The modulation of B7.2 and B7.1 on B cells by immunosuppressive agents

Abstract

Several recent studies demonstrate that B7.2, but not B7.1, play an important role in allergic inflammation and IgE production. Agents that down-regulate B7.2 may therefore be of benefit for the treatment of Th2-driven allergic diseases. Our current study was carried out to investigate the effect of immunosuppressive agents, cyclosporin A (CsA) and dexamethasone, on B7.2 and B7.1 expression on B cells stimulated with the superantigen, toxic shock syndrome toxin-1 (TSST-1). The analysis of B7.2 and B7.1 on the same cells by flow cytometry demonstrated that TSST-1 up-regulated B7.2+B7.1- but not B7.1+B7.2- on B cells in a dose-dependent fashion. CsA and dexamethasone significantly down-regulated B7.2+B7.1- but up-regulated B7.2-B7.1+ B cells in the presence or absence of TSST-1 (100 ng/ml). Interestingly, the combination of CsA and dexamethasone was much more potent in the inhibition of B7.2 expression than either of these agents alone. As CD40 is known to up-regulate B7.2 expression on B cells, the mechanism of B7.2 down-regulation by CsA and dexamethasone was further studied by investigating the effect of these agents on CD40 expression on B cells. TSST-1 significantly increased CD40 expression on B cells. However, the addition of CsA or dexamethasone significantly down-regulated CD40 expression. Anti-CD40 MoAb significantly reversed the effects of CsA or dexamethasone on B7.2 and B7.1 expression, suggesting that T cell engagement of CD40 plays a role in the mechanisms by which CsA and dexamethasone acts on B cells. These data demonstrate the modulatory effect of CsA and dexamethasone on B7.2 and B7.1 expression on B cells and the potential role of CD40 in mediating this effect.

Fig. 1

Toxic shock syndrome toxin-1 (TSST-1) up-regulates total B7.2 and B7.1 expression on B cells. Peripheral blood mononuclear cells (PBMC) from six normal donors were cultured with a range of TSST-1 concentrations for 48 h and analysed for B7.2 and B7.1 expression on B cells by flow cytometry. Results from each individual and geometric mean values are shown.

Fig. 2

Toxic shock syndrome toxin-1 (TSST-1) up-regulates B7.2⁺B7.1⁻ but not B7.2⁻B7.1⁺ B cells. Peripheral blood mononuclear cells (PBMC) from six normal donors were analysed by flow cytometry for the expression of B7.2 and B7.1 on the same B cells after culture for 48 h with a range of TSST-1 concentrations. Results from each individual and geometric mean values are shown.

Fig. 3

Cyclosporin A (CsA) significantly decreases B7.2⁺B7.1⁻ but not B7.2⁻B7.1⁺ B cells. Peripheral blood mononuclear cells (PBMC) from six normal donors were cultured for 48 h with or without 100 ng/ml toxic shock syndrome toxin-1 (TSST-1) in the presence or absence of 1 μg/ml CsA. Cells were analysed by flow cytometry for the expression of B7.2 and B7.1 on the same B cells. Results from each individual and geometric mean values are shown.

Fig. 4

Dexamethasone significantly decreases B7.2⁺B7.1⁻ but not B7.2⁻B7.1⁺ B cells. Peripheral blood mononuclear cells (PBMC) from six normal donors were cultured for 48 h with or without 100 ng/ml toxic shock syndrome toxin-1 (TSST-1) in the presence or absence of 10⁻⁶m dexamethasone. Cells were analysed by flow cytometry for the expression of B7.2 and B7.1 on the same B cells. Results from each individual and geometric mean values are shown.

Fig. 5

Cyclosporin A (CsA) and dexamethasone have synergistic effects on the down-regulation of B7.2⁺B7.1⁻ B cells. Toxic shock syndrome toxin-1 (TSST-1; 100 ng/ml) was added in cell culture with or without 100 ng/ml CsA in the absence or presence of 10⁻⁶m dexamethasone (left panel), and with or without 1 μg/ml CsA in the absence or presence of 10⁻⁷m dexamethasone (right panel). In both panels, the combination of both agents showed significant synergistic effects compared with either agent alone in the down-regulation of B7.2⁺B7.1⁻ B cells and up-regulation of B7.2⁻B7.1⁺ B cells. The synergistic effect can also be demonstrated in B7.2⁺B7.1⁺ B cells, which were significantly down-regulated by the combination of both agents. Results from individuals and geometric mean values are shown.

Fig. 6

Cyclosporin A (CsA) and dexamethasone decrease CD40 expression on B cells. Peripheral blood mononuclear cells (PBMC) were cultured for 48 h with or without 100 ng/ml toxic shock syndrome toxin-1 (TSST-1) in the presence or absence of 1 μg/ml CsA (a) and in the presence or absence of 10⁻⁶m dexamethasone (b). These experiments demonstrate that CD40 was up-regulated by 100 ng/ml TSST-1, and 1 μg/ml CsA as well as dexamethasone significantly decreased the effect of TSST-1-induced CD40 expression on B cells. Results from individuals and geometric mean values are shown.

Fig. 7

Anti-CD40 MoAb significantly increases B7.2⁺B7.1⁻ and decreases B7.2⁻B7.1⁺ B cells in culture with cyclosporin A (CsA) or dexamethasone. Peripheral blood mononuclear cells (PBMC) were cultured for 48 h with 100 ng/ml toxic shock syndrome toxin-1 (TSST-1) in the presence or absence of 1 μg/ml CsA or 10⁻⁶m dexamethasone. Anti-CD40 MoAb at 1 μg/ml was added to activate CD40 molecule on B cells. These experiments demonstrate that anti-CD40 MoAb significantly reversed the effect of CsA and dexamethasone on B7.2⁺B7.1⁻ and B7.2⁻B7.1⁺ B cells in the presence of TSST-1. Results from individuals and geometric mean values are shown.