SPECIFIC RECOGNITION OF EXTRACELLULAR VESICLES BY ANTIBODIES MODULATES THE FUNCTION OF IMMUNE CELLS IN A FC-GAMMA RECEPTOR-DEPENDENT MANNER

OTERO, ADRIÁN; PEREZ P; RUSSO C; SEERY V; SANANEZ, I; OSTROWSKI M; ARRUVITO L

Congreso; Reunión Anual de SAI 2023; 2023

Background: Extracellular vesicles (EVs) are nanometer-sized, lipid membrane enclosed vesicles involved in cell-to-cell communication that influence both physiological and pathological conditions. Owing to their ability to transfer bioactive components and surpass biological barriers, EVs are increasingly explored as therapeutics. However, whether EVs can form immune complexes binding antibodies (IC), and if so, what function might exert, is still unknown. Aim: To determine the functional consequences of antibody recognition of an EV-surface antigen on Fc-γ receptor (FcγR) carrying immune cells. Methods: A B-cell lymphoma cells (Ramos cells) that release EVs carrying CD20 and the therapeutic anti-CD20 antibody Rituximab (RTX) were used to generate IC (EVs+RTX). EVs were isolated by ultracentrifugation technique and characterized by western blot, beads-based flow cytometry, Nanoparticle-tracking analysis (NTA) and Transmission Electron Microscopy (TEM). Formation of the IC was confirmed by flow cytometry and labeling with protein A–immunogold microscopy. Purified neutrophils were exposed to EVs and/or IC and reactive oxygen species (ROS) production was analyzed. NK cell-mediated cytotoxicity was evaluated on labeled-K562 cells in presence of EVs and/or IC by flow cytometry. Results: Isolated EVs derived from Ramos cells presented bona fide characteristics of EVs, ie, cup-shaped appearance in electron microscopy, enrichment for EVs markers proteins ALIX, CD63, CD81, HLA-DR and CD107a and a size distribution of 90,53 ± 27,01 nm. Importantly, the EVs also carried the B-cell plasma membrane protein CD20 and mirrored the expression of this protein in the parental cells. We confirmed that these EVs, upon exposure to the therapeutic anti-CD20 antibody, formed immune complexes, as revealed by immunogold/TEM (n=1) and beads-based flow cytometry (n=5). We also found that the exposure to EVs promoted both spontaneous and PMA-induced ROS production in neutrophils (p