B-cell surface receptor–specific mAbs interact with and activate FcγRIIb in a cis and trans fashion. (A) FcγRIIb-positive Daudi cells (left) or FcγRIIb-negative Ramos cells (right) were incubated with 5 µg/mL A488-labeled mAb at 5 × 104 cells/mL at 4°C. After extensive washing, the cells were mixed 1:1 with FcγRIIb-negative Ramos cells (left) or FcγRIIb-positive Daudi cells (right) at a final concentration of 1 × 105 cells/mL for 30 minutes at 37°C. Lysates were blotted for pFcγRIIb and α tubulin as a loading control. Sample lanes: (1) NT, (2) RTX, (3) Tosit, (4) F3.3 (MHC II), (5) AT13/5h (CD38), (6) M15/8 (BCR). (B) A488-mAb labeled FcγRIIb-negative Ramos cells (left) or FcγRIIb-positive Daudi cells (right) were mixed 1:1 with FcγRIIb-positive Daudi cells (left) or FcγRIIb-negative Ramos cells (right) at a final concentration of 4 × 106 cells/mL then lysates were blotted as described in panel A. (C) Ramos cells B-cell surface receptors were transfected with empty vector, or Tr FcγRIIb and assessed for expression by flow cytometry using AT10-PE. Clones expressing low (solid gray line) or high (dotted line) were compared with control cells (filled histogram). (D) Ramos Tr FcγRIIb-low (left) or FcγRIIb-positive Daudi cells (right) were incubated with 5 µg/mL A488-labeled mAb at 4 × 106 cells/mL at 4°C. After washing, the cells were mixed 1:1 with untransfected Daudi cells (left) or Ramos Tr FcγRIIb-low (right) cells at 4 × 106 cells/mL for 30 minutes at 37°C. Lysates were then blotted as in panel A. (E) As in panel D but using Ramos Tr FcγRIIb-high cells. In all blots, a positive control of A488-RTX–labeled FcγRIIb-positive Daudi cells mixed 1:1 with FcγRIIb-negative Ramos cells at a final concentration of 4 × 106/mL was included. Two representative blots are shown for each condition. Ctl, control.
