Lena Ekman

Interferon-gamma receptor-deficient mice exhibit impaired gut mucosal immune responses but intact oral tolerance.

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.

Strong Differential Regulation of Serum and Mucosal IgA Responses as Revealed in CD28-Deficient Mice Using Cholera Toxin Adjuvant.

In this study, we show that costimulation required for mucosal IgA responses is strikingly different from that needed for systemic responses, including serum IgA. Following oral immunization with cholera toxin (CT) adjuvant we found that whereas CTLA4-Hγ1 transgenic mice largely failed to respond, CD28−/− mice developed near normal gut mucosal IgA responses but poor serum Ab responses. The local IgA response was functional in that strong antitoxic protection developed in CT-immunized CD28−/− mice. This was in spite of the fact that no germinal centers (GC) were observed in the Peyer’s patches, spleen, or other peripheral lymph nodes. Moreover, significant somatic hypermutation was found in isolated IgA plasma cells from gut lamina propria of CD28−/− mice. Thus, differentiation to functional gut mucosal IgA responses against T cell-dependent Ags does not require signaling through CD28 and can be independent of GC formations and isotype-switching in Peyer’s patches. By contrast, serum IgA responses, similar to IgG-responses, are dependent on GC and CD28. However, both local and systemic responses are impaired in CTLA4-Hγ1 transgenic mice, indicating that mucosal IgA responses are dependent on the B7-family ligands, but require signaling via CTLA4 or more likely a third related receptor. Therefore, T-B cell interactions leading to mucosal as opposed to serum IgA responses are uniquely regulated and appear to represent separate events. Although CT is known to strongly up-regulate B7-molecules, we have demonstrated that it acts as a potent mucosal adjuvant in the absence of CD28, suggesting that alternative costimulatory pathways are involved.

The ADP-Ribosylating CTA1-DD Adjuvant Enhances T Cell-Dependent and Independent Responses by Direct Action on B Cells Involving Anti-Apoptotic Bcl-2- and Germinal Center-Promoting Effects

We recently developed a novel immunomodulating gene fusion protein, CTA1-DD, that combines the ADP-ribosylating ability of cholera toxin (CT) with a dimer of an Ig-binding fragment, D, of Staphylococcus aureus protein A. The CTA1-DD adjuvant was found to be nontoxic and greatly augmented T cell-dependent responses to soluble protein Ags after systemic as well as mucosal immunizations. Here we show that CTA1-DD does not appear to form immune complexes or bind to soluble Ig following injections, but, rather, it binds directly to B cells of all isotypes, including naive IgD+ cells. No binding was observed to macrophages or dendritic cells. Immunizations in FcεR (common FcRγ-chain)- and FcγRII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that FcγR-expressing cells are not required for the adjuvant function. Whereas CT failed to augment Ab responses to high m.w. dextran B512 in athymic mice, CTA1-DD was highly efficient, demonstrating that T cell-independent responses were also enhanced by this adjuvant. In normal mice both CT and CTA1-DD, but not the enzymatically inactive CTA1-R7K-DD mutant, were efficient enhancers of T cell-dependent as well as T cell-independent responses, and both promoted germinal center formation following immunizations. Although CT augmented apoptosis in Ag receptor-activated B cells, CTA1-DD strongly counteracted apoptosis by inducing Bcl-2 in a dose-dependent manner, a mechanism that was independent of the CD19 coreceptor. However, in the presence of CD40 stimulation, apoptosis was low and unaffected by CT, suggesting that the adjuvant effect of CT is dependent on the presence of activated CD40 ligand-expressing T cells.

Splenic Marginal Zone Dendritic Cells Mediate the Cholera Toxin Adjuvant Effect: Dependence on the ADP-Ribosyltransferase Activity of the Holotoxin

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.

ADP-Ribosylation Controls the Outcome of Tolerance or Enhanced Priming Following Mucosal Immunization

Accumulating evidence suggests that the dichotomy between tolerance and active IgA immunity in mucosal immune responses is regulated at the APC level. Therefore, immunomodulation of the APC could be an effective mechanism to control the two response patterns. In this study, we demonstrate that ADP-ribosylation controls the outcome of tolerance or active effector T cell immunity to an internal peptide p323–339 from OVA inserted into the cholera toxin (CT)-derived CTA1-OVA-DD adjuvant. We found that a single point mutation, CTA1R7K-OVA-DD, resulting in lack of enzymatic activity, promoted peptide-specific tolerance in TCR transgenic CD4+ T cells following a single intranasal (i.n.) treatment. The CTA1R7K-OVA-DD–induced tolerance was strong, long-lasting, and impaired the ability of adoptively transferred naive peptide-specific CD4+ T cells to respond to Ag-challenge, irrespective if this was given i.p or i.n. The tolerance correlated with induction of regulatory T cells of the regulatory T type 1 characterized by CD25−Foxp3−CD4+ T cells producing IL-10. In contrast, in IL-10–deficient mice, no peptide-specific tolerance was observed, and these mice exhibited unimpaired CD4+ T cell responsiveness to recall Ag irrespective of if they were untreated (PBS) or treated i.n. with CTA1R7K-OVA-DD. Thus, for the first time, we can provide unequivocal proof that ADP-ribosylation can control the outcome of mucosal Ag exposure from tolerance to an enhanced effector CD4+ T cell response. The exploitation of this system for clinical treatment of autoimmune diseases is discussed.