Characterization of B Regulatory Cell-Derived-Interleukin-35 Containing Exosomes and Their Immunosuppressive Effect

Abstract

Background: IL-35 has emerged as a potent immunosuppressive cytokine that plays a role in regulating autoimmune and inflammatory diseases. IL-35 production was thought to be restricted to T regulatory cells (Tregs) until the discovery of IL-35 producing B regulatory cells (i35-Bregs). The difficulty of ex-vivo production of bioactive heterodimeric IL-35 has impeded the use of IL-35 for immunotherapy. On the other hand, i35-Bregs provide advantages over therapeutic administration of IL-35 because they proliferate in vivo and can sustain delivery of IL-35 in target tissues. Therefore, understanding cues required for the development of i35-Bregs will facilitate optimization of their expansion ex-vivo for therapeutic purposes. Although the role of B-cell receptor (BCR) in the development of regulatory B cells including B10 and LAG-3 expressing natural plasma cells is well established, little is known about the role of BCR in the development of i35-Bregs. In this study, we examined different stimuli including BCR stimulation to induce i35-Breg expansion. We also characterized IL-35 expression by i35-Bregs to better understand the influence of different signals on the expression of IL35 subunits. There are many unsolved questions regarding IL-35 biology and i35-Breg mechanisms of action. Unlike IL-12 and IL-23, whose subunits are linked by disulfide bond, IL-35 subunits (EBI3 and IL-12p35) were presumed to be secreted independently and bind to each other by unknown mechanisms. Recently, it was reported that Tregs express cell-surface IL-35 and release IL-35 bound to CD81 in exosomes. This observation raised the question of whether IL-35 subunits are also expressed on the surface of i35-Bregs in association with CD81, and if IL-35 is secreted in exosomes, that contribute to i35-Bregs’ mechanism of action. Exosomes are small extracellular vesicles (50-150 nm in diameter) that readily cross the blood brain barrier (BBB) or brain-retina-barrier (BRB) and are therefore desirable vehicles to deliver biologics into CNS tissues. In this study, we investigated; (i) the expression of IL35 on the surface of i35-Bregs, (ii) the association of EBI3 and IL-12p35 subunits with CD81, (iii) the secretion of IL-35 containing exosomes (i35-exosomes) by i35-Bregs, (iv) therapeutic potential of i35-exosomes in CNS autoimmune diseases. Methods: To determine the most optimal method for inducing i35-Breg expansion, we stimulated B cells with LPS, LPS + anti-CD40, anti-CD40, or anti-IgM (BCR) + anti-CD40, and examined i35-Breg induction by flow cytometry. We characterized the expression of IL-35 and the association of IL-35 subunits with CD81 by immunofluorescence staining and proximity ligation assay (PLA). Exosomes were isolated from the supernatant of i35-Breg culture by using ExoQuick ULTRA kit and their sizes were characterized by Nanoparticle Tracking Analysis and Tunable Resistive Pulse Sensing. To determine whether the exosomes contain IL-35, we used Western blotting to detect IL-35 subunits (IL-12p35 and EBI3). Finally, we assessed the effect of heterodimeric IL-35 and i35-exosome treatment on encephalitogenic T cells and regulatory lymphocytes by flow cytometry. Results: Stimulating of B cells from naïve mice with a low concentration of anti-IgM (BCR) in combination with anti-CD40 induced the expansion of i35-Bregs up to ~44% of total B cells, underscoring the role of BCR signaling strength in the induction of i35-Bregs. The i35-Bregs showed a polarized expression of IL-35 and colocalization of IL-35 subunits with CD81 on their plasma membrane. Supernatant of B cells stimulated with anti-IgM+anti-CD40 contained extracellular vesicles (EVs) of 50-150 nm in size that express the exosomes markers CD81, CD9, and CD63. Importantly, the exosomes contained IL-35 (i35-exosomes). While i35-Bregs/i35-exosomes generated in response to activation by anti-IgM+ anti-CD40 contain more IL-12p35, i35-Bregs/i35-exosomes generated in response to stimulation by LPS are enriched with EBI3 subunit, suggesting differential expression of IL-35 subunits depending on the stimuli. Importantly, IL-35 or i35-exosomes inhibited encephalitogenic T cells while inducing the expansion of regulatory lymphocytes. Conclusions: Data presented in this study show that stimulating B cells from naïve mice with a combination of anti-IgM+anti-CD40 is optimal for the expansion of i35-Bregs compared to other stimuli, establishing role of BCR signaling in i35-Breg development. Furthermore, IL-35 was found to be a membrane-bound cytokine in i35-Breg cells suggesting that cell-cell contact might underlie immunomodulatory functions mediated by i35-Bregs. i35-exosomes-mediated suppression of encephalitogenic T cells further indicates that i35-Bregs and the exosomes they secrete can be exploited for the treatment of CNS autoimmune diseases.