TLR-5 ligand conjugated with Per a 10 and T cell peptides potentiates Treg/Th1 response through PI3K/mTOR axis

Novel strategies are needed to enhance efficacy of immunotherapy for allergic diseases. The present study evaluates role of conjugating flagellin with allergen, Per a 10 and its T cell epitopes in DC mediated polarization. Fusion proteins (FPer a 10, FPT1, FPT2 and FPT3) were sub-cloned, expressed and purified to homogeneity. FlaA fused proteins showed TLR-5 binding in TLR5⁺HEK293 cells as observed by enhanced NF-κB dual luciferase assay and IL-8 secretion in culture supernatant. Fusion proteins upregulate co-stimulatory molecules CD80, CD83 and CD86 on BMDCs and enhanced IL-6, IL-β, IL-12p70 and IL-10 secretion. Inhibition studies suggest fusion proteins mediate IL-10 secretion via activation of PI3K/mTOR/NF-κB pathway. FPT1 and FPT3 deviate Th2 cytokine milieu towards Th1/Treg by upregulating the expression of IFN-γ, IL-12p70, IL-10 and TGF-β in BMDCs co-culture with allergen specific T cells. Flow cytometric examination of BMDC:T cell co-culture revealed CD4⁺ T cell differentiation towards Th1/Treg by FPT1 and FPT3. Thus, our results indicate that flagellin fused with T cell peptides are efficient immunomodulators and may possess potential for allergy therapeutics.