Chaitrali Saha1, Amy Zhang1, Kun Leng2, Chloe Gerungan2, Ahmed Abdelhak2, Refujia Gomez2, Meagan Harms2, Asritha Tubati2, Tiffany Cooper2, Martineau Louine2, Joseph Sabatino2, Martin Kampmann2, Ari Green2, Jorge Oksenberg2, Bruce Cree3, Michael Wilson2, Stephen Hauser1
1UCSF Weill Institute for Neurosciences, 2University of California San Francisco, 3UCSF, Multiple Sclerosis Center
Objective:
To assess the effects of secreted factors from human B cells on neuronal viability
in vitro.
Background:
In multiple sclerosis (MS), B cell rich lymphoid aggregates form in the meninges adjacent to cerebral sulci, and contribute to underlying cortical neurodegeneration, demyelination, and clinical progression. Little is known regarding how these B cells produce pathology.
Design/Methods:
CD19+ B cells were isolated from peripheral blood of treatment-naïve MS patients and age/sex matched healthy controls (HC), and cultured under unstimulated conditions, or following stimulation with anti-IgM/G/A and recombinant human CD40L with or without CpG. After 3 days, supernatants were added to human induced pluripotent stem cell (iPSC) derived day 14 glutamatergic neurons (iNeurons). Neurotoxicity was quantitated by thiazol red staining and neurofilament light chain levels. Size fractionation, Ig depletion, and blocking experiments were performed to identify factors responsible for the B cell mediated toxicity.
Results:
Unstimulated B cell supernatants were consistently neurotoxic to iNeurons. In approximately half of MS subjects, levels of neurotoxicity were higher than in HC and were present under unstimulated culture conditions, as reported earlier using different assays (Lisak et al. J. Neuroimmunol 309:88, 2017). Selective depletion and fractionation studies indicated that B cell mediated neurotoxic activities were predominantly present in the low molecular weight (e.g. lower than 30kD) fractions and were absent from Ig-containing or high molecular weight (including exosome) supernates. Increased B cell mediated neurotoxicity in MS was associated with production of higher levels of proinflammatory molecules, including TNF-α, IL-8, RANTES, MDC, and MIP-1β, and neurotoxicity could be partially blocked by inhibitors of TNF-α.
Conclusions:
B cells can directly injure nearby neurons through secreted factors including TNF-α. These data build upon evidence that the B cell transcriptome is fundamentally dysregulated in MS, and demonstrate that one consequence may be a more neurotoxic secretome.