The endoplasmic reticular heat shock protein gp96 is transcriptionally upregulated in interferon-treated cells.

A cDNA clone complementary to an interferon (IFN)-induced mRNA approximately 3 kb in length was identified and sequenced revealing homology with the endoplasmic reticular heat shock protein/ATPase gp96. Both IFN-alpha and -gamma transcriptionally upregulate expression of this gene. gp96 transcripts, protein, and ATPase activity are shown to be enhanced as a result of IFN treatment in two human cell lines and this effect requires de novo protein synthesis. gp96 molecules have recently been implicated in the presentation of endogenous antigens. A number of the key elements in this pathway, the transporter proteins, the major histocompatibility complex (MHC)-linked units of the proteasomes and the MHC class I molecules are known to be IFN inducible. Our results show that yet another molecule suggested to play an accessory role in the endogenous presentation pathway is IFN inducible. Further, our studies represent the first demonstration of modulation of expression of a heat shock protein by a cytokine and identify a new enzymatic activity upregulated in IFN-treated cells.

Heat shock protein-peptide complexes, reconstituted in vitro, elicit peptide-specific cytotoxic T lymphocyte response and tumor immunity.

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.

Coupling and uncoupling of tumor immunity and autoimmunity.

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.

Immunity against cancer: lessons learned from melanoma.

Most major advances in human cancer immunology and immunotherapy have come from studies in melanoma. We are beginning to understand the immune repertoire of T cells and antibodies that are active against melanoma, with recent glimpses of the CD4(+) T cell repertoire. The view of what the immune system can see is extending to mutations and parts of the genome that are normally invisible.

Evaluation of Elispot assays: influence of method and operator on variability of results.

In this study, a comprehensive comparative analysis of different evaluation methods of Elispot plates was performed. Three investigators using three different evaluation approaches read 50 randomly selected wells at three different time points. The methods were: (1) manual evaluation using a stereomicroscope, (2) automated evaluation using an image analysis reader system with reading parameters established by each investigator, and (3) automated evaluation using a reader system with preset reading parameters using assay-specific controls. We demonstrate that manual evaluation had the highest variability both within the same method and when comparing all methods, followed by automated evaluation with investigator-dependent parameters. The variability was low only when all investigators used the same parameters established using assay-specific controls. This variability was independent of operator or spot number per well. Based on this study, recommendations for standardization and validation procedures of Elispot assay performance and evaluation procedures are presented.

Alternative roles for interferon-gamma in the immune response to DNA vaccines encoding related melanosomal antigens.

Tyrosinase-related proteins-1 and -2 (gp75/TRP-1 and TRP-2) are melanosomal membrane glycoproteins recognized by antibodies and T-cells from patients with melanoma. Xenogeneic DNA immunization against gp75/TRP-1 generates antibody-dependent tumor immunity and autoimmune depigmentation. In contrast xenogeneic TRP-2 DNA immunization induces immunity mediated by CD8+ T-cells. The role of IFN-gamma in the generation of tumor immunity and autoimmune depigmentation in these two models was investigated. No tumor protection and minimal depigmentation was observed after immunization with human TRP-2 DNA in mice deficient in IFN-gamma ligand. Repletion with recombinant murine IFN-gamma restored tumor immunity. Experiments using IL4 deficient mice demonstrated that tumor immunity was unaffected but that autoimmune depigmentation was potentially accelerated, consistent with down-modulation of autoimmunity against TRP-2 by IL4. In contrast, IFN-gamma was not required for the generation of immunity to gp75/TRP-1. In fact, exogenous IFN-gamma ablated autoantibody responses against gp75/TRP-1 after xenogeneic DNA immunization, consistent with a down-regulatory effect of IFN-gamma. These results show that immunity to TRP-2 following DNA immunization uses an IFN-gamma-dependent Th1 pathway, but immunity to gp75/TRP-1 is down-regulated by IFN-gamma.

Protective cellular immunity against a spontaneous mammary carcinoma from ras transgenic mice.

Mammary carcinomas of v-Ha-ras transgenic mice closely resemble human breast cancer in their multi-step nature and in the requirement of genetic, hormonal and somatic mutational events for full-scale malignancy. We demonstrate that spontaneous breast cancers derived from v-Ha-ras transgenic FVB (H-2q) mice are highly immunogenic and that they elicit a protective T cell response. A continuous tumor cell line OM-2 has been established from a progressively growing mammary tumor and three sublines OM-10, OM-12 and OM-14 have been derived by in vivo passage of OM-2. All lines express the v-Ha-ras gene product and surface MHC class I. The parental OM-2 line is highly immunogenic and behaves like a regressor tumor. The regression of OM-2 is mediated by CD8+ T lymphocytes, although CD4+ lymphocytes also appear to play a limited role. Cytotoxic T lymphocytes (CTLs) obtained from mice immunized with OM-2 show MHC class I-restricted, specific T cell cytotoxicity against OM-2 but not normal fibroblasts derived from ras transgenic mice. The anti-OM-2 CTLs lyse the OM-2 sublines OM-12 and OM-14, although to a lesser degree than OM-2, and do not lyse OM-10, in spite of the fact that all cell lines express comparable levels of activated ras and MHC class I. Our studies represent the first analysis of protective T cell response to breast cancer and demonstrate that contrary to expectation, the spontaneous breast cancers are highly immunogenic and that the immune response does not appear to be directed to activated ras.

Effective antigen cross-presentation by prostate cancer patients' dendritic cells: implications for prostate cancer immunotherapy.

Despite the potency with which dendritic cells (DCs) are able to utilize the exogenous MHC I antigen cross-presentation pathway to cross-present antigen for the activation of killer T cells in model systems, concern about defects in immune function in cancer patients has led to uncertainty regarding whether immune cells derived from patients can effectively be used to generate tumor vaccines. We have undertaken a careful analysis of the potency of using DCs obtained from prostate cancer patients to cross-present antigen derived from human prostate tumor cells for the activation of antigen-specific T cells. Such DCs can be matured ex vivo into functionally active cells and are capable of cross-presenting influenza antigen derived from internalized apoptotic prostate tumor cells. Importantly, we demonstrate effective stimulation of both CD4+ and CD8+ T cells, as evident by production of IFN-gamma, and the ability of CD8+ T cells to differentiate into effector CTLs. These results, defining conditions in which prostate cancer patient DCs can efficiently utilize the cross-presentation pathway and in which apoptotic tumor can serve as a source of antigen for DCs to activate T cells, demonstrate that this system warrants clinical study as a potential immunotherapy.

Heat shock protein-based cancer vaccines and related thoughts on immunogenicity of human tumors.

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

Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by major histocompatibility complex I molecules.

The studies reported here bear on the events in the cytosol that lead to trafficking of peptides during antigen processing and presentation by major histocompatibility complex (MHC) I molecules. We have introduced free antigenic peptides or antigenic peptides bound to serum albumin or to cytosolic heat shock proteins hsp90 (and its endoplasmic reticular homologue gp96) or hsp70 into the cytosol of living cells and have monitored the presentation of the peptides by appropriate MHC I molecules. The experiments show that (i) free peptides or serum albumin-bound peptides, introduced into the cytosol, become ligands of MHC I molecules at a far lower efficiency than peptides chaperoned by any of the heat shock proteins tested and (ii) treatment of cells with deoxyspergualin, a drug that binds hsp70 and hsp90 with apparent specificity, abrogates the ability of cells to present antigenic peptides through MHC I molecules, and introduction of additional hsp70 into the cytosol overcomes this abrogation. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing.

Generation of tumor-specific cytotoxic T lymphocytes and memory T cells by immunization with tumor-derived heat shock protein gp96.

Tumor-derived heat shock proteins have been shown previously to elicit specific prophylactic immunity to cognate tumors. Here we establish that administration of gp96 results in generation of tumor-specific cytotoxic T lymphocyte response in addition to protective immunity. In one instance, gp96 preparations are shown to elicit an immune response against a tumor, where intact tumor cells are unable to do so. Finally, our results indicate that gp96 preparations elicit a memory T cell response that is capable of being recalled.

Heat shock protein vaccines against cancer.

Vaccination of mice with heat shock proteins (HSPs) derived from a tumor makes the mice resistant to the tumor from which the HSP was obtained. This phenomenon has been demonstrated with three HSPs--gp96, hsp90, and hsp70. Vaccination with HSPs also elicits antigen-specific cytotoxic T lymphocytes (CTLs). The specific immunogenicity of HSPs derives apparently, not from the HSPs per se, but from the peptides bound to them. These observations provide the basis for a new generation of vaccines against cancer. The HSP-based cancer vaccines circumvent two of the most intractable hurdles to cancer immunotherapy. One of them is the possibility that human cancers, like cancers of experimental animals, are antigenically distinct. The prospect of identification of immunogenic antigens of individual cancers from patients is daunting to the extent of being impractical. The observation that HSPs chaperone antigenic peptides of the cells from which they are derived circumvents this extraordinary hurdle. Second, most current approaches to cancer immunotherapy focus on determining the CTL-recognized epitopes of cancer cell lines. This approach requires the availability of cell lines and CTLs against cancers. These reagents are unavailable for an overwhelming proportion of human cancers. In contrast, the HSP-based vaccines do not depend on the availability of cell lines or CTLs nor do they require definition of the antigenic epitopes of cancer cells. These advantages, among others, make HSPs attractive and novel immunogens against cancer.

Heat shock proteins transfer peptides during antigen processing and CTL priming.

Recently emerging evidence indicates that the heat shock proteins (HSPs) gp96, hsp90, and hsp70 associate with antigenic peptides derived from cellular proteins. This evidence forms the basis of the following two hypotheses: 1) that HSPs constitute a relay line in which the peptides, after generation in the cytosol by the action of proteases, are transferred from one HSP to another, until they are finally accepted by MHC class I molecules in the endoplasmic reticulum, and 2) that the binding of peptides by HSPs constitutes a key step in the priming of cytotoxic T lymphocytes (CTLs) in vivo. The following chain of events is suggested: HSPs are released from virus-infected cells or tumor cells in vivo during lysis of cells during infection or by the action of antibodies or nonspecific effectors. The HSPs, which are now complexed with antigenic peptides derived from the cognate cells, are taken up by macrophage or other specialized antigen-presenting cells, possibly by a receptor-mediated mechanism. The HSP-borne peptide is then routed to the endogenous presentation pathway in the antigen-presenting cell and is displayed in the context of that cell's MHC class I, where it is finally recognized by the precursor CTLs. Thus it is suggested that, as with antigen presentation by MHC class II molecules, presentation by MHC class I molecules is also carried out primarily by the host antigen-presenting cells. This mechanism explains the phenomenon of cross-priming and has implications for the development of immunological strategies against cancer and infectious diseases.

Membranes activate tumor- and virus-specific precursor cytotoxic T lymphocytes in vivo and stimulate tumor-specific T lymphocytes in vitro: implications for vaccination.

Plasma membranes contain the entire antigenic repertoire of a cell in the form of processed antigens presented as peptides by major histocompatibility complex (MHC) class I molecules. We report here that plasma membranes but not internal membranes of cognate tumors stimulate murine fibrosarcoma and human melanoma-specific cytotoxic T lymphocyte (CTL) clones in vitro in an antigen-specific. MHC class I-restricted manner. Although stimulation of CTLs by class I-peptide complexes on reconstituted membranes has been documented before, this is the first demonstration of stimulation of cloned CTLs by natural, endogenously processed MHC class I-peptide complexes on plasma membranes. In addition to their ability to stimulate CTLs in vitro, immunization of syngeneic mice with membranes derived from ultraviolet-induced tumor cells, SV40 transformed fibroblasts, or influenza-infected fibroblasts elicits an antigen-specific, MHC class I restricted primary CTL response. To the best of our knowledge, this is also the first demonstration of the ability of cellular membranes to prime an MHC class I-restricted CTL responses in vivo. The ability of membranes to prime a CTL response in vivo suggests that they may be used as T-cell vaccines against tumors or infectious viruses. This approach circumvents the difficulties in generation of human tumor cell lines and identification of CTL-recognized determinants for vaccination and avoids some of the risks associated with whole-cell vaccination such as inoculation of patients with immunosuppressive factors, transforming DNA, or infectious viruses.

Injection of DNA encoding granulocyte-macrophage colony-stimulating factor recruits dendritic cells for immune adjuvant effects.

An important issue for effective vaccines is the development of potent adjuvants that can facilitate induction or augmentation of immunity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for myeloid progenitors of monocytes and dendritic cells (DC), which upon maturation are antigen-presenting cells (APC). The adjuvant effects of inoculation of DNA encoding GM-CSF into skin were studied. Initial experiments examined whether the GM-CSF gene injected into the skin of mice could affect the density of epidermal DC (Langerhans cells). DNA encoding GM-CSF delivered by particle bombardment into skin resulted in a significant increase of epidermal DC at the inoculation site. Kinetic analysis of epidermal recruitment after GM-CSF inoculation showed an increase in DC that peaked at seven days. This increase was accompanied by recruitment of DC into draining lymph nodes. The adjuvant effects of DNA encoding GM-CSF inoculated into skin were measured by the ability to augment antibody and T-cell responses against poorly immunogenic tumor antigens. Peptide immunization at skin sites containing epidermal DC newly recruited by GM-CSF DNA elicited T-cell responses against mutant p53, whereas peptide immunization of control skin sites did not elicit any detectable T-cell responses. Likewise, generation of antibodies following immunization with DNA encoding human gp75TRP1, a tyrosinase family member expressed by melanomas, was accelerated and protection from tumor challenge augmented by GM-CSF DNA.