Cholera toxin B subunit linked to glutamic acid decarboxylase suppresses dendritic cell maturation and function

Abstract

Dendritic cells are the largest population of antigen presenting cells in the body. One of their main functions is to regulate the delicate balance between immunity and tolerance responsible for maintenance of immunological homeostasis. Disruption of this delicate balance often results in chronic inflammation responsible for initiation of organ specific autoimmune diseases such as rheumatoid arthritis, multiple sclerosis and type I diabetes. The cholera toxin B subunit (CTB) is a weak mucosal adjuvant known for its ability to stimulate immunity to antigenic proteins. However, conjugation of CTB to many autoantigens can induce immunological tolerance resulting in suppression of autoimmunity. In this study, we examined whether linkage of CTB to a 5kDa C-terminal protein fragment of the major diabetes autoantigen glutamic acid decarboxylase (GAD(35)), can block dendritic cell (DC) functions such as biosynthesis of co-stimulatory factor proteins CD86, CD83, CD80 and CD40 and secretion of inflammatory cytokines. The results of human umbilical cord blood monocyte-derived DC-GAD(35) autoantigen incubation experiments showed that inoculation of immature DCs (iDCs), with CTB-GAD(35) protein dramatically suppressed levels of CD86, CD83, CD80 and CD40 co-stimulatory factor protein biosynthesis in comparison with GAD(35) alone inoculated iDCs. Surprisingly, incubation of iDCs in the presence of the CTB-autoantigen and the strong immunostimulatory molecules PMA and Ionomycin revealed that CTB-GAD(35) was capable of arresting PMA+Ionomycin induced DC maturation. Consistent with this finding, CTB-GAD(35) mediated suppression of DC maturation was accompanied by a dramatic decrease in the secretion of the pro-inflammatory cytokines IL-12/23p40 and IL-6 and a significant increase in secretion of the immunosuppressive cytokine IL-10. Taken together, our experimental data suggest that linkage of the weak adjuvant CTB to the dominant type 1 diabetes autoantigen GAD strongly inhibits DC maturation through the down regulation of major co-stimulatory factors and inflammatory cytokine biosynthesis. These results emphasize the possibility that CTB-autoantigen fusion proteins enhance DC priming of naïve Th0 cell development in the direction of immunosuppressive T lymphocytes. The immunological phenomena observed here establish a basis for improvement of adjuvant augmented multi-component subunit vaccine strategies capable of complete suppression of organ-specific autoimmune diseases in vivo.