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Keywords:

  • Heat shock protein 70;
  • Tumor immunity;
  • APC;
  • Internalization;
  • Immunogenic peptides

Abstract

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

Extracellular heat shock protein 70 (HSP70) is a potent agent for tumor immunotherapy, which can break tolerance to tumor-associated antigens and cause specific tumor cell killing by cytotoxic CD8+ T cells. The pro-immune effects of extracellular HSP70 are, to some extent, extensions of its molecular properties as an intracellular stress protein. The HSP70 are characterized by massive inducibility after stress, preventing cell death by inhibiting aggregation of cell proteins and directly antagonizing multiple cell death pathways. HSP70 family members possess a domain in the C terminus that chaperones unfolded proteins and peptides, and a N-terminal ATPase domain that controls the opening and closing of the peptide binding domain. These properties not only enable intracellular HSP70 to inhibit tumor apoptosis, but also promote formation of stable complexes with cytoplasmic tumor antigens that can then escape intact from dying cells to interact with antigen-processing cells (APC) and stimulate anti-tumor immunity. HSP70 may be released from tumors undergoing therapy at high local extracellular concentrations, and send a danger signal to the host leading to APC activation. Extracellular HSP70 bind to high-affinity receptors on APC, leading to activation of maturation and re-presentation of the peptide antigen cargo of HSP70 by the APC. The ability of HSP70-peptide complexes (HSP70-PC) to break tolerance and cause tumor regression employs these dual properties as signaling ligand and antigen transporter. HSP70-PC thus coordinately activate innate immune responses and deliver antigens for re-presentation by MHC class I and II molecules on the APC cell surface, leading to specific anti-tumor immunity.

Abbreviations:
HSC:

heat shock cognate

PC:

peptide complexes

Introduction: extracellular HSP70 and its role in anti-tumor immunity

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

HSP70 and other molecular chaperones are finding increasing and effective use in tumor immunotherapy 14. One of the remarkable aspects of these approaches is the variety of contexts in which extracellular HSP are deployed in tumor immunotherapy. HSP70 in some approaches has been employed as an adjuvant in combination with other treatments to activate APC, and break tolerance to tumor-associated antigens, while in others it is used primarily as a carrier protein to deliver extracellular antigens to MHC class I and II molecules on APC 1, 57. The antigens presented by HSP70 can be individualized to patients’ tumors, or may be common to many tumor types 2, 8. The variety of ways in which the HSP70 may be deployed appears to reflect the molecular properties of HSP70, and the multiple ways in which it can interact with APC. Here, we discuss the unique properties of the HSP70 family that make them effective agents in capturing and cross-presenting tumor antigens.

The HSP70 family is intrinsic to cellular life, permitting proteins to perform essential enzymic, signaling and structural functions within the tightly crowded milieu of the cell, and working to avert the catastrophe of protein aggregation during stress 9, 10. HSP70 is induced to extremely high levels by stress along with a cohort of other HSP through powerful transcriptional activation, mRNA stabilization and preferential translation 9. For molecular chaperone function, HSP70 family members are equipped with two major functional domains, including a C-terminal region that binds peptides and denatured proteins, and an N-terminal ATPase domain that controls the opening and closing of the peptide binding domain (for review see 11). These two domains play important roles in the function of HSP70 in tumor immunity, mediating the acquisition of cellular antigens and their delivery to immune effector cells 7, 12. There are at least 12 members of the human HSP70 family, including proteins expressed in the cytoplasm, endoplasmic reticulum (ER) and mitochondria 9, 11, 13. Both constitutive [such as human heat shock cognate (HSC) 73] and stress-inducible HSP family members (human HSP72) exist 13, 14. HSP70 expression becomes dysregulated in many types of cancer, leading to elevated HSP70 levels under non-stress conditions that protect emerging cancer cells from the apoptosis that accompanies many steps in transformation, but also creates an opportunity for vaccine design 13, 1517. A pro-immune function for HSP70 family members may also be inferred from the fact that the immunosuppressive drug 15-deoxyspergualin binds with high affinity to HSP70 proteins 18.

Role of HSP70 family members in antigen processing and capture

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

In most mammalian cells, damaged, denatured or superfluous proteins are degraded through the ubiquitin proteasome pathway to small peptides, subsequent to release and complete breakdown to amino acids (Fig. 1) 19, 20. A fraction of the peptides released from the proteasome are, however, not degraded further and are instead used for immune surveillance purposes 1921. Such peptides are taken up into the ER through an ABC family transport system that involves the transporters associated with antigen processing 1 (TAP1) and TAP2 proteins (Fig. 1). TAP1 and TAP2 form a complex that transports peptides across the ER membrane and delivers them to MHC class I protein complexes 22, 23. Peptides of suitable size and sequence are then bound by MHC class I, transported to the cytoplasm via the vesicular system and displayed on the cell surface where they are subject to surveillance by cytotoxic, CD8+ lymphocytes 24. The purpose of such cell surface display of antigens via the MHC class I pathways permits identification of non-self foreign antigens in microorganism-infected cells, which are then targeted for lysis by CTL. However, this pathway is a potential target in cancer therapy if tumor antigens can be targeted for recognition by CTL 12, 2527. HSP70 family members have the potential to participate in this pathway for antigen processing and presentation due to their ability to bind peptides using the C-terminal polypeptide binding domain 11, 2830. It has been assumed that HSP70 proteins might bind peptides released into the cytoplasm from the proteasome in a similar way to their acquisition by the TAP1/TAP2 complex. This is largely inferred from the fact that HSP70 extracted from tumors can be used to cross-present (see Fig. 1) tumor antigens on APC, which are recognized by specific clones of cytotoxic CD8+ lymphocytes 6, 12, 31. The peptides that bind to the TAP complex and MHC class I, although of varying sequence, have some common properties; such peptides are 8–10 amino acids in length 32, 33. Most MHC class I ligands bind in extended conformation to the MHC binding groove and have an anchor residue at the C terminus that is either hydrophobic or basic, and such sequences are also preferred by TAP 26, 33. MHC class I-peptide binding is of fairly low affinity (KD 10–6 M), but is almost irreversible in the intact MHC class I-peptide complex. The binding domain of HSP70 has common properties including a similar affinity for peptides (KD approx 10–6 M; L. Mannheim-Rodman and S. K. Calderwood, unpublished) and accommodates peptides in extended conformation of 7–15 amino acids 3436. A number of studies have addressed the peptide sequence binding preferences of HSP70 proteins and indicated a similar peptide binding preference for MHC class I, indicating roles for hydrophobic and basic amino acids 30, 3436. Inferences derived from these studies, however, are complicated by the findings that substrate preferences in HSP70 family proteins are determined not solely by the properties of the peptide binding domain of HSP70 itself, but by J-domain proteins, co-factors that localize HSP70 to target molecules and effect binding by modulating the ATPase activity of HSP70 (Fig. 1) 37, 38. A range of J domain proteins with different substrate preferences have been found 11, 3941. Thus, peptide binding by HSP70 proteins may be determined at least partially by the J domain protein partner 38. There is an interesting confluence in properties between MHC class I, MHC class II and HSP70 in that each protein requires the assistance of a co-factor for peptide association; TAP1 is required for MHC class I-peptide binding, HLA-DM for MHC class II and J domain proteins for HSP70. It is currently unknown whether HSP70 binds preferentially to any class of intracellular peptides. However, in an in vivo proteomic study, Grossmann et al. 42 suggest that HSP70 binds to peptides of generally 8–26 amino acids in length. Furthermore, HSP70 favorably interacts to a 5-amino acid core sequence, which likely contains some acidic residues 42. One compelling hypothesis is that a fraction of the intracellular HSP70 binds peptides released into the cytoplasm from the proteasome, protects them from further degradation and passes them to the TAP1/TAP2 complex. It is known that HSP70 proteins function “upstream” in the processing of proteins. HSP70 can bind to damaged target proteins, while associated with CHIP (C terminus of HSC70-interacting protein), which functions as an HSC70 binding ubiquitin E3 ligase, marking the target protein for proteasomal degradation by ligation of its side chains with ubiquitin 4345. This complex is then transported to the proteasome in the company of another HSP70 binding protein Bag-I 44. HSP70 may then perform an “end-around” and accept and chaperone peptides extruded from the exit tunnel of the proteasome.

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Figure 1. Potential role of HSP70 in antigen processing and presentation on MHC class I molecules. HSP70 appears to play at least two roles in antigen processing including: (1) delivery of ubiquitinated proteins to the proteasome, and (2) chaperoning of peptides exiting the proteasome. Chaperoned peptides may thus be protected from endopeptidases, and (3) passed onto TAP1/TAP2. The TAP complex can then load peptides onto the MHC class I complex, and (4) the MHC class I-PC can then traverse the ER and be inserted into the plasma membrane.

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In addition, HSP70 family member HSC73 takes part in another form of antigen processing; HSC73 is evidently required for the processing of external antigens in the lysosome proximal endosome compartments, and is thus involved in the processing of peptides that associate with MHC class II proteins in APC and activate immune response through CD4 T lymphocytes 46. HSC73 co-associates with MHC class II in spherical organelles in macrophages and leads to the presentation of external antigens to MHC class II-restricted T lymphocytes 46. A role for HSC73 in protecting peptides generated by proteolysis in MHC class II-containing endosomes from further breakdown has been suggested, which is similar to the role proposed for the HSP70 family in protecting cytoplasmic peptides 4749. The presence of HSC73 in clathrin-coated pits present in endosomes and lysosomes is well established and HSC73 has been shown by Dice and co-workers 4749 to participate in targeting proteins for degradation through recognition of a consensus sequence (KFERQ). A common theme for HSP70, of mobilizing target proteins towards the sites of degradation while sparing a fraction of the partially digested peptide for immune surveillance, is suggested for both main pathways of protein degradation in the cytoplasm (proteasome) and lysosome. As the lysosomal pathway can be stimulated by serum starvation of tissue culture cells, it may thus play a role in generation of tumor immunity in nutritionally deprived tumor cells, which could potentially be captured in the production of anti-tumor HSP70 based vaccines 48. A major unknown in these studies is, whether, HSP70 proteins capture a nonspecific sample of intracellular peptide or actively select classes of peptides along structural lines, guided perhaps by associated J-domain proteins. In this context it has been shown recently that immunosuppressive drug 15-deoxyspergualin (DSG) specifically binds with high affinity to HSC70 and inhibits interactions that do not require DnaJ co-chaperone activity 18, 50. Although these studies are not definitive, as the properties of DSG are not completely understood, they do indicate a need to understand the mechanisms of molecular chaperone-peptide interactions in antigen processing.

Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

The findings that HSP70 is released into the blood stream and stimulates the production of anti-HSP70 antibodies after a number of pathological conditions was the first indication that extracellular HSP70 may play some physiological role 5153. It has been suggested that circulating HSP70 is (a) derived from cells dying under a number of pathological conditions or (b) actively released from intact cells 53, 54. Approaches to cancer therapy aim to destroy tumors locally by a gradual necrotic mechanism with the aim of producing HSP70 release at high concentrations 1, 4, 55, 56. The rationale behind this approach rests on the high constitutive levels of tumor HSP70 and abundant induction of HSP70 gene transcription (an unusually efficient process) by the stress of therapy 13. The terminal stages of necrosis may also favor antigen uptake by HSP70, as intracellular ATP levels decline and peptides become locked onto the ADP-associated HSP70 57. The extracellular presence of HSP70 can be enhanced by heat shock in prostate cancer cells 58. HSP70-peptide complexes (HSP70-PC) released locally in the tumor microenvironment may thus constitute a “danger signal” and attract and activate APC 59, 60. Such HSP70 is likely released along with other molecular chaperones with pro-immune activity and other danger signals, such as the high-mobility group-1 protein (HMG-b1) (59 and S. Mambula and S. K. Calderwood, in preparation). HSP-PC are, however, not merely danger signals but likely carry samples of tumor-associated antigens, which may be fragments of mutated proteins or re-expressed embryonic or developmental antigens for potential cross-presentation to immune effector cells 61. Injection of HSP70 or multi-chaperone based vaccines may mimic these effects 2, 62. These mechanisms are, to some extent an over-simplification, as tumor cell populations under therapy rarely die from a single form of cell death and necrosis, apoptosis and other pathways likely occur in concert. However, it has also been shown that HSP70 overexpression in the presence of slow necrotic death is an extremely potent approach to breaking tolerance and induction of specific immune destruction of tumors 1.

As mentioned above, little is known about how HSP70, a protein with no signal sequence for secretion, exits cells by mechanisms other than escape from cells undergoing necrotic lysis. However, Dice et al.63, 64 showed that proteins can be released from a late endosomal lysosomal location where HSC70 participates in protein degradation (HSC70 is the murine homologue of human HSC73). In addition, HSC70 can associate with the transferrin receptor in exosomes, suggesting a potential exosomal mechanism for HSC70 release 65. Secretion and/or detergent-resistant microdomain fractions with associated HSP70 have also been observed in epithelial cells in normal conditions, and a lipid raft-based mechanism has been suggested for membrane delivery and release of HSP70. 66. Further studies are currently needed to define the mechanisms of HSP70 release from cells under stress or non-toxic conditions.

In addition to release into the tumor microenvironment, HSP70 also becomes associated with the extracellular face of the plasma membrane 6770. Cell surface HSP70-PC in tumor cells could interact with receptors on APC in this context, or be released into the microenvironment. A number of studies show that HSP70-PC associated with the cell surface of tumors interacts with T cell receptors on CD3+, CD4, CD8 natural killer (NK) cells and suggest that cell surface HSP70 can present processed antigen to such receptors 70. It may thus be significant that a granzyme B-mediates NK cell killing by mechanisms involving membrane-bound HSP70-PC of tumor cells, thus supporting a significant role of HSP70 in NK cell function 69. Recent attempts to characterize the global profile of the cell surface proteome of cancer cells also reveal the abundant presence of at least two HSP70 members on the cancer cell surface 71.

Binding of HSP70-PC to the cell surface of APC

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

HSP-PC released from tumor cells undergoing necrosis will be of necessity diluted as they enter the interstitial fluid, and APC response to such low concentrations of HSP70-PC implies the existence of high-affinity receptors. Extracellular HSP70-PC have at least two major enhancing effects on APC function: (i) induction of innate immune function through activation of the APC maturation program 12, 72, and (ii) induction of the adaptive immune response, leading to the transport of peptide antigens into APC cells and delivery to MHC molecules 6. Whether these two effects are achieved through binding to one or more cell surface receptors is not clear. A major emphasis has therefore been placed on identifying HSP70 receptors on the APC surface. Previous studies indicated HSP-PC-mediated uptake of tumor antigens through receptor-mediated endocytosis because of the low (nanomolar) concentrations of HSP-PC involved and the saturability of the process 7375. Although the nature of the high affinity receptors for HSP70-PC binding to the APC cell surface has still not been fully defined, four main classes of cell surface structure have been suggested, including: (i) the CD14/TLR 2/4 complex, (ii) the CD91 receptor, (iii) CD4O, and (iv) scavenger receptors, most notably the lectin-like receptor for oxidized low-density lipoprotein (also known as LOX-1) 6, 57, 73, 76. The CD14/TLR 2 or 4 pathway, which is known to mediate cellular responses to bacterial danger signals, such as LPS and peptidoglycans, appears to play a role in activation of the innate immune response gene (TNF-α, IL-1β, IL-6) expression program by HSP70, 12, 73, 77, 78. However, there is currently only evidence for a functional interaction between HSP70, CD14, TLR2 and TLR4. Others have suggested a role for binding of HSP70 and other immune-effective stress proteins to APC cells through CD91, the α2-macroglobulin receptor, and have proposed this structure as the sole receptor for HSP70-PC uptake and presentation to MHC molecules in immune effector cells 79. In addition, a recent study by Becker et al. 57 also shows evidence for binding of HSP70-PC to another receptor, the TNF receptor family protein CD40. This interaction is stabilized by the presence of ADP and peptide bound to HSP70 57. However, other studies indicate instead that only mycobacterial HSP70 is able to bind CD40 and that mammalian HSP70-PC does not directly interact with CD40, casting doubt on a role for CD40 as a direct receptor for mammalian HSP70 76, 80. Finally, Delneste et al.57 have shown a significant role for the c-type lectin LOX-1 in HSP70 binding and antigen cross-presentation, and this receptor seems a convincing candidate for mediation of immune effects of HSP70. We have directly compared HSP70 binding to each class of receptor after overexpressing them in non-APC cells 81. Our studies indicate that only the scavenger receptors, as exemplified by LOX-1, bind with high avidity to mammalian HSP70, while TLR2, TLR4, CD40 or CD91 expression failed to support binding 81.

Clearly, despite these promising findings, much has to be learned regarding the nature of the proximal HSP70 receptor or receptors responsible for the immune effects of HSP70. One perplexing question is how cells of the immune system recognize the wide range of stress proteins from a number of unrelated families evidently capable of activating APC. One possibility is that the peptide component of the HSP70-PC complex plays an important role in recognition by HSP70 receptors, in a similar way to recognition of MHC-PC by T cell receptors. In this context, the finding that cell surface expressed HSC70 can interact with the rat Vδ6 T cell receptor on NK cells and present peptides to these receptor cells may be of significance for HSP70-APC interactions 70, 82. Another possibility is that the nature of the adenosine phosphate moiety bound to HSP70 (ATP vs. ADP) may be important in the interaction with a receptor. However, scavenger receptors can bind a wide array of unrelated ligands by unknown mechanisms (and have been described as “molecular flypaper”) making the promiscuous association with molecular chaperones from different families perhaps more understandable 83. As will be gathered, understanding of HSP70 recognition by cell surface structures in APC is still not completely understood and may await a more complete determination of authentic HSP70 receptors.

Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

HSP70-induced APC maturation and pro-inflammatory signaling

Binding of HSP70-PC is succeeded by the activation of transmembrane signaling mechanisms, internalization and the delivery of the peptide cargo of HSP70 to MHC molecules. Whether a single receptor carries out these processes or whether multiple receptors mediate these effects is not clear.

HSP70 can initiate a potent innate immune response resulting in APC maturation as well as a pro-inflammatory response (Fig. 2) 1, 2, 84. Dendritic cell (DC) maturation requires the activation of a gene expression program that leads to production of co-stimulatory molecules including CD40, OX40L, B7.1 and B7.2 on the cell surface for effective interaction with CD8+ T lymphocytes (Fig. 2) 85. Direct HSP70 binding to the CD40 receptor would be an attractive hypothesis as ligation of CD40 plays an important role in DC maturation 85. In fact, a key functional role for CD40 in breaking tolerance in an autoimmune form of diabetes by HSP70 has been demonstrated 72. However, as mentioned earlier, it is not clear whether direct HSP70-CD40 binding to the APC cell surface is or is not involved. It seems likely that that HSP70-induced DC maturation involves CD40 and/or CD40L up-regulation, and probably requires activation of the transcription factor NF-κB 86. Most of the genes involved in DC maturation require the activation of NF-κB and, therefore, receptors that are involved in DC maturation by HSP70 likely cause downstream activation of NF-κB (Fig. 2) 86. Some intriguing studies have shown that extracellular HSP70 induces NF-κB through the activation of the CD14/TLR signaling pathway in a CD14-dependent manner in APC 73, 87. This results in the expression of the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 as well as co-stimulatory molecule B7.1 73, 87. Although the role of this pathway in the innate immune response to HSP70 is not clear, it can be activated equally effectively by HSP70-PC and free HSP70 88. However, this effect seems indirect since there is no evidence for high-affinity binding of extracellular HSP70 to TLR 2, TLR 4 or CD14 81. In addition, some skepticism has been directed at the role of CD14 or TLR due to potential HSP70 contamination by endotoxin in these effects 89. In addition, other potential HSP70 receptors are coupled to NF-κB, and LOX-1 has recently been shown to activate the NF-κB pathway in endothelial cells stimulated by oxidized-LDL, while ligation of CD40 can lead to NF-κB activation in APC 90, 91. Oxidized-LDL can also induce CD40 expression through LOX-1 in the same cells suggesting a potential role for LOX-1 in APC maturation by HSP70 92.

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Figure 2. Mechanism of HSP70-induced innate immune response (pro-inflammatory and co-stimulatory molecule expression). ATP- or ADP-bound HSP70 binds to signaling receptor(s) present on APC. All signaling pathways converge to the activation of the NF-κB pathway. The result of this activation is the stimulation of cytokine (TNF-α, IL-1β and IL-6), co-stimulatory molecule (B7.1, B7.2, CD40 and MHC class II) and nitric oxide (NO) release.

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HSP70-PC internalization by APC and peptide association with cell surface MHC molecules

A receptor-mediated mechanism appears to be involved in antigen cross-presentation by HSP 7, 93. After binding HSP70-PC, such a receptor would mediate internalization of complexes and delivery of the peptide cargo to MHC class I and perhaps MHC class II molecules. Receptor-mediated protein internalization is mainly governed through specific motifs located in the cytoplasmic tails of receptors which engage the protein coats of receptor-rich regions of membranes that ultimately give rise to the coated vesicles that mediate internalization 94, 95. Tyrosine-based or di-leucine-based sorting signals within the C termini of receptors represent the sorting codes for the vast majority of receptor endocytosis and target liganded receptors to specific organelles 94, 95. Tyrosine-based sorting signals usually involve short consensus internalization motifs such as YXXΘ (Θ represents an amino acid with a bulky hydrophobic side chain, X is any amino acid) and NPXY 94, 95. Each motif is recognized by components of protein coats and is targeted to specific compartments such as endosomes 94, 95. YXXΘ and di-leucine motifs are recognized by the adaptor protein complexes (AP-1,2) and NPXY motif through clathrin, AP-2 and Dab2 94, 95. In some circumstances, phosphorylation of the single tyrosine present on these motifs is observed, and can regulate the signal recognition to endocytic component as well as the initiation of specific signal transduction pathways 94, 95. Among the proposed HSP70 receptors, only CD40 has been shown directly to take part in HSP70-PC uptake 57. Nevertheless, CD91 and LOX-1 also mediate internalization of various extracellular ligands 9698. Since both receptors are involved in HSP70-mediated antigen cross-presentation, CD91 and LOX-1 may also possess the capacity to facilitate internalization of HSP70-PC. Interestingly, the uptake of one CD91 ligand, receptor-associated protein (RAP), is mediated mainly through a YXXL motif 98. While it has been shown that LOX-1 can internalize oxidized LDL, the mechanisms that underlie LOX-1 triggering of oxidized LDL uptake are not clear 99. However, some information regarding LOX-1-mediated internalization may be partially deduced from ligand uptake studies of the closely related c-type lectin receptor Dectin-1. Dectin-1 utilizes the YXXL motif to endocytose yeast zymosan 100 and thus, by implication, YXXL motif could play a role in HSP70-PC uptake.

Following receptor-induced extracellular HSP70-PC internalization by APC, complexes may traffic through a number of intracellular compartments, leading to peptide release into the cytoplasm and re-presentation on the cell surface associated with MHC proteins (Fig. 3). HSP70-PC may be taken up by the endocytic pathway and lead to peptide presentation to MHC class II molecules by the standard extracellular pathway of antigen presentation 101. In addition, HSP70 can also deliver peptides to MHC class I molecules through the re-presentation or cross-presentation pathways 102. After internalization of HSP70-PC, peptides may be released into the cytoplasm and processed, as previously mentioned in the Introduction to MHC class I molecules by the classical antigen re-presentation pathways. However, cross-presentation may proceed through a newly discovered alternative route involving a specialized MHC class I structure known as the ER/phagosome fusion compartment, which seems auto-sufficient to induce antigen cross-presentation (Fig. 3; discussed in 103). HSP70 and HSP90 have been located in this compartment, supporting a potential role for HSP in this pathway of antigen cross-presentation 103. The possibility that HSP70 receptor(s) can also co-localize with components located in this structure during HSP70-mediated cross-presentation is thus worth exploring. Intriguingly, cross-presentation of cell-associated antigens to MHC class I is significantly impaired in cells devoid of the hsf1 gene 104. In this context, reduction of intracellular HSP70 and HSP90 content by hsf1 inactivation correlates with loss of antigen-mediated cross-presentation, re-enforcing a key role for HSP70 and/or HSP90 in this process 104. So far, no definitive studies have addressed the pathways of HSP70-PC internalization involved in tumor antigen cross-presentation. Nonetheless, the internalization pathway of another HSP family member, gp96, has been studied in the past few years 93, 105. Gp96-PC is rapidly internalized, after interacting with undefined cell surface receptor(s), into a pre-endosomal compartment 93, 105. Such endocytosed Gp96 was found to co-localize with FcR and MHC class I but not with other receptors such as CD91, transferrin, rab5a or lysosomal markers such as LAMP-1 or LAMP-2 74, 105.

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Figure 3. Proposed model for HSP70-mediated antigen cross-presentation (adaptive response). A repertoire of HSP70-associated antigens (HSP70-PC) elicits specific adaptive immune responses. HSP70-PC bound to endocytic receptors is internalized by APC through the activation of an intracellular signaling module. In APC endocytic compartment, HSP70-PC is transported to the proteasome or the ER/phagosome compartment where the peptide is processed and transferred onto MHC class I molecule for representation on the cell surface. The MHC class I/antigen complex is recognized by cytotoxic T lymphocyte bearing an antigen-specific T cell receptor.

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Conclusion

  1. Top of page
  2. Abstract
  3. Introduction: extracellular HSP70 and its role in anti-tumor immunity
  4. Role of HSP70 family members in antigen processing and capture
  5. Escape of HSP70 proteins from the cytoplasm; insertion onto the membrane and release into the external milieu
  6. Binding of HSP70-PC to the cell surface of APC
  7. Internalization of HSP70-PC by APC, activation of transmembrane signaling cascades and presentation of peptides to cell surface MHC molecules
  8. Conclusion
  9. Acknowledgements

Extracellular HSP70-PC is an effective agent for breaking tolerance to tumor antigens and eliciting specific CD8+ tumor-specific immunity. Targeting HSP70 is thus a highly effective approach to tumor immunotherapy. The pro-immune effects of extracellular HSP70 appear to reflect its ability to act as both a signaling ligand to induce APC maturation and inflammatory processes, and as a carrier protein to chaperone and transport tumor antigens for re-presentation by APC. However, most of the molecular mechanisms that underlie these effects are not completely understood and further progress in the field will likely require a clearer understanding of HSP70-PC release from tumor cells and interaction with immune effector cells. Areas that require development include: the role of HSP70 and co-chaperones in antigen processing in tumor cells, mechanisms of release of HSP70-PC from tumors, receptor-mediated uptake of HSP70-PC by APC, and mechanisms of HSP70-mediated re-presentation of tumor antigens to immune effector cells. Understanding these processes may permit us to manipulate more effectively the use of HSP70 in tumor immunotherapy.

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