Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein

www.nature.com/scientificreports OPEN Novel B cell-dependent multiple sclerosis model using extracellular domains of myelin proteolipid protein Alexander W. Boyden1,2, Ashley A. Brate1,2 & Nitin J. Karandikar1,2* Therapeutic success of B cell-targeting approaches in multiple sclerosis (MS) has intensified research into the pathogenic and regulatory roles these cells play in demyelinating disease. Dissecting the function of B cells in the MS mouse model experimental autoimmune encephalomyelitis (EAE) is largely confined to induction with either the myelin oligodendrocyte glycoprotein epitope MOG35–55 or the fulllength recombinant human MOG protein, the latter representing the most-used B cell-dependent EAE model. There is a clear need to investigate B cell function in additional myelin antigen contexts. Unlike MOG35–55, where lack of B cells yields more severe disease, we show here that the immunodominant myelin proteolipid protein epitope (PLP178–191) elicited identical EAE in WT and μMT mice, suggesting an absence of B cell engagement by this peptide. We hypothesized that a longer PLP antigen may better engage B cells and designed a peptide encompassing the extracellular domains (ECD) of PLP. We demonstrate here that PLPECD-immunized B cell-deficient mice failed to exhibit EAE. In contrast, PLPECD induced EAE not only in WT mice, but in B cell-sufficient mice incapable of secreting antibodies, suggesting a predominant antigen presentation role. These results establish a novel, efficient B celldependent EAE model. Recent clinical trials involving the targeted deletion of B cells1–3 have reinvigorated intense interest in the role of this lymphocyte in immune-mediated demyelinating diseases such as multiple sclerosis (MS). It is essential to have multiple models, whereby various angles of B cell biology (such as their potential pathogenic or regulatory roles) can be deciphered in the context of disease. Historically, myelin oligodendrocyte glycoprotein (MOG) sequence 35–55 has been used to great success in inducing demyelinating disease in WT B6 mice4,5, where knockout capabilities and genetic tools like the 2D2 system (mice harboring a TCR transgene specific for the MOG35–55 epitope) are available6. B cell studies in MOG-induced demyelinating disease have led to a contradictory literature, in that there is evidence for both pathogenic and regulatory roles, as well as models that require and yet do not require the presence of B cells for EAE. These discrepancies can largely be explained by differences in model type, animal strain, and inciting antigen used. For instance, antibody titers and B cell numbers in the cerebrospinal fluid (CSF) have been shown to correlate with disease severity in mice and humans7–10. Likewise, full length recombinant human MOG (hMOG)-reactive serum transfers from B6 mice have been shown to cause disease in healthy animals11. Decreased demyelination in B cell-deficient versus WT B6 mice has been observed (yet both exhibited disease)12 while other data demonstrate that induction of EAE failed altogether in B cell-deficient mice13. Interestingly, these studies utilized rodent MOG versus human MOG, respectively. It has further been demonstrated that deleting B cells from MOG35–55 EAE mice during peak disease ameliorates clinical symptoms14. In addition to pathogenic antibody studies, B cells have been shown to contribute to rodent MOG-induced EAE in C3H mice as well as hMOG-induced EAE in B6 mice by reactivating CD4 T cells in the CNS through a likely antigen presentation function15–18. These reports of B cell pathogenicity notwithstanding, it has also been demonstrated that B cell-deficient mice cannot recover from myelin basic protein-induced EAE disease in B10.PL mice19 and have a quicker and more robust MOG35–55 and rodent MOG disease onset compared to their WT B6 counterparts20,21. 1 Department of Pathology, University of Iowa Health Care, Iowa City, IA, USA. 2Iowa City Veterans Affairs Medical Center, Iowa City, IA, USA. *email: Scientific Reports | (2020) 10:5011 | https://doi.org/10.1038/s41598-020-61928-w 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Further, deletion of B cells prior to MOG35–55 disease onset led to exacerbated disease14, adding to studies attributing regulatory function to B cells during EAE. These data clearly suggest that B cell subsets have varying functional capacities (IL-10-producing Bregs20 versus CD4 T cell-reactivating CNS B cells15, for example). However, the general discrepancies seen in these reports, particularly regarding experiments involving B cell-deficient mice, can be explained by differences in the length of MOG antigen used for immunization (short peptide versus full-length recombinant protein), and the origin of MOG antigen (rodent versus human). It turns out that a single amino acid at position 42 (serine in the rodent, proline in the human) in the MOG sequence22,23 can have striking consequences for the role of B cells in MOG-induced EAE models. Ultimately, it is understood that recombinant hMOG is necessary to induce a robust B cell-dependent mouse model of MS13,22,23. Yet it is still unclear whether this is due to pathogenic antibody production or requirement for antigen processing and presentation. In addition, production of the hMOG protein is cumbersome and represents a significant rate-limiting step in the study of B cell functionality in vivo during demyelinating disease. Finally, heavy reliance on a single model may bias our understanding of the role of these cells in complex human disease. Investigating the role of B cells in myelin proteolipid protein (PLP)-induced EAE models is needed. PLP is highly conserved, where complete amino acid sequence homology is shared between mice and humans, and is very abundant in the CNS, comprising 50% of total myelin protein24. Indeed, understanding immune responses in this context is extremely relevant and may provide insights into the pathogenicity and regulation of MS. Whereas PLP-targeted responses are a focus in the relapsing-remitting SJL mouse model of EAE25,26, this protein or its peptides are underutilized in the context of B6 models, where there is a greater availability of genetic tools to dissect the function of various cells and molecules. We have recently utilized PLP178–191-induced EAE in B6 mice to demonstrate the robust disease regulatory role of PLP178–191-induced CD8 T cell responses27–30. We therefore decided to focus on PLP-induced EAE to develop a model wherein B cell function could also be delineated. Interestingly, our findings here suggest that B cells are ancillary during the immunodominant PLP178– 191-incduced form of EAE in B6 mice. We hypothesized that designing a longer peptide may provide a processable antigen for B cells to engage and present to T cells, thus alleviating the need for intricate whole protein production methodology. Here, we utilized a novel designed peptide (PLPECD, an 8 (...truncated)