committee of Shanghai Jiao Tong University School of Medicine.

Recombinant murine IFN-γ and TNF-α were purchased from PeproTech (Rocky Hill, USA). Antibodies against β-actin, iNOS, NF-κB p65, phospho-NF-κB p65, IκBα, histone H3, STAT1, phospho-STAT1 (Tyr701), AKT, phospho-AKT (Ser473), phospho-AKT (Thr308), LAMP1, PPCA, and LAMP-2s (ABL93) were purchased from Cell Signaling Technology (Danvers, USA). Antibodies against LAMP-2A, HSPA8, NFAT1, and IKKα+β were purchased from Abcam (San Francisco, USA). L-NMMA, Fludarabine, MK2206, PDTC, Nifuroxazide, and LY294002 were purchased from Selleckchem (Houston, USA). BAY11-7082 was purchased from MedChemExpress (Shanghai, China). ConA, Griess reagents, and the lysosome isolation kit were from Sigma (St Louis, USA). C57BL/6 mice were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences and maintained under specific pathogen-free conditions in the vivarium of the Shanghai Institute of Nutrition and Health of the Chinese Academy of Sciences. All animal experiments were performed according to the guidance of the Institutional Animal Care and Use Committee of the Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, and complied with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health.

Splenocytes, which were used as responder cells, were derived from naive BALB/C mice and labeled with carboxyfluorescein succinimidyl ester (CFSE) (5 μM; Invitrogen). The CFSE-labeled splenocytes (2 × 10⁵ cells/well) were cocultured with purified G-MDSCs and M-MDSCs, and stimulated with anti-mouse CD28 (0.5 μg/ml, eBioscience) and anti-mouse CD3e (10 μg/ml, eBioscience) in a flat-bottomed 96-well plate. The ratio of splenocytes to MDSCs was 2:1. N^G-hydroxy-L-arginine (an inhibitor of arginase, Selleck Chemicals) or L-NMMA (an inhibitor of NOS activity, Selleck Chemicals) were added to the wells at a final concentration of 500 μM. After 3 days of co-culture, the proliferation of CD4⁺ T cells was evaluated by flow cytometry and analyzed as follows:
T-cell inhibition (%) = (1-proliferation rate with MDSC/proliferation rate without MDSC) × 100%