LMP1-mediated glycolysis induces myeloid-derived suppressor cell expansion in nasopharyngeal carcinoma
Fig 3
LMP1 promoted NPC-induced MDSC differentiation and the expression of MDSC-related molecules in NPC cells.
CD33+ cells were isolated from healthy PBMCs using human CD33 MicroBeads and were co-cultured with NPC or NPC-LMP1 cells in a Transwell system for 48 h. The percentage of CD33+CD11b+HLA-DR- MDSCs was measured by FACS staining. (A) Expression of the NLRP3, ASC, caspase-1, IL-1β, COX-2, Arg-1 and iNOS mRNAs was determined via real-time qRT-PCR, and the results were normalized against GAPDH expression. (B) NPC cells, including CNE2-vector, CNE2-LMP1, TW03-vector, and TW03-LMP1 cells, were cultured overnight following stimulation with LPS (0.1 μg/mL) and ATP (5 mM, 30 min) or no stimulation. Then, the culture supernatants were collected, and the concentrations of IL-1β, IL-6 and GM-CSF were measured by ELISA. (C) Representative density plots are shown as the CD33+CD11b+cells in the HLA-DR- gate induced by NPC or NPC-LMP1 cells in different combinations. Representative data from 1 of 5 experiments are presented. (D) Statistical analysis of the percentage of MDSCs mediated by NPC cells in different combinations. CFSE-labeled PBMCs were co-cultured with M-MDSCs, CNE2-vector-MDSCs, CNE2-LMP1-MDSCs, TW03-vector-MDSCs or TW03-LMP1-MDSCs at a ratio of 1:1 in OKT3-coated 96-well plates. After 3 days, the cells were collected, stained with anti-human mAbs against CD4 and CD8 and quantified by flow cytometry. Representative FACS density plots (E) and the statistical data (F) showed that the proliferation of PBMCs and CD4+ and CD8+ T cells was markedly suppressed by CNE2-LMP1-MDSCs or TW03-LMP1-MDSCs compared with the effects of CNE2-vector-MDSCs or TW03-vector-MDSCs. Data are presented as the means ± SEM of representative experiments performed in triplicate. *P < 0.05, **P < 0.01 compared with the control treatment.