Figure 1.
Binding interactions of YB2/0- and NS0-produced Fog-1 G1 and G1Δnab antibodies with human FcγRIIIa.
A–D CHO cells expressing FcγRIIIa of allotypes 158F (A, C) and 158V (B, D) were incubated with pre-complexed (A, B) or monomeric (C, D) Fog-1 IgG and binding detected with fluorescent reagents and flow cytometry. Graphs show mean fluorescence of ≥12 000 cells at each antibody concentration and are typical of the results obtained in at least three experiments with each receptor. E–H Examples of the rosetting of FcγRIIIa 158V-expressing cells by RBC sensitised with Fog-1 G1 (YB2/0) at 10 µg/ml (E) and 1.1 µg/ml (F) or with Fog-1 G1 (NS0) at 100 µg/ml (G) and 11 µg/ml (H). Images are typical of eight independent experiments.
Figure 2.
Binding of YB2/0- and NS0-produced Fog-1 G1 antibodies to human FcγR.
A Binding of monomeric IgG was measured for FcγRI using the B2KA cell line and flow cytometry. Fog-1 IgG2 antibody, produced in the YB2/0 cell line, was used as the non-binding control antibody for this receptor. B–F Binding of pre-complexed IgG was measured using CHO cell lines expressing the low affinity receptors which were FcγRIIa, allotypes 131R (B) and 131H (C), FcγRIIb (D) and FcγRIIIb, allotypes NA1 (E) and NA2 (F). The level of background binding is given by the negative control antibody, IgA,κ. Graphs show mean fluorescence of ≥12 000 cells at each antibody concentration and are typical of the results obtained in at least three experiments with each receptor.
Figure 3.
Functional responses of NK cells to RBC sensitised with Fog-1 antibodies.
A The specific lysis of sensitised RBC by NK-cell mediated ADCC is presented as mean±SD of triplicate samples. This experiment used effector cells pooled from 6 donors but similar results were obtained in four experiments with individual donors of PBMCs. B–D The activation of NK cells in response to sensitised RBC as visualised by the level of CD54 on the surface of CD3−, CD56+ lymphocytes. Each graph shows the mean±SD of triplicate samples for each data point. Donors of PBMC were of the following genotypes: FcγRIIIa 158V/V, FcγRIIc 13Q/13Q (B), FcγRIIIa 158F/V, FcγRIIc 13STP/13STP (C) and FcγRIIIa 158F/F, FcγRIIc 13STP/13STP (D). CD54 signals for samples with no test antibody were 14.2±0.1 (B), 17.0±2.5 (C) and 13.0±1.1 (D). CD54 signals for samples incubated with irrelevant IgG1 were 13.3±0.4 (B), 18.7±4.1 (C) and 12.1±0.9 (D).
Figure 4.
Interactions of Fog-1 IgG-sensitised RBC with monocytes and macrophages.
A The numbers of adherent (ext) and phagocytosed (int) RBC per macrophage were determined for RBC sensitised with saturating concentrations of Fog-1 IgG. Results are shown for macrophages from three different donors. The numbers of unsensitised RBC associating with macrophages were typically 15- to 20-fold lower than the numbers of Fog-1 G1 (YB2/0)-sensitised RBC. B The mean chemiluminescent response of monocytes to sensitised RBC is plotted with the error bars indicating the range of the duplicate samples.