Clinical Spectrum, Pathology, and Mechanisms of Anti-LGI4 Antibody-Positive Autoimmune Nodopathy

Abstract

Background and objectives: The aim of this study was to elucidate the clinical spectrum and mechanisms of autoimmune nodopathy (AN) with autoantibodies against leucine-rich repeat leucin-rich glioma inactivated 1 (LGI) family member 4 (LGI4) localized at juxtaparanodes and satellite glia in the dorsal root ganglion.

Methods: We developed a live cell-based assay for LGI4-immunoglobulin (Ig) G and surveyed 131 patients with chronic inflammatory demyelinating polyneuropathy. LGI4-IgG, anti-LGI4 antibody-negative CIDP-IgG, or healthy control (HC)-IgG were applied to Schwann cells, and cell proliferation was assayed using 5-bromo-2'-deoxyuridine labeling. Quantitative real-time PCR was used to assess the expression of Krox20 and Prx, both of which independently control peripheral nerve myelination. LGI4-IgG or HC-IgG was intraneurally injected into mouse sciatic nerves, and the effects were immunohistochemically and morphometrically assessed.

Results: Eight anti-LGI4 antibody-positive (LGI4⁺) patients were identified; dominant tissue-binding IgG subclasses were IgG4 in 4 patients and IgG2 and IgG3 in 2 patients each. Patients had a relatively old onset age (median 72 years, range 41-90 years). Four patients had acute/subacute monophasic courses, and 4 had chronic progressive/relapsing courses. Predominant symptoms/signs were motor weakness (8 cases), muscle atrophy (4), deep and superficial sensory impairment (8 and 7, respectively), Romberg sign (4), and hand/finger tremor (5). CSF showed extremely high protein levels (median 253 mg/dL). Three chronic cases had nerve hypertrophy. IVIg and efgartigimod were partially (7/7 patients) and markedly (1/1 patient) effective, respectively. Notably, 1 chronic patient with predominant tissue-binding LGI4-IgG2 but without LGI4-IgG4 showed a moderate reduction in myelinated fibers with endoneurial edema, numerous onion bulbs, and few inflammatory cell infiltrates in the biopsied sural nerve. Electron microscopy demonstrated onion bulb formations around the axons and subtle detachment of myelin terminal loops from axonal membranes in the paranodes. In Schwann cell culture, proliferation was significantly higher, and Krox20 but not Prx mRNA levels were lower after incubation with LGI4-IgG from chronic (but not acute-onset) cases compared with HC-IgG incubation. With intraneural injection, both LGI4-IgG4 and LGI4-IgG2 deposited mainly at the nodes extending toward the paranodes and caused nodal/paranodal alterations.

Discussion: LGI4⁺ AN manifests as acute/subacute monophasic and chronic progressive/relapsing diseases, sometimes exhibiting nerve hypertrophy with onion bulbs through nonmyelinating Schwann cell proliferation.

Figure 1. Live CBA for Anti-LGI4 Antibodies and LGI4-IgG Subclass Analysis

(A) Schematic illustration of the live CBA. LGI4-FLAG plasmids and ADAM22-HA plasmids were cotransfected into HEK293T cells. ADAM22 was expressed on the membranes of transfected cells, whereas the secreted FLAG-tagged LGI4—attached to ADAM22 on the cell surface—was stained by anti-FLAG antibodies. (B and C) Representative live CBA and LGI4-IgG subclass analysis (Cases 6 and 8). The predominant IgG subclass was determined according to staining intensity by the visual inspection of a blinded examiner. ADAM22 = a disintegrin and metalloprotease domain–containing protein 22; CBA = cell-based assay; CIDP = chronic inflammatory demyelinating polyneuropathy; HC = healthy control; HEK = human embryonic kidney; Ig = immunoglobulin; LGI4 = leucine-rich repeat LGI family member 4; NF = neurofascin.

Figure 2. Cervical and Lumbosacral Magnetic Resonance Neurography of Patients With LGI4+ AN

(A) Image of cervical roots from 1 representative patient with LGI4^- CIDP. (B) Image of cervical roots from Case 7, a patient with LGI4-IgG2–predominant AN. (C) Image of lumbar roots from 1 patient with representative LGI4^- CIDP. (D) Image of lumbar roots from the same patient with LGI4+ AN (Case 7) as in (B). Note that asymmetric and focal hypertrophy of the cervical and lumbar spinal roots (arrow) was visible in this case. AN = autoimmune nodopathy; CIDP = chronic inflammatory demyelinating polyneuropathy; Ig = immunoglobulin; LGI4 = leucine-rich repeat LGI family member 4.

Figure 3. Pathology of Biopsied Sural Nerve Specimens From a Patient With LGI4⁺ AN

(A) Hematoxylin and eosin staining of the biopsied sural nerve specimens from Case 7. Neither vasculitis nor inflammatory cell infiltrates were present. (B and C) Toluidine blue staining of the sural nerve. Panel C shows a higher magnification of (B). Moderate endoneurial edema, a moderate reduction of myelinated fiber density, and frequent moderate-sized onion bulbs (arrowhead) were observed. Scale bars: (A) = 100 µm; (B) = 200 µm; (C) = 20 µm. AN = autoimmune nodopathy; LGI4 = leucine-rich repeat LGI family member 4.

Figure 4. Electron Micrograph of Biopsied Sural Nerve Specimens From a Patient With LGI4⁺ AN

(A and B) Electron micrograph showing onion bulb formation on axial (A) and longitudinal (B) sections of biopsied sural nerve specimens from Case 7. An asterisk indicates the axon. (C) The cytoplasm of macrophages containing myelin debris (red arrow) was observed adjacent to axons and Schwann cells. A demyelinated axon is indicated by asterisks. (D) Mild detachment of myelin terminal loops from axolemma was observed at the paranodes (red arrows). Yellow arrows indicate regions with no detachment. Scale bars: (A and B) = 5 µm; (C) = 1 µm; (D) = 1 µm. AN = autoimmune nodopathy; LGI4 = leucine-rich repeat LGI family member 4.

Figure 5. Intraneural Injection of LGI4-IgG

Serum IgG samples from LGI4-IgG2–predominant Case 7 (LGI4-IgG2), LGI4-IgG4–predominant Case 2 (LGI4-IgG4), and a healthy control (HC-IgG) were injected twice into mouse sciatic nerves. Teased nerve fibers were immunolabeled for human IgG (green) and CASPR (red). LGI4-IgG diffused into the paranodal regions. LGI4-IgG2 infiltrated the juxtaparanodes (red arrowhead) through the disrupted nodes (red arrow) more extensively than LGI4-IgG4. NF155-IgG4 also penetrated the paranodes (white arrow) and reduced CASPR immunostaining (white arrowhead). Regarding IgG deposition and CASPR immunostaining (red arrow and white arrowhead), an LGI4 and NF155 double-positive IgG-injected nerve showed a combined pattern of LGI4-IgG and NF155-IgG injections. After injection, HC-IgG did not deposit in either paranodes or juxtaparanodes. Scale bar = 20 µm. CASPR = contactin-associated protein 1; Ig = immunoglobulin; LGI4 = leucine-rich repeat LGI family member 4; NF155 = neurofascin 155.