Figure 1 Central B cell tolerance is compromised in patients with NMOSD. Recombinant antibodies (rIgGs) derived from ... 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Brain, Volume 142, Issue 6, 05 May 2019, Pages 1598–1615, https://doi.org/10.1093/brain/awz106 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 2 Accumulation of polyreactive mature naïve B cells in the blood of patients with NMOSD. Recombinant antibodies ... 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Brain, Volume 142, Issue 6, 05 May 2019, Pages 1598–1615, https://doi.org/10.1093/brain/awz106 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 3 The peripheral B cell tolerance checkpoint is impaired in patients with NMOSD. Recombinant antibodies (rIgGs) ... 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Brain, Volume 142, Issue 6, 05 May 2019, Pages 1598–1615, https://doi.org/10.1093/brain/awz106 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 4 Anti-AQP4 autoantibodies and their unmutated revertants contain both polyreactive and autoreactive clones. ... 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Brain, Volume 142, Issue 6, 05 May 2019, Pages 1598–1615, https://doi.org/10.1093/brain/awz106 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 5 Unmutated precursors of the anti-AQP4-specific autoantibodies do not bind to AQP4. Anti-AQP4 autoantibodies ... 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Brain, Volume 142, Issue 6, 05 May 2019, Pages 1598–1615, https://doi.org/10.1093/brain/awz106 The content of this slide may be subject to copyright: please see the slide notes for details.

Figure 6 Schematic diagram illustrating the potential consequence of defective B cell tolerance checkpoints in the ... 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. Unless provided in the caption above, the following copyright applies to the content of this slide: © The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.comThis article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) Brain, Volume 142, Issue 6, 05 May 2019, Pages 1598–1615, https://doi.org/10.1093/brain/awz106 The content of this slide may be subject to copyright: please see the slide notes for details.