Selective knockdown of A2AR in CD8+ T cells using CD8-targeting nanoliposomes

Abstract

Abstract Adenosine accumulates in the tumor microenvironment and contributes to the inhibition of T cell function via the A2A receptor (A2AR). Blockade of the A2AR has been shown to rescue T cell function and decrease tumor burden. However, A2AR is present in a variety of cells and thus a limitation of non-selective A2AR pharmacological blockers is the development of side effects. Our goal is to selectively knockdown the A2AR in cytotoxic CD8+ T cells through lipid nanoparticles (NPs) decorated with anti-CD8 antibody and containing A2AR siRNAs. The A2AR knockdown by A2AR siRNAs was verified in human peripheral blood T cells (PBT). Nucleofection of PBTs with A2AR or scramble siRNAs downregulated mRNA and protein by 61% and 14–42%, respectively (as determined by RT-qPCR and flow cytometry). A functional assay for IL-2 production (a cytokine produced by activated PBTs and suppressed by the A2AR pathway) in PBTs treated with siRNAs and activated in the presence of CGS21680 (an A2AR agonist) was used to verify the A2AR knockdown. Treatment of PBTs with A2AR siRNAs prevents the CGS21680-induced inhibition of IL-2 production by 27%. Additionally, NPs were fabricated using biotinylated lipids as previously described (Hajdu et al., Biomaterials 2013) and labeled with anti-CD8 antibody. Their selectivity and internalization were determined. Flow cytometry revealed that CD8-labeled NPs are specific to CD4− PBTs and suggested that the NPs are subsequently internalized. Thus, these data indicate the feasibility of fabrication and potential efficacy of CD8-specific A2AR siRNAs-loaded NPs that should restore the functionality selectively in CD8+ tumor infiltrating lymphocytes—a potential targeted cancer therapy with limited side effects.