Immunolocalization of CD1d in human intestinal epithelial cells and identification of a beta(2)-microglobulin-associated form

In order to better understand the role of intestinal CD1d, we sought to def ine the cellular localization and further characterize the biochemical stru cture of CD1d in human intestinal epithelial cells (IEC), Using a CD1d-spec ific rabbit anti-gst-CD1d antibody, immunoprecipitation of radiolabeled cel l surface proteins detected a previously identified 37 kDa protein as well as a 48-50 kDa protein which were confirmed by Western blotting with a CD1d -specific mAb, D5, Immunoprecipitation of protein lysates with the CD1d-spe cific mAb, D5 and 51.1.3, and the beta(2)-microglobulin (beta(2)m)-specific mAb, BBM.1, followed by N-glycanase digestion and Western blotting with th e D5 mAb showed that the 48-50 kDa protein was a beta(2)m-associated, CD1d glycoprotein. CD1d was immunolocalized to the apical and lateral regions of native small and large intestinal IEC as defined by confocal laser microsc opy using the D5 mAb and the rabbit anti-gst-CD1d antibody. In addition, a large apical intracellular pool of CD1d was identified. Identical observati ons were made with polarized T84 cells. Selective biotin labeling of apical and basolateral cell surfaces followed by immunoprecipitation with the D5 mAb, N-glycanase digestion and avidin blotting confirmed the presence of gl ycosylated CD1d on both cell surfaces and immunolocalization of the 37 kDa non-glycosylated form of CD1d to the apical cell surface. These studies sho w that CD1d is located in an ideal position for luminal antigen sampling an d presentation to subjacent intraepithelial lymphocytes.