Interferon‐γ (IFN‐γ) receptor knock‐out (IFN‐γR−/−) mice were used to analyse the role of IFN‐γ in mucosal immune responses following oral immunization. We found that the IFN‐γR−/− mice demonstrated 50% reduced spot‐forming cell (SFC) responses in the gut lamina propria and spleen after oral immunization with keyhole limpet haemocyanin (KLH) plus cholera toxin (CT) adjuvant. The IFN‐γR−/− mice exhibited 10‐fold reduced total serum KLH‐specific antibody levels compared with wild‐type mice after oral immunization, while after intravenous immunization, no such difference was seen, suggesting a selective impairment of mucosal immune responses. Moreover, oral immunizations resulted in impaired interleukin‐4 (IL‐4), IL‐10 and IFN‐γ production by spleen T cells from IFN‐γR−/− mice, indicating that no reciprocal up‐regulation of Th2‐activities had occurred despite the lack of IFN‐γR function. No reduction in Th1 or Th2 cytokines was observed following systemic immunizations. Despite potentially strong modulating effects of IFN‐γ on epithelial cell IgA transcytosis and electrolyte barrier functions, CT‐immunized IFN‐γR−/− mice demonstrated unaltered protection against CT in ligated intestinal loops together with normal anti‐CT IgA and total IgA levels in gut lavage. Oral feeding with KLH followed by parenteral immunization resulted in strongly suppressed SFC numbers and reduced cell‐mediated immunity in both wild‐type and IFNγR−/− mice. CT‐adjuvant abrogated induction of oral tolerance in both IFN‐γR−/− and wild‐type mice. Collectively, our data argue that the two major response patterns induced by oral administration of protein antigen, i.e. active IgA immunity and oral tolerance, are differently regulated. Thus, IFN‐γR−/− mice have impaired mucosal immune responses while induction of oral tolerance appears to be unaffected by the lack of IFN‐γ functions.

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The in vivo mechanisms of action of most vaccine adjuvants are poorly understood. In this study, we present data in mice that reveal a series of critical interactions between the cholera toxin (CT) adjuvant and the dendritic cells (DC) of the splenic marginal zone (MZ) that lead to effective priming of an immune response. For the first time, we have followed adjuvant targeting of MZ DC in vivo. We used CT-conjugated OVA and found that the Ag selectively accumulated in MZ DC following i.v. injections. The uptake of Ag into DC was GM1 ganglioside receptor dependent and mediated by the B subunit of CT (CTB). The targeted MZ DC were quite unique in their phenotype: CD11c+, CD8α−, CD11b−, B220−, and expressing intermediate or low levels of MHC class II and DEC205. Whereas CTB only delivered the Ag to MZ DC, the ADP-ribosyltransferase activity of CT was required for the maturation and migration of DC to the T cell zone, where these cells distinctly up-regulated CD86, but not CD80. This interaction appeared to instruct Ag-specific CD4+ T cells to move into the B cell follicle and strongly support germinal center formations. These events may explain why CT-conjugated Ag is substantially more immunogenic than Ag admixed with soluble CT and why CTB-conjugated Ag can tolerize immune responses when given orally or at other mucosal sites.

SUMMARYWe used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4 and CD8 T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3T-cell frequencies in CD4 ÿ /ÿ and CD8 ÿ /ÿ mice such that in CD4 ÿ /ÿ mice the CD8 and double-negative (DN) T cells were increased. In CD8 ÿ /ÿ mice the CD4 T cells were increased, with the exception that in the intraepithelial compartment the CD3 T cells were predominantly DN g d T-cell receptor (TCR) T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8 ÿ /ÿ mice consistently exhibited three-to fivefold stronger gut mucosal responses compared to normal C57Bl/6 mice. By contrast, no difference was observed for systemic responses between CD8 ÿ /ÿ and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8 ÿ /ÿ mouse gut, the normal gut mucosa may host CD8 T cells that exert a local suppressive effect on T-and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8 ÿ /ÿ and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8 T cells. On the other hand, the adjuvant effect of CT required CD4 T cells, because no or poor anti-KLH responses were observed in CD4 ÿ /ÿ mice. In both normal and CD8 ÿ /ÿ mice CT adjuvant promoted KLH-specific CD4 T-cell priming without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4 T cells expressing a memory phenotype, i.e. CD44high , LECAM-1 low and CD45RB low . We have shown, using gene-targeted mice, that CD8 T cells are not required for the adjuvant effect of CT, and that CD8 T cells may exert local mucosal down-regulation of intestinal immune responses.

Adjuvants that can improve mucosal vaccine efficacy are much warranted. In this comparative study between cholera toxin (CT) and immune-stimulating complexes (ISCOM) we found that, contrary to CT, ovalbumin (OVA)-ISCOM were poor inducers of mucosal anti-OVA IgA responses, but induced similar or better systemic immunity following oral immunizations. The addition of CT to the oral OVA-ISCOM protocol did not stimulate local anti-OVA IgA immunity, nor did it change the quality or magnitude of the systemic responses. Both vectors recruited strong innate immunity, but only OVA-ISCOM could directly induce IL-12, demonstrable at the protein and mRNA levels. CT had no inhibitory effects on lipopolysaccharide/IFN-gamma-induced IL-12 mRNA expression or IL-12 production. Furthermore, adjuvanticity of CT was unaffected in IL-12-deficient mice, while OVA-ISCOM showed partly impaired adjuvant effects by the lack of IL-12. CT abrogated the induction of oral tolerance stimulated by antigen feeding in these mice. In addition, CT did not alter TGF-beta levels, suggesting that the immunomodulating effect of CT was independent of IL-12 as well as TGF-beta production. Taken together, these findings indicate that mucosal adjuvanticity of CT and ISCOM are differently dependent on IL-12, suggesting that separate and distinct antigen-processing pathways are involved.