Experiments used multiple B-cell lines: Raji, RM3 (MHC class II deficient derivative of the Raji line), two Epstein-Barr virus (EBV)-transformed B-cell lines (LAD and JY), EBV-negative Burkitt's lymphoma Ramos cells (germinal centre B cells), and Daudi cells, which are EBV-transformed but do not express the EBV-encoded latent infection membrane protein (LMP)-1. In addition, B cells from normal human tonsils were examined.
Immunoprecipitation experiments using biotin-labelled cells and two anti-CD40 monoclonal antibodies (mAbs) were performed. Co-precipitation experiments were also performed with mAb specific for CD23, which is known to physically associate with HLA-DR. Western blotting with anti-HLA-DR mAb was used to analyse immunoprecipitates obtained with CD40, CD23 mAbs or surface immunoglobulin M. Daudi cells were used to exclude the possibility of EBV-encoded LMP1 causing aggregation of CD40 and HLA-DR. Complementary experiments were performed using HLA-DR for immunoprecipitation and CD40 mAb in western blots.
To assess whether crosslinking of CD40 triggered association of CD40 and HLA-DR with the cytoskeleton, murine anti-CD40 antibodies, followed by anti-mouse IgG, were used to crosslink CD40 molecules on the cell surface. Fluorescein isothiocyanate (FITC)-labelled anti-DR antibodies were used to label DR molecules and the labelled cells were lysed with NP-40. Flow cytometry was used to assess the percentage of HLA-DR molecules associated with the NP-40 insoluble cell matrix. Finally, using Raji cells, the interaction of MHC class II with the ligand staphylococcal enterotoxin A (SEA) was used to determine whether ligand binding influenced the formation of the CD40/MHC class II complex.