Autophagy induced macrophages by α-alumina(α-AL2O3) conjugated cysteine peptidase, enhances the cytotoxic activity of CD8+ T lymphocytes against Leishmania major.

Introduction: Induction of a protective immune response against Leishmania major requires the activation of both TH1 and CD8⁺ T lymphocytes. Because L. major is an intra-phagosomal parasite, its antigens do not have access to MHC-I. The present study aimed to evaluate the effect of cysteine peptidase A (CPA)/cysteine peptidase B (CPB) conjugated to α-AL2O3 on autophagy induction in L. major infected macrophages and subsequent activation of cytotoxic CD8⁺ T lymphocytes.

Methods: Recombinant CPA and CPB of L. major were produced in expression vectors and purified. Aldehyde functionalized α-AL2O3 were conjugated to hydrazine-modified CPA/CPB by a chemical bond was confirmed by Fourier-transform infrared spectroscopy (FTIR). The High efficient internalization of α-AL2O3 conjugated CPA/CPB to macrophages was confirmed using a fluorescence microscope and flowcytometry. Induction of the acidic autophagosome and LC3 conversion in macrophages was determined by acridine orange (AO) staining and western blot. Autophagy-activated macrophages were used for CD8⁺ T cell priming. Cytotoxic activity of the primed CD8⁺ T cell against L. major infected macrophages was measured using apoptosis assay.

Results: α-AL2O3 conjugated CPA/CPB enhances macrophages antigen uptake and increases acidic vacuole formation and LC-3I to LC-3II conversion. Co-culture of autophagy-activated macrophages with CD8⁺ T cells augmented CD8⁺ T cells priming and proliferation more than in other study groups. These primed CD8⁺ T cells induce significant apoptotic death of L. major infected macrophages compared with non-primed CD8⁺ T cells.

Conclusion: α-AL2O3 nanoparticles enhance the cross-presentation of L. major antigens to CD8⁺ T cells by inducing autophagy. This finding supports the positive role of autophagy and encourages the use of α-AL2O3 in vaccine design.