T follicular helper (Tfh) cells are a subset of CD4⁺ T cells that promote antibody production during vaccination. Conventional dendritic cells (cDCs) efficiently prime Tfh cells; however, conclusions regarding which cDC instructs Tfh cell differentiation have differed between recent studies. We found that these discrepancies might exist because of the unusual sites used for immunization in murine models, which differentially bias which DC subsets access antigen. We used intranasal immunization as a physiologically relevant route of exposure that delivers antigen to all tissue DC subsets. Using a combination of mice in which the function of individual DC subsets is impaired and different antigen formulations, we determined that CD11b⁺ migratory type 2 cDCs (cDC2s) are necessary and sufficient for Tfh induction. DC-specific deletion of the guanine nucleotide exchange factor DOCK8 resulted in an isolated loss of CD11b⁺ cDC2, but not CD103⁺ cDC1, migration to lung-draining lymph nodes. Impaired cDC2 migration or development in DC-specific Dock8 or Irf4 knockout mice, respectively, led to reduced Tfh cell priming, whereas loss of CD103⁺ cDC1s in Batf3^−/− mice did not. Loss of cDC2-dependent Tfh cell priming impaired antibody-mediated protection from live influenza virus challenge. We show that migratory cDC2s uniquely carry antigen into the subanatomic regions of the lymph node where Tfh cell priming occurs—the T-B border. This work identifies the DC subset responsible for Tfh cell–dependent antibody responses, particularly when antigen dose is limiting or is encountered at a mucosal site, which could ultimately inform the formulation and delivery of vaccines.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors using its B subunit (CTB). The ganglioside (glycolipid) GM1 is thought to be the sole CT receptor; however, the mechanism by which CTB binding to GM1 mediates internalization of CT remains enigmatic. Here we report that CTB binds cell surface glycoproteins. Relative contributions of gangliosides and glycoproteins to CTB binding depend on cell type, and CTB binds primarily to glycoproteins in colonic epithelial cell lines. Using a metabolically incorporated photocrosslinking sugar, we identified one CTB-binding glycoprotein and demonstrated that the glycan portion of the molecule, not the protein, provides the CTB interaction motif. We further show that fucosylated structures promote CTB entry into a colonic epithelial cell line and subsequent host cell intoxication. CTB-binding fucosylated glycoproteins are present in normal human intestinal epithelia and could play a role in cholera.

Cholera toxin (CT) enters and intoxicates host cells after binding cell surface receptors via its B subunit (CTB). We have recently shown that in addition to the previously described binding partner ganglioside GM1, CTB binds to fucosylated proteins. Using flow cytometric analysis of primary human jejunal epithelial cells and granulocytes, we now show that CTB binding correlates with expression of the fucosylated Lewis X (Le^X) glycan. This binding is competitively blocked by fucosylated oligosaccharides and fucose-binding lectins. CTB binds the Le^X glycan in vitro when this moiety is linked to proteins but not to ceramides, and this binding can be blocked by mAb to Le^X. Inhibition of glycosphingolipid synthesis or sialylation in GM1-deficient C6 rat glioma cells results in sensitization to CT-mediated intoxication. Finally, CT gavage produces an intact diarrheal response in knockout mice lacking GM1 even after additional reduction of glycosphingolipids. Hence our results show that CT can induce toxicity in the absence of GM1 and support a role for host glycoproteins in CT intoxication. These findings open up new avenues for therapies to block CT action and for design of detoxified enterotoxin-based adjuvants. The disease cholera, caused by cholera toxin produced by Vibrio cholerae, is responsible for over 100 000 deaths every year. When taken up by cells in the intestine, the toxin causes diarrhea, and the ganglioside GM1 (a glycolipid) has long been considered the main receptor for cholera toxin. We here present data showing that cholera toxin binds to fucose within the trisaccharide blood group antigen Lewis X. Lewis X is expressed on human granulocytes as well as intestinal cells and correlates with cholera toxin binding. Furthermore, cell lines deficient in GM1 expression and mice lacking GM1 treated to further reduce glycolipid synthesis are intoxicated by cholera toxin. This indicates an active role of glycoproteins, and our results thereby provide new insights on how cholera toxin is internalized into cells in the human intestine. This knowledge can be used to design novel drug candidate inhibitors of cholera toxin. Another way to utilize our findings is to modify cholera toxin to be less toxic so that it can be used as an adjuvant in vaccines.

To generate vaccines that protect mucosal surfaces, a better understanding of the cells required in vivo for activation of the adaptive immune response following mucosal immunization is required. CD11c^high conventional dendritic cells (cDCs) have been shown to be necessary for activation of naive CD8⁺ T cells in vivo, but the role of cDCs in CD4⁺ T cell activation is still unclear, especially at mucosal surfaces. The activation of naive Ag-specific CD4⁺ T cells and the generation of Abs following mucosal administration of Ag with or without the potent mucosal adjuvant cholera toxin were therefore analyzed in mice depleted of CD11c^high cDCs. Our results show that cDCs are absolutely required for activation of CD4⁺ T cells after oral and nasal immunization. Ag-specific IgG titers in serum, as well as Ag-specific intestinal IgA, were completely abrogated after feeding mice OVA and cholera toxin. However, giving a very high dose of Ag, 30-fold more than required to detect T cell proliferation, to cDC-ablated mice resulted in proliferation of Ag-specific CD4⁺ T cells. This proliferation was not inhibited by additional depletion of plasmacytoid DCs or in cDC-depleted mice whose B cells were MHC-II deficient. This study therefore demonstrates that cDCs are required for successful mucosal immunization, unless a very high dose of Ag is administered.

To limit Cholera severity, blockers mimicking the canonical Cholera toxin (CT) ligand ganglioside GM1 have been made. However, efficacy of these blockers has been evaluated in non-cellular systems that lack ligands other than GM1. Importantly, CT B subunit (CTB) has a non-canonical site that binds fucosylated structures, which in contrast to GM1, are highly expressed in the human intestine. Here we evaluate the capacity of norbornene polymers displaying galactose and/or fucose to block CTB binding to primary human small intestine epithelial cells. We show that binding of CTB is largely dependent on the non-canonical binding site while interference with the canonical site has limited effect. However, pre-incubation of CT with galactose/fucose polymer only partially blocked toxic effects on cultured human enteroid cells, while pre-incubation with GM1 completely blocked CT-mediated secretion. Our results support a model whereby binding of fucose to the non-canonical site places CT in close vicinity to scarcely expressed galactose receptors such as GM1 to enable binding via the canonical site leading to CT internalization and intoxication.

Cholera toxin (CT) binds to GM1‐ganglioside receptors present on all nucleated cells. Despite this, it is a very potent mucosal adjuvant that has a dramatic impact on immune cells, as well as nerve and epithelial cells, causing diarrhea. This fact has hampered our understanding of whether the adjuvanticity of CT is direct or indirect, as cells that bind CT may or may not be involved in its adjuvant function. The mucosal barrier is maintained by tight junctions between epithelial cells but dendritic cells (DCs) can protrude luminal dendrites. Here we investigated which cells are involved in the immune augmenting effect of CT. We explored oral immunizations with ovalbumin (OVA) and CT in bone marrow chimeric mice deficient in GM1‐ganglioside in defined cellular subsets. We found that chimeric mice lacking GM1 in nonhematopoietic cells, including epithelial cells, mounted an unaltered intestinal IgA response. In contrast, chimeric mice lacking GM1‐expressing hematopoietic cells in general, or specifically GM1‐expressing conventional DCs (cDCs), largely failed to elicit anti‐OVA adaptive immune responses. Therefore, the adjuvanticity of CT does not require epithelial activation, but is directly dependent on the binding of CT to gut cDCs via GM1‐ganglioside. These results could have important implications for the generation of novel oral adjuvants.