Human papillomavirus type 16 (HPV16) infection has been linked to the development of cervical and anal dysplasia and cancer. One hallmark of persistent infection is the synthesis of the viral E7 protein in cervical epithelial cells. The expression of E7 in dysplastic and transformed cells and its recognition by the immune system as a foreign antigen make it an ideal target for immunotherapy. Utilizing the E7-expressing murine tumor cell line, TC-1, as a model of cervical carcinoma, an immunotherapy based on the administration of an adjuvant-free fusion protein comprised of Mycobacterium bovis BCG Hsp65 linked to HPV16 E7 (HspE7) has been developed. Initial in vitro analyses indicate that immunization with HspE7 results in the induction of a type 1 immune response based on the pattern of secreted cytokines and the presence of cytolytic activity following antigenic recall. It has been previously shown that prophylactic immunization with HspE7 protected mice against challenge with TC-1 cells and that these tumor-free animals are also protected against rechallenge with TC-1 cells. The present report shows that a single therapeutic immunization with HspE7 induces regression of palpable tumors, confers protection against tumor rechallenge, and is associated with long-term survival (>253 days). In vivo studies using mice with targeted mutations in CD8 or MHC class II or depleted of CD8 or CD4 lymphocyte subsets demonstrate that tumor regression following therapeutic HspE7 immunization is CD8 dependent and CD4 independent. These studies extend previous observations on the induction of CTL by Hsp fusion proteins and are consistent with the clinical application of HspE7 as an immunotherapy for human cervical and anal dysplasia and cancer.

Heat shock protein 70 (HSP70) is a molecular chaperone involved in protein folding and resistance to the deleterious effects of stress. Here we show that HSP70 suppresses transcription of c-fos, an early response gene that is a key component of the ubiquitous AP-1 transcription factor complex. HSP70 repressed Ras-induced c-fos transcription only in the presence of functional heat shock factor1 (HSF1). This suggests that HSP70 functions as a corepressor with HSF1 to inhibit c-fos gene transcription. Therefore, besides its known function in the stress response, HSP70 also has the property of a corepressor and combines with HSF1 to antagonize Fos expression and may thus impact multiple aspects of cell regulation.

In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.

Variances, particularly single nucleotide polymorphisms (SNP), in the genomic sequence of individuals are the primary key to understanding gene function as it relates to differences in the susceptibility to disease, environmental influences, and therapy. In this report, the HSP70B′ gene is the target sequence for mutation detection in biopsy samples from human prostate cancer patients undergoing combined hyperthermia and radiation therapy at the Dana-Farber Cancer Institute, using temperature-modulated heteroduplex analysis (TMHA). The underlying principles of TMHA for mutation detection using DHPLC technology are discussed. The procedures involved in amplicon design for mutation analysis by DHPLC are detailed. The melting behavior of the complete coding sequence of the target gene is characterized using WAVEMAKER^TM software. Four overlapping amplicons, which span the complete coding region of the HSP70B′ gene, amenable to mutation detection by DHPLC were identified based on the software-predicted melting profile of the target sequence. TMHA was performed on PCR products of individual amplicons of the HSP70B′ gene on the WAVE® Nucleic Acid Fragment Analysis System. The criteria for mutation calling by comparing wild-type and mutant chromatographic patterns are discussed.

We recently elucidated a novel function for the 70-kDa heat shock protein (HSP70) as a chaperone and a cytokine, a chaperokine in human monocytes. Here we show that peptide-bearing and peptide-negative HSP70 preparations isolated from EMT6 mammary adenocarcinoma cells (EMT6-HSP70) act as chaperokines when admixed with murine splenocytes. EMT6-HSP70 bound with high affinity to the surface of splenocytes recovered from naive BALB/c mice. The [Ca² ]i inhibitor BAPTA dose dependently inhibited HSP70- but not LPS-induced NF-κB activity and subsequent augmentation of proinflammatory cytokine TNF-α, IL-1β, and IL-6 production. Taken together, these results suggest that presence of peptide in the HSP70 preparation is not required for spontaneous activation of cells of the innate immune system.

Heat shock transcription factor 1(HSF1) activation is a multistep process. The conversion of a latent cytoplasmic form to a nuclear, DNA binding state appears to be activated by nonsteroidal anti-inflammatory drugs. In previous studies, we showed that HSF 1 is phosphorylated by the protein kinase RSK2 in vitro and that this effect is inhibited by nonsteroidal anti-inflammatory drugs at the concentration that leads to the activation of HSF1 in vivo (Stevenson et al 1999). In the present study, using cells from a patient with Coffin-Lowry syndrome (deficient in RSK2), we demonstrate that RSK2 slightly represses activation of HSF1 in vivo at 37°C. In Coffin-Lowry syndrome cells, HSF1-HSE DNA binding activity after treatment with sodium salicylate was slightly higher than that in untreated cells, indicating that although RSK2 is involved in HSF1 regulation, it is not the unique protein kinase that suppresses HSF1-HSE binding activity at 37°C. However, heat shock treatment resulted in significantly higher HSF1-HSE binding activity in Coffin-Lowry syndrome cells as compared with normal controls, suggesting that RSK2 represses HSF1-HSE binding activity during heat shock.

A tumor-selective cell surface localization of heat shock protein 70 (Hsp70), the major heat-inducible member of the Hsp70 group, correlates with an increased sensitivity to lysis mediated by human natural killer (NK) cells and, therefore, might be of clinical relevance. With the exception of mammary carcinomas, an Hsp70 plasma membrane expression was found on freshly isolated human biopsy material of colorectal, lung, neuronal, and pancreas carcinomas, liver metastases, and leukemic blasts of patients with acute myelogenous leukemia. Since normal tissues and bone marrow of healthy human individuals do not express Hsp70 on the cell surface, Hsp70 can be considered as a tumor-selective structure in vivo. Furthermore, we demonstrate that autologous, Hsp70-positive leukemic blasts can be killed by NK cells stimulated with low doses of interleukin 2 plus recombinant Hsp70 protein.

The ability of heat shock proteins to (1) chaperone peptides, including antigenic peptides; (2) interact with antigen-presenting cells through a receptor; (3) stimulate antigen-presenting cells to secrete inflammatory cytokines; and (4) mediate maturation of dendritic cells, makes them a unique starting point for generation of immune responses. These properties also permit the use of heat shock proteins for development of a new generation of prophylactic and therapeutic vaccines against cancers and infectious diseases.

Immunization with heat shock proteins has protective effects in models of induced arthritis. Analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer and immunization with selected T cell epitopes (synthetic peptides) have indicated the protection to be mediated by T cells directed to conserved hsp epitopes. This was shown first for mycobacterial hsp60 and later for mycobacterial hsp70. Fine specificity analysis showed that such T cells were cross-reactive with the homologous self hsp. Therefore protection by microbial hsp reactive T cells can be by cross-recognition of self hsp overexpressed in the inflamed tissue. Preimmunization with hsp leads to a relative expansion of such self hsp cross-responsive T cells. The regulatory nature of such T cells may originate from mucosal tolerance maintained by commensal flora derived hsp or from partial activation through recognition of self hsp as a partial agonist (Altered Peptide Ligand) or in the absence of proper costimulation. Recently, we reported the selective upregulation of B7.2 on microbial hsp60 specific T cells in response to self hsp60. Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Also, regulatory T cells produced IL10.

Inflammation of the skin and systemic fever, both of which occur with injury or infection, include a hyperthermic component that many believe constitutes a physiological stress. Such increases in local or systemic body temperature may also have a regulatory effect on immune function. Langerhans cells (LCs), the dendritic cells of the skin, continuously monitor the extracellular matrix of the skin by taking up particles and microbes that they then carry to draining lymph nodes for presentation to T lymphocytes. We hypothesize that the thermal element of inflammation and/or fever may help regulate the activation and migration of LCs out of the epidermis. To test this hypothesis, Balb/c mice were exposed to a mild (39.8°C ± 0.2°C), long-duration (6 hours) whole body hyperthermia (WBH) treatment, which mimics the thermal component of fever. The number of LCs and their morphology were analyzed at various time points up to 7 days after the initiation of WBH. The LCs of the ear epidermis were visualized using a fluorescein isothiocyanate–conjugated antibody specific for the major histocompatibility complex (MHC) class II molecule and confocal microscopy. Although MHC class II staining was diffuse on the surface of the LC body and dendritic extensions of both WBH and control samples, the WBH-treated LCs exhibited a more punctate morphology with fewer dendritic processes compared with control LCs. A significant decrease in the number of LCs was also observed 1 to 5 days after WBH treatment. Furthermore, in vitro heating of Balb/c ear skin cultures at 40°C for 6 to 8 hours enhanced the numbers of viable LCs that migrated into the culture wells. These results suggest that WBH treatment stimulates epidermal LCs in the absence of foreign antigen.

Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by up-regulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-α and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.

The heat shock response is an ancient and highly conserved process that is essential for surviving environmental stresses, including extremes of temperature. Fever is a more recently evolved response, during which organisms temporarily subject themselves to thermal stress in the face of infections. We review studies showing that fever is beneficial in the infected host. We show that core temperatures achieved during fever can activate the heat shock response and discuss some of the biochemical consequences of such an effect. We present data suggesting 4 possible mechanisms by which fever might confer protection: (1) directly killing or inhibiting growth of pathogens; (2) inducing cytoprotective heat shock proteins (Hsps) in host cells; (3) inducing expression of pathogen Hsps, an activator of host defenses; and (4) modifying and orchestrating host defenses. Two of these mechanisms directly involve the heat shock response. We describe how heat shock factor-1, the predominant heat-induced transcriptional enhancer not only activates transcription of Hsps but also regulates expression of pivotal cytokines and early response genes. The relationship between fever and the heat shock response is an illuminating example of how a more recently evolved response might exploit preexisting biochemical pathways for a new function.