Cross-presentation of glycoprotein 96-associated antigens on major histocompatibility complex class I molecules requires receptor-mediated endocytosis

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated prot ein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) re sponses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic c ells (DCs), macrophages, and B cells, but not T cells, are able to bind gp9 6, The binding is saturable and can be inhibited using unlabeled gp96 molec ules. Receptor binding by APCs leads to a rapid internalization of gp96, wh ich colocalizes with endocytosed major histocompatibility complex (MHC) cla ss I and class II molecules in endosomal compartments. Incubation of gp96 m olecules isolated from cells expressing an adenovirus type 5 E1B epitope wi th the DC line D1 results in the activation of E1D-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that o nly receptor-mediated endocytosis of gp96 molecules leads to MHC class I-re stricted re-presentation of gp96-associated peptides and CTL activation, no n-receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the c ross-presentation of antigens from cellular origin.