Alum impairs tolerogenic properties induced by allergoid-mannan conjugates inhibiting mTOR and metabolic reprogramming in human DCs.

ABSTRACT

BACKGROUND: Polymerized allergoids conjugated to mannan (PM) are suitable vaccines for allergen-specific immunotherapy (AIT). Alum remains the most widely used adjuvant in AIT, but its way of action is not completely elucidated. The better understanding of the mechanisms underlying alum adjuvanticity could help to improve AIT vaccine formulations. OBJECTIVE: We sought to investigate the potential influence of alum in the tolerogenic properties imprinted by PM at the molecular level. METHODS: Flow cytometry, ELISAs, cocultures, intracellular staining and suppression assays were performed to assess alum and PM effects in human dendritic cells (DCs). BALB/c mice were immunized with PM alone or adsorbed to alum. Allergen-specific antibodies, splenocyte cytokine production and splenic forkhead box P3 (FOXP3)+ regulatory T (Treg) cells were quantified. Metabolic and immune pathways were also studied in human DCs. RESULTS: Alum decreases PD-L1 expression and IL-10 production induced by PM in human DCs and increases pro-inflammatory cytokine production. Alum impairs PM-induced functional FOXP3+ Treg cells and promotes Th1/Th2/Th17 responses. Subcutaneous immunization of mice with PM plus alum inhibits in vivo induction of Treg cells promoted by PM without altering the capacity to induce functional allergen-specific blocking antibodies. Alum inhibits mTOR activation and alters metabolic reprogramming by shifting glycolytic pathways and inhibiting reactive oxygen species (ROS) production in PM-activated DCs, impairing their capacity to generate functional Treg cells. CONCLUSION: We uncover novel mechanisms by which alum impairs the tolerogenic properties induced by PM, which might well contribute to improve the formulation of novel vaccines for AIT.