Antibodies to neurofascin, contactin-1, and contactin-associated protein 1 in CIDP

Patients with anti-Nfasc155 IgG4 antibodies

Of patients with anti-Nfasc155 IgG4 antibodies, 4 were men and 3 were women, with an average age at onset of 31 ± 18 years (table e-1, links.lww.com/NXI/A164). The onset of neuropathy was subacute in 5 patients. One case was initially diagnosed with GBS.

All patients had symmetric 4-limb muscle weakness, which was mild to moderate in the upper limbs and proximal muscles of the lower limbs but severe in 5 patients in the lower limb distal muscles. Pinprick and position sense at the hallux were reduced or abolished in all patients. Tremor was also invariably present, and 6 had sensory ataxia. One patient reported neuropathic pain at onset, and 1 had dysphagia. One of them presented optic neuritis during steroid tapering. Brain MRI was thus performed but did not show evidence of disseminated inflammatory CNS disease. The neuropathy was moderately to severely disabling with an ONLS at onset of 5.8 ± 2.2. Two patients required a wheelchair 1 month after the disease onset. The patients received IVIG (n = 6), steroids (n = 7), azathioprine (n = 1), or rituximab (n = 1). Only 1 patient responded to IVIG and steroids (ONLS 4 → 2), 1 patient showed response to steroids (ONLS 6 → 2), and 1 patient unresponsive to IVIG and steroids had sustained response after rituximab (ONLS 9 → 2).

Patients with anti-CNTN1 IgG4 antibodies

Of patients with anti-CNTN1 IgG4 antibodies, 3 were men with a mean age at onset of 56 ± 26 years (table e-1, links.lww.com/NXI/A164). The onset of neuropathy was subacute in 2 patients. Strength was impaired in both proximal and distal muscles in the upper and lower limbs, often to a severe degree, and 2 of them required a wheelchair. Pinprick sensation and proprioception were either severely reduced or abolished, and all of them had sensory ataxia. One patient showed cranial nerve involvement and respiratory failure. One patient with anti-CNTN1 IgG3/IgG4 antibodies had a concurrent onset of CIDP and nephrotic syndrome because of membranous glomerulonephritis. A kidney biopsy showed subepithelial deposits of immune complexes and complement deposition. CNTN1 patients had the highest disability at onset with an ONLS of 7.6 ± 2.5. One patient died of cardiac disease 6 months after the disease onset. The patients were treated with IVIG (n = 3), steroids (n = 3), plasma exchange (n = 2), and immunosuppressive drugs (n = 2). None of them had a sustained response to either IVIG or steroids. Cyclophosphamide was effective in 2 patients, leading to significant improvement of the neuropathy in 1 case and complete remission of the neuropathy together with regression of the nephrotic syndrome in the other case, who did not require further treatment.

Patients with anti-Caspr1 IgG4 antibodies

Of seropositive patients with anti-Caspr1 IgG4 antibodies, 2 were men and 1 was a woman, with a mean age at onset of 43 ± 17 years (table e-1, links.lww.com/NXI/A164). The onset was subacute in 2 patients. Weakness was mild to moderate in proximal muscle groups and moderate to severe in distal muscle groups in both upper and lower limbs. Pinprick sensation was reduced, and position sense was abolished at the hallux in all of them. None of them reported neuropathic pain. One patient complained of dysphagia, and 1 patient had tremor. The disease was severe, with a mean ONLS of 8.3 ± 0.6. The patients were treated with IVIG (n = 3), steroids (n = 3), plasma exchange (n = 1), methotrexate (n = 1), and interferon-alfa (n = 1). A sustained response was observed only in 1 patient receiving steroids and methotrexate (ONLS 9 → 6) and 1 patient treated with interferon-alfa (ONLS 8 → 7).

Seropositive patients with an IgG1-3 isotype or undetectable IgG isotype

Six patients had IgG1 or IgG3 antibodies or an undetectable IgG isotype against Nfasc155 (n = 3) or Caspr1 (n = 3) (table e-1, links.lww.com/NXI/A164). One of these patients presented antibodies reacting to both Nfasc155 and Nfasc140/186. The clinical features of this patient, mainly consisting in a very early onset IVIG-responsive neuropathy, have been previously detailed.¹⁶ We will thus focus here on the 5 other patients, 2 men and 3 women. In 2 of them, both reactive against Caspr1, the disease started in the first decade of life. The mode of onset was subacute in 1 patient only. If present, weakness was graded as mild to moderate in upper limbs and proximal muscles of lower limbs. All 5 patients had weakness in lower limb distal muscles, which was severe in 2 of them. Pinprick sensation was reduced in 3 and position sense was reduced at the hallux in 2 patients, but none had absent proprioception. Sensory ataxia was reported in 4 patients, 2 with anti-Nfasc155 antibodies and 2 with anti-Caspr1 antibodies, whereas tremor was observed in 2 patients with anti-Caspr1 antibodies. Disability was usually low with a mean ONLS at onset of 3.2 ± 1.3. Only 1 case with anti-Caspr1 antibodies needed walking aids. The patients were treated with IVIG (n = 4) and steroids (n = 2). One patient with minimal disability did not require treatment. Three of 4 patients treated with IVIG had a good response.

Comparison of seropositive vs seronegative patients

We compared the clinical features and responses to treatments of patients with IgG4 antibodies against Nfasc155, CNTN1, or Caspr1 with those of 64 randomly selected patients with CIDP who were negative for antibodies against Nfasc155, Nfasc140/186, CNTN1, or Caspr1. We also compared seropositive patients with an IgG1, IgG3, or undetectable IgG isotype with seronegative patients (table 1).

Anti-Nfasc155 and anti-Caspr1 IgG4 seropositive patients had more frequently an early disease onset, before 30 years of age, as opposed to that of anti-CNTN1 IgG4 seropositive patients and seronegative patients. A subacute onset was reported in up to two-thirds of anti-Nfasc155, anti-CNTN1, and anti-Caspr1 IgG4 seropositive patients compared with 28% of seronegative patients. According to the EFNS/PNS criteria, CIDP phenotype was typical in all IgG4-seropositive patients. Tremor was invariably present in patients with anti-Nfasc155 IgG4 antibodies but was also observed in anti-CNTN1 and anti-Caspr1 IgG4 seropositive patients. Severe proprioceptive loss and sensory ataxia were significantly more frequent in IgG4-seropositive patients and were observed in all patients with anti-CNTN1 antibodies, 2 patients with anti-Caspr1 IgG4 antibodies, and 3 patients with anti-Nfasc155 IgG4 antibodies (table 1). In anti-CNTN1 and anti-Caspr1 IgG4 seropositive patients, weakness was usually graded as moderate to severe across proximal and distal muscle groups, whereas in patients with anti-Nfasc155 IgG4 antibodies, motor impairment showed a characteristic distal predominance in the lower limbs.

A higher level of CSF total protein and the presence of temporal dispersion on nerve conduction studies were also characteristic features of patients with autoantibodies of the IgG4 isotype. IgG4-seropositive patients had a higher disability at onset: 5 patients needed a walking aid, and 5 patients required a wheelchair. In particular, patients with anti-CNTN1 or anti-Caspr1 IgG4 antibodies showed the highest disability at onset.

Patients with anti-Nfasc155, CNTN1, or Caspr1 IgG4 antibodies had a lower response rate to IVIG compared with seronegative patients (8% vs 67%, p < 0.001), but there was no remarkable difference in the response to steroids or immunosuppressive treatment. In particular, 1 patient with anti-Nfasc155 IgG4 antibodies was treated with rituximab, 1 patient with anti-Nfasc155 IgG4 antibodies was treated with azathioprine, 2 patients with anti-CNTN1 IgG4 antibodies were treated with cyclophosphamide, and 1 patient with anti-Caspr1 IgG4 antibodies was treated with methotrexate. Four of them who were started on immunosuppressors within 1 year from disease onset showed long-lasting good response.

Patients with antibodies to Nfasc155, CNTN1, or Caspr1 of the IgG1, IgG3, or undetectable isotype did not show distinct clinical features or response to treatments compared with seronegative patients, except for more frequent temporal dispersion on nerve conduction studies.

Morphological changes of the nodes of Ranvier in skin biopsy of seropositive patients

We evaluated skin biopsies from 3 patients with anti-Nfasc155 IgG4 antibodies, 1 patient with anti-CNTN1 IgG3/IgG4 antibodies, 1 patient with anti-Caspr1 IgG4 antibodies, 1 patient with anti-Nfasc155 antibodies of an undetectable isotype, and 6 seronegative patients with CIDP. Analysis of myelinated fibers from patients with anti-Nfasc155 and anti-Caspr1 IgG4 showed elongation of the nodes of Ranvier and loss of paranodal Nfasc155 and Caspr1 staining. Moderate elongation of the nodes of Ranvier and loss of Nfasc155 paranodal staining were also observed in myelinated fibers of the patient with anti-CNTN1 IgG3/IgG4 antibodies. Contrarily, we did not observe similar changes in the patient with anti-Nfasc155 antibodies with an undetectable isotype, seronegative patients CIDP, or HCs (figure 2).

Figure 2 Morphological alterations of the nodes of Ranvier in patients with CIDP with IgG4 autoantibodies

We evaluated skin biopsies from 3 patients with IgG4 anti-Nfasc155 antibodies, 1 patient with IgG3/IgG4 anti-CNTN1 antibodies, 1 patient with IgG4 anti-Caspr1 antibodies, 1 patient with undetectable isotype IgG anti-Nfasc155, and 6 seronegative patients with CIDP. Analysis of myelinated fibers showed elongation of the nodes of Ranvier and loss of paranodal Nfasc155 staining in skin biopsies from patients with anti-Nfasc155 (C) and Caspr1 (E) IgG4. Moderate elongation of the nodes of Ranvier and loss of Nfasc155 paranodal staining were also observed in myelinated fibers of a CNTN1 IgG3/IgG4-positive patient (D). Contrarily, we did not observe similar changes in the patient with undetectable isotype IgG anti-Nfasc155 antibodies (F), in seronegative patients with CIDP (B), or in HCs (A). A complete loss of Caspr1 staining was observed in biopsies from patients with IgG4 antibodies to paranodal proteins (I, L, M), but not in an Nfasc155 seropositive patient with an undetectable isotype (N) and in seronegative CIDP (H) or healthy patients (G). Caspr1 = contactin-associated protein 1; CIDP = chronic inflammatory demyelinating polyradiculoneuropathy; CNTN1 = contactin-1; HC = healthy control; Nfasc155 = neurofascin-155.

Pathogenic effects of anti-Caspr1 IgG4 and IgG1 antibodies

Anti-CNTN1 IgG4 antibodies were previously found to have function-blocking activity and to disrupt the interaction between CNTN1 and its glial partner Nfasc155.¹² We have previously shown that anti-CNTN1 IgG4 and anti-Nfasc155 IgG4 antibodies are pathogenic and disrupt the paranodal regions.^11,21 We thus examined whether anti-Caspr1 antibodies also affect the integrity of the CNTN1/Caspr1/Nfasc155 complex. In particular, we purified IgG1 and IgG4 and tested the effects of the different antibody isotypes. First, we tested the impact of IgG4 and IgG1 fractions from Caspr1-positive patients on the interaction of CNTN1/Caspr1 with Nfasc155 by a cell aggregation assay (figure 3). Human embryonic kidney cells were transfected with Nfasc155 and mCherry and incubated for 2 hours with cells coexpressing CNTN1/Caspr1 and green fluorescent protein (GFP). The percentage of cell clusters showing contacts between red and green cells and the percentage of green and red cells per cell clusters were quantified. As a negative control, Nfasc155-mCherry-transfected cells were incubated with cells expressing GFP only. In the absence of CNTN1/Caspr1, Nfasc155-expressing cells do not form significant contact with GFP-expressing cells, and most clusters express only green or red cells. The expression of CNTN1/Caspr1 significantly increases the number of clusters with contacts, and most cell clusters show an average of 50% of CNTN1/Caspr1- and 50% of Nfasc155-expressing cells. The presence of 10 μg of IgG4 from a Caspr1-positive patient, but not IgG1, abolished the interaction between CNTN1/Caspr1- and Nfasc155-expressing cells to the level of negative controls, thus supporting a blocking function of anti-Caspr1 IgG4 antibodies.

Figure 3 IgG4 to Caspr1 disrupt the interaction between Nfasc155 and CNTN1/Caspr1

(A–D) HEK cells transfected with CNTN1 and Caspr1 (green) or Nfasc155 (red) were incubated together for 2 hours in the presence of control IgG4 (B), anti-Caspr1 IgG1 (C), or anti-Caspr1 IgG4 (D). As negative controls, HEK cells transfected with Nfasc155 (red) were incubated with cells transfected with GFP (A). Anti-Caspr1 IgG4, but not anti-Caspr1 IgG1, abrogated the aggregation of Nfasc155-transfected cells with CNTN1/Caspr1. Scale bar: 10 μm. (E–F) The graphs represent the relative frequency of green cells per aggregates (n = 4 experiments for each condition). The percentage of cell clusters with contacts between green and red cells was quantified (F). The percentage of contacts was significantly decreased in the presence of anti-Caspr1 IgG4 (p < 0.005 by unpaired 2-tailed Student t tests and by one-way ANOVA, followed by Bonferroni post hoc tests). Bars represent mean and SEM. ANOVA = analysis of variance; Caspr1 = contactin-associated protein 1; CNTN1 = contactin-1; GFP = green fluorescent protein; HEK = human embryonic kidney; Nfasc155 = neurofascin-155.

To further confirm these findings, mouse sciatic nerve segments were incubated in vitro with 10 μg of IgG4 or IgG1 fractions from Caspr1-positive patients for 3 hours, and IgG deposition was monitored. IgG4 but not IgG1 antibodies were found to penetrate the paranodal regions (figure 4). These findings were replicated in vivo by performing intraneural injections. Again, only IgG4 antibodies were found to penetrate the paranodal regions. Of interest, the level of antibody penetration across the paranodal region was similar at 1 or 3 days after intraneural injection, and IgG4 deposition was detected only at the border of the nodes of Ranvier (figure 4). No paranodes presented a complete invasion by IgG4 at 1 or 3 days postinjection.

Figure 4 Anti-Caspr1 IgG4, but not IgG1, invades the paranodal regions

(A–C) Sciatic nerve fibers were incubated in vitro with purified control IgG4 (A), anti-Caspr1 IgG1 (B), or anti-Caspr1 IgG4 (C) for 3 hours and immunolabeled for IgG (green) and CNTN1 (red). (D–F) Sciatic nerves were fixed 1 or 3 days after intraneural injections of purified control IgG4 (D), anti-Caspr1 IgG4 (E–F), immunolabeled for CNTN1 (red), and human IgG (green). Note that only anti-Caspr1 IgG4 penetrated the paranodes. One or 3 days after injection, IgG4 antibodies were detected at the paranode borders (arrows). Images are representative of 3 independent experiments. Scale bar: 10 μm. Caspr1 = contactin-associated protein 1; CNTN1 = contactin-1.