Antibodies Against Glutamic Acid Decarboxylase 65 Are Locally Produced in the CSF and Arise During Affinity Maturation

Abstract

Background and objectives: Antibodies (Abs) against the cytoplasmic protein glutamic acid decarboxylase 65 (GAD65) are detected in patients with neurologic syndromes together referred to as GAD65-Ab spectrum disorders. The response of some of these patients to plasma exchange or immunoglobulins indicates that GAD65-Abs could contribute to disease pathogenesis at least at some stages of disease. However, the involvement of GAD65-reactive B cells in the CNS is incompletely understood.

Methods: We studied 7 patients with high levels of GAD65-Abs and generated monoclonal Abs (mAbs) derived from single cells in the CSF. Sequence characteristics, reactivity to GAD65, and the role of somatic hypermutations of the mAbs were analyzed.

Results: Twelve CSF-derived mAbs were generated originating from 3 patients with short disease duration, and 7/12 of these mAbs (58%) were GAD65 reactive in at least 1 detection assay. Four of 12 (33%) were definitely positive in all 3 detection assays. The intrathecal anti-GAD65 response was polyclonal. GAD65-Abs were mostly of the IgG1 subtype and had undergone affinity maturation. Reversion of 2 GAD65-reactive mAbs to their corresponding germline-encoded unmutated common ancestors abolished GAD65 reactivity.

Discussion: GAD65-specific B cells are present in the CNS and represent a sizable fraction of CSF B cells early in the disease course. The anti-GAD65 response in the CSF is polyclonal and shows evidence of antigen-driven affinity maturation required for GAD65 recognition. Our data support the hypothesis that the accumulation of GAD65-specific B cells and plasma cells in the CSF is an important feature of early disease stages.

Figure 1. Evaluation of GAD65 Reactivity of Generated mAbs by ELISA

(A) Overview of the technical workflow to generate CSF-derived mAbs (created with BioRender). (B–D) Patient-derived mAbs were tested for GAD65 reactivity in ELISA. The horizontal dashed line indicates 4× OD of the negative control. Data are shown as the mean of at least 3 independent experiments; error bars indicate the SEM. Kd values were calculated using nonlinear regression analysis. (B) MAbs were tested at a concentration of 40 μg/mL and undiluted (concentrations ranging from 0.35 to 5.29 mg/mL). (C and D) MAbs positive in the screening-ELISA and the mAb HK3 and the myelin oligodendrocyte glycoprotein-specific mAb 8.18C5 as controls were tested in different dilutions with high-affinity GAD65-reactive Abs presented in (C) and low-affinity GAD65-reactive Abs in (D). GAD = glutamic acid decarboxylase; Kd = dissociation constant; mAb = monoclonal antibody; na = nonapplicable; OD = optical density.

Figure 2. Evaluation of GAD65 Reactivity of Generated mAbs by Tissue- and Cell-Based Assay

(A and B) Cell-based assay using HEK293 cells transfected with GAD65 or mock transfected was performed. MAbs were used at a concentration of 50 μg/mL and in a first step diluted 1:10 followed by dilutions of 1:100/1:400/1:1,600/1:6,400/1:25,600 if positive and applied undiluted if negative. (A) Representative pictures for mAb 7A2 are shown. (B) The highest dilutions for all mAbs that revealed a positive staining are depicted. (C) Tissue-based assay with indirect immunofluorescence on primate cerebellar slices was performed to detect anti–GAD65-specific staining patterns using the patient-derived mAbs (50 μg/mL). The mAb HK3 was used as a negative control. A condition with only secondary antibody was used as an additional negative control. #Marks mAbs that show a GAD65-specific staining pattern characterized by a leopard-like granular layer staining. GAD = glutamic acid decarboxylase; mAb = monoclonal antibody.

Figure 3. Unmutated Common Ancestors (UCAs) of 2 GAD65-Reactive mAbs

(A) The differences of the amino acid (AA) sequences of heavy and light chains between the patient-derived sequences (PDS) and their corresponding UCAs are illustrated. The differing AAs are depicted in white letters and the AA positions in black numbers. (B) GAD65 reactivity of PDS of mAb 7A2 and the corresponding UCA were compared by GAD65 ELISA. MAb 5D1 is not included due to weak reactivity in ELISA with positivity only at high concentrations (>350 μg/mL); however, the corresponding UCA was not producible at such a high concentration. (C) Cell-based assay using HEK293 cells transfected with GAD65 was performed. MAbs representing PDS or UCAs were used at a concentration of 50 μg/mL and in a first step diluted 1:10 followed by dilutions of 1:100/1:400/1:1,600/1:6,400/1:25,600 if positive and applied undiluted if negative. The highest dilutions that revealed a positive staining are depicted. (D) Comparison of binding to primate cerebellar and pancreas tissue of patient-derived sequences (PDS) and unmutated common ancestors (UCAs). For the mAbs 5D1 and 7A2, binding of the PDS and UCAs to primate cerebellar tissue and pancreas tissue was compared by indirect immunofluorescence with concentrations of 50 μg/mL of the mAbs. Left panel: Binding to primate cerebellar tissue is illustrated. The dashed white lines confine the leopard-like staining pattern of the granular layer that is specific for GAD65-Abs. Right panel: Binding to primate pancreas islets is illustrated. Pancreas islet cells are marked with a dashed white circle. GAD65-specific staining pattern is marked with #. CDR = complementarity determining region; ctrl = control; FR = framework region; GAD = glutamic acid decarboxylase; HC = heavy chain; mAb = monoclonal antibody; neg = negative; pos = positive; κC = kappa light chain; λC = lambda light chain.