Binding of staphylococcal enterotoxin A to HLA-DR on B cell lines.
Staphylococcal enterotoxin A (SEA) is a potent polyclonal T cell activator. Its activating effect is entirely dependent upon its binding to accessory cells. Monocytes, B cells, and B lymphomas can bind SEA and support activation of T cells. We have earlier found that Raji cells are particularly efficient as accessory cells for SEA-induced T cell proliferation. In the present investigation we have used this cell line for the isolation and characterization of the membrane molecule to which SEA binds. Flow cytometric analysis of cells dually stained with SEA and anti-HLA-DR mAb showed that the amount of bound SEA was proportional to the HLA-DR expression. Electrophoresis of detergent extracts of Raji cells revealed one distinct SEA-binding band with a Mr of 60 to 65 kDa. This band had the same electrophoretic mobility as the MHC class II molecules. A mAb (G8) with the ability to block SEA binding to Raji cells was established. This mAb was shown to bind to the HLA-DR molecule. Both the G8 mAb and an anti-HLA-DR mAb 9-49 inhibited SEA binding to accessory cells and also inhibited SEA-induced, but not PHA-induced, T cell proliferation and production of IL-2. Immunoprecipitation with specific anti-HLA-DR and anti-HLA-DQ mAb demonstrated that SEA binds to the HLA-DR molecule but not to the HLA-DQ molecule. Binding SEA to Raji cells followed by cross-linking and detergent solubilization of cell membranes, electrophoresis, and Western blotting resulted in two SEA-containing bands corresponding to a Mr of 90 and 105 kDa, respectively. Both these bands also contained the HLA-DR molecule and their appearance could be blocked by preincubation of the Raji cells with the G8 mAb. Collectively the results show that the HLA-DR molecule is the main functional molecule for binding of SEA to accessory cells and that this binding of SEA to HLA-DR is a necessary requirement for SEA-induced T cell activation.
Fischer
H
H
Department of Tumor Immunology, University of Lund, Sweden.
Dohlsten
M
M
Lindvall
M
M
Sjögren
H O
HO
Carlsson
R
R
D000911:Antibodies, Monoclonal / Q000502:physiology; D000938:Antigen-Presenting Cells / Q000378:metabolism; D001402:B-Lymphocytes / Q000378:metabolism*; D001667:Binding, Competitive; D002460:Cell Line; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D004768:Enterotoxins / Q000302:isolation & purification / Q000378:metabolism*; D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008213:Lymphocyte Activation; D008565:Membrane Proteins / Q000378:metabolism; D013211:Staphylococcus aureus / Q000378:metabolism*; D013601:T-Lymphocytes / Q000276:immunology
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D004768:Enterotoxins; D006683:HLA-DQ Antigens; D006684:HLA-DR Antigens; D008565:Membrane Proteins; C026066:enterotoxin A, Staphylococcal