| ObjectiveCytotoxic T-Lymphocyte Antigen-4 (CTLA-4) is one of the critical inhibitory regulators of early stages of T cell activation and proliferation which opposes the actions of CD28-mediated co-stimulation. Anti-CTLA-4 therapy has been effective clinically in enhancing immunity and improving survival in patients with metastatic cancer. Sepsis is a lethal condition which shares many of the same mechanisms of immune suppression with cancer. Given the similarities in immune defects in cancer and sepsis, we examined the ability of anti-CTLA-4 antibody to block apoptosis, reverse the immunosuppression of sepsis, and improve survival in the cecal ligation and puncture (CLP) model in mice.Measurements1.CD1 mice were randomly divided into sham group (n = 16), 1 d (n = 10), 4 d (n = 16), 7 d (n = 20) after surgery. Mice of each group were scarified at corresponding time points. Spleen was harvested and stained with CTLA-4, CD4, CD8, CD25, and/or Foxp3. The expressions of CTLA-4 on CD4~+, CD8~+, and CD4~+CD25~+ Foxp3~+ T cells were examined by flow cytometry.2.CLP model of sepsis and 2-hit model of sepsis, CLP followed by Candida Albicans were constructed. 36 CD1 mice were randomly divided into 2 groups, CLP group (n=18) and control group (n=18). 1.5 hrs after CLP, mice were injected with 50μg of anti-CTLA-4 antibody or PBS per mouse. A second injection of the anti-CTLA-4 antibody or PBS was administered at 24 h after surgery. Survival was recorded for 7 days. 14 CD1 mice were randomly divided into 2 groups. The effect of anti-CTLA-4 antibody in a CLP followed 4 days later by i.v. injection of Candida Albicans was conducted. Three doses of (33 ug/mouse of anti-CTLA-4 antibody were injected i.p. 2, 5 and 7 days after Candida Albicans.3.CD1 mice were randomly divided into sham group (n = 11), sham ~+ anti-CTLA-4 antibody group (n = 11), CLP ~+ PBS control group (n = 16), CLP ~+ anti-CTLA-4 antibody group (n = 16). Mice were injected with 50μg anti-CTLA-4 antibody or PBS per mouse at 6 h, 24 h after surgery. Mice were sacrificed at 48 h after CLP. Expressions of Caspase-3 in CD4~+, CD8~+ T cells were examined by flow cytometry. Cytokines, i.e., TNF-α, IL-6, IL-10 and IFN-γwere quantiated after stimulation with anti-CD3/CD28 antibody.4.CD1 mice were randomly divided into sham group (n = 6), sham ~+ anti-CTLA-4 antibody group (n = 6), CLP ~+ PBS control group (n = 8), CLP ~+ anti-CTLA-4 antibody treatment group (n = 8). 50μg/mouse anti-CTLA-4 antibody was given intraperitonealy at 1, 3, and 5 days after CLP per mouse. Mice were sacrificed at 7 days after CLP. Total number of spleen cells, CD4~+, CD8~+ T cells, na?ve (CD44low/CD62Lhigh), central memory (CD44high/CD62Lhigh ), and effector memory (CD44high/CD62Llow) T cells were measured by flow cytometry.Results:1.The expression of CTLA-4 on CD4~+ T cells of CLP mice increased by ~ 2.5-fold, 1.9-fold and 3.4-fold by day 1, day 4 and day 7 respectively (P<0.05). The expression of CTLA-4 on CD8~+ T cells in CLP mice was significantly higher at day 4 and day 7 (P<0.05).2.CLP-operated CD-1 mice treated with 50μg/mouse of anti-CTLA-4 had a marked improvement in survival compared to controls (33.3% vs. 5%, P<0.05). Anti-CTLA-4 did cause an improvement in survival compared to controls in the 2-hit sepsis model as well (35% vs. 0%, P<0.05).3.Mice treated with anti-CTLA-4 had an approximately 50% reduction in sepsis-induced apoptosis in CD4~+ and CD8~+ T cells (P<0.05). Anti-CTLA-4 therapy decreased sepsis-induced apoptosis but had little effect on pro- or anti-inflammatory cytokines(P>0.05).4.Anti-CTLA-4 therapy did not affect the percentage of na?ve, central and effector memory T cells after sepsis(P>0.05).ConclusionThe expression of CTLA-4 on CD4 ~+, CD8 ~+ Tcells increased significantly during sepsis. There is an increase of percentage of CTLA-4~+ CD4 ~+ CD25 ~+ Foxp3 ~+ T cells after sepsis. Anti-CTLA-4 antibody can significantly improve the survival of two murine models with sepsis, i.e., CLP and CLP plus Candida albicans. Anti-CTLA-4 antibody can significantly reduce the expression of Caspase-3 on CD4 ~+, CD8 ~+ T cells. However, anti-CTLA-4 antibody did not change the expression of cytokines and T cells subtypes. These findings suggest that the protective effect of anti-CTLA-4 antibody in CLP-induced sepsis of mice might be achieved by inhibiting the apoptosis pathway. |
PDF Full Text Request