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

Abstract

Background and objectives: IgG4 autoantibodies to neurofascin-155 (Nfasc155) are associated with a subgroup of patients with chronic inflammatory demyelinating polyneuropathy (CIDP), currently named autoimmune nodopathy. We previously demonstrated that those antibodies alter conduction along myelinated axons by inducing Nfasc155 depletion and paranode destruction. In blood, IgG4 have the potency to exchange their moiety with other unrelated IgG4 through a process called Fab-arm exchange (FAE). This process results in functionally monovalent antibodies and may affect the pathogenicity of autoantibodies. Here, we examined this issue and whether FAE is beneficial or detrimental for Nfasc155 autoimmune nodopathy.

Methods: The bivalency and monospecificity of anti-Nfasc155 were examined by sandwich ELISA in 10 reactive patients, 10 unreactive CIDP patients, and 10 healthy controls. FAE was induced in vitro using reduced glutathione and unreactive IgG4, and the ratio of the κ:λ light chain was monitored. To determine the pathogenic potential of bivalent anti-Nfasc155 IgG4, autoantibodies derived from patients were enzymatically cleaved into monovalent Fab and bivalent F(ab')₂ or swapped with unreactive IgG4 and then were injected in neonatal animals.

Results: Monospecific bivalent IgG4 against Nfasc155 were detected in the serum of all reactive patients, indicating that a fraction of IgG4 have not undergone FAE in situ. These IgG4 were, nonetheless, capable of engaging into FAE with unreactive IgG4 in vitro, and this decreased the levels of monospecific antibodies and modulated the ratio of the κ:λ light chain. When injected in animals, monovalent anti-Nfasc155 Fab did not alter the formation of paranodes; by contrast, both native anti-Nfasc155 IgG4 and F(ab')₂ fragments strongly impaired paranode formation. The promotion of FAE with unreactive IgG4 also strongly diminished the pathogenic potential of anti-Nfasc155 IgG4 in animals and decreased IgG4 clustering on Schwann cells.

Discussion: Our findings demonstrate that monospecific and bivalent anti-Nfasc155 IgG4 are detected in patients and that those autoantibodies are the pathogenic ones. The transformation of anti-Nfasc155 IgG4 into monovalent Fab or functionally monovalent IgG4 through FAE strongly decreases paranodal alterations. Bivalency thus appears crucial for Nfasc155 clustering and paranode destruction.

Figure 1. Anti-Nfasc155 IgG4 Cross-Link Nfasc155 and Undergo Fab-Arm Exchange In Vitro

(A) Principle of Fab-arm exchange (FAE) and monospecificity assay. Reduced glutathione (GSH) favors FAE of monospecific IgG4 to generate bispecific IgG4. Monospecific antibodies cross-link untagged Nfasc155 with biotinylated Nfasc155. Bispecific antibodies are unable to cross-link these proteins. (B) Purified IgG4 from healthy controls (HC; n = 10), seronegative CIDP (CIDP−; n = 10) and Nfasc155+ autoimmune nodopathy (AN+; n = 10) were tested by ELISA against Nfasc155 and then incubated with a biotinylated Nfasc155 and revealed with streptavidin-HRP. IgG4 from HCs or seronegative patients did not cross-link Nfasc155. By contrast, IgG4 from Nfasc155+ patients cross-linked Nfasc155 (***p < 0.001 by one-way ANOVA followed by Bonferroni post hoc tests). The percentage of monospecific Nfasc155 antibodies correlated with the titers of anti-Nfasc155 IgG4. p Value, Spearman correlation coefficient (r), R square (R²), and 95% confidence band (dotted lines) are indicated on the graph. (C) To determine whether anti-Nfasc155 IgG4 can undergo FAE, IgG4 from Nfasc155+ patients (n = 9) were incubated with GSH in the absence (1:0) or presence of a 3-fold excess of HC IgG4 (1:3). The addition of GSH decreased IgG4 ability to cross-link Nfasc155. This effect was exacerbated by the addition of 3-fold excess of HC IgG4. The decrease in monospecific antibodies is shown on the right as ratio (**p < 0.005 compared with control condition in the absence of GSH or HC IgG4 using paired Student t tests). (D) As controls, IgG1 from Nfasc155+ patients (n = 7) were tested against Nfas155 with the monospecificity ELISA assay. Purified IgG1 cross-linked Nfasc155. GSH and 3-fold IgG4 excess did not affect IgG1-mediated cross-linking (p > 0.005 using paired Student t tests). Gray shades delineate positivity limit values. Bars represent mean and SEM, CIDP = chronic inflammatory demyelinating polyneuropathy; ns = nonsignificant; Nfasc155 = neurofascin-155.

Figure 2. Fab-Arm Exchange Modulates the Ratio of κ:λ Light Chain in Nfasc155-Reactive IgG4

(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.

Figure 3. Bivalent F(ab’)² From Nfasc155-Reactive IgG4 Abrogate Paranode Formation but Not Monovalent Fab

(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.

Figure 4. Bivalent Antibodies Cluster on the Schwann Cell Surface of Myelinated Fibers

(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.

Figure 5. Fab-Arm Exchange Decreases the Pathogenicity of Nfasc155-Reactive IgG4

(A) Fab-arm exchange (FAE) was induced in vitro between IgG4 from patient AN1 and healthy donor IgG4. New-born rat pups were injected at birth with control IgG4 (250 µg per animal), IgG4 from patient AN1 (250 µg per animal), or swapped IgG4 from patient AN1 (FAE; 1 mg per animal) and killed after 2 days. Teased fibers were immunostained for voltage-gated sodium channels (Nav; green) and CASPR1 (red) (n = 4 animals for each condition and age). (B and C) The percentage of Nav clusters lacking CASPR1-positive paranodes (arrows) and Nav clusters with 1 or 2 flanking paranodes (double arrowheads) was counted in each group. The injection of native Nfasc155-reactive IgG4 reduced the formation of CASPR1-positive paranodes and significantly decreased the mean length of paranodes. By contrast, swapped reactive Nfasc155 IgG4 did not significantly affect paranode formation. Nonetheless, paranodal length was decreased in animals treated with swapped IgG4 (****p < 0.0001, ***p < 0.001, **p < 0.005, and *p < 0.05 by one-way ANOVA followed by Bonferroni post hoc tests) (n = 100–200 nodes or paranodes for each condition). Scale bars: 10 μm. Bars represent mean and SEM. CASPR1 = contactin-associated protein 1; ns = nonsignificant; Nfasc155 = neurofascin-155.