Cholera toxin subunit B peptide fusion proteins reveal impaired oral tolerance induction in diabetes‐prone but not in diabetes‐resistant mice

Abstract

The B subunit of cholera toxin (CTB) has been used as adjuvant to improve oral vaccine delivery in type 1 diabetes. The effect of CTB/peptide formulations on antigen-specific CD4 T cells has remained largely unexplored. We investigated by tetramer analysis how oral delivery of CTB fused to 2 CD4 T cell epitopes, the BDC-2.5 T cell 2.5mi mimotope and glutamic acid decarboxylase (GAD) 286–300, affected diabetogenic CD4 T cells in NOD mice. CTB-2.5mi activated 2.5mi+ T cells when administered intraperitoneally and generated Ag-specific Foxp3+ Treg and Th2 cells following intragastric delivery. While 2.5mi+ and GAD-specific T cells were tolerized in diabetes resistant NODxB6.Foxp3EGFP F1 and NOR mice, this did not occur in NOD mice. This indicated NOD mice had a recessive genetic resistance to induce oral tolerance to both CTB-fused epitopes. Contrarily to NODxB6.Foxp3EGFP F1 mice, oral treatment in NOD mice lead to strong 2.5mi+ T cell activation and the sequestration of these cells to the effector-memory pool. Oral treatment of NOD mice with CTB-2.5mi failed to prevent diabetes. These findings underline the importance of investigating the effect of oral vaccine formulations on diabetogenic T cells as in selected cases they may have counterproductive consequences in human patients.

Use of Autoantigen-Loaded Phosphatidylserine-Liposomes to Arrest Autoimmunity in Type 1 Diabetes

“…We next examined the generation of antigen-specific Foxp3 + CD4 + T after PS2.5mi-liposome administration using NOD.Foxp3 EGFP reporter mice. It has been shown that these mice generate 2.5mi + T cells as well as Foxp3 + Treg with the same specificity [ 17 ]. Antigen-specific CD4 + T cells, both Foxp3 - T cells and Foxp3 + T cells, were expanded in the spleen, PLN and MDLN from mice treated with PS2.5mi-liposomes ( S3 Fig ).…”

“…Only prediabetic 8–12 week old females were included in the study. NOD.Foxp3 EGFP mice were generated in-house using a speed congenic approach by backcrossing B6.Foxp3 EGFP animals into the NOD background while monitoring 15 independent Idd loci as described [ 17 ], and used to study the expansion of Foxp3 + CD4 + T cells. Mice were sacrificed by cervical dislocation.…”

Cholera Toxin Subunit B as Adjuvant––An Accelerator in Protective Immunity and a Break in Autoimmunity

“…The protein is well expressed, with typical yields of purified protein exceeding 100 mg/L of bacterial culture volume in our laboratory. Proper refolding and formation of the pentameric structure needs to be verified e.g., by size exclusion chromatography and by SDS-PAGE under native conditions in which the pentamer maintains its conformation [ 17 ]. The functionality of the pentamer can be tested by its binding capacity to immobilized D-galactose [ 18 ].…”

“…CTB activates murine bone marrow derived dendritic cells and macrophages cells via Toll-like receptor signaling pathways, leading to the expression of chemokines, mainly Th2 (IL-4, IL-10) but also Th1 cytokines (IFN-γ), and inflammatory cytokines (IL-1β) in vitro and in vivo [ 53 , 59 ]. The generation of Th2 versus Th1 cytokines is independent from the route of immunization or the Ag as in several studies using one route and one Ag both types of cytokines were observed simultaneously [ 17 , 59 ]. When CTB and a DNA vaccine encoding for HIV Env were coinjected intramuscularly into mice, the response generated against Env was enhanced 10-fold, approximately, compared to the DNA vaccine administered alone [ 54 ].…”

“…However, in diabetes-resistant F1 mice generated by crossing diabetes prone NOD mice with disease-resistant C57BL/6 mice, oral tolerance, i.e. , the inability of Ag-specific T cells to proliferate in vivo after parenteral immunization, was restored using the above-mentioned prime-boost regimen [ 17 ]. These results show that when analyzing oral tolerance induction by CTB to treat autoimmune diseases, a background truly prone to autoimmunity such as the NOD mouse is needed in order to gain meaningful insights.…”