9E9 binds to and blocks both FcγRIII and FcγRIV in vivo. (A) Adoptive transfer studies in WT or FcγR^−/− mice. Mice were injected IV with a 1:1 mix of target hCD20 Tg (T) and WT nontarget (nT) splenocytes differentially stained with CFSE (50 and 5 µM, respectively) as previously described.⁸  Approximately 20 hours later, mice were injected with irrelevant mAbs or Ritm2a (10 µg) and then 24 hours later spleens were stained with anti-mouse CD19 (1D3) before assessment by flow cytometry and the T:nT ratio of B cells in the spleen calculated. (B) Adoptive transfer studies comparing FcγRI^−/− and FcγRIII^−/− mice in combination with 9E9. Mice were treated with 9E9 (400 µg) 3 to 4 hours before injection of Ritm2a (10 µg) as in panel A. (C) SPR data demonstrating the binding affinity of 9E9 or de-gly9E9 to (i) FcγRI, (ii) FcγRIIb, (iii) FcγRIII, and (iv) FcγRIV. Anti-His antibody (R&D Systems) was bound to a CM5 sensor chip and His-tagged mFcγR (R&D Systems) captured. 9E9 or de-gly9E9 antibodies were then injected (200 nM) at 30 µL per minute. Association was monitored for 5 minutes and dissociation monitored for 10 minutes. (D) Adoptive transfer studies using de-gly9E9 in either FcγRI^−/− or FcγRIII^−/− mice. To produce de-gly9E9, 9E9 was treated with 0.05 U of PNGase F per µg at 37°C overnight. Deglycosylation was confirmed by SDS-PAGE and/or SPR. Purification of antibody from enzyme was achieved through size-exclusion chromatography using Sephadex 200. Adoptive transfer assay was carried out as in panel B; however, mice were treated with de-gly9E9 (400 µg) 3 to 4 hours before injection of Ritm2a (N = 4). Statistical analysis was performed using 2-way ANOVA. Experiments were cleared through local ethical committees and performed under home office license PPL30/2964. ANOVA, analysis of variance; CFSE, carboxyfluorescein succinimidyl ester; RU, resonance units; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. ***P = .0003; ****P < .0001 (2-way ANOVA, multiple comparisons using Tukey's test).