Antigen binding to G_M1 ganglioside results in delayed presentation: minimal effects of G_M1 on presentation of antigens internalized via other pathways

Abstract

SUMMARYPlasma membrane rafts are sphingolipid-and cholesterol-rich patches that function as membrane trafficking and surface signalling regions. Ganglioside G M1 is an integral component of these microdomains, and Escherichia coli enterotoxin B subunit (EtxB) is a pentamer that binds with high affinity to G M1 resulting in G M1 cross-linking. We previously demonstrated that antigen coupled directly to EtxB resulted in enhanced presentation relative to antigen taken up by fluid-phase endocytosis. Here we demonstrate a new role for G M1 in antigen presentation by examining the effects of cross-linking G M1 on the kinetics of presentation and processing of antigen by the B-cell receptor (BCR), fluid-phase endocytosis and G M1 -targeted antigen. EtxB bound to B cells does not augment the subsequent kinetics or magnitude of presentation of either BCR-internalized antigen or soluble antigen. Moreover, presentation of G M1 -bound antigen is significantly slower than antigen presentation following BCR-mediated uptake. In contrast to the rapid internalization of BCR-bound antigen (which has a half life of 60 min), the majority of EtxB-bound antigen forms a plasma membrane depot detectable for many hours after initial incubation (and with a half life of 12 hr). We conclude that cross-linking of G M1 by EtxB minimally affects the processing and presentation of antigens internalized via other pathways. Nevertheless, binding of antigens to G M1 results in delayed presentation that has important implications for in vivo immunization using G M1 -targeted adjuvants.

The B subunit of Escherichia coli heat‐labile toxin alters the development and antigen‐presenting capacity of dendritic cells

“…The combination of increased cell viability and reduced activation could prolong the antigen presentation capacity of DC and consequently manifest as an adjuvant effect because of increased T cell activation. Indeed, this interpretation is not without precedent, as similar reports of EtxB increasing the viability and survival of B cells and macrophages for delayed antigen presentation have been reported [14,39]. An alternative interpretation of the in vitro results could be that an increase in cell viability and a reduction in expression of activation markers is because of increased maturation of DC.…”

Cell clustering and delay/arrest in T-cell division implicate a novel mechanism of immune modulation by E. coli heat-labile enterotoxin B-subunits

“…For OVA in the fluid phase, such an impact on the cytoskeleton would conceivably enhance its uptake and presentation. However, in a previous study it was found that incubation of A20 WT B cell with LT-IB did not noticeably enhance OVA presentation when added in the fluid phase [19]. In the latter study, the response of DO.11.10 T cells to OVA was evaluated by measuring the level of proliferation of IL-2-responsive HT-2 cells.…”

“…LT-IB and LT-IIaB were used at 5μg/ml and 2.5μg/ml, respectively, unless indicated otherwise. The choice of LT-IB and LT-IIaB doses in this study is based on their direct or indirect stimulatory effect on immune cells and their products, as shown previously [13, 14, 19, 22]. LT-IIbB and LT-IIbB(S74D) were used at 10 μg/ml.…”

“…LT-IB also modulates cytokine secretion by dendritic cells [13]. Further, the targeting of Ag which is chemically coupled or fused to LT-IB to the surface of APCs significantly enhances the presentation of that Ag to T cells and its immunogenicity [13, 19]. These findings are explained by the high affinity binding of LT-IB to G M1 on surface of APCs and the efficient delivery of the Ag to MHC-I and MHC-II compartments of Ag processing and presentation [13, 20].…”

“…In this regard, early studies on the adjuvanticity of CT to sheep red blood cells (SRBCs) showed that the remarkable expansion of the immune response to SRBCs was delayed as it occurred several days after a subsequent boost with the Ag [25]. A delay in the uptake of LT-IB conjugated to ovalbumin (OVA) has also been shown in B cells when compared to the binding of OVA alone to the BCR, which suggests it may play a role in its adjuvanticity [19]. Moreover, maximal expression of MHC class II on B cells occurred after a 48 hr incubation with LT-IB [14] compared to only 24 hrs with LPS or anti-BCR antibody.…”