Studies on induction of mucosal immunity using gene targeted mice

Abstract

The gut mucosal immune system directs powerful immune responses against harmful microorganisms, while the normal gut flora and the food constituents are tolerated without causing adverse immune activation or hypersensitivity reactions. This thesis focuses on certain regulatory phenomena that determine whether active immunity or tolerance ensues after antigen administration via the oral route. By use of various cytokine deficient gene targeted mice, along with well-defined immunization regimens, the prerequisites for induction of mucosal immune responses were studied at the levels of antigen presentation and T helper helper cell differentiation. In genetically engineered fusion proteins, the non-immunogenic heat-stable enterotoxin (STa) of enterotoxigenic E. coli was linked to single or multiple heterologous ovalbumin T cell epitopes. A construct containing four, rather than one or eight, T cell epitopes was found to have optimum immunogenicity, stimulating strong anti-STa B cell responses, as well as anti-ovalbumin T cell responses. The enhanced immunogenicity of the construct was due to efficient antigen processing, leading to an expanded pool of epitope specific T cells. Moreover, by the incorporation of an Ig-binding moeity, the fusion proteins effectively targeted B cells. Thus, an optimally constructed fusion protein vaccine should not only contain immunogenic epitopes, but also be designed to facilitate uptake and processing in the antigen presenting cell (APC). The Peyerís patches (PP) are generally regarded as the inductive site of gut mucosal IgA responses. However, other locations, such as the gut epithelium, may have regulatory functions in gut immunity. While different cell types act as APC in the PP, the immunomodulating role of the intestinal epithelial cells (IEC) is poorly defined. Here we show that cholera toxin (CT) enhances the ability of IEC to present alloantigen by enhancing interleukin (IL)-1 and IL-6 co-stimulation, suggesting that IEC may have a role in CTís adjuvant effect on gut mucosal immune responses. The differentiation of CD4+ T cells is affected by the cytokines to which these cells are exposed during antigenic stimulation. IL-4 deficient (IL-4-/-) and interferon-( deficient (IFN-(-/-) mice were used to analyze the roles of T helper (Th) 1 and Th2 cells in the induction of gut mucosal IgA responses. IL-4-/- mice had strongly impaired mucosal IgA and Th2 responses after oral immunization with protein antigen given together with CT adjuvant, implying an important function for IL-4 and Th2 cells in the induction of intestinal secretory IgA responses. However, mucosal IgA responses in IL-4-/- mice could be restored by coupling of antigen to CT, indicating that the conjugates had access to an inductive pathway that was independent on IL-4 associated functions. IFN-( deficiency appeared to have different effects in different mouse strains. Thus, C57BL/6 IFN-(-/- mice exhibited strong mucosal IgA responses and increased Th2 responses, while IFN-( receptor-deficient 129/Sv mice showed impaired IgA responses and reduced Th1 as well as Th2 responses. Double deficient 129/Sv mice, lacking functions of both IL-4 and IFN-(, had reduced IgA responses and decreased Th1 and Th2 responses. Taken together, these results underscore the important role for IL-4 functions and Th2 cells in the induction of mucosal IgA responses. Since in 129/Sv mice, both IFN-( deficiency and IFN-(/IL-4 double deficiency resulted in impaired IgA induction and decreased Th2 responses, the existence of a classical Th1/Th2 cross-regulation in the intestinal mucosa was questioned. In contrast, the induction of tolerance to ingested protein antigen was unaffected in C57BL/6 IL-4-/- and 129/Sv IFN-(R-/- mice, suggesting that induction of tolerance and active IgA immunity are differently regulated.