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The Journal of Immunology, 2002, 168: 1697-1703.
Copyright © 2002 by The American Association of Immunologists

Minor Histocompatibility Antigen-Specific MHC-Restricted CD8 T Cell Responses Elicited by Heat Shock Proteins1

Jacques Robert2, Jennifer Gantress, Laura Rau, Alisa Bell and Nicholas Cohen

Department of Microbiology and Immunology, University of Rochester Medical Center, Rochester, NY 14642


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In mammals, the heat shock proteins (HSP) gp96 and hsp70 elicit potent specific MHC class I-restricted CD8+ T cell (CTL) response to exogenous peptides they chaperone. We show in this study that in the adult frog Xenopus, a species whose common ancestors with mammals date back 300 million years, both hsp70 and gp96 generate an adaptive specific cellular immune response against chaperoned minor histocompatibility antigenic peptides that effects an accelerated rejection of minor histocompatibility-locus disparate skin grafts in vivo and an MHC-specific CD8+ cytotoxic T cell response in vitro. In naturally class I-deficient but immunocompetent Xenopus larvae, gp96 also generates an antitumor immune response that is independent of chaperoned peptides (i.e., gp96 purified from normal tissue also generates a significant antitumor response); this suggests a prominent contribution of an innate type of response in the absence of MHC class I Ags.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
An increasing body of data supports the idea that certain heat shock proteins (HSPs)3 play a role in both innate and adaptive immunity (reviewed in Refs. 1, 2, 3). On one hand, HSPs independent of chaperoned peptides may evoke innate immunity by recruiting NK cells (4, 5) and by stimulating APCs (e.g., macrophages and dendritic cells (DC)) to produce cytokine such as IL-1{beta}, TNF-{alpha}, and IFN-{gamma} (6). APC stimulation by HSPs involves the NF-{kappa}B pathway (6). On the other hand, immunization with microgram quantities of HSPs (e.g., hsp70 or gp96) elicit potent Ag-specific cellular adaptive immune responses that depend on the ability of the HSP to chaperone antigenic peptides (1, 7). The effector response generated by HSPs is primarily mediated by CD8+ CTL; it can be abrogated by depletion with anti-CD8 mAbs (4, 8). Specific CTL responses have been obtained against a large variety of chaperoned antigenic peptides. Such antigenic peptides include those from tumors (1, 4, 9) and virus-infected cells (10, 11), minor histocompatibility (12) and model Ags (12, 13, 14), and purified native (1, 4, 9) as well as in vitro reconstituted (10, 15) HSP-peptide complexes. In vitro, HSPs are able to interact with APC via a receptor (16, 17, 18, 19), and antigenic peptides chaperoned by HSP, after internal processing in the APC, can be re-presented in the context of MHC class I of APCs to CD8 T cells (13, 14). A surface receptor that interacts with gp96 and is expressed by DC and a subset of macrophages has been identified as CD91, the receptor for {alpha}2-macroglobulin (19). Upon binding to CD91, gp96-peptide complex is rapidly internalized by endocytosis (19).

Given the high degree of structural conservation of HSPs among vertebrates and invertebrates, it has been proposed that some of their immunological properties may also have been conserved during evolution (20, 21). Indeed, we have shown that the Xenopus gp96 protein homolog stably and noncovalently complexes peptides in vitro, and that peptides chaperoned by Xenopus gp96 can be efficiently represented by MHC class I of mouse macrophages to specifically stimulate murine class I-restricted CTL lines (22). Moreover, immunization of Xenopus with gp96 purified from a highly tumorigenic class I-negative Xenopus thymic tumor generates potent antitumor immunity as measured by a significant delay in the appearance and diminished size of tumors after challenge. No such effect has been observed with gp96 from normal tissues. Similar antitumor responses result from priming with a purified Xenopus hsp70-tumor peptide complex, but not with tumor-derived hsp70 that is free of peptide (22).

The immune system of the adult African clawed frog Xenopus is fundamentally similar to that of mammals (e.g., rearranging TCR and Ig genes, and MHC class I- and class II-restricted T cell recognition; reviewed in Refs. 21 and 23). However, MHC-restricted Ag-specific T cell clones or lines are not yet available in Xenopus, and the transplantable tumors we use do not express the MHC class I molecule (24). Therefore, to study the phylogenetic conservation of HSP T cell adjuvanticity properties in our Xenopus model, we assessed the ability of HSPs to generate an MHC-restricted CD8 T cell-mediated response against chaperoned exogenous antigenic peptides encoded for by Xenopus minor histocompatibility (H) genes. The involvement of MHC class I representation was further evaluated by studying tumor-derived gp96-induced immunity in naturally MHC class I-deficient larvae. We report in this study that both Xenopus gp96 and hsp70 generate specific in vivo cellular immune responses and in vitro CTL responses against chaperoned minor H-Ag peptides. Furthermore, in naturally MHC class I-deficient larvae, gp96 elicits a significant antitumor response even when it is purified from normal (non-tumor) tissue, suggesting it can evoke a non-MHC-restricted innate type of antitumor response. The finding that immunological properties of HSPs have been conserved since amphibian and mammalian lineages diverged some 300 million years ago provides, by itself, a strong "evolutionary" validation of their biological importance.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and tumor lines

Outbred (OB) and isogenetic (cloned; Ref. 25) MHC heterozygous LG-15, LG-6, and LG-46 (MHC haplotype a/c) Xenopus were obtained from our breeding colony. The 15/0 lymphoid tumor line, derived from spontaneously arising thymic tumors of LG-15 animals, is MHC class I and class II negative (24, 26).

Purification of Xenopus gp96 and hsp70

Gp96 was purified as previously described (15) by 50–70% ammonium sulfate fractionation, concanavalin A-Sepharose, and DEAE chromatography. hsp70 was purified as previously described (27) by Blue-Sepharose chromatography to remove albumin contaminants and either an ADP-agarose or ATP-agarose column (Sigma-Aldrich, St. Louis, MO), followed by DEAE chromatography. Purity was assessed by SDS-PAGE and silver staining, and by Western blotting (data not shown).

Flow cytometry

Cells (105 cells) were stained with undiluted hybridoma supernatants specific for Xenopus CD8 (AM22, Ref. 28 ; F17, Ref. 29), CD5 (2B1; Ref. 30), pan T cell (XT-1; Ref. 31), IgM (10A9; Ref. 32), or isotype-matched mAb controls followed by fluorescein-labeled goat anti-mouse Ig F(ab')2 that had been preadsorbed twice on Xenopus erythrocytes. The stained cells were then analyzed by flow cytometry on a FACSCalibur (BD Biosciences, San Jose, CA), 10,000 events were collected, and dead cells stained by propidium iodide were gated out.

Immunization and skin grafting

LG-6 isogenetic Xenopus were injected two or three times (2-wk interval) with either irradiated (50 Gy) 15/0 tumor (LG-15-derived), LG-15 total proteins (100,000 x g supernatant from Xenopus liver), or with purified gp96 in 0.3 ml of amphibian PBS (APBS). Two weeks after the last immunization, LG-6 animals were grafted according to published methods (33). Briefly, a piece of ventral skin (5 mm2) was inserted under the dorsal skin of a recipient, and 48 h later the overlying host skin was removed. The onset of rejection was marked by initiation of pigment cell death, and rejection was considered complete when all pigment cells in the graft were destroyed.

In vitro killing assay

Splenocytes from naive or immunized frogs were harvested and restimulated (5 x 106 cells/ml) in vitro for 6 days with one-half as many irradiated LG-15 splenocytes in medium containing 0.25% Xenopus normal serum (medium supplementation with Xenopus serum considerably improves the survival of cytotoxic effectors). Cultures were supplemented at day 3 with a T cell growth factor-rich supernatant (34). Killing activity was determined by the "just another method" technique (35). Targets were labeled for 2 h at 26°C with 5 mC/ml [3H]TdR (6-day-old LG-15 normal splenic PHA-induced blast targets were labeled for 20 h), washed, incubated for 4 h at different E:T ratios, and harvested with a 96-well harvester (Betaplate; PerkinElmer Wallac, Gaithersburg, MD); thymidine uptake was determined by scintillation spectrometry.

Larval tumor assay

Tadpoles were blocked at premetamorphic stage 55 with sodium perchlorate (1 g/L) in their aquarium water, and they were primed i.p. with two injections of one of the following: 5 µl of APBS; 103, 104, or 105 irradiated tumor cells in 5 µl of culture medium; 5 µl of 15/0 whole protein lysate (60 ng each); or 5 µl of purified 15/0 gp96, or normal LG-15 (12 or 60 ng each). The second injection was given 2 wk after the first, and larvae were challenged i.p. with 5 x 104 15/0 tumor cells (<5% cell death) 2 wk after the second injection. On day 14 after challenge, peritoneal fluid from each tadpole was collected and the tumor cells were counted. Host survival after tumor challenge was monitored daily.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Generation of anti-minor H Ag responses in vivo by gp96 and hsp70

Thymus dependency and Ag specificity of skin allograft rejection are well characterized in Xenopus thanks to MHC-defined inbred strains and isogenetic clones (33, 36, 37, 38, 39, 40). LG-15, LG-6, and LG-46 isogenetic Xenopus clones all display the identical heterozygous (a/c) MHC genotype (25). However, these clones differ from each other by multiple minor H loci (24, 40, 41, 42). Skin grafts transplanted between these clones are rejected significantly more slowly (30–50 days at ~21°C; see Fig. 1Go) than are MHC disparate allografts (20 ± 2 days at ~21°C). Gp96 purified from LG-15, LG-46, or LG-6 liver was used to immunize LG-6 adult animals. Immunization with 10 µg of LG-15-derived gp96 twice (a 2-wk interval) significantly and consistently accelerated rejection of LG-15 skin grafts relative to control LG-6 injected with either APBS, total LG-15 protein lysate, or autologous LG-6-derived gp96 (Fig. 1Go, A–C). Immunized animals did not reject autologous LG-6 skin (survival, >100 days), whereas they did reject MHC-disparate LG-5 (b/c haplotype) skin in 20 ± 2 days (data not shown). Similar accelerated rejection of LG-46 skin was obtained by immunizing LG-6 recipients with LG-46-derived gp96 (Fig. 1GoD). In this experiment, LG-6 animals were simultaneously grafted with both LG-46 and LG-15 skin. Only rejection of LG-46 skin was accelerated in immunized animals, whereas the mean rejection times (±SD) for LG-15 grafts were comparable between control and immunized groups (45.4 ± 3.5 and 47 ± 2.8, respectively).



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  		FIGURE 1. In vivo immune responses to minor H-Ags induced by HSP-peptide complex. LG-6 Xenopus isogenetic adults (2 years old) were immunized two or three times at 2-wk intervals with APBS (c, control); 5 x 106 15/0 irradiated tumor cells; 10 µg of liver-derived gp96 from LG-15, LG-6, or LG-46 frogs; 10 µg of LG-15 liver-derived ADP-purified peptide-loaded hsp70; or 10 µg of LG-15 liver-derived ATP-purified peptide-free hsp70. Two weeks after the last immunization, skin from LG-15 (A–C and E) or LG-46 (D) minor H-Ag disparate animals was grafted to the dorsal surface of the different LG-6 hosts. Time of complete rejection for each graft together with group means and p values (Student’s t test) are shown. D, PBS (C2) and gp96-immunized LG-6 recipients were simultaneously grafted with both LG-46 and LG-15 skin. Only rejection of LG-46 skin was accelerated in immunized animals, whereas the mean rejection times (±SD) for LG-15 grafts were comparable between control and immunized groups (45.4 ± 3.5 and 47 ± 2.8, respectively).

 
To better determine the need for chaperoned peptides in the generation of a specific anti-minor H-Ag response, we compared the immunogenicity of hsp70-peptide complexes (purified by ADP-agarose chromatography) with hsp70 without peptide (purified by ATP-agarose chromatography; Refs. 14, 22). Immunization with LG-15-derived hsp70-peptide complexes induced an accelerated rejection comparable with LG-15-derived gp96, whereas hsp70 without peptide had no effect on skin graft survival (Fig. 1GoE).

Effect of HSP immunization on cell-mediated cytotoxicity

Although thymus-dependent, MHC-specific cytotoxic activity against both major (43, 44) and minor (45) H-Ags has been described in Xenopus, the involvement of MHC-restricted CD8 cytotoxic T cells in this alloreactivity has not been formally demonstrated. To examine whether Xenopus CD8 T cells are CTL, Xenopus CD8 T cells were separated from splenocytes using a Xenopus-specific anti-CD8 mAb (46) and magnetic microbeads (MACS); cytotoxic activity of the positively and negatively selected cells was then studied in vitro. As determined by flow cytometry, the MACS-sorted population contained >90% CD8+ cells (Fig. 2GoA). This population also stained positive for the pan-T cell marker, XT-1 (Ref. 30 and data not shown) and CD5 (T cell marker in Xenopus; Ref. 30) but was negative for surface IgM (Fig. 2GoA). The splenocytes remaining after selection for CD8+ cells (referred to as CD8-depleted) contained some CD8+ cells (10–20%), CD5+ T cells, and IgM+ B cells.



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  		FIGURE 2. CTL characterization against major and minor-H Ags. A, Flow cytometry of CD8+ T cells positively selected with magnetic microbeads, 10,000 events (<5% IgM+ B cells, >90% are CD8+ and CD5+, which is a T cell marker in Xenopus). B, Phenotype of remaining cells after sorting (10–20% CD8+, 20% IgM+ B cells, and 30% CD5+). C, Cytotoxic activity of CD8-selected anti-LG-15 effectors from OB (MHC-disparate) frogs. D, Cytotoxic activity of CD8-depleted anti-LG-15 effectors from OB frogs. E, Cytotoxic activity of CD8-selected LG-6 anti-LG-46 (minor H-disparate) effectors. F, Cytotoxic activity of CD8-depleted LG-6 anti-LG-46 (minor H-disparate) effectors. {blacktriangleup}, OB PHA-lymphoblasts; {diamondsuit}, LG-6 PHA-lymphoblasts; {circ}, LG-15 PHA-lymphoblasts; {square}, LG-46 PHA-lymphoblasts; •, class I-negative 15/0 tumor targets. Splenocytes from OB animals that had rejected MHC-disparate LG-15 (a/c) skin (C and D) and from LG-6 animals that had rejected minor H-Ag disparate LG-46 skin (E and F) were restimulated in vitro for 6 days with irradiated LG-15 or LG-46 spleen cells, respectively. CD8+ T cells were sorted with anti-CD8 AM22 mAb and anti-mouse IgM magnetic microbeads and were cultured overnight before being assayed for killing. 15/0 tumor (10,000) or PHA-blast targets (25,000) labeled with [3H]TdR were incubated with various number of effectors, in triplicate, for 4 h at 26°C in 96-well plates. Thymidine uptake was determined by scintillation spectrometry. The percentage of killing activity was calculated by determining loss of apoptotic DNA relative to target alone control ([cpm control - experimental]/[cpm control] x 100). Variation within triplicate groups was <10% of group mean cpm. The cpm value for 10,000 tumor targets ranged between 5,000 and 6,000 cpm, and the cpm value for 25,000 PHA-lymphoblasts ranged between 1,000 and 1,200 cpm.

 
To determined the cytotoxic activity of Xenopus CD8+ T cells purified from alloimmunized frogs, splenocytes harvested from OB animals that had been immunized against LG-15 Ags by multiple LG-15 skin grafts were restimulated in vitro with irradiated LG-15 spleen cells (43). When tested for cytotoxic activity using the "just another method" technique (a more sensitive DNA fragmentation assay than the classical 51Cr-release assay; Ref. 34), these CD8+ T cells (determined by flow cytometry) showed a strong and specific cytotoxic activity against LG-15 splenocytes but not against MHC-disparate (non-a/c) PHA-lymphoblast targets. NK-like activity against MHC-negative tumor targets was minimal (Fig. 2Go, C and D). Similarly, CD8-selected T cell effectors from LG-6 animals immunized against LG-46 minor H-Ags by multiple skin grafts specifically killed LG-46 but not cognate LG-6 PHA-lymphoblast targets (Fig. 2Go, E and F). Specific killing activity against LG-15 lymphoblasts targets by CD8 purified T cells was also obtained by immunizing LG-6 recipients with multiple skin grafts from LG-15 minor H-Ag disparate donors; splenocytes from these LG-6 animals specifically killed LG-15 but not cognate LG-6 PHA-lymphoblast targets (data not shown).

Given this capacity of Xenopus to generate a MHC-restricted CD8-T cell response, we next investigated whether the immune response elicited against minor H-alloantigens by HSP-peptide complexes also involved CD8 cytotoxic T cells. CD8+ T cell effectors from LG-6 animals immunized with hsp70-peptide complexes purified from LG-15 tissue, but not control CD8+ lymphocytes from LG-6 immunized with peptide-free hsp70 or from naive LG-6, showed specific reproducible killing against LG-15 blast targets (Fig. 3Go, A–C). The remaining CD8-depleted effectors showed reduced killing activity against minor H-Ag disparate class I+ targets, but a strong NK-like activity against class I-negative 15/0 tumor targets derived from LG-15 animals (Fig. 3Go, D–F). Anti-minor H-Ag-specific CTL activity was also obtained by immunizing LG-6 animals with gp96-peptide complexes purified either from LG-15 liver (Fig. 4Go, A and D) or LG-46 (Fig. 4Go, C and G). The higher killing activity of anti-LG-46 effectors observed against cognate LG-6 target cells was presumably due to some nonspecific autokilling because it did not titrate with the E:T ratio. In each case, sorted CD8+ effector cells killed the minor H-Ag disparate targets, but did not kill the cognate LG-6 PHA-lymphoblast targets or MHC-disparate OB targets (data not shown). No CTL activity was obtained when cognate LG-6 splenocyte stimulators were used instead of irradiated minor H-Ag disparate LG-15 stimulators (data not shown). More importantly, immunization of LG-15 animals with cognate LG-15 gp96 did not generate any detectable CTL (Fig. 4GoB). These experiments were repeated several times, both for hsp70 and gp96, with comparable results. They strongly suggest that the CTL responses elicited by gp96, like those elicited by hsp70, were directed against HSP-chaperoned minor H-antigenic peptides.



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  		FIGURE 3. Generation of anti-minor H-Ag-specific CTL by immunization with hsp70-peptide complexes. Splenocytes from naive (A and D), immunized LG-6 with 10 µg (three times s.c.) of liver LG-15-derived ATP-purified (peptide-depleted) hsp70 (B and E), or 10 µg of LG-15 liver-derived ADP-purified (peptide-loaded) hsp70 (C and F). LG-6 splenocytes were restimulated in vitro with irradiated LG-15 spleen cells. Killing activity of CD8 selected (upper line) and CD8-depleted (lower line) effectors was assayed against LG-15 PHA-lymphoblasts ({circ}), LG-6 PHA-lymphoblasts ({diamondsuit}), and class I-negative 15/0 tumor (•) targets as described for Fig. 2Go.

 


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  		FIGURE 4. Generation of anti-minor H-Ag-specific CTL by immunization with gp96-peptide complexes. A and D, Splenocytes from LG-6 animals immunized (three times s.c.) with 10 µg of LG-15 liver-derived gp96, and restimulated in vitro with irradiated LG-15 splenocytes. B and F, Splenocytes from LG-6 animals immunized (three times s.c.) with 10 µg of cognate LG-6 liver-derived gp96, and restimulated in vitro with irradiated LG-6 splenocytes. C and G, Splenocytes from LG-6 animals immunized (three times s.c.) with 10 µg of LG-46 liver-derived gp96 were restimulated in vitro with irradiated LG-46 cells. Killing activity of CD8+ selected (upper line) and the remaining CD8-depleted (lower line) effectors was assayed against the following targets: LG-15 ({circ}), LG-46 ({square}), and LG-6 ({diamondsuit}) PHA-lymphoblasts as described for Fig. 2Go.

 
Immunogenicity of gp96-peptide complexes in MHC class I-negative tadpoles

Larval Xenopus are immunocompetent, but cell surface expression of MHC class I molecules does not occur in most tissues, including the thymus, until metamorphosis (47, 48). Furthermore, because LMP7 gene expression also does not occur until metamorphosis (49), MHC class I-restricted peptide presentation in tadpoles is likely to be absent or, at best, inefficient. We took advantage of this unique natural MHC deficiency to further explore the requirement of MHC class I presentation in the context of HSP immunogenicity. LG-15 larvae were blocked by sodium perchlorate at premetamorphic stage 55; this delays class I expression for several weeks (50). Before challenge with 15/0 tumor cells, larvae were primed with APBS, irradiated 15/0 tumor cells, 15/0 total protein lysates, or different concentrations of tumor- or normal tissue-derived gp96. Growth of tumor in animals injected with irradiated 15/0 cells or with 15/0 whole protein lysate did not differ from tumor growth in APBS-injected controls (p > 0.05; Fig. 5Go, A and B). By contrast, injection of gp96 from either tumor or normal tissue resulted in significantly decreased tumor growth compared with controls. The partial tumor growth inhibition of tumor-derived and normal tissue-derived gp96 did not differ statistically (Student’s t test; p > 0.5). In addition to retarding tumor growth, normal- and tumor-derived gp96 were associated with prolonged host survival. Nonparametric statistical analysis (Mantel-Cox; StatView; Abacus Concepts, Berkeley, CA) indicates that the survival of tadpoles immunized with either with tumor- or normal tissue-derived gp96 and subsequently challenged with viable tumor cells was significantly prolonged relative to nonimmunized control tadpoles (p = 0.001 and 0.027, respectively). However, the survival of tadpoles in these two experimental groups did not differ (p > 0.46).



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  		FIGURE 5. Immunogenicity of tumor-derived gp96 in naturally MHC class I-deficient larval hosts. A–C, Effect of gp96 immunization on tumor growth after challenge. LG-15 larvae blocked at premetamorphic stage 55 with sodium perchlorate were primed i.p. with APBS (C, control), 1,000–100,000 irradiated 15/0 tumor cells, 15/0 total protein (T; 200 ng), 15/0 tumor-derived gp96, or normal LG-15 tissue-derived gp96 (24 or 120 ng). Doses as described in the figure were administered twice after a 2-wk interval. Two weeks after the last injection, larvae were challenged i.p. with 5 x 104 15/0 tumor cells. Two weeks later, the number of tumor cells per microliter of collected peritoneal fluid was determined for each animal. Group means (horizontal bars) and Student’s t test p values are given. D, Effect of gp96 immunization on survival after tumor challenge. Survival of LG-15 larvae used in panel A3 was monitored daily until all animals died. The percentage of group survival at each day postchallenge is shown (control, 12 individuals; experimental, 12 individuals). Differences in survival were statistically significant (Mantel-Cox nonparametric test; StatView) for control versus LG-15 liver-derived gp96 (p = 0.027) and control versus 15/0-derived gp96 (p = 0.001), but not between the two gp96 immunized groups (p > 0.46).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Cell-mediated cytotoxicity has been characterized in vitro in a few nonmammalian taxa, including teleosts (51) and Xenopus, a species where both NK-like (52, 53) and MHC-specific CTL-like (43, 45, 54) killing has been documented. Xenopus CD8 T cells have been characterized by two mAbs (known as F17 (29) and AM22 (28, 46)); each stains 85–90% of total thymocytes and 20–30% of splenic lymphocytes. The anti-CD8 mAb AM22 binds a polypeptide of 30–32 kDa as determined by Western blotting (28), and it immunoprecipitates a dimeric complex of 65 kDa under nonreducing conditions that resolves to a 35-kDa band and a 30- to 32-kDa band under reducing conditions (26). Xenopus CD8 T cells stained with either of the two anti-CD8 mAbs express a pan-T cell marker (XT-1), high levels of CD5, and CD45 (55, 56, 57); these cells are not detectable in animals that had been thymectomized at early developmental stages before the migration of stem cells into the thymus (55). Finally, in vitro studies suggest that the mammalian type of thymocyte differentiation (i.e., immature double-positive CD4CD8 cells giving rise to more mature single positive CD4+CD8- and CD4-CD8+) also occurs in both MHC class I-negative larvae and class I-expressing adults (56, 57). The formal demonstration in Xenopus of an effector cell that is the phenotypic and functional equivalent of the mammalian CD8+ cytotoxic T cell was prerequisite to assessing whether the ability of some HSPs to function as natural T cell adjuvants is a fundamental property of HSP that has been maintained during evolution. Our data show for the first time in a cold-blooded vertebrate the existence of MHC-restricted cytotoxic effectors that are pan-T cell+, CD8+, CD5+, and IgM-.

In mice, antigenic peptides chaperoned by hsp70 or gp96 elicit potent CTL responses against a large variety of chaperoned antigenic peptides, including those from tumors (1, 4, 9), viruses (10, 11), skin grafts (12), and model Ags (13, 14). The proposed mechanism by which such a robust response is generated involves internalization of the HSP-peptide complex by APCs after its interaction with a surface receptors, and the channeling of the peptides into the class I presentation pathway (1, 7). Recently, a surface receptor (CD91) expressed by DC and a subset of macrophages has been shown to bind gp96-peptide and lead to its rapid internalization (19). Receptor-mediated uptake of hsp70 by APC has also been documented (18). We have reported that Xenopus gp96 complexed in vitro with vesicular stomatitis virus antigenic peptides can interact with murine macrophages, leading to the cross-representation of the antigenic peptide by class I molecules of murine macrophages and peptide-specific activation of a MHC-restricted mouse CD8+ T cell line (22). We show in this study that both Xenopus hsp70 and gp96 can generate a CTL response specific against minor H-Ags. As in the mouse, neither Xenopus hsp70 that is free of peptide nor cognate gp96-peptide complexes elicit any CTL activity. In addition, CTL generated by gp96 or hsp70-peptide complexes kill only MHC-compatible targets derived from the same minor H-locus disparate genotype as the clone from which the HSPs were purified. This strongly suggests that the CTL response has been generated against chaperoned minor-H antigenic peptides and that these exogenous peptides have been channeled in the Ag presentation pathway of the host.

Strikingly, gp96 is able to generate responses against MHC class I-negative tumor in naturally class I-deficient larvae. However, in this case, the response does not appear particularly dependent on the presence of antigenic chaperoned peptide because immunization with gp96 purified from normal tissue is as potent as tumor-derived gp96 in evoking inhibition of tumor growth. In addition, immunization with either tumor-derived or non-tumor-derived gp96 results in a significant prolonged survival of tumor-challenged tadpoles.

We have shown previously in adult Xenopus that the immune responses against the MHC class I-negative 15/0 tumor, elicited by gp96 or hsp70, were specific for the complexed peptide, whereas gp96 purified from normal tissue was not immunogenic (22). The less-specific antitumor responses generated by tumor-derived gp96 in larvae are agreement with the idea that the MHC class I representation pathway of exogenous HSP-chaperoned peptides is critical to elicit a specific adaptive cellular immune response. MHC class I molecules are not expressed at the surface of most tissues, including the thymus, until metamorphosis (14, 15, 16). Furthermore, because LMP7 gene expression also does not occur until metamorphosis (17), MHC class I-restricted peptide presentation is likely to be absent, or at least inefficient, in tadpoles. Because such effects are not obtained after priming with either total protein lysate or irradiated tumor cells, it is tempting to speculate that gp96 itself elicits a long-lasting (>2 wk) NK-like response in tadpoles. Although in mice immunization with gp96 primarily stimulates CD8+ T cells, CD4 T cells (8) and NK cells (4) also appear to be recruited. These data and other in vitro studies (5, 6) suggest that HSP per se could induce nonpeptide-specific innate immune responses mediated by enhanced cytokine production and nonspecific killing. The recent characterization of a mouse B cell line that is deficient in gp96 is of interest in this regard. This gp96-deficient line is unresponsive to bacterial endotoxin (owing to a lack of Toll receptor surface expression), but it is unaffected by other stress conditions like temperature or glucose deprivation (58). This suggests that, at least in higher organisms, gp96 may have become specialized by responding to stressors associated with pathogens rather than physical parameters.

Although the immunogenicity of tumor- and non-tumor-derived gp96 in the present experiments with larvae does not appear to be statistically different, a more potent response of HSP complexed with tumor-derived peptide cannot be completely excluded. In contrast to hsp70, peptides complexed to gp96 can only be eluted by harsh treatments (e.g., heat or acid treatment) that profoundly affect its conformation and its immunological properties. Our recent cloning of Xenopus gp96 cDNA (22) will be useful in developing a recombinant strategy to overcome this problem.


   Acknowledgments
 
We thank David Albright for his expert animal husbandry. We also thank Dr. Gregory Maniero for his help in the statistical analyses of the data.


   Footnotes
 
1 This research was supported by the National Institutes of Health (RO1 Grants AI-44011 and CA-76312) and by the National Science Foundation (Integrated Research Challenges in Environmental Biology Grant). 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: robert{at}uhura.cc.rochester.edu Back

3 Abbreviations used in this paper: HSP, heat shock protein; H, histocompatibility; OB, outbred; APBS, amphibian PBS; DC, dendritic cell. Back

Received for publication September 18, 2001. Accepted for publication December 7, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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