Mutations in the substrate binding site of human heat‐shock protein 70 indicate specific interaction with HLA‐DR outside the peptide binding groove

Heat‐shock protein 70 (Hsp70)–peptide complexes are involved in MHC class I‐ and II‐restricted antigen presentation, enabling enhanced activation of T cells. As shown previously, mammalian cytosolic Hsp70 (Hsc70) molecules interact specifically with HLA‐DR molecules. This interaction might be of significance as Hsp70 molecules could transfer bound antigenic peptides in a ternary complex into the binding groove of HLA‐DR molecules. The present study provides new insights into the distinct interaction of Hsp70 with HLA‐DR molecules. Using a quantitative binding assay, it could be demonstrated that a point mutation of amino acids alanine 406 and valine 438 in the substrate binding pocket led to reduced peptide binding compared with the wild‐type Hsp70 whereas HLA‐DR binding remains unaffected. The removal of the C‐terminal lid neither altered the substrate binding capacity nor the Hsp70 binding characteristics to HLA‐DR. A truncated variant lacking the nucleotide binding domain showed no binding interactions with HLA‐DR. Furthermore, the truncated ATPase subunit of constitutively expressed Hsc70 revealed similar binding affinities to HLA‐DR compared with the complete Hsc70. Hence, it can be assumed that the Hsp70–HLA‐DR interaction takes place outside the peptide binding groove and is attributed to the ATPase domain of HSP70 molecules. The Hsp70‐chaperoned peptides might thereby be directly transferred into the binding groove of HLA‐DR, so enabling enhanced presentation of the peptide on antigen‐presenting cells and leading to an improved proliferation of responding T cells as shown previously.     

Compartmentalization of class II antigen presentation: contribution of cytoplasmic and endosomal processing

Summary: During antigen processing, peptides are generated and displayed in the context of major histocompatibility complex (MHC) class II molecules on the surface of antigen‐presenting cells (APCs) to modulate immune responses to foreign antigens and guide self‐tolerance. Exogenous and cytoplasmic antigens are processed by distinct routes within APCs to yield class II ligands. Exogenous antigens are internalized, processed, and bound to class II molecules within endosomal and lysosomal compartments of APCs. Studies reviewed here demonstrate the importance of reduction in regulating exogenous antigen presentation. The differential expression of a γ‐interferon‐inducible lysosomal thiol reductase in professional APCs and melanomas is discussed in the context of tumor immune evasion. Cytoplasmic autoantigens, by contrast, are degraded by the proteasome and other enzymes in the cytosol, with the resulting peptides translocating to endosomal and lysosomal compartments for intersection with class II molecules. Processing and editing of these antigenic peptides within endosomes and lysosomes may be critical in regulating their display via class II proteins. Multiple pathways may regulate the transit of cytosolic peptides to class II molecules. The role of lysosome‐associated membrane protein‐2a and heat‐shock cognate protein 70 in promoting cytoplasmic peptide presentation by MHC class II molecules is discussed.     

Heat shock protein 70 moderately enhances peptide binding and transport by the transporter associated with antigen processing

Hsp70 molecules are capable of binding antigenic peptides and eliciting CTL responses to the bound peptide. However, the precise mechanism for the induction of CTL has not been determined. One possibility is that hsp molecules can directly shuttle peptides in the MHC class I antigen processing and presentation pathway, as previously postulated. Here, we have addressed this issue by testing the effect of purified hsp70 molecules on peptide binding and transport by the transporter associated with antigen processing (TAP). Our results indicate that purified hsp70 molecules moderately enhance TAP function. In addition, we detect a physical association between hsp70 molecules and TAP, as well as the homologous drug transporter P-glycoprotein. We conclude that while hsp70 molecules may not be directly involved in the delivery of peptide to TAP, they may play an important role in TAP transport by binding to TAP and promoting its function.     

Recombinant complexes of antigen with stress proteins are potent CD8 T-cell-stimulating immunogens

Heat shock proteins (Hsp) of the Hsp70/90 families facilitate cellular immune responses to antigenic peptides or proteins bound to them and have therefore been used as vaccine vehicles. We developed an expression system in which chimeric proteins with an Hsp-capturing, viral J domain fused to diverse antigen-encoding sequences form stable complexes with eukaryotic (Hsp70, Hsp73) or bacterial (DnaK) stress proteins and accumulate to high steady-state levels. J domains from different species (viruses/SV40, bacteria/Chlamydia trachomatis or plants/Arabidopsis thaliana) efficiently capture murine or human stress proteins in this system, thus making different J domains available for vaccine production. A novel expression and purification method was developed to produce native Hsp/antigen complexes in transfectants. These purified Hsp/antigen complexes efficiently elicited antigen-specific CD8 T cell responses in mice when delivered as vaccines without adjuvants. In situ complex formation of antigen with Hsp was critical for CD8 T cell priming. Because the described expression system supports the flexible design of multivalent vaccines, it is an attractive strategy to elicit CD8 T cell responses either to recombinant proteins or to selected antigenic domains of these molecules.     

Involvement of CD91 and scavenger receptors in Hsp70‐facilitated activation of human antigen‐specific CD4+ memory T cells

Hsp70 plays several roles in the adaptive immune response. Based on the ability to interact with diverse peptides, extracellular Hsp70:peptide complexes exert profound effects both in autoimmunity and in tumor rejection by evoking potent T cell responses to the chaperoned peptide. The interaction with receptors on APC represents the basis for the immunological functions of Hsp70 and a critical point where the immune response can be regulated. Various surface proteins (e.g. CD91, scavenger receptors (SR)) have been implicated in binding of Hsp70. In this study, antigenic peptides from tetanus toxin and influenza hemagglutinin complexed to human stress‐inducible Hsp70 were found to enhance the proliferation and cytokine production of human antigen‐specific CD4⁺ T cells. This was demonstrated in proliferation experiments using human monocytes as APC. Proliferated antigen‐specific cells were detected combining HLA‐DRB1^*0401 or HLA‐DRB1^*1101 tetramer and CFSE staining. Treating monocytes with CD91 siRNA diminished these effects. Additional blocking of SR by the SR ligand fucoidan completely abolished enhanced proliferation and production of Th1 and Th2 cytokines. Taken together, our data indicate that in the human system, CD91 and members of the SR family efficiently direct Hsp70:peptide complexes into the MHC class II presentation pathway and thus enhance antigen‐specific CD4⁺ T cell responses.     

Processing‐dependent and ‐independent pathways for recognition of iodinated contrast media by specific human T cells

Background One to three percent of patients exposed to intravenously injected iodinated contrast media (CM) develop delayed hypersensitivity reactions. Positive patch test reactions, immunohistological findings, and CM‐specific proliferation of T cells in vitro suggest a pathogenetic role for T cells. We have previously demonstrated that CM‐specific T cell clones (TCCs) show a broad range of cross‐reactivity to different CM. However, the mechanism of specific CM recognition by T cell receptors (TCRs) has not been analysed so far. Objective To determine how T cells specifically recognize CM. Methods CM‐specific TCCs were generated from human blood of three CM‐allergic patients and a specific TCR was transfected into a mouse T cell hybridoma. Functional analysis such as proliferation assays, IL‐2 secretion assays, and calcium influx experiments were performed using irradiated, glutaraldehyde‐fixed, CM‐pre‐incubated, human leucocyte antigen (HLA)‐DR‐matched or ‐mismatched antigen‐presenting cells (APCs), and HLA‐blocking antibodies. Results We identified two mechanisms of T cell stimulation: some TCCs and the transfectant reacted to CM independent of uptake by APCs because proliferation/IL‐2 secretion occurred in the presence of glutaraldehyde‐fixed APCs, and intracellular calcium increased within seconds after drug addition. Other TCCs required functional APCs, compatible with uptake and presentation of CM on MHC‐class II molecules, as implied by three findings: (1) glutaraldehyde fixation of APCs abrogated presentation; (2) CM could not be washed away from CM‐pre‐incubated APCs; and (3) the optimal pulsing time was 10–20 h. Because allogeneic, MHC‐matched, CM‐pulsed APCs could induce proliferative responses as well, the ability of CM uptake and presentation is not unique to APCs from patients with CM‐induced delayed hypersensitivity. Conclusion Our data suggest that CM may be stimulatory for T cells either by direct binding to the MHC–TCR complex or by binding after uptake and processing by APCs. This questions the assumed inert nature of CM. Cite this as: M. Keller, M. Lerch, M. Britschgi, V. Tâche, B. O. Gerber, M. Lüthiuthi, P. Lochmatter, G. Kanny, A. J. Bircher, C. Christiansen and W. J. Pichler, Clinical & Experimental Allergy, 2010 (40) 257–268.     

Activation of cytotoxic T cells in vitro by recombinant gp96 fusion proteins irrespective of the 'fused' antigenic peptide sequence.

Heat shock proteins (HSP) like Hsp70 and gp96 are potent molecules to induce MHC class I-restricted cytotoxic T cells against antigens present in the cells from which the HSP were isolated. Fusion proteins consisting of mycobacterial Hsp70 covalently linked to antigenic peptide sequences are also capable of generating CTL specific for the peptide-encoded antigens. For effective CTL induction direct binding of the peptide or covalent association of the peptide in the case of antigenic fusion proteins is required. Since mycobacterial Hsp70 and eukaryotic Hsp70 differ significantly in their primary structure, and since gp96 compared to Hsp70 is more efficient in priming antigen specific CTL in our hands, we created fusion proteins consisting of His-tagged eukaryotic gp96 fused C-terminally to various peptide antigens. Here, we used antigenic sequences derived from the established ovalbumin (OVA) and beta-galactosidase (beta-GAL) model systems. We show that in vitro established OVA and beta-GAL specific CTL clones release TNF-alpha and IFN-gamma when incubated with recombinant gp96 irrespective of the antigenic peptide sequences hooked to the C-terminus of gp96. In contrast to gp96 preparations purified from beta-GAL expressing cell lines, recombinant gp96/beta-GAL fusion proteins were not able to generate beta-GAL-specific T cells in vivo. Possible explanations for the lack of antigen-specific immunogenicity of gp96 fusion proteins in vivo are discussed.     

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.     

Uptake and processing of glycosylated mycolates for presentation to CD1b-restricted T cells

Antigen presenting cells (APCs) expressing CD1b mediate the specific T cell recognition of mycobacterial lipid antigens. These lipid antigens require internalization by APCs prior to presentation, but the detailed mechanisms of uptake and intracellular processing are not known. Here we have examined several steps in the presentation of two related classes of CD1b-presented antigens, free and glycosylated mycolates. T cell recognition of glucose monomycolate (GMM) was blocked by agents that fix APC membranes or neutralize the pH of endosomes, indicating a requirement for GMM uptake into an acidic compartment prior to recognition. Different T cell lines responded to free mycolate or GMM without crossreactivity, yet both antigens were taken up by APCs at the same rate. This demonstrated that differential recognition of these antigens resulted from T cell specificity for their hydrophilic caps and that APCs were unable to interconvert these antigens by enzymatic or chemical deglycosylation or glycosylation. APCs were also unable to cleave mycobacterial trehalose dimycolate (TDM) at its most chemically labile linkages to yield antigenic free mycolates or GMM. Our results indicate that these mycolate-containing antigens are resistant to chemical or enzymatic cleavage by APCs, suggesting that molecular trimming is not a universal feature of lipid antigen processing.     

Mycobacterium avium ssp. paratuberculosis recombinant heat shock protein 70 interaction with different bovine antigen-presenting cells

Abstract Heat shock proteins (Hsp) can deliver antigen into the major histocompatibility complex class I presentation pathway of antigen-presenting cells (APC), a process called cross priming, thus stimulating antigen-specific CD8+ T-cell reactions. Hsp were shown to elicit proinflammatory responses in APC. Both processes require interaction of Hsp with APC via specific receptors. This study describes the interaction of recombinant Hsp70 (rHsp70) of Mycobacterium avium subspecies paratuberculosis with bovine peripheral blood mononuclear cells that was restricted to CD14+ cells. Characterized monocyte-derived macrophages, monocyte-derived dendritic cells (DC) and BoMac, an immortalized bovine macrophage cell line, were used to investigate the interaction of rHsp70 with different bovine APC. Saturation of immature DC with high concentrations of rHsp70 is demonstrated, and it was found that interaction of rHsp70 with DC was related to the maturation stage of the DC. Involvement of CD91 as a cellular receptor for rHsp70 was demonstrated; however, competition studies with immature DC demonstrated that other receptors exist on bovine APC. These data suggest that rHsp70-based vaccines may be useful for the successful immunization of cattle.     

IgE: an immunoglobulin specialized in antigen capture?

IgE bound to the low-affinity receptor for immunoglobulin E (Fc epsilon R) on antigen-presenting cells (APCs) may permit binding of antigens to APCs prior to their processing and presentation. Here, G. C. Mudde and colleagues argue that the unique characteristics of IgE serological responses together with the distribution of Fc epsilon RII (CD23) on APCs are consistent with this novel function of IgE. The concept of IgE involvement in antigen capture is discussed in relation to Langerhans cells, B cells and follicular dendritic cells.     

A 14-mer peptide from HSP70 protein is the critical epitope which enhances NK activity against tumor cells in vivo

Heat shock protein 70 (Hsp70) has been found to play key roles in tumor immunity due its chaperone function of binding antigenic peptides. Here we report it can also stimulate NK cells in vivo, which is another role in Hsp70s' anti-tumor response. Injecting Hsp70 into mice increased splenic NK cell populations, which may be reason for anti-tumor effect of Hsp70. The Hsp70 14-mer peptide (aa450-463, TRD) was identified as the critical epitope for this stimulatory activity. It was the murine Hsp70 14-mer peptide TRD instead of the corresponding human Hsp70 14-mer peptide TKD that functioned in the mouse experimental model.     

Dynamics of the membrane–cytoskeleton interface in MHC class II-restricted antigen presentation

Antigen presentation refers to the ability of cells to show MHC-associated determinants to T lymphocytes, leading to their activation. MHC class II molecules mainly present peptide-derived antigens that are internalized by endocytosis in antigen-presenting cells (APCs). Here, we describe how the interface between cellular membranes and the cytoskeleton regulates the various steps that lead to the presentation of exogenous antigens on MHC class II molecules in the two main types of APCs: dendritic cells (DCs) and B lymphocytes. This includes antigen uptake, processing, APC migration, and APC–T cell interactions. We further discuss how the interaction between APC-specific molecules and cytoskeleton elements allows the coordination of antigen presentation and cell migration in time and space.     

A 14-mer Peptide from HSP70 Protein is the Critical Epitope Which Enhances NK Activity Against Tumor Cells in vivo

Heat shock protein 70 (Hsp70) has been found to play key roles in tumor immunity due its chaperone function of binding antigenic peptides. Here we report it can also stimulate NK cells in vivo, which is another role in Hsp70s' anti-tumor response. Injecting Hsp70 into mice increased splenic NK cell populations, which may be reason for anti-tumor effect of Hsp70. The Hsp70 14-mer peptide (aa450–463, TRD) was identified as the critical epitope for this stimulatory activity. It was the murine Hsp70 14-mer peptide TRD instead of the corresponding human Hsp70 14-mer peptide TKD that functioned in the mouse experimental model.     

Heat shock protein and innate immunity

In addition to serving as molecular chaperones,heat shock proteins (HSPs) have been implicated inautoimmune diseases,antigen presentation and tumor immunity.Extensive work in the last 10 years has alsosuggested that HSPs such as Hsp60,Hsp70,Hsp90 and gp96,may be potent activators of the innate immunesystem capable of inducing the production of pro-inflammatory cytokines by the monocyte-macrophage system,and the activation and maturation of dendritic cells via the Toll-like receptor 2 and 4 signal transductionpathways.However,recent evidence suggests that the reported cytokine effects of HSPs may be a result of thecontaminating bacterial cell-wall products.This concise review summarizes the current controversy over therole of HSPs in innate immunity.Cellular & Molecular Immunology.2004;1(4):274-279.     

Antigen-presentation capacity of dendritic cells is impaired in ongoing enterovirus myocarditis

Coxsackievirus B3 (CVB3)-infection is a frequent cause of acute myocarditis, which may result in chronic myocarditis and virus persistence. Investigation of the initial immune responses to CVB3 may shed light on the mechanisms that contribute to ongoing disease. DCs, as key professional APCs, were investigated in two MHC-matched hosts: while C57BL/6 mice are resistant to chronic CVB3-myocarditis, the A.BY/SnJ mouse strain exhibits susceptibility. DC maturation and activation were critically impaired in A.BY/SnJ mice, as reflected by the failure of DCs to induce co-stimulatory molecules and cytokine/chemokine responses. MHC class I-restricted antigen presentation via the cross-presentation pathway was also affected in DCs from A.BY/SnJ mice. DC maturation involves the accumulation of DC aggresome-like induced structures (DALISs) and the transient storage of defective ribosomal products (DRiPs). DCs from A.BY/SnJ mice showed permanent DALIS accumulation; the detection of poly-ubiquitinylated CVB3 proteins in these DALISs suggested a limitation in the MHC class I antigenic supply in this host. In conclusion, ongoing chronic disease in A.BY/SnJ mice due to a failure to clear the virus may be attributed to defects in DC maturation/activation and DC MHC class I antigen processing.     

Detection of heat shock protein 70 (HSP70) and anti-HSP70 antibodies in the serum of normal individuals

Heat shock or stress proteins (Hsp) are typically regarded as being intracellular proteins that have a range of functions including the maintenance of cellular integrity. Members of the Hsp70 family of molecules have been implicated in the processing and presentation of antigen and the cross reactivity of lymphocytes specific for pathogen-derived heat shock proteins with self Hsp70 has been suggested to be an underlying cause of certain autoimmune diseases. This study reports the presence of soluble Hsp70 in the peripheral circulation of normal individuals. Concentrations of soluble Hsp70 in females were approximately twice those in males. Circulating anti-Hsp70 antibodies were detected in all individuals assessed, but there were no differences between males and females. However, there was a significant correlation between soluble Hsp70 concentration and antibody levels in males, but not females. The physiological role for circulating heat shock proteins is intriguing, but currently unknown. These findings extend our previous observations that Hsp60 is present in the peripheral circulation and support the proposition that soluble heat shock proteins may play a regulatory role in either the prevention or protection of pathophysiological processes involving inadvertent immunorecognition or cross-recognition of heat shock proteins.     

Detection of heat shock protein 70 (HSP70) and anti-HSP70 antibodies in the serum of normal individuals

Heat shock or stress proteins (Hsp) are typically regarded as being intracellular proteins that have a range of functions including the maintenance of cellular integrity. Members of the Hsp70 family of molecules have been implicated in the processing and presentation of antigen and the cross reactivity of lymphocytes specific for pathogen-derived heat shock proteins with self Hsp70 has been suggested to be an underlying cause of certain autoimmune diseases. This study reports the presence of soluble Hsp70 in the peripheral circulation of normal individuals. Concentrations of soluble Hsp70 in females were approximately twice those in males. Circulating anti-Hsp70 antibodies were detected in all individuals assessed, but there were no differences between males and females. However, there was a significant correlation between soluble Hsp70 concentration and antibody levels in males, but not females. The physiological role for circulating heat shock proteins is intriguing, but currently unknown. These findings extend our previous observations that Hsp60 is present in the peripheral circulation and support the proposition that soluble heat shock proteins may play a regulatory role in either the prevention or protection of pathophysiological processes involving inadvertent immunorecognition or cross-recognition of heat shock proteins.     

Redundancy renders the glycoprotein 96 receptor scavenger receptor A dispensable for cross priming in vivo

CD8(+) T cells (T(CD8+)) differentiate into effector cells following recognition of specific peptide-major histocompatibility complex (MHC) class I complexes (pMHC-I) on the surface of professional APCs (pAPCs), such as dendritic cells. Antigenic pMHC-I can be generated from two spatially distinct sources. The direct presentation pathway involves generation of peptide from protein substrate synthesized within the cell that is presenting the pMHC-I. Alternatively, the cross presentation pathway involves presentation of antigen that is not synthesized within the presenting cell, but is derived from exogenous proteins synthesized within other donor cells. The mechanisms by which cross presentation of exogenous antigens occur in vivo remain controversial. The C-type lectin scavenger receptor A (SR-A) has been implicated in a number of potential cross presentation pathways, including the presentation of peptide bound to heat shock proteins, such as glycoprotein 96 (gp96), and the transfer of pMHC-I from a donor cell to the pAPC. We demonstrate here that initiation of T(CD8+) responses is normal in mice lacking SR-A, and that the redundancy of ligand binding exhibited by the SR family is likely to be an important mechanism that ensures cross presentation in vivo. These observations emphasize the requirement to target multiple receptors and antigen-processing pathways during the rational design of vaccines aimed at eliciting protective T(CD8+).     

Membrane Translocation of Binary Actin-ADP-Ribosylating Toxins from Clostridium difficile and Clostridium perfringens Is Facilitated by Cyclophilin A and Hsp90

Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.