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Stress proteins and immunity mediated by cytotoxic T lymphocytes
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Stress proteins and immunity mediated by cytotoxic T lymphocytes Hansjörg Schild, Danièle Arnold-Schild, Eckhard Lammert and Hans-Georg Rammensee Chaperone molecules, including members of the heat shock protein family, are able to stimulate αβ and γδ T cells as well as natural killer cells. For αβ T cells, specificity is induced by chaperone-assisted peptides; this has lead to detailed investigations of peptides that bind to these chaperones and their possible role in antigen presentation. Address Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany Current Opinion in Immunology 1999, 11:109–113 http://biomednet.com/elecref/0952791501100109 © Elsevier Science Ltd ISSN 0952-7915 Abbreviations CTL cytotoxic T lymphocyte ER endoplasmic reticulum HSP heat shock protein NK natural killer PDI protein disulfide isomerase TAP transporter associated with antigen presentation
Introduction The search for tumor antigens led to the isolation of proteins that were derived from tumor cells and were able to protect mice against a subsequent challenge with the same tumor cells from which the proteins were isolated [1].These proteins were identified as stress proteins and they include the heat shock proteins (HSPs) — HSP70, HSP90 and the endoplasmic reticulum (ER)-resident molecule gp96 (also known as glucose-regulated protein 94). Surprisingly, the genes coding for these proteins in tumor cells and normal tissues displayed no nucleotide sequence differences [2].To provide an explanation for the specificity of the immune response induced by the immunization with HSPs, it was postulated that peptides derived from intracellular proteins are associated with HSPs — thus providing specificity [3]. Because of this, HSP molecules attracted the attention of immunologists, like ourselves, who had previously been fascinated mainly by the cytotoxic T lymphocyte (CTL)-mediated recognition of MHC–peptide complexes. In the meantime, this hypothesis has been validated not only by numerous examples of specific CTL induction [4] but also, firstly, by autologous immunotherapy protocols [5••], secondly, by the finding that HSPs deprived of peptides do not induce specific immunity [6,7] and, thirdly, by the direct characterization of peptides associated with HSP70 and the ER-resident HSP gp96 [8,9]. This review summarizes the latest studies dealing with the induction of CTL responses, the peptide binding and the activation of γδ T cells or natural killer (NK) cells by HSP molecules.
Induction of CTL responses The immunogenic potential of HSP molecules was first demonstrated in immunotherapy experiments by Srivastava and co-workers (reviewed in [4]). Tumor-specific protection was mediated by CD8+ T cells as shown both by in vivo cell depletion studies [10] and by the ability to generate CTL lines specific for a variety of antigens from mice immunized with HSP molecules [11–13]. The HSPs that mediated this effect include the cytosolic HSP70 and HSP90 and the ER-resident chaperone gp96 [14]. It was subsequently shown that the immunization with gp96 molecules also resulted in the induction of memory T cells [15]; furthermore, gp96 molecules have been shown to induce CTL cross-priming against viral and minor histocompatibility antigens, which supports the hypothesis that gp96 molecules are associated with a large repertoire of peptides not influenced by the cellular MHC haplotype [12,13]. This peptide repertoire is, however, influenced by the expression of functional transporter associated with antigen presentation (TAP) molecules [16•]. Recent experiments have also revealed that HSP molecules can provide an immunogenic context to synthetic peptides that are complexed to HSP70 or gp96 molecules in vitro [17••,18]. Because of this, HSP molecules were called adjuvants of mammalian origin [4]. This ability is not only limited to mammalian HSP molecules, however; it is also observed when mycobacterial HSP70 molecules carrying covalently attached CTL epitopes are used [19•]. Interestingly, the same mycobacterial HSP70 molecules either carrying a covalently attached HIV-1 p24 protein or complexed with mycobacterial peptides bearing CD4+ T cell epitopes were able to elicit both antigen-specific humoral responses and specific immune responses that were mediated by CD4+ T cells [20–22]; however, it remains to be determined whether or not this feature is due to the xenogenic nature of the mycobacterial HSP70, which might activate existing CD4+ memory T cells induced by a previous encounter of the organism with pathogens expressing related HSP molecules. The ER-resident chaperone calreticulin is also able to induce specific immunity in vivo (PK Srivastava, personal communication). A general application in immunotherapy protocols might, however, be questionable as it was demonstrated that calreticulin binds only glycosylated peptides [23••]. The efficient induction of immune responses by HSP–peptide complexes, formed in vivo or in vitro, swiftly led to the proposal that these complexes are taken up by specialized cells [3]. This hypothesis was supported, firstly, by experiments utilizing the depletion of phagocytic
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cells in vivo [10], secondly, by the presentation of HSPassociated peptides in a manner that is restricted by MHC class I by activated macrophages incubated with gp96 molecules [13] and, finally, by the ability of gp96-pulsed bone-marrow-derived dendritic cells to induce protective immunity against tumor cells [24]. The mechanism of this efficient peptide presentation has been shown by Suto and Srivastava [13] to be sensitive to Brefeldin A but a detailed understanding of the intracellular events involved in the transfer of HSP-associated peptides onto MHC class I molecules requires further investigation. Independent experiments (R Binder, A Ménoret, PK Srivastava, personal communication; D Arnold-Schild et al., unpublished data) have led to the identification of a receptor-mediated uptake of HSP molecules by macrophages and dendritic cells, as postulated earlier [3]. This was not observed if controls — bovine serum albumin or F(ab) fragments — were used instead; furthermore, we were able to demonstrate that endocytosed gp96 molecules colocalize in endosomal compartments with recycled MHC class I molecules in a mouse dendritic cell line (D Arnold-Schild et al., unpublished data). These findings now provide a basis for understanding, firstly, the high efficiency of HSP–peptide complexes in inducing CTL-mediated immune responses in vivo and, secondly, the observation that the immunogenicity of tumor cells cosegregates with the expression of HSP molecules [25,26•].
Peptide-binding by stress proteins Peptide binding by members of the HSP70 family has been investigated in detail; it entails the association of peptide segments in an extended form, reminiscent of peptide binding to MHC molecules, but requires ATP hydrolysis. Both interactions share generic hydrogenbonding to the backbone and specificity-determining interactions with pockets located in the peptide-binding region. The existence of these pockets in the HSP70-family members DnaK, HSC70 and BiP was observed in peptide binding studies [27] and the detailed analysis of the crystal structure of DnaK in a complex with a peptide [28] revealed a sandwich-like peptide-binding domain, that indeed contained one specificity-determining pocket. The experiments addressing the ATPase activity and the potential substrate-binding regions of HSP90 molecules have recently been summarized [24,29]. For gp96, neither the structure of the peptide-binding domain nor the peptide-binding properties are known; however, it has been shown that gp96 can bind TAP-translocated peptides [23••,30•,31•]; moreover, an immunodominant viral peptide has been eluted from gp96 in infected cells [8] and the H2-Kb epitope SIINFEKL (using single-letter code for ovalbumin amino acids 257–264) has been found associated with both HSP70 and gp96 in ovalbumin-expressing cells [9]. It has also been demonstrated recently that gp96 cannot efficiently bind peptides with charged amino acids at positions 2 and 9 (P2 and P9) in nonamer peptides [23••] and that it has a hydrophobic peptide-binding site [32]. The necessity to transiently heat or to chemically denature
and renature gp96 in order to obtain notable peptide-binding in vitro [17••,32] and the fact that adenine nucleotides do not influence peptide binding [33•] might argue for the existence of proteins inside the ER that promote peptidebinding to gp96. Using photoreactive peptides, additional ER-resident peptide-binding proteins were identified [23••,31•] and among these the most notable is protein disulfide isomerase (PDI) [34•]. Interestingly, no peptide binding to BiP was observed in these studies. Whether or not the peptide binding to ER-resident proteins such as gp96 or PDI has any immunological function is still a matter of speculation. Srivastava et al. [3] proposed that gp96 transfers peptides from TAP to MHC class I molecules; however, evidence is accumulating that gp96 is not generally involved in this process. The first indication came from antisense experiments in which a strong reduction in gp96 expression was found not to influence the ability of cells to present peptides to CTLs [35]. Secondly, the finding that gp96 cannot bind peptides with charged amino acids at P2 and P9 [23••] and that it has a hydrophobic peptide-binding domain [32] suggest a selectivity in peptide binding that might interfere with a general participation in peptide-loading of MHC class I; moreover — in contrast to tapasin, calreticulin and ERp60 [36] — gp96 cannot be immunoprecipitated with TAP or MHC molecules in digitonin lysates, arguing against a physical association with the ER-resident key players of class-I-restricted antigen presentation. Even though PDI — in contrast to gp96 — is capable of binding all TAP-translocated peptides analyzed to date [23••,34•] and coprecipitates with calreticulin [37], its participation in class-I-restricted antigen presentation could not be demonstrated either. In addition, an allosteric inhibitor of peptide-binding to PDI does not affect peptide binding to ER-resident H-2L d and HLA-A2 molecules in photolabelling experiments [34•]. Thus, both PDI and gp96 are unlikely to be generally involved in MHC peptide-loading despite their remarkable ability to bind peptides translocated into the ER. During the search for alternative functions of these ER-resident chaperones, we have postulated that both PDI and gp96 might assist in peptide export from the ER. Especially PDI, with its capacity to bind virtually all TAP-translocated peptides, might be an ideal candidate. This hypothesis, however, still awaits confirmation. As regards the function of gp96 and PDI, an alternative possibility is that these molecules are antigen-presenting molecules —similar to CD1 and nonclassical MHC molecules. The surface expression of HSP molecules, as reported for HSP72 [38] and gp96 (reviewed in [29]), is in line with this speculation.
Stress proteins and immune responses that are independent of αβ T cells Besides their ability both to chaperone peptides inside the cytosol and ER and to induce specific CTL responses by
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αβ T cells, HSPs are also known to be target structures recognized by γδ T cells and NK cells. The recognition of HSPs by γδ T cells was first described in the late 1980s and several groups observed the stimulation of human and mouse γδ T cells by mycobacterial protein extracts [39–41]. Further experiments proved the main component to be bacterial HSP65 and demonstrated that there was crossreactivity to human HSP65. As of now, it is not clear how recognition of these HSPs takes place. It is possible either that HSP-derived peptides are presented by MHC or by nonclassical MHC molecules [42,43] or that intact HSP molecules on the cell surface are recognized [44,45]. The latter model became increasingly attractive when the notion was put forward that many HSPs previously believed to be exclusively located to intracellular structures were also present on the surface of numerous cells, including tumor cells [38,46–48]. Soon thereafter, the recognition of surface-bound HSPs by γδ T cells was described and, in one case, a grp75-restricted presentation of a peptide derived from the immunoglobulin λ chain was reported (grp75 is a mitochondrial HSP that can also be expressed on the surface of some cells and can present peptides to γδ T cells) [49]. This recognition, which supported a possible role of HSPs as peptide-presenting molecules, could be inhibited with a grp75-specific antiserum. The recognition of HSPs, directly or as peptide presenting molecules, is, however, not limited to γδ T cells. Multhoff et al. [50] reported the recognition of HSP72 on heat-shocked human sarcoma cells by CD3– NK cells. This recognition was independent of antibodies blocking MHC class I molecules but could be inhibited by antibodies against HSP72 [50]. At a similar time, Fujieda et al. [51] observed an increased lysis of heat-treated thyroid cancer cells by lymphokine-activated killer cells. The involvement of HSPs in this process, however, was not demonstrated [51]. Subsequent studies on the recognition of HSP molecules by NK cells revealed that the induction of HSP72 expression on the cell surface is limited to certain tumor cells and that the increased lysis correlated precisely with an increase of surface, but not intracellular, levels of HSP72 after heat shock [52]. The part of the HSP70 molecule that is recognized seems to be located at the carboxyl terminus of the HSP molecule, which is predicted to be located extracellularly [53]. Interestingly, the recognition of HSP70 molecules is limited to the inducible HSP70 and HSP70hom proteins (HSP70hom is one allele of the human HSP70 family). The constitutive HSP70 protein and the bacterial homologue of HSP70, DnaK, can neither induce proliferation of NK cells nor interfere with NK-cell-mediated cytotoxicity (G Multhoff, personal communication). On the other hand, Blom et al. [54] reported an upregulation of HSP70 on the surface of uveal melanoma cells after heat shock that did not correlate with an increased susceptibility to lysis by NK cells. Apparently, not only the expression and upregulation of surface HSP70 but also the recognition of HSP70 molecules by NK cells seem to
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be tumor-specific. These findings might provide an explanation for the experiments of von Ardenne [55] in the 1970s; these experiments included hyperthermia treatment as one measure that was used to induce tumor regression. Whether or not the recognition of HSP70-expressing tumor cells by NK cells depends on the expression of certain HLA haplotypes on the tumor cells and/or on the expression of certain killer-cell inhibitory/activating receptors on NK cells still remains to be seen; so far clonal NK cell populations have not been used.
Conclusions HSPs are becoming increasingly attractive for use in the development of immunotherapy protocols. Their ability to induce specific immune responses against peptides that associate with this class of proteins inside any given cell allows the induction of tumor immunity without the need to identify the tumor-specific antigens; however, the potential hazard of inducing autoimmunity needs to be taken into consideration, even though experiments in the mouse model did not reveal any signs of autoimmune reactions. The mechanisms leading to the induction of tumor immunity also require more detailed investigation. Both the ability of HSPs to induce antitumor immunity in vivo more efficiently than CTL responses in vitro and the finding that immunizations with HSP70 molecules that are not associated with antigenic peptides lead to increased frequencies of ovalbumin-specific CTLs (A von Bonin, personal communication) possibly suggest that antigenspecific mechanisms are not the only ones that contribute to the immunogenicity of HSPs. Whether or not the binding of peptides to HSPs inside cells is a prerequisite for their presentation by MHC class I molecules is also an open question that needs to be answered in the future.
Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft (Leibnizprogram to H-G Rammensee [Ra369/41], Sonderforschungsbereich 510, C1 to H Schild) and the European Union (Biomed 95-1627). The authors thank L Yakes for reading the manuscript and G Multhoff, A von Bonin, P Srivastava and A Ménoret for providing unpublished results.
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Stress proteins and immunity mediated by cytotoxic T lymphocytes Hansjörg Schild, Danièle Arnold-Schild, Eckhard Lammert and Hans-Georg Rammensee Chaperone molecules, including members of the heat shock protein family, are able to stimulate αβ and γδ T cells as well as natural killer cells. For αβ T cells, specificity is induced by chaperone-assisted peptides; this has lead to detailed investigations of peptides that bind to these chaperones and their possible role in antigen presentation. Address Department of Immunology, Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany Current Opinion in Immunology 1999, 11:109–113 http://biomednet.com/elecref/0952791501100109 © Elsevier Science Ltd ISSN 0952-7915 Abbreviations CTL cytotoxic T lymphocyte ER endoplasmic reticulum HSP heat shock protein NK natural killer PDI protein disulfide isomerase TAP transporter associated with antigen presentation
Introduction The search for tumor antigens led to the isolation of proteins that were derived from tumor cells and were able to protect mice against a subsequent challenge with the same tumor cells from which the proteins were isolated [1].These proteins were identified as stress proteins and they include the heat shock proteins (HSPs) — HSP70, HSP90 and the endoplasmic reticulum (ER)-resident molecule gp96 (also known as glucose-regulated protein 94). Surprisingly, the genes coding for these proteins in tumor cells and normal tissues displayed no nucleotide sequence differences [2].To provide an explanation for the specificity of the immune response induced by the immunization with HSPs, it was postulated that peptides derived from intracellular proteins are associated with HSPs — thus providing specificity [3]. Because of this, HSP molecules attracted the attention of immunologists, like ourselves, who had previously been fascinated mainly by the cytotoxic T lymphocyte (CTL)-mediated recognition of MHC–peptide complexes. In the meantime, this hypothesis has been validated not only by numerous examples of specific CTL induction [4] but also, firstly, by autologous immunotherapy protocols [5••], secondly, by the finding that HSPs deprived of peptides do not induce specific immunity [6,7] and, thirdly, by the direct characterization of peptides associated with HSP70 and the ER-resident HSP gp96 [8,9]. This review summarizes the latest studies dealing with the induction of CTL responses, the peptide binding and the activation of γδ T cells or natural killer (NK) cells by HSP molecules.
Induction of CTL responses The immunogenic potential of HSP molecules was first demonstrated in immunotherapy experiments by Srivastava and co-workers (reviewed in [4]). Tumor-specific protection was mediated by CD8+ T cells as shown both by in vivo cell depletion studies [10] and by the ability to generate CTL lines specific for a variety of antigens from mice immunized with HSP molecules [11–13]. The HSPs that mediated this effect include the cytosolic HSP70 and HSP90 and the ER-resident chaperone gp96 [14]. It was subsequently shown that the immunization with gp96 molecules also resulted in the induction of memory T cells [15]; furthermore, gp96 molecules have been shown to induce CTL cross-priming against viral and minor histocompatibility antigens, which supports the hypothesis that gp96 molecules are associated with a large repertoire of peptides not influenced by the cellular MHC haplotype [12,13]. This peptide repertoire is, however, influenced by the expression of functional transporter associated with antigen presentation (TAP) molecules [16•]. Recent experiments have also revealed that HSP molecules can provide an immunogenic context to synthetic peptides that are complexed to HSP70 or gp96 molecules in vitro [17••,18]. Because of this, HSP molecules were called adjuvants of mammalian origin [4]. This ability is not only limited to mammalian HSP molecules, however; it is also observed when mycobacterial HSP70 molecules carrying covalently attached CTL epitopes are used [19•]. Interestingly, the same mycobacterial HSP70 molecules either carrying a covalently attached HIV-1 p24 protein or complexed with mycobacterial peptides bearing CD4+ T cell epitopes were able to elicit both antigen-specific humoral responses and specific immune responses that were mediated by CD4+ T cells [20–22]; however, it remains to be determined whether or not this feature is due to the xenogenic nature of the mycobacterial HSP70, which might activate existing CD4+ memory T cells induced by a previous encounter of the organism with pathogens expressing related HSP molecules. The ER-resident chaperone calreticulin is also able to induce specific immunity in vivo (PK Srivastava, personal communication). A general application in immunotherapy protocols might, however, be questionable as it was demonstrated that calreticulin binds only glycosylated peptides [23••]. The efficient induction of immune responses by HSP–peptide complexes, formed in vivo or in vitro, swiftly led to the proposal that these complexes are taken up by specialized cells [3]. This hypothesis was supported, firstly, by experiments utilizing the depletion of phagocytic
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cells in vivo [10], secondly, by the presentation of HSPassociated peptides in a manner that is restricted by MHC class I by activated macrophages incubated with gp96 molecules [13] and, finally, by the ability of gp96-pulsed bone-marrow-derived dendritic cells to induce protective immunity against tumor cells [24]. The mechanism of this efficient peptide presentation has been shown by Suto and Srivastava [13] to be sensitive to Brefeldin A but a detailed understanding of the intracellular events involved in the transfer of HSP-associated peptides onto MHC class I molecules requires further investigation. Independent experiments (R Binder, A Ménoret, PK Srivastava, personal communication; D Arnold-Schild et al., unpublished data) have led to the identification of a receptor-mediated uptake of HSP molecules by macrophages and dendritic cells, as postulated earlier [3]. This was not observed if controls — bovine serum albumin or F(ab) fragments — were used instead; furthermore, we were able to demonstrate that endocytosed gp96 molecules colocalize in endosomal compartments with recycled MHC class I molecules in a mouse dendritic cell line (D Arnold-Schild et al., unpublished data). These findings now provide a basis for understanding, firstly, the high efficiency of HSP–peptide complexes in inducing CTL-mediated immune responses in vivo and, secondly, the observation that the immunogenicity of tumor cells cosegregates with the expression of HSP molecules [25,26•].
Peptide-binding by stress proteins Peptide binding by members of the HSP70 family has been investigated in detail; it entails the association of peptide segments in an extended form, reminiscent of peptide binding to MHC molecules, but requires ATP hydrolysis. Both interactions share generic hydrogenbonding to the backbone and specificity-determining interactions with pockets located in the peptide-binding region. The existence of these pockets in the HSP70-family members DnaK, HSC70 and BiP was observed in peptide binding studies [27] and the detailed analysis of the crystal structure of DnaK in a complex with a peptide [28] revealed a sandwich-like peptide-binding domain, that indeed contained one specificity-determining pocket. The experiments addressing the ATPase activity and the potential substrate-binding regions of HSP90 molecules have recently been summarized [24,29]. For gp96, neither the structure of the peptide-binding domain nor the peptide-binding properties are known; however, it has been shown that gp96 can bind TAP-translocated peptides [23••,30•,31•]; moreover, an immunodominant viral peptide has been eluted from gp96 in infected cells [8] and the H2-Kb epitope SIINFEKL (using single-letter code for ovalbumin amino acids 257–264) has been found associated with both HSP70 and gp96 in ovalbumin-expressing cells [9]. It has also been demonstrated recently that gp96 cannot efficiently bind peptides with charged amino acids at positions 2 and 9 (P2 and P9) in nonamer peptides [23••] and that it has a hydrophobic peptide-binding site [32]. The necessity to transiently heat or to chemically denature
and renature gp96 in order to obtain notable peptide-binding in vitro [17••,32] and the fact that adenine nucleotides do not influence peptide binding [33•] might argue for the existence of proteins inside the ER that promote peptidebinding to gp96. Using photoreactive peptides, additional ER-resident peptide-binding proteins were identified [23••,31•] and among these the most notable is protein disulfide isomerase (PDI) [34•]. Interestingly, no peptide binding to BiP was observed in these studies. Whether or not the peptide binding to ER-resident proteins such as gp96 or PDI has any immunological function is still a matter of speculation. Srivastava et al. [3] proposed that gp96 transfers peptides from TAP to MHC class I molecules; however, evidence is accumulating that gp96 is not generally involved in this process. The first indication came from antisense experiments in which a strong reduction in gp96 expression was found not to influence the ability of cells to present peptides to CTLs [35]. Secondly, the finding that gp96 cannot bind peptides with charged amino acids at P2 and P9 [23••] and that it has a hydrophobic peptide-binding domain [32] suggest a selectivity in peptide binding that might interfere with a general participation in peptide-loading of MHC class I; moreover — in contrast to tapasin, calreticulin and ERp60 [36] — gp96 cannot be immunoprecipitated with TAP or MHC molecules in digitonin lysates, arguing against a physical association with the ER-resident key players of class-I-restricted antigen presentation. Even though PDI — in contrast to gp96 — is capable of binding all TAP-translocated peptides analyzed to date [23••,34•] and coprecipitates with calreticulin [37], its participation in class-I-restricted antigen presentation could not be demonstrated either. In addition, an allosteric inhibitor of peptide-binding to PDI does not affect peptide binding to ER-resident H-2L d and HLA-A2 molecules in photolabelling experiments [34•]. Thus, both PDI and gp96 are unlikely to be generally involved in MHC peptide-loading despite their remarkable ability to bind peptides translocated into the ER. During the search for alternative functions of these ER-resident chaperones, we have postulated that both PDI and gp96 might assist in peptide export from the ER. Especially PDI, with its capacity to bind virtually all TAP-translocated peptides, might be an ideal candidate. This hypothesis, however, still awaits confirmation. As regards the function of gp96 and PDI, an alternative possibility is that these molecules are antigen-presenting molecules —similar to CD1 and nonclassical MHC molecules. The surface expression of HSP molecules, as reported for HSP72 [38] and gp96 (reviewed in [29]), is in line with this speculation.
Stress proteins and immune responses that are independent of αβ T cells Besides their ability both to chaperone peptides inside the cytosol and ER and to induce specific CTL responses by
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αβ T cells, HSPs are also known to be target structures recognized by γδ T cells and NK cells. The recognition of HSPs by γδ T cells was first described in the late 1980s and several groups observed the stimulation of human and mouse γδ T cells by mycobacterial protein extracts [39–41]. Further experiments proved the main component to be bacterial HSP65 and demonstrated that there was crossreactivity to human HSP65. As of now, it is not clear how recognition of these HSPs takes place. It is possible either that HSP-derived peptides are presented by MHC or by nonclassical MHC molecules [42,43] or that intact HSP molecules on the cell surface are recognized [44,45]. The latter model became increasingly attractive when the notion was put forward that many HSPs previously believed to be exclusively located to intracellular structures were also present on the surface of numerous cells, including tumor cells [38,46–48]. Soon thereafter, the recognition of surface-bound HSPs by γδ T cells was described and, in one case, a grp75-restricted presentation of a peptide derived from the immunoglobulin λ chain was reported (grp75 is a mitochondrial HSP that can also be expressed on the surface of some cells and can present peptides to γδ T cells) [49]. This recognition, which supported a possible role of HSPs as peptide-presenting molecules, could be inhibited with a grp75-specific antiserum. The recognition of HSPs, directly or as peptide presenting molecules, is, however, not limited to γδ T cells. Multhoff et al. [50] reported the recognition of HSP72 on heat-shocked human sarcoma cells by CD3– NK cells. This recognition was independent of antibodies blocking MHC class I molecules but could be inhibited by antibodies against HSP72 [50]. At a similar time, Fujieda et al. [51] observed an increased lysis of heat-treated thyroid cancer cells by lymphokine-activated killer cells. The involvement of HSPs in this process, however, was not demonstrated [51]. Subsequent studies on the recognition of HSP molecules by NK cells revealed that the induction of HSP72 expression on the cell surface is limited to certain tumor cells and that the increased lysis correlated precisely with an increase of surface, but not intracellular, levels of HSP72 after heat shock [52]. The part of the HSP70 molecule that is recognized seems to be located at the carboxyl terminus of the HSP molecule, which is predicted to be located extracellularly [53]. Interestingly, the recognition of HSP70 molecules is limited to the inducible HSP70 and HSP70hom proteins (HSP70hom is one allele of the human HSP70 family). The constitutive HSP70 protein and the bacterial homologue of HSP70, DnaK, can neither induce proliferation of NK cells nor interfere with NK-cell-mediated cytotoxicity (G Multhoff, personal communication). On the other hand, Blom et al. [54] reported an upregulation of HSP70 on the surface of uveal melanoma cells after heat shock that did not correlate with an increased susceptibility to lysis by NK cells. Apparently, not only the expression and upregulation of surface HSP70 but also the recognition of HSP70 molecules by NK cells seem to
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be tumor-specific. These findings might provide an explanation for the experiments of von Ardenne [55] in the 1970s; these experiments included hyperthermia treatment as one measure that was used to induce tumor regression. Whether or not the recognition of HSP70-expressing tumor cells by NK cells depends on the expression of certain HLA haplotypes on the tumor cells and/or on the expression of certain killer-cell inhibitory/activating receptors on NK cells still remains to be seen; so far clonal NK cell populations have not been used.
Conclusions HSPs are becoming increasingly attractive for use in the development of immunotherapy protocols. Their ability to induce specific immune responses against peptides that associate with this class of proteins inside any given cell allows the induction of tumor immunity without the need to identify the tumor-specific antigens; however, the potential hazard of inducing autoimmunity needs to be taken into consideration, even though experiments in the mouse model did not reveal any signs of autoimmune reactions. The mechanisms leading to the induction of tumor immunity also require more detailed investigation. Both the ability of HSPs to induce antitumor immunity in vivo more efficiently than CTL responses in vitro and the finding that immunizations with HSP70 molecules that are not associated with antigenic peptides lead to increased frequencies of ovalbumin-specific CTLs (A von Bonin, personal communication) possibly suggest that antigenspecific mechanisms are not the only ones that contribute to the immunogenicity of HSPs. Whether or not the binding of peptides to HSPs inside cells is a prerequisite for their presentation by MHC class I molecules is also an open question that needs to be answered in the future.
Acknowledgements This work was supported by grants of the Deutsche Forschungsgemeinschaft (Leibnizprogram to H-G Rammensee [Ra369/41], Sonderforschungsbereich 510, C1 to H Schild) and the European Union (Biomed 95-1627). The authors thank L Yakes for reading the manuscript and G Multhoff, A von Bonin, P Srivastava and A Ménoret for providing unpublished results.
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