Background: B cells mediate pathogenesis in multiple sclerosis (MS) by mechanisms unrelated to immunoglobulin (Ig) production. Supernatants (Sup) from cultured MS B cells but not controls are cytotoxic to oligodendrocytes (OL) and neurons (Lisak et al. 2012, 2017). Killing is independent of complement, and does not correlate with Sup levels of IgG, IgM or cytokines tested. Death of OL and neurons involves apoptosis (Lisak et al. 2017) and is mediated by factors in exosome-enriched fractions (Ex-En) (Benjamins et al. 2019).
Objective: To investigate how Ex-En released by cultured unstimulated peripheral blood B cells from MS patients kill OL.
Methods: B cells were cultured in exosome-depleted serum-free medium. Ex-En were prepared from Sup by ultracentrifugation. Sup or Ex-En were diluted 1:4 with OL culture medium and tested for toxicity on rat OL. Proteomic analysis was performed on Sup and Ex-En.
Results: MS B cell Sup kill OL primarily by caspase-dependent pathways and are toxic to OL in both mixed glial and OL-enriched cultures, suggesting direct action on OL. Toxicity is reduced by activation of melanocortin and sigma-1 receptors, implicating cAMP and IP3 pathways in protection. We developed methods for reliable proteomic analysis of the low amounts of protein in Ex-En, and a strategy for RNASeq, lipidomic and integrated bioinformatic analyses. Feasibility studies in progress will give a sample-size estimate based on analysis of variability for detection of significant differences between MS and control.
Conclusions: A multi-omics approach may allow identification of candidates responsible for toxicity to OL in Ex-En from MS B cells.