NMDAR encephalitis: passive transfer from man to mouse by a recombinant antibody

Abstract

Objective: Autoimmune encephalitis is most frequently associated with anti-NMDAR autoantibodies. Their pathogenic relevance has been suggested by passive transfer of patients' cerebrospinal fluid (CSF) in mice in vivo. We aimed to analyze the intrathecal plasma cell repertoire, identify autoantibody-producing clones, and characterize their antibody signatures in recombinant form.

Methods: Patients with recent onset typical anti-NMDAR encephalitis were subjected to flow cytometry analysis of the peripheral and intrathecal immune response before, during, and after immunotherapy. Recombinant human monoclonal antibodies (rhuMab) were cloned and expressed from matching immunoglobulin heavy- (IgH) and light-chain (IgL) amplicons of clonally expanded intrathecal plasma cells (cePc) and tested for their pathogenic relevance.

Results: Intrathecal accumulation of B and plasma cells corresponded to the clinical course. The presence of cePc with hypermutated antigen receptors indicated an antigen-driven intrathecal immune response. Consistently, a single recombinant human GluN1-specific monoclonal antibody, rebuilt from intrathecal cePc, was sufficient to reproduce NMDAR epitope specificity in vitro. After intraventricular infusion in mice, it accumulated in the hippocampus, decreased synaptic NMDAR density, and caused severe reversible memory impairment, a key pathogenic feature of the human disease, in vivo.

Interpretation: A CNS-specific humoral immune response is present in anti-NMDAR encephalitis specifically targeting the GluN1 subunit of the NMDAR. Using reverse genetics, we recovered the typical intrathecal antibody signature in recombinant form, and proved its pathogenic relevance by passive transfer of disease symptoms from man to mouse, providing the critical link between intrathecal immune response and the pathogenesis of anti-NMDAR encephalitis as a humorally mediated autoimmune disease.

Figures

Figure 1

Intrathecal B‐cell and plasma cell accumulation in patients with anti‐NMDAR encephalitis corresponds to the clinical disease course. (A–C) Representative multiparameter flow cytometry analysis of CD19+ B cells and CD19+ CD138+ plasma cells in peripheral blood (PB, left panels) and cerebrospinal fluid (CSF, right panels) in a healthy control (A) and a patient (Pt. 1) with anti‐NMDAR encephalitis before (B) and after (C) immunotherapy. (D) Representative CD138+ plasma cell staining of a biopsy specimen of a newly occurring cerebral MRI lesion in a patient with later on established anti‐NMDAR encephalitis (scale bar represents 50 μm; insert scale bar represents 10 μm). (E–H) Multiparameter flow cytometry analysis of absolute numbers (E) and relative fractions (F) of CD19+ B cells and absolute numbers (G) and relative fractions (H) of CD138+ CD19+ plasma cells within peripheral blood (PB, left panels) and cerebrospinal fluid (CSF, right panels) in four patients with anti‐NMD‐R encephalitis and 25 controls. Data are given as whisker plots. Statistical testing was performed using the Wilcoxon rank sum test with an α error of 0.05. Levels of significance are indicated by n.s. (not significant) for all P > 0.05, *P < 0.05 and **P < 0.01. (I–K) Time course of absolute numbers of CD19+ B cells (I), CD19+ CD138+ plasma cells (J), and NMDAR IgG titers (K) in cerebrospinal fluid of anti‐NMDAR encephalitis before, during, and after treatment. (L) Comprehensive results of detailed neuropsychological assessments (total rank sum of test of attention, memory and executive functions) of patients with anti‐NMDAR encephalitis 1–16 weeks after admission (pre) and after 8–48 months (post).

Figure 2

Presence of clonally expanded plasma cells with hypermutated antigen receptors indicates an intrathecal antigen‐driven immune response in patients with anti‐NMDAR encephalitis. Shown are unrooted phylogenetic trees of the IgG‐VH (B), V lambda (C), and V kappa (D) gene family repertoires of sequences obtained from CSF plasma cells of a patient with anti‐NMDAR encephalitis by single cell RT‐PCR. Each colored branch of the tree represents a single V family. The external node of a branch represents a “leaf” or a “sequence.” The branch length and distance between sequences correspond to sequence similarity/dissimilarity, shorter and closer branches relate to a greater sequence similarity. The bootstrap support values for each branch were calculated based on 100 resampling of the original dataset. Ovals indicate clonally expanded plasma cells (cePc) 1–7, from which recombinant monoclonal antibodies SSM 1–7 were derived. The gate (R5) in the FACS panel (A) shows the cell population used for single cell analysis.

Figure 3

The pattern of brain tissue immunostaining of rhuMab SSM5 is characteristic of the NMDAR and blocked by serum antibodies from patients with anti‐NMDAR encephalitis. (A, B) Patient‐derived rhuMab reacts with brain tissue. Representative immunoreactivity of a patient‐derived rhuMab (SSM5) (A) and control rhuMab (12D7) (B) with sagittal sections of rat brain. SSM5 antibody, but not the control antibody shows a robust immunostaining of brain in a pattern characteristic of the NMDAR. Scale bar 2 mm. (C, D, E) Patient‐derived rhuMab competes for similar epitopes recognized by serum antibodies from patients with anti‐NMDAR encephalitis. Reactivity of biotinylated rhuMab SSM5 (diluted 1:20) with rat hippocampus preincubated with serum from a healthy subject (C) and serum from a patient with anti‐NMDAR encephalitis (diluted 1:2) (D). (E) Preincubation with sera of anti‐NMDAR encephalitis patients substantially blocks the reactivity of SSM5. In this experiment, the tissue sections were preincubated with sera from seven different patients (091, 095, 112, 125, 138, 141, 215) and one control without antibodies (670). There is competition of reactivity by each of the patients' sera, while control serum (top left) does not reduce rhuMab SSM5 reactivity (as expected). Case to case variation of competition suggests that anti‐NMDAR antibodies against epitopes recognized by rhuMab SSM5 are present in each of the patients but differ in concentration and/or affinity.

Figure 4

RhuMab SSM5 targets the same restricted epitope of the GluN1 subunit of the NMDAR as patient's CSF antibodies. (A–C) Patient‐derived rhuMab shows the same staining pattern as CSF from a patient with anti‐NMDAR encephalitis in immunohistochemistry with HEK cells expressing full‐length or mutant GluN1/GluN2B. Immunofluorescence with HEK293 cells expressing full‐length GluN1/GluN2B (A) or mutant GluN1 with full‐length GluN2B (B, C) and stained for human IgG (green) and with a commercial GluN1‐specific antibody (red); nuclear counterstaining with DAPI (blue). Cells were treated with human CSF (diluted 1:16), patient‐derived rhuMab (SSM5, 4.3 μg/mL) or control rhuMab (isotype, rhuMab 12D7, 4.3 μg/mL). The epitope‐disrupting G369I mutant (B) does not show staining by patients' CSF or rhuMab, while a mutant with less epitope disruption G369S (C) does not affect staining. Scale bar represents 10 μm. (D) Confirmation of antigen specificity of patient‐derived rhuMab by immunoprecipitation (IP). Proteins precipitated from brain membrane lysates were separated by PAGE electrophoresis and blotted onto PVDF membranes. Western blot detection was performed with a commercial GluN1‐specific antibody. A band of around 120 kDa corresponding to the GluN1 subunit of the NMDA receptor was identified using SSM5 rhuMab (5 μg, covalently coupled to Dynabeads) in the membrane fraction of brain tissue. Commercially available antibodies to the respective target antigen were used as positive control in IPs and in western blot detections. A rhuMab specific for an irrelevant target (12D7) was included as a negative control (NC) for IP experiments.

Figure 5

RhuMab SSM5 decreases the density of cell surface and synaptic NMDAR in cultured hippocampal neurons. (A) Representative dendrites from cultures of primary rat hippocampal neurons showing the density of NMDAR clusters (top panels), PSD95, an excitatory postsynaptic protein (middle panels), and synaptic NMDAR (colocalization of NMDAR with PSD95, lower panels) after 24 h treatment with control rhuMab 12D7 (4.5 μg/mL), CSF from a patient with anti‐NMDAR encephalitis (“patient's CSF,” 1:20 dilution), patient‐derived rhuMab SSM5 (4.5 μg/mL) or not treated; scale bar represents 10 mm. The quantification of these effects is shown in (B), demonstrating the density of surface NMDAR (left plot), PSD95 (middle plot) and synaptic NMDAR (right plot). Patient‐derived rhuMab SSM5, but not the control rhuMab 12D7 causes a significant reduction of surface and synaptic NMDAR clusters similarly to that of the CSF from a patient with anti‐NMDAR encephalitis. No effects occurred on PSD95. All graphs represent mean ± SD, n = 60 cells per condition. Significance of treatment effect was assessed by one‐way ANOVA (P < 0.0001 for NMDAR, synaptic NMDAR) with Bonferroni post hoc correction. **P < 0.001, ****P < 0.0001.

Figure 6

RhuMab SSM5 reduces NMDAR‐mediated currents in vitro and induces severe memory deficits in mice. (A, B) RhuMab SSM5 reduces NMDAR‐mediated currents in transfected Xenopus oocytes. (A) Representative current traces measured in Xenopus oocytes expressing GluN1‐1a plus GluN2B in response to superfusion with a solution containing 100 μmol/L glutamate plus 10 μmol/L glycine in Ba2+ Ringer. All currents were measured at −70 mV. GluN1‐1a/GluN2B expressing oocytes were preincubated with 4 μg/mL patient‐derived rhuMab in bath medium for 1 h prior to measurement (“antibody”), whereas GluN1‐1a/GluN2B expressing control oocytes were not preincubated with antibody (“control”). Control oocytes were treated the same way, either preincubated with patient rhuMab (“uninjected, antibody,” n = 5) or not preincubated (“uninjected,” n = 7). Uninjected oocytes revealed no measurable currents. (B) GluN1‐1a/GluN2B amplitudes in oocytes either without (“control”) or with (“antibody”) preincubation with autoantibody were measured and are shown normalized to control currents. Data are given as mean ± SEM. Statistical testing was performed by two‐tailed t‐test with an α error of 0.05. Significant difference is indicated by *P < 0.05; n indicates number of oocytes measured. (C–E) Cerebroventricular infusion of anti‐NMDAR rhuMab SSM5 causes severe reversible memory deficits. (C) Infusion of rhuMab SSM5 into the cerebroventricular system of mice for a total of 14 days causes deficits of memory. Novel object recognition index in mice treated with the patient‐derived rhuMab (SSM5, red circles) and the control rhuMab (12D7, green circles) (each at 90 μg/mL in saline solution). A high index indicates better object recognition memory. Note that mice infused with patient‐derived rhuMab showed a progressive decrease of memory that was maximal on Day 18. The total time of exploration of both objects was similar in both groups (not shown). Data are presented as mean ± SEM. Number of animals: SSM5: n = 7, 12D7: n = 7. Significance of treatment effect was assessed by two‐way ANOVA with an α error of 0.05 and post hoc testing with Bonferroni adjustment (asterisks). ***P < 0.001. (D, E) Animals infused with rhuMab SSM5 have a progressive increase of human IgG bound to hippocampus. (D) Immunostaining of human IgG in sagittal brain sections showing the hippocampus of representative animals infused with anti‐NMDAR monoclonal antibody (SSM5) (right panels) and control antibody (12D7) (left panels), sacrificed at the indicated experimental days. In animals infused with the SSM5 antibody, a gradual increase of IgG immunostaining until Day 18, followed by decrease of immunostaining is observed. Scale bar represents 200 μm. (E) Quantification of intensity of human IgG immunolabeling in hippocampus of mice infused with SSM5 antibody (dark gray columns) and control CSF (pale gray columns) sacrificed at the indicated time points. Mean intensity of IgG immunostaining in the group with the highest value (animals treated with anti‐NMDAR monoclonal antibody (SSM5) and sacrificed at Day 18) was defined as 100%. Data are presented as mean ± SEM. Number of animals: 5 animals per condition and time point. Significance of treatment effect was assessed by two‐way ANOVA with an α error of 0.05 (o) and post hoc testing with Bonferroni adjustment (*). °°°P < 0.001, **P < 0.01, ****P < 0.0001.

Figure 7

RhuMab SSM5 decreases the density of synaptic NMDAR density in mice hippocampus after intraventricular infusion. (A, B) Anti‐NMDAR monoclonal antibody (SSM5) mediates a reduction of total and synaptic NMDAR in mice hippocampus. (A) 3D projection of the density of total clusters of NMDAR, PSD95, and synaptic clusters of NMDAR (defined as NMDAR clusters colocalizing with PSD95) in a hippocampal region from a representative animal of each experimental group. Merged images (merge: PSD95 [green]/NMDAR [red]) were postprocessed and used to calculate the density of clusters (density = spots/μm3). Scale bar represents 2 μm. (B) Quantification of the density of total NMDAR, total PSD95, and synaptic NMDAR clusters at Day 18 in a pooled analysis of hippocampal areas (CA1, CA2, CA3, and dentate gyrus) in animals treated with anti‐NMDAR monoclonal antibody (SSM5; dark gray) and control antibody (12D7; pale gray). Mean density of clusters in control antibody treated animals was defined as 100%. Data are presented as scatterplot plus mean ± SD. Number of animals: 5 animals per condition (18 hippocampal areas per animal = 90 hippocampal areas per condition). Significance of treatment effect was assessed by two‐tailed t‐test with an α error of 0.05. ****P < 0.0001.