Intrathecal IgG and IgM synthesis correlates with neurodegeneration markers and corresponds to meningeal B cell presence in MS

Abstract

Intrathecal synthesis of immunoglobulins (Igs) is a key hallmark of multiple sclerosis (MS). B cells are known to accumulate in the leptomeninges of MS patients and associate with pathology in the underlying cortex and a more severe disease course. However, the role of locally produced antibodies in MS brain pathology is poorly understood. Here, we quantified the protein levels of IgA, IgM, IgG and albumin in serum and cerebrospinal fluid (CSF) samples of 80 MS patients and 28 neurological controls to calculate Ig indices. In addition, we quantified presence of meningeal IgA⁺, IgM⁺ and IgG⁺ B cells in post-mortem brain tissue of 20 MS patients and 6 controls using immunostainings. IgM and IgG, but not IgA, indices were increased in CSF of MS patients compared to controls, with no observed differences between MS disease types. Both IgM and IgG indices correlated significantly with neurofilament light (NfL) levels in CSF, but not with clinical or radiological parameters of disease. Similarly, IgG⁺ and IgM⁺ B cells were increased in MS meninges compared to controls, whereas IgA⁺ B cells were not. Neuronal loss did not differ between sections with low or high IgA⁺, IgM⁺ and IgG⁺ B cells, but was increased in sections with high numbers of all CD19⁺ meningeal B cells. Similarly, high presence of CD19⁺ meningeal B cells and IgG⁺ meningeal B cells associated with increased microglial density in the underlying cortex. Taken together, intrathecal synthesis of IgG and IgM is elevated in MS, which corresponds to an increased number of IgG⁺ and IgM⁺ B cells in MS meninges. The significant correlation between intrathecal IgG and IgM production and NfL levels, and increased microglial activation in cortical areas adjacent to meningeal infiltrates with high levels of IgG⁺ B cells indicate a role for intrathecal IgM- and IgG-producing B cells in neuroinflammatory and degenerative processes in MS.

Keywords: Antibodies; B cells; Meningeal inflammation; Multiple sclerosis; Neuroinflammation.

Fig. 1

Intrathecal immunoglobulin production in MS patients. (a) IgA, IgM and IgG concentration in CSF of MS patients and controls. (b) IgA, IgM and IgG indices in MS patients and controls, which reflect possible intrathecal production of immunoglobulins. * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Bars represent mean ± SEM. CSF cerebrospinal fluid, ctrl control, MS multiple sclerosis, NINC non-inflammatory neurological control, INC inflammatory neurological control, RRMS relapsing-remitting multiple sclerosis, SPMS  secondary progressive multiple sclerosis, PPMS  primary progressive multiple sclerosis.

Fig. 2

Intrathecal immunoglobulin production correlates with NfL levels in CSF of MS patients. (a) Spearman’s correlation matrix showing correlations between IgA, IgM and IgG indices and clinical and MRI parameters. Colour key represents Spearman’s rho coefficient. (b) Scatter plots between IgM and IgG index and NfL levels in the CSF of MS patients. Plot statistics correspond to all MS patients. Statistically significant correlations were assessed using Spearman’s correlations with false-discovery rate (FDR) correction for multiple testing. * = P ≤ 0.05. EDSS  expanded disability status scale, NfL neurofilament light, CSF cerebrospinal fluid, MS multiple sclerosis, RRMS relapsing-remitting multiple sclerosis, progMS progressive MS.

Fig. 3

IgG- and IgM-producing B cells are enriched in MS leptomeninges. (a) Representative images of MS leptomeninges stained for CD19 (B cells; magenta) and IgA, IgM or IgG (cyan) (IgA, n = 11 ctrl, n = 33 MS; IgM, n = 12 ctrl, n = 26 MS; IgG, n = 12, n = 30 MS). (b) Percentage of total number of meningeal cells that are Ig⁺ CD19⁺ in MS and controls. (c) Percentage of total number of meningeal cells that are CD19⁺ in MS and controls (n = 12, n = 30 MS). (d) Percentage of total number of CD19⁺ cells that are IgA⁺, IgM⁺ and IgG⁺ in MS (IgA, n = 15, IgM = 13, IgG = 21). Each dot represents one tissue section. Statistically significant differences between groups were tested using Mann-Whitney test (b, c) or Kruskal-Wallis followed by Dunn’s multiple comparison test (d). * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001. Bars represent mean ± SEM. Scalebars 25 μm (a left), 10 μm (a right).

Fig. 4

B cells in MS meninges associate with pathology in the underlying cortex in MS. (a) Representative image of MS cortex stained for MOG (myelin; brown) showing a partially demyelinated cortical area. (b) Percentage of cortex that is demyelinated in MS tissue sections with low/absent meningeal CD19⁺ and Ig⁺ CD19⁺ cells and MS tissue sections with high/present CD19⁺ and Ig⁺ CD19⁺ cells. (c) Representative image of MS cortical layer 3 stained for HuC/D (neurons; magenta). (d) Neuronal density in cortical layer 3 in MS tissue sections with low/absent meningeal CD19⁺ and Ig⁺ CD19⁺ cells and MS tissue sections with high/present CD19⁺ and Ig⁺ CD19⁺ cells. (e) Representative image of MS cortex stained for IBA1 (microglia; magenta) and HLA-DR (cyan). (f) Percentage of cortex that is demyelinated in MS tissue sections with low/absent meningeal CD19⁺ and Ig⁺ CD19⁺ cells and MS tissue sections with high/present CD19⁺ and Ig⁺ CD19⁺ cells. (g) Microglial density in the cortex of MS tissue sections with low/absent meningeal CD19⁺ and Ig⁺ CD19⁺ cells and MS tissue sections with high/present CD19⁺ and Ig⁺ CD19⁺ cells. Each dot represents one tissue section; CD19⁺ and IgG, low: n = 13, high: n = 13; IgA⁻: n = 16, IgA⁺: n = 13; IgM⁻: n = 13, IgM⁺: n = 13. Statistically significant differences between groups were tested using Mann-Whitney test and Student’s T-test. * = P ≤ 0.05. Bars represent mean ± SEM. Scalebars 250 μm (a) 100 μm (c, e left), 25 μm (e right). MFI = mean fluorescence intensity.