Phylogenetic Conservation of Glycoprotein 96 Ability to Interact with CD91 and Facilitate Antigen Cross-Presentation 1 Jacques Robert, 2 * Thaminda Ramanayake,* Gregory D. Maniero, † Heidi Morales,* and Asiya S. Chida* Although the ability of gp96 to activate APCs and generate CD8 CTLs against peptides they chaperone through interaction with the endocytic receptors CD91 is supported by solid evidence, its biological relevance in immune surveillance is debated. We have used an evolutionary approach to determine whether gp96 interacts with receptors expressed on APCs and promotes MHC class I cross-presentation of minor histocompatibility Ags (H-Ags) to CTLs in the frog Xenopus. We show that in Xenopus gp96 binds the CD91 homolog at the surface of peritoneal leukocytes, and that this binding is inhibited by molar excess of unlabeled gp96 or the CD91 ligand  2 -macroglobulin, by anti-CD91 Ab and by the specific CD91 antagonist receptor-associated protein. Surface binding followed by internalization of gp96 was confirmed by fluorescent microscopy. Furthermore, adoptive transfer of peritoneal leukocytes pulsed with as little as 800 ng of gp96 chaperoning minor H-Ags, but not minor H-Ag-free gp96, induces potent CD8 T cell infiltration and Ag-specific accelerated rejection of minor H-locus disparate skin grafts. Inhibition of gp96-CD91 interaction by pretreatment with anti-CD91 Ab and receptor-associated protein impairs both CD8 T cell infiltration and acute skin graft rejection. These data provide evidence of the conserved ability of gp96 to facilitate cross-presentation of chaperoned Ags by interacting with CD91. The persistence of this biological process for >350 million years that separate mammals and amphibians from a common ancestor strongly supports the proposition that gp96 and CD91 are critically involved in immune surveillance. The Journal of Immunology, 2008, 180: 3176 –3182. I n mammals (reviewed in Ref. 1), as well as in the frog Xe- nopus (2), compelling evidence indicates that the stress pro- tein gp96, also known as glucose-regulated protein 94, elicits potent adaptive CTL responses against the various antigenic pep- tides it chaperones. gp96-driven CTL responses appear to reside from two main properties: 1) the capacity of gp96 independent of associated peptides to stimulate professional APCs to produce proinflammatory cytokines and chemokines and to up-regulate co- stimulatory molecules (3–5) and 2) the capacity of gp96 to chap- erone and shuttle antigenic peptides, through receptor-mediated endocytosis, into the MHC class I presentation pathway of APCs (6, 7). This process is referred to as cross-presentation (8, 9) and it is thought to be important to prime naive CD8 T cells (10, 11). One endocytic receptor expressed at the surface of APCs that interacts with gp96 has been identified as CD91, the receptor for  2 -macroglobulin ( 2 -M 3 ; Refs. 12 and 13). CD91 has been shown to be critically involved in mediating cross-presentation of antigenic peptides carried by gp96 by competition with  2 -M, in- hibition with anti-CD91 Abs or the natural CD91 inhibitor recep- tor-associated protein (RAP; in the case of gp96), and by RNA interference (14). More recently, the scavenger receptor type A (SR-A) has been reported to also bind gp96 and mediate transfer of peptides to the class I presentation pathway (15). In addition to endocytic receptors, several signaling receptors appear to interact with gp96 and subsequently activate APCs; these include CD36 (16) and TLR2/4 (17). The multiple essential cellular functions performed by gp96 make it hard to evaluate the importance of its role in immune function. Recent evidence for the biological relevance of gp96 in cross-presentation and ultimately in immune surveillance comes from the demonstration that the depletion of three major heat shock proteins (hsp; including gp96) from lysate of cell lines ex- pressing the model Ags -galactosidase and OVA eliminate their cross-priming activity (18). The relevance of this study is, how- ever, challenged by another study showing efficient cross-priming response from the subcellular fraction that lacks major species of hsp (11). In addition, although a peptide-binding site has been identified in the N-terminal 355 aa (19, 20), the modalities of pep- tide binding, especially in vivo, are still not well-characterized. Despite the identification of a broad range of immunodominant class I peptide epitopes associated with gp96 (21, 22), the relative abundance of such peptides is extremely low (23), and in some cases the peptide dependence of the response generated has been questioned (24). Finally, the involvement or requirement of dif- ferent receptors in gp96-mediated immune response is still unknown. Our approach is to investigate the potential immunological role of gp96 from an evolutionary perspective using the frog Xenopus. We postulate that given the high degree of phylogenetic conser- vation of the structure of gp96, its role in immune responses should have been conserved during evolution. In this regard, we have *Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642; and † Department of Biology, Stonehill College, Eas- ton, MA 02357 Received for publication November 7, 2007. Accepted for publication January 2, 2008. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported by T32-AI 07285 (to H.M.), R01-CA-108982-02, and R24- AI-059830 from the National Institutes of Health and MCB-0445509 from the National Science Foundation. 2 Address correspondence and reprint requests to Dr. Jacques Robert, Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642. E-mail address: Jacques_robert@urmc.rochester.edu 3 Abbreviations used in this paper:  2 -M,  2 -macroglobulin; SR-A, scavenger recep- tor type A; RAP, receptor-associated protein; hsp, heat shock protein; PL, peritoneal leukocyte; APBS, amphibian PBS; H-Ag, histocompatibility Ag. Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00 The Journal of Immunology www.jimmunol.org