Specific recognition of extracellular vesicles by antibodies modulates the function of immune cells in a Fc-gamma receptor-dependent manner

OTERO, ADRIÁN; PÉREZ, PAULA; RUSSO, CONSTANZA; SEERY, VANESA; SANANEZ, INÉS; OSTROWSKI, MATÍAS; ARRUVITO, LOURDES

San Luis

Congreso; Reunión Anual SAI 2023; 2023

SAI

Background:Extracellular vesicles (EVs) are nanometer-sized, lipid membraneenclosed vesicles involved in cell-to-cell communication thatinfluence both physiological and pathological conditions. Owing totheir ability to transfer bioactive components and surpass biologicalbarriers, EVs are increasingly explored as therapeutics. However,whether EVs can form immune complexes binding antibodies (IC), and ifso, what function might exert, is still unknown. Aim:To determine the functional consequences of antibody recognition ofan EV-surface antigen on Fc-γ receptor (FcγR) carrying immunecells. Methods:A B-cell lymphoma cells (Ramos cells) that release EVs carrying CD20and the therapeutic anti-CD20 antibody Rituximab (RTX) were used togenerate IC (EVs+RTX). EVs were isolated by ultracentrifugationtechnique and characterized by western blot, beads-based flowcytometry, Nanoparticle-tracking analysis (NTA) and TransmissionElectron Microscopy (TEM). Formation of the IC was confirmed by flowcytometry and labeling with protein A–immunogold microscopy.Purified neutrophils were exposed to EVs and/or IC and reactiveoxygen species (ROS) production was analyzed. NK cell-mediatedcytotoxicity was evaluated on labeled-K562 cells in presence of EVsand/or IC by flow cytometry. Results:Isolated EVs derived from Ramos cells presented bona fidecharacteristics of EVs, ie, cup-shaped appearance in electronmicroscopy, enrichment for EVs markers proteins ALIX, CD63, CD81,HLA-DR and CD107aand a size distribution of 90,53 ± 27,01 nm.Importantly,the EVs also carried the B-cell plasma membrane protein CD20 andmirrored the expression of this protein in the parental cells. Weconfirmed that these EVs, upon exposure to the therapeutic anti-CD20antibody, formed immune complexes, as revealed by immunogold/TEM(n=1) and beads-based flow cytometry (n=5). We also found that theexposure to EVs promoted both spontaneous and PMA-induced ROSproduction in neutrophils (p<0.01, n=9) that was enhanced in thepresence of the IC (n=4). While isolated EVs impaired the NKcell-mediated cytotoxicity (p<0.001, n=9), the addition of the ICpromoted it (p<0.01). Blocking NK cells with CD32 and CD16neutralizing antibody previous to the IC stimulation diminished thecytotoxicity rate showing that the effect observed is, at least inpart, due to CD32 and CD16 specific activation.Conclusions:Collectively, our data describe that specific recognition of an EVantigen by antibodies modify the functionality of the vesicles uponinteraction with Fc-gamma receptor bearing cells. Further studies areneeded to assess how this novel concept combines the effect of EVs asdelivered agents with monoclonal antibody therapy. p { line-height: 115%; text-align: left; orphans: 2; widows: 2; margin-bottom: 0.25cm; direction: ltr; background: transparent }p.western { font-family: "Calibri", serif; so-language: en-US }p.cjk { font-family: "Calibri"; so-language: es-AR }p.ctl { font-family: "Calibri" }