IgG4 Valency Modulates the Pathogenicity of Anti–Neurofascin-155 IgG4 in Autoimmune Nodopathy

(A and B) The levels of λ and κ light chains of anti-Nfasc155 IgG4 were measured by ELISA against Nfasc155 in 10 reactive patients (A). The levels of λ (green dots) and κ (blue dots) light chains were also quantified by sandwich ELISA in the purified IgG4 fractions from healthy controls (n = 10), unreactive CIDP patients (CIDP−; n = 10), and reactive autoimmune nodopathy patients (AN+; n = 10) and were compared with those found in anti-Nfasc155 IgG4 (B). The levels of κ light chain strongly correlated with those of λ light chain in Nfasc155+ patients. No significant differences in the levels of λ and κ light chains were found between the groups (p > 0.01 by one-way ANOVA followed by Bonferroni post hoc tests). p Value, Spearman correlation coefficient (r), R square (R²), and 95% confidence band (dotted lines) are indicated on the graph. (C and D) IgG4 from Nfasc155+ patients (n = 10) were incubated with of a 3-fold excess of monoclonal IgG4/λ (C) or IgG4/κ (D) in the presence or absence of reduced glutathione (GSH). The levels of κ and λ light chains were then measured by ELISA against Nfasc155, and the ratio κ/λ (red dots) was calculated. Fab-arm exchange (FAE) with IgG4/λ significantly increased the levels of λ light chains and decreased the levels of κ light chains in anti-Nfasc155 IgG4 and resulted in a decrease of the κ/λ ratio. Reversely, FAE with FAE with IgG4/κ significantly increased the levels of κ light chains and decreased those of λ light chains in anti-Nfasc155 IgG4 and resulted in an increase of the κ/λ ratio (*p < 0.05; **p < 0.005; ***p < 0.001 compared with control condition in the absence of GSH using paired Student t tests). The percentage of increase or decrease of the κ/λ ratio following FAE (gray dots) with IgG4/λ or IgG4/κ was quantified for each patient. Changes in the κ/λ ratio were significantly different following FAE with IgG4/λ or IgG4/κ (***p < 0.001 using paired Student t tests). CIPD = chronic inflammatory demyelinating polyneuropathy; Nfasc155 = neurofascin-155.

(A and B) New-born rat pups received an intraperitoneal injection of 250 µg of anti-Nfasc155 IgG4, Fab, or F(ab’)₂ from patient AN1 (n = 4 animals for each condition and age). As controls, animals received 250 µg of IgG4 from healthy donors. Two days after injection, animals were killed, and sciatic nerve fibers were fixed. Teased sciatic nerve fibers were then immunolabeled for voltage-gated sodium channels (Nav; green) to label nodes and heminodes and for CASPR1 (red) to label paranodes. (C and D) The percentage of Nav clusters lacking CASPR1-positive paranodes (arrowheads) or with 1 or 2 flanking CASPR1 positive paranodes (double arrowheads) was quantified, as well as the paranodal length (D) (n = 100–200 nodes or paranodes for each condition). The injection of native IgG4 or F(ab’)₂ fragments from patient AN1 strongly abrogated the formation of CASPR1-positive paranodes and resulted in a higher percentage of heminodes lacking paranodes (****p < 0.0001, ***p < 0.001, **p < 0.005, and *p < 0.05 by one-way ANOVA followed by Bonferroni post hoc tests). The mean length of paranodes was also shorter after treatment with native IgG4 or F(ab’)₂ fragments reactive to Nfasc155 (****p < 0.0001 and ***p < 0.001 by one-way ANOVA followed by Bonferroni post hoc tests). By contrast, the injection of the monovalent Fab fragment of IgG4 did not affect the formation or the length of paranodes (G and H). Scale bar: 10 μm. Bars represent mean and SEM CASPR1 = contactin-associated protein 1; ns = nonsignificant; Nfasc155 = neurofascin-155.

(A–D) Rat sciatic nerve were incubated in vitro for 3 hours with control IgG4 (A) or with Nfasc155-reactive IgG4 (B), Fab fragment (C), or F(ab’)₂ fragment (D) from patient AN1. Fibers were then immunostained for IgG (green) and contactin-1 (CNTN1; red) to label paranodes. By contrast to control IgG4, Nfasc155-reactive IgG4 accumulated at paranode vicinity (double arrowheads) and along the outer mesaxon (arrows). The cleavage into the Fab fragment inhibited IgG4 accumulation at the mesaxon and around paranodes (C). By contrast, the cleavage into the F(ab’)₂ fragment did not alter the ability to aggregate at the mesaxon and paranode vicinity despite the absence of the Fc region (D). Scale bars: 10 μm. Nfasc155 = neurofascin-155.