Potential receptor mechanism underlying the effect of high mobility group box-1 protein on expression of cytotoxic T lymphocyte-associated antigen 4 in regulatory T cells in mice

Abstract

Objective To investigate the effect of high mobility group box-1 protein (HMGBI)on the expression of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) in regulatory T cells (Treg)and its potential receptor mechanism in mice.Methods CD4+ CD25+ Tregs isolated from the spleens of C3H/HeN [ Toll-like receptor 4 (TLR4) wild type ] mice by magnetic beads were seeded on 96-well cell culture plates coated with 1 mg/L anti-CD3 and soluble CD28.By treatment with or without anti-mouse TLR4 antibody,the time-and dose-dependent responses between HMGBI stimulation and CTLA-4 expres-sion on CD4+ CD25+ Tregs were studied.Results Compared to normal controls, the expression of CTLA-4 on CD4+ CD25+ Tregs stimulated by 0.1 mg/L HMGBI was significantly down-regulated at 24,48,and 72 h (47.56±5.49,35.83±4.96, and 31.94±4.65, respectively) (all P<0.01), especially at 48 and 72 h.Meanwhile, markedly decreased expression of CTLA-4 was found CD4+ CD25+ Tregs treated with HMGB1 at various concentrations (0.01,0.1 and 1 mg/L) for 48 h (58.87±6.70,33.34±4.00 and 29.90±4.30, respectively, all P<0.01).After pretreatment with TLR4 antibody, however, the CTLA-4 expression on CD4+ CD25+ Tregs was significantly enhanced by HMGB1 (0.1 Mg/L) stimulation at 24,48 and 72H(90.39±8.80,93.13±4.80 and 80.and 80.29±4.31,respectively,P<0.05 or P,0.01).After treatment with different doses of HMGB1 for 48 h,the CTLA-4 expression on CD4+CD25+Tregs was much higher in 0.1 mg/L HMGB1 group(94.98±6.35)than in mormal controls(P<0.01).Conclusion With HMGB1 stimulation,TLR4 can markedly down-regulate CTLA-a expression levels on CD4+CD25+Tregs,thereby influencing the immunlolgical activity of Tregs. Key words: High mobility group box 1 protein; Toll like reecreptor; Regulatory T cells