Antoine Ménoret

Heat Shock Proteins Come of Age: Primitive Functions Acquire New Roles in an Adaptive World

During the analysis of representation of HSP-peptide complexes by APCs, another facet of HSP-APC interaction came to light. Exposure of APCs to gp96 preparations was observed to lead to stimulation of APCs to secrete low levels of cytokines regardless of the peptides associated with gp96 (Suto and Srivastava 1995xA mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Suto, R and Srivastava, P.K. Science. 1995; 269: 1585–1588Crossref | PubMedSee all ReferencesSuto and Srivastava 1995; Figure 3Figure 3). This was surprising but consistent with the observation that immunization with cancer-derived HSP-peptide complexes elicits, in addition to the CD8+ and CD4+ T cell response, an NK response that is crucial for eliciting protective immunity (Tamura et al. 1997xImmunotherapy of tumors with autologous tumor-derived heat shock protein preparations. Tamura, Y, Peng, P, Kang, L, Daou, M, and Srivastava, P.K. Science. 1997; 278: 117–120Crossref | PubMed | Scopus (572)See all ReferencesTamura et al. 1997). In light of the fact that HSPs are abundant components of pathogens as well as hosts, this observation may indicate a basic role for HSP-APC interaction in innate immunity in addition to the crucial role of such interactions in adaptive, peptide-dependent immunity discussed thus far (73xHeat shock proteins transfer peptides during antigen processing and CTL priming. Srivastava, P.K, Udono, H, Blachere, N.E, and Li, Z. Immunogenetics. 1994; 39: 93–98Crossref | PubMed | Scopus (456)See all References, 81xCellular requirements for tumor-specific immunity elicited by heat shock proteins (tumor rejection antigen gp96 primes CD8+ T cells in vivo) . Udono, H, Levey, D.L, and Srivastava, P.K. Proc. Natl. Acad. Sci. USA. 1994; 91: 3077–3081Crossref | PubMedSee all References, 75xA mechanism for the specific immunogenicity of heat shock protein-chaperoned peptides. Suto, R and Srivastava, P.K. Science. 1995; 269: 1585–1588Crossref | PubMedSee all References). Contemplation of these ideas leads to some interesting insights. HSPs are among the most primitive proteins in living systems, and macrophage-like cells of one kind or another are present in the earliest multicellular living systems (Beck and Habicht 1996xImmunity and invertebrates. Beck, G and Habicht, G.S. Sci. Am. 1996; 275: 60–63Crossref | PubMedSee all ReferencesBeck and Habicht 1996). It is conceivable that in a less polymorphic era, when adaptive immune response was but a distant gleam in the evolutionary eye, the interaction of HSPs with macrophage-like cells, leading to stimulation of the macrophage-like cells to secrete IL-1 and other messengers, was the primary “innate” defense mechanism. As HSPs are the most abundant soluble molecules in cells, they can be reasonably expected to be the most reliable messengers of cell death or, at any rate, cell lysis, which would result if the organism were in stress or danger, e.g., being consumed by a predator. This chain of events would also explain the otherwise strange presence of a surface receptor for HSPs, which are normally intracellular. In this view, the HSPs would have been transformed from being protectors of cells from cellular stress in the single-celled organisms to being messengers of stress and protectors against such stress in the first multicellular organisms or, at any rate, among those in which a differentiated macrophage-like cell had evolved. By all accounts, this happened quite early, as phagocytes of echinoderms, mollusks, annelids, and tunicates had already acquired the ability to secrete IL-1 like molecules (seeOttaviani and Franceschi 1997xThe invertebrate phagocytic immunocyte (clues to a common evolution of immune and neuroendocrine systems) . Ottaviani, E and Franceschi, C. Immunol. Today. 1997; 18: 169–174Abstract | Full Text PDF | PubMed | Scopus (130)See all ReferencesOttaviani and Franceschi 1997). From this initial point, it is relatively straightforward to see how the HSP-APC interaction could have been incorporated into future versions of the developing defense mechanisms until, with the arrival of specific immunity and its paraphernalia of specificity of peptide-binding and T cell receptors, the primal peptide-binding proteins, i.e., the HSPs, would become accessories in antigen presentation by MHC molecules. The evolutionary implications of a convergence of the peptide-binding functions of the HSPs and the MHC molecules has been discussed in detail elsewhere (Srivastava and Heike 1991xTumor-specific immunogenicity of stress-induced proteins (convergence of two evolutionary pathways of antigen presentation?) . Srivastava, P.K and Heike, M. Semin. Immunol. 1991; 3: 57–64PubMedSee all ReferencesSrivastava and Heike 1991).In the proposed role of HSPs as harbingers of cell death and, therefore, of danger to an organism (seeMatzinger 1994xTolerance, danger, and the extended family. Matzinger, P. Annu. Rev. Immunol. 1994; 12: 991–1045Crossref | PubMedSee all ReferencesMatzinger 1994), a kind of death that results in an encounter of HSPs with the immune system will be more productive, i.e., communicative of danger, and protective than a “silent death” that does not lead to HSP-APC interaction. It is therefore our belief that immunogenicity of cells, infectious agents, and cancers is defined to a significant degree upon the extent to which cell death leads to induction of HSPs and consequent HSP-APC interaction. Studies that show a cosegregation of immunogenicity of cancers with the expression of molecules of the hsp60 and hsp70 family (39xCo-segregation of tumor immunogenicity with expression of inducible but not constitutive hsp70 in rat colon carcinomas. Menoret, A, Patry, Y, Burg, C, and Le Pendu, J. J. Immunol. 1995; 155: 740–747PubMedSee all References, 85xRestoration of MHC class I surface expression and endogenous antigen presentation by a molecular chaperone. Wells, A.D, Rai, S.K, Salvato, M.S, Band, H, and Malkovsky, M. Scand. J. Immunol. 1997; 45: 605–612Crossref | PubMedSee all References, 38xTumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Melcher, A, Todryk, A, Hardwick, N, Ford, M, Jacobson, M, and Vile, R.G. Nat. Med. 1998; 4: 581–587Crossref | PubMed | Scopus (368)See all References) are consistent with this belief. It may be tempting in this context to imagine that immunologically unproductive and productive modes of death will correlate neatly with apoptotic and necrotic types of death; however, recent results indicate that this is not necessarily so (7xProcessing of engulfed apoptotic bodies yields T cell epitopes. Bellone, M, Iezzi, G, Rovere, P, Galati, G, Ronchetti, A, Protti, M.P, Davoust, J, Rugarli, C, and Manfredi, A.A. J. Immunol. 1997; 159: 5391–5399PubMedSee all References, 1xDendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Albert, M.L, Sauter, B, and Bhardwaj, N. Nature. 1998; 392: 86–89Crossref | PubMed | Scopus (1839)See all References, 38xTumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Melcher, A, Todryk, A, Hardwick, N, Ford, M, Jacobson, M, and Vile, R.G. Nat. Med. 1998; 4: 581–587Crossref | PubMed | Scopus (368)See all References). Other factors yet to be discovered must influence the immunological productivity of dying cells. In any case, it is safe to suggest that the recent fascination of immunologists with death has atavistic origins in the evolutionary past, when the presence of extracellular HSPs signaled physical disintegration.*To whom correspondence should be addressed (e-mail: srivastava@nso2.uchc.edu).

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.

Purification of immunogenic heat shock protein 70–peptide complexes by ADP-affinity chromatography

Adenosine triphosphate (ATP)-affinity chromatography has been widely used to purify molecules of the Hsp70 family. This procedure leads to dissociation of peptides from Hsp70 molecules, resulting in Hsp70 preparations devoid of immunological activity. We report that substitution of ATP-affinity chromatography by ADP-affinity chromatography results in isolation of Hsp70 molecules which are still associated with peptides and are immunogenic. The Hsp70 preparations thus obtained contain the constitutive Hsp73 and the inducible Hsp72 molecules. These observations furnish a basis for an analysis of the structural heterogeneity among the members of the Hsp70 family and of the antigenic peptides associated with individual members of this family. They also provide a practical method for the isolation of large quantities of immunologically active Hsp70-peptide preparations.

Saturation, Competition, and Specificity in Interaction of Heat Shock Proteins (hsp) gp96, hsp90, and hsp70 with CD11b + Cells

Heat shock proteins (hsp(s)) have been postulated to interact with APCs through specific receptors, although the receptors are yet to be identified. Specificity, saturation, and competition are the three defining attributes of a receptor-ligand interaction. We demonstrate here that the interaction of the heat shock proteins gp96 and hsp90 with CD11b+ cells is specific and saturable and that gp96 can compete with itself in gp96-macrophage interaction. Interestingly, the phylogenetically related hsp90 also competes quite effectively with gp96 for binding to macrophages, whereas the unrelated hsp70 does so relatively poorly, although it binds CD11b+ cells just as effectively. These data provide evidence that the heat shock proteins interact with APCs with specificity and for the existence of at least two distinct receptors, one for gp96 and hsp90 and the other for hsp70.

CD134 Plus CD137 Dual Costimulation Induces Eomesodermin in CD4 T Cells To Program Cytotoxic Th1 Differentiation

Cytotoxic CD4 Th1 cells are emerging as a therapeutically useful T cell lineage that can effectively target tumors, but until now the pathways that govern their differentiation have been poorly understood. We demonstrate that CD134 (OX40) costimulation programs naive self- and virus-reactive CD4 T cells to undergo in vivo differentiation into cytotoxic Th1 effectors. CD137 (4-1BB) costimulation maximized clonal expansion, and IL-2 was necessary for cytotoxic Th1 differentiation. Importantly, the T-box transcription factor Eomesodermin was critical for inducing the cytotoxic marker granzyme B. CD134 plus CD137 dual costimulation also imprinted a cytotoxic phenotype on bystanding CD4 T cells. Thus, to our knowledge, the current study identifies for the first time a specific costimulatory pathway and an intracellular mechanism relying on Eomesodermin that induces both Ag-specific and bystander cytotoxic CD4 Th1 cells. This mechanism might be therapeutically useful because CD134 plus CD137 dual costimulation induced CD4 T cell-dependent tumoricidal function in a mouse melanoma model.

Combination of Imatinib Mesylate with Autologous Leukocyte-Derived Heat Shock Protein and Chronic Myelogenous Leukemia

Purpose: To test the feasibility, safety, immunogenicity, and clinical efficacy of an autologous vaccine of leukocyte-derived heat shock protein 70-peptide complexes (Hsp70PC), in conjunction with imatinib mesylate, in patients with chronic myeloid leukemia (CML) in chronic phase. Experimental Design: Patients had cytogenetic or molecular evidence of disease, despite treatment with imatinib mesylate for all except one patient, at the beginning of study. Hsp70PCs were purified from the leukopheresed peripheral blood mononuclear cells and were administered in eight weekly intradermal injections at 50 μg/dose without adjuvant. Clinical responses were assessed by bone marrow analysis before and after vaccinations. An IFN-γ enzyme-linked immunospot assay was used to estimate the effect of treatment on natural killer cells and T cells against CML. Results: Twenty patients were treated. The manufacturing of Hsp70PCs was successful and the administration was safe for all patients. Minimal or no side effects were reported. Clinical responses were seen in 13 of 20 patients as measured by cytogenetic analysis of bone marrow Philadelphia chromosome–positive cells in metaphases and/or, when possible, the level of Bcr/Abl transcript by PCR. Immunologic responses were observed in 9 of 16 patients analyzed, characterized by an increase in the frequency of CML-specific IFN-γ-producing cells and IFN-γ-secreting natural killer cells in the blood. A significant correlation between clinical responses and immunologic responses was observed. Conclusions: Autologous Hsp70PC vaccination is feasible and safe. When combined with imatinib mesylate, it is associated with immunologic and possible clinical responses against CML in chronic phase.

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.

Selective PGE2 suppression impairs colon carcinogenesis and modifies local mucosal immunity

Prostaglandin E2 (PGE2) is a bioactive lipid that mediates a wide range of physiologic effects and plays a central role in inflammation and cancer. PGE2 is generated from arachidonic acid by the sequential actions of the COX and terminal synthases (PGES). Increased levels of COX-2, with a concomitant elevation of PGE2, are often found in colorectal cancers (CRC), providing the rationale for the use of COX-2 inhibitors for chemoprevention. Despite their proven efficacy in cancer prevention, however, COX-2 inhibitors exhibit dose-dependent toxicities that are mediated in part by their nonspecific reduction of essential prostanoids, thus limiting their chemopreventive benefit. To achieve enhanced specificity, recent efforts have been directed toward targeting the inducible terminal synthase in the production of PGE2, microsomal PGES (mPGES-1). In the present study, we show that genetic deletion of mPGES-1 affords significant protection against carcinogen-induced colon cancer. mPGES-1 gene deletion results in an about 80% decrease in tumor multiplicity and up to a 90% reduction in tumor load in the distal colon of azoxymethane (AOM)-treated mice. Associated with the striking cancer suppression, we have identified a critical role for PGE2 in the control of immunoregulatory cell expansion (FoxP3-positive regulatory T cells) within the colon-draining mesenteric lymph nodes, providing a potential mechanism by which suppression of PGE2 may protect against CRC. These results provide new insights into how PGE2 controls antitumor immunity. Cancer Prev Res; 4(8); 1198–208. ©2011 AACR.

The inducible Hsp70 as a marker of tumor immunogenicity.

Abstract Growing evidence indicates that the stress response in general and heat shock proteins (Hsps) in particular have a profound impact on tumor immunogenicity. In this study, we show that tumor cells subjected to a nonlethal heat shock stress are unable to form tumors in syngenic mice, whereas they do so in athymic nude mice. Moreover, heat-shocked MethA immunity is tumor specific. Enhancement of T-cell–mediated immunogenicity correlates with the expression of the inducible Hsp70 but not the constitutive Hsc70. These observations have a bearing on the proposed functional role of Hsp-peptide association in antigen processing and presentation by major histocompatibility complex I molecules under normal and stressful conditions.

Purification of multiple heat shock proteins from a single tumor sample

Heat shock protein-based vaccines have been shown to immunize against cancer and infectious diseases in both prophylactic and therapeutic protocols. So far, four classes of heat shock proteins (HSPs) preparation: gp96, HSP90 (hsp86, hsp84), HSP70 (hsc70, hsp70) and calreticulin have been used successfully. The methods for purifying them individually are now readily available. However, since tumors are not always available in large quantity, a major challenge remains the development of a procedure to simultaneously isolate these HSPs from the same sample. We report here that hsp40, hsp60, hsc70, hsp70, hsp84, hsp86, and gp96 (grp94) but not BiP (grp78) and calreticulin can be separated from a single tumor sample in one step using heparin-agarose chromatography. Interestingly this procedure separates the HSP70 isoforms hsp70 from hsc70, but not the HSP90 isoforms hsp84 and hsp86. The three main immunogenic HSPs, gp96, hsp86/84, and hsc70 can be further isolated to homogeneity using additional purification methods. In addition, we have shown that the interaction of the chaperoned peptides with hsc70 and gp96 is not compromised during heparin chromatography. These observations provide a new method for preparation of multiple HSP-based vaccines, circumventing the sample size limitation, as well as providing the possibility to study how multiple HSPs can synergize in eliciting immunity.