Purification of recombinant and endogenous HSP70s

Heat shock proteins (HSPs) are powerful immunogens against the antigenic peptides they chaperone. The antigenic peptides are MHC I-binding peptides and their elongated precursors derived from tumor antigens, viral antigens, minor histocompatibility antigens, or model antigens. HSP-peptide complexes can immunize against tumors and pathogen-infected cells. Remarkably, HSPs do not immunize after elution of the peptides they chaperone, demonstrating that HSPs are not immunogenic per se, whereas HSP-peptide complexes are. Additionally, HSPs activate professional antigen presenting cells (APC) through specific receptor(s) to stimulate secretion of pro-inflammatory cytokines, up-regulation of co-stimulatory molecules and activation of dendritic cells. The mechanistic exploration of the role of the HSPs on the innate and adaptive component of the immune system requires their isolation in large quantity. On one hand, isolation of naturally formed HSP-peptide complexes is key to study their specific immunogenicity. On the other hand, purification of HSPs free of endotoxin contamination is an absolute requirement for the analysis of their ability to activate APC in vitro. This chapter describes a convenient and fast method of purification of endogenous and recombinant HSP of 70 kDa (HSP70) that addresses these two considerations.     

The role of heat shock proteins and their receptors in the activation of the immune system

Heat shock proteins (HSPs) have been described as potent tumor vaccines in animal models and are currently studied in clinical trials. The underlying immune response relies on immunogenic peptides that the HSPs have acquired intracellularly by interfering with the classical antigen processing pathways. There have been numerous reports shedding light on how HSPs are able to gain this function and a number of important requirements for HSP-mediated specific immunity have been described: first, the ability of HSPs to bind immunogenic peptides. Second, the acquisition of HSPs by specialized antigen presenting cells with efficient antigen processing pathways capable of inducing cellular immune responses. Third, the existence of specific receptors on the surfaces of antigen presenting cells, allowing efficient and rapid uptake of HSP-peptide complexes from the extracellular fluid. And fourth, the ability of heat shock proteins to activate antigen presenting cells, enabling the latter to prime cytotoxic T cell responses against the peptides associated to HSPs.     

Heat shock protein-peptide interaction as the basis for a new generation of vaccines against cancers and infectious diseases

Heat shock proteins (HSPs) are associatedin vivo with the entire repertoire of peptides (antigenic and otherwise) generated within that cell. Immunization with such HSP-peptide complexes is unusually efficient in eliciting cellular immune responses against the antigenic peptides associated with the HSPs. This broad and general principle is the basis for a new generation of vaccines against cancers and infectious diseases and circumvents the need for identification of the T cell epitopes for any given cancer or infectious agent.     

Structure and function: heat shock proteins and adaptive immunity

Heat shock proteins (HSPs) have been implicated in the stimulation and generation of both innate and adaptive immunity. The ability of HSPs to bind antigenic peptides and deliver them to APCs is the basis of the generation of peptide-specific T lymphocyte responses both in vitro and in vivo. The different HSP families are genetically and biochemically unrelated, and the structural basis of peptide binding and the dynamic models of ligand interaction are known only for some of the HSPs. We examine the contribution of HSP structure to its immunological functions and the potential "immunological repertoire" of HSPs as well as the use of biophysical techniques to quantify HSP-peptide interactions and optimize vaccine design. Although biochemical evidence for HSP-mediated endogenous processing of Ag has now emerged, the issue of whether HSP-peptide complexes act as physiological sources of Ag in cross-presentation is controversial. We assess the contribution of biochemical studies in this field.     

Dendritic cells pulsed with gp96-peptide complexes derived from human hepatocellular carcinoma (HCC) induce specific cytotoxic T lymphocytes

Dendritic cells (DCs) are one of the most potent antigen-presenting cells (APCs) capable of activating immune responses. Different forms of tumor antigens have been used to load DCs to initiate tumor-specific immune responses. Heat shock proteins (HSPs) are considered natural adjuvants which have the ability to chaperone peptides associated with them presented efficiently by interaction with professional APCs through specific receptors. In the present study, we used HSP, gp96-peptide complexes, derived from human hepatocellular carcinoma (HCC) cells as antigens for pulsing DCs. We found that gp96-peptide complexes derived from HCC cells induced the maturation of DCs by enhancing expression of human leukocyte antigen class II, CD80, CD86, CD40, and CD83. The matured DCs stimulated a high level of autologous T cell proliferation and induced HCC specific cytotoxic T lymphocytes, which specifically killed HCC cells by a major histocompatability complex (MHC) class I restricted mechanism. These findings demonstrate that DCs pulsed with gp96-peptide complexes derived from HCC cells are effective in activating specific T cell responses against HCC cells.     

Cutting edge: receptor-mediated endocytosis of heat shock proteins by professional antigen-presenting cells

Immunization with heat shock proteins (HSPs) induces Ag-specific CTL responses. The specificity of the immune response is based on peptides associated with HSPs. To investigate how exogenous HSP/peptide complexes gain access to the MHC class I-restricted Ag presentation pathway, we incubated the monocytic cell line P388D1 and the dendritic cell line D2SC/1 with gold-labeled HSPs gp96 and HSC70. We show that HSPs bind specifically to the surface of these APCs and are internalized spontaneously by receptor-mediated endocytosis, demonstrating the existence of specific receptors for HSPs on these cells. In addition, we observe colocalization of internalized HSPs and surface MHC class I molecules in early and late endosomal structures. These findings provide possible explanations for the immunogenicity of HSP/peptide complexes and for the transfer of HSP-associated peptides onto MHC class I molecules.     

Heat shock proteins and the antitumor T cell response

Heat shock proteins (HSP) have been shown to participate in the antitumor T cell response. First, HSP play a crucial role in the intracellular pathway for antigen processing where HSP can make complexes with a broad spectrum of cellular proteins and peptides through their chaperone functions. In this pathway, macrophages are required for processing the chaperoned peptides to make stable molecules with the major histocompatibility complex (MHC) class I molecules, even when HSP-peptide complexes are exogenously administered. Through this pathway, vaccination with HSP-peptide complexes is thus able to elicit the response of CD8⁺ T cells specific for the chaperoned peptides. These findings suggest an essential role of HSP in ‘cross-priming’ and their usefulness for antitumor vaccination with tumor peptides. Second, HSP have been suggested to be expressed on the cell surface by transformation and, in addition, to function as antigen-presenting molecules for double negative T cells. Third, HSP derived from tumor cells have reportedly been recognized by T cells with either T cell receptor (TCR)-αβ or TCR-γδ. These lines of evidence therefore indicate that HSP may be potentially promising target molecules for antitumor T cell immunotherapy.     

Immunotherapy of Tumors with α2-Macroglobulin-Antigen Complexes Pre-Formed In Vivo

The cell surface receptor CD91/LRP-1 binds to immunogenic heat shock proteins (HSP) and α₂M ligands to elicit T cell immune responses. In order to generate specific immune responses, the peptides chaperoned by HSPs or α₂M are cross-presented on MHC molecules to T cells. While the immunogenic HSPs naturally chaperone peptides within cells and can be purified as an intact HSP-peptide complex, the peptides have had to be complexed artificially to α₂M in previous studies. Here, we show that immunogenic α₂M-peptide complexes can be isolated from the blood of tumor-bearing mice without further experimental manipulation in vitro demonstrating the natural association of tumor antigens with α₂M. The naturally formed immunogenic α₂M-peptide complexes are effective in prophylaxis and therapy of cancer in mouse models. We investigate the mechanisms of cross-presentation of associated peptides and co-stimulation by APCs that interact with α₂M. These data have implications for vaccine design in immunotherapy of cancer and infectious disease.     

Bacterial heat shock proteins promote CD91-dependent class I MHC cross-presentation of chaperoned peptide to CD8+ T cells by cytosolic mechanisms in dendritic cells versus vacuolar mechanisms in macrophages

APCs process mammalian heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC (MHC-I) molecules to CD8(+) T cells. HSPs are also expressed in prokaryotes and chaperone microbial peptides, but the ability of prokaryotic HSPs to contribute chaperoned peptides for Ag presentation is unknown. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-I presentation by both murine macrophages and dendritic cells. HSP-enhanced MHC-I peptide presentation occurred only if peptide was complexed to the prokaryotic HSP and was dependent on CD91, establishing CD91 as a receptor for prokaryotic as well as mammalian HSPs. Inhibition of cytosolic processing mechanisms (e.g., by transporter for Ag presentation deficiency or brefeldin A) blocked HSP-enhanced peptide presentation in dendritic cells but not macrophages. Thus, prokaryotic HSPs deliver chaperoned peptide for alternate MHC-I Ag processing and cross-presentation via cytosolic mechanisms in dendritic cells and vacuolar mechanisms in macrophages. Prokaryotic HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD8(+) T cells.     

Glycoprotein 96 can chaperone both MHC class I- and class II-restricted epitopes for in vivo presentation, but selectively primes CD8+ T cell effector function

The ability of mature T lymphocytes to develop effector capacity after encounter with cognate Ag is generally dependent upon inflammatory signals associated with infection that induce dendritic cell activation/maturation. These inflammatory signals can derive directly from pathogens or can be expressed by host cells in response to infection. Heat shock proteins (HSPs) are a class of host-derived inflammatory mediators that perform the dual function of both chaperoning MHC class I-restricted epitopes into the cross-presentation pathway of DCs and inducing the activation/maturation of these DCs to allow priming of cognate CD8(+) T cell effector responses. Although the ability of HSPs to elicit effector CD8 cell responses has been well established, their potential to prime CD4 cell effector responses has been relatively unexplored. In the current study we compared the ability of the endoplasmic reticulum-resident HSP gp96 to prime CD4 vs CD8 cells using TCR transgenic adoptive transfer systems and soluble gp96-peptide complexes. As expected, gp96 facilitated the cross-presentation of a class I-restricted peptide and priming of effector function in cognate CD8 cells. Interestingly, gp96 also facilitated the in vivo presentation of a class II-restricted peptide; however, the resulting CD4 cell response did not involve the development of effector function. Taken together, these data suggest that gp96 is an inflammatory mediator that selectively primes CD8 cell effector function.     

Heat shock proteins and DNA repair mechanisms: an updated overview

Heat shock proteins (HSPs), also known as molecular chaperones, participate in important cellular processes, such as protein aggregation, disaggregation, folding, and unfolding. HSPs have cytoprotective functions that are commonly explained by their antiapoptotic role. Their involvement in anticancer drug resistance has been the focus of intense research efforts, and the relationship between HSP induction and DNA repair mechanisms has been in the spotlight during the past decades. Because DNA is permanently subject to damage, many DNA repair pathways are involved in the recognition and removal of a diverse array of DNA lesions. Hence, DNA repair mechanisms are key to maintain genome stability. In addition, the interactome network of HSPs with DNA repair proteins has become an exciting research field and so their use as emerging targets for cancer therapy. This article provides a historical overview of the participation of HSPs in DNA repair mechanisms as part of their molecular chaperone capabilities.     

Development of cancer vaccines using autologous and recombinant high molecular weight stress proteins

High molecular weight heat shock proteins (HSPs), hsp110 and grp170, derived from cancer cells have been previously shown to elicit tumor-specific immunity. This phenomenon is attributed to the antigenic peptides associated with the HSPs. Based on the unique chaperoning properties of these HSPs, a new vaccination strategy has been recently developed to elicit antigen-specific antitumor immunity. This approach utilizes tumor-associated antigens naturally complexed to these highly efficient molecular chaperones under heat shock conditions. This chapter focuses on the methodologies of these two vaccine strategies: I. purification of hsp110 and grp170 from tumor tissue or cell lines; II. generation and characterization of in vitro HSP-antigen complexes by heat shock using recombinant HSPs derived from a baculovirus protein expression system.     

Limited Promiscuity of HLA-DRB1 Presented Peptides Derived of Blood Coagulation Factor VIII

The formation of inhibitory antibodies directed against coagulation factor VIII (FVIII) is a severe complication in the treatment of hemophilia A patients. The induction of anti-FVIII antibodies is a CD4⁺ T cell-dependent process. Activation of FVIII-specific CD4⁺ T cells is dependent on the presentation of FVIII-derived peptides on MHC class II by antigen-presenting cells. Previously, we have shown that FVIII-pulsed human monocyte-derived dendritic cells can present peptides from several FVIII domains. In this study we show that FVIII peptides are presented on immature as well as mature dendritic cells. In immature dendritic cells half of the FVIII-loaded MHC class II molecules are retained within the cell, whereas in LPS-matured dendritic cells the majority of MHC class II/peptide complexes is present on the plasma membrane. Time-course studies revealed that presentation of FVIII-derived peptides was optimal between 12 and 24 hours after maturation but persisted for at least 96 hours. We also show that macrophages are able to internalize FVIII as efficiently as dendritic cells, however FVIII was presented on MHC class II with a lower efficiency and with different epitopes compared to dendritic cells. In total, 48 FVIII core-peptides were identified using a DCs derived of 8 different donors. Five HLA-promiscuous FVIII peptide regions were found – these were presented by at least 4 out of 8 donors. The remaining 42 peptide core regions in FVIII were presented by DCs derived from a single (30 peptides) or two to three donors (12 peptides). Overall, our findings show that a broad repertoire of FVIII peptides can be presented on HLA-DR.     

Bacterial heat shock proteins enhance class II MHC antigen processing and presentation of chaperoned peptides to CD4+ T cells

APCs process heat shock protein (HSP):peptide complexes to present HSP-chaperoned peptides on class I MHC molecules, but the ability of HSPs to contribute chaperoned peptides for class II MHC (MHC-II) Ag processing and presentation is unclear. Our studies revealed that exogenous bacterial HSPs (Escherichia coli DnaK and Mycobacterium tuberculosis HSP70) delivered an extended OVA peptide for processing and MHC-II presentation, as detected by T hybridoma cells. Bacterial HSPs enhanced MHC-II presentation only if peptide was complexed to the HSP, suggesting that the key HSP function was enhanced delivery or processing of chaperoned peptide Ag rather than generalized enhancement of APC function. HSP-enhanced processing was intact in MyD88 knockout cells, which lack most TLR signaling, further suggesting the effect was not due to TLR-induced induction of accessory molecules. Bacterial HSPs enhanced uptake of peptide, which may contribute to increased MHC-II presentation. In addition, HSPs enhanced binding of peptide to MHC-II molecules at pH 5.0 (the pH of vacuolar compartments), but not at pH 7.4, indicating another mechanism for enhancement of MHC-II Ag processing. Bacterial HSPs are a potential source of microbial peptide Ags during phagocytic processing of bacteria during infection and could potentially be incorporated in vaccines to enhance presentation of peptides to CD4+ T cells.     

Strategy for Identifying Dendritic Cell-Processed CD4(+) T Cell Epitopes from the HIV Gag p24 Protein

Mass Spectrometry (MS) is becoming a preferred method to identify class I and class II peptides presented on major histocompability complexes (MHC) on antigen presenting cells (APC). We describe a combined computational and MS approach to identify exogenous MHC II peptides presented on mouse spleen dendritic cells (DCs). This approach enables rapid, effective screening of a large number of possible peptides by a computer-assisted strategy that utilizes the extraordinary human ability for pattern recognition. To test the efficacy of the approach, a mixture of epitope peptide mimics (mimetopes) from HIV gag p24 sequence were added exogenously to Fms-like tyrosine kinase 3 ligand (Flt3L)-mobilized splenic DCs. We identified the exogenously added peptide, VDRFYKTLRAEQASQ, and a second peptide, DRFYKLTRAEQASQ, derived from the original exogenously added 15-mer peptide. Furthermore, we demonstrated that our strategy works efficiently with HIV gag p24 protein when delivered, as vaccine protein, to Flt3L expanded mouse splenic DCs in vitro through the DEC-205 receptor. We found that the same MHC II-bound HIV gag p24 peptides, VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ, were naturally processed from anti-DEC-205 HIV gag p24 protein and presented on DCs. The two identified VDRFYKTLRAEQASQ and DRFYKLTRAEQASQ MHC II-bound HIV gag p24 peptides elicited CD4(+) T-cell mediated responses in vitro. Their presentation by DCs to antigen-specific T cells was inhibited by chloroquine (CQ), indicating that optimal presentation of these exogenously added peptides required uptake and vesicular trafficking in mature DCs. These results support the application of our strategy to identify and characterize peptide epitopes derived from vaccine proteins processed by DCs and thus has the potential to greatly accelerate DC-based vaccine development.     

Molecular chaperones: proposal of a systematic computer-oriented nomenclature and construction of a centralized database

Molecular chaperones are a wide group of unrelated protein families whose role is to assist others proteins. Comparably, under environmental stress, stress proteins behave as biocatalysts of protein stabilization. Stress proteins include a large class of proteins that were originally termed heat shock proteins (HSPs) due to their initial discovery in tissues exposed to elevated temperatures. Many, but not all, stress proteins and HSPs are molecular chaperones. Moreover, not all HSPs are derivable from stress. HSPs are structurally diversified by the contribution of various domains having specific roles. HSPs have been grouped, mainly on the basis of their molecular masses, into specific families that include small HSPs (sHSPs)/alpha-crystallins, HSP10s, HSP40s, HSP60s, HSP70s, HSP90s, HSP100s and HSP110s. The names of these major families are historical artefacts with limited information content. Using the current databases, names and proteic domains of many molecular chaperones in different species were analyzed. Although traditional names of HSPs are trivial, it is unrealistic to suggest replacing them, because they are preferred and widely used. Here we suggest that these traditional names be chaperoned, in silico, by a systematic nomenclature. Thus, for example, with the same intent of use of [trioxygen: O3] for ozone, we propose here C7HSP70[Ehsa]ER-P11021 for GRP78 (78 kDa endoplasmic Human molecular chaperone in HSP70 superfamily with P11021 as its accession number in the database of the National Center for Biotechnology Information (NCBI)). The proposed systematic computer-oriented naming and classification method is designed for HSPs and also their partners based on the number of amino acids, domain structure, phylogenetic domain, localization in the cell and accession number as stated in the NCBI. Arabidopsis thaliana was analyzed as a model, because it contains a large number of various HSPs localized in several organelles. Overall, this naming system helps in building, optimizing and managing a novel online database entirely devoted to HSPs. The purported taxonomy, coupled with the newly constructed database, can contribute to studies involving large amounts of stored data on HSPs.     

Heat shock protein 70 (Hsp70): membrane location, export and immunological relevance

Stress or heat shock proteins (HSPs) are remarkably conserved in all living organisms. Their expression is induced in response to a variety of physiological and environmental insults. In the cytosol these proteins play an essential role as molecular chaperones by assisting the correct folding of nascent and stress-accumulated misfolded proteins, preventing protein aggregation, transport of proteins, and supporting antigen processing and presentation. Following stress, intracellularly located HSPs fulfill protective functions and thus prevent lethal damage. In contrast, membrane-bound or extracellularly located HSPs act as danger signals and elicit immune responses mediated either by the adaptive or innate immune system. Here, HSPs act as carriers for immunogenic peptides, induce cytokine release or provide recognition sites for natural killer (NK) cells. This article will discuss methods for the detection of membrane-bound and extracellular HSPs and methods for determining their immunological functions.     

Developing strategies to enhance and focus humoral immune responses using filamentous phage as a model antigen

Filamentous bacteriophage are commonly used as immunogenic carriers for peptides and proteins displayed on the phage surface. Previously, we showed that immunization with phage to which peptides had been chemically conjugated can elicit a focused anti-peptide antibody response compared with traditional carrier molecules bearing the same peptide, perhaps due to the low surface complexity of the phage. The regularity of its surface also gives the phage other advantages as a carrier, including immunological simplicity and thousands of well-defined sites for chemical conjugation. More recently, we showed that focusing of antibody responses against 'target' peptides was enhanced when the phage's molecular surface was simplified by removal of immunodominant B-cell epitopes present on the minor coat protein, pIII. The pIII-truncated variant elicits an antibody response that is largely restricted to the exposed N-terminus of the major coat protein, pVIII, and to phage-associated bacterial lipopolysaccharide, and a significant fraction of this response cross-reacts with a 12-residue peptide covering the surface-exposed region of pVIII. This allows one to track antibody responses against the phage (and any associated haptens) as they develop over time, and characterize them using a combination of serological, flow cytometric, cellular and immunogenetic assays. The filamentous phage thus provides an excellent model system for studying various aspects of the antibody response, all with the goal of targeting antibody production against weakly immunogenic peptides, proteins and carbohydrates.     

Biophysical analysis of the endoplasmic reticulum-resident chaperone/heat shock protein gp96/GRP94 and its complex with peptide antigen

Animals vaccinated with heat shock protein (HSP)--peptide complexes develop specific protective immunity against cancers from which the HSPs were originally isolated. This autologous specific immunity has been demonstrated using a number of HSP--peptide antigen complexes. A prototypical HSP-based cancer vaccine is the gp96--peptide antigen complex, which is currently undergoing human clinical trials. Here, we analyzed the structure of a recombinant wild-type and a mutant gp96 protein and their peptide complexes using a number of biophysical techniques. Gel filtration chromatography, dynamic light scattering, and equilibrium analytical ultracentrifugation demonstrated that both a wild-type gp96 and a gp96 mutant lacking a dimerization domain formed higher order structures. More detailed analysis using scanning transmission electron microscopy indicated that both the wild-type and dimerization deletion mutant gp96 protein were organized, unexpectedly, into large aggregates. Size distributions ranged from dimers to octamers and higher. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 dimerization domain deletion mutant protein was more compact than the wild-type gp96. A fluorescent peptide antigen was synthesized, and the peptide-binding properties of wild-type and the dimerization domain deletion mutant gp96 were studied. Fluorescence lifetime and anisotropy decay showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for the rotation of the probe. Deletion of the dimerization domain affected the peptide-binding microenvironment, although peptide-binding affinity was reduced by only a small extent. Peptide--gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide--gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.     

Mycobacterial heat shock proteins as vaccines - a model of facilitated antigen presentation

Heat shock proteins (hsps) are a highly conserved family of proteins, first recognized by their upregulated expression in response to host exposure to raised temperatures. Further study has revealed that they have numerous functions in the cell, primarily as chaperones mediating both the correct folding of nascent polypeptide chains and the dissolution of aggregated protein complexes. The energy requirement for this chaperone activity is provided by the ATPase activity found in most families of hsps and thus the peptide binding capacity is controlled by ATP hydrolysis. The structural consequence of this is that hsps isolated in situ are found complexed to chaperoned peptides (hspCs). Much previous work has implicated hsps in the immune response to pathogens and recent studies have shown that the interaction of hsps with antigen presenting cells, such as dendritic cells (DCs), mediates the integration of the innate and acquired immune responses. This central role for hspCs in immunity is facilitated by their dual function in both innate immunity, with the induction of cytokines and the maturation of DCs mediated by the hsp component, and acquired immunity, with the trafficking of antigens chaperoned in hspCs for antigen presentation by the mature DCs.