N-methyl-D-aspartate receptor antibody production from germinal center reactions: Therapeutic implications

Mateusz Makuch, Robert Wilson, Adam Al-Diwani, James Varley, Anne-Kathrin Kienzler, Jennifer Taylor, Antonio Berretta, Darren Fowler, Belinda Lennox, M Isabel Leite, Patrick Waters, Sarosh R Irani, Mateusz Makuch, Robert Wilson, Adam Al-Diwani, James Varley, Anne-Kathrin Kienzler, Jennifer Taylor, Antonio Berretta, Darren Fowler, Belinda Lennox, M Isabel Leite, Patrick Waters, Sarosh R Irani

Abstract

Introduction: N-methyl-D-aspartate receptor (NMDAR) antibody encephalitis is mediated by immunoglobulin G (IgG) autoantibodies directed against the NR1 subunit of the NMDAR. Around 20% of patients have an underlying ovarian teratoma, and the condition responds to early immunotherapies and ovarian teratoma removal. However, despite clear therapeutic relevance, mechanisms of NR1-IgG production and the contribution of germinal center B cells to NR1-IgG levels are unknown.

Methods: Clinical data and longitudinal paired serum NR1-reactive IgM and IgG levels from 10 patients with NMDAR-antibody encephalitis were determined. Peripheral blood mononuclear cells from these 10 patients, and two available ovarian teratomas, were stimulated with combinations of immune factors and tested for secretion of total IgG and NR1-specific antibodies.

Results: In addition to disease-defining NR1-IgG, serum NR1-IgM was found in 6 of 10 patients. NR1-IgM levels were typically highest around disease onset and detected for several months into the disease course. Moreover, circulating patient B cells were differentiated into CD19+ CD27++ CD38++ antibody-secreting cells in vitro and, from 90% of patients, secreted NR1-IgM and NR1-IgG. Secreted levels of NR1-IgG correlated with serum NR1-IgG (p < 0.0001), and this was observed across the varying disease durations, suggestive of an ongoing process. Furthermore, ovarian teratoma tissue contained infiltrating lymphocytes which produced NR1-IgG in culture.

Interpretation: Serum NR1-IgM and NR1-IgG, alongside the consistent production of NR1-IgG from circulating B cells and from ovarian teratomas suggest that ongoing germinal center reactions may account for the peripheral cell populations which secrete NR1-IgG. Cells participating in germinal center reactions might be a therapeutic target for the treatment of NMDAR-antibody encephalitis. Ann Neurol 2018;83:553-561.

© 2018 The Authors Annals of Neurology published by Wiley Periodicals, Inc. on behalf of American Neurological Association.

Figures

Figure 1
Figure 1
NR1‐subunit–specific IgG and IgM antibodies. (A) Live cell‐based assays expressing surface NR1/enhanced green fluorescent protein (EGFP) as a fusion protein (green), as described previously.2 IgG and IgM antibodies were detected among patients with NMDAR‐antibody encephalitis (NMDAR‐AbE; 1:20 serum dilution, red). One of 116 of the disease controls (DC) and healthy controls (HC) showed NR1‐IgM antibodies. No DC/HC showed NR1‐IgG. Scale bar, 10 μm. (B) Both NR1‐reactive IgM and IgG (red) colocalized well with an anti‐NR1 commercial antibody (green). Scale bar, 10 μm. (C) NR1‐IgM reactivity was specifically depleted with anti‐IgM precipitation, whereas protein G depletion of IgG abrogated all NR1‐IgG binding in live NR1‐expressing HEK cells. Scale bar, 10 μm. (D) Similarly, these reciprocal serum depletions confirmed specificity of both high‐titer NR1‐specific IgM and IgG antibodies which bound the surface of live hippocampal neurons (microtubule associated protein 2, MAP2, in red). Scale bar, 20 μm. Depletion was > 99%. (E–G) are representative figures of three observed trends in individual patients, and dotted line at serum dilution of 1:20 indicates cutoff for positivity. IgG is represented in blue and IgM in red, throughout. (E) Two of 10 patients showed parallel reductions of both NR1‐IgM and NR1‐IgG. (F) Four of 10 patients had NR1‐IgG without NR1‐IgM. (G) Four of 10 showed low‐level sustained fluctuations in NR1‐IgM levels. (H) Overall, the mean serum NR1‐IgM and IgGs of the 6 patients with NR1‐IgM were compared by end‐point dilutions. Values of neighboring points were used to smooth data (GraphPad Prism v7; 0th order polynomial). CyP = cyclophosphamide; DAPI = 4',6‐diamidino‐2‐phenylindole; Ig = immunoglobulin; IVIG = intravenous immunoglobulin; MMF = mycophenolate mofetil; NMDAR = N‐methyl‐D‐aspartate receptor; PLEX = plasma exchange.
Figure 2
Figure 2
In vitro production of NR1‐directed antibodies from PBMC cultures. (A) Antibody‐secreting cells (ASCs) reach a peak at day 7. (B) This CD19+CD27++CD38++ population of ASCs correlated best with the total IgG produced in culture. From (A), total IgG and IgM production (µg/ml) plateaued at around day 14, and NR1 antibodies could be detected in some culture supernatants. Time course is plotted for experiments from 5 patients using soluble CD40‐ligand, interleukin‐2, and the toll‐like receptor 7/8 agonist, R848 (mean and SD shown). Scale bar, 10 μm. EGFP = enhanced green fluorescent protein; Ig = immunoglobulin; PBMC = peripheral blood mononuclear cell.
Figure 3
Figure 3
NR1‐specific IgG and total IgG production in vitro. Production of total IgG (upper heatmaps; ng/ml), NR1‐specific IgG (middle heatmaps), and NR1‐IgM (lower heatmaps) from 10 patients with NMDAR antibody encephalitis. Patients (numbers corresponding to the Table) ordered in rows by descending serum NR1‐IgG levels at time of PBMC sampling. *Serum NR1‐IgG and supernatant NR1‐IgG and ‐IgM levels were determined by live cell‐based assay end‐point dilutions. Culture conditions are listed below each column and varied in use of CD40‐ligand (nil in black; soluble CD40‐ligand in blue; membrane CD40‐ligand in red). Those with CD40‐ligand, interleukin‐2, and R848 most frequently generated NR1‐antibodies. Heatmaps show gamma‐transformed data for visualization, and absolute levels are shown in the bars. Limited cell numbers meant 1 patient had unavailable data in soluble CD40‐ligand conditions (dots). BAFF = B‐cell activating factor; CD40L = CD40‐ligand; Ig = immunoglobulin; IL = interleukin; NMDAR = N‐methyl‐D‐aspartate receptor; PBMC = peripheral blood mononuclear cell; TNF‐α = tumor necrosis factor alpha.
Figure 4
Figure 4
NR1‐IgG antibody generation from ovarian teratomas. (A) An ovarian teratoma from patient number 2 (scale bar, 0.2cm), showed dense B cell (CD20) infiltration, with more sparse T cells (CD3) and few plasma cells (CD138, inset × 80 magnification). T and B cells express CD27, whereas CD38 is more restricted to the B cell regions. The NR1 subunit is densely expressed throughout this region. Scale bar, 100 μm. (B) Cystic aspirate from another teratoma (a 28‐year‐old female recruited after patients 1–10) was studied by flow cytometry and found to contain CD19+ B cells, which (C) expressed surface IgM, IgG, and IgD. (D) In addition, some CD19+ cells were CD27++CD38++ antibody‐secreting cells (ASCs). (E,F) Culture of these cells with an ASC‐maintenance condition (interleukin‐6), but not B cell proliferative condition (R848, CD40‐ligand, and interleukin‐2), resulted in NR1‐IgG generation (E,F, lower panel). Scale bar, 5 μm. NR1‐IgG was also detected from supernatants of the teratoma explants of patient 2 (F, upper panel) after incubation with B cell proliferative conditions or interleukin‐6. In addition, NR1‐IgG was detected in aspirates directly from the teratoma at higher levels than in the serum from these 2 patients (F). DAPI = 4',6‐diamidino‐2‐phenylindole; EGFP = enhanced green fluorescent protein; Ig = immunoglobulin.

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