Anti–neurofascin-155 IgG4 antibodies prevent paranodal complex formation in vivo
We next investigated whether anti-Nfasc155 IgG4 may affect the interaction between Nfasc155 and its axonal partners CNTN1 and CASPR1 using a cell aggregation assay. For that purpose, HEK293 cells were transfected with mCherry-conjugated rat Nfasc155, or with CNTN1 and GFP-tagged CASPR1. Cells were then mixed together and incubated for 2 hours under gentle agitation to enable cell aggregate formation. The percentage of cell aggregates showing contacts between mCherry-expressing (red) and GFP-expressing (green) cells was then quantified, as well as the percentage of green cells per aggregate. As negative controls, Nfasc155-expressing cells were incubated with cells expressing GFP alone. Under such conditions, minimal interaction was observed between Nfasc155-and GFP-expressing cells (Figure By contrast, Nfasc155-expressing cells formed numerous interactions with CNTN1/CASPR1-expressing cells, and most cell aggregates contained 50% green and red cells (Figure To test the efficacy of anti-Nfasc155 IgG4 to dismantle these aggregates, 10 g of IgG4 was added to the cell mixture during the 2-hour incubation step. For comparison, anti-CNTN1 IgG4 antibodies were also tested. As previously described (28), anti-CNTN1 IgG4 potently inhibited the interaction of Nfasc155-expressing cells with CNTN1/ CASPR1. By contrast, anti-Nfasc155 IgG4 from the 3 distinct CIDP patients did not decrease cell-cell interaction (Figure Interestingly, cell aggregates incubated with anti-Nfasc155 IgG4 presented a higher percentage of Nfasc155-expressing cells. This indicated that anti-Nfasc155 IgG4 antibodies do not prevent the interaction between Nfasc155 and its axonal partners CNTN1/ CASPR1, but may favor Nfasc155 clustering.