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The Journal of Immunology, 2008, 180, 3176 -3182
Copyright © 2008 by The American Association of Immunologists, Inc.
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Phylogenetic Conservation of Glycoprotein 96 Ability to Interact with CD91 and Facilitate Antigen Cross-Presentation1

Jacques Robert2,*, Thaminda Ramanayake*, Gregory D. Maniero{dagger}, Heidi Morales* and Asiya S. Chida*

* Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642; and {dagger} Department of Biology, Stonehill College, Easton, MA 02357


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
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 {alpha}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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
In mammals (reviewed in Ref. 1), as well as in the frog Xenopus (2), compelling evidence indicates that the stress protein gp96, also known as glucose-regulated protein 94, elicits potent adaptive CTL responses against the various antigenic peptides 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 costimulatory molecules (3, 4, 5) and 2) the capacity of gp96 to chaperone 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 {alpha}2-macroglobulin ({alpha}2-M3; 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 {alpha}2-M, inhibition with anti-CD91 Abs or the natural CD91 inhibitor receptor-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 expressing the model Ags β-galactosidase and OVA eliminate their cross-priming activity (18). The relevance of this study is, however, 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 peptide 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 different 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 conservation of the structure of gp96, its role in immune responses should have been conserved during evolution. In this regard, we have shown that the ability of gp96 to chaperone antigenic peptides and generate class Ia-restricted CTL responses against them has been conserved between amphibians and mammals (25). Moreover, we have demonstrated that in Xenopus, gp96 elicits a robust cellular immune response against tumor (26). Furthermore, we have shown that Xenopus gp96, complexed in vitro with exogenous antigenic peptides, can interact as efficiently as murine gp96 to facilitate cross-presentation of the antigenic peptide by class I molecules of murine macrophages and activation of a MHC-restricted mouse CD8 T cell line (26). Finally, we have recently characterized the Xenopus homolog of CD91 and demonstrated its up-regulation in adult peritoneal leukocytes (PLs) during an immune response (27).

Taking advantage of a minor histocompatibility Ag (H-Ag) transplantation system in Xenopus isogenic clones, we have demonstrated here the evolutionary conserved ability of gp96 to facilitate Ag cross-presentation through interaction with CD91.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals and reagents

LG-15 and LG-6 Xenopus clones were obtained from our breeding colony (www.urmc.rochester.edu/smd/mbi/xenopus/index.htm). All animals were handled under strict laboratory and University Committee on Animal Resources regulations (approval number 2004-199), minimizing discomfort at all times. Rabbit anti-CD91 polyclonal Ab and full-length human RAP were provided by Antigenic. {alpha}2-M (Sigma-Aldrich) was converted into a thiol-ester-cleaved activated derivative ({alpha}2-M*) that binds to CD91, by incubation with 0.2 M NH4HCO3 (28).

Purification of gp96

gp96 was purified from liver tissue of LG-6 and LG-15 Xenopus clones by ammonium sulfate fractionation and Con A-Sepharose and DEAE column chromatography as previously described (29). Purity was verified by silver staining after SDS-PAGE (see Fig. 1). His-tagged gp96 recombinant protein was purified from overnight Escherichia coli culture using nickel-agarose chromatography (yield: 10 µg of gp96/ml of bacterial culture). gp96 labeling was done either by biotinylation with sulfosuccinimidyl-6'-(biotinamido)-6-hexanamido hexanoate (sulfo-NHS-LC-biotin) (0.5 mg/ml)/amphibian PBS (APBS) solution or with Alexa Fluor 488 (Molecular Probes) for 30 min according to the manufacturer’s protocols. Labeled gp96 was separated from residual product by chromatography on Sephadex 50.


Figure 1
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  		FIGURE 1. Purification of normal and recombinant Xenopus gp96. gp96 purified from liver tissue according to Ref. 25 or from overnight E. coli culture using nickel-agarose chromatography (see Materials and Methods), were resolved by SDS-PAGE using 10% gel under reducing condition and were silver stained to assess purity (S). Separate gels were transferred onto polyvinylidene difluoride membranes, incubated with rat anti-gp96 mAb, HRP-conjugated goat-anti-rat followed by chemoluminescence development (W).

 
Cell-binding assay

A total of 5 x 105 PLs obtained by lavage from frogs either untreated or 3–4 days after elicitation by injection with heat-killed E. coli bacteria (27) were incubated with labeled gp96 (5–10 µg/ml) in 100 µl of washing buffer (1% BSA in APBS) for 30 min on ice followed by three washes in ice-cold APBS. Cells were analyzed by flow cytometry on a FACSCalibur; 10,000 events were collected. PLs were also stained with rabbit anti-mouse CD91 polyclonal Ab followed by goat anti-rabbit conjugated to a FITC label. For two-color flow cytometry, cells were first stained with Alexa-gp96 or anti-CD91 followed by FITC-conjugated goat anti-rabbit Ab, then with Xenopus-specific anti-class I TB17 mAb followed by allophycocyanin-conjugated goat anti-mouse Ab. Both goat secondary Abs were preadsorbed twice on Xenopus splenocytes. Other Xenopus-specific mAbs recognizing class II (AM20), CD8 (AM22), CD5 (2B1), and IgM (10A9) have been described elsewhere (29).

Internalization assay

PLs were incubated with Alexa-gp96 and washed as before, then further incubated 15–30 min either on ice or at 27°C. Cells were fixed with 2% paraformaldehyde for 15 min on ice, washed twice, and stained 15 min with Alexa 594 wheat germ agglutinin (5 µg/ml) and blue-fluorescent Hoechst 33342 dye (2 mM) for selective staining of plasma membrane and nucleus, respectively. Cells were cytocentrifuged in 400 µl of 1:1 (v/v) 20% BSA in APBS and mounted in Mowial. Samples were visualized with a Leica DMIRB inverted fluorescence microscope with a cooled charge-coupled device (Cooke) controlled by Image-Pro Plus software (Media Cybernetics).

Cross-presentation assay

PLs were pulsed with gp96 (0.8–1.0 µg) for 1 h on ice then washed three times with cold APBS, before adoptive transferred into isogenetic recipients by i.p. injection (0.5–5 x 106 cells). Two to 5 days after transfer, frogs were skin-grafted as previously described (30). Briefly, a piece of ventral skin (5 x 5 mm) from a donor was inserted under the dorsal skin of a recipient and, 24 h later, the overlying host skin was removed. The extent of tissue rejection was estimated based on the percent of the skin graft surface that no longer had silvery iridophore (30).

Whole-mount immunohistochemistry

Transplanted skin tissues were directly stained as previously published (30) without fixation at 4°C for 3 h in 0.2 ml of APBS-BSA containing 25 µg/ml of either anti-CD8 (AM22), or anti-class II (AM20) mAbs, followed by PE-conjugated anti-mouse IgM (Southern Biotechnology Associates) or goat anti-mouse IgG (Jackson ImmunoResearch Laboratories) secondary Abs (25 µg/ml, 0.2 ml). Mouse IgM and IgG1 isotypes were used as negative controls. The stained skin samples were gently pressed with a coverslip on a microscope slide, and observed under a fluorescent microscope set for PE detection (excitation filter D546/10x, emission filter D580/30m; Chroma Technologies). Images were captured in 8-bit black and white format, and analyzed with Image-Pro Plus software (version 5.0; Media Cybernetics).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Specific binding of gp96 to Xenopus macrophage-like cells

We have recently identified a Xenopus CD91 gene homolog at the cDNA and protein level (27) and showed its expression by immune cells including PLs. To further characterize this receptor and determine whether it interacts with gp96, we generated a Xenopus-recombinant gp96 (Rec-gp96) with its KDEL replaced by a His-tag. Rec-gp96 purified from E. coli culture displays similar molecular mass form dimers and is recognized by both anti-gp96 mAb (Fig. 1) and polyclonal Ab (data not shown). Rec-gp96 either biotinylated or conjugated to Alexa was then used for cell surface binding assays on PLs obtained by lavage of frogs untreated or 3–4 days after elicitation by injection with heat-killed E. coli before harvest (27). These elicited PLs are in majority (50–60%) mononucleated cells with multiples pseudopods typical of macrophages. Compared with PLs from untreated frogs bacteria-elicited PLs display an increased size (i.e., high forward scatter), are enriched for nonlymphocytic leukocytes (e.g., low signal for B and T cell markers), and still express, albeit at a lower level, surface MHC class II and class I (Fig. 2).


Figure 2
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  		FIGURE 2. Comparison by flow cytometry of PLs elicited by heat-killed E. coli and untreated. PLs obtained by lavage from frogs either untreated (gray peaks) or 3 days after injection with heat-killed E. coli bacteria (white peaks with thick line) were stained with Xenopus-specific anti-IgM (10A9), anti-class I (TB17), anti-class II (AM20), anti-CD5 (2B1), anti-CD8 (AM22), followed by FITC-conjugated secondary goat anti-mouse Ab. Ten thousand events were collected and analyzed on a FACSCalibur. Upper dot plots, The leukocyte gate based on forward and side scatter. Histograms of the forward scatter (second lane) show the change in relative size of cells.

 
Cell surface binding of gp96 was determined by incubating PLs with labeled rec-gp96 on ice. Flow cytometry reveals that, similar to mammals, labeled Xenopus rec-gp96 consistently binds at the surface of a fraction of PLs (Fig. 3A). The fraction of PLs binding gp96 varied from 10 to 30% from one animal to another. Nonrecombinant gp96 purified from Xenopus outbred livers showed similar binding capacity (Fig. 3D). No significant binding was obtained with labeled BSA or OVA (Fig. 3, A, D, and E). In addition, the binding of labeled gp96 was saturable (Fig. 3F), and was competed by molar excess of unlabeled rec-gp96 (Fig. 3, A, D, and E), two features suggesting interaction with a surface receptor.


Figure 3
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  		FIGURE 3. gp96 binding to CD91 at the surface of PLs. A, Flow cytometry of PLs (0.5 x 106) incubated for 20 min on ice with 5 µg/ml biotinylated rec-gp96 (bold-lined peak), biotinylated OVA (gray-filled peak), or biotinylated rec-gp96 with a 50x molar excess of nonbiotinylated rec-gp96 (dot-lined peak), followed by FITC-conjugated streptavidin. Cell were washed three times with ice-cold APBS-BSA before analysis on a FACSCalibur; 10,000 events were collected. B, PLs incubated with 10 µg/ml biotinylated rec-gp96 (bold-lined peak), activated {alpha}2-M* (dash-lined peak), or biotinylated OVA (negative control; dot-lined peak). C, Competition of gp96 binding (bold-lined peak) with 10x molar excess of activated {alpha}2-M* (gray-filled peak) on PLs. D, PLs incubated with 10 µg/ml nonrecombinant biotinylated gp96 purified from liver tissues of outbred frogs (bold-lined peak), from biotinylated OVA (gray-filled peak), or biotinylated nonrecombinant gp96 with a 50x molar excess of nonbiotinylated nonrecombinant gp96 (dot-lined peak). E, Competition of 5 µg/ml Alexa-conjugated rec-gp96 binding on PLs (2) with either 10x (3), 20x (4), or 100x (5) molar excess of unlabeled rec-gp96, or 100x molar excess of OVA (6). F, Saturation of gp96 binding by flow cytometry (mean fluorescence intensity (MFI)). PLs (0.5 x 106) were incubated with 5, 10, 20, 50, or 80 µg/ml Alexa-conjugated rec-gp96 (squares) or Alexa-OVA (gray diamonds). G, Inhibition of Alexa-conjugated rec-gp96 binding on PLs by preincubation with anti-CD91 Ab (dilution 1/100) or RAP (5 and 2.5 µg), or by competition with 10x molar excess of {alpha}2-M*. Data from three different experiments are expressed as the average (±SD) fold decrease of the signal obtained with 5 µg of Alexa-conjugated rec-gp96 alone. NRS showed no change and replacing rec-gp96 by Alexa-conjugated OVA showed some increase in nonspecific background signal.

 
Because {alpha}2-M is a natural ligand of CD91, we tested whether activated {alpha}2-M ({alpha}2-M*) can bind at the surface of Xenopus cells and compete with the binding of gp96. In several experiments (one is shown in Fig. 3B), {alpha}2-M* was able to bind to a fraction of PLs comparable to that of gp96 (e.g., 15–30%). Furthermore, the binding of gp96 was sensitive to the presence of a 10-fold molar excess of {alpha}2-M* (Fig. 3, C and G). To obtain further evidence of CD91 involvement in gp96 binding, we next used a rabbit polyclonal anti-CD91 Ab that specifically cross-reacts with Xenopus CD91 (27). Preincubation with this anti-CD91 Ab, but not the preimmune rabbit serum, inhibits the binding of gp96 (Fig. 3F). Similarly, preincubation with a human recombinant of the natural CD91 antagonist RAP, markedly decreased rec-gp96 binding (Fig. 3F). Interestingly, two-color flow cytometry analysis reveals that a fraction of PLs expressing a high level of MHC class I at their surface coexpresses surface CD91 and binds Alexa-gp96 (Fig. 4A). CD91+ PLs are also class II+ (Fig. 4B).


Figure 4
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  		FIGURE 4. gp96 binding, CD91, and MHC class I expression profiles of PLs by two-color flow cytometry. A, PLs (0.5 x 106) were incubated with rabbit anti-CD91 Ab followed by FITC-conjugated anti-rabbit secondary Ab (1) or with 5 µg/ml Alexa-gp96 (2). Cells were then stained with Xenopus anti-class I mAb TB17, followed by allophycocyanin-conjugated anti-mouse secondary Ab. Both secondary Abs were preadsorbed on Xenopus leukocytes, and show minimal cross-reaction. Ten thousand events were collected; leukocytes were gated based on forward and side scatter profiles. B, PLs (0.5 x 106) were stained either (1) with preimmune rabbit serum followed by FITC-conjugated anti-rabbit secondary Ab, and mouse IgG1 isotype control followed by allophycocyanin-conjugated anti-mouse secondary Ab; (2) rabbit anti-CD91 Ab, followed by FITC-conjugated anti-rabbit secondary Ab, and Xenopus anti-class I mAb TB17, followed by allophycocyanin-conjugated anti-mouse secondary Ab; (3) rabbit anti-CD91 Ab, followed by FITC-conjugated anti-rabbit secondary Ab, and biotinylated Xenopus anti-class II mAb flowed by allophycocyanin-conjugated streptavidin.

 
gp96 was also found to bind a fraction (10–15%) of PBMC and splenocytes (20–25%) but not to purified T cells; excess of unlabeled rec-gp96 or {alpha}2-M* inhibited gp96 binding on PBMC (data not shown). Overall, these data indicate that CD91 is a receptor that binds gp96 at the surface of a fraction of Xenopus leukocytes in the peritoneal cavity and in the blood.

gp96 internalization by PLs

To further validate that the receptor bound by gp96 is endocytic, we monitored gp96 internalization by fluorescence microscopy. Cells were allowed to bind Alexa-conjugated rec-gp96, washed, and then either incubated on ice or warmed to 27°C for various times. In several experiments, 8–10% cells incubated on ice displayed a surface positive staining pattern, while cells with intracellular staining pattern were rarely observed (<1%) and were usually dead cells. In contrast, following incubation for 15–30 min at 27°C, the majority of positively stained cells showed an intracellular staining pattern (Fig. 5). These results strongly suggest that receptor(s) expressed by PL cells and binding gp96 is endocytic.


Figure 5
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  		FIGURE 5. Internalization of rec-gp96 by PLs. Fluorescence microscopy of PLs incubated 30 min on ice with 10 µg/ml Alexa-conjugated rec-gp96 (A) or at 27°C to allow internalization (B). Following treatment, cells were fixed and stained with WGA and Hoechst. Images are representative of ~10% positively stained cells taken at single focal plan under 6.3x oil objective. Bar, 10 µm.

 
Responses elicited by adoptively transferred PLs pulsed with gp96/minor H-Ag complexes

To functionally characterize the receptor-mediated cross-presentation of Ags chaperoned by gp96 in Xenopus, we took advantage of a minor H-Ag-mismatched system (25, 31). LG-15 and LG-6 isogenetic Xenopus clones display the identical heterozygous (a/c) MHC haplotype, but differ from each other by multiple minor H loci (31). Skin grafts transplanted between these clones are rejected more slowly (30–50 days at ~21°C) than are MHC-disparate allografts (20°C ± 2 days at ~21°C). We have shown that immunization with gp96 generates a specific cellular immune response against chaperoned minor H-Ags that effects an accelerated rejection of minor H-locus disparate skin grafts in vivo and an MHC-specific CD8+ cytotoxic T cell response in vitro (25, 32).

To determine whether the T cell responses generated by gp96/minor H-Ag complex involve cross-presentation of minor H-Ags by APCs, bacteria-elicited PLs from isogenetic LG-15 animals were pulsed with minor H-disparate LG-6-derived gp96 and adoptively transferred into isogenetic LG-15 recipients; these recipients were grafted with minor H-disparate LG-6 skin 1 wk later. In multiple experiments (one representative is shown in Fig. 6), adoptively transferred LG-15 PLs pulsed with as little as 0.8 µg of LG-6 gp96 induced an accelerated rejection of LG-6 skin by LG-15 recipients, with an average time for complete rejection of 19.0 ± 1.2 days (Table I), a rejection rate similar to an acute secondary response due to priming with a minor H-disparate graft or gp96 derived from a minor H-Ag-disparate donor (25). Similar responses were obtained in the reverse combination where LG-6 PLs, but not CD8 T cells, pulsed with LG-15-derived gp96 and transferred into LG-6 frogs generated acute rejection of LG-15 skin (Table I). In this case, PLs were not obtained by bacterial stimulation, which further support that the response is Ag-specific and not due to PL activation by bacterial products.


Figure 6
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  		FIGURE 6. Cross-presentation of minor H-Ag by gp96. LG-15 PLs (0.5 x 106) were incubated for 1 h at 27°C with 1 µg of gp96 purified from minor H-Ag disparate LG-6 liver (A), Xenopus-recombinant gp96 (B), preincubated 10 min with rabbit preimmune serum (C), or anti-CD91 antiserum (D) followed by incubation with LG-6-derived gp96, or with APBS (E). After three washes, cells were adoptively transferred to LG-6 adult recipient (1 x 106/individual) that were then grafted, 1 wk later, with skin from LG-15 and LG-7 (third-part) donors. Percent of rejection was monitored three times per week for 70 days. LG-7 skin grafts were rejected within 2 mo with no significant difference between the different groups (data not shown), whereas LG-15 skins were rejected faster by recipients of adoptively transferred LG-15 gp96-primed PLs.

 

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  		Table I. Compilation of gp96-mediated minor H-Ag cross-presentationa

 
The response elicited against the skin transplant was Ag-specific because PLs incubated with APBS, rec-gp96 (Fig. 6), or LPS (data not shown) did not result in any change in the onset of the rejection as compared with naive recipients. Furthermore, no sign of rejection was observed with skin isograft and rejection of a third-part LG-7 skin graft with minor H-Ags different from LG-6 and LG-15 was not accelerated significantly (Table I). Finally, as few as 50,000 gp96-minor Ag-complex PLs can induce a detectable, albeit weaker, acceleration of skin graft rejection (Table I).

We conclude from these results that PLs are able to acquire minor H-Ags chaperoned by gp96 and elicit a potent response in the recipient leading to a specific accelerated rejection of skin expressing the same Ags.

Involvement of CD91 in responses elicited by adoptively transferred PLs pulsed with gp96/minor H-Ag complexes

We further investigated the involvement of CD91 in the acquisition anti-minor H-Ags chaperoned by gp96 by interfering with its ligand binding using anti-CD91 Ab. The acceleration of skin graft rejection induced by transferring bacteria-elicited PLs pulsed with LG-6 gp96/minor H-Ag complex was abrogated by preincubating PLs with anti-CD91 Ab, but not with preimmune serum (Fig. 6; Table I). To obtain additional evidence of CD91 involvement, PLs were incubated with the CD91-specific antagonist RAP before gp96 pulse. As in the case of anti-CD91, the anti-minor H-Ag response by RAP-treated PLs was markedly decreased (Table I). These results strongly suggest that in Xenopus CD91 contribute to the cross-presentation by APCs of minor H-Ags chaperoned by gp96.

CD8 T cell infiltration induced by APCs pulsed with gp96/minor H-Ag complexes

We have recently adapted a whole-mount immunohistology procedure that enabled us to visualize leukocyte infiltration into unfixed transplanted skin tissue using fluorescent Abs (30). We used this technique to determine whether PLs pulsed with gp96-minor H-Ag increased CD8 T cell infiltration in the minor H-Ag-disparate skin transplant. Skin tissues were excised 12 days postgrafting and stained with the Xenopus-specific anti-CD8 mAb AM22. Consistent with the extent of rejection already occurring in skin transplant of the recipients, adoptively transferred with gp96-minor H-Ag complex-pulsed APCs, marked CD8 T cell infiltration was observed (Fig. 7). In contrast, frogs that received RAP-treated PLs pulsed with gp96/minor H-Ag complexes had far less CD8 T cell infiltration. These results further substantiate the conserved ability of CD91 expressed by Xenopus PLs to cross-present minor H-Ags chaperoned by gp96 and elicit a CD8 T cell response.


Figure 7
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  		FIGURE 7. CD8 T cell infiltration in minor H-Ag-disparate Xenopus skin undergoing rejection. Representative whole-mount immunohistology of LG-6 skin tissues 12 days posttransplantation stained for mouse IgM isotype control (left panel) or Xenopus-specific anti-CD8 mAb AM22 followed by a PE-conjugated mouse mu-specific Ab (middle and right panel). LG-6 skins were grafted onto LG-15 recipients 1 wk after adoptive transfer of LG-15 PLs (0.5 x 106) that were pulsed 1 h with 0.8 µg of LG-6-derived gp96 (left and middle panel) or preincubated with 1.5 µg of RAP 10 min before incubation with LG-6 gp96. Whole-mount immunohistology of each transplant was performed 12 days postgrafting (30–35% rejection) as described in Materials and Methods. Scale bar, 50 µm.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The results of this study show that the ability of gp96 to chaperone naturally derived skin Ags, to interact with the endocytic receptor CD91, and to promote cross-presentation leading to a specific potent T cell response has been conserved between the amphibian Xenopus and mammals whose ancestors diverged from a common ancestor 350 million years ago. This finding by itself strongly supports the proposition by Srivastava (1) that gp96 and CD91 are part of an immune surveillance system involving both innate and adaptive elements of the immune system. In addition, this study provides the first evidence that cross-presentation, the ability of APCs to acquire and present exogenous Ags, in the context of MHC class I to CD8 T cells, is a conserved and therefore fundamental element of the vertebrate adaptive immune system.

In mammals, the ability of gp96 to generate a robust CD8 T cell response against minute amounts of chaperoned antigenic peptides is generally considered to result from its efficient internalization by APCs through receptor-mediated endocytosis and the subsequent channeling of chaperoned peptides into the APC class I presentation pathway (6, 7, 12, 33). In addition, gp96 activates APCs independent of any bound peptides (3, 4, 5). The comparative model we have developed in Xenopus has been instrumental to show the evolutionary conservation of gp96’s ability to chaperone Ags and generate CTLs against tumor (26) and minor H-Ags (25). In addition, the Xenopus gp96 protein homolog can complex synthetic peptides in vitro as stably as human or mouse gp96, and peptides chaperoned by Xenopus gp96 can be represented by MHC class I of mouse macrophages to specifically stimulate murine class I-restricted CTL lines as efficiently as mouse gp96 (26).

We have reported recently that a Xenopus CD91 gene homolog displays a high sequence identity (>65%) with other CD91 homologs and contains the additional distinctive cytoplasmic NPXY motif. Phylogenetic analysis indicates that CD91 homologs branch as a monophyletic group distinct from other low density lipoprotein receptor, which suggests an origin of CD91 contemporary with that of metazoans. We have shown here that CD91 expressed at the surface of PLs can bind gp96. On average, ~15–30% of PLs exhibited surface binding of gp96, or of the CD91 natural ligand {alpha}2-M, or were stained by an anti-CD91 Ab. This corresponds to the fraction of mouse peritoneal macrophages estimated from earlier cross-presentation experiments (6) as well as from the first characterization of CD91 (12). Interestingly, our two-color flow cytometry analysis reveals that a similar fraction (20–30%) of PLs in Xenopus coexpress CD91 and high level of surface MHC class I. This class Ibright PL fraction also appears to bind to Alexa-gp96. Whether CD91+/class Ibright represents a particular cell subset of Xenopus APC remains to be determined. As in mammals, gp96 binding on PLs is saturable and can be competed by a molar excess of unlabeled gp96 but not an irrelevant protein such as OVA, indicative of a surface receptor-ligand interaction. Furthermore, prevention or reduction of Xenopus gp96 binding at the surface of PLs either by competition with a molar excess of the CD91 natural ligand {alpha}2-M, with anti-CD91 Ab, or with the CD91-specific antagonist RAP, provide convergent evidence of involvement of the endocytic receptor CD91. It is important to remark, however, that these data do not preclude that gp96 may also bind other receptors. In mice, another endocytic receptor, SR-A, has been reported to bind gp96 and mediate cross-presentation of the chaperoned peptide. So far, no SR-A homolog has been identified in Xenopus laevis or even in Xenopus tropicalis whose full genome sequence is now available.

The detailed process of gp96 interaction with CD91, its internalization followed by the channeling and processing of associated Ags, is not fully understood in mammals. As is true with most scavenger receptors, CD91 has many ligands, whose binding characteristics (e.g., binding affinity, domain of the receptor involved in the interaction) are poorly defined. In addition to its role in lipoprotein metabolism, CD91 has been reported to interact with as many as 25 different ligands (34) and is postulated to have multiple functions including a role in immunity. CD91 has been shown previously to mediate endocytosis of the serum protease inhibitor {alpha}2-M (35, 36), a phylogenetically conserved element of the innate immune system (37). Importantly, despite the current lack of knowledge of a mechanism involved in gp96/CD91-mediated interaction, the immunological consequences are remarkable. In this study, we show that in Xenopus as in mammals, the acquisition of low amounts of gp96, and therefore even scantier amounts of gp96-bound Ags, by APCs is sufficient to generate potent and Ag-specific T cell response in vivo. In contrast to immunization that requires two injections at a 2-wk interval of at least 10 µg of the gp96-minor H-Ag complex (25, 26), adoptive transfer of PLs pulsed only once with 800 ng of the same gp96 preparation is sufficient to generate similar acceleration of minor H-disparate skin graft rejection. This rules out possible passive immunization from gp96 remaining attached to cells after washes, and implies by itself that the response generated by pulsed PL cells result from an active process and therefore that some of these cells function as APCs. In contrast, T cells pulsed with the same amount of gp96 did not induce any response.

To our knowledge, these results provide the first functional characterization of an APC in ectothermic vertebrates. Macrophage/monocytes constitute the major fraction of Xenopus PLs (27, 38). They display a typical monocytic morphology and express surface MHC class I and class II molecules. Other minor cell populations present in a peritoneal lavage include basophils, eosinophils, and lymphocytes. Unfortunately, reagents are limited in Xenopus for a detailed phenotypic characterization. However, our two-color flow cytometry analysis reveals the presence of fraction of large granular PL expressing a high level of surface MHC class I molecules that are expressing CD91, and bind Alexa-gp96. Although further investigation will be needed to determine whether anti-CD91 and Alexa-gp96 target the same class Ihigh PLs representing a particular APC subset, it is reasonable to propose that gp96-minor H-Ag complexes are mainly taken up by a fraction of PLs functioning as APCs. In the absence of lymph node in Xenopus, adoptively transferred PLs are likely to drain into the spleen where they can encounter and activate T cells. Phenotypic and functional characterization of anti-minor H-Ag splenic CTLs generated by priming either with skin graft or s.c. gp96 immunization is consistent with this view (25, 26, 32). In addition, we have shown previously that skin graft rejection requires CD8 T cells in vivo (39) and that the kinetics of CD8 T cell infiltration correlate with the onset of rejection (30).

In the last few years, it has become clear that cross-presentation plays an important role in immune surveillance by allowing APCs to take up exogenous Ags and prime CD8 T cells (11). The pathways of Ag uptake and intracellular trafficking leading to class I presentation and CD8 T cell activation is an active research area. Besides apoptotic bodies, immune complexes, whole particulate or soluble proteins or peptides, hsp-chaperoned antigenic peptides constitute a well-documented form of Ag acquired by APCs that has the advantage of being efficiently internalized and of activating APCs (1). Our study in Xenopus gives evolutionary evidence of the biological relevance of gp96/CD91-mediated Ag cross-presentation in immune surveillance.


   Acknowledgments
 
The expert animal husbandry provided by Tina Martin and David Albright is gratefully appreciated. We thank Dr. Brian Ward for his technical help for internalization assays, as well as Dr. Edward Pope, Shauna Marr, and Jennifer Gantress for their technical contribution. We also thank Ana Goyos for critically reading the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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. Back

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{at}urmc.rochester.edu Back

3 Abbreviations used in this paper: {alpha}2-M, {alpha}2-macroglobulin; SR-A, scavenger receptor type A; RAP, receptor-associated protein; hsp, heat shock protein; PL, peritoneal leukocyte; APBS, amphibian PBS; H-Ag, histocompatibility Ag. Back

Received for publication November 7, 2007. Accepted for publication January 2, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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