Molecular Level Characterization of Circulating Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder

Abstract

Objective: To determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD.

Methods: We combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro.

Results: Multiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01).

Conclusions: The composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones.

Figure 1. Human AQP4 Production, Experimental Design, and AQP4-IgG Detection by Cell-Based Assay

(A) Typical elution profile of hAQP4 from a nickel affinity chromatography column, visualized by stain-free imaging technology. Individual fraction from the elution is loaded in each lane. The hAQP4-containing fractions, 3–14, were pooled and concentrated. (B) Experimental design: For each neuromyelitis optica spectrum disorder patient, both B cell receptor sequences (BCR-seq) and immunoglobulin sequences (Ig-seq) were acquired. Peripheral B cells were isolated and half was used for constructing VH library as a custom database for heavy chain peptide identification, the other half for VH:VL pairing to acquire the endogenous light chain sequences for the heavy chains. Serum AQP4-IgG were purified by affinity chromatography using AQP4-immobilized column and analyzed by LC-MS/MS. The repertoire was analyzed and recombinant mAbs were expressed for functional characterization. (C) A cell-based, immunofluorescence assay confirmed the presence of AQP4-IgG in select human serum and/or plasma samples. A FITC-labeled secondary antibody was used to detect the presence of AQP4-IgG in human serum or plasma samples. Patient 2 plasma tested positive for AQP4-IgG while patient 1 exhibited negative result. AHC = healthy control; P1 = Patient 1; P2 = Patient 2; NEG = negative control; POS = positive control; QP4 = aquaporin-4. All images were taken at 20× magnification using a Nikon T2 microscope.

Figure 2. The AQP4-IgG Serum Repertoire of NMOSD Patients

(A) A representative BCR lineage of Patient 1 showing IgG peptides identified by Ig-seq. Green lines indicate peptides that are unique to the lineage, and numbers refer to the ratio of a peptide's abundance in the elution vs the flow-through chromatography fractions. Red indicates the CDR-H1, CDR-H2, and CDR-H3 regions. (B) Relative abundance of AQP4-IgG lineages in Patient 1 (B.a) and Patient 2 (B.b). Each bar represents AQP4-binding IgG clones mapping to a BCR CDR-H3 antibody lineage. Red *, VH:VL matched and recombinant mAb was generated. Blue *, VH:VL paired identified from yeast display library. (C) AQP4-IgG V-gene somatic hypermutation rates for NMOSD Patient 1 and Patient 2. (D) V-gene usage in the serologic repertoire of NMOSD patients. AQP4 = aquaporin-4; BCR = B-cell receptor; NMOSD = neuromyelitis optica spectrum disorder.

Figure 3. Serum IgG Clones Identified by B-cell Receptor-Seq/Ig-Seq Platform Bind to AQP4 When Expressed as Recombinant mAbs

(A) Western blot of mAbs P1, P21, and P22 binding to recombinant human AQP4 and cell lysate of U87 cell line expressing AQP4 (1: hAQP4; 2: U87-AQP4). (B) Monoclonal Abs binding to hAQP4 was evaluated by ELISA. Values represent mean ± SD (n = 3). (C) Binding of the mAbs to the surface of U87-AQP4 analyzed by flow cytometry. 10,000 events/sample were collected and each sample was run in triplicates. Values represent mean ± SD (n = 3). (D) Histogram of mAb P1 binding to U87-AQP4 at different concentrations. Negative control mAb shown for comparison. AQP4 = aquaporin-4; mAb = monoclonal antibody.

Figure 4. Complement-Mediated Cytotoxicity Can Be Induced by Recombinant Anti-AQP4 mAbs

CHO cells expressing AQP4 (CHO-AQP4) were incubated with the recombinant mAbs (P1, P21, P22, or P2_κ) or irrelevant control mAb, in combination with serum as complement source or complement-depleted serum (CDS). Cell dyes were added and cell viability was visualized by fluorescent inverted microscope and quantified using ImageJ (n = 6 per group). The black dots represented outliers of replicates that were not included in the statistics. P1, P22, and P2_κ mAbs showed statistically significant (p < 0.01) killing of cells compared to irrelevant mAb control. AQP4 = aquaporin-4; CHO = Chinese hamster ovary; mAb = monoclonal antibody.