Phylogenetic conservation of the molecular and immunological properties of the chaperones gp96 and hsp70

The heat shock proteins (HSP) gp96 and hsp70 have been shown to have a critical role in eliciting adaptive immune responses to cancers and viruses. This role derives from (i) their ability to chaperone antigenic peptides generated in the cells from which the HSP are isolated, and (ii) their capacity to interact with antigen presenting cells (APC) which re-present the HSP-chaperoned peptides in context of MHC I molecules. We have asked whether the immunological properties of HSP extend beyond the mammals to other phyla. We report here the serological, biochemical, genetic, and immunological characterization of the Xenopus gp96. Like mammalian gp96, Xenopus gp96 forms non-covalent complexes with peptides. Immunization with gp96 and hsp70 purified from Xenopus tumors, elicits potent and specific anti-tumor immunity, which is dependent on their ability to chaperone peptides in vivo. An immunogenic peptide chaperoned by the Xenopus gp96 can be processed and presented by mouse APC, to antigen-specific CD8+ T cells of mice. The remarkable conservation of these essential immunological properties of gp96 and hsp70 between amphibians and mammals suggests the importance of HSP in the evolution of the vertebrate immune system.     

CD91: a receptor for heat shock protein gp96

Antigen presenting cells (APCs) can take up exogenous antigenic peptides chaperoned by heat shock protein gp96 and re-present them through the endogenous pathway on their major histocompatibility class I molecules. The high efficiency of this process has been attributed previously to a receptor for gp96 on APCs. The CD91 molecule (also called alpha 2-macroglobulin receptor or the low density lipoprotein-related protein) is shown here to be a cell surface receptor for the heat shock protein gp96. CD91 binds gp96 directly, rather than through another ligand for CD91. The previously known CD91 ligand, alpha 2-macroglobulin, inhibits re-presentation of gp96-chaperoned antigenic peptides by macrophages, as do antibodies to CD91. As gp96 is exclusively intracellular and is released as a result of necrotic but not apoptotic cell death, we propose that CD91 acts as a sensor for necrotic cell death.     

The heat shock protein gp96 induces maturation of dendritic cells and down-regulation of its receptor

Peptides associated with the heat shock protein gp96 induce a specific T cell response against cells from which gp96 is isolated. Recently, we have shown that gp96 binds to a yet unknown receptor present on dendritic cells (DC) and that receptor-mediated uptake is required for cross-presentation of gp96-associated peptides by DC. We now describe that gp96 mediates maturation of DC as determined by up-regulation of MHC class II and CD86 molecules, secretion of the cytokines IL-12 and TNF-alpha and enhanced T cell stimulatory capacity. Heat-denatured gp96 is not able to induce DC maturation and cytokine secretion. Furthermore, we show that mature DC are no longer able to bind gp96 molecules. Hence, the gp96 receptor is down-regulated on mature DC, suggesting that this receptor behaves similar to other receptors involved in antigen uptake like the scavenger receptor CD36, the mannose receptor or the integrins alpha(v)beta(3) and alpha(v)beta(5). Together, our findings provide an additional explanation for the remarkable immunogenicity of gp96 as a cross-priming antigen carrier and direct activator of DC.     

Heat shock proteins 70 and 60 share common receptors which are expressed on human monocyte-derived but not epidermal dendritic cells.

Priming of CTL by means of heat shock proteins (hsp) is dependent on antigen-presenting cells (APC), which present the hsp-associated peptides, via their cell surface MHC class I molecules, toCD8(+) T cells. It has not yet been established how human (hu) hsp70 interacts with the major (hu)APC, the dendritic cells (DC). Here we show that (hu)hsp70 is specifically internalized intoCD14(-), Toll-like receptor 4(-) monocyte-derived (hu)DC by receptor-mediated endocytosis. We further demonstrate that (hu)hsp70 and (hu)hsp60 share the same receptors on (hu)monocyte-derived DC. Both molecules as well as MHC class I molecules are spontaneously internalized and reach the MHC class II-enriched compartments. Finally, freshly isolated (hu) epidermal Langerhans cells (LC), the DC of the skin, as well as CD34(+)-derived LC do not bind hsp60 or hsp70. Given the likely importance of the internalization of hsp70 by APC in the induction of the immune responses, the finding that hsp60 and hsp70 are internalized through the same receptor(s) may explain why microbial hsp60 represents a major T cell antigen. This may rationalize the use of microbial hsp60 to prime immune responses against microbes. The lack of hsp60/70 receptors on epidermal LC raises the crucial question as to whether absence of priming of the skin and mucosal immune systems by hsp-polypeptide complexes could account for some tissue-specific diseases. This work also points to a potential advantage of using monocyte-derived DC in human immunotherapeutic applications of hsp60/70.     

Autoimmune intestinal pathology induced by hsp60-specific CD8 T cells.

Due to their ubiquitous distribution and high degree of structural similarity, heat shock proteins (hsp) are potential target antigens in autoimmune diseases. Here, we describe induction of intestinal inflammation following transfer of hsp60-reactive CD8 T cells into mice. Inflammatory reactions were MHC class I dependent and developed primarily in the small intestine. IFN gamma and TNF alpha, as well as gut-derived hsp60, were elevated at sites of T cell infiltration. Intestinal lesions were drastically reduced in mice lacking receptors for TNF alpha. Pathology also developed in germ-free mice, indicating recognition of host-derived hsp60 by CD8 T cells. This report demonstrates that CD8 T cells with defined antigen specificity cause intestinal inflammation, emphasizing a link between infection and autoimmune disease.     

A proposed mechanism for the induction of cytotoxic T lymphocyte production by heat shock fusion proteins

A 65 kDa mycobacterial heat shock protein (hsp65), fused to a polypeptide that contains an octapeptide (SIYRYYGL) agonist for a particular T cell receptor (2C TCR), stimulated C57BL/6 mice as well as CD4-deficient mice to produce CD8+ cytolytic T lymphocytes (CTL) to the fusion partner's octapeptide. This and other hsp65 fusion proteins but not native hsp65 itself stimulated dendritic cells in vitro and in vivo to upregulate the levels of MHC (class I and II) and costimulatory (B7.2) molecules. The results suggest a mechanism for the general finding that hsp fusion proteins, having fusion partners of widely differing lengths and sequences, elicit CD8 CTL to peptides from the fusion partners without requiring exogenous adjuvants or the participation of CD4+ T cells.     

TAP-translocated peptides specifically bind proteins in the endoplasmic reticulum,including gp96, protein disulfide isomerase and calreticulin

The endoplasmic reticulum (ER) membrane-embedded transporter associated with antigen processing (TAP) associates with peptides in the cytosol and translocates these into the ER lumen. Here, MHC class I molecules bind a subset of these peptides and the remainder is either removed or degraded, or may be retained in the ER in association with other proteins. We have visualized peptide-binding proteins in the ER using radioactive peptides with a photoreactive group. Besides TAP, two proteins were identified as gp96 and protein disulfide isomerase (PDI). Calreticulin, previously found in complex with TAP, only binds glycosylated peptides. In addition, two as yet unidentified, ER luminal glycoproteins (gp120 and gp170) were visualized. The effects of peptide size and sequence on binding to the ER-resident proteins were studied by using partially degenerated peptides with photoreactive side chains. All identified proteins were able to bind peptides within the size range of peptides translocated by TAP, from 8 to more than 20 amino acids. Whereas PDI associated with all peptides tested, gp96 and gp120 showed a clear sequence preference for non-charged amino acids at positions 2 and 9 in 9mer peptides. Thus various ER proteins, other than the MHC class I heterodimer and TAP, are able to interact with peptides albeit with a different substrate selectivity.     

Hsp90–peptide complexes stimulate antigen presentation through the class II pathway after binding scavenger receptor SREC-I

Molecular chaperones such as heat shock protein 90 (Hsp90) have been shown to form complexes with tumor antigens and can be used to prepare anticancer vaccines largely due to this property. Earlier studies had suggested that mice immunized with a molecular chaperone-based vaccine derived from tumors became immune to further vaccination and that both CD8⁺ and CD4⁺ T cells were activated by the chaperone vaccine in a manner dependent on scavenger receptor SREC-I. Here we have investigated mechanisms whereby SREC-I might facilitate uptake of Hsp90-conjugated peptides by APC into the MHC class II pathway for presentation to CD4⁺ T cells. Our studies showed that antigenic peptides associated with Hsp90 were taken up into the class II pathway by a mechanism dependent on SREC-I binding and internalization and presented to CD4⁺ T cells. In addition our studies showed that SREC-I could associate with MHC class II molecules on the cell surface and in intracellular endosomes, suggesting a mechanism involving facilitated uptake of peptides into the MHC class II pathway. These studies in addition to our earlier findings showed SREC-I to play a primary role in chaperone-associated antigen uptake both through cross priming of MHC class I molecules and entry into the class II pathway.     

Carrier-mediated uptake and presentation of a major histocompatibility complex class I-restricted peptide

Antigenic peptides derived from endogenous or viral proteins can associate with class I or class II major histocompatibility complex (MHC) molecules, while exogenous antigens are endocytosed, processed intracellularly and presented on MHC class II molecules. Here we describe a method that allows the presentation of an MHC class I-restricted antigenic peptide on MHC class I molecules, although it was taken up from the outside. The HLA-A2-restricted influenza virus matrix protein-derived peptide (flu, 57–68) was used either in soluble form or coupled via an S-S bridge to transferrin (Tf-flu). Target cells were incubated with flu or Tf-flu and the effective antigen presentation was detected in a cytotoxicity assay using flu peptide-specific, HLA-A2-restricted CD8⁺ cytotoxic T lymphocytes. Sensitization of target cells with Tf-flu required 5 to 10 times higher molar concentrations of peptide compared to sensitization with soluble free peptide. The Tf-flu construct was taken up by the cells via the Tf receptor (CD71) as the binding of Tf-flu was blocked by an excess of Tf. In contrast to the flu peptide, cytotoxicity elicited by Tf-flu was blocked by brefeldin A but not by chloroquine nor inhibitors of intracellular reducing steps, like 1-buthionine-(s, r)-sulfoximine or n-ethylmaleimide. Presentation of the flu peptide derived from Tf-flu construct is not hindered in the mutant T2 cell line, which lacks genes coding for transporter proteins for antigenic peptides (TAP1/TAP2) and proteasomes subunits, suggesting that the processing pathway described in this report may involve TAP-independent steps.     

MHC presentation via autophagy and how viruses escape from it

T cells detect infected and transformed cells via antigen presentation by major histocompatibility complex (MHC) molecules on the cell surface. For T cell stimulation, these MHC molecules present fragments of proteins that are expressed or taken up by the cell. These fragments are generated by distinct proteolytic mechanisms for presentation on MHC class I molecules to cytotoxic CD8⁺ and on MHC class II molecules to helper CD4⁺ T cells. Proteasomes are primarily involved in MHC class I ligand and lysosomes, in MHC class II ligand generation. Autophagy delivers cytoplasmic material to lysosomes and, therefore, contributes to cytoplasmic antigen presentation by MHC class II molecules. In addition, it has been recently realized that this process also supports extracellular antigen processing for MHC class II presentation and cross-presentation on MHC class I molecules. Although the exact mechanisms for the regulation of these antigen processing pathways by autophagy are still unknown, recent studies, summarized in this review, suggest that they contribute to immune responses against infections and to maintain tolerance. Moreover, they are targeted by viruses for immune escape and could maybe be harnessed for immunotherapy.     

Hsp90alpha chaperones large C-terminally extended proteolytic intermediates in the MHC class I antigen processing pathway

Intracellular proteins are degraded in the antigen processing pathway to generate peptide-loaded MHC I complexes (pMHC I) for immune surveillance. The characteristics of the final pMHC I are clear but those of their precursors and their potential binding partners remain poorly defined. By using a unique method to biochemically detect preprocessed ovalbumin-derived antigenic peptides, we find that cells generate large, C-terminally extended proteolytic intermediates that are associated with the alpha isotype of hsp90 chaperone. Knockdown of hsp90alpha expression by siRNA resulted in the loss of these intermediates and decreased presentation of the final pMHC I on the cell surface. Generation of pMHC I was also inhibited by knockdown of the cochaperone CHIP that interacts with heat shock proteins, ubiquitinates their clients, and delivers them to the proteasome. Thus, hsp90alpha can serve as a chaperone for precursors of pMHC I at an early stage in the antigen processing pathway.     

Lysis of interferon-gamma activated Schwann cell by cross-reactive CD8+ alpha/beta T cells with specificity for the mycobacterial 65 kd heat shock protein.

Heat shock protein (hsp) 65 is a major T cell antigen of Mycobacterium leprae. The hsp 65 of M. leprae is nearly identical in M. bovis/M. tuberculosis (greater than 95% protein sequence homology) and surprisingly similar in man (65% protein sequence homology). Recently, we had provided evidence in a murine model that CD8+ T cells recognize and lyse Schwann cells presenting M. leprae antigen in the context of major histocompatibility (MHC) class I gene products. Because murine Schwann cells are class I negative, antigen presentation requires prior stimulation with interferon-gamma (IFN-gamma). CD8+ T cells were activated against tryptic fragments of mycobacterial hsp 65. These T cells recognized epitopes of hsp 65 which had been generated through the cytoplasmic class I processing pathway. They were also capable of lysing Schwann cells which had been activated by IFN-gamma and not primed with nominal hsp 65 peptides. In contrast, T cells activated against tryptic ova peptides only lysed Schwann cells which had been both stimulated with IFN-gamma and primed with ova peptides. Evidence is presented that class I (H-2D) restricted, CD8+ alpha/beta T lymphocytes with specificity for the mycobacterial hsp 65 recognize IFN-gamma-stimulated Schwann cells probably because they are specific for a(n) epitope(s) shared by the bacterial hsp and a host cognate. Activation of autoreactive T cells with specificity to shared epitopes could contribute to nerve damage in tuberculoid leprosy which is characterized by low to absent M. leprae in Schwann cells.     

Melanoma cell necrosis facilitates transfer of specific sets of antigens onto MHC class II molecules of dendritic cells

Vaccine strategies that target dendritic cells (DC) in order to elicit immunity against tumors are the subject of intense research. For the induction and maintenance of anti-tumor immunity, CD4+ helper T cells are often required, which need to see appropriate MHC class II-peptide complexes on DC. So far, it remained widely unclear what type of tumor cells can feed the MHC class II processing pathway of DC with what type of antigens. Here, we report that peptide loading onto MHC class II molecules of myeloid DC is facilitated by melanoma cells undergoing necrotic rather than apoptotic cell death. Importantly, the set of MHC class II-associated peptides induced by necrotic tumor cells differed from those found upon engagement of apoptotic tumor cells. This may be due to the fact that only necrotic cells liberated heat shock proteins, which bind tumor-derived peptides and thereby may promote processing by DC. The potential of DC to activate T cells was kinetically controlled through their antigen receptivity: CD4+ T cells were easily stimulated upon encountering antigen early in DC maturation, whereas antigen capture at later maturation stages favored activation of CD8+ T cells. These findings may aid in designing future vaccination strategies and in identifying novel tumor-specific helper T cell antigens.     

Roles of heat-shock proteins in antigen presentation and cross-presentation.

Heat-shock proteins chaperone antigenic peptides that are generated within cells. Such chaperoning is a part of the endogenous pathway of antigen presentation by MHC class I molecules. In addition, peptides that are chaperoned by heat-shock proteins, or are released by cell stress or death, are taken up by antigen-presenting cells and re-presented by their MHC molecules.     

MHC class I antigen presentation--recently trimmed and well presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is the key to the cellular immune response. Non-self intracellular proteins are processed into short peptides and transported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by several chaperone proteins to form trimeric complex. MHC class I complex loaded with optimised peptides travels to the cell surface of antigen presentation cells to be recognised by T cells. The cells presenting non-self peptides are cleared by CD8 positive T cells. In order to ensure that T cells detect an infection or mutation within the target cells the process of peptide loading and class I expression must be carefully regulated. Many of the cellular components involved in antigen processing and class I presentation are known and their various functions are now becoming clearer.     

Xenopus, a unique comparative model to explore the role of certain heat shock proteins and non-classical MHC class Ib gene products in immune surveillance

The heat shock proteins (HSPs) gp96 and hsp70 can elicit potent anti-tumor responses and as such have significant clinical potential. Besides cytotoxic CD8 T cell (CTLs) effectors, evidence suggests that natural killer (NK) cells and other less well-characterized cell types also play a critical role in HSP-mediated anti-tumor responses. Owing to their high degree of phylogenetic conservation, we have proposed that HSPs are ancestral agents of immune surveillance; and postulated that their immunological properties, if important, should have been conserved during evolution. We are investigating this issue using a unique non-mammalian comparative tumor-immunity model in the frog Xenopus, which allows us to focus on the relationship between HSPs, classical MHC class Ia, and non-classical MHC class Ib molecules. In addition to a transplantable lymphoid tumor in genetically defined cloned Xenopus, we are generating transgenic frogs with inducible or knocked-down (RNAi) gene expression.     

MHC Class Ⅰ Antigen Presentation-Recently Trimmed and Well Presented

Presentation of antigenic peptide to T cells by major histocompatibility complex (MHC) class I molecules is thekey to the cellular immune response.Non-self intracellular proteins are processed into short peptides andtransported into endoplasmic reticulum (ER) where they are assembled with class I molecules assisted by severalchaperone proteins to form trimeric complex.MHC class I complex loaded with optimised peptides travels to thecell surface of antigen presentation cells to be recognised by T cells.The cells presenting non-self peptides arecleared by CD8 positive T cells.In order to ensure that T cells detect an infection or mutation within the targetcells the process of peptide loading and class I expression must be carefully regulated.Many of the cellularcomponents involved in antigen processing and class I presentation are known and their various functions arenow becoming clearer.Cellular & Molecular Immunology.2004;1(1):22-30.     

Generation of cytotoxic T lymphocytes by MHC class I ligands fused to heat shock cognate protein 70

Immunization with gp96 and heat shock cognate protein 70 (hsc70) purified with in vivo bound naturally occurring peptides or bound to synthetic peptides by in vitro reconstitution has been shown to induce peptide-specific cytotoxic T lymphocytes (CTL). In addition, mycobacterial heat shock protein 70 covalently fused to ovalbumin (OVA)-derived fragments has been shown to generate MHC class I-restricted CTL responses. Here, we genetically fused five different CTL epitopes, including peptides derived from Plasmodium yoelii circumsporozoite protein, tumor antigens, HY antigen and OVA, to either the N- or C-terminus of murine hsc70 and expressed the resulting proteins in Escherichia coli. Vaccination with all five fusion proteins induced peptide-specific CTL, indicating that no cognate flanking regions of CTL epitopes are necessary for the immune response. The point of injection was crucial for CTL induction. CD4(+) T cells were not required for the priming of CD8(+) T cells and vaccination with bone marrow-derived dendritic cells pulsed with hsc70 fusion proteins also elicited CTL responses. Furthermore, by using deletion mutants of hsc70, we identified amino acid residues 280-385 of hsc70 as the region most critical for inducing the CTL response.     

Enhanced susceptibility to cytotoxic T lymphocytes without increase of MHC class I antigen expression after conditional overexpression of heat shock protein 70 in target cells

Antigenic peptides have been found associated with heat shock proteins (HSP) including cytoplasmic HSP70 and heat shock cognate protein 70 as well as the endoplasmic reticulum-resident glucose-regulated protein 94. Recently, HSP70 transfection has been reported to increase MHC class I cell surface expression and antigen presentation on mouse melanoma B16 cells (Wells et al., Int. Immunol. 1998. 10: 609). To analyze the effect of HSP70 on MHC class I cell surface expression and lysability of target cells we transfected a human melanoma cell line with the rat Hsp70-1 gene using the Tet-On system for conditional overexpression of HSP70. Induction of HSP70 did not increase cell surface expression of HLA class I molecules in general or individual HLA-A and B antigens in particular. Nonetheless, induction of HSP70 enhanced susceptibility of these cells to lysis by allospecific CTL. The same effect was observed using an HLA-A2-restricted tyrosinase-specific CTL clone after pulsing the tyrosinase-negative target cells with the specific peptide. Thus, HSP70 induction can increase killing by CTL without affecting MHC class I cell surface expression or antigen processing. This effect of HSP70 appears to be different from the commonly found protection exerted by HSP70 against stress like heat shock, and might be mediated by improving CTL-induced apoptosis.     

All the peptides that fit: the beginning, the middle, and the end of the MHC class I antigen-processing pathway

Summary: The end result of the antigen‐processing pathway is the display of peptide‐bound major histocompatibility complex I (pMHC I) molecules. The pMHC I molecules are expressed on the cell surface where they can be surveyed by CD8⁺ T cells for abnormal proteins. MHC I molecules present a large repertoire of peptides that fit perfectly in their binding grooves and represent the otherwise hidden intracellular contents. Many peptides originate as defective ribosomal products in the cytoplasm. In a stepwise manner, the antigen‐processing pathway generates and protects the proteolytic intermediates until they yield the final peptides that can fit the MHC I in the endoplasmic reticulum.