Early B cell tolerance defects in neuromyelitis optica favour anti-AQP4 autoantibody production

Abstract

Neuromyelitis optica spectrum disorders (NMOSD) constitute rare autoimmune disorders of the CNS that are primarily characterized by severe inflammation of the spinal cord and optic nerve. Approximately 75% of NMOSD patients harbour circulating pathogenic autoantibodies targeting the aquaporin-4 water channel (AQP4). The source of these autoantibodies remains unclear, but parallels between NMOSD and other autoantibody-mediated diseases posit compromised B cell tolerance checkpoints as common underlying and contributing factors. Using a well established assay, we assessed tolerance fidelity by creating recombinant antibodies from B cell populations directly downstream of each checkpoint and testing them for polyreactivity and autoreactivity. We examined a total of 863 recombinant antibodies. Those derived from three anti-AQP4-IgG seropositive NMOSD patients (n = 130) were compared to 733 antibodies from 15 healthy donors. We found significantly higher frequencies of poly- and autoreactive new emigrant/transitional and mature naïve B cells in NMOSD patients compared to healthy donors (P-values < 0.003), thereby identifying defects in both central and peripheral B cell tolerance checkpoints in these patients. We next explored whether pathogenic NMOSD anti-AQP4 autoantibodies can originate from the pool of poly- and autoreactive clones that populate the naïve B cell compartment of NMOSD patients. Six human anti-AQP4 autoantibodies that acquired somatic mutations were reverted back to their unmutated germline precursors, which were tested for both binding to AQP4 and poly- or autoreactivity. While the affinity of mature autoantibodies against AQP4 ranged from modest to strong (Kd 15.2-559 nM), none of the germline revertants displayed any detectable binding to AQP4, revealing that somatic hypermutation is required for the generation of anti-AQP4 autoantibodies. However, two (33.3%) germline autoantibody revertants were polyreactive and four (66.7%) were autoreactive, suggesting that pathogenic anti-AQP4 autoantibodies can originate from the pool of autoreactive naïve B cells, which develops as a consequence of impaired early B cell tolerance checkpoints in NMOSD patients.

Keywords: AQP4; B cells; autoantibodies; neuromyelitis optica spectrum disorder (NMOSD); tolerance.

Figure 1

Central B cell tolerance is compromised in patients with NMOSD. Recombinant antibodies (rIgGs) derived from new emigrant/transitional B cells from three NMOSD patients were compared to those derived from 15 healthy controls. Antibodies were tested for polyreactivity on a solid-phase ELISA against three structurally distinct antigens: double-stranded DNA (dsDNA), lipopolysaccharide (LPS), and insulin. The recombinant IgGs were tested at a maximum concentration of 1.0 µg/ml (shown here) and three additional 4-fold serial dilutions (Supplementary Fig. 2). (A) Representative ELISA data from the NMOSD and healthy control groups are shown. Polyreactivity results for six individuals are summarized in the 3D plots. LPS and dsDNA absorbance values are plotted along the axes; insulin absorbance is indicated by diamond size. Each point represents a mean of experimental duplicates. Boxed area mark the positive reactivity cut-off at OD₄₀₅ 0.5. Polyreactive recombinant IgGs were defined as those that bound all three antigens (dsDNA, LPS, insulin) above the cut-off. Filled diamonds represent polyreactive recombinant IgGs; non-polyreactive recombinant IgGs are represented by open diamonds. (B) The frequency of polyreactive recombinant IgGs per subject is represented in the corresponding pie charts. Black shading indicates the polyreactive antibody frequency (%). The number in the centre of the pie chart represents the total number of individual recombinant IgGs tested. Data from the three NMOSD subjects are compared to three representative examples from the HD cohort. (C) Polyreactive antibody frequencies in the NMOSD and healthy control cohorts. The frequency of polyreactive antibodies was plotted for each subject along with the mean and standard deviation for each subject group. Statistical differences are shown when significant. (D) Frequencies of polyreactive new emigrant/transitional B cells in seven distinct autoimmune diseases and healthy control cohorts. Proportions of polyreactive antibodies expressed by new emigrant/transitional B cells were plotted for each subject group along with the mean and standard deviation for each subject group. Statistical differences are shown when significant (****P < 0.0001; ***P ≤ 0.001; **P ≤ 0.01). MG = myasthenia gravis; MS = multiple sclerosis; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; SS = Sjögren’s syndrome.

Figure 2

Accumulation of polyreactive mature naïve B cells in the blood of patients with NMOSD. Recombinant antibodies (rIgGs) derived from mature naïve B cells from three NMOSD patients were compared to those derived from 15 healthy controls. Antibodies were tested for polyreactivity on a solid-phase ELISA against three structurally distinct antigens: double-stranded DNA (dsDNA), lipopolysaccharide (LPS), and insulin. The recombinant IgGs were tested at a maximum concentration of 1.0 µg/ml (shown here) and three additional 4-fold serial dilutions (Supplementary Fig. 3). (A) Representative ELISA data from the NMOSD and healthy control groups are shown. Polyreactivity results for six individuals are summarized in the 3D plots. LPS and dsDNA absorbance values are plotted along the axes; insulin absorbance is indicated by diamond size. Each point represents a mean of experimental duplicates. Boxed areas at OD₄₀₅ 0.5 mark the positive reactivity cut-off. Filled diamonds represent polyreactive recombinant IgGs; non-polyreactive recombinant IgGs are represented by open diamonds. (B) The frequency of polyreactive recombinant IgGs per subject is represented in the corresponding pie charts. Black shading indicates the polyreactive antibody frequency (%). The number in the centre of the pie chart represents the total number of individual recombinant IgGs tested. Data from the three NMOSD subjects are compared to three representative examples from the HD cohort. (C) Polyreactive antibody frequencies in the NMOSD and healthy control cohorts. The frequency of polyreactive antibodies was plotted for each subject along with the mean and standard deviation for each subject group. Statistical differences are shown when significant. (D) Frequencies of polyreactive mature naïve B cells in seven distinct autoimmune diseases and healthy controls. Proportions of polyreactive antibodies expressed by mature naïve B cells were plotted for each subject group along with the mean and standard deviation for each subject group. Statistical differences are shown when significant (****P < 0.0001; ***P ≤ 0.001; **P ≤ 0.01). MG = myasthenia gravis; MS = multiple sclerosis; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; SS = Sjögren’s syndrome.

Figure 3

The peripheral B cell tolerance checkpoint is impaired in patients with NMOSD. Recombinant antibodies (rIgGs) derived from mature naïve B cells from the three NMOSD patients were compared to those derived from 15 healthy controls. Purified antibodies were tested for autoreactivity on a solid-phase ELISA against human epithelial type 2 (HEp-2) cell lysate. (A) Representative ELISA data from the three NMOSD patients and healthy control groups are shown. Antibody reactivity to HEp-2 lysate is illustrated by the binding curves. ED38, a monoclonal antibody cloned from a VpreB+L+ peripheral B cell, was used as a positive control and shown by the dotted line curves. Solid line curves represent patient and control-derived antibodies. Each data point represents the mean of experimental duplicates. Dotted horizontal lines mark the positive reactivity cut-off at OD₄₀₅ 0.5. For each subject, the total number of antibodies tested and the percentage of which displayed autoreactivity, as determined through HEp-2 lysate binding, is displayed in the corresponding pie charts. (B) Autoreactive antibody frequencies in the NMOSD and healthy control cohorts. The frequency of autoreactive antibodies was plotted for each subject along with the mean and standard deviation for each subject group. Statistical differences are shown when significant. (C) Frequencies of autoreactive mature naïve B cells in seven distinct autoimmune diseases and healthy controls. Proportions of polyreactive antibodies expressed by mature naïve B cells were plotted for each subject group along with the mean and standard deviation for each subject group. Statistical differences are shown when significant (****P < 0.0001; ***P ≤ 0.001; **P ≤ 0.01). MG = myasthenia gravis; MS = multiple sclerosis; RA = rheumatoid arthritis; SLE = systemic lupus erythematosus; SS = Sjögren’s syndrome.

Figure 4

Anti-AQP4 autoantibodies and their unmutated revertants contain both polyreactive and autoreactive clones. Anti-AQP4 autoantibodies (mAb) and their unmutated revertants (mAb-R) were tested for polyreactivity on a solid-phase ELISA against three structurally distinct antigens: double-stranded DNA (dsDNA), lipopolysaccharide (LPS), and insulin. Antibodies were tested at a maximum concentration of 1.0 µg/ml (shown here) and three additional 4-fold serial dilutions (Supplementary Fig. 7). Polyreactivity results are summarized in the 3D plots. LPS and dsDNA absorbance values are plotted along the axes; insulin absorbance is indicated by diamond size. Each point represents a mean of experimental duplicates. Boxed areas at OD₄₀₅ 0.5 mark the positive reactivity cut-off. Polyreactive recombinant IgGs were defined as those that bound all three antigens (dsDNA, LPS, insulin) above the cut-off. Filled diamonds represent polyreactive recombinant IgGs; non-polyreactive recombinant IgGs are represented by open diamonds. The frequency of polyreactive antibodies is represented in the corresponding pie charts. Black shading indicates the polyreactive antibody frequency (%). The number in the centre of the pie chart represents the total number of unique antibodies tested. (A) mutated anti-AQP4 autoantibodies; (B) unmutated reverted antibodies. Purified anti-AQP4 autoantibodies (mAb) and their unmutated revertants (mAb-R) were tested for autoreactivity on a solid-phase ELISA against human epithelial type 2 (HEp-2) cell lysate. Antibody reactivity to HEp-2 lysate is illustrated by the binding curves. Solid line curves represent AQP4 mAbs and mAb-Rs. ED38, a monoclonal antibody cloned from a VpreB+L+ peripheral B cell, was used as a positive control; L50, a monoclonal antibody cloned from a mature naïve B cell of a uracil N-glycosylase-deficient patient was used as a negative control. Dotted line curves represent ED38 and L50. Each data point represents the mean of experimental duplicates. Dotted horizontal lines mark the positive reactivity cut-off at OD₄₀₅ 0.5. For both the mAbs and the mAb-Rs, the total number of antibodies tested and the percentage of which displayed autoreactivity, as determined through HEp-2 lysate binding, is displayed in the corresponding pie charts. (C) Mutated anti-AQP4 autoantibodies; (D) unmutated reverted antibodies.

Figure 5

Unmutated precursors of the anti-AQP4-specific autoantibodies do not bind to AQP4. Anti-AQP4 autoantibodies (mAb) and their unmutated revertants (mAb-R) were tested for surface binding to the AQP4 M23 isoform on AQP4-transfected U87MG cells. Representative binding curves from which affinity values were calculated for two autoantibodies (mAb) and their respective unmutated revertants are shown. The y-axis represents ratios of bound antibody to cell surface AQP4 (rAb/AQP4) as means with their respective standard errors. The x-axis represents the various concentrations (nM) at which mAbs and mAb-Rs were tested. Data are fit using a single site total binding model. Detailed K_d results for all of the autoantibodies and their unmutated revertants are presented in Table 2.

Figure 6

Schematic diagram illustrating the potential consequence of defective B cell tolerance checkpoints in the development of NMOSD autoantibodies. During early B cell development, immunoglobulin variable region gene segments are stochastically recombined to generate functional antibodies (B cell receptors) that are expressed on the cell surface. This process is fundamental for the generation of the wide diversity of the immunoglobulin repertoire but also generates self-reactive B cells (red cells) alongside those that comprise the non-self-reactive naïve repertoire (green cells). To evade the development of an immune response against self, two separate tolerance mechanisms remove autoreactive B cells during their development. The first is a central tolerance checkpoint in the bone marrow between the early immature and immature B cell development stages, which removes a large population of B cells that express self-reactive/polyreactive antibodies (shown as red cells). The second checkpoint selects against self-reactive new emigrant/transitional B cells before they enter the long-lived mature naïve B cell compartment. Deficiencies in the integrity of these tolerance mechanisms can be demonstrated through quantifying the frequency of both polyreactive and self-reactive B cells downstream of each checkpoint. A number of autoimmune diseases, including NMOSD, have central and peripheral B cell checkpoints that fail to enforce B cell tolerance and proper counterselection. Thus, these patients include an abnormally high frequency of polyreactive and/or self-reactive new emigrant/transitional and mature naïve B cells. Our study findings suggest that the reservoir of new emigrant/transitional or mature naïve B cells that develop in the presence of defective B cell tolerance checkpoints can supply clones that become pathogenic anti-AQP4 autoantibodies after acquiring somatic hypermutations.