The low-affinity receptor for IgE (CD23) on B lymphocytes is spatially associated with HLA-DR antigens

Two hybridomas that produce the mAbs 135 and 449 B4 were obtained that inhibited the binding of IgE to the Fc epsilon RL/CD23 on the EBV- transformed B cell line RPMI 8866. mAb 135 was obtained from a mouse immunized with RPMI 8866 cells, whereas mAb 449B4 was obtained from a mouse immunized with a partially purified preparation of Fc epsilon RL/CD23 obtained as the eluate of an IgE immunoabsorbent loaded with a soluble extract of RPMI 8866 cells. These two mAbs bound to Fc epsilon RL/CD23- cell lines and precipitated two polypeptides with 36,000 Mr and 28,000 Mr, which were the HLA-DR alpha and beta chains, respectively. Immunoprecipitation with mAb 135 of NP-40 lysates from dithio-bis(succinimidyl propionate) (DSP) crosslinked 125I-labeled RPMI 8866 or normal B cells incubated with rIL-4 showed three polypeptides with 42,000, 36,000, and 28,000 Mr. The 42,000 Mr polypeptide is identical to the Fc epsilon RL/CD23 since it could be precipitated by the anti-Fc epsilon RL/CD23 mAb 25 after resolubilization from the SDS- PAGE gel. Immunoprecipitations of the crosslinked cell extracts carried out with the anti-Fc epsilon RL/CD23 mAb 25 yielded the same three polypeptides. Furthermore, when RPMI 8866 or rIL-4 preincubated normal B cells were solubilized with a digitonin buffer, which prevents the dissociation of noncovalently linked polypeptide complexes, mAb 135 and mAb 25 precipitated complexes composed of three molecules with 42,000, 36,000, and 28,000 Mr. The well-characterized anti-HLA-DR mAb L243 was unable to block the binding of either IgE or mAb 135 to RPMI 8866 cells, although it could immunoprecipitate the complex (HLA-DR-Fc epsilon RL/CD23) from crosslinked cell lysates. Since mAb 135 and L243 were able to both bind the RPMI 8866 cells, it demonstrates that they bind to different epitopes of the HLA-DR complex, the mAb 135 epitope of the HLA-DR molecule being close to the IgE binding site of the Fc epsilon RL/CD23. These data demonstrated that the Fc epsilon RL/CD23 and HLA-DR antigens are spatially associated on the B cell membrane.