Fig 1. Functional modulation of PlxnA1 by monoclonal antibodies.

(A) Sema3A dose dependency. Fluorescent images of adherent DCs (inset) w re recorded after incubation with the indicated concentrations of Sema3A, followed by image analysis to assess the collapse morphology of individual cells. The activity was scored as described in the Methods. (B) The effect of anti-PlxnA1 antibodies on Sema3A-induced DC collapse. Various antibodies were added to DCs at the indicated concentrations, together with (red circles) or without (blue circles) 0.8 μg/ml Sema3A, and DC morphology was evaluated after 4–5 h. The collapse response was determined as in (A) and is presented as a “collapse index”, wherein the response obtained with Sema3A was set to 100%. (C) Divalence is required for functional activity of agonistic but not antagonistic antibodies. Three rabbit-mouse chimeric antibodies, in the form of intact IgG (filled circles with solid line) or monovalent Fab fragments (open circles with dotted line), were added to DCs together with (red) or without (blue) 0.8 μg/ml Sema3A, and the collapse response was evaluated as in (B). Antibody concentrations are presented as molar concentrations, assuming molecular mass values of 150 kDa (IgG) and 50 kDa (Fab), respectively. All data are presented as the mean ± SD (n = 3) from one representative experiment out of at least two independent sets of experiments.