Exogenous stress proteins enhance the immunogenicity of apoptotic tumor cells and stimulate antitumor immunity.

We have previously reported that apoptotic tumor cells can be either immunogenic or nonimmunogenic in vivo, depending on whether or not these cells are heat stressed before induction of apoptosis. Stressed apoptotic cells express heat shock proteins on their plasma membranes and dendritic cells are capable of distinguishing them from nonstressed apoptotic cells. Here we provide evidence that when purified heat shock protein 70 or chaperone-rich cell lysate (CRCL) from syngeneic normal tissue is used as an adjuvant with nonimmunogenic apoptotic tumor cells in vaccination, potent antitumor immunity can be generated. This antitumor immunity is mediated by T cells because antitumor effects are not observed in either severe combined immunodeficiency or T cell-depleted mice. We further demonstrate that vaccination of mice with apoptotic tumor cells mixed with liver-derived CRCL as adjuvant were capable of enhancing the production of T(H)1 cytokines, inducing specific cytotoxic T lymphocytes and eliciting long-lasting antitumor immunity. Stress proteins from autologous normal tissue components therefore can serve as danger signals to enhance the immunogenicity of apoptotic tumor cells and stimulate tumor-specific immunity

Related papers:

title: CD36 is required for phagocytosis of apoptotic cells by human macrophages that use either a phosphatidylserine receptor or the vitronectin receptor (alpha v beta 3).
abstract: In vivo, apoptotic cells are efficiently removed by professional or nonprofessional phagocytes, a process thought to be essential for tissue remodeling and resolution of inflammation. Macrophages recognize apoptotic cells by several mechanisms, including recognition of exposed phosphatidylserine (PS); however, PS recognition on apoptotic cells has not been identified as a feature of human macrophages. The purpose of this study was to determine whether human monocyte-derived macrophages could be stimulated to recognize PS, defined as inhibition of phagocytosis by PS-containing liposomes. We also assessed the potential roles for scavenger receptors, CD14, and lectins. Uptake of apoptotic neutrophils into unstimulated macrophages was blocked about 50% by Arg-Gly-Asp-Ser and anti-alpha(v), and up to 20% by oxidized low density lipoprotein and N-acetylglucosamine, implying a major role for integrin and minor roles for scavenger and lectin receptors. Uptake into macrophages stimulated with beta-1,3-glucan was blocked 50% by PS liposomes and 40% by oxidized low density lipoprotein, suggesting that the macrophages had switched from using integrin to recognition of PS. MEM-18 and 61D3 (anti-CD14 mAbs) were poor inhibitors of apoptotic neutrophil uptake, but good inhibitors of apoptotic lymphocyte uptake. The switch to PS recognition was accompanied by down-regulation of alpha(v)beta3 expression and function. Anti-CD36 blocked uptake into unstimulated or stimulated macrophages, suggesting CD36 involvement not only with the alpha(v)beta3 integrin mechanism (as previously reported) but also with PS recognition. A maximum of 70% inhibition was achieved by combining anti-CD36 with either anti-a(v) or PS liposomes.

title: CD14-dependent clearance of apoptotic cells: relevance to the immune system.
abstract: Until very recently, the function of CD14 was thought to be limited to innate immune responses to bacterial and other microbial structures. It is now known that macrophage CD14 serves an additional unexpected function, namely as a receptor involved in the recognition and phagocytosis of cells undergoing apoptosis. In stark contrast to its role in eliciting pro-inflammatory responses following binding of microbial ligands, macrophage CD14 mediates clearance of apoptotic cells without inciting inflammation. Increasing interest in the profoundly important final stage of apoptosis - the engulfment process - together with significant advances in knowledge of the immunological consequences of apoptotic-cell clearance and of the means by which signal transduction may be achieved following CD14-ligand binding have begun to produce a clearer picture of the role of CD14 in the immune system.

title: Processing of engulfed apoptotic bodies yields T cell epitopes.
abstract: Programmed death via apoptosis is the metazoan physiologic mode of cell death. Apoptotic cells are recognized by scavenger phagocytes via a number of membrane receptors and engulfed. Thereafter, little is known of their fate, or that of phagocytes. Here, we have traced apoptotic cells upon their engulfment by macrophages. After 3 h, apoptotic cells were contained in discrete well-defined vacuoles. Upon overnight chase, several small vesicles, possibly originating from the fragmentation of original vacuoles, were evident all over the macrophage body. Furthermore, Ags were diffused in the cytosol of some cells, which raises the possibility that epitopes from engulfed apoptotic cells may associate with macrophage MHC class I molecules and be recognized by T lymphocytes. Indeed, Ag-specific CTLs recognize and specifically lyse syngeneic macrophages upon phagocytosis of MHC class I-positive or -negative apoptotic cells, provided that they contain the relevant Ags. Synthesis and membrane expression of class I molecules by macrophages, together with functional transporters associated with Ag presentation, were necessary for recognition and lysis. The indirect presentation of epitopes from engulfed apoptotic cells by scavenger Ag-presenting phagocytes may, in the absence of "danger" signals, have implications for the establishment of central and peripheral self-tolerance.

title: Building better vaccines: how apoptotic cell death can induce inflammation and activate innate and adaptive immunity.
abstract: The immunological consequences of apoptosis have been hotly debated. Apoptosis was originally described as a set of cellular morphological changes that occur in the absence of inflammation but the term has been redefined on the basis of a set of conserved molecular events that include the activation of caspases. Though the apoptosis occurring during normal development is immunologically bland or even tolerizing, the apoptotic death after viral infection or after the ligation of Fas can trigger powerful innate and adaptive immune responses. The molecular machinery at the nexus of apoptosis and inflammation includes caspase-1 --an activator of IL-1beta and IL-18 - as well as the double-stranded-RNA-dependent protein kinase pathway and RNaseL pathway, which are key effectors of antiviral immunity. New proapoptotic vaccines induce immune responses that may be able to prevent or treat infectious disease and cancer.

title: The macrophage haunted by cell ghosts: a pathogen grows.
abstract: Apoptosis is induced in the course of immune responses to infectious agents. The last step of apoptosis is recognition and ingestion of the dying cells by phagocytes. Here, Marcela F. Lopes and colleagues discuss recent studies and argue that phagocytosis of apoptotic cells plays a previously unrecognized role in regulating the nature of immune responses against pathogens.

title: Stressed apoptotic tumor cells express heat shock proteins and elicit tumor-specific immunity.
abstract: In attempting to develop effective anticancer immunotherapies, the relative ability of apoptotic cells to induce an immune response remains an important but controversial consideration. A novel gene-transfer approach was used by which rapid induction of pure apoptosis can be selectively achieved in a transfected tumor cell population following exposure to a semisynthetic dimerizing ligand, AP20187. Inoculation of BALB/c mice with apoptotic and viable 12B1-D1 leukemia cells, at a 12:1 ratio subcutaneously, led to early tumor growth. Heat stress up-regulated the expression of membrane heat shock proteins (HSP72 and HSP60) on apoptotic 12B1-D1 cells, and stressed apoptotic cells were capable of generating a T-cell-mediated specific antitumor response. Pulsing of stressed apoptotic leukemia cells onto syngeneic dendritic cells resulted largely in rejection of coinjected viable 12B1-D1 cells. Mice rejecting the primary 12B1-D1 inoculum were immune to the same but not to a different leukemia challenge. Our findings indicate that tumor immunogenicity is dependent on whether cells are stressed before apoptosis induction and suggest that the immune system is capable of distinguishing between stressed and nonstressed cells undergoing programmed cell death.

title: Stressed apoptotic tumor cells stimulate dendritic cells and induce specific cytotoxic T cells.
abstract: We have previously reported that stressed apoptotic tumor cells are more immunogenic in vivo than nonstressed ones. Using confocal microscopy we have confirmed our previous observation that heat-stressed apoptotic 12B1-D1 leukemia cells (BCR-ABL(+)) express HSP60 and HSP72 on their surface. To explore how the immune system distinguishes stressed from nonstressed apoptotic tumor cells, we analyzed the responses of dendritic cells to these 2 types of apoptotic cells. We found that nonstressed and heat-stressed apoptotic 12B1-D1 cells were taken up by dendritic cells in a comparable fashion. However, when stressed apoptotic 12B1-D1 cells were coincubated with immature dendritic cells for 24 hours, this resulted in greater up-regulation of costimulatory molecules (CD40, CD80, and CD86) on the surface of dendritic cells. Moreover, stressed apoptotic 12B1-D1 cells were more effective in stimulating dendritic cells to secrete interleukin-12 (IL-12) and in enhancing their immunostimulatory functions in mixed leukocyte reactions. Furthermore, we demonstrated that immunization of mice with stressed apoptotic 12B1-D1 cells induced the secretion of T helper-1 (T(H)1) profile of cytokines by spleen cells. Splenocytes from mice immunized with stressed apoptotic cells, but not nonstressed ones, were capable of lysing 12B1-D1 and the parental 12B1 line, but not a B-cell leukemia line, A20. Our data indicate that stressed apoptotic tumor cells are capable of providing the necessary danger signals, likely through increased surface expression of heat shock proteins (HSPs), resulting in activation/maturation of dendritic cells and, ultimately, the generation of potent antitumor T-cell responses.

title: Heat shock proteins come of age: primitive functions acquire new roles in an adaptive world.
abstract: 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;  1585–1588Crossref | PubMedSee all ReferencesSuto and Srivastava 1995; . 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.  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.  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;  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,  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.  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,  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.  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,  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,  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.  1998;  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,  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).

title: Immunoprotective activities of multiple chaperone proteins isolated from murine B-cell leukemia/lymphoma.
abstract: Although the use of tumor-derived heat shock/chaperone proteins (HSPs) as anticancer vaccines is gaining wider study and acceptance, there have thus far been no reports concerning chaperone antitumor activities against disseminated hematological malignancies. We have devised an efficient and effective method for purification of the chaperone proteins grp94/gp96, HSP90, HSP70, and calreticulin from harvested A20 murine leukemia/lymphoma tumor material. We have demonstrated that these purified proteins, when used as vaccines, can induce potent and specific immunity against a lethal tumor challenge. Individual chaperone proteins were differentially effective in their abilities to provide immune protection. The increase in survival generated by the most effective chaperone vaccine, HSP70, resulted from at least a 2-log reduction in tumor burden. Syngeneic granulocyte macrophage colony-stimulating factor producing fibroblasts were injected at the site of vaccination in an attempt to augment the immune response. Surprisingly, localized granulocyte macrophage colony-stimulating factor production inhibited the protective effects of chaperone vaccination. These studies provide evidence that chaperone proteins can be isolated from B-cell tumors and used effectively to immunize against disseminated lymphoid malignancies.

title: Tumor-derived chaperone-rich cell lysates are effective therapeutic vaccines against a variety of cancers
abstract: With the clinical use of purified, tumor-derived chaperone proteins as anti-cancer vaccines already in clinical trial stages, we have focused our attention on the utility of chaperone-rich cell lysates (CRCL) in cancer immunotherapy. CRCL, as prepared from tumor lysates via a free solution-isoelectric focusing (FS-IEF) technique, is a high-yield vaccine enriched for numerous chaperone proteins. We have compared the efficacy of CRCL vaccines to that of individual chaperone protein vaccines in in vivo settings, including ELISPOT assays, tumor-growth assays and survival assays. In all experiments, CRCL vaccines were at least as effective, and in some settings perhaps even more effective, than either of the two most heavily studied components of CRCL, HSP70 and GRP94/gp96, in reduction in tumor growth and prolongation of survival in both prophylactic and pre-existing tumor settings against tumors of diverse origin and genetic background. Combining CRCL preparations with dendritic cells ex vivo resulted in a cellular vaccine that could eradicate pre-existing tumors in a high percentage of cases. The high yields of CRCL vaccines from small quantities of starting materials, the relative ease of its procurement and the functional data presented here suggest that CRCL vaccines are worthy of evaluation in pilot clinical trial cancer immunotherapy protocols.

title: Tumor-derived multiple chaperone enrichment by free-solution isoelectric focusing yields potent antitumor vaccines
abstract: We have utilized a free-solution/isoelectric focusing technique (FS-IEF) to obtain fractions rich in multiple chaperone proteins from clarified A20 tumor lysates. Vaccines prepared from chaperone-rich fractions are capable of providing protective immunity in mice subsequently challenged intravenously with the same A20 B cell leukemia cells. This protection is at least equal to that provided by purified, tumor-derived heat-shock protein 70, which was the best chaperone immunogen in our hands against this aggressive murine leukemia model. Dosage escalation studies, however, revealed that increasing vaccine dosages actually abrogated the protective effects. The physical nature of the enriched chaperones indicates that they are associated in complexes, which may have implications for their function. FS-IEF is relatively simple, rapid, and efficient, thus making combined multi-chaperone therapy feasible.

title: Priming of T cells by heat shock protein-peptide complexes as the basis of tumor vaccines.
abstract: A number of heat shock proteins, or stress proteins, have been shown functionally as potent 'tumor rejection antigens', despite the fact that there are no single tumor-specific mutations in these proteins. Current studies have solved many aspects of this puzzle biochemically and immunologically. The proposal that heat shock proteins are not antigenic per se, but chaperone and present antigenic peptides to MHC class I molecules for recognition by T lymphocytes, has been mechanistically defined. This significance of this discovery is elaborated in the context of the importance of MHC molecules. The immunological principle and the practical vaccination strategy of the two major mammalian heat shock proteins, gp96/grp94 and hsp70 are discussed. It is also argued that extracellular heat shock proteins signal cell stress and tissue damage, and are involved in presenting peptides to the MHC class I pathway through professional antigen presenting cells. These results and ideas form the basis of a new generation of T-cell vaccines against tumors and intracellular organisms.

title: Heat shock protein--peptide complexes as immunotherapy for human cancer.
abstract: Heat shock proteins (Hsps), ubiquitous in nature, act as chaperones for peptides and other proteins. They have been implicated in loading immunogenic peptides onto major histocompatibility complex molecules for presentation to T cells. When isolated from tumor cells, Hsps are complexed with a wide array of peptides, some of which serve as tumor-specific antigens. Animal studies have demonstrated that heat shock protein--peptide complexes (HSPPCs) from tumor cells can act as vaccines to prevent or treat tumors. Potent and specific tumor antigens have long been the holy grail in cancer immunotherapy; HSPPCs from tumor cells could become a safe and reliable source of tumor-specific antigens for clinical application.

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

title: Stress protein/peptide complexes derived from autologous tumor tissue as tumor vaccines.
abstract: Vaccination of inbred mice with tumor-derived stress proteins hsp70, hsp90, and gp96/grp94 elicits a protective immunity to the tumor from which the vaccine was purified. There is now comprehensive experimental evidence that the antigenicity of tumor-derived hsp70, hsp90, and gp96 preparations results from diverse arrays of endogenous peptide antigens complexed with these stress proteins. Vaccination with tumor-derived stress protein/peptide complexes leads to their uptake and processing by professional antigen-presenting cells and to presentation of associated tumor peptide antigens to cytotoxic T cells. This induces a tumor-specific cytotoxic T cell response. The attractiveness of the concept of using tumor-derived stress proteins as vaccines is derived from two observations: (i) tumor stress protein vaccines mirror the individual antigenicity of a tumor, which results from random mutations due to genetic instability; and (ii) stress proteins represent powerful adjuvants for the peptide antigens complexed to them.

title: Heat shock proteins refine the danger theory
abstract: Deficiencies of the self–non-self paradigm in explaining immune phenomena, such as the existence and breaking of immune tolerance, have lead the likes of Janeway1 and Matzinger2 to suggest additional levels of control of antigen-specific immune responses. In simple terms, the immune system is thought to be turned on by ‘danger’ associated with certain molecules of infectious organisms, or cell products released during tissue damage or stress. Antigen-presenting cells (APC) possess pattern-recognition receptors that can recognize such molecules. This recognition is thought to cause upregulation of costimulatory molecules on local APC, which is the second signal [in addition to antigenic peptide with major histocompatibility complex (MHC)] that is required to initiate an antigen-specific immune response. Candidate danger signals from infectious organisms include lipopolysaccharide, mannans, glycans and CpG DNA motifs, whilst those from mammalian cells include heat shock proteins (hsp; inducible and constitutive), mitochondria, mannose, RNA and DNA. With respect to hsp, recent evidence now suggests that their role in signalling danger may be wide ranging and, not surprisingly for immunology, more complicated than first thought.

title: Interaction of heat shock proteins with peptides and antigen presenting cells: chaperoning of the innate and adaptive immune responses.
abstract: Heat shock proteins are abundant soluble intracellular proteins, present in all cells. Members of the heat shock protein family bind peptides including antigenic peptides generated within cells. Heat shock proteins also interact with antigen presenting cells through CD91 and other receptors, eliciting a cascade of events including re-presentation of heat shock protein-chaperoned peptides by MHC, translocation of NF kappa B into the nuclei and maturation of dendritic cells. These consequences point to a key role of heat shock proteins in fundamental immunological phenomena such as activation of antigen presenting cells, indirect presentation (or cross-priming), and chaperoning of peptides during antigen presentation. Heat shock proteins appear to have been involved in innate immune responses since the emergence of phagocytes in early multicellular organisms and to have been commandeered for adaptive immune responses with the advent of specificity. These properties of heat shock proteins also allow them to be used for immunotherapy of cancers and infections in novel ways.

title: Heat-shock proteins as activators of the innate immune system.
abstract: Peptides bound or linked to heat-shock proteins (HSPs) of microbial or mammalian origin have been shown to elicit potent antigen-specific immunity. Some members of the HSP family, such as hsp60, hsp70, hsp90 and gp96, are able also to stimulate cells of the innate immune system directly and thus, act as 'danger'-signaling molecules. This effect is independent of HSP-associated peptides and, in many respects, resembles the effect of lipopolysaccharide (LPS). Here, we discuss the similarities between the responses to HSPs and LPS and also, emphasize that care must be taken when working with preparations of HSPs in experimental settings and interpreting experimental data.

title: IgG or IgM monoclonal antibodies reactive with different determinants on the molecular complex bearing Lyt 2 antigen block T cell-mediated cytolysis in the absence of complement.
abstract: Five rat monoclonal antibodies have been derived that express specificities for determinants present on the molecular complex bearing the Lyt 2 antigen. SDS-polyacrylamide gel electrophoresis of 125I-labeled polypeptides precipitated by each of these antibodies reveal 3 components (150,000, 75,000, and 33,000 daltons), and 2 components (44,000 and 33,000 daltons) when analyzed under nonreducing and reducing conditions, respectively. Two of these antibodies are IgG and are specific for the Lyt 2.2 determinant; the other 3 are IgM and react with determinants other than Lyt 2.2, which are nonpolymorphic. Each of the 5 antibodies can block the cytolytic activities of 5-day MLC cells or of cloned cytolytic T cells in the absence of C. Treatment of responding spleen cells with any of these antibodies and C inhibits the generation of cytolytic activity in MLC.

title: Tumor antigens recognized by T lymphocytes
abstract: In the last five years, knowledge of human tumor antigens recognized by autologous cytolytic T lymphocytes (CTL) has increased considerably. So far, genetic and biochemical approaches have led to the molecular identification of three classes of antigens. Most of these antigens consist of peptides that are presented to T cells by HLA molecules. The first class comprises antigens encoded by genes such as MAGE, BAGE, and GAGE, which are expressed in various tumors of different histological origins, but not in normal tissues other than testis. The second class represents differentiation antigens encoded by genes that are only expressed in melanoma and normal melanocytes like tyrosinase, Melan-A/MART-1, gp100 and gp75. The third class includes antigens produced by unique point mutations in genes that are ubiquitously expressed. In most cases, the antigenic peptide is encoded by the mutated region of the gene. A number of these antigens provide promising targets for new protocols of specific cancer immunotherapy.

title: Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs
abstract: CD8+ cytotoxic T lymphocytes (CTLs) mediate resistance to infectious agents and tumours. Classically, CTLs recognize antigens that are localized in the cytoplasm of target cells, processed and presented as peptide complexes with class I molecules of the major histocompatibility complex (MHC). However, there is evidence for an exogenous pathway whereby antigens that are not expected to gain access to the cytoplasm are presented on MHC class I molecules. The most dramatic example is the in vivo phenomenon of cross-priming: antigens from donor cells are acquired by bone-marrow-derived host antigen-presenting cells (APCs) and presented on MHC class I molecules. Two unanswered questions concern the identity of this bone-marrow-derived cell and how such antigens are acquired. Here we show that human dendritic cells, but not macrophages, efficiently present antigen derived from apoptotic cells, stimulating class I-restricted CD8+ CTLs. Our findings suggest a mechanism by which potent APCs acquire antigens from tumours, transplants, infected cells, or even self-tissue, for stimulation or tolerization of CTLs.

title: The immune system evolved to discriminate infectious nonself from noninfectious self.
abstract: Here, Charles Janeway argues that the requirement for two signals to initiate the adaptive immune response may reflect the evolutionary history of host defences. Early phases of host defence involve receptors and ligands that may have controlled immune responses prior to the development of clonally-distributed receptors encoded in rearranging genes. The former receptors persist in contemporary vertebrates both to trigger innate or nonclonal responses and to signal to lymphocytes that a particular antigen is associated with a microorganism.

title: Decoding the Patterns of Self and Nonself by the Innate Immune System
abstract: The innate immune system evolved several strategies of self/nonself discrimination that are based on the recognition of molecular patterns demarcating infectious nonself, as well as normal and abnormal self. These patterns are deciphered by receptors that either induce or inhibit an immune response, depending on the meaning of these signals.

title: Tolerance, danger, and the extended family.
abstract: For many years immunologists have been well served by the viewpoint that the immune system's primary goal is to discriminate between self and non-self. I believe that it is time to change viewpoints and, in this essay, I discuss the possibility that the immune system does not care about self and non-self, that its primary driving force is the need to detect and protect against danger, and that it does not do the job alone, but receives positive and negative communications from an extended network of other bodily tissues.

title: The Danger Model: A Renewed Sense of Self
abstract: For over 50 years immunologists have based their thoughts, experiments, and clinical treatments on the idea that the immune system functions by making a distinction between self and nonself. Although this paradigm has often served us well, years of detailed examination have revealed a number of inherent problems. This Viewpoint outlines a model of immunity based on the idea that the immune system is more concerned with entities that do damage than with those that are foreign.

title: Consequences of Cell Death
abstract: Cell death by necrosis is typically associated with inflammation, in contrast to apoptosis. We have identified additional distinctions between the two types of death that occur at the level of dendritic cells (DCs) and which influence the induction of immunity. DCs must undergo changes termed maturation to act as potent antigen-presenting cells. Here, we investigated whether exposure to apoptotic or necrotic cells affected DC maturation. We found that immature DCs efficiently phagocytose a variety of apoptotic and necrotic tumor cells. However, only exposure to the latter induces maturation. The mature DCs express high levels of the DC-restricted markers CD83 and lysosome-associated membrane glycoprotein (DC-LAMP) and the costimulatory molecules CD40 and CD86. Furthermore, they develop into powerful stimulators of both CD4+ and CD8+ T cells. Cross-presentation of antigens to CD8+ T cells occurs after uptake of apoptotic cells. We demonstrate here that optimal cross-presentation of antigens from tumor cells requires two steps: phagocytosis of apoptotic cells by immature DCs, which provides antigenic peptides for major histocompatibility complex class I and class II presentation, and a maturation signal that is delivered by exposure to necrotic tumor cells, their supernatants, or standard maturation stimuli, e.g., monocyte-conditioned medium. Thus, DCs are able to distinguish two types of tumor cell death, with necrosis providing a control that is critical for the initiation of immunity.

title: Natural adjuvants: Endogenous activators of dendritic cells
abstract: Dendritic cells, the most potent antigen-presenting cells, need to be activated before they can function to initiate an immune response. We report here that, in the absence of any foreign substances, dendritic cells can be activated by endogenous signals received from cells that are stressed, virally infected or killed necrotically, but not by healthy cells or those dying apoptotically. Injected in vivo with an antigen, the endogenous activating substances can function as natural adjuvants to stimulate a primary immune response, and they may represent the natural initiators of transplant rejection, spontaneous tumor rejection, and some forms of autoimmunity.

title: Recognizing death: the phagocytosis of apoptotic cells.
abstract: Although apoptotic cell death is widespread, dying cells are rarely seen in situ because of their rapid clearance by neighbouring phagocytes. Phagocytic recognition of apoptotic cells is less well understood than the death programme itself, but an increasing number of recent studies are highlighting its importance. This review discusses the nature of the receptors that have been implicated in apoptotic cell phagocytosis, the mechanisms of uptake and the immunological consequences of apoptotic cell ingestion.

title: Induction of Anti-Human Immunodeficiency Virus Type 1 (HIV-1) CD8+ and CD4+ T-Cell Reactivity by Dendritic Cells Loaded with HIV-1 X4-Infected Apoptotic Cells
abstract: T-cell responses to X4 strains of human immunodeficiency virus type 1 (HIV-1) are considered important in controlling progression of HIV-1 infection. We investigated the ability of dendritic cells (DC) and various forms of HIV-1 X4 antigen to induce anti-HIV-1 T-cell responses in autologous peripheral blood mononuclear cells from HIV-1-infected persons. Immature DC loaded with HIV-1 IIIB-infected, autologous, apoptotic CD8− cells and matured with CD40 ligand induced gamma interferon production in autologous CD8+ and CD4+ T cells. In contrast, mature DC loaded with HIV-1 IIIB-infected, necrotic cells or directly infected with cell-free HIV-1 IIIB were poorly immunogenic. Thus, HIV-1-infected cells undergoing apoptosis serve as a rich source of X4 antigen for CD8+ and CD4+ T cells by DC. This may be an important mechanism of HIV-1 immunogenicity and provides a strategy for immunotherapy of HIV-1-infected patients on combination antiretroviral therapy.

title: Salmonella-Induced Apoptosis of Infected Macrophages Results in Presentation of a Bacteria-Encoded Antigen after Uptake by Bystander Dendritic Cells
abstract: Salmonella typhimurium is a gram-negative bacterium that survives and replicates inside vacuolar compartments of macrophages. Infection of macrophages with S. typhimurium grown under conditions allowing expression of the type III secretion system results in apoptotic death of the infected cells. Here, we show that infection of bone marrow–derived macrophages (MΦ) with wild-type S. typhimurium 14028 results in presentation of epitopes derived from a bacteria-encoded antigen on major histocompatibility complex (MHC) class I and MHC class II molecules after internalization of apoptotic MΦ by bystander dendritic cells (DCs). In contrast, infection of MΦ with the phoP constitutive mutant strain CS022, which does not induce apoptosis in infected MΦ, does not result in presentation of a bacteria-derived antigen by bystander DCs unless the infected MΦ are induced to undergo apoptosis by treatment with lipopolysaccharide and ATP. DCs appear to be unique in their ability to present antigens derived from MΦ induced to undergo apoptosis by Salmonella, as bystander MΦ are not capable of presenting the bacteria-derived antigen despite the fact that they efficiently internalize the apoptotic cells. These data suggest that apoptosis induction by bacterial infection of MΦ may not be a quiescent death that allows the bacteria to escape recognition by the immune system, but rather may contribute to an antimicrobial immune response upon engulfment by bystander DCs.

title: Heat Shock Protein–Peptide Complexes, Reconstituted In Vitro, Elicit Peptide-specific Cytotoxic T Lymphocyte Response and Tumor Immunity
abstract: Heat shock protein (HSP) preparations derived from cancer cells and virus-infected cells have been shown previously to elicit cancer-specific or virus-specific immunity. The immunogenicity of HSP preparations has been attributed to peptides associated with the HSPs. The studies reported here demonstrate that immunogenic HSP–peptide complexes can also be reconstituted in vitro. The studies show that (a) complexes of hsp70 or gp96 HSP molecules with a variety of synthetic peptides can be generated in vitro; (b) the binding of HSPs with peptides is specific in that a number of other proteins tested do not bind synthetic peptides under the conditions in which gp96 molecules do; (c) HSP–peptide complexes reconstituted in vitro are immunologically active, as tested by their ability to elicit antitumor immunity and specific CD8+ cytolytic T lymphocyte response; and (d) synthetic peptides reconstituted in vitro with gp96 are capable of being taken up and re-presented by macrophage in the same manner as gp96– peptides complexes generated in vivo. These observations demonstrate that HSPs are CD8+ T cell response–eliciting adjuvants.

title: Immunization with a Lymphocytic Choriomeningitis Virus Peptide Mixed with Heat Shock Protein 70 Results in Protective Antiviral Immunity and Specific Cytotoxic T Lymphocytes
abstract: Heat shock proteins (hsp's) isolated from murine cancer cells can elicit protective immunity and specific cytotoxic T lymphocytes (CTLs) by channeling tumor-derived peptides bound to hsp's to the major histocompatibility class I antigen presentation pathway. Here we have investigated if hsp70 can be used in a novel peptide vaccine for the induction of protective antiviral immunity and memory CTLs. A CTL epitope from the well-defined lymphocytic choriomeningitis virus (LCMV) system was mixed with recombinant hsp70 in vitro under conditions that optimize peptide binding to hsp70. Mice were immunized with the hsp70–peptide mixture and challenged with LCMV. Virus titers were reduced 10–100-fold in these mice compared to control mice. Immunization with the hsp70–peptide mixture resulted in the development of CTL memory cells that could be reactivated during LCMV infection, and that in a 51Cr-release assay could lyse cells pulsed with the same peptide, but not cells pulsed with another LCMV peptide. These results show that hsp70 can be used with CTL epitopes to induce efficient protective antiviral immunity and the generation of peptide-specific CTLs. The results also demonstrate the usefulness of hsp70 as an alternative to adjuvants and DNA vectors for the delivery of CTL epitopes to antigen-presenting cells.

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

title: The heat shock protein gp96 induces maturation of dendritic cells and down‐regulation of its receptor
abstract: 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‐α 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 αvβ3 and αvβ5. Together, our findings provide an additional explanation for the remarkable immunogenicity of gp96 as a cross‐priming antigen carrier and direct activator of DC.

title: CD91: a receptor for heat shock protein gp96
abstract: 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 α2-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, α2-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.

title: Cutting Edge: Heat Shock Protein 60 Is a Putative Endogenous Ligand of the Toll-Like Receptor-4 Complex1
abstract: Human heat shock protein 60 (hsp60) elicits a potent proinflammatory response in cells of the innate immune system and therefore has been proposed as a danger signal of stressed or damaged cells. We report here that macrophages of C3H/HeJ mice, carrying a mutant Toll-like-receptor (Tlr) 4 are nonresponsive to hsp60. Both the induction of TNF-α and NO formation were found dependent on a functional Tlr4 whereas stimulation of macrophages by CpG DNA was Tlr4 independent. We conclude that Tlr4 mediates hsp60 signaling. This is the first report of a putative endogenous ligand of the Tlr4 complex.

title: Endocytosed HSP60s Use Toll-like Receptor 2 (TLR2) and TLR4 to Activate the Toll/Interleukin-1 Receptor Signaling Pathway in Innate Immune Cells*
abstract: Heat shock proteins (HSPs) require no adjuvant to confer immunogenicity to bound peptides, as if they possessed an intrinsic “danger” signature. To understand the proinflammatory nature of HSP, we analyzed signaling induced by human and chlamydial HSP60. We show that both HSP60s activate the stress-activated protein kinases p38 and JNK1/2, the mitogen-activated protein kinases ERK1/2, and the I-κB kinase (IKK). Activation of JNK and IKK proceeds via the Toll/IL-1 receptor signaling pathway involving MyD88 and TRAF6. Human fibroblasts transfected with TLR2 or TLR4 plus MD-2 gain responsiveness to HSP60, while TLR2- or TLR4-defective cells display impaired responses. Initiation of signaling requires endocytosis of HSP60 that is effectively inhibited by serum component(s). The results revealed that adjuvanticity of HSP60 operates similar to that of classical pathogen-derived ligands.

title: Human 60-kDa heat-shock protein: a danger signal to the innate immune system.
abstract: Mammalian 60-kDa heat-shock protein (hsp60) is a key target of T cell and Ab responses in chronic inflammation or atherosclerosis. We show in this study that human hsp60 is also an Ag recognized by cells of the innate immune system, such as macrophages. Both mouse and human macrophages respond to contact with exogenous human hsp60 with rapid release of TNF-alpha; mouse macrophages in addition produce nitric oxide. The proinflammatory macrophage response is hsp60 dose dependent and similar in kinetics and extent to LPS stimulation. Human hsp60 was found to synergize with IFN-gamma in its proinflammatory activity. Finally, human hsp60 induces gene expression of the Th1-promoting cytokines IL-12 and IL-15. These findings identify autologous hsp60 as a danger signal for the innate immune system, with important implications for a role of local hsp60 expression/release in chronic Th1-dependent tissue inflammation.

title: HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine
abstract: Here, we demonstrate a previously unknown function for the 70-kDa heat-shock protein (HSP70) as a cytokine. HSP70 bound with high affinity to the plasma membrane, elicited a rapid intracellular calcium flux, activated nuclear factor (NF)-κB and upregulated the expression of pro-inflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β and IL-6 in human monocytes. Furthermore, two different signal transduction pathways were activated by exogenous HSP70: one dependent on CD14 and intracellular calcium, which resulted in increased IL-1β, IL-6 and TNF-α; and the other independent of CD14 but dependent on intracellular calcium, which resulted in an increase in TNF-α but not IL-1β or IL-6. These findings indicate that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of an previously unrecognized function for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as chaperone and cytokine.

title: Immunosuppressive effects of apoptotic cells
abstract: Apoptotic cell death is important in the development and homeostasis of multicellular organisms and is a highly controlled means of eliminating dangerous, damaged or unnecessary cells without causing an inflammatory response or tissue damage,. We now show that the presence of apoptotic cells during monocyte activation increases their secretion of the anti-inflammatory and immunoregulatory cytokine interleukin 10 (IL-10) and decreases secretion of the proinflammatory cytokines tumour necrosis factor-α (TNF-α), IL-1 and IL-12. This may inhibit inflammation and contribute to impaired cell-mediated immunity in conditions associated with increased apoptosis, such as viral infections, pregnancy, cancer and exposure to radiation.

title: A receptor for phosphatidylserine-specific clearance of apoptotic cells
abstract: The culmination of apoptosis in vivo is phagocytosis of cellular corpses. During apoptosis, the asymmetry of plasma membrane phospholipids is lost, which exposes phosphatidylserine externally. The phagocytosis of apoptotic cells can be inhibited stereospecifically by phosphatidylserine and its structural analogues, but not by other anionic phospholipids, suggesting that phosphatidylserine is specifically recognized. Using phage display, we have cloned a gene that appears to recognize phosphatidylserine on apoptotic cells. Here we show that this gene, when transfected into B and T lymphocytes, enables them to recognize and engulf apoptotic cells in a phosphatidylserine-specific manner. Flow cytometric analysis using a monoclonal antibody suggested that the protein is expressed on the surface of macrophages, fibroblasts and epithelial cells; this antibody, like phosphatidylserine liposomes, inhibited the phagocytosis of apoptotic cells and, in macrophages, induced an anti-inflammatory state. This candidate phosphatidylserine receptor is highly homologous to genes of unknown function in Caenorhabditis elegans and Drosophila melanogaster, suggesting that phosphatidylserine recognition on apoptotic cells during their removal by phagocytes is highly conserved throughout phylogeny.

title: Immunobiology of dendritic cells.
abstract: Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. DCs capture and transfer information from the outside world to the cells of the adaptive immune system. DCs are not only critical for the induction of primary immune responses, but may also be important for the induction of immunological tolerance, as well as for the regulation of the type of T cell-mediated immune response. Although our understanding of DC biology is still in its infancy, we are now beginning to use DC-based immunotherapy protocols to elicit immunity against cancer and infectious diseases.

title: Primary Tumor Tissue Lysates Are Enriched in Heat Shock Proteins and Induce the Maturation of Human Dendritic Cells1
abstract: Upon exposure to lysates or supernatants of necrotic transformed cell lines, human dendritic cells (DCs) undergo maturation. In contrast, DCs exposed to apoptotic transformed cell lines or necrotic lysates of primary cells remain immature. Analysis of supernatants of necrotic transformed cell lines showed them to be enriched in the heat shock proteins (hsp)70 and gp96, in contrast to supernatants of primary cells. Likewise, cells from a variety of primary human tumors contained considerably higher levels of hsp than their normal autologous tissue counterparts. Of the majority of human tumors enriched in hsps (hsp70 and/or gp96), their corresponding lysates matured DCs. The maturation effect of tumor cell lysates was abrogated by treatment with boiling, proteinase K, and geldanamycin, an inhibitor of hsps, suggesting that hsps rather than endotoxin or DNA were the responsible factors. Supporting this idea, highly purified, endotoxin-depleted hsp70, induced DC maturation similar to that seen with standard maturation stimuli LPS and monocyte conditioned medium. These results suggest that the maturation activity inherent within tumor cells and lines is mediated at least in part by hsps. The release of hsps in vivo as a result of cell injury should promote immunity through the maturation of resident DCs.

title: Macrophages orchestrate the immune response to tumor cell death.
abstract: The mechanisms by which the immune system distinguishes normal developmental cell death from pathological immunogenic cell killing are central to effective cancer immunotherapy. Using HSVtk suicide gene therapy, we showed that macrophages can distinguish between tumor cells dying through classical apoptosis and tumor cells engineered to die through nonapoptotic mechanisms, resulting in secretion of either immunosuppressive cytokines (interleukin 10 and transforming growth factor beta) or inflammatory cytokines (tumor necrosis factor alpha or interleukin 1beta), respectively. Additionally heat shock protein 70 acts as one component of a bimodal alarm signal that activates macrophages in the presence of stressful, immunogenic tumor cell killing. These differential responses of macrophages can also be used to vaccinate mice against tumor challenge, using adoptive transfer, as well as to cure mice of established tumors.

title: Apoptosis A Link between Cancer Genetics and Chemotherapy
abstract: Defects in apoptosis underpin both tumorigenesis and drug resistance, and because of these defects chemotherapy often fails. Understanding the molecular events that contribute to drug-induced apoptosis, and how tumors evade apoptotic death, provides a paradigm to explain the relationship between cancer genetics and treatment sensitivity and should enable a more rational approach to anticancer drug design and therapy.

title: The Induction of Tolerance by Dendritic Cells That Have Captured Apoptotic Cells
abstract: What makes a protein immunogenic, particularly for strong T cell–mediated immunity? To a first approximation, this determination seems to be made by dendritic cells (DCs). Immature DCs, as in skin ([1][1])([2][2])([3][3])([4][4]), lung ([5][5]), blood ([6][6])([7][7]), and spleen ([7][7])([8][8