Matrix protein 2 ectodomain (M2e) is considered an attractive component of a broadly protective, universal influenza A vaccine. Here we challenge the canonical view that antibodies against M2e are the prime effectors of protection. Intranasal immunizations of Balb/c mice with CTA1-3M2e-DD-generated M2e-specific memory CD4 T cells that were I-A d restricted and critically protected against infection, even in the complete absence of antibodies, as observed in JhD mice. Whereas some M2e-tetramer-specific memory CD4 T cells resided in spleen and lymph nodes, the majority were lung-resident Th17 cells, that rapidly expanded upon a viral challenge infection. Indeed, immunized IL-17A −/− mice were significantly less well protected compared with wild-type mice despite exhibiting comparable antibody levels. Similarly, poor protection was also observed in congenic Balb/B (H-2 b ) mice, which failed to develop M2e-specific CD4 T cells, but exhibited comparable antibody levels. Lung-resident CD69 + CD103 low M2e-specific memory CD4 T cells were αβ TCR + and 50% were Th17 cells that were associated with an early influx of neutrophils after virus challenge. Adoptively transferred M2e memory CD4 T cells were strong helper T cells, which accelerated M2e- but more importantly also hemagglutinin-specific IgG production. Thus, for the first time we demonstrate that M2e-specific memory CD4 T cells are broadly protective.

We have developed a novel platform for intranasal treatment of autoimmune diseases in which ADP-ribosylation determines whether immunity or tolerance is induced. Hence, cholera toxin A1-subunit based immunomodulation through the CTA1-peptide-DD fusion protein promotes enhancement, while inactive mutants induce suppression. Whereas the target population after intranasal administration of both constructs was CD103+CD11blow DC’s, both active and inactive mutants induced strong CD4 T cell priming, but they differentially affected CD4 T cell differentiation. This way inactive mutant constructs generated regulatory CD4 T cells producing IL-10 and effectively preventing experimental autoimmune encephalitis (EAE) and collagen-induced arthritis (CIA), when carrying relevant peptides. Targeted DCs expressed low levels of CD80, CD86 and CD40, while, by contrast, ADP-ribosylating CTA1-peptide-DD constructs gave significantly enhanced co-stimulation. Suppression was global in that regulatory T cells following treatment were able to suppress adoptively transferred naïve CD4 T cells and could subsequently maintain tolerance. The use of specifically tolerance-inducing fusion proteins may be a way forward in the search of effective treatments against autoimmune diseases. The immunomodulating effect on the CD103+CD11blow DCs will be described and the requirement for CTA1-binding to Gsa evaluated in detail, using Cre-lox Gsa-deficient mice.

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40−/−, Batf3−/−, and IL-17A−/− mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3−/− mice, lacking CD8α+ DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b+ DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.

Despite an extensive literature on the mechanism of action of cholera toxin (CT), we still lack critical information about how the toxin acts as an adjuvant and, especially, which dendritic cells (DCs) are the target cells. Although a T helper type 2 (Th2)-skewing effect of CT is most commonly reported, effective priming of Th17 cells as well as suppression of Th1 responses are well documented. However, the ability of CT to block interferon regulatory factor 8 (IRF8) function and interleukin (IL)-12 production in DCs, which blocks CD8α DC and Th1 cell development, is inconsistent with priming of Th1 and CD8 T cells in many other reports. This prompted us to investigate the adjuvant effect of CT in wild-type, IL-12p40−/−, Batf3−/−, and IL-17A−/− mice and in mice that selectively lack the Gsα target protein for CT adenosine diphosphate (ADP)-ribosylation in DCs. We found that CT promoted Th1 priming independently of IL-12, and whereas Th2 and also Th17 responses were augmented, the gut IgA responses did not require IL-17A. Adjuvanticity was intact in Batf3−/− mice, lacking CD8α + DCs, but completely lost in mice with Gsα-deficient CD11c cells. Thus, our data demonstrate that the adjuvant effect requires Gsα expression in CD11b + DCs, and that priming of mucosal IgA and CD4 T cells appears unbiased and is independent of IL-12 and IL-17A.

Earlier studies have reported on both proinflammatory and anti-inflammatory activities of cholera toxin (CT). As CT is a powerful adjuvant, we were interested in identifying genes with a possible involvement in these functions. A global gene expression analysis in mouse B cells showed that CT regulated <100 annotated genes, which encoded transcription factors, G proteins, cell-cycle regulators, and immunoregulating molecules. Interestingly, CT regulated the expression of the signal transducer and activator of transcription (STAT)3 gene and influenced the level and activation of both isoforms STAT3α and STAT3β, in vitro in a B-cell line and in Peyer's patch (PP) B cells and in vivo in freshly isolated splenic B cells from CT-treated mice. This effect was cAMP dependent and was not seen with CTB. B cells pre-exposed to CT were significantly more susceptible to the activation of STAT3 by interleukin (IL)-6 and IL-10. This exerted a stronger inhibitory effect of IL-10 on lipopolysaccharide (LPS)-stimulated B-cell proliferation and cytokine production (IL-6). Moreover, IgG1 and IgA production induced by LPS and IL-10 were enhanced by the addition of CT to cultures of PP or splenic B cells. This is the first study to provide a molecular mechanism that can reconcile previous findings of proinflammatory and anti-inflammatory effects by CT adjuvant.

Safe and efficacious adjuvants are much needed to facilitate the development of mucosal vaccines. Here, we have asked whether our nontoxic vaccine adjuvant, CTA1‐DD, can enhance protective immunity against Helicobacter pylori infection. Intranasal immunizations with H. pylori lysate together with CTA1‐DD‐adjuvant induced significant protection in C57Bl/6 mice, almost as strong as similar immunizations using cholera toxin (CT)‐adjuvant. Protection remained strong even at 8 weeks postchallenge and the bacterial colonization was reduced by 20‐fold compared to lysate‐immunized controls. Although CTA1‐DD was designed to bind to B cells, µMT mice developed significant, but lower, level of protection following immunization. Intranasal immunizations with CT adjuvant in C57Bl/6 mice resulted in the development of severe postimmunization gastritis at 2 and 8 weeks postchallenge, whereas the degree of gastritis was substantially lower in the CTA1‐DD‐immunized mice. Protection induced by both CTA1‐DD‐ and CT adjuvant was associated with a strong local infiltration of CD4+ T cells in the gastric mucosa, and recall responses to specific Ag elicited substantial IFN‐γ production, indicating Th1‐dominance. These findings clearly demonstrate that CTA1‐DD adjuvant is a promising candidate to be further exploited in the development of a mucosal vaccine against H. pylori infection.

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(+), CD8alpha(-), 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.

We recently reported that Helicobacter pylori-specific Abs impair the development of gastritis and down-regulate resistance against H. pylori infection. In this study, we asked whether IgA Abs specifically can have an impact on H. pylori colonization and gastric inflammation. To obtain a sensitive model for the study of inflammation we crossed IgA- and IL-10-deficient mice. We found that IL-10(-/-)/IgA(-/-) mice were significantly less colonized than IL-10(-/-)/IgA(-/-) mice, which in turn were less colonized than wild-type (WT) mice. The IL-10(-/-)/IgA(-/-) mice exhibited a 1.2-log reduction in bacterial counts compared with that in IL-10(-/-)/IgA(+/+) mice, suggesting that IgA Abs rather promoted than prevented infection. The reduced colonization in IL10(-/-)/IgA(-/-) mice was associated with the most severe gastritis observed, albeit all IL-10(-/-) mice demonstrated more severe gastric inflammation than wild-type mice. The gastritis score and the infiltration of CD4(+) T cells into the gastric mucosa were significantly higher in IL-10(-/-)/IgA(-/-) mice than in IL-10(-/-)/IgA(+/+) mice, arguing that IgA Abs counteracted inflammation. Moreover, following oral immunization, IL-10(-/-)/IgA(-/-) mice were significantly better protected against colonization than IL10(-/-)/IgA(+/+) mice. However, the stronger protection was associated with more severe postimmunization gastritis and gastric infiltration of CD4(+) T cells. There was also a clear increase in complement receptor-expressing cells in IL-10(-/-)/IgA(-/-) mice, though CM-fragment deposition in the gastric mucosa was comparable between the two. Finally, specific T cell responses to recall Ag demonstrated higher levels of IFN-gamma production in IL-10(-/-)/IgA(-/-) as compared with IL-10(-/-)/IgA(+/+) mice. Thus, it appears that IgA and IL-10 help H. pylori bacteria evade host resistance against infection.

In recent years, Abs have been considered a correlate rather than an effector of resistance against Helicobacter pylori infection. However, it is still poorly understood to what extent Ab production correlates with gastric immunopathology. Here we report that Abs not only are dispensable for protection, but they are detrimental to elimination of the bacteria and appear to impair gastric inflammatory responses. We found that the initial colonization with H. pylori bacteria was normal in the B cell-deficient (μMT) mice, whereas at later times (>8 wk) most of the bacteria were cleared, concomitant with the development of severe gastritis. In contrast, wild-type (WT) mice exhibited extensive bacterial colonization and only mild gastric inflammation, even at 16 wk after inoculation. Oral immunizations with H. pylori lysate and cholera toxin adjuvant stimulated comparable levels of protection in μMT and WT mice. The level of protection in both strains correlated well with the severity of the postimmunization gastritis. Thus, T cells were responsible for the gastritis, whereas Abs, including potentially host cell cross-reactive Abs, were not involved in causing the gastritis. The T cells in μMT and WT mice produced high and comparable levels of IFN-γ to recall Ag at 2 and after 8 wk, whereas IL-4 was detected after 8 wk only, indicating that Th1 activity dominated the early phase of protection, whereas later a mixed Th1 and Th2 activity was seen.

Protective immunity against Helicobacter pylori infection in mice has been associated with a strong Th1 response, involving IL-12 as well as IFN-gamma, but recent studies have also demonstrated prominent eosinophilic infiltration, possibly linked to local Th2 activity in the gastric mucosa. In this study we investigated the role of IL-18, because this cytokine has been found to be a coregulator of Th1 development as well as involved in Th2-type responses with local eotaxin production that could influence gastric eosinophilia and resistance to infection. We found that IL-18(-/-) mice failed to develop protection after oral immunization with H. pylori lysate and cholera toxin adjuvant, indicating an important role of IL-18 in protection. Well-protected C57BL/6 wild-type (WT) mice demonstrated substantial influx of CD4(+) T cells and eosinophilic cells in the gastric mucosa, whereas IL-18(-/-) mice had less gastritis, few CD4(+) T cells, and significantly reduced numbers of eosinophilic cells. T cells in well-protected WT mice produced increased levels of IFN-gamma and IL-18 to recall Ag. By contrast, unprotected IL-18(-/-) mice exhibited significantly reduced gastric IFN-gamma and specific IgG2a Ab levels. Despite differences in gastric eosinophilic cell infiltration, protected WT and unprotected IL-18(-/-) mice had comparable levels of local eotaxin, suggesting that IL-18 influences protection via Th1 development and IFN-gamma production rather than through promoting local production of eotaxin and eosinophilic cell infiltration.

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-H1 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-Hgamma1 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 regulatory roles of Th1 and Th2 cells in immune protection against Helicobacter infection are not clearly understood. In this study, we report that a primary H. pylori infection can be established in the absence of IL-12 or IFN-gamma. However, IFN-gamma, but not IL-12, was involved in the development of gastritis because IFN-gamma(-/-) (GKO) mice exhibited significantly less inflammation as compared with IL-12(-/-) or wild-type (WT) mice. Both IL-12(-/-) and GKO mice failed to develop protection following oral immunization with H. pylori lysate and cholera toxin adjuvant. By contrast, Th2-deficient, IL-4(-/-), and WT mice were equally well protected. Mucosal immunization in the presence of coadministered rIL-12 in WT mice increased Ag-specific IFN-gamma-producing T cells by 5-fold and gave an additional 4-fold reduction in colonizing bacteria, confirming a key role of Th1 cells in protection. Importantly, only protected IL-4(-/-) and WT mice demonstrated substantial influx of CD4(+) T cells in the gastric mucosa. The extent of inflammation in challenged IL-12(-/-) and GKO mice was much reduced compared with that in WT mice, indicating that IFN-gamma/Th1 cells also play a major role in postimmunization gastritis. Of note, postimmunization gastritis in IL-4(-/-) mice was significantly milder than WT mice, despite a similar level of protection, indicating that immune protection is not directly linked to the degree of gastric inflammation. Only protected mice had T cells that produced high levels of IFN-gamma to recall Ag, whereas both protected and unprotected mice produced high levels of IL-13. We conclude that IL-12 and Th1 responses are crucial for H. pylori-specific protective immunity.

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 non-toxic and greatly augmented T cell dependent and independent responses. Following injection it binds to both naïve and memory B cells and up-regulates co-stimulatory molecules as well as prevents apoptosis of activated B cells. Here we show that CTA1-DD is a potent mucosal adjuvant administered intranasally. A dose-response analysis revealed that the adjuvant effect of CTA1-DD given intranasally was equally strong to that observed after systemic immunizations. The adjuvant effect was independent of any possible contamination with endotoxin as indicated by the similar enhancing effects of CTA1-DD in C3H/HeN and the LPS-insensitive C3H/HeJ mice. Contrary to many other adjuvants CTA1-DD induces an immune response to itself. However, despite the presence of high serum titers of pre-existing anti-CTA1 antibodies we observed no reduction of the adjuvant function of CTA1-DD when given either intranasally or systemically. These results support the notion that the CTA1-DD adjuvant can repeatedly be used in the clinic without loss of efficacy even when pre-existing anti-CTA1 antibody levels are high.

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA323–339 peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

Mucosally active vaccine adjuvants that will prime a full range of local and systemic immune responses against defined antigenic epitopes are much needed. Cholera toxin and lipophilic immune stimulating complexes (ISCOMS) containing Quil A can both act as adjuvants for orally administered Ags, possibly by targeting different APCs. Recently, we have been successful in separating the adjuvant and toxic effects of cholera toxin by constructing a gene fusion protein, CTA1-DD, that combines the enzymatically active CTA1-subunit with a B cell-targeting moiety, D, derived from Staphylococcus aureus protein A. Here we have extended this work by combining CTA1-DD with ISCOMS, which normally target dendritic cells and/or macrophages. ISCOMS containing a fusion protein comprising the OVA(323-339) peptide epitope linked to CTA1-DD were highly immunogenic when given in nanogram doses by the s.c., oral, or nasal routes, inducing a wide range of T cell-dependent immune responses. In contrast, ISCOMS containing the enzymatically inactive CTA1-R7K-DD mutant protein were much less effective, indicating that at least part of the activity of the combined vector requires the ADP-ribosylating property of CTA1. No toxicity was observed by any route. To our knowledge, this is the first report on the successful combination of two mechanistically different principles of adjuvant action. We conclude that rationally designed vectors consisting of CTA1-DD and ISCOMS may provide a novel strategy for the generation of potent and safe mucosal vaccines.

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.

Allograft rejection involves T-cell activation, requiring T-cell receptor interactions with major histocompatibility complex (MHC) molecules and costimulatory signals delivered through the B7-CD28 pathway. We evaluated the effect of blocking this pathway on graft rejection and survival, in a rat experimental model of small bowel transplantation. Heterotopic small bowel transplantation was performed between PVG donor rats and DA recipient rats. The recipient animals were treated with CTLA4-Ig or irrelevant immunoglobulin (Ig)G as control and followed for 18, 30 or 90 days. The survival rate and degree of inflammation and accumulation of CD4(+) T cells and macrophages were determined in the transplanted bowels. We found that administration of CTLA4-Ig significantly improved the survival rate compared to control rats: after 30 days 73% of the treated rats had survived and at 90 days 5/8 rats were still living, whereas in the control group only 2/8 rats had survived. The grafts showed preserved mucosal structure with only a mild degree of subacute inflammation and the accumulation of CD4(+) T cells and macrophages was noticeably reduced in treated animals as compared to control rats. Necrosis was extensive in control rats, whereas CTLA4-Ig treated animals had grafts with at least some preserved villus morphology and no necrotic tissue. Although small bowel transplantation has proven exceptionally difficult, in this study we have shown that CTLA4-Ig treatment may provide a promising strategy to prevent rejection and induce long term tolerance and graft survival.

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 round 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, father, it binds directly to B cells of all isotypes, including naive IgD(+) cells, No binding was observed to macrophages or dendritic cells. Immunizations in Fc is an element of R (common FcR gamma-chain)- and Fc gamma RII-deficient mice demonstrated that CTA1-DD exerted unaltered enhancing effects, indicating that Fc gamma R-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 Bel-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.

Recent publications have provided confusing information on the importance of the J chain for secretion of dimeric IgA at mucosal surfaces, Using J chain-deficient (J chain(-/-)) mice, we addressed whether a lack of J chain had any functional consequence for the ability to resist challenge with cholera toxin (CT) in intestinal loops. J chain(-/-) mice had normal levels of IgA plasma cells in the gut mucosa, and the Peyer's patches exhibited normal IgA B cell differentiation and germinal center reactions. The total IgA levels in gut lavage were reduced by roughly 90% as compared with that in wild-type controls, while concomitantly serum IgA levels were significantly increased, Total serum IgM levels were depressed, whereas IgG concentrations were normal. Following oral immunizations with CT, J chain(-/-) mice developed 10-fold increased serum antitoxin IgA titers, but gut lavage anti-CT IgA levels were substantially reduced. However, anti-CT IgA spot-forming cell frequencies in the gut lamina propria were normal. Anti-CT IgM concentrations were low in serum and gut lavage, whereas anti-CT IgG titers were unaltered. Challenge of small intestinal ligated loops with CT caused dramatic fluid accumulation in immunized J chain(-/-) mice, and only 20% protection was detected compared with unimmunized controls. In contrast, wild-type mice demonstrated 80% protection against CT challenge, Mice heterozygous for the J chain deletion exhibited intermediate gut lavage anti-CT IgA and intestinal protection levels, arguing for a J chain gene-dosage effect on the transport of secretory IgA, This study unequivocally demonstrates a direct relationship between mucosal transport of secretory SIgA and intestinal immune protection.