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Glucose-regulated stress proteins and antibacterial immunity Ulrike K. Rapp and Stefan H.E. Kaufmann Max Planck Institute for Infection Biology, Schumannstrasse 21-22, 10117 Berlin, Germany

The role of stress proteins in immunity and their feasibility as vaccine vehicles against infectious disease have been the focus of intensive examination. Endoplasmic reticulum (ER)-resident stress proteins in particular are interesting model proteins as they perform crucial functions in an organelle that responds promptly to cell stress. We describe transcriptional regulation of ER-resident stress proteins, their involvement in the cellular response to infection and discuss their potential as vaccine candidates against infectious diseases. The immunologic functions of heat shock proteins (HSP) and their effective application in prevention and therapy of infectious diseases have gained increasing attention. HSP are frequently referred to as stress proteins, because they are upregulated during cell stress and are crucial in reinstating cellular homeostasis. This review focuses on endoplasmic reticulum (ER)-restricted stress proteins, most of which belong to the family of glucose-regulated stress proteins (GR-HSP), because the cell-stress response in the ER is both crucial and well studied. Transcriptional upregulation of stress proteins probably induces ER stress, via the unfolded protein response (UPR) or the ER overload response (EOR) pathways, as a consequence of accelerated protein synthesis. Similarly, an infected cell involved in pathogen replication might exhibit an ER-associated stress response, as a result of upregulation of stress proteins and pathogen-induced protein synthesis. Transfer of antigenic peptides from stress proteins to major histocompatibility complex (MHC) class I molecules has been investigated using GR-HSP as a model, among others [1 – 4]. Insights gained from these studies could provide indications for novel approaches to therapy and prophylaxis of autoimmune and infectious diseases. We will introduce physiological and immunologic aspects of GR-HSP function and point out where this can be extended to HSP in general. On the basis of this, we will discuss first, the unique qualities of ER-restricted GR-HSP in comparison with other HSP, and second, the potential immunologic relevance and application of these proteins in therapy and prevention of infectious diseases. Disparate observations in the current literature regarding cellular uptake and processing, as well as characterization of specific cell-surface receptors, reveal areas of study that require further attention. Despite current controversies, this review aims to provide an integrated view of the Corresponding authors: Ulrike K. Rapp ([email protected]), Stefan H.E. Kaufmann ([email protected]).

physiological and immunologic roles of GR-HSP and the regulatory circuits involved in their regulation. Stress proteins: chaperoning and beyond HSP were first identified as a family of proteins that are produced in response to various types of cellular stress, including heat shock, osmotic shock, exposure to free radicals, nutrient deprivation, infection, inflammation and malignancy [5– 9]. HSP are highly conserved, have remarkable cross-species homology, and are present in all organisms from prokaryotes to eukaryotes. They are also residents of many different cellular compartments, including the cytosol, mitochondria and ER. Numerous ER-resident HSP belong to the family of glucose-regulated proteins (Grp). This is reflected in their nomenclature, for example, gp96 and Bip are synonymous to Grp94 and Grp78, respectively. Because of their role as scaffolding proteins during protein biosynthesis in the cell, HSP are also referred to as molecular chaperones. HSP bind a wide range of proteins. Their primary function is the chaperoning of intracellular proteins during protein folding at the ribosome [10– 12]. During cellular stress, rapid upregulation of HSP expression helps prevent extensive protein degradation and reinstate cellular homeostasis [10– 12]. Three basic regulatory pathways involved in ‘protein homeostasis’ are the ubiquitin-mediated pathway, the UPR and the EOR pathways [13 – 16]. In the ubiquitin-mediated pathway, molecular chaperones are involved in the selection of denatured, misfolded, foreign and overexpressed proteins for targeted proteolysis in the proteasome. Targeting is preceded by the addition of ubiquitin molecules to an internal lysine side-chain of the protein. Chaperones bind unassembled or misfolded proteins in the cytosol and in the ER, thus preventing their aggregation and promoting their transport to the cytosol, where they are degraded [10 –12]. For the UPR pathway, the accumulation of unfolded proteins in the ER induces an inter-organelle signaling pathway that links the ER with the nucleus and results in the rapid transcriptional upregulation of ER chaperones and other essential folding enzymes [13 – 16] (Figure 1). ER stress has also been linked to activation of the NF-kB transcription factor complex [15]. This ER-nuclear signal transduction pathway, known as EOR, is independent of the UPR pathway [15]. The EOR pathway induces NF-kB activation as a consequence of the accumulation of proteins within the ER membrane via two second-messengers: calcium ions and reactive oxygen

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Figure 1. Endoplasmic reticulum (ER) Stress: the ER overload response (EOR) and the unfolded protein response (UPR) pathways. ER stress induced by one or multiple factors ultimately leads to the activation of the EOR and the UPR pathways. The EOR pathway induces NF-kB activation and an inflammatory response. The UPR pathway results in transcriptional upregulation of ER chaperones, translational attenuation of protein synthesis, and removal of misfolded or aggregated proteins from the ER.

intermediates (ROI) [15]. The EOR is distinguished from the UPR in that it is not caused by the accumulation of unfolded proteins and does not induce expression of GR-HSP. Finally, the basal expression of ER chaperones is regulated by various growth and differentiation factors via mitogenic signaling pathways [16]. This ensures that chaperone synthesis is coordinated with the proteintranslation activity of the cell. Thus, two essential cellular functions of HSP are regulation of protein homeostasis and protein quality control. In addition, HSP are markers of cell stress as a result of their upregulation in stressed cells [5 – 9], and also their release by stressed cells as dangersignaling molecules [17– 20]. HSP have been applied successfully as vaccine vehicles in experimental models of infectious and autoimmune diseases [1– 9,18,21– 24]. HSP-based vaccines applied in infection models induce an inflammatory response, whereas, application of HSP in the autoimmune model induces an anti-inflammatory response. The association between upregulation of HSP during cell stress and the induction of an inflammatory response might be threshold-dependent, and a regulatory circuit could control the induction of either an inflammatory or an anti-inflammatory response to immunization with HSP. Two additional aspects of chaperone function that suggest an immunologic role have been the focus of recent attention: the release of HSP into extracellular space [17], and the observation that chaperones transport polypeptides between intracellular compartments to promote transfer of these peptides to MHC molecules [1– 9,25]. Although supporting the idea of HSP and GR-HSP involvement in innate and adaptive immunity with http://www.trends.com

broad implications for vaccine development, these issues raise several questions regarding the underlying mechanisms and the regulation of these processes. GR-HSP promoter structure and genetic control The regulatory network involved in the induction of GR-HSP transcription reveals important insights into the stress response of the cell. GR-HSP encoding genes serve as a prototype of a class of genes regulated by signal transduction pathways that originate in the ER and lead to the nucleus [9,13– 15]. Induction of GR-HSP genes during stress is regulated primarily at the transcriptional level. Evidence suggests that different stress stimuli use distinct signaling pathways to activate GR-HSP genes, and also that activation patterns are tissue specific [9,13 –15]. The mammalian GR-HSP promoter is characterized by a CCAAT element flanked by GC-rich sequences [26 – 29]. These represent repetitive units of the ER stress-response element (ERSE), an evolutionarily conserved sequence that is comparable, albeit more complex, to the unfolded protein response element (UPRE), first identified in yeast [26 –29]. Several proteins have been identified which bind the CCAAT motif, and are required for basal and stressinduced transcription from the GR-HSP promoter (Figure 2). The proteins that enhance transcription of GR-HSP proteins during cell stress include: the basic leucine zipper protein ATF6 [30], the transcription factor TFII-I, known to facilitate protein –protein interactions [30 – 34], and the CCAAT binding factor NF-Y [30 –34]. GR-HSP induction can be inhibited specifically by another basic leucine zipper protein, known as CREB (c-AMP response-element binding protein) [26 –34]. ATF6

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Figure 2. Cellular signaling associated with the unfolded protein response (UPR) pathway and endoplasmic reticulum (ER) chaperone upregulation. Multiple signaling proteins are activated in response to ER stress. The basic leucine zipper transcription factors, ATF6 (activating transcription factor 6) and CREB (c-AMP response-element binding proteins), both undergo stress-induced cleavage. This generates small cytoplasmic fragments that translocate to the nucleus and form complexes with other transcription factors, leading to the activation (ATF6) or suppression (CREB) of GR-HSP (glucose-regulated stress proteins) transcription. The cytoplasmic fragments of ATF6 and CREB also positively regulate XBP-1 (a basic leucine zipper protein originally identified as a protein that binds to the promoter regions of human major histocompatibility complex [MHC] class II genes) transcription and induction of CHOP, a C– EBP (CCAAT-enhancer binding protein) homologous transcription factor. ER-resident kinases, PERK (protein kinase-like ER kinase) and Ire1 a/b are also activated in response to ER stress. PERK activation induces attenuation of protein synthesis via phosphorylation of eukaryotic initiation factor (elF)-2a. Activated Ire1 has endoribonuclease activity and leads to mRNA splicing of XBP-1, resulting in a fragment that interacts directly with the ER stress response element (ERSE), followed by activation of ER chaperone transcription. Activation of Ire1 might also induce phosphorylation of c-Jun and induction of CHOP. The balance between CHOP induction and induction of ER GR-HSP determines the ultimate decision for cell death or survival.

(activating transcription factor 6) and CREB both undergo stress-induced cleavage, generating small cytosolic fragments that enter the nucleus and interact with the ERSE through complex formation with NF-Y [30 – 34]. Finally, it is important to note that the activities of both ATF6, a substrate of p38 MAP (mitogen activated protein) kinase, and TFII-I are modulated by phosphorylation and could provide a link between GR-HSP transcription and other cellular signaling pathways [30 – 35]. Those HSP that do not belong to the GR-HSP family are regulated in a different manner. Transcription of HSP genes is induced by the interaction of heat shock factors (HSF1, HSF2) with heat shock elements in the HSP gene promoter regions [36]. Cell stress causes activation of the inactive cytosolic HSF1 molecule. Phosphorylated HSF1 trimers then translocate to the nucleus where they induce HSP gene transcription [37]. The regulation of HSP gene expression and signaling involved has been reviewed extensively [38]. Stress proteins and innate immunity HSP, including GR-HSP, are released into the extracellular space either by secretion during cell stress or as a http://www.trends.com

consequence of necrotic cell death [19,20]. Given that this release is biologically relevant, cell-surface receptors must exist that bind HSP or a HSP-associated structure. Furthermore, ligand binding must initiate differentiated signal transduction that allows cells to respond to specific types of stress. Several receptors on antigen presenting cells (APC) have been suggested to bind HSP, including the scavenger receptors CD91 and CD36, [39 –43] as well as members of the Toll-like receptor (TLR) family, TLR2 and TLR4 [44– 47] and, most recently, the co-stimulatory molecule CD40 [48]. Receptor-mediated internalization of HSP complexes is thought to initiate NF-kB activation, leading to pro-inflammatory cytokine upregulation [41,42,44,45,48]. Elucidation of the activation of signal transduction pathways in conjunction with the respective cell surface receptor is the focus of current investigation. Most conclusive data have been obtained for TLR, where NF-kB activation induced by interaction of HSP complexes with the TLR2 or TLR4 receptor [46– 47] depends on signaling via MyD88 [49]. In the case of the CD91 and CD36 scavenger receptors, analysis has not yet revealed a distinct signaling pathway, and association between HSP– receptor interaction and NF-kB activation is based

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on indirect evidence [39 – 42]. Interactions of HSP complexes with cell-surface receptors on APC and internalization of HSP by macrophages and dendritic cells (DC), induce cellular maturation, subsequent activation and migration, cytokine secretion and upregulation of co-stimulatory molecules and MHC II [50,51]. In fact, observations imply that HSP are expressed on the cell surface, thus functioning as co-stimulatory molecules [18,52,53]. Together these observations confirm the ability of HSP to interact with cells of the innate immune system and the interaction of HSP with cell-surface receptors known to associate with microbial molecules or immunostimulatory cytokines. This supports the hypothesis that HSP function both as a ‘danger signal’ and a natural adjuvant [5,17,18]. Stress proteins and adaptive immunity in infection Many of the functions attributed to HSP and GR-HSP have been modified or expanded owing to the observation that HSP bind polypeptides in the cell, and that these HSP –peptide complexes remain stable when released [1 – 3,19,20]. Initial studies focused on the ability of HSP –peptide complexes to induce an adaptive immune response specific for the peptide associated with the HSP [1 – 9,18]. HSP bind polypeptides in the cytosol and shuttle these between cellular compartments, such as the cytosol and the ER [1 –9,18]. This applies specifically to self- and foreign-polypeptides acquired during, or as a consequence of, protein processing. In a virus-infected cell, this might include foreign proteins produced by the host cell. Several independent observations in viral, bacterial and parasitic infection models confirm the ability of HSP – peptide complexes to induce peptide-specific T-cell responses, and in some cases protective immunity [5 –9,18]. The protective T-cell response achieved is almost entirely independent of CD4 þ T-cells [5– 9,18]. This is an important hallmark of HSP-immune activation and implies the preferred involvement of the MHC I antigen processing pathway [1– 4,9]. It also provides a basis for understanding cellular trafficking and processing of HSP –peptide complexes. Processing and antigen presentation through the MHC I pathway are usually reserved for cytosolic or newly synthesized antigens, as opposed to phagosomal antigens. Current hypotheses diverge suggesting that receptor-mediated uptake is essential [54,55], and that cellular processing of HSP complexes involves the proteasome, an alternative cytosolic route, or the early endosome (Figure 3) [1 –4,54– 57]. Original investigations that form the groundwork for these diverging hypotheses are discussed in the following. Further studies were directed towards elucidating when, and where, peptides are bound by HSP. Most data suggest that peptide-binding occurs only intracellularly, predominantly in the cytosol, and not after release of HSP into the extracellular space, which occurs as a consequence of cell stress or damage [19,20,54,55]. But, what are the cellular processing pathways of exogenous and endogenous HSP –peptide complexes and how are peptides transferred to the MHC class I molecule? Are there differences between complexes that are internalized by receptor-mediated endocytosis and macropinocytosis? Do http://www.trends.com

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these processing pathways converge and how is signaling affected? These are important questions, some of which have been addressed in recent studies [1 –4,13,25,54 – 58] and will be discussed below. Nevertheless, further investigation is required to clarify these issues. Stress proteins and adaptive immunity in autoimmunity Another important question refers to differences in the adaptive immune response to self- and non-self-HSP complexes. Despite, or perhaps on account of, the extensive sequence homology between HSP of prokaryotic and eukaryotic origin, both have been applied successfully in experimental models of infectious and autoimmune disease [21– 24]. This has led to the assumption that nonself-HSP are recognized as self-HSP and that all HSP and GR-HSP function equally as natural adjuvants. If this is the case, it could explain the observation that major T-cell responses against bacterial chaperones are mounted during infection [21 – 24]. Cellular stress induces a controlled upregulation of self-HSP. It is plausible that anti-inflammatory T cells participate in a negative-feedback-regulated circuit that monitors and coordinates inflammation and HSP expression (Figure 4). Accordingly, cross-reactivity between self- and non-self-HSP should not represent a major cause of autoimmune pathology, as assumed initially [21– 24]. In addition, administration of HSP prevents or alleviates disease in animal models of autoimmune arthritis and non-obese autoimmune diabetes [23,24]. Further analyses have revealed that antiself-HSP T-cells are positively selected in the thymus of healthy animals and that these T cells are activated as a consequence of inflammation [23,24]. The anti-inflammatory response induced by the abundance of self- and nonself- HSP is considered responsible for suppression of some autoimmune disease symptoms [21 – 24]. As a result, HSP must be able to induce both inflammatory and antiinflammatory responses, depending on the cellular response cascades activated. To date, it is unresolved which signaling pathways are involved in mediating these effects and what regulates the switch between inflammatory and anti-inflammatory responses. Collectively, these observations lead to the conclusion that the activation of self-HSP-reactive T-cells serves to downregulate the overexpression of HSP and subsequent induction of an inflammatory response at a given time-point during cell stress or infection (Figure 4). This supports the function of HSP as universal markers of cell stress, and suggests an intriguing regulatory circuit in which HSP play crucial roles as target molecules through which the immune system can monitor and regulate cellular conditions (Figure 2). Cellular uptake and processing of GR-HSP: diverging opinions Recent attention has been directed towards the molecular and biochemical characterization of unique GR-HSPassociated functions, GR-HSP receptor interactions, and cellular trafficking. Srivastava and co-workers published seminal observations in the ‘90’s, based on the analysis of a specific GR-HSP-family member, gp96 (Grp94) [1 – 3]. Preliminary biochemical analyses suggested enzymatic

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Figure 3. Cellular uptake and processing of GR-HSP (glucose-regulated stress proteins)–peptide complexes. Cellular uptake of GR-HSP– peptide complexes by antigen presenting cells might involve the CD91 scavenger receptor, a member of the Toll-like receptor (TLR) family, or an unknown receptor. Two cellular processing pathways have been suggested. Pathway 1 involves endosomal or lysosomal processing of the GR-HSP– peptide complex. This initiates transport of peptide to the endoplasmic reticulum (ER), where it is loaded onto major histocompatibility complex (MHC) class I molecules and translocated to the cell surface for re-presentation. The peptide can enter the ER using either the TAP (transporters associated with antigen processing) transporter or retrograde transport from the Golgi system. Pathway 2 involves dissociation of the GR-HSP –peptide complex in the early endosome, transfer of the peptide onto recycling MHC class I molecules in the same compartment, and translocation of this MHC I –peptide complex to the cell surface for re-presentation.

activities including ATPase activity, aminopeptidase activity, and heparanase activity [25,59,60]. Studies aimed at clarifying the mechanisms of uptake of gp96 – peptide complexes and the induction of signaling cascades implied an indirect association of receptor-mediated uptake with NF-kB activation [1 – 3,41,42]. The exact pathway linking gp96 –receptor interactions and NF-kB activation has not yet been elucidated. Also, it remains unclear how gp96 complexes interact directly with their putative receptor: there might be one or several GR-HSPassociated molecules responsible for mediating GR-HSP receptor interactions. Further studies suggest specific interactions of gp96 with the CD91 scavenger receptor [42]. Moreover, the CD91 receptor was proposed to be a common receptor for gp96, HSP90, HSP70 and calreticulin [41,42]. Identification of CD40 as a receptor for HSP70 raises the question whether this co-stimulatory molecule is also involved in GR-HSP uptake [48]. To classify the mechanisms by which stress-protein-associated peptides are introduced to MHC class I molecules, the cellular trafficking of GR-HSP– peptide complexes in macrophages was analyzed in the presence or absence of the proteasome inhibitor lactacystin [42]. These studies showed that a http://www.trends.com

functional proteasome is necessary for MHC I presentation of GR-HSP-associated peptides [42]. However, the cellular localization of gp96– peptide complexes and the mechanism of peptide transfer remains unclear. The use of TAP (transporters associated with antigen processing)knockout macrophages and mice illustrated that gp96 – peptide complexes induce peptide-specific T-cell stimulation in a TAP-dependent manner [42]. Accordingly, it was presumed that gp96-chaperoned peptides must enter the ER using TAP proteins before MHC loading. Independent studies focusing on the same questions have revealed a different picture. Nicchitta et al. examined the enzymatic activities previously ascribed to gp96 [25,58 – 60]. Extensive purification of GR-HSP preparations by chromatographic fractionation revealed several separate proteins with ATPase, aminopeptidase and protein phosphorylation activities [61]. These proteins were shown to co-purify with gp96. None of the previously described enzymatic activities was found to be associated with the gp96 preparation itself. It is not clear whether the co-purifying proteins associated with gp96 in vivo, or if their association occurred only during protein purification in vitro [61]. A subsequent study investigated cellular

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Figure 4. Integrating the stress-related and immunologic roles of GR-HSP (glucose-regulated stress proteins). Cellular insult leads to endoplasmic reticulum (ER) stress, acute inflammation, and release of GR-HSP–peptide complexes from the cell. Release of these complexes from the cell constitutes a ‘danger signal’. Surrounding cells engulf and process the complexes. This leads to the presentation of peptides by major histocompatibility complex (MHC) class I molecules on the cell surface and the induction of a peptide-specific T-cell response. ER stress induces upregulation of ER chaperone expression and induction of an inflammatory response. Higher concentrations of GR-HSP might activate anti-self-HSP reactive T cells. This results in downregulation of the inflammatory response and expression of ER chaperones.

uptake of gp96 – peptide complexes [61,62]. Despite previous investigations including re-presentation and inhibition assays, a direct role for the CD91 receptor in the re-presentation of chaperone-bound peptides has not been demonstrated unequivocally [61,62]. Competition binding assays and inhibition assays involving the CD91 receptor and gp96 – peptide complexes did not confirm a role for CD91 in cellular uptake of the complexes [61,62]. Although a candidate receptor was not identified, gp96 – peptide processing was found to occur independently of the CD91 receptor [62]. In parallel, the trafficking requirements of these complexes were analyzed and compared with those of an obligate CD91 ligand [62]. The ligand and the gp96 – peptide complexes were internalized in a receptormediated fashion. However, the gp96 –peptide complex co-localized to cellular compartments distinct from both the CD91 receptor and the obligate CD91 ligand [62,63]. Under no condition was co-localization of CD91, or the CD91 ligand, with the gp96 – peptide complex observed. Finally, the cellular compartment to which the gp96 – peptide complex localized was characterized with the aim of identifying a potential mechanism for peptide transfer from HSP to the MHC class I molecule [62,63]. Receptormediated uptake of gp96 – peptide complexes appear to be independent of CD91 [62,63]. Examination of subcellular trafficking and kinetics of peptide re-presentation revealed that internalized gp96 localized to an endocytic compartment. This endocytic compartment is further classified as one that contains MHC I and FcR IgG, but does not contain transferrin, CD1 or Rab5a [62,63]. Kinetic analysis of MHC I biogenesis and chaperone-bound http://www.trends.com

peptide re-presentation suggested a post-ER endosomal compartment as the predominant site of peptide exchange from gp96 to MHC I [62,63]. These findings are in contrast to previous hypotheses that suggest TAP-dependent gp96-associated peptide re-presentation and peptide transfer from gp96 to MHC class I molecules in the ER [62,63]. It was also noted that established CD91 ligands traffic to lysosomes [62,63]. Together with the observation that gp96 did not co-localize with CD91 in the cell, the marked differences in trafficking pathways argue against the CD91 receptor as prime candidate for the gp96 receptor [62,63]. Integrating the stress-related and immunological roles of GR-HSP The following discussion aims to integrate various facets of GR-HSP action by emphasizing correlations in signaling pathways and to suggest from these what renders it unique. Constitutive roles of stress proteins include primarily their functions as chaperones for protein folding and quality control. These roles can be directly associated with the ability of stress proteins to signal cell stress or damage caused by increased concentrations of aggregated, unfolded or misfolded proteins. Signaling of cell stress leads to upregulation of GR-HSP expression and ultimately to an inflammatory response [41,42,44,45,48]. The described effects are regulated through distinct signaling cascades; the UPR, the EOR, and signal transduction pathways induced by interactions between stress proteins and cell-surface receptors [13– 16]. The abundance of stress proteins in the extracellular space, in conjunction

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with acute inflammation, could stimulate anti-self-reactive T cells, which then downregulate the inflammatory response. In addition, stressed cells release stress proteins into the extracellular space. These proteins are then recognized and internalized by surrounding cells (danger signal). Cellular uptake of stress – protein complexes then induces an adaptive immune response directed at antigenic peptides associated with the protein. This must take place in a manner that is coordinated with the elimination of stress proteins by self-reactive T cells (Figure 4). Outlook Several candidate cell-surface receptors for GR-HSP and HSP have been described along with specific cellular processing and signaling pathways. Diverging observations regarding specific interactions between GR-HSP and receptor remain to be clarified. Accordingly, the cellular uptake and processing of GR-HSP, characterization of specific cell-surface receptors and the mechanism of peptide transfer between these proteins and MHC class I molecules remain the focus of intensive investigation. Moreover, immunostimulatory effects of GR-HSP immunization need to be re-evaluated taking into consideration possible contributions of contaminants such as LPS or ConA. The presence of contaminants in semipurified protein solutions can affect cellular uptake of GR-HSP via their specific receptors and subsequent signal transduction. So far evidence shows that GR-HSP are unique in their ability to mediate among multiple regulatory signaling cascades. This makes GR-HSP intriguing candidates for gene therapy. In immunotherapy, stress proteins have been applied successfully in vaccine formulations in viral and parasitic model systems [5– 9,18,21– 24]. Stress protein-based vaccines are non-toxic, non-mutagenic and the quantity required for effective immunization is logscales lower than that of other adjuvants [5 –9]. Their ability to cross-prime and to exert broad influence on cellular response cascades make them particularly effective vaccine vehicles, and highlights their potential in a broad spectrum of applications for prophylaxis and therapy. A deeper understanding of their regulation on a genetic level and of the mechanisms through which they function in multiple regulatory circuits will provide indications for basic mechanisms underlying autoimmune pathology. Finally, insight into the evolutionary development of stress proteins and their functions will lead to a deeper understanding of their structure and physiological as well as pathological relevance. Acknowledgements We would like to thank Prof. Dr. W. Goebel and M. Ingersoll for critical reading of the manuscript, and D. Schad for assistance with the graphics. This work is supported by the DFG Priority Program, Novel Vaccination Strategies (KA 573/4).

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3 Srivastava, P.K. et al. (1994) Heat shock proteins transfer peptides during antigen processing and CTL priming. Immunogenetics 39, 93 – 98 4 Robert, J. et al. (2002) Minor histocompatibility antigen specific MHC-restricted CD8 T cell responses elicited by HSP. J. Immunol. 168, 1697– 1703 5 Harmala, L.A. et al. (2002) The adjuvant effects of Mycobacterium tuberculosis heat shock protein 70 result from the rapid and prolonged activation of antigen-specific CD8 þ T cells in vivo. J. Immunol. 169, 5622– 5629 6 Liu, D.W. et al. (2000) Recombinant adeno-associated virus expressing human papillomavirus type 16 E7 peptide DNA fused with heat shock protein DNA as a potential vaccine for cervical cancer. J. Virol. 74, 2888– 2894 7 Ciupitu, A.M. et al. (1998) Immunization with a lymphocytic choriomeningitis virus peptide mixed with heat shock protein 70 results in protective antiviral immunity and specific cytotoxic T lymphocytes. J. Exp. Med. 187, 685– 691 8 Oglesbee, M.J. et al. (2002) Role for heat shock proteins in the immune response to measles virus infection. Viral Immunol. 15, 399– 416 9 Lee, A. (2001) The glucose regulated proteins: stress induction and clinical applications. Trends Biochem. Sci. 26, 504 – 510 10 Horwich, A.L. et al. (2001) Protein folding takes shape. EMBO Rep. 2, 1068– 1073 11 Hartl, F.U. and Hayer-Hartl, M. (2002) Molecular chaperones in the cytosol: from nascent chain to folded protein. Science 295, 1852– 1858 12 Meunier, L. et al. (2002) A subset of chaperones and folding enzymes form multiprotein complexes in the ER to bind nascent proteins. Mol. Biol. Cell 13, 4456– 4469 13 Patil, C. and Walter, P. (2001) Intracellular signaling from the ER to the nucleus: the unfolded protein response in yeast and mammals. Curr. Opin. Cell Biol. 13, 349 – 356 14 Ma, Y. and Hendershot, L.M. (2001) The unfolding tale of the unfolded protein response. Cell 107, 827– 830 15 Pahl, H.L. and Baeuerle, P.A. (1995) A novel signal transduction pathway from the ER to the nucleus is mediated by transcription factor NFkB. EMBO J. 14, 2580 – 2588 16 Brewer, J.W. et al. (1997) A pathway distinct from the mammalian unfolded protein response regulates expression of ER chaperones in non-stressed cells. EMBO J. 16, 7207 – 7216 17 Matzinger, P. (2002) The danger model: a renewed sense of self. Science 296, 301 – 305 18 Lehner, T. et al. (2000) Heat shock proteins generate beta-chemokines which function as innate adjuvants enhancing adaptive immunity. Eur. J. Immunol. 30, 594 – 603 19 Basu, S. et al. (2000) Necrotic but not apoptotic cell death releases HSP, which deliver a partial maturation signal to dendritic cells and activate the NFkB signaling pathway. Int. Immunol. 12, 1539– 1546 20 Berwin, B. et al. (2001) Virally induced lytic cell death elicits the release of immunogenic Grp94/gp96. J. Biol. Chem. 276, 21083 – 21088 21 Van Eden, W. et al. (2003) Immunopotentiating heat shock proteins: negotiators between innate danger and control of autoimmunity. Vaccine 21, 897 – 901 22 Van Eden, W. et al. (1998) Do HSP control the balance of T-cell regulation in inflammatory diseases? Immunol. Today 19, 303 – 307 23 Cobelens, P.M. et al. (2000) Treatment of adjuvant induced arthritis by oral administration of Mycobacterial HSP65 during disease. Arthritis Rheum. 43, 2694 – 2702 24 Van Eden, W. et al. (1998) HSP T-Cell epitopes trigger a spreading regulatory control in a diversified arthritogenic T cell response. Immunol. Rev. 164, 169– 174 25 Menoret, A. et al. (2001) An ER protein implicated in chaperoning peptides to MHC I is an aminopeptidase. J. Biol. Chem. 276, 33313 – 33318 26 Liu, E.S. and Lee, A.S. (1991) Common sets of nuclear factors binding to the conserved promoter sequence motif of two coordinately regulated ER protein genes, GRP 78 and GRP 94. Nucleic Acids Res. 19, 5425 – 5431 27 Yoshida, H. et al. (1998) Identification of the cis-acting ER stress response element responsible for transcriptional induction of mammalian Grps. Involvement of basic leucine zipper transcription factors. J. Biol. Chem. 273, 33741 – 33749

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