FIGURE 5.

CD81 is expressed on the surface of DCs and is bound to MHC-II molecules. A, DCs were stained with the anti-CD81 mAb MT81 and analyzed by flow cytometry. In the upper panel, a representative histogram of CD81 staining of LPS-activated DCs from either CD81^+/− mice (heterozygous (Het), black solid line) or CD81^−/− mice (KO, black dotted line) is shown. Also shown is staining for isotype control antibody (gray filled). The inset shows a CD81 immunoblot of Brij-58 lysates of DCs isolated from wild-type (WT), CD81 heterozygous (Het), and CD81-deficient (KO) mice. In the lower panel, a representative histogram of CD81 staining of untreated (immature, gray solid line) or LPS-activated (mature, black solid line) DCs from B10.BR mice is shown. Also shown are isotype controls for immature (gray filled) and mature (black dotted line) DCs. B, MHC-II molecules were immunoprecipitated from Brij-58 (B) or Triton X-100 (T) lysates of activated DCs using isotype control, I-A-, or I-E-specific mAb and analyzed by immunoblotting using I-E α-chain, I-A β-chain, or CD81 antibodies as indicated. An aliquot of the cell lysate was also analyzed. A representative gel is shown. C, DCs were prepared from CD81 heterozygous (control) and CD81 KO mice crossed onto the H-2^k background. MHC-II molecules were immunoprecipitated (IP) from Brij-58 lysates of activated DCs using I-A- or I-E-specific mAb and analyzed by immunoblotting as indicated. An aliquot of the cell lysate was also analyzed. The relative amount of I-A or I-E present in each immunoprecipitate was determined by densitometry and expressed as a fraction of the amount of I-A or I-E present in the cell lysate, and the binding of I-E to I-A and I-A to I-E in CD81 KO DCs were normalized to that obtained in the heterozygous controls. The mean ± S.D. from three independent experiments is shown.