Abstract
Although B cell depletion is an effective therapy of multiple sclerosis (MS), the pathogenic functions of B cells in MS remain incompletely understood. We asked whether cerebrospinal fluid (CSF) B cells in MS secrete different cytokines than control-subject B cells and whether cytokine secretion affects MS phenotype. We blindly studied CSF B cells after their immortalization by Epstein-Barr Virus (EBV) in prospectively-collected MS patients and control subjects with other inflammatory-(OIND) or non-inflammatory neurological diseases (NIND) and healthy volunteers (HV). The pilot cohort (n = 80) was analyzed using intracellular cytokine staining (n = 101 B cell lines [BCL] derived from 35 out of 80 subjects). We validated differences in cytokine production in newly-generated CSF BCL (n = 207 BCL derived from subsequent 112 prospectively-recruited subjects representing validation cohort), using ELISA enhanced by objective, flow-cytometry-based B cell counting. After unblinding the pilot cohort, the immortalization efficiency was almost 5 times higher in MS patients compared to controls (p < 0.001). MS subjects' BCLs produced significantly more vascular endothelial growth factor (VEGF) compared to control BCLs. Progressive MS patients BCLs produced significantly more tumor necrosis factor (TNF)-α and lymphotoxin (LT)-α than BCL from relapsing-remitting MS (RRMS) patients. In the validation cohort, we observed lower secretion of IL-1β in RRMS patients, compared to all other diagnostic categories. The validation cohort validated enhanced VEGF-C production by BCL from RRMS patients and higher TNF-α and LT-α secretion by BCL from progressive MS. No significant differences among diagnostic categories were observed in secretion of IL-6 or GM-CSF. However, B cell secretion of IL-1β, TNF-α, and GM-CSF correlated significantly with the rate of accumulation of disability measured by MS disease severity scale (MS-DSS). Finally, all three cytokines with increased secretion in different stages of MS (i.e., VEGF-C, TNF-α, and LT-α) enhance lymphangiogenesis, suggesting that intrathecal B cells directly facilitate the formation of tertiary lymphoid follicles, thus compartmentalizing inflammation to the central nervous system.
Highlights
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) thought to be mediated by myelin-specific CD4+ T cells [1, 2]
After unblinding the diagnostic codes, the n = 80 pilot cohort consisted of 47 MS patients (35 RRMS, 9 PPMS and 3 secondary-progressive MS (SPMS)) and 33 controls (14 other inflammatory neurological diseases (OIND) and 19 non-inflammatory neurological diseases (NIND))
From cytokines that were not measured in the pilot cohort, we found no significant differences among diagnostic categories in the B-cell secretion of GM-cerebrospinal fluid (CSF) (Table 3)
Summary
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) thought to be mediated by myelin-specific CD4+ T cells [1, 2]. Whereas depletion of CD4+ T cells does not reduce formation of MS lesions [3], depletion of B cells is strikingly more effective [4]. Intrathecal activation of B cells and their differentiated, antibody (Ab)-producing counterparts (plasmablasts and plasma cells) is the hallmark of MS [7, 8]. Pathologists identified B cells in the meningeal tissue, in the form of diffuse infiltrates in most MS subjects [9] or tertiary lymphoid follicles in some secondary-progressive MS (SPMS) patients [10, 11]. The same authors demonstrated that meningeal follicles harbor Epstein-Barr Virus (EBV) [12, 13], a finding not reproduced by others [14]. EBV remains one of the best documented environmental links in MS [15] with a possible causal role [16, 17]
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