Abstracts from the Second International Conference on Heat-Shock Proteins in Immune Response, 8-12 October 2000 Farmington, CT USA

Active specific immunotherapy in patients with renal cell carcinoma using autologus tumor derived heat shock protein peptide complex 96 vaccine

Robert J. Amato¹,Cherylyn Savary², Steve Tomasovic², Dirk Reitsma³, Elma Hawkins³, and Pramod K. Srivastava⁴.

¹Baylor University, TX; ²MD Anderson Cancer Center, Houston, TX; ³Antigenics, Inc., NY, NY; ⁴University of CT School of Medicine, Farmington, CT.

Preparations of stress proteins gp96 from tumor cells are active as tumor vaccines by eliciting immune responses against mixtures of individual tumor peptide antigens which are complexed to gp96. Due to the individual antigenicity of tumors, a vaccine consisting of tumor derived gp96 has to be prepared individually for each patient from autologous tumor tissue. The gp96 is nonpolymorphic but acts as a chaperone for tightly bound immunogenic peptides, which are believed to represent a full repertoire of immunogens. Thus, HSP peptide complexes or gp96-peptide complexes (HSPPC-96) isolated from a patient's tumor may present the unique opportunity to deliver a vaccine specific to that patient's tumor.

We have previously observed, HSPPC-96 preparations isolated from a patient's primary renal tumor was safe and well tolerated, and demonstrated clinical activity in 24% of the patients for a duration of 14 + months. This current study was designed with two primary objectives; first, to verify the results of HSPPC-96 alone; second, to document the activity of adding IL-2 to patients failing HSPPC-96 vaccine. Vaccination begins 4 weeks after the nephrectomy to obtain the tumor sample. All patients were to receive 4 intradermal doses of 25 micrograms of HSPPC-96 at weekly intervals, followed by 2 doses every 2 weeks. Patients were then evaluated for tumor response at week 10. Responding or stable patients continued with 4 more 25 microgram doses of vaccine at 2 week intervals. Patients that progressed continued with vaccine plus 4 consecutive 5 day per week courses of 11 million units of IL-2 subcutaneously. The next evaluation is at week 18. Seventy patients were enrolled between March 1999 and November 1999. Twenty-one patients were inevaluable. Twenty-four patients have not reached first evaluation point. Presently there are 25 evaluable patients. Eight patients (32%) have continued on HSPPC-96 alone, one complete response and one partial response. Six patients remain stable. Nine patients (36%) went on to vaccine plus IL-2. Six patients remain stable at week 18 and 3 patients are presently receiving their first course. Eight patients (32%) are off study. Vaccine preparation in patients with RCC is feasible. Vaccine alone or vaccine plus IL-2 has resulted in no significant toxicity. Preliminary results confirm that HSPPC-96 alone has promising clinical activity reflected in 32% of the patients continuing vaccine without the addition of IL-2. At present it is too early to comment on HSPPC-96 plus IL-2. Laboratory correlates are being analyzed.

Mammalian stress granules allow the preferential expression of heat shock proteins in cells subjected to environmental stress

Paul Anderson and Nancy Kedersha*

*Brigham and Women's Hospital, Boston, MA 02115

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Heat shock transcripts are selectively excluded from SGs, allowing their preferential translation in stressed cells. TIA-1 and TIAR are RNA-binding proteins that recruit non-heat shock mRNAs to SGs in response to eIF-2_ phosphorylation. A truncation mutant of TIA-1 (TIA-1_RRM) that functions as a transdominant inhibitor of SG assembly also promotes the expression of co-transfected reporter genes, suggesting that TIA-1+ SGs regulate stress-induced translational arrest. Drugs that stabilize polysomes (e.g. emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (e.g. puromycin) promote the assembly of SGs. Moreover, emetine dissolves pre-formed SGs and promotes the assembly of polysomes, suggesting that these mRNA species (i.e. SGs and polysomes) are in equilibrium. We used green fluorescent protein(GFP)-tagged SG-associated RNA-binding proteins (e.g. TIA-1 and polyA binding protein (PABP-I)) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching (FRAP) indicates that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that SGs are not passive depots of untranslated mRNPs. This unexpected and highly dynamic behavior leads us to propose that SGs are sites of mRNA triage that sort individual transcripts for reinitiation, degradation, or packaging into stable non-polysomal mRNP complexes.

Necrotic but not apoptiotic cell death releases heat shock proteins, which deliver a partial maturation signal to dendritic cells and activate the NFκB pathway

Sreyashi Basu¹, Robert J. Binder¹, Ryuichiro Suto², Kirstin Anderson¹, and Pramod K. Srivastava¹

¹Center for Immunotherapy of Cancer and Infectious Diseases, University of CT School of Medicine, Farmington, CT, USA; ²Apt. A101 Osaki Houfu, Yamaguchi 747, Japan

Dendritic cells are key components of innate and adaptive immune responses. The identity of endogenous signals that activate dendritic cells is a crucial and unresolved question. We report here that heat shock proteins, the most abundant and conserved mammalian molecules, constitute such an internal signal. Necrotic but not apoptotic cell death leads to release of heat shock proteins gp96, calreticulin, hsp 90 and hsp70. Heat shock proteins stimulate macrophages to secrete cytokines and induce expression of antigen presenting and co-stimulatory molecules on the dendritic cells. The heat shock proteins gp96 and hsp70 act differentially and each induces some but not all molecules. Heat shock proteins interact with these antigen presenting cells through the highly conserved NFκB pathway. As heat shock proteins are intracellular, abundant and soluble, their presence in the extra-cellular milieu and the consequent activation of antigen presenting cells constitutes an excellent mechanism for response to cell death. As heat shock proteins are conserved from bacteria to mammals, the ability of heat shock proteins to activate antigen presenting cells provides a unified mechanism for response to internal and external stimuli.

Heat shock protein-chaperoned peptides but not free peptides introduced into the cytosol are presented efficiently by MHC I molecules

Robert J. Binder, Nathalie E. Blachere and Pramod K. Srivastava

University of CT School of Medicine, Center for Immunotherapy of Cancer and Infectious Diseases, Farmington, CT USA

The studies reported here bear on the events in the cytosol which lead to trafficking of peptides during antigen processing and presentation by 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, (ii) treatment of cells with deoxyspergualin, a drug which 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, (iii) Presentation of heat shock protein-chaperoned peptides by MHC I molecules requires proteasomal function, regardless of whether the peptides are precise ligands for a given MHC I allele, or are extended on the amino or the carboxy termini. These results suggest for the first time a functional role for cytosolic chaperones in antigen processing and hint at a role for proteasomes beyond degradation of proteins and precursor peptides.

HSP70 binds to the cell surface of monocytes and induces cytokine expression

Alexzander A. Asea and Stuart K. Calderwood

Dana Farber Cancer Institute, Department of Radiation Oncology 4 Binney Street, Boston, MA 02115-6013 USA

The HSP family function as molecular chaperones and intracellular regulators in all cell types. We have demonstrated a previously unknown cytokine-like function for HSP70 when added to human monocytes. HSP70 binds with high affinity to monocytic membranes and elicits a rapid intracellular calcium flux, activates nuclear factor κ-B and upregulates expression of the proinflammatory cytokines tumor necrosis factor- α, interleukin-1β and interleukin-6. Furthermore two different signals appeared to be activated by HSP70: one dependent on CD14 and calcium which resulted in increased TNF, IL1 and IL6 and the other CD14 independent which resulted in increased TNF only. These findings suggest that CD14 is a co-receptor for HSP70-mediated signaling in human monocytes and are indicative of a previously unrecognized role for HSP70 as an extracellular protein with regulatory effects on human monocytes, having a dual role as a chaperone and cytokine.

HUMAN HSP70 EFFICIENTLY LOAD MONOCYTES WITH ANTIGENIC PEPTIDES AND ALLOW SPECIFIC RECOGNITION BY ANTI-TUMOR T CELLS

Castelli C.¹, Dalerba P.¹, Ciupitu A.T.², Rini F.¹, Rivoltini L.¹, Mazzocchi A.³, Kiessling R.², Parmiani G¹.

(1) Unit of Immunotherapy of Human Tumors, Istituto Nazionale Tumori, Milano, Italy; (2) Department of Oncology MTC, Karolinska Institute, Stockholm, Sweden; (3) Unit of Immunohematology, Istituto Nazionale Tumori, Milano, Italy.

Heat Shock Proteins (HSP) are able to bind small peptides and to “chaperone” them into the MHC antigen processing and presentation pathways. Among HSP, members of the HSP70 subfamily show a broad distribution within different cell compartments, and could potentially vehicle a wide repertoire of peptides from proteins with different subcellular localizations. We have used HSP70 purified from human melanoma cell lines to load monocytes derived from peripheral blood mononuclear cells (PBMC), and we tested whether this procedure allowed recognition by autologous tumor-specific T cells directed against melanoma differentiation antigens. Monocytes loaded with autologous melanoma-derived HSP70 were efficiently recognized by both CD8 and CD4 T cell clones, and their recognition was blocked by antibodies directed against HLA class I and class II molecules, respectively. Moreover, HSP70 were successfully used to load antigenic peptides also in non-autologous systems. HSP70 purified from allogenic melanoma cells when loaded on MHC matched monocytes specifically stimulated anti-melanoma T cells in a MHC dependent fashion. This observation suggests that the HSP70 peptide repertoire is not dependent by the MHC alleles expressed by the cells used as source of HSP. Finally, monocytes loaded with HSP70 purified from antigen-negative cells or with HSP70 devoid of endogenous complexed peptides were still able to trigger low levels of IFN-γ release from tumor-specific T cells in an HLA-unrestricted manner. The molecular mechanism leading to this non-specific activation is still to be defined, but is probably due to an adjuvant effect of HSP70 on monocytes, since HSP70 alone exerts no effect on T cells.

Taken together our data are consistent with the hypothesis that HSP70 are able to transfer precursor peptides into both the HLA class I and class II presentation pathways of the antigen presenting cells. The finding that HSP70 can efficiently activate anti-tumor T cells across MHC barries suggests that these proteins can be exploited as common vectors of shared tumor antigens in the development of anti-cancer vaccines.

Ancient functions of HSP70: gamete self discrimination in Protochordates

Rosaria De Santis,* Rita Marino* and Maria Rosaria Pinto*,^§

* Laboratory of Cell Biology, Stazione Zoologica “A. Dohrn”, Villa Comunale, 80121 Napoli, Italy; ^§ Institute of Protein Biochemistry and Enzymology, CNR, Via Marconi, 10, 80125 Napoli, Italy

The phylogenetic position of Tunicates, Protochordates, at the bases of Vertebrates makes these animals particularly suitable for studies in evolutionary biology. The observations of Oka (1), Watanabe (2) and Scofield et al. (3,4) on the presence of a tissue-based kin recognition in the hermaphrodite colonial tunicate Botryllus, lead the attention on the possible presence in these animals of a vertebrate MHC-like system. In fact, genetic studies indicated that the fusion or rejection between two colonies is mediated by hemocytes and is governed by a single highly polymorphic Mendelian locus, bearing a strong resemblance with the behaviour of the MHC genes (3). In Botryllus somatic polymorphism is maintained by gamete self-incompatibility, a process that prevents fertilization between gametes belonging to kin colonies (1, 4). Based on experimental observations, the Authors suggested that the genes that regulate gamete self-incompatibility are linked or identical to the fusibility genes (4).

In this context, the study of gamete self-discrimination is pivotal. The hermaphrodite self-sterile tunicate ascidian Ciona intestinalis is a good model to approach this problem since it provides amounts of gametes that can be easily handled, and information is available on the basic features of sperm-egg interaction in this animal. Mature oocytes collected from the gonoduct are strictly self-sterile and the vitelline coat (VC) is the site at which self-discrimination of the spermatozoa occurs (5). Self-sterility is established in the late stages of oogenesis: in fact, young oocytes, which after germinal vesicle breakdown (GVBD) can be fertilized by both heterologous and autologous spermatozoa become self-sterile within several hours. This process can be followed in vitro, thus allowing the study of structures and the molecules that are responsible for the functional changes of the vitelline coat (6).

It has been proved that the follicle cells, elongated and highly vacuolated cells attached at the outer surface of the VC, are responsible for the onset of self-sterility. In fact, once the follicle cells are removed from the maturing self-fertile oocytes, these never become self-sterile. Detached follicle cells exert their action when they are left in the medium, and even when they are separated from the oocyte by a dialysis membrane (10,000 molecular weight cut off). This suggests that self-sterility-endowing molecules of small molecular mass are secreted by the follicle cells and apposed onto the VC (6,7).

The fact that attempts to identify MHC-related molecules both in Botryllus and in Ciona have been unsuccessful, lead us to consider genes that are present in the MHC and may be endowed with ancestral MHC-like functions. HSP70 is the most ancient protein with a peptide-binding domain (8) and an HSP70 gene is present in the MHC class III. Furthermore, an HSP70 gene belongs to a syntenic group of genes found in the mouse, that could represent a “primordial MHC” (9). An HSP70 gene (CiHSP70) has been isolated and characterized in Ciona intestinalis. This gene, a member of family of the heat-inducible HSPs, is constitutively expressed exclusively in the ovary and specifically in the follicle cells of the maturing oocyte. Interestingly, the gene and its product are expressed in these cells at developmental stages that correspond to the timing of the acquisition of self-sterility, while silenced in the mature oocyte. An antibody against HSP70 is able to block the onset of self-sterility and experiments, designed to define its specific target, indicate that the action of the antibody is exterted specifically on the follicle cells. The interpretation of these data is helped by immunoelectronmicroscopy indicating that CiHSP70 is present at the membrane of the follicle cells in the area facing the VC. Together with the data from the physiology of the onset of self-sterility, this has been interpreted as a possible action of the HSP70 in shuttling individual-specific molecules, possibly peptides, from the internal compartments to the plasma membrane of the follicle cells. The peptides would be then transferred to the VC, thus making it able to discriminate self-sperm (10).

Further information is provided by studies on the involvement of a proteasome activity in the onset of self-sterility. It is known that proteasomes have a key function in the production of peptides and that they have been recruited by the immune system of vertebrates (11). Clasto-lactacystin β-lactone (CLβL), a specific and irreversible inhibitor of all the proteasome catalytic β-subunits, is able to prevent the onset of self-sterility. In fact, oocytes that have not undergone germinal vesicle breakdown, incubated in the presence of the inhibitor reach full maturation, but never become self-sterile. The inhibitor is not effective if applied after germinal vesicle breakdown, thus indicating that only in the previous stage the substrate for CLβL is available. Even if a final proof that the peptides produced by the action of the proteasomes are directly linked to the function of the HSP70 cannot be provided, the data clearly show that a proteasome activity is involved in the pathway leading to self-sterility (12).

The data obtained until now in the study of the molecular basis of gamete self-discrimination point to the presence of common elements between this function and the immune system of vertebrates. In fact, if widely known that HSPs transport peptides within the cytoplasm, recent observations indicate that these proteins have a function in the presentation of peptides at the cell surface in cancer (13) and virus-infected cells (14). This function of the HSPs as “peptide-presenting” molecules could be reminiscent of a primordial function of these proteins in the pathway of expression of self-specific peptides in Protochordates. This, together with the observations on the involvement of a proteasome activity in the establishment of self-discrimination, is highly suggestive of the presence of primordial mechanism based on ancestral antigen-presenting molecules that would exert their action also in reproductive systems.

REFERENCES

1. Oka K. Colony specificity in compound ascidians: the genetic control of fusibility. In: H. Yukawa (ed.); Profiles of Japanese Science and Scientists. Kodansha, Tokyo; 1970: 195–206.

2. Watanabe H, Taneda Y. Am Zool 1982; 22: 775–782.

3. Scofield V L, Schlumpberger JM, Weissman IL. Nature 1982; 295: 499–503.

4. Scofield V L, Schlumpberger JM, Weissman IL 1982. Am Zool 1982; 22: 783–794.

5. Rosati F, De Santis R. Exp Cell Res 1978; 121: 111–119.

6. De Santis R, Pinto M R. Mol. Reprod Dev 1991: 29: 47–50.

7. Pinto M R, De Santis R, Marino, R. Develop Growth Differ 1995; 37: 287–291.

8. Salter-Cid, L., and Flajnik, F. M. Crit Rev Immunol.1995; 15: 31–75.

9. Kasahara M, Hayashi M, Tanaka K, Inoko H, Sugaya K, Ikemura T, Ishibashi T. Proc. Natl. Acad. Sci. USA 1996; 93: 9096–9101.

10. Marino R, Pinto MR, Cotelli F, Lora Lamia C, De Santis R. Development 1998; 125: 899–907.

11. Niederman G, Grimm R, Geier E, Maurer, M, Realini C, Gartmann C, Solt J, Omura S, Rechsteiner MC, Baumeister W, et al. J Exp Med 1997;1: 209–220.

12. Marino R, De Santis R, Giuliano P, Pinto MR. Proc Natl Aca. USA 1999; 96: 9633–9636.

13. Multhoff G, Botzler C, Meier T, Wiesnet M, Issels LD.. Int J Cancer. 1995; 61: 272–279.

14. Suto R, Srivastava PK. A mechanism for the specific immunogenicity of heat shock protein-chaperone peptides. Science 1995; 269: 1585–1588.

Double-stranded RNA and/or heat-shock as initiators of chaperone mode switches in diseases associated with protein aggregation?

Donald R. Forsdyke

Department of Biochemistry, Queen's University, Kingston, Ontario, Canada K7L3N6

Molecular chaperones participate in discrimination between (i) self proteins that can be refolded and/or disaggregated, (ii) self proteins that are irremediably denatured and fated to enter proteolytic pathways, (iii) not-self proteins that, having been recognized as such by aggregation, are fated to enter the MHC-peptide presentation pathway, and (iv) self proteins selected from a repertoire of polymorphic individual-specific proteins which coaggregate with (iii), and share their fate. Diseases associated with protein aggregates (e.g. trinucleotide repeat diseases, prion diseases) may help us understand the role of protein aggregation, chaperones and heat-shock in these intracellular self/not-self discriminations (leading to detection and elimination of a cell with foreign nucleic acid or with a mutated oncogene). ^1–3

Foreign (not-self) nucleic acid, by way of double-stranded RNA (dsRNA), can trigger post-transcriptional gene silencing ⁴, which can be temperature-dependent ⁵. Indeed, self-RNAs are “purine loaded” apparently to prevent inadvertent formation of self-dsRNA, an adaptation which is very apparent in thermophiles ⁶. dsRNA can trigger a protein kinase (PKR) and upregulate expression of various interferon and MHC protein genes. Trinucleotide repeats can form dsRNA hairpins, which also activate PKR ⁷. If some disease features were the result of intracellular alarms triggered by dsRNA, this would explain the difficulty of correlating these features with protein aggregation. Formation of a sufficient length of dsRNA might lead to cell death either by apoptosis, or by switch of molecular chaperones to peptide-presentation mode, allowing cytotoxic T-cell recognition of MHC-peptide complexes.

Human and yeast prion-precursor genes encode RNAs containing repeats which can adopt dsRNA structures ⁸, but it is not known if these can activate PKR. Some prion diseases require both internal prion-precursor genes and exogenous factors ⁹. These factors might induce pyrexia and/or activate PKR resulting in chaperone-mode switching, so permitting presentation of peptides from prion aggregates. The mode switch might be evident as a change from one type of chaperone (e.g. constitutive hsp70) to another (e.g. inducible hsp70 ¹⁰). Intriguingly, in bacteria a single protein has the capacity to switch from chaperone mode to proteolytic mode as temperature increases. ¹¹

1. Forsdyke DR (1991). Early evolution of MHC polymorphism. J Theor Biol 150, 451–456.

2. Forsdyke DR (1995). Entropy-driven protein self-aggregation as the basis for self/not-self discrimination in the crowded cytosol. J Biol Sys 3, 273–287.

3. Forsdyke DR (1999). Heat shock proteins as mediators of aggregation-induced ‘danger’ signals: implications of the slow evolutionary fine-tuning of sequences for the antigenicity of cancer cells. Cell Stress & Chaperones 4, 205–210.

4. Fire A (1999). RNA-triggered gene silencing. Trends Genet 15, 358–363.

5. Fortier E and Belote JM (2000). Temperature-dependent gene silencing by an expressed inverted repeat in Drosophila. Genesis 26, 240–244.

6. Lao PJ and Forsdyke DR (2000). Thermophilic bacteria strictly obey Szybalski's transcription direction rule and politely purine-load RNAs with both adenine and guanine. Genome Res 10, 228–236.

7. Tian B, White RJ, Xia T, Welle S, Turner DH, Mathews MB and Thornton CA (2000). Expanded CUG repeat RNAs form hairpins that activate the double-stranded RNA-dependent protein kinase PKR. RNA 6, 79–87.

8. Poisson G and Barrette I (2000). Les maladies du prion pourraient être un vestige d'un ancien monde de l'ARN. Unpublished communication to Congrès 68 de l'Association Canadienne-Française pour l'Avancement des Sciences, Montreal.

9. Hunter N, Cairns D, Foster JD, Smith G, Goldmann W and Donnelly K (1997). Is scrapie solely a genetic disease? Nature 386, 137.

10. Ménoret A, Patry Y, Burg C and Le Pendu J (1995) Co-segregation of tumor immunogenicity with expression of inducible but not constitutive hsp70 in rat colon carcinomas. J Immunol 155, 740–747.

11. Spiess C, Bell A and Ehrmann M (1999). A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 339–347.

A Phase I Trial of Heat Shock Protein Heat Shock Protein-gp96 Peptide Complex-96 (HSPPC-96) as autologous tumor vaccine after surgery for gastric cancer

M. Heike, C. Hertkorn, D.J. Reitsma,² H. A. Lehr, N. Simiantonaki, T. Wölfel, T. Junginger, W.G. Dippold,¹ P.R. Galle, P. K. Srivastava³

Johannes Gutenberg-University-Hospital, ¹St. Vincenz Hospital, Mainz, Germany, ²Antigenics L.L.C., NY, ³University of Connecticut, Farmington, CT

The objectives of this study are to establish the feasibility of autologous HSPPC-96 tumor vaccine preparation from gastric cancer tissue, to evaluate the safety and tolerability of the vaccine in patients after surgery for gastric cancer and to study its capacity to induce a systemic anti-tumor immune response. Patients with diagnosis of gastric carcinoma underwent gastric surgery with curative intent. At least 1.5 grams of resected tumor were procured and HSPPC-96 was purified under GMP conditions. Four to eight weeks after surgery patients were vaccinated intradurmally four to nine times in weekly or two-week intervals with one of three possible doses of HSPPC-96: 2.5, 25 or 100 μg. Patients were monitored for toxicity and immune reponses.

Vaccine preparation was successful in 12 of 15 patients, showing the feasibility of vaccine preparation from resected gastric carcinoma. However, a relevant proportion of patients were not eligible for vaccination because of unresectability, small tumor size, incomplete recovery after surgery or withdrawal of consent.

So far as of May 2000, 6 patients received 4 x 2.5 μg, 2 patients 4 x 25 μg, 1 patient 8 x 25 μg and 1 patient 9 x 100 μg HSPPC-96. All 5 stage IIIb and IV patients and 1 stage IIIa patient developed tumor progression after a median of 6.5 months after surgery. Four patients with tumor stages II and IIIa remain without evidence of disease. Five out of six patients with tumor progression were in the lowest vaccine dose level of 2.5 mg, while the three of four patients remaining disease free were vaccinated with 25 mg and 100 mg vaccine doses. In conclusion, the lowest vaccine dose of 2.5 mg did not change the natural course of locally advanced stage III and IV gastric cancer after R0/R1 surgery. The vaccine was well-tolerated. No significant toxicity or autoimmunity was observed.

Tumor tissue was analyzed by immunohistochemistry. Each tumor sample expressed gp96 and HLA-class I and cytokeratins. Glandular parts of the carcinomas showed enhanced gp96 expression compared to surrounding stroma. Ratios of tumor and stroma contents of the tumor specimen ranged from 0.2 to 2.1. There was no obvious correlation between the tumor/stroma ratio and the yield of the vaccine.

T cell responses against autologous tumor cells and NK-cell acvtivity were evaluated using IFN-gamma ELISPOT assays and cytotoxicity assays. PBMC and T cell subpoulations of patients were monitored by FACS analysis. The vaccination did not induce increased frequencies of autoreactive CD8+ T cells reacting with autologous monocytes. Autologous tumor cells or tumor cell membranes were not reliable as antigenic substrates to test frequencies of tumor reactive autologous T cells in ELISPOT-Assays in this study. HSPPC-96 vaccination did not lead to increased frequencies of patient CD8+ T cells reactive with certain defined tumor peptide antigens from CEA, p53 or Her2neu. However, the patients CD8+ T cells did react with recall antigens from EBV and influenza and could be stimulated with PHA lectin. An increase of NK-like cytotoxicity could be observed in the 2 of 4 patients with no evidence of disease which got the intermediate or highest vaccine dose and underwent extended vaccination. Preliminary data show increases of CD8+CD45RO+ memory T cells and decreases of CD3-CD56+ NK cells during vaccination. Further analysis will focuss on these changes of the immune effector cells of patients induced by vaccination. Clonal expansions of CD8-positive T-cells in patients during vaccination will be tested by TCR-Vbeta-spectratyping.

The Heat Shock Response: A Brake on Inflammation

L.E. Hightower,¹ G.A. Perdrizet,² M. Rewinski,² P.T. Guidon Jr.,³ T. Mistry³ and S.D. House³

¹Dept. of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269–3044; ²Trauma Program, Hartford Hospital, Hartford, CT 06102 ; ³Dept. of Biology, Seton Hall University, South Orange, NJ 07079

The heat shock response was so named because heat was the first known inducer and the first indication that the response changed cellular physiology; cells became transiently thermotolerant. Now much more is known about this protected state, frequently called cytoprotection. Cells in this state are nonproliferative, anti-apoptotic and anti-inflammatory. A major reason for the unresponsiveness of cytoprotected cells is that products of stress-inducible genes block signal transduction pathways. For example, Sherman and coworkers showed that Hsp70 prevents the activation of JNK and p38 kinases and inhibits heat-induced apoptosis in human tumor cell lines (Gabai et al 1997). The mechanism involves increased rate of inactivation of stress kinase JNK (Volloch et al 2000). Wong and coworkers obtained data suggesting that heat induction of the inhibitory protein I-κB inhibits the activation of the pro-inflammatory transcription factor NF-κB (Wong et al 1999). Calderwood and colleagues found another anti-inflammatory effect of the heat shock response: transcription factor Hsf1 acts as a transcriptional repressor of genes encoding several pro-inflammatory cytokines including IL1β and TNF-α (Xie et al 1999). How does this response work in an animal? Barbara Polla was among the first to suggest that inflammation is a major venue for the heat shock response and that it may serve as a brake on inflammatory responses (Polla 1988). I will present new findings that stress-conditioning rats with either heat or stannous chloride blocks extravasation of neutrophils across venules in response to a proinflammatory stimulus. Since white blood cell flux decreased significantly in response to the pro-inflammatory peptide FMLP in both conditioned and placebo animals, we concluded that the initial low affinity interactions between lymphocytes and endothelium were not blocked. However, firm attachment, measured in a leukocyte-endothelial adhesion assay, was blocked in conditioned animals. Our working hypothesis is that vascular endothelial cells and/or neutrophils are in a cytoprotected state in which they do not respond to signals that would normally up-regulate cellular adhesion molecules involved in the firm attachment of neutrophils to the vascular endothelium. Blood vessels are now returning to center stage in studies of cytoprotection in intact animals and tissues. We say ‘returning’ because the studies of Fredric White done twenty years ago (White 1980b; White 1980a) showed that cells associated with the brain microvasculature are among the most stress-responsive cells in explants and in heat shocked rats.

Gabai V, Meriin A, DD M, Caron A, Rits S. 1997. Hsp70 prevents activation of stress kinases. J Biol Chem 272: 18033–18037.

Polla BS. 1988. A role for heat shock proteins in inflammation. Immunol Today 9: 134–137.

Volloch V, Gabai V, Rits S, Force T, Sherman M. 2000. Hsp72 can protect cells from heat-induced apoptosis by accelerating the inactivation of stress kinase JNK. Cell Stress Chaperones 5: 139–147.

White FP. 1980a. Differences in protein synthesized in vivo and in vitro by cells associated with the cerebral microvasculature. A protein synthesized in response to trauma? Neuroscience 5: 1793–1799.

White FP. 1980b. The synthesis and possible transport of specific proteins by cells associated with brain capillaries. J Neurochem 35: 88–94.

Wong H, Ryan M, Menendez I, Wispe J. 1999. Heat shock activates the I-κBα promoter and increases I-κBα mRNA expression. Cell Stress Chaperones 4: 1–7.

Xie Y, Cahill C, Asea A, Auron P, Calderwood S. 1999. Heat shock proteins and regulation of cytokine expression. Infect Dis Obstet Gynecol 7: 26–30.

Interaction of hsp60 with innate immune cells

Hubert Kolb and Volker Burkart

German Diabetes Research Institute at the University of Düsseldorf, Auf'm Hennekamp 65, 40225 Düsseldorf, Germany

Heat shock protein 60 is a major target of the immune response during infection, chronic inflammation or autoimmunity. We have suggested that hsp60 is special in that it is recognised by invariable receptors of the innate immune system and thereby facilitates antigen presentation and an adaptive immune response. Human hsp60 was found to induce a pro-inflammatory response in human or mouse macrophages. Exposure to human hsp60 elicited the production of TNFα, NO₂^-, IL-12 and IL-10, with similar release kinetics as seen in response to LPS.

Further studies revealed that hsp60 signals via Toll-like receptor-4 (TLR4). I.e., macrophages of C3H/HeJ mice which express a mutant TLR4 or cells of C57BL/10.ScCr which carry a tlr4 null mutation were unresponsive to hsp60 as well as to LPS, but not to other stimuli. We conclude that exogenous hsp60 interacts with membrane receptors of innate immune cells, which may contribute to the regulation of inflammatory immune responses.

Clinical Trials Update

Jonathan J. Lewis

Antigenics, Inc. New York, NY

We have vaccinated over 250 patients with 6 different cancer-types. This has been done in collaboration with investigators at Memorial Sloan-Kettering Cancer Center and MD Anderson Cancer Center in the USA, Instituto dei Tumori in Milan, Italy and Johannes Gutenberg University Hospital, Mainz, Germany. The cancers treated include: renal cell carcinoma, melanoma, pancreatic adenocarcinoma, gastric cancer, colorectal carcinoma and lymphoma.

In this presentation, we will review and summarize the results in these Phase I and II studies. The basic science and translational implications will be discussed. We will also review upcoming clinical trials, including the Phase III renal cell cancer trial being performed at multiple centers in the USA and Europe.

CD40 independent priming of protective T cell immunity against tumor cells engineered to secrete a heat shock protein gp96

Jie Dai¹, Diliana Stoilova¹, Sreyashi Basu¹, Robert J Binder¹, Marissa Cardill², Eckhard R Podack² and Zihai Li¹*

¹Center for Immunotherapy of Cancer and Infectious Disease, MC 1601, University of Connecticut Health Center, Farmington, CT 06030–1601, USA. ²Department of Microbiology and Immunology, University of Miami School of Medicine, Miami, FL33101, USA. *Correspondence should be addressed to Z.L. (zli@up.uchc.edu)

The secretion and cell surface expression of an endoplasmic reticular (ER) heat-shock protein (HSP) gp96 has been observed in various conditions. The significance of this phenomenon on tumor immunogenicity is studied in light of the fact that soluble gp96 can activate antigen presenting cells (APC) such as dendritic cells (DC) and the peptides associated with gp96 can be re-presented to MHC class I molecules through cross-priming. By deleting or modifying the c-terminal ER retention signal KDEL, we have constructed several mammalian expression vectors for either cell surface expression or secretion. By transfection and stable expression into Meth A fibrosarcoma, we have obtained several stable cell lines. The immunological properties of the engineered tumor cells are studied in parallel with the parental cells.

We have observed that constitutive secretion of gp96 by Meth A increases drastically its immunogenicity. Mechanistic studies demonstrated that the engineered Meth A primes CD8+ T lymphocytes in a CD40—independent manner. In addition, we have also observed that, (1) Soluble gp96 can stimulate normal bone marrow-derived immature DCs to upregulate MHC class II, B7.1 and B7.2, but not CD40 on the cell surface; (2) DCs from CD40 knockout mice can be activated by gp96; (3) A defined peptide epitope chaperoned by gp96 can be re-presented to MHC class I by macrophages from peritoneal exudates of CD40 knockout mice to stimulate the cognate peptide-specific T cell clones.

Taken together, we conclude that priming of T cells by gp96-engineered tumor cells is independent of CD40-CD40L interaction. The role of recently identified gp96 receptor, CD91 or α2 macroglobulin receptor in this process is under active investigation. The above finding is consistent with the observation that priming of CD8+ T cells by HSP immunization is independent of CD4+ T cells.

CD91 is involved in MHC class II presentation of gp96-chaperoned peptides

Toyoshi Matsutake and Pramod K. Srivastava

Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA

We have shown in the last HSP meeting that exogenous gp96-peptide complexes can re-present their peptides through the MHC class II pathway (1) as well as MHC class I pathway (2). Recently, we have identified CD91 (α₂M receptor) as a receptor for gp96 (3). Exogenous gp96-peptide complexes can enter the MHC class I pathway after internalization of gp96-peptide complexes by CD91. We will now show that CD91 might also be involved in the MHC class II presentation pathway of gp96-peptide complexes. We have isolated a CD4⁺ T- cell clone which specifically recognize a cytosolic protein of methylcholanthrane induced sarcoma, Meth A, in the presence of splenic antigen presenting cells (APCs). This T-cell clone proliferates in the presence of APCs and Meth A -gp96 but not without APCs nor with gp96 purified from other tumors or normal tissue. This response was inhibited by addition of anti-CD4 or anti MHC class II monoclonal antibody, or by treatment of splenic APCs with chloroquine, cytochalasin D, or BFA. In addition to that, this response was also inhibited by addition of irrelevant liver gp96, α₂M, or anti-CD91 monoclonal antibody. Thus peptides that bind to gp96 are processed and presented on the MHC class II molecules as well as MHC class I after internalization of exogenous gp96-peptide complexes by CD91. These results have implications for the use of HSPs for immunization to elicit not only antigen specific CD8⁺ T-cells, but also CD4⁺ T-cells.

1. Matsutake, T. and P.K. Srivastava.1998. Tumor antigens chaperoned by gp96 gain access to the MHC class II pathway. Cell Stress & Chaperone.(suppl 1):5

2. Suto, R. and P.K. Srivastava (1995). Science 269,1585.

3. Binder, R.J., D.K. Han, and P.K. Srivastava. 2000. CD91: a receptor for heat shock protein gp96. Nature Immunology.1:151

Immunogenicity of the inducible hsp70

Antoine Ménoret, Delphine Chaillot, Ursula Munoz-Najar, Hong Zheng, Margaret Callahan and Paul Clark

University of CT School of Medicine, Center for Immunotherapy of Cancer and Infectious Diseases, Farmington, CT USA

Apparently identical tumor clones and variants derived from the same tumor display distinct immunological potentials when injected in syngenic host. We have shown that the immunogenicity of these tumors co-segregated with the expression of the inducible hsp70 but not with the expression of the constitutive hsc70, nor with classical immunologic markers (Ménoret et al. J. Immunol. 1995, 155:740). This observation has been further tested in the mouse colon carcinoma CT26. We observed an immunological heterogeneity of the CT26-derived clones that cannot be explained by the expression of MHC class I expression and other markers. The expression of 10 HSPs regulated by different stresses has been measured under non-stressful condition, under nutritional stress, under hypoxic stress, and under hyperthermic stress. We observed that only the expression of the inducible hsp70 is associated with the immunogenicity of the clones whereas the expression of all the other HSPs is similar in all the clones.

We and others have shown that physiological or artificial up-regulation of the inducible hsp70 increases tumor immunogenicity. Altogether these observations suggest that the inducible hsp70 possesses a specific and unknown immunological property that is different from most of the other HSPs. We have explored a series of non-exclusive hypothesis that may explain this phenomenon: Based on the fine structural dissimilarities between the hsp70 and hsc70 we have tested the peptide binding capacities of these two HSPs in pre-apoptotic and necrotic stressful environments. Considering the possibility that the inducible hsp70 can gain access to the extracellular space it might stimulate the innate immune system (Macrophage, Dendritic cells and Natural Killer) and provoke an autoimmune reaction. Finally, a differential interaction with the putative HSP70-receptor on antigen presenting cells might be of first interest.

The Molecular Regulation of gp96/GRP94: Heat, Ligands and Conformational Consequences

Christopher V. Nicchitta

Duke University Medical Center, Durham, NC, USA Phone: 919.684.8948; Fax: 919. 684–5481; E-Mail: c.nicchitta@cellbio.duke.edu

A series of landmark observations from the realm of structural biology have served to initiate a new era in the regulatory biology of the Hsp90s. Beginning with the observations that the Hsp90s contain a highly conserved domain that serves as the target for geldanamycin and radicicol, subsequent studies have demonstrated that this domain serves as a binding site for ATP and ADP. In extending these observations to gp96/grp94, we report that the ligand binding properties of this nucleotide binding domain (NBD) differ between Hsp90 and gp96/GRP94. Whereas gp96/GRp94 displays high affinity, saturable binding of the adenosine derivative, N-ethylcarboxamidoadenosine (NECA), Hsp90 exhibits no stable interactions with this ligand. In NECA displacement assays, the NBD of gp96/GRP94 displayed high selectivity for adenosine-bearing ligands, but poor discrimination between ATP, ADP, and AMP.

To define a mechanistic consequence to ligand interactions in the NBD of gp96/GRP94, the effects of ligand binding on gp96/GRP94 conformation and activity were investigated. Heat shock elicits a tertiary conformational change associated with an activation of peptide binding and molecular chaperone activity; ATP, ADP, geldanamycin, radicicol, in contrast, bind to gp96/GRP94 and inhibit the heat shock-dependent conformational activation. Such data suggest that, with respect to gp96/GRP94, ATP and ADP, geldanamycin and radicicol serve as inhibitors of gp96/GRP94 function. We report that bisANS, a fluorophore known to interact with NBDs, binds to the NBD-bearing region of gp96/GRP94 and elicits a conformational change identical to that occurring in response to heat shock. From these data, we postulate that gp96/GRP94 activity is regulated through ligand interactions in the NBD and speculate on the existence of adenosine-bearing ligands, other than ATP/ADP, that serve to activate gp96/GRP94. A model describing the conformational consequences of heat shock and ligand interactions, and the functional readout of such interactions, is included below and will be discussed.

Regulation of Immune Activity and Anti-Tumor Immune Responses by Mild (Fever-Range) Whole Body Hyperthermia

JR Ostberg, SS Evans, R Patel, MT Pritchard, J Subjeck, EA Repasky.

Departments of Immunology, Molecular & Cellular Biology and Molecular & Cellular Biophysics, Roswell Park Cancer Institute, Buffalo, NY 14263 USA

We have shown that exposure of mice to mild whole body hyperthermia (WBH) similar to that obtained during a normal febrile episode (ie: a temperature of 39.8+0.2^oC for 6–8hrs) results in increased heat shock protein expression in a tissue specific manner and significant tumor growth delay in SCID mice bearing human tumors and Balb/c mice bearing syngeneic tumors. This anti-tumor effect of mild WBH appeared to be a result of lymphocyte and/or NK cell activity as indicated by increased tumor cell apoptosis, increased numbers of leukocytes in the tumors, and inhibition of tumor apoptosis upon antibody mediated inactivation of immune cell function. WBH also appeared to induce changes in tumor vasculature that might be responsible for the anti-tumor effects. To help clarify the mechanisms by which fever-range WBH causes its anti-tumor effects, further studies have been performed which specifically identify several WBH induced changes in lymphocytes that are indicative of altered activation levels. These include alterations in the organization of the spectrin-based cytoskeleton and formation of uropods, activation and reorganization of several PKC isoforms, and induction of heat shock proteins. Other studies have revealed that fever-range WBH induces a transient decrease in the numbers of lymphocytes in the blood and spleen, suggesting their movement into the periphery, as well as a loss of epidermal Langerhans cells in the skin, suggesting their activation and migration to draining lymph nodes. Fever-range hyperthermia has also been shown to enhance L-selectin dependent adhesion of lymphocytes to high endothelial venules, resulting in increased migration to the lymph node. These data, as well as the ability of WBH to enhance localization of immune cells to the tumor microenvironment, provide strong evidence for the capacity of this mild hyperthermic treatment to alter immune cell motility and homing potential. WBH treatment of mice has also been shown to enhance LPS induced TNF-α and IL-6 levels in the serum. Perhaps most interestingly, while fever-range hyperthermia has been found to enhance mitogen induced production of the Th1-type cytokines IFN-γ and IL-2 by in vitro cultured lymph node cells, it also appears to reduce production of the Th2-type cytokine IL-4. This suggests that the mild WBH protocol helps drive Th1-type, or cellular, immune responses. Furthermore, in a murine contact hypersensitivity model for cellular immunity, WBH treatment after application of the eliciting antigen dose was found to enhance the ear swelling response. Overall, these data suggest that this physiological “heat stress” can stimulate beneficial anti-tumor responses at the level of the organism's immunity.

Supported by the CRI and NIH grants CA71599, CA16056 and CA79765.

GP96 AND THE EVOLUTION OF ADAPTIVE IMMUNITY

Jacques Robert,¹ Laura Rau,¹ Jennifer Strong,¹ Antoine Ménoret,² Pramod K. Srivastava² and Nicholas Cohen¹

¹University of Rochester Medical Center, Rochester, NY 14642, USA; ²Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA.

In mammals, gp96 complexed to antigenic peptides elicits potent T-cell adaptive immunity; gp96 alone can also evoke innate immunity. We are investigating the role of gp96 as an ancestral agent in antigen presentation and danger signaling in the evolution of adaptive immunity (Robert and Cohen, Immunol. Rev. 1998, 166: 231).

Our molecular, biochemical, and serological characterization of gp96 homologues in all vertebrate classes (including the Agnatha which lack an adaptive immune system) confirm the extensive structural conservation of this chaperone. The evolutionary relationship between gp96 structure and immunological properties was further studied in the Amphibia, vertebrates that occupy a pivotal position in the evolution of tetrapods. The frog model (Xenopus laevis) we have developed allows us to study immunity during two distinct developmental periods: adult life, which is comparable to mammals in terms of MHC class I expression, and larval life, where tadpoles are naturally MHC class I deficient.

Antigenic peptides can be complexed in vitro to Xenopus gp96, processed, and presented by mouse macrophage; immunization with gp96 purified from Xenopus tumors, elicits potent and specific anti-tumor immunity; and Xenopus gp96 effects in vivo MHC-restricted thymus-dependent immunity against anti-minor histocompatibililty antigens. Thus, the capacity of gp96 to chaperone antigenic peptides and to interact with antigen-presenting cells has been phylogenetically conserved.

Recent data also reveal additional immunological activity of gp96 in Xenopus. For example, anti-tumor immune response generated by tumor-derived gp96, both in vitro and in vivo, is MHC-unrestricted and can be obtained in naturally class I deficient, but immunocompetent, larvae. We have also observed active surface expression of gp96 by Xenopus T-cell tumors, a subset of non-transformed Xenopus sIgM⁺ B cells, and agnathan leukocytes (Robert et al., J. Immunol.1999,163:4133).

In summary, our studies strongly validate a crucial role of gp96 in adaptive immune response and also suggest a diversified complex involvement of this chaperone in the evolution of vertebrate immunity.

Research supported by RO1 AI-44011 and CA-76312 (N.C) and CA-64394 and CA-44786 and by a research agreement with Antigenics, L.L.C (P.S).

Structure and mechanism of peptide loading by gp96

Srin Sastry

Assistant Professor, Molecular Genetics, Box 174 The Rockefeller University, 1230 York Ave, New York, N.Y. 10021 USA

Animals vaccinated with heat shock protein (HSP)-peptide complexes develop specific protective immunity against cancers. A prototypical HSP-based cancer vaccine is the gp96-peptide antigen complex, which is currently undergoing human clinical trials. We have analyzed the structure and mechanism of recombinant gp96-peptide complex. A vesicular stomatitis virus capsid-derived peptide ligand bearing a photoreactive azido group was specifically bound by and cross-linked to murine HSP gp96. The minimal peptide-binding site was mapped to amino acid residues 624–630 in a highly conserved region of gp96. A model of the peptide-binding pocket of gp96 predicted that the peptide ligand is held in a groove formed by alpha-helices and lies on a surface consisting of antiparallel beta-sheets. Interestingly, in this model, the peptide-binding pocket abuts the dimerization domain of gp96. Scanning transmission electron microscopy indicated that gp96 protein was organized into large aggregates. Size distributions ranged from dimers to octamers and higher. Interestingly, gp96 lacking a dimerization sequence (DDD mutant protein) was also organized into higher order structures. Circular dichroism and intrinsic Trp fluorescence suggested that the gp96 DDD mutant protein was more compact than wild type gp96. A fluorescent peptide antigen was synthesized and the peptide-binding properties of wild type and DDD gp96 were studied. The pyrene-conjugated peptide bound gp96 with a remarkably strong affinity (Kd ∼100–150 nM). At high gp96 to peptide molar ratios we observed pyrene excimer formation, suggesting that the peptides were assembled as dimers in complexes with gp96. These observations indicated that peptides were probably assembled in gp96 dimers in close proximity to each other, Fluorescence lifetime and anisotropy decay studies showed that the bound antigenic peptide was located in a hydrophobic pocket, with considerable free space for rotation. Deletion of the dimerization domain affected the peptide-binding microenvironment although peptide-binding affinity was reduced by only a small extent. Peptide-gp96 complexes were extremely stable, persisting for many days in the cold. The extraordinary stability of peptide-gp96 complexes and the plasticity of the peptide-binding pocket support the proposed relay of diverse peptides to MHC and/or other molecules via molecular recognition.

Regulation of tumor CTL epitope generation by molecular chaperones

Noriyuki Sato, Yasuaki Tamura and Toshihiko Torigoe

Department of Pathology Sapporo Medical University School of Medicine, Sapporo 060–8556, Japan

Major histocompatibility complex (MHC) class I -bound antigenic peptides generated in the cytosol are translocated into the endoplasmic reticulum (ER) by transporters associated with antigen processing (TAP). In our study, the physical association of 73 kD heat shock cognate protein (HSC73) with TAP in human lymphoblastoid T1 cells was demonstrated. The dissociation was induced in the presence of 10 mM ATP, indicating that the ADP-binding form of HSC73 might be associated with TAP. We found that HS C73-binding immunosuppressant, methyldeoxyspergualin (MeDSG) down-regulaed MHC class I expression on the cell surface and disrupted the HSC73-TAP association, while it did not affect the binding of HSC73 to a substrate protein. Then, the effect of MeDSG on the TAP-mediated ER translocation was examined using two homologous model peptides, NGT-Bw4 and NGT-Bw6, which had distinct binding affinity to HSC73. Though high affinity peptide NGT-Bw4 was translocated by TAP, low affinity peptide NGT-Bw6 was not. The TAP-dependent translocation of NGT-Bw4 was abolished in the presence of MeDSG. Similar results were demonstrated for the presentation of HLA-A31-restricted natural antigenic peptide of gastric carcinoma, F4.2, which had high affinity to HSC73. It was indicated that disruption of the HSC73-TAP association resulted in inhibition of TAP-dependent translocation of HSC73-bound peptides. Our findings highlight the important role of HSC73 for feeding antigenic peptides to TAP, and reveal a novel possibility that the repertoire of MHC class I peptides and the tumor peptide antigenicity may be limited, at least in part, in the cytosol by their affinity to molecular chaperone HSC73.

Gp96 delivers receptor-mediated signals for the innate and adaptive immune system

Harpreet Singh-Jasuja*, Norbert Hilf,*, Hans Ullrich Scherer*, Pieter Spee#, Christian Münz¶, Stephen P. Schoenberger#, Paola Ricciardi-Castagnoli^**, Jacques Neefjes#, Hans-Georg Rammensee*, René E.M. Toes§, Danièle Arnold-Schild*, Hansjörg Schild*

^* Institute for Cell Biology, Dept. of Immunology, University of Tübingen, Auf der Morgenstelle 15, D-72076 Tübingen, Germany; ^§ Dept. of Immunohematology and Bloodbank, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands; ^# Div. of Tumor Biology, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands; ^¶ Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York 10021, U. S. A.; ^** Dept. of Biotechnology and Bioscience, University of Milano-Bicocca, Pz. della Scienza 2, 20126 Milan, Italy

Heat-shock proteins (HSPs) like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides specific, receptor-mediated uptake of gp96 by antigen presenting cells (APC) is required. Moreover, we show that both in humans and mice only professional APCs like dendritic cells (DCs), macrophages and B cells, but not T cells are able to bind gp96. The binding is saturable and can be inhibited using unlabelled gp96 molecules. Receptor-binding by APCs leads to a rapid internalization of gp96 which co-localizes with endocytosed MHC class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an Adenovirus type 5 (Ad5) E1B epitope with the dendritic cell line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. We further demonstrate that gp96 mediates maturation of DCs 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. Heated, and therefore denatured gp96 is not able to induce DC maturation and cytokine secretion. In addition, we find that mature DCs are no longer able to bind gp96 molecules. Hence, the gp96 receptor is down-regulated on mature DCs suggesting that this receptor behaves similar to other receptors involved in antigen uptake like the scavenger receptor CD36, the mannose receptor or the integrins α_vβ₃ and α_vβ₅.Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I-restricted re-presentation of gp96-associated peptides and CTL activation; nonreceptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence for gp96 acting as a cross-priming antigen carrier and direct activator of dendritic cells.

Characterization of Hsp110 and Grp170 as Cancer Vaccines and the Effect of Fever-Range Hyperthermia on Vaccine Activity

Xiang-Yang Wang, Latif Kazim, Elizabeth A Repasky*, and John R Subjeck

Department of Molecular and Cellular Biophysics and *Department of Immunology, Roswell Park Cancer Institute, Buffalo, New York 14263.

Several studies have confirmed that certain stress proteins can function as potent vaccines against a specific cancer when purified from the same tumor. Recent studies have of two long recognized but unstudied stress proteins, hsp110 and grp170, have shown them to be efficient peptide chain binding proteins. The present investigation examines the vaccine potential of hsp110 and grp170. Firstly, it is shown that prior vaccination with hsp110 or grp170 purified from Meth A fibrosarcoma caused the complete regression of this tumor in BALB/c mice. In a second tumor model, hsp110 or grp170 vaccines purified from Colon 26 tumors lead to a significant growth inhibition of this tumor; whilst similar preparations from normal livers of the same animals had no effect on tumor growth. In addition, hsp110 or grp170 immunization significantly extended the life span of Colon 26 tumor-bearing mice when applied after tumor transplantation. In each tumor, a tumor specific cytotoxic T lymphocyte response developed in the mice immunized with tumor derived hsp110 or grp170. Furthermore, treatments of the mice with bone marrow derived dendritic cells pulsed with hsp110 or grp170 from tumor also elicited a strong anti-tumor response, implying that dendritic cells are able to re-present hsp110 or grp170 chaperoned peptides. Lastly, we showed that mild, fever-like hyperthermic conditions, which have been shown to stimulate other immunological functions, also stimulate the vaccine efficiency of hsp110 as well as hsc70, but not grp170. These studies indicate that hsp110 and grp170 can be used in heat shock protein-based cancer immunotherapy, that antigen-presenting dendritic cells can be used to efficiently mediate this therapeutic approach, and that fever-level hyperthermia as would occur during a natural febrile episode can significantly enhance the vaccine efficiency of heat shock proteins hsc70 and hsp110.

Introduction of PA28α lacking C-terminal residues act as dominant-negative form and reveals a novel function of hsp90 in MHC I antigen processing

Heiichiro Udono, Taketoshi Yamano, Katsuyuki Yui.

Department of Medical Zoology and Immunology, Nagasaki University School of Medicine, Nagasaki Japan.

MHC I ligands recognized by CD8⁺ T cells originate from degradation products of endogenous proteins. The degradation events are mostly catalyzed by proteasome. A regulatory complex, PA28, greatly stimulates the peptidase activity of the 20S proteasome but does not stimulate the degradation of intact proteins. The crystal structure of PA28α revealed a barrel-shaped heptamer comprising each subunit. There are four long helices forming the core of each subunit and two distinct loops. A loop is between Pro⁶⁴-Gly¹⁰² on the upper surface of each unit containing KEKE motif, which is presumably responsible for protein-protein interaction. The last nine amino acids in the C-terminus have been shown to be important in binding to the proteasome. We have made PA28α lacking KEKE motif or C-terminal several residues. These deletion mutants along with OVA-derived pepitdes OVA248–269 were osmotically introduced into the target cells. And the processing of OVA peptides was monitored by specific CTLs and mAb specific for Kb-OVA257–264 complex. The results shows that PA28α lacking five to nine residues in C-terminus decreased the processing of the peptide, while C-terminal two amino acids and KEKE motif are dispensable. Interestingly, PA28α deleting three or four residues neither enhance nor decrease the processing. The inhibitory effect by exogenously introduced PA28α deletion mutants appears to be associated with functional disruption of endogenous PA28. Even after the introduction of a dominant-negative PA28α, there remained the processing of the peptide which was abolished by geldanamycin, a specific inhibitor of hsp90, and augmented by co-introduction of exogenous hsp90. Conversely, geldanamycin inhibited the processing, which was restored by introduction of exogenous PA28α. Thus, contributions of PA28 and hsp90 upon the processing are mutually exclusive.

HSP REACTIVE T CELLS AS ANTI-INFLAMMATORY REGULATORS

Willem van Eden, Liesbeth Paul, Uwe Wendling, Peter van Kooten, Judy Bos-de Ruijter, Ruurd van der Zee and Berent Prakken*.

Dept. of Immunology, Veterinary Faculty, Utrecht University, Yalelaan 1, 3584 CL Utrecht. Tel 31–30 2534358 Fax 31–30 2533555 e-mail: w.eden@vet.uu.nl. *University Hospital for Children and Youth, Lundlaan 6, 3584EA Utrecht, The Netherlands.

Immunization with heat shock proteins has protective effects in many different models of experimentally induced arthritis. Immunohistological analysis has shown a reduced synovial inflammation in such protected animals. Adoptive transfer studies 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 (JEM 181: 943;1995) and later for mycobacterial hsp70 (JI 163: 5560;1999 and 164: 2711; 2000). Fine specificity analysis of the protective T cells responding to conserved microbial sequences showed that such T cells were cross-reactive with the homologous (inflammatory stress upregulated) self hsp. Therefore the protective anti-inflammatory effect of microbial hsp reactive T cells is possibly mediated through the cross-recognition of self hsp when overexpressed in the inflamed tissue. Preimmunisation with hsp leads to a relative expansion of such potentially self hsp cross-responsive T cells. The regulatory nature of such T cells may have originated 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 have reported the selective upregulation of the costimulatory molecule B7.2 on microbial hsp60 specific T cells in response to self hsp60 (Int Imm 12: 1041; 2000). Through a preferred interaction with CTLA-4 on proinflammatory T cells this may constitute an effector mechanism of regulation. Furthermore, the regulatory T cells were found to produce the anti-inflammatory cytokine IL10.

Stressful Death is Important to Enhance Tumour Immunogenicity

Michael Gough, Alan Melcher, Atique Ahmed, Marka Crittenden, Lisa Emiliusen, and Richard Vile.

Molecular Medicine Program, Mayo Clinic, 200 First Street, Rochester, Minnesota.

Previously, we have demonstrated that the killing of tumour cells in vivo is more immunogenic if death occurs through non-apoptotic mechanisms. A major mediator of this enhanced immunogenicity is the inducible form of hsp70 which acts to recruit dendritic cells and enhances their ability to take up antigens for later presentation. Therefore, we and others, have proposed that efficient removal of apoptotic cell debris is crucial to avoid the generation of inflammatory responses and autoimmune responses to normal cells. Similarly, the inhibition of such clearance mechanisms within tumours would be expected to increase the amount of necrotic cell death and therefore the immunogenicity of tumours. Since macrophages are a principal component of such clearance mechanisms we investigated whether these cells can sense different mechanisms of cell killing and can respond to them. Our data demonstrate that macrophages are able to sense the relative balance of apoptotic/necrotic death within a tumour and respond by differential profiles of cytokine secretion to reflect this balance. In addition, these cells are very sensitive to the presence of hsp70 which acts to suppress the immunosuppressive response that the macrophages display in the presence of high levels of apoptotic cell death. In this respect, our data also suggest that a two component alarm signal is required to activate macrophages in response to cell death: a first signal (release of cell contents) is made immunologically active by a second signal (presence of stress proteins such as hsp70).

We are exploiting these findings to deliver genes to tumours which both kill and lead to manifestations of a stressful death, such that macrophages and other important immune mediators recognize the tumour cell killing as an appropriate signal for the generation of vigorous anti-tumour immune responses. On a cautionary note, we have observed that induction of cellular stress per se is not necessarily immunostimulatory and may even be immunoprotective unless it is accompanied by aggressive cell killing. Taken together, these observations support the hypothesis that tumour immunogenicity is optimally enhanced through a two component signal involving both forced cell killing and a co-induced stress response.

Heat shock proteins as molecular links between innate and adaptive immunity

Arne von Bonin

Bernhard-Nocht-Institute for Tropical Medicine Bernhard-Nocht-Str.74, 20359 Hamburg, Germany

Heat shock proteins (HSPs) with their specific peptide-ligands are capable of inducing CD8+ positive T cells in vivo and in vitro without the addition of further adjuvants. We are interested in the interaction of dendritic cells (DC), the most powerful antigen-presenting cells (APC), and HSPs and what the consequences of DC/HSP interactions are, e.g., for the activation of T cells in vivo and in vitro. It seems likely that there is more than one possibility how HSPs can potentially modulate an evoking immune-response. Besides the already established “peptide-delivery” into the antigen-presenting cell, HSPs are also able to activate cytotoxic T-cells independently of the HSP-associated peptides. Moreover, certain members of the HSP family directly activate APC and macrophages leading to the secretion of a defined set of cytokines. To analyze whether T cells are influenced by HSP as a class of “danger-signalling” molecules, we compared the activation of naive and effector T cells derived from DO11.10 T cell receptor (TCR) transgenic mice in the presence or absence of recombinant HSP. The antigenic peptide induced release of IFN-g could be dramatically increased and accelerated by the addition of certain members of the HSP family when added to purified populations of naive T cells and APC. This accelerated activation of T cells is also reflected by the upregulation of the surface markers CD25 and CD69. In contrast, effector T cells displayed an almost unchanged activation kinetic in the presence of HSP. Taken together our data show that the presence of certain HSP has significant consequences for the activation of naive T cells, probably via a crosstalk of soluble factors induced in professional APC.

Expression profiling human macrophage and dendritic cell responses to pathogens and heat shock proteins

Richard Allen Young

Whitehead Institute, Biomedical Research, Nine Cambridge Center Cambridge, MA 02142 USA

Abstract not available

WE ARE GRATEFUL TO OUR CORPORATE SPONSORS:

ANTIGENICS, INC.

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IRELAND

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IMMUNEX CORP.

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SMITHKLEIN BEECHAM

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Special thanks and Acknowledgement

We also acknowledge, with gratitude, the many and extensive efforts of Mr. Gamil deChadarevian, for helping us obtain many of the corporate sponsorships. Mr. deChadarevian has a deep interest in the medical application of heat-shock proteins, and in healthcare in general.

Macrophages from diabetes prone mice exhibit hsp60 hyperreactivity

Volker Burkart, Hidehiko Akiyama, Christian Herder, Hubert Kolb.

German Diabetes Research Institute at the University of Düsseldorf, Düsseldorf, Germany. Tel. +49 211 3382 641; FAX +49 211 3382 606; e-mail burkart@dfi.uni-duesseldorf.de

During the pathogenesis of human type 1 diabetes cells of the innate immune system seem to exert critical regulatory and cytotoxic activities, which are important for the initiation and progression of the inflammatory processes leading to the destruction of autologous insulin producing pancreatic beta cells. The present study aimed at the identification of beta cell derived antigens which might directly activate cells of the innate immune system. Since previous studies indicate that heat shock protein 60 (hsp60) associated with insulin granula is able to modulate beta cell directed immune reactivity, the experiments focused on the effects of hsp60 on macrophages of non-obese diabetic (NOD) mice, which serve as animal model of human type 1 diabetes. In our experiments bone marrow derived macrophages from diabetes prone NOD mice, diabetes resistant NON mice and healthy C57BL/6J mice were exposed to recombinant human hsp60, lipopolysaccharide (LPS, 10 ng/ml) and/or recombinant mouse interferon γ (IFNγ, 50 U/ml). After 6h of incubation TNFα concentrations were determined by ELISA, and after 24h the concentrations of IL-12(p70) and of nitrite (NO₂^-) as a measure of nitric oxide (NO) release were determined by ELISA and by the Griess reaction in the culture supernatants. In C57BL/6J-, NON- and NOD-derived cells comparable levels of TNFα were induced by LPS (260–980 pg/ml TNFα). Increased levels of NO₂^- were detected after exposure to LPS/IFNγ (890–1800 pg/ml TNFα) and to 3 μg/ml hsp60 (750–1580 pg/ml TNFα). NO₂^- production from cells of the three mouse strains was in a range of 6–21 μM after LPS treatment. LPS/IFNγ exposure strongly increased nitrite production to a range of 34–57 μM. NO₂^- formation by hsp60 was only detectable at a concentration of 30 μg/ml hsp60, inducing 4 ± 1 μM NO₂^- in C57BL/6J cells and significantly (p<0.01 compared to C57BL/6J cells) increased levels in NON cells (30 ± 2 μM) and NOD cells (17 ± 4 μM). LPS-induced IL-12 levels in C57BL/6J- and NON-derived macrophages were in a low range of 0–50 pg/ml whereas NOD cells produced increased levels of 106 ± 42 pg/ml IL-12. Exposure to LPS/IFNγ induced 1131 ± 404 pg/ml IL-12 in C57BL/6J cells and 443 ± 33 pg/ml IL-12 in NON cells but very high levels of 4436 ± 1638 pg/ml IL-12 in NOD macrophages (p<0.001 compared to C57BL/6J and NON). In C57BL/6J and NON cells IL-12 production after hsp60 (3 μg/ml) exposure was variable in a range of 0–150 pg/ml. In contrast, NOD macrophages produced strongly increased levels of 919 ± 307 pg/ml IL-12 (p<0.001 compared to C57BL/6J and NON). Taken together, our studies showed that hsp 60, which is present in insulin granula of beta cells, strongly activates macrophages to release proinflammatory mediators such as TNFα or NO. We conclude that hsp60 represents a unique autoantigen in that it has a strong proinflammatory potential, similar to LPS. Macrophages of spontaneously diabetic NOD mice “hyperreact” to hsp60 as evident from the significantly increased production of the Th1 associated cytokine IL-12.

Redox state is a novel aspect of peptide binding capacity for HSP70

Margaret Callahan, Claire Jacquin, Antoine Ménoret

Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA. Tel: 860 679 4248, Fax: 860 679 6512, e-mail: menoret@up.uchc.edu

Heat shock protein 70 (HSP70) purified from tumor cells can protect against challenge with whole tumor cells in a T-cell dependent manner. This activity is dependent upon HSP70‘s association with immunogenic peptides.¹ The physiological circumstances under which HSP70 chaperones peptides are not clear, however, one emerging theme is that heat shock proteins most potently activate the immune system under conditions of cell stress and death.² The most salient intracellular change that occurs during cell death is an alteration in the redox state where the normally reducing environment of the cytosol becomes oxidized by release of protons from the mitochondria. Here we explore the ability of HSP70 to bind peptides under conditions that mimic the oxidation related to cell death. We have observed several features of HSP70 that appear to depend upon its redox state. (i) We show a titratable increase in the formation of HSP70 oligomers over a range of conditions from reducing to oxidizing. A subset of HSP70 treated with hydrogen peroxide and subject to boiling migrates in bands in the range of 100 to 300 kDa instead of the expected 70 kDa band. In contrast, this same protein treated with the reducing agent DL-Dithiothreitol migrates in a single 70 kDa band. (ii) Purified HSP70 complexed in vitro with radiolabeled peptides retains these peptides under oxidative conditions, but dissociates from these peptides under reducing conditions. (iii) Purified HSP70 retains a subset of natively complexed peptides under oxidative condition that can be eluted with DL-Dithiothritol. We conclude that HSP70‘s chaperoning capacity is sensitive to redox states in vitro. The immunological significance of this finding in vivo may contribute to the elucidation of the relationship between cell death and immunogenicity.

1. Udono, H. and Srivastava, P.K. (1993). Heat Shock Protein 70-associated Peptides Elicit Specific Cancer Immunity. Journal of Experimental Medicine. 178: 1391–1396.

2. Melcher et al. (1998). Tumerogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nature Medicine. 4(5): 581–587.

Molecular Modeling of Mouse hsc70 and hsp70

Delphine Chaillot and Antoine Ménoret

Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA. e-mail: chaillot@up.uchc.edu, menoret@up.uchc.edu

The 70-kDa heat shock proteins family (HSP70), including the inducible hsp70 and the constitutive hsc70, has been involved in primordial functions of the cell as the protection against different kinds of stress, chaperoning of peptides and folding of nascent and denatured polypeptides. We previously observed that these proteins are also immunologically active in that the immunogenicity of tumor cells cosegregates with the expression of the inducible hsp70 but not with the constitutive hsc70 (Ménoret et al., 1995).

In order to understand the structural origin of that difference, we modeled the mouse hsp70 and the hsc70 domain structures. We used previous HSP70 domains structures from DnaK (1BPR, 2BPR, 1DKX), bovine hsp70 (1ATR, 3HSC), rat hsc70 (7HSC) and Human hsc70 (1HJO) to model the entire mouse hsp70 and hsc70. The sequences of these crystallized domains bear a high identity to the mouse hsp70 and hsc70 sequences such that the built molecular models should resemble to the real proteins (Chothia and Lesk, 1986). The hsp70 and hsc70 are composed of three domains: the 44 kDa N-terminal ATPase domain, the 17 kDa peptide-binding domain and the 10 kDa C-terminal part. The mouse HSP70 domains were linked together based on some previous site-directed mutagenesis results (Burchberger et al., 1999; Davies et al., 1999; Montgomery et al., 1999; Mayer et al., 2000) and on the crystallized complex structure of GrpE with the DnaK ATPase domain (1DKG, Harrison et al., 1997).

Modeled ATPase and peptide-binding domains structures from mouse hsc70 and hsp70 are alike with minor differences mainly located in the orientation of the side-chain residues of the loops, the backbone structure being conserved. All the α-helixes and the β-sheets are conserved between the both hsc70 and hsp70 structures. The highest overall differences of residues and structures are located in the flexible C-terminal part that contains the chaperonin function (residues EEVD). The last thirty residues of the C-terminal part show the more flexible structure of the whole protein and include a stretch of five supplementary residues (GMPGG in hsc70) that could confer a different flexibility to the concerned protein domain. Molecular dynamics studies will enable us to compare this plasticity. The visualization of these differential structures of these two HSP70s may contribute to the understanding of their respective immunological talents.

Buchberger A., Gassler C.S., Buttner M., McMacken R., Bukau B. (1999) J. Biol. Chem. 274:38017–26

Chothia C. and Lesk A.M. (1986) Embo J. 5:823

Davies J.E., Voisine C., Craig E.A. (1999) Proc. Natl. Acad. Sci. USA 96:9269–76

Harrison C.J., Hayer-Hartl M., Di Liberto M., Hartl F.U. and Kuriyan J. (1997) Science 276:431–435

Mayer M.P., Schroder H., Rudiger S., Paal K., Laufen T., Bukau B. (2000) Nat. Struct. Biol. 7:586–93

Ménoret A., Patry Y., Burg C., Le Pendu J. (1995) J. Immunol. 155:740–747

Montgomery D.L., Morimoto R.I., Gierasch L.M. (1999) J. Mol. Biol. 286:915–32

USE OF HSP70 FOR DIAGNOSIS AND TREATMENT OF PATIENTS OF SENSORINEURAL AUTOIMMUNE HEARING LOSS

Sheila Charchat ¹, Luiz Lavinsky², Elisa Cohen¹, Carlos Alberto vohn Muhlen ¹, and Cristina Bonorino ^1.

¹Instituto de Pesquisas Biomédicas, Pontificia Universidade Católica do Rio Grande do Sul; ² Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Brazil. Phone nr. 55 51 320 3000 ext. 2725; Fax nr. 320 3312; email cbonorino@pucrs.br

Although hearing loss can be caused by a variety of conditions, it can sometimes be reversed. This is the case for autoimmune sensorineural hearing loss (ASHL). Patients with ASHL, if diagnosed and treated with corticosteroids early enough, can completely recover hearing. Diagnosis of this syndrome, however, is difficult, involving a number of laboratory tests that most of the time are inconclusive. A western blot for detection of antibodies to bovine HSP70 has been described as the best predictive test for responsiveness to corticosteroid therapy in patients with ASHL. However, the test lacks sensitivity. Also, it is not known if the presence of these antibodies is associated specifically with the disease, and no studies have yet determined it́s diagnostic value. We have analyzed patients with either ASHL, Menierés disease, diverse autoimmune disorders or healthy control individuals for the presence of antibodies to bovine and human and HSP70, both by western blot and ELISA, before corticosteroid therapy was initiated. Basically, EVERY individual had IgG to both of these proteins, although in low titers, something that had not been described previously. An expressive percentage (30 to 50%) of the Meniere and AHL patients show significantly higher titers of antibodies to both HSPs, suggesting these are autoantibodies. We are monitoring the clinical evolution of these patients upon treatment, and testing their levels of autoantibodies to HSP70 periodically, in a double blind study. Finally, we will determine if the ELISA for antibodies to human HSP70 is a more accurate and sensitive test to predict responsiveness to treatment.

The inducible hsp70 as a marker of tumor immunogenicity

Paul R. Clark and Antoine Ménoret

Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA. Tel: 860 679 4248, Fax: 860 679 6512, e-mail: menoret@up.uchc.edu

Growing evidence indicates that the stress response in general and the heat shock proteins (HSPs) in particular have a profound impact on tumor immunogenicity. In this study we show that tumor cells subjected to a non-lethal heat shock stress (42⁰C for 20, 40 and 60 minutes) are unable to form tumors in syngenic mice whereas they do so in athymic nude mice. The enhancement of T cell mediated immunogenicity correlates with the expression of the inducible hsp70 but not the constitutive hsc70. The immune response against the heat shocked MethA (60 min at 42⁰ C) was further studied by ELISPOT assay of TNFα release. Spleen cells derived from mice injected with non-heat shocked MethA produce low amounts of TNFα. On the contrary, more than 70 spots/10⁶ spleen cells were detected in the culture of the spleen cells of mice injected with live heat shocked MethA and stimulated with either culture medium (no stimulation) or live MethA cells. These observations have a bearing on the proposed functional role of HSP-peptide association in antigen processing and presentation by MHC I molecules in normal and stressful conditions.

DISTRESS AND IMMUNE FUNCTION IN RENAL CELL CARCINOMA PATIENTS RECEIVING HEAT SHOCK PROTEIN-PEPTIDE COMPLEX-96 (HSPPC-96) VACCINE TREATMENT

Lorenzo Cohen, Cherylyn Savary, Carl de Moor, Stephen P. Tomasovic, Robert J. Amato

Department of Behavioral Science, U.T. M. D. Anderson Cancer Center, Houston, TX, USA

Stress, social support, and immune indices were measured in 30 newly diagnosed stage IV renal cell carcinoma patients prior to immunotherapy using an autologous tumor vaccine, at the end of treatment, and 4 weeks later. Immune measures included cytotoxicity to K562 target and autologous tumor target, and CD4, CD8, and NK cell numbers.

After controlling for disease status variables (number of metastases, site of metastases, and calcium levels), perceived stress was negatively associated with cytotoxicity to K562 cells (p< 0.003) and to autologous tumor cells (p< 0.005), and positively associated with CD8 cell numbers (p< 0.008) at the start of treatment. Importantly, perceived stress was still negatively associated with cytotoxicity to autologous tumor cells even after controlling for cytotoxicity to K562 target cells and the other covariates (p< 0.02). This suggests that perceived stress was associated with cytotoxicity to autologous tumor cells independent of a more general immune response. Perceived stress levels at the end of treatment and 4 weeks later were also negatively associated with cytotoxicity to K562 target cells on those days (p< 0.03; p< 0.02). Social support was also a significant moderator of the perceived stress/cytotoxicity to autologous tumor relationship (p< 0.05), in that as the perceived stress levels increased, autologous cell killing decreased, but less so in patients reporting high levels of social support.

These results may be especially relevant to the clinical course of disease progression given that these patients received an autologous tumor vaccine. Importantly, psychosocial interventions that reduce distress may affect treatment response and disease progression in cancer patients receiving vaccine treatment.

HSP-27, a new preferential anti-inflammatory monokine stimulus

A De, K Kodys, K Laudanski, C Miller-Graziano

Univ. Mass. Med. School, Worcester,MA.Tel:(508)856–3288,F:(508)856–6636, Carol.Miller-Graziano@UMassMed.edu.

Monocyte (M∅) HSP-27 is known as an essential substrate of mitogen activated protein kinase 2 (MAPKAPK2), activated in the p38 signal transduction cascade which triggers many monokine genes. Here, exogenous HSP-27 is newly demonstrated to induce a predominant human M∅ IL-10 response versus zymosan or muramyl dipeptide + Staphylococcal enterotoxin B(M+S) stimuli. Although stimulating M∅ production of IL-10 (anti-inflammatory), TNF_α (proinflammatory) and IL-12(immunostimulatory), HSP-27 induced mean 4.6 fold more IL-10 than M+S and 5 fold more than zymosan. However, HSP-27 induced identical M∅ TNF_α levels to M+S but 24 fold less than zymosan. Surprisingly, HSP-27 also induced 3.1 fold more M∅ IL-12 than M+S but statistically similar levels to zymosan. HSP 27‘s predominant IL-10 induction may reflect its prolonged phosphorylation (>180 min) of the p38 MAP kinase pathway components, but transient JNK activation. P38 phosphorylation is required for M∅ ΙL-10 induction while JNK activation is inhibitory. Since HSP-27 differentially induces M∅ IL-10,TNF_α and IL-12, HSP-27 was assessed for stimulation of trauma patients' M∅ with depressed IL-10 and IL-12 (> 50% normal). Selected patients had M+S induced M∅ IL-10 and IL-12 production which was 15–40% of normal's. These patients' M∅ responded to HSP-27 with IL-10 levels which were increased to 27–100% of the normal's M+S levels. These results reflect both the higher IL-10 induction potential of HSP-27 and the patients' M∅ capacity to still produce IL-10. These patients' M∅‘s IL-12 levels concomitantly induced by HSP-27 were still equal to or greater than the normals' MDP+SEB induced levels. These data imply that HSP-27 can potently stimulate IL-10 without down regulation of IL-12 in both normals' and trauma patients' M∅.

Permanent Hsp70 overexpression in tumor cell lines does not affect MHC class I expression and lysability by cytotoxic cells

Ralf Dressel, Husnia Baraki, *Matthias Kreiss, *Dirk Lindemann, *Thomas Herrmann, Eberhard Günther

Division of Immunogenetics, University of Göttingen, Göttingen, *Institute for Virology and Immunobiology, University of Würzburg, Würzburg, Germany

Hsp70 has been shown to protect tumor cell lines against immune mechanisms of cytotoxicity like TNF-α, but has also found to be associated with increased MHC class I expression and enhanced tumor regression.

We have observed that the human melanoma cell line Ge becomes more susceptible to CTL one day after acute overexpression of Hsp70 using the conditional, doxycycline-dependent Tet-On system (1), and that Hsp70 prevents heat shock-induced resistance to CTL in a rat myeloma cell line (2). These data indicate that Hsp70 is not generally cytoprotective.

In the present study Ge cells that overexpress rat Hsp70 permanently due to retroviral transduction, were analyzed. No change of their lyzability by CTL was observed compared to Ge cells permanently transduced with TCRβ or GFP. MHC class I cell surface expression was also not altered. Thus, increased lysability after conditional overexpression of Hsp70 (1) appears to be due to the acuteness of increasing Hsp70 rather than to overexpression per se. Similarly, also in rat Y3, mouse L929 and human K562 cells permanent overexpression of Hsp70 did not change MHC class I expression and lysability by CTL or LAK cells, respectively.

To further analyze the difference between acute and permanent overexpression of Hsp70 in Ge cells, Hsc70, the constitutively expressed member of the Hsp70 family, was tested. Immunoblot and flow cytometry revealed a reduction of Hsc70 in Hsp70 overexpressing cells. Induction of Hsp70 by heat shock was in the normal range in these transductants. Thus, permanent overexpression of the otherwise inducible Hsp70 seems to lead to an adaptation of the chaperone network that may explain different effects of acute and permanent overexpression of Hsp70.

1. Dressel et al., Eur J Immunol 29: 3925–3935, 1999>

2. Dressel et al., J Immunol, 164: 2362–2371, 2000

Supported by DFG (SFB 500, Gu 105/16–1).

IMMUNOLOGICAL ROLE OF STRESS PROTEIN DURING HYPOXIA

LILLY GANJU, S. CHNADA, D. KARAN, KK SRIVASTAVA

Defence Institute Of Physiology & Allied Sciences, Lucknow Road, Timarpur, Delhi 110054 INDIA

The expression of hsp in response to physical & chemical stresses has long been appreciated. Prior to our studies, the possibility that hypoxic stress could elicit a stress protein response in kidney had not been considered. Our findings that hypoxia, an environmental stress akin to stresses encountered by humans at high altitude, induced synthesis of 65kd stress protein in rodents provide such an evidence. Though it is yet to be demonstrated that this primitive cellular response to stress is intimately linked to neurohormonal stress response in mammals and is therefore, likely to play an important homeostatic role in coping with the stress of hypoxia. This could have important implications with respect to general health & well being as physiological and environmental disorders are associated with perturbation in kidney and hormone system function. Hypobaric hypoxia at 25,000ft does induce (Erythropoeitin) EPO production in both lymphocytes and macrophages. Tissue hypoxia serves as a stimulus for EPO production and is related to humoral environment & hsp and is known to be attached to steroid receptors. Therefore the modulation of hypoxic response in terms of stress proteins and EPO developed our interest. We have demonstrated that the macrophages & lymphocytes produce immunologically identifiable EPO in a regulated manner in response to hypoxia. Experiments are in progress to better understand the intracellular and molecular events by which hypoxia induces increased production of EPO. It would be of interest to study whether the induced stress protein inhibits EPO production and if there is any temporal relationship between the stress protein and EPO production in kidneys.

Correlation of Hsp70 and p53 expression, DNA damage in HEL cells induced by benzo(a)pyrene and high temperature

Yajuan Gao, Tangchun Wu, Sheng Chen, Chengfeng Xiao, Hanzhen He

Institute of Occupational Medicine, Tongji Medical University, Wuhan 430030 P.R.China. Phone number: 86-27-83692347, Fax: 86-27-83628553 E-mail: gaoyajuan@hotmail.com

Heat shock proteins (hsp) are molecular chaperones that are increased by heat, patho-physiological stimuli and various environmental xenobiotics or chemical stressors. Some members of the Hsp70, Hsp27, and Hsp90 families have been suggested to play a defined role in cancinogenesis. They can interact with the mechanisms of gene expression and regulation as molecular chaperones and can regulate the cell growth, related to cell proliferation and differentiation, oncogene and tumor-suppressor gene products and DNA damage repair system. Hsp can bind to mutant/wild-type p53 in tumors and, consequently, could not only regulate p53 accumulation or localization but also modulate its biological effects on cells. However, there is little information available on the significance of hsp and p53 expression in human embryonic lung cells induced by benzo(a)pyrene and high temperature. The combined existence of high temperature and benzo(a)pyrene (BaP) are commonly seen in working or living environments. Many studies seem to indicate that the occupationally exposed to BaP is the major reason of coke oven worker lung cancer. The aim of our study was to investigate the combined effects of heat and benzo(a)pyrene on HSP70 synthesis, p53 expression, and DNA damage in human embryonic lung cells (HEL). HELs were selected and divided into unheated groups at selected BaP dose (10, 50, 100, 200 μM) for 3 h incubation and heated group at 41° for 1 h, recovered at 37° for 2 h, stimulated by BaP at the same concentrations. The DNA damage, hsp70 and p53 expression were determined by single cell gel assay, Western blotting and immuno-histochemistry (double-labeling methods, using scanning confocal microtechnic). HEL cells DNA damage induced by BaP were detected at 50μM in untreated group, 10μM in heated group. BaP-induced DNA damage does not correlate with HSP induction in each group. These results suggested that the preventive effects of Hsp70 on HEL cells DNA damage induced by BaP are not significant. Hsp70 expression was related to cytoplasmic p53 expression in HEL cells but not to nuclear p53 expression. Hsp-p53 interaction may lead to increased tumorgenesis. Future studies are required to elucidate the biological nature of hsp70 and p53 and the roles of their interaction in oncogenesis and offer some ideas about the immunotherapy of occupational lung cancer,.

Keywords: The combined effects; heat; benzo(a)pyrene (BaP); heat stress protein(HSP70) ; human embryonic lung cell (HEL)

1. Hanelt S, Helbig R, Hartmann A, Lang M, Seidel A, Speit G (1997) A comparative investigation of DNA adducts, DNA strand breaks and gene mutations induced by benzo(a)pyrene and (¨¤)-anti-benzo(a)pyrene−7,8 -diol 9,10-oxide in cultured human cells. Mutation Res 390:179–188.

2. Singh NP, McCoy MT, Tice RR (1988) A simple technique for quantification of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191.

3. Tice RR (1995) The single cell gel/comet assay: a microgel electrophoretic technique for detection of DNA damage and repair in individual cells,in:DH phillips,S Venitt (Eda.),Environmental mutagenesis,Biox,Oxford,pp.315–339.

* This work was supported by the National Natural Science Foundation of China (NNSFC)

ELEVATED LEVELS OF ANTI-HSP70 AND ANTI-HSP90 ANTIBODIES ARE ASSOCIATED WITH DEVELOPMENT OF GRAFT-VERSUS-HOST DISEASE IN PERIPHERAL BLOOD STEM CELL TRANSPLANT RECIPIENTS

J. Goral¹, S. Shenoy², T. Mohanakumar² and J. Clancy, Jr.¹

¹Loyola University Medical Center, Maywood, IL 60153, ²Washington University School of Medicine, St. Louis, MO 63110.

Heat shock proteins (hsps) often induce strong humoral and cellular immune responses and play an important role in a number of diseases including immune system disorders. We have shown that increased synthesis of hsp70 and the presence of circulating anti-hsp70 antibodies are associated with acute graft-versus-host disease (aGVHD) in (DAxLEW)F1 rats. The study was extended to examine the relationship between antibodies to hsp70, hsp90 and hsp60 and the development of GVHD in human recipients of allogeneic stem cells. 29 recipients with high risk hematologic malignancies, who received G-CSF mobilized peripheral blood stem cell (PBSCs) from HLA matched family donors after myeloablative conditioning, were evaluated. Plasma antibody levels against hsps were determined by ELISA between 4 and 37 months after transplantation. 22 of the 29 recipients were evaluated at two different time points. The analysis also included 8 healthy controls and 3 stem cell donors. 2/29 recipients developed no GVHD, 17 developed aGVHD, followed by chronic GVHD (cGVHD) and 9 developed only cGVHD. Levels of anti-hsp antibodies did not differ between healthy controls, normal donors before and after G-CSF mobilization, and transplant recipients without GVHD. In patients with aGVHD, a significant increase (p<0.001) in anti-hsp70 and anti-hsp90 but not in anti-hsp60 antibodies was present early following PBSCT (1–4 months). These levels returned to normal in the subsequent 8 months following PBSCT in the majority of the patients (13/16). The occurrence of isolated cGVHD without aGVHD (7 patients) did not result in a significant increase of anti-hsp antibody levels (6/7 analyzed 8–25 months after PBSCT). This study strongly suggests that the development of aGVHD in humans is accompanied by an increase in antibodies to hsp70 and hsp90. Thus, hsp70 and hsp90 may contribute to the development of aGVHD and its pathology in humans. Monitoring levels of anti-hsp70 and anti-hsp90 antibodies in stem cell transplant recipients may serve as a diagnostic tool and help to predict the onset of aGVHD.

TUMOR-DERIVED MULTIPLE CHAPERONE PROTEIN ENRICHMENT BY FREE-SOLUTION ISOELECTRIC FOCUSING (FS-IEF) YIELDS POTENT ANTI-TUMOR VACCINES

Michael Graner, Amy Raymond, Emmanuel Akporiaye, and Emmanuel Katsanis

University of Arizona, Tucson, Arizona, USA, 85724-5073 520/626-6527; fax 520/626-4220 graner@u.arizona.edu; katsanis@peds.arizona.edu

We have utilized a free solution-isoelectric focusing technique (FS-IEF) whose fractionation enriches for multiple chaperone proteins from clarified A20 murine 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 HSP70, which was the most effective chaperone immunogen in our hands against this aggressive murine leukemia model. Dosage escalation studies, however, revealed that increasing FS-IEF 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. We are currently characterizing the repertoire of peptides that are escorted by the FS-IEF enriched, tumor-derived chaperones. Additionally, we are characterizing potential immune inhibitors that are also enriched with chaperone proteins, as well as examining the role of immunostimulatory cytokines in ameliorating vaccine efficacy. Vaccines derived from tumors via FS-IEF are relatively simply, rapidly, and efficiently obtained, thus making multi-chaperone combinatorial therapy feasible.

Hsp70-Induced Activation of Human Dendritic Cells In Vitro

Lisa A. E. Harmala and Christopher A. Pennell

University of Minnesota Cancer Center and Department of Laboratory Medicine & Pathology, University of Minnesota, Minneapolis, MN, USA Telephone: (612) 625-0453 (L.A.E.H) and (612) 625-7138 (C.A.P.) Facsimile: (612) 626-5135 E-mail: lengen@lenti.med.umn.edu (L.A.E.H) and penne001@tc.umn.edu (C.A.P.)

We are interested in developing vaccines to human cancers using heat shock proteins (HSPs). Several studies have revealed an association between HSP expression and tumor immunogenicity in both human and murine models. The immune responses generated in these models were not specific for the HSPs, but rather were specific for the peptides with which the HSPs were associated. Additional data support three, not mutually exclusive mechanisms by which HSPs contribute to tumor immunogenicity: 1) activating antigen-presenting cells (APCs) to secrete pro-inflammatory cytokines, 2) cross-priming, and 3) modulation of tumors to present and process antigens more efficiently. To understand better the mechanisms by which HSPs activate APCs, we have used flow cytometry, cytokine ELISAs, and in vitro cell activation assays to investigate the phenotypic and functional changes that occur following exposure of human dendritic cells (DCs) to human hsp70. We find that in the absence of antigen, hsp70 induces immature DCs to differentiate and become CD83⁺, and to express high levels of CD40 and CD86. Hsp70 causes a dose-dependent increase in IL-12p40 secretion by DCs whether or not CD40 is ligated. We are currently investigating the functional consequences of hsp70-induced maturation of DCs with respect to activating antigen-specific cytotoxic T cells in vitro.

The heat shock protein gp96 binds specifically to human platelets

Hilf N, Singh-Jasuja H, Scherer U, Gouttefangeas C, Rammensee HG and Schild H

University of Tuebingen, Institute for Cell Biology, Department of Immunology, Tuebingen, Germany Tel. 49 7071 29 87623; Fax 49 7071 29 5653; norbert.hilf@uni-tuebingen.de

Platelets are small, nonnucleated cells which have a key function in hemostasis. Activation and aggregation is triggered by many different stimuli e.g. thrombin, collagen and ADP. The later two are tissue components or intracellular molecules which are normally not present in the plasma or on vascular endothelium and serve as indicators of injury and necrotic cell death.

Similarly, the ER-resident heat shock protein gp96 signals necrotic cell death to bone-marrow derived dendritic cells and leads to their activation^1,2. In addition, gp96 is associated with peptides which originate from the donor cell where the heat shock protein has been synthesized. After receptor mediated uptake of gp96:peptide complexes the peptide can be represented by MHC class I molecules and trigger an immune response against pathogens or tumors which might have led to the death of the donor cells.

In this work we investigated whether gp96 binds to human platelets and whether it might also indicate necrotic cell death in hemostasis in a similar way as ADP. Fluorescine-labeled gp96 bound specifically and saturable to human platelets. Moreover, thrombin activated platelets showed increased binding. The binding on resting and activated platelets could be competed by unlabeled gp96 but not by control proteins. The functional implications of gp96 binding on platelet activation and aggregation was examined.

1. Singh-Jasuja, H., Scherer HU., Hilf, N., et al. Eur J Immunol 30, 2211–2215 (2000).

2. Binder, R.J., Han, D.K. & Srivastava, P.K. Nature Immunology 1, 151–155 (2000).

Hsc70 genetically fused with a helper- epitope conferred a reduced CTL activity, resulting in a failure to keep hosts away from tumor growth in vivo

Hiroshi Ishikawa¹², Taketoshi Yamano¹, Ichirou Katayama², Katuyuki Yui¹, and Heiichiru Udono¹

¹Dept. Med. Zool. & Immunol., Nagasaki Univ. Sch. Med, ²Dept. Dermatol., Nagasaki Univ. Sch. Med

It has been demonstrated that vaccination with HSP-peptide complexes induce the peptide specific cellular immunity in vivo and generates specific CTL responses in vitro.The manners in which HSP associates with peptides are either an ATP-sensitive formation or a covalently fused protein, and both have been shown effective in inducing the specific immunity. Priming of CD8+ T cells with HSP-peptide complex is independent of CD4+ T cell, however, synergistic effect between a CTL epitope and a helper epitope both of which are fused with HSP, may augument the protective immunity against cancers and infectious diseases.

To examine this possibility, mouse heat shock cognate protein 70 (hsc70) was covalently fused with OVA-derived K^b-restricted peptide OVA257–264 (dominant epitope) and I-Ab restricted helper epitope (OVA265–280) at C- and N-terminus (hsc70 D, H). Also, OVA-derived subdominant epitope OVA176–183 (K^b-restricted) was fused to hsc70 alone or with dominant epitope (hsc70-S,hsc70-D,S). Surprisingly, immunization with hsc70-D,H diminished a protective effect against the in vivo growth of OVA-expressing tumor E.G7,which was in contrast with hsc70-D or hsc70-D,S or hsc70-S, which showed significant protective immunities in prophyraxis and therapy models. One of the reasons was the reduced ability of CTL induction with hsc70-D,H compared with hsc70-D. Since immunization with synthetic OVA257–264(D) and OVA265–280(H) peptides emulsified with IFA also reduced CTL activity, the phenomena should not be attributed to the regulatory effect of hsc70 but to the nature of the helper epitope used. These findings may provide a new insight for the use of helper and subdominant epitope against cancer immunity.

GM and IgG antibodies to pancreatic hsp60; inverse balance between IgG3 and IgG4 subclass antibodies in prediabetic patients with cystic fibrosis

Per Jensen and Niels Høiby.

Department of Clinical Microbiology, National University Hospital, Copenhagen, Denmark. Tel +45 35327890, Fax +45 35456412, e-mail: pej@biobase.dk

To investigate the adaptive immune response towards a human pancreatic hsp60 in diabetic vs. nondiabetic patients with cystic fibrosis (CF), hsp60-specific immunoglobulin G antibodies, IgG-subclass antibodies and Ig allotypes were measured in a large population of CF patients. An ELISA technique was used for cross-sectional quantification of hsp60-specific IgG antibodies in the serum of 230 CF patients and 725 healthy controls. Ig allotypes (Gm and Km) were determined by haemagglutination inhibition and typed for G1m(1,2,3,17), G2m(23), G3m(5,13,21), and Km(1,3) in 239 CF patients, of which 42 (18%) were diabetic. In longitudinal and cross-sectional studies, hsp60-specific IgG antibodies and IgG-subclass antibodies (IgG1–4) were measured in prediabetic and nondiabetic CF patients. These studies show that in CF the hsp60-specific IgG antibody increases with age, which is not found in healthy persons. The genotype G3m(13) is less prevalent in diabetics (16.7%) as compared to nondiabetic CF patients (47.2%), whereas the genotype Km(1,3) is more prevalent in diabetics (23.8%) as compared to nondiabetic CF patients (10,7%). Diabetic CF patients show an increase of the hsp60-specific IgG antibody of 28.2% (P=.008) in an one-year period prior to onset of diabetes. In this one-year period we found an inverse correlation between hsp60-specific levels of IgG3 (5.43 vs. 15.06 units/ml, p=.039) and IgG4 (0.48 vs. 0.42 units/ml, p=.049) in prediabetics vs. nondiabetic CF patients, respectively. The genetic differences in Ig allotypes between CF patients may explain the found variance in prediabetic hsp60-specific subclass antibodies and perhaps cause different susceptibility to development of diabetes.

Human T-cell proliferation is down-regulated by self-hsp60 and by the bacterial 60-kDa GroELs of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Burkholderia cepacia.

Per Jensen¹, Jens Mølvig² and Niels Høiby¹.

¹Department of Clinical Microbiology, The National University Hospital, Copenhagen and ²Steno Diabetes Center, Gentofte, Denmark. Tel +45 35327890, Fax +45 35456412, e-mail: pej@biobase.dk

The human hsp60 has in previous studies been shown to be involved in autoimmunity especially in development of diabetes^1,2 and arthritis^3,4 in animal models. Our aim was to evaluate in vitro human mononuclear cell response to a human pancreatic hsp60 and to 4 bacterial 60-kDa GroELs from Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Burkholderia cepacia, all produced recombinant. The study showed that proliferation of human peripheral blood mononuclear cells (PBMC) to a protein derivative of tuberculin (Tuberculin PPD) was inhibited by 17–29%, 65–67%, 57–69% and 96% by the GroELs of P. aeruginosa, S. aureus, E. coli and B. cepacia, respectively. By the use of an antigen-specific (PPD) T-cell line (SEI15; HLA DF0401 restricted, isolated from a type-1 diabetic patient)⁵, proliferation to PPD was reduced 43% to 72% by pancreatic hsp60. This study also showed that proliferation of SEI15 to PPD was down-regulated by 51–87% by the GroEL of P. aeruginosa. We examined 10 healthy persons for PMBC proliferation to PPD with and without hsp. This showed an inhibitory effect of hsp60 on the PPD response of 21.0–75.8%, and an inhibitory effect of 4–56%, 24–79%, 38–71% and 26–72% by the GroELs of P. aeruginosa, S. aureus, E. coli and B. cepacia, respectively. The inhibitory effect was accompanied by a reduction in supernatant IFNg, and hsp alone induced a production of IL-10. These results show that human hsp60 and bacterial GroELs have down-regulatory effect on human PPD-specific T-cell proliferative response. This corresponds well with the finding of Elias et al. of an induction of IL-10 by the hsp60 peptide p277 in non-obese diabetic mice⁶, and with Yi et al. who found an induction of IL-10 by the hsp60 in CBA mice⁷. The present study shows that several bacterial 60-kDa GroEls and the human hsp60 have capacity to down-regulate a T-cell proliferative response to PPD, and suggest an effect of hsps to modulate the cellular immune response. This modulation may explain the inhibitory effect of hsps in various models of autoimmune disorders⁴.

1. Elias, D., Markovits, D., Reshef, T., van der Zee, R. & Cohen, I.R. Induction and therapy of autoimmune diabetes in the non-obese diabetic (NOD/Lt) mouse by a 65-kDa heat shock protein. Proc Natl Acad Sci U S A 87, 1576–80 (1990).

2. Elias D., et al. Vaccination against autoimmune mouse diabetes with a T-cell epitope of the human 65-kDa heat shock protein. Proc Natl Acad Sci U S A 88, 3088–91 (1991).

3. van Eden, W. Heat-shock proteins in autoimmune arthritis: a critical contribution based on the adjuvant arthritis model. Apmis 98, 383–94 (1990).

4. van Eden, W., et al. Do heat shock proteins control the balance of T-cell regulation in inflammatory diseases? Immunol Today 19, 303–7 (1998).

5. Mølvig, J., et al. Characterization of PPD-specific T-cell lines generated in type I (insulin-dependent) diabetic and healthy individuals. Scand J Immunol 30, 615–29 (1989).

6. Elias, D., et al. Hsp60 peptide therapy of NOD mouse diabetes induces a Th2 cytokine burst and downregulates autoimmunity to various beta-cell antigens. Diabetes 46, 758–764 (1997).

7. Yi, Y., Yang, X & Brunham, R.C. Autoimmunity to heat shock protein 60 and antigen-specific production of interleukin-10. Infect Immun 65, 1669–74 (1997).

TISSUE SPECIFIC INDUCTION OF HSP70 BY MILD, FEVER-RANGE HYPERTHERMIA

K.C. Kaplan, J.R. Subjeck, E.A. Repasky, J.R. Ostberg

Department of Molecular Immunology, Roswell Park Cancer Institute, Buffalo, New York. Phone:(716) 845-3350 Fax:(716) 845-8906 JulieOstberg@RoswellPark.org

When exposed to environmental stress, cells undergo many physiological changes that increase their chances of survival. Such changes include the up-regulation of heat shock proteins that help to prevent protein denaturisation. Previous studies have utilized classical heat shock protocols that investigate the effects of very high temperatures for short periods of time (e.g. 42–45°C for 10–30 min). In higher organisms, these temperatures do not occur physiologically. We have become interested in whether there are differences in the regulation of the heat shock response if the heating process more closely mimics the hyperthermia that can be experienced physiologically (e.g. fever). To characterize the ability of fever-range hyperthermia (39.5–40°C for 6 hrs) to induce heat shock proteins, normal or tumor bearing (CT26) Balb/C mice were subjected to 6 hours of mild, whole body hyperthermia (WBH) after which, eleven different tissues were extracted for examination of HSP70 expression. Cytosolic HSP70 was determined by Western analysis using β-actin as an internal control for gel loading. Our data show that the spleen, liver and lymph node have an increased expression of HSP70 immediately following the WBH treatment. In many instances, high levels of HSP70 were still found 24 hours after the onset of the WBH period. Interestingly, detectable levels of HSP70 were not observed in the cerebrum at any time before or after WBH treatment. Variable induction of HSP70 has been observed in tissues such as the thymus, heart, lung, muscle, kidney, serum and CT26 tumor and the results from this data will also be presented.

This work was supported by CRI and NIH grants CA71599, CA16056.

ROLE OF STRESS RESPONSE PROTEINS IN THE REGULATION OF CELL DEATH PROCESSES

Ashok Khar, Usha K Srinivas, B.V.V. Pardhasaradhi, A.S. Sreedhar, Ch. Varalakshmi and A.M. Ali

Centre for Cellular & Molecular Biology, Hyderabad 500 007, India

Heat shock response is a universal phenomenon and is triggered when the cells experience any kind of stress. Rat histiocytic cell line AK-5, fails to mount a protective stress response and therefore is induced to undergo apoptosis. Heat stress also induced expression of CD95 on these cells which may be involved in the apoptotic process. Curcumin the pigment from curcuma longa, induced generation of reactive oxygen species (ROS) in all the human tumor cell lines tested but induced stress protein synthesis only in some cell lines. Cell lines from different malignancies like lung, kidney, prostrate, cervix, CNS and melanomas that synthesized hsps in response to curcumin were found to be resistant to apoptosis whereas, other cell lines like leukemia, breast, colon, hepatocellular and ovarian carcinomas which fail to synthesize hsps in response to curcumin treatment underwent apoptosis which was induced through the generation of ROS. In the curcumin resistant cell lines the expression of hsp70 is probably involved in scavenging superoxide anions, thereby imparting resistance to apoptosis in these cells. These observations yield clues towards understanding the regulation of the cell death machinery by the stress proteins.

Progressive bovine paratuberculosis is associated with a loss of intestinal CD4+-T-cells, an increased frequency of γδ-T-cells, and changes in Hsp specific T-cell function

Ad Koets¹, Victor Rutten¹, Merel Langelaar¹, Aad Hoek¹, Frans van Mil², Kerstin Müller³, Erik Gruys², Willem van Eden¹

¹Department of Immunology (a.p.koets@vet.uu.nl, Phone 31302534608, Fax 31302533555), ²Department of Pathology, ³ Department of Farm Animal Health, Faculty of Veterinary Medicine, Utrecht University, The Netherlands>

Bovine paratuberculosis is caused by infection of young calves with Mycobacterium avium ssp. paratuberculosis resulting in a chronic granulomatous infection of the ileum. In some of the chronically infected cows a fatal protein losing enteropathy develops (after 2–4 years) with a loss of systemic cell mediated immune responses. The aim of the present study was to compare systemic and local changes in Hsp specific immune responsiveness.

Lymphocytes were isolated from blood, mesenteric lymph nodes and intestinal wall (lamina propria lymphocytes (LPL)) of 22 cows in different stages of disease. The lymphocytes were phenotyped and enumerated by FACS and in-situ immunohistochemistry. Lymphoproliferation assays, using M. a. paratuberculosis derived antigens (PPD, 70kD and 65kD Heat shock proteins (Hsp70, Hsp60)) and whole bacteria (WB) used for functional evaluation.

Results indicated that progressive bovine paratuberculosis is associated with a loss of intestinal CD4+-T-cells, and an increased frequency of γδ-T-cells. The observed systemic decrease in lymphocyte responsiveness to PPD, WB and Hsp was also reflected in mesenteric lymph nodes and jejunal LPL. However, the ileum LPL had increased responses to Hsp70 as compared to those from asymptomatic animals.

In conclusion the findings in this study indicated that both peripheral and intestinal cell mediated responses are decreased in progressive bovine paratuberculosis, which is likely related to a loss of CD4+-T-cells. The hypothesis that activated Hsp70 specific T-cells regulate this process is subject of ongoing investigations.

Primary and Postoperative Adjuvant Immunotherapy of Cancer using Tumor-Derived Heat Shock Proteins: Determinants of Efficacy

Joseph T Kovalchin BS, Ananth S Murthy MD, Mark C Horattas MD, Daniel P Guyton MD and Rajiv Y Chandawarkar MD

Kenneth Calhoun Research Center (JTK) & Department of Surgery (ASM, MCH, DPG) Akron General Medical Center 400 Wabash Avenue, Akron OH 44307 and Division of Plastic and Reconstructive Surgery (RYC), Summa Health System, Akron OH

INTRODUCTION: For anti-cancer immunotherapy to be successful the host must be able to recognize tumor antigens and be capable of eliciting a powerful cell-mediated response that can overcome tumor immune-inhibitory factors. Tumor-derived Heat Shock Protein (HSP) gp96 chaperones tumor antigens and when injected into the host, evokes potent T-cell-mediated immunity capable of tumor regression.

OBJECTIVES: In order to define biological determinants that would optimize HSP-based anti-cancer immunotherapy, we tested its efficacy with respect to the following parameters: primary and metastatic cancer; dosage requirements; antigen-specificity; tumor stage and as a postoperative adjuvant following partial resection of the tumor.

KEY FINDINGS: Tumor-derived gp96 is effective in the treatment of both, primary as well as metastatic cancer. Early tumors, which were inherently smaller than advanced tumors, responded better to treatment. Gp96 therapy is antigen specific, its effects are titratable and dose dependent. Intradermal injections of smaller doses of gp96 are as effective as subcutaneous administration of larger quantities. When optimal doses are exceeded, the anti-tumor effect diminishes thereby arguing for the presence of a dose-window for its use in anti-cancer therapy. Used as post-operative adjuvant therapy in animals that underwent partial resection of their tumors, gp96 can stabilize tumor growth. Metastatic tumors respond to gp96 derived from primary tumors underscoring its importance in adjuvant therapy in a clinical setting.

CONCLUSIONS: Determinants for successful immunotherapy with tumor-derived gp96 are early treatment of smaller tumor volumes with optimally defined doses of gp96. In a postoperative setting, gp96 used as an adjuvant immunotherapy halts tumor growth in animals with a substantially large residual tumor volume or metastatic disease.

CTL generation by DNA vaccination is effected through enhanced production of peptide bound stress proteins

Udayasankar Kumaraguru, Gouffon,C., Bruce,B^*., and Rouse,B.T.

Dept. of Microbiology; ^*Dept. of Biochemistry,Cellular & Molecular Biology, University of Tennessee, Knoxville, TN-37996, USA. Tel. 865-974-6810, Fax. 865-974-4007, e mail udayk@utk.edu

Analysis of the macrophage released factor (MRF) induced by a DNA (encoding ovalbumin or HSVgB protein) pulsed macrophage, reveals it to be an ATP dependent peptide chaperone. While the MRF itself used as an immunogen did not protect C57BL/6 mice from Zosteriform challenge by HSV, recombinant human hsp70 complexed with SSIEFARL (HSVgB498–505) offered protection.

However hsp70 class chaperones obtained from other species including plant(CSS1 and hsc70) and bacteria (DnaK), loaded in vitro with SSIEFARL peptide had adjuvant properties and induced a reasonable proliferative response but were inferior in affording protection from lethal challenge. In addition ova or gB protein mixed with bioactive CpG ODNs induced MRF production that drove a DC stimulated naïve T cell culture to become a CTL. Cytochalasin D and methods that prevented hsp70 production prevented the release of stimulatory MRF.

Initial studies indicate that these hsp70 bound to, as yet unidentified receptor on the APC cell surface as evidenced by confocal microscopic studies. Treatment of recipient DC with brefeldin A prevented the presentation of the peptide on its MHC.

These findings indicate the role of stress proteins in the cross-priming or cross-presentation especially after DNA immunization.

A feasibility study of autologous tumor-derived HSP70 vaccination against chemotherapy-resistant metastatic breast cancer

Zihai Li^1,2*, Syed Bilgrami¹, Richard L Edwards¹, Robert D Bona¹, Diane Gran², Arpita Aggarwal², Antoine Ménoret², Pramod K. Srivastava², and Peter J Tutschka¹

¹Bone Marrow Transplant Program, ²Center for Immunotherapy of Cancer and Infectious Disease, MC 1601, University of Connecticut Health Center, Farmington, CT 06030–1601, USA. *Correspondence should be addressed to Z.L. (zli@up.uchc.edu)

Over 600,000 new cases of breast cancer are diagnosed worldwide each year. Breast cancer ultimately will be the cause of death in approximately half of these patients. Despite the encouraging trend of decreasing mortality for the first time in the 1990s, the median survival of all patients with clinically detectable metastatic breast cancer remains approximately two years. Therefore, an effective approach to control or eradicate metastatic diseases is required. The ability of vaccination with heat shock protein (HSP)-peptide complexes to generate immunity against the peptides, but not against HSPs, has been established. Since tumors are antigenically unique in each individual, this vaccine is only effective against the tumor from which the HSP-peptide complexes originate. This principle has been applied effectively for cancer immunotherapy in multiple preclinical tumor models. In order to translate this concept into clinical practice for the management of metastatic breast cancer, patients have to undergo surgery and a sufficient amount of intact HSP-peptide complexes has to be generated. We have initiated a pilot study to determine the feasibility of autologous HSP70 vaccination. Metastatic breast cancer patients who have failed 1st and 2nd line conventional therapy were enrolled in this study. A total of 7 patients with chemo-resistant metastatic breast cancer underwent surgery for harvesting tumors. HSP70 was purified by 2-step chromatography: ADP-agarose column and DEAE-Sepharose column from 5 patients. Immunization was carried out intradermally. Four patients became ineligible due to rapid disease progression prior to the vaccination including one patient with extensive brain metastasis. Three patients eventually received HSP70 injections. Two of these patients had significant pre-existing liver metastasis, and succumbed rapidly to the diseases without completion of vaccination. One patient demonstrated a biopsy-proven Her2/neu- negative liver metastasis after sequential therapy with high dose chemotherapy supported by autologous stem cell transplant, adriamycin, and taxol-based regimen. She completed a total of 6 weekly intradermal HSP70 vaccinations at 1 μg per dose. Remarkably, she is currently free of disease 10 months after the last immunization. This pilot study has shown that, (1) HSP70 can be purified successfully from breast cancer cells. (2) The yield of HSP70 is extremely variable and cannot be predicted accurately based on the size of the tumor due to significant heterogeneity of histology and patient's prior history of chemotherapy. (3) HSP70 vaccination is safe and well-tolerated.

A Reversed-Phase HPLC Method for Quantifying Peptide Loading onto HSP70

Chuanliang Liu, James Zabrecky, Aimee Cestra, Tami Velasco, Mina Hassan-Zahraee and Neal Gordon

Antigenics, Inc., 34 Commerce Way, Woburn, MA 01801, USA

Peptide exchange onto HSP molecules has become an increasingly important tool for understanding their chaperone activities. We report here a reversed phase HPLC method for quantifying peptide loading onto HSP70. Peptides were loaded onto HSP70 by incubating peptides with protein at different molar ratios. The loading mixture was then processed to remove excess free peptide by either repetitively diluting and concentrating sample solution using a Centricon device, gel filtration spin column or a combination of both. Once the expected free peptide was below ∼0.1 μg/mL, 10x molar excess of ATP was added into the loading mixture and the solution was incubated at 37^oC for 30 minutes to release the loaded peptide. The ATP-treated loading mixture was split into two equal fractions. One fraction was mixed with 0.5% TFA to a final TFA concentration of 0.1%, while the other fraction was filtered through a MWCO 10K Microcon device and the filtrate was then mixed with TFA similarly in order to differentiate specifically bound peptides from nonspecifically associated peptides. Both fractions were injected onto a C18 reversed phase column and separated using an acetonitrile/H₂O/TFA gradient. The amount of peptide released from the loaded HSP70 was calculated from a peak area standard curve.

Results of peptide loading onto HSP70 using different peptides under different loading conditions are presented. Correlations between HPLC results with in vivo representation studies are also discussed. This method is relatively simple, easy to use and does not involve radioactive material commonly used for peptide loading studies. Although our studies have focused on HSP70, this method should have broad applicability to other HSP molecules.

MHC class I molecules accumulate in the late-endosomes of human Langerhans cells

Paul A. MacAry, Margaret Lyndsey, Mike Scott, Jenny Craig, J. Paul Luzio and Paul J.Lehner

Division of Immunology, Addenbrooke¹s Hospital, Hills Rd., Cambridge, UK.

Langerhans cells (LC) are a subset of dendritic cells (DC) found in the human epidermis with unique morphological and molecular properties enabling their function as Osentinels¹ of the immune system. DCs are pivotal in the initiation and regulation of primary MHC class I restricted T lymphocyte immune responses, and are able to present both endogenous and exogenous antigen onto class I molecules. Here we studied the MHC class I presentation pathway following activation of immature LC by LPS. LPS induces an increase in all components of the MHC class I pathway including the Transporter for Antigen Presentation (TAP), tapasin and calnexin, and the immunoproteasome subunits LMP 2 and LMP 7. Moreover, in LC, the rapid expression of MHC class I molecules seen at the cell surface following LPS activation is due to mobilization of MHC class I molecules from HLA-DM positive endosomal compartments, a pathway not seen in monocyte-derived DCs. Mobilization of class I from this compartment is primaquine sensitive and brefeldin A insensitive. These data demonstrate the regulation of the class I pathway in concert with the maturation of the DCs and suggest the MIIC as a potential site for exogenous antigen loading in LC.

The expression of the inducible hsp70 but not other HSPs is associated with the immunological rejection of CT-26 derived clones

Ursula Muñoz-Najar, Hong Zheng, Yahui Zang and Antoine Ménoret

Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA. Tel: 860 679 4248, Fax: 860 679 6512, e-mail: menoret@up.uchc.edu

In this study we explored the mechanism of spontaneous tumor rejection by an immuno-competant host. Such rejections have been commonly reported in animals and humans as a rare but well-established phenomenon for almost all kinds of tumors without clear understanding of the mechanism involved. We have tested the ability of 28 clones derived from a parental mouse tumor CT26 to form tumors in syngenic BALB/c mice. We observed a continuum of tumorigenicity among the clones; some are highly tumorigenic (HTC: highly tumorigenic clones) while others are poorly tumorigenic (LTC: low tumorigenic clones) or intermediate (ITC: intermediate tumorigenic clones). LTC injected in nude mice mimic the growth of HTC showing that the rejection in syngenic host is due to the T cell compartment of the immune system. Moreover, mice rejecting LTC developed long term immunological memory against the parental line. The difference of Immunogenicity of these clones cannot be explained by the expression of MHC of class-I since HTC, ITC and LTC express similar level of H2-L^d, H2-K^d and H2-D^d. The expression of 9 HSPs regulated by different stresses has been tested in HTC, ITC and LTC and in the parental CT26. Under non-stressful conditions and under stressful conditions generally present in solid tumors, i.e.: glucose starvation, 2% oxygen, and glucose starvation plus 2% oxygen, all the clones expressed similar constitutive and inducible amounts of hsp10, hsp27, hsp40, hsc70, BiP, gp96 and hsp110. In contrast, after exposure to thermal stress, 42⁰C for 1 hour, only LTC have the ability to synthesize the inducible hsp70. Interestingly, the hsp27 subjects to the same regulation as the hsp70 is expressed at the same level in all clones. These results confirm (1–3) and extend the phenomenon that tumor cells expressing the inducible hsp70 become more immunogenic.

1. Ménoret A., Patry Y., Burg., Le Pendu J. Co-segregation of tumor immunogenicity with expression of inducible but not constitutive hsp70 in rat colon carcinomas. J. Immunol. 1995. 155: 740–747.

2. Wells A.D., Rai S.K., Salvato M.S., Band H., Malkovsky M. Hsp72-mediated augmentation of MHC class I surface expression and endogenous antigen presentation. Int. Immunol. 1998. 10: 609–617.

3. Melcher A., Todryk S., Hardwick N., Ford M., Jacobson M., Vile R.G. Tumor immunogenicity is determined by the mechanism of cell death via induction of heat shock protein expression. Nature Medicine. 1998. 4: 581–587.

Intracellular polyamine regulates MHC class I antigen presentaiton by affecting the interaction between 73 kD heat shock protein and TAPI

S. Ohshima^1,2, T. Torigoe¹, H. Yoshida¹, K. Kamiguchi¹, O. Fujiwara^1,2, Y. Tamura¹, Y. Hirohashi¹, H. Sahara¹, T. Nakagawa² and B N. Sato¹.

¹Dept. Pathol. & ²Dept. Ophthalmol. Sapporo Med. Univ. Sch. Med., Sapporo, Japan.

MHC class I-presentable antigenic peptides are produced in the cytosol, and translocated by transporters associated with antigen processing (TAP) into the endoplasmic reticulum (ER), where MHC class I resides. We have previously shown that 73 kDa heat shock cognate protein (HSC73) is associated with TAP in lymphoblastoid cell line T1 cells in an ATP-dependent manner. Deoxyspergualin (DSG) is a synthetic polyamine compound which is known to bind to HSC73 in the cytosol. We found that one of the stable DSG derivatives, methyl-DSG, inhibited the association of HSC73 with TAP1, and thus down-regulated cell surface MHC class I level of T1 cells as low as the level of T2 cells. Methyl-DSG could inhibit the presentation of HLA-A31-bound natural antigenic peptide, F4.2, which had high binding affinity to HSC73. Moreover, methyl-DSG inhibited the TAP-dependent translocation of HSC73-binding peptide in semi-permeabilized T1 cells. Our data indicates that the association of HSC73 with TAP1 is functionally important for the TAP-dependent translocation of cytosolic peptides into the ER and that intracellular polyamine may affect the molecular interaction. Then, we transfected cDNA encoding ornithin decarboxylase (ODC) into Hela cells to establish cell lines containing high endogenous polyamine levels. We found that MHC class I levels were down-regulated in such cell lines, and the levels were partially recovered by treatment with ODC inhibitor. These data indicate that intracellular polyamine level may affect the MHC class I antigen presentation. Our study provides a novel insight into the field of immune escape of cancer, since low immunogenic adenocarcinomas such as prostatic cancer and pancreatic cancer are known to have high endogenous polyamine levels.

The CD36 Scavenger Receptor as a Receptor for gp96

Naveed N. Panjwani*, Lana Popova* Maria Febbraio^# and Pramod K Srivastava⁺.

*Division of Molecular Immunology, Antigenics, Inc. 34 Commerce Way, Woburn, MA 01801. ^#Div. Of Haematology/Oncology, Dept. of Medicine, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021. ⁺Center for Immunotherapy of Cancer and Infect. Diseases, Univ of Connecticut Health Center, 263 Farmington Ave Office 6018, Farmington CT 06030.

Scavenger Receptors (SRs) are functionally related by their ability to bind modified LDL and other ligands such as anionic phospholipids and dsDNA. CD36 is a member of the scavenger receptor B family, which is expressed on dendritic cells and macrophages in addition to micro-vascular endothelium. CD36 has been shown to bind apoptotic bodies and to be required for MHC class I restricted representation of apoptotic body antigens. Structurally, CD36 has a large (463 aa) extracellular domain of which all known ligands have mapped to regions between aa 28–183, a single transmembrane domain and a short cytoplasmic tail containing a putative tyrosine kinase docking site. CD36 can, therefore, potentially signal gene activation events downstream of ligand binding, and has been found to be associated with a number of tyrosine kinases in vivo. Its tissue distribution, specificity for ligands with predominant negative charge, antigen representation and putative signalling activity allow CD36 to fulfill a number of criteria for a candidate receptor for gp96. Our studies have shown that CD36 null primary murine macrophages bind 52% less gp96 than wildtype controls, indicating that some part of gp96 binding to murine APCs is via CD36. Also, transgenic expression of human CD36 on the surface of an otherwise non-expressing cell line confers upon that cell line the ability to bind several-fold greater quantities of gp96. This binding was not competed for by anti-hCD36 monoclonal antibodies and modified LDL ligands which map to a.a. 155–183 and a.a. 28–93 of hCD36, respectively. This indicates that gp96 binding to CD36 may occur via regions of the extracellular domain other than these.

Activation of Nitric Oxide Production in gp96-stimulated antigen presenting cells

Naveed N. Panjwani*, Lana Popova* and Pramod K Srivastava⁺.

* Division of Molecular Immunology, Antigenics, Inc. 34 Commerce Way, Woburn, MA 01801. ⁺ Center for Immunotherapy of Cancer and Infect. Diseases, Univ of Connecticut Health Center, 263 Farmington Ave Office 6018, Farmington CT 06030.

Nitric Oxide is produced by activated macrophages and can function as both a cytotoxic effector and immunomodulatory cytokine. The enzyme responsible (inducible Nitric Oxide Synthase, iNOS) is induced upon activation by immunomodulators such as LPS and IFN-γ. The cytotoxic functions of NO have been shown to be directly tumoricidal. At high levels of production, deleterious effects of NO may result as tissue damage during inflammation and immunosuppression by T cell cytostasis and apoptosis. Our studies have shown that gp96 stimulation results in the production of NO in primary murine macrophages, murine macrophage cell line RAW264 and human myeloid cell lines U937 and THP1 at both monocyte and macrophage stages. This NO production increases with increasing gp96 dose, is inhibited by the iNOS inhibitor N-monomethyl arginine, and is not seen with heat-denatured gp96. Gp96 stimulated NO production is not due to LPS contamination as (1) it is unaffected by an LPS competitive inhibitor and (2) there is no residual NO production above basal levels with boiled gp96. The phenomenon of NO production by gp96 stimulated macrophages has implications for the direct involvement of macrophages as tumoricidal effectors in vivo during immunotherapeutic anti-cancer vaccination. It also offers a possible explanation for the immunosuppressive outcome of high dose gp96 vaccination as NO production appears to increase in direct proportion to gp96 dose in vitro and higher concentrations of NO are immunosuppressive in vivo.

Production of chemokines by hsp70 and gp96 stimulated antigen presenting cells

Naveed N. Panjwani*, Lana Popova* and Pramod K Srivastava⁺.

* Division of Molecular Immunology, Antigenics, Inc. 34 Commerce Way, Woburn, MA 01801. ⁺ Center for Immunotherapy of Cancer and Infect. Diseases, Univ of Connecticut Health Center, 263 Farmington Ave Office 6018, Farmington CT 06030.

Heat Shock Protein stimulation of antigen presenting cells results in a panel of inflammatory products being produced by those cells. Here we show that the chemokines, a group of small proteins involved in site-directed recruitment and activation of leukocytes, are among this panel of products. HSP70 and gp96 stimulation of primary peritoneal macrophages and ex-vivo generated bone marrow dendritic cells results in a dose-dependent production of the CC monocyte-macrophage targeted chemokine MCP-1. In addition, the CXC neutrophil targeted chemokines MIP-2 and KC/gro are also produced by macrophages in response to HSP70 stimulation. These activities are not exhibited upon heat denaturation of either heat shock protein. These findings further broaden understaning of the cytokine-like functions of HSPs, and suggest functional consequences in line with the proposed inflammatory outcome of interaction of APCs with necrosis-derived or otherwise in vivo liberated HSP.

Suppression of stimulating effect of human HSP90 on antigen specific T Cell responses by natural anti-HSP90 antibodies

Pashov A.¹, A. Kenderov¹, S. Kyurkchiev¹, M. Kazatchkine², S. Kaveri²

1 IBIR, Bulgarian Academy of Sciences, Sofia, BulgariaPhone/Fax: 359 2 720 925; e-mail: ibir@netbg.com; 2 INSERM U430, Hopital Broussais, Paris, France; Phone: 33 1 43959586; Fax: 33 1 45459059; e-mail: srini.kaveri@brs.ap-hop-paris.fr

We have recently reported the existence of anti-HSP90 natural autoantibodies in preparations of pooled human IgG for intravenous use (IVIg). It has been established previously that both parasite and host HSP70 and HSP60 stimulate immune responses in a nonspecific way. Recently a similar effect of parasitic HSP90 was reported. In preliminary experiments we found that submicromolar concentrations of human HSP90 caused an increase of the specific response of human peripheral T cells to tetanus toxoid (TT). The objective of the present study was to determine whether the natural anti-HSP90 autoantibodies could modulate the adjuvanticity of self-HSP90. The stimulating effect of HSP90 on antigen-specific T cell responses was almost completely suppressed by micromolar concentrations of affinity purified natural anti-HSP90 autoantibodies. Natural anti-TG autoantibodies did not affect stimulation by HSP90 of antigen specific T cell responses. At the same time human HSP90 itself stimulated proliferation of PBMC only in the presence of micromolar concentrations of natural anti-HSP90 antibodies but not when added alone to the cell culture.

These results point to a new regulatory effect of natural antibodies that may modulate the natural adjuvant activity of self and non-self HSP. A network of natural immunity mechanisms is considered whereby HSPs act as adjuvants or autoantigens, dependent on the concentration and accessibility of anti-HSP natural autoantibodies.

Identification and Quantitation of a Virus-Specific Epitope Associated with Heat Shock Proteins in vivo

Ping Peng and Pramod K. Srivastava

Antigenics Inc., Woburn, MA 01801 and Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut, Farmington, CT 06001

A monoclonal antibody specific for a known CTL epitope of the Herpes Simplex Virus-2 (HSV-2) glycoprotein D (gD) has been used to identify and quantitate this virus-specific epitope associated with HSP-peptide complexes generated in vivo. We have determined the detection limit of this antibody for the virus-specific epitope using immunoblotting analysis of a radio-labeled synthetic peptide containing the epitope sequence that was reconstituted with gp96 and hsp70 in vitro. Then, we immunoprecipitated hsp70-peptide complexes from HSV-2-infected cell lysate, and evaluated the presence and quantity of this virus-specific epitope that is associated with endogenously-derived hsp70. This study allows us to assess quantitatively the quantity of antigenic peptides chaperoned by HSPs in vivo.

Complement activation by heat-shock proteins: a possible link between the ancestral danger signal and the cellular arm of innate immunity

Zoltán Prohászka and George Füst

3rd Department of Medicine, Faculty of Medicine, Semmelweis University, Budapest, Hungary

According to new hypotheses extracellular heat-shock proteins may represent an ancestral danger signal of cellular death or lysis activating innate immunity¹. Recent studies demonstrating a dual role for hsp70, as both a chaperone and cytokine², inducing potent pro-inflammatory response in human monocytes, provided support for the hypothesis that extracellular hsp is a messenger of stress. Our previous work focused on the complement activating ability of human hsp60³. We demonstrated that hsp60 complexed with specific antibodies induces a strong classical pathway (CP) activation. Here we show that other chaperone molecules also possess complement activating ability. Solid phase ELISA method was applied for the experiments. Like human hsp60 mycobacterial hsp65 and E. coli GroEL proteins were found to induce CP activation leading to cleavage and covalent attachment of the C4b and C3b complement protein fragments to hsp. In contrast to hsp60, however, human hsp70 activated the CP independently of antibodies. No complement activation was found in the case of human hsp90. Our data further support the hypothesis that chaperones may messenger stress to other cells. Complement like molecules and primitive immune cells appear together early in evolution. A joint action of these arms of innate immunity in response to free chaperones, the most abundant cellular proteins displaying a stress signal, may further strengthen the effectiveness of immune reactions.

1. Srivastava PK et al.: Heat-shock proteins come of age: Primitive functions acquire new roles in an adaptive world. Immunity 1998, 8:657–665

2. Asea A et al.: Hsp70 stimulates cytokine production through a CD14-dependent pathway, demonstrating its dual role as a chaperone and cytokine. Nat. Med. 2000, 6:435–442

3. Prohászka Z et al.: Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement activating ability. Internat. Immunol. 1999, 11:1363–1370

Supported by OTKA F029030 grant.

EVOLUTION OF IMMUNOLOGICAL PROPERTIES OF HSP

Laura Rau, Jennifer Strong, Nicholas Cohen, and Jacques Robert

University of Rochester Medical Center, Rochester, NY 14642, USA; phone (716) 275-5359, email robert@mail.rochester.edu

Heat shock proteins (HSPs) are phylogenetically highly conserved structures. Our molecular and biochemical characterization of gp96 homologues in all vertebrate classes including the Agnatha confirm the extensive structural conservation of this HSP, suggesting that its function is both highly specialized and essential. GP96 is postulated to be an important member of antigen presentation and/or danger signaling pathway that predates the evolution of adaptive immunity, a hypothesis best tested by evaluating a corresponding preservation of gp96 immunological properties.

The frog, Xenopus, has two developmentally distinct immune systems that can be exploited for this study, the larval functioning without MHC class I presentation or expression, even though both adult and larval systems have CD8 T-cells. We have determined that, as in mammals, Xenopus gp96 chaperones peptides. Specifically antigenic peptide can be complexed in vitro to Xenopus gp96, processed and presented by mouse macrophage. Xenopus gp96 also effects MHC-restricted T-cell adjuvanticity in that gp96 immunization elicits thymus-dependent anti-minor histocompatibililty antigen immune response. These observations reveal remarkable conservation of gp96‘s immunological properties between amphibians and mammals whose ancestors diverged about 350 million years ago.

However, other evidence indicates additional immunological activity of gp96 in Xenopus. Our data show generation by tumor-derived gp96 of MHC-unrestricted specific anti-tumor cellular immune response both in vitro and in vivo in MHC class I expressing adults, as well as in vivo in naturally class I deficient (but immunocompetent) larvae. We have also observed specific and active surface expression of gp96 by tumors and some normal non-transformed Xenopus sIgM⁺ B cells (Robert et al., J. Immunol.1999,163:4133). Finally, MHC class I–negative tumors appear more rapidly after challenge in CD8-depleted frogs. These data suggest uncharacterized effector recognition, hinting at a possible niche for an alternative surveillance molecule such as gp96.

Research supported by NIH grants R37 HD-07901 and RO1 CA-76312

MOLECULAR AND IMMUNOLOGICAL PROPERTIES OF THE CHAPERONES GP96 AND HSP70 ARE PHYLOGENETICALLY CONSERVED ACROSS DISTANT SPECIES

Jacques Robert¹, Antoine Ménoret², Sreyashi Basu², Nicholas Cohen¹, and Pramod K. Srivastava²

¹ University of Rochester Medical Center, Rochester, NY 14642, USA. ² Center for Immunotherapy of Cancer and Infectious Diseases, University of Connecticut School of Medicine, Farmington, CT 06030, USA.

The heat shock proteins (HSPs) gp96 and hsp70 are highly conserved structures that have been shown to induce potent specific T cell-mediated immunity against antigenic peptides they chaperone. We have investigated the relationship between their structure and immune function by an evolutionary approach (Robert et al., submitted).

We report the serological, biochemical, genetic and immunological characterization of the Xenopus gp96. Like human and mouse gp96, Xenopus gp96 forms stable non-covalent complexes with peptides in vitro. Immunization with gp96 and hsp70 purified from Xenopus tumors, elicit in syngenic frog hosts a potent and specific anti-tumor immunity, which is dependent on their ability to chaperone peptides in vivo. Moreover, we show that an immunogenic peptide complexed to Xenopus gp96 can be processed and presented in the context of MHC class I by mouse macrophage, to antigen-specific CD8⁺ T cells of mice.

The remarkable conservation of the immunological properties of gp96 and hsp70 in between amphibian and mammals suggests the importance of hsp in the evolution of the vertebrate immune system.

Research supported by NIH grants R37 HD-07901 and RO1 CA-76312

The exchange of one single amino acid at position 71 of the DR4 β-chain leads to significant differences in antigen processing and presentation of a human autoantigen chaperoned by a member of the HSP70-family

Sabine Roth,¹ Nicholas Willcox², Matthias P. Mayer³, and Inga Melchers⁴

¹Clinical Research Unit for Rheumatology, Albert-Ludwigs-University Medical Center, Breisacher Str. 64, 79106 Freiburg, Germany, Tel.: 49-761-270-5297, Fax: 49-761-270-5298, email:rothsa@nz11.ukl.uni-freiburg.de; ²Neurosciences Group, Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, UK; ³Institute of Biochemistry and Molecular Biology, Albert-Ludwigs-University, Freiburg, Germany; ⁴Clinical Research Unit for Rheumatology, Albert-Ludwigs-University Medical Center, Freiburg, Germany, Tel.: 49-761-270-5295, Fax: 49-761-270-5298, email:melchers@nz11.ukl.uni-freiburg.de

The immune response to protein antigens depends on processing by antigen-presenting cells (APC) and subsequent presentation of peptides to specific T-cells by molecules of the major histocompatibility complex (MHC class-I and class-II). In rheumatoid arthritis (RA) there is much evidence implicating a positive genetic association of the disease with several alleles of the DRB1 gene, characterized by a common amino acid sequence at position 70–74 in the α-helical peptide-binding domain of these class-II molecules (the shared epitope). The most frequent of these alleles is DRB1*0401 with the sequence QKRAA. The same amino acid sequence was also detected in microbial molecules, notably chaperones related to the E. coli DnaJ. According to publications by the group of Roudier the QKRAA-motif in E. coli is involved in the binding of DnaJ to its partner chaperone DnaK, and in human cells the binding of some DR β-chains (including DRB1*0401) to the human heat shock cognate protein Hsc70 [Auger and Roudier (1997), J.Clin.Invest. 99, 1818].

We work with T-cell clones with restriction patterns similar to RA to define whether functional differences are due to differences in peptide binding or in protein processing involving specific interactions of the DR β-chain with additional molecules (chaperones). We here present data concerning one T-cell clone specific for an epitope derived from the human α-subunit of the acetylcholine receptor. It reacts with either recombinant protein or synthetic peptides presented by APC expressing DRB1*0401 (71Lys) or DRB1*0408 (71Arg). If APC present the peptide epitope, both alleles have the same capacity to stimulate the T-cells. However, using a recombinant α-chain (r1–437), 0401 presents much better than 0408 [Nicolle et al. (1995), Eur.J.Immunol., 25, 2119]. We proved that this difference in processing entirely depends on the amino acid sequence difference by using murine P388.D1 cells transfected with genes coding for DRB1*0401 or DRB1*0408. We now show for DRB1*0401-positive APC that i) most of the preparations of recombinant antigen or muscle extracts contained E. coli or human HSP70 molecules, ii) removal of HSP70 resulted in reduced T-cell activation, iii) reconstitution of the HSP70-free preparation with HSP70 molecules resulted in increased T-cell activation.

Our interpretations of these results are as follows: efficient processing of r1–437 by DRB1*0401 requires the presence of a member of the HSP70-family. In contrast the DRB1*0408-positive APC do not properly activate T-cells apparently due to a non-efficient interaction between the class-II molecule and the HSP70 molecule during antigen loading.

Supported by a grant of DAAD.

MYCOBACTERIAL HSP70 CAN SWITCH THE CYTOKINE PROFILE OF INFLAMATORY SINOVIAL CELLS

Ana Sabrito¹, Thiago Detanico ¹, Carlos vohn Muhlen ¹, Mauro Keiserman ¹, e Cristina Bonorino ¹.

¹Instituto de Pesquisas Biomédicas, Pontificia Universidade Católica do Rio Grande do Sul, Brazil. Phone nr. 55 51 320 3000 ext. 2725; Fax nr. 320 3312; email cbonorino@pucrs.br

Reactive arthritis (ReA) is a chronic, debilitating disease, treated basically with corticosteroid therapy. We have isolated cells from the sinovial liquid of arthritis patients in inflamatory flare, cultured these cells in the presence of phytohemaglutinin (PHA) or human or mycobacterial LPS-free HSP70, and analyzed their cytokine profile by ELISA. Acute ReA patientś cells incubated with PHA tend to make pro-inflamatory cytokines, such as IFN gamma and IL-12, while TNF-alpha is induced by incubation with human HSP70. Surprisingly, incubation with mycobacterial HSP70 tends to induce in the same cells a complete switch in this phenotype, inducing TH2-type cytokines, such as IL-10 and TGF-beta. The target cells for TB hsp70 seem to be among the adherent population, while clearly PHA stimulates the non-adherent cells, more specifically T cells. We are currently doing a dose-dependent analysis of this parameter, seeking possible applications for HSP70-based anti-inflamatory therapy.

The Heat Shock Protein Gp96–a receptor-targeted Cross-priming carrier and activator of dendritic cells

Harpreet Singh-Jasuja¹, Norbert Hilf¹, Hans Ulrich Scherer¹, Danièle Arnold-Schild¹, Hans-Georg Rammensee¹, René E.M. Toes², Hansjörg Schild¹

¹ Institute for Cell Biology, Dept. of Immunology, University of Tübingen, Auf der Morgenstelle 15, 72076 Tübingen, Germany, Tel. +49–7071–2987623, Fax +49–7071–295653, singh@uni-tuebingen.de; ² Dept. of Immunohematology and Blood Transfusion, Leiden University Medical Center, 2300 RC Leiden, The Netherlands, Tel. +31–71–5263174, Fax +31–71–5216751, r.e.m.toes@immunohematology.medfac.leidenuniv.nl

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 professional 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.

We are now able to show that 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 stimulatory capacity. Furthermore, we describe that 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: firstly, the existence of specific gp96 receptors on APCs and secondly, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.

1. Singh-Jasuja, H., Toes, R.E.M., Spee, P., et al. J Exp Med 191, 1965–1974 (2000).

2. Singh-Jasuja, H., Scherer, H.U., Hilf, N., et al. Eur J Immunol 30, 2211–2215 (2000).

Heat shock proteins and dendritic cell immunity

Selin Somersan*, Marie Larsson*, J.F. Fonteneau*, Sreyashi Basu^#, Pramod Srivastava^#, Nina Bhardwaj*

* The Rockefeller University, NY, NY and the ^# University of Connecticut Medical School, Farmington, CT.

The maturation state of dendritic cells (DCs) is critical for T cell antigen presentation. Mature DCs rather than immature DCs induce the activation of naive T cells. Some of the known stimuli that induce DC maturation are LPS, MCM, CD40L, CpG DNA, Poly I:C. We have previously shown that exposure to different forms of dying cells also yield different consequences of DC maturation. When exposed to necrotic transformed cell line lysates dendritic cells undergo maturation, as seen by the upregulation of maturation markers such as CD86, CD83, DC LAMP and an increase in costimulatory function. However when exposed to apoptotic transformed cell lysates, these DCs remain immature. Here we show that HSPs account for this difference. The supernatants of necrotic cell lysates from transformed cell lines contain high levels of HSPs (HSC 70, gp96), while supernatants of apoptotic cell lines have very low levels of HSPs as detected by Western Blotting. Interestingly, primary cells do not induce maturation of DCs with either method of death, and their supernatants do not contain high levels of HSPs. Strikingly, most of the primary tumor tissue samples we have tested have higher levels of HSPs (HSC 70 and gp96) compared to their autologous normal tissues; and their corresponding lysates matured human DCs. HSP 70 free of endotoxin also induces DC maturation in a similar way to that seen with conventional stimuli. Finally we show that necrotic cell lysates can provide antigens for cross-presentation by DCs to CD8+ T cells These results suggest that HSPs are involved in both maturation of human DCs caused by tumor lines and possibly for providing antigens from tumor cell lysates for activation of CD8+ T cells.

H2M3 restricted listerial peptides associated with gp96

A.M. Sponaas, R. Hurwitz,, P. Jungblut, S. Weber, R. Winter, S. Lamer and S.H.E. Kaufmann

Max-Planck Institute for Infectionsbiology; Monbijoustrasse 2; 10117 Berlin Germany; Tel 49 3028460545; Fax 49 3028460501; Email sponaas@mpiib-berlin.mpg.de

N- formylated (N-f-met) peptides derived from proteins of the intracellular bacterium Listeria monocytogenes generate a protective, H2M3 restricted CD8 T cell response in mice. (1)

At present, 3 N-f- met peptides have been described; fMIVIL, fMIVIL, fMIGWII and fMIVTLF (2,3,4). When C57BL/6 mice were immunized with gp96 from infected mice, CTLs specific to each of the 3 N-f-met peptides could be generated. No peptide specific CTLs were observed in mice immunized with gp96 from uninfected mice. The CTLs generated showed cross-reaction between the 3 N-f-met peptides.

Masses corresponding to one of these, fMIVIL, was found by MALDI-MS on gp96 isolated from spleens and livers from C57BL7/6 mice infected with L. monocytogenes,but not on gp 96 from un infected mice.

Radioactively labelled N-f met peptide fMIGWII was found to bind in a detergent resistant manner to gp96. Addition of fMIVIL and fMIVTLF competes with this binding.

Bacterial N-f-met peptides from intracellular bacteria can therefore bind to gp96 in infected mice, and gp96 loaded with these peptides can generate N-f-met-peptide specific T cells.

1. S.H.E Kaufmann (1998). Immunity to Intracellular Bacteria., (B.Paul ed) Foundations in Immunology

2. Lenz et al 1996 Immunity 5:63

3. Gulden et al 1996 Immunity 5:73

4. Princiotta et al 1998 J.E.M. 187:1711

TREATMENT OF PATIENTS WITH LOW-GRADE B-CELL NEOPLASMS WITH AUTOLOGOUS HSP70-TUMOR PROTEIN COMPLEX

Sporn J, Laska E, Gran D, Aggarwal A, Siegel R, Byeff P, Bona R, Ménoret A., and Pramod K Srivastava.

Univ of Connecticut Health Center, Farmington, CT.

Low-grade B-cell neoplasms, while incurable with standard therapies, are often indolent. Because the malignant B-cells have been shown to be recognizable as targets for cell-mediated immunity, they are a potential model system for tumor vaccine therapy. Tumor cells are readily available from the peripheral blood in patients with CLL, or from lymph nodes in patients with lymphoma, facilitating vaccine strategies which depend on autologous tumor-derived proteins. We have initiated a clinical trial of tumor-derived HSP70 in patients with these diseases. Eligible adult patients had either low-grade B-cell lymphoma or B-cell CLL and had stable disease which was either previously untreated or recurrent after therapy, and ECOG PS 0–1. Tissue was obtained from patients with CLL by leukapheresis and subsequent gradient centrifugation, and from patients with low-grade lymphoma by peripheral node biopsy and subsequent preparation of a cell suspension. HSP70-tumor protein was prepared using ADP-affinity chromatography, and purified for clinical administration. Patients received intradermal injection of either 10, 25, or 50 μg weekly for 4 weeks and then monthly for 3 months. DTH to HSP70-tumor protein, free HSP70, and tumor lysate were measured before and 2 weeks after completion of the vaccine sequence. The concentration of tumor-cell reactive CD8+ cells in peripheral blood was measured by elispot assay of interferon-γ production before treatment, 4 weeks after the fifth injection, and 2 weeks after the final injection. Tumor response was assessed by measurement of adenopathy on physical exam and/or CT scan, and by the concentration of circulating malignant B-cells in patients with lymphocytosis. Six patients (4 with CLL and 2 with lymphoma) have completed the full vaccine sequence. Toxicities have been minimal, including mild chills, fatigue, and in one patient the flare of a pre-existing rash. DTH to HSP70-tumor protein has not been observed either before or after vaccination. All patients had clinically stable disease when re-evaluated after completion of therapy. HLA-restricted tumor-reactive CD8+ cells have been detected.

Haplosufficiency or functional redundancy of a heat shock protein gp96 gene in the adaptive immune response

Diliana Stoilova¹, Jie Dai¹, Rini de Crom², Rien van Haperen ² and Zihai Li¹*

¹Center for Immunotherapy of Cancer and Infectious Disease, MC 1601, University of Connecticut Health Center, Farmington, CT 06030-1601, USA.; ²Department of Cell Biology and Genetics, Erasmus University, 3000 DR Rotterdam, The Netherlands.*Correspondence should be addressed to Z.L. (zli@up.uchc.edu)

Gp96 is a constitutively expressed endoplasmic reticulum (ER) heat shock protein (HSP). It is a major peptide acceptor in the lumen of ER, and it has been shown to bind antigenic peptides directly. Moreover, gp96 chaperoned peptides can be re-presented to MHC class I through CD91 on the surface of antigen presenting cells (APC). Thus gp96 may play important roles in antigen presentation in the ER as well as in the initiation of adaptive immunity. Since there is only one single gene of gp96 in mouse, we undertook a genetic approach to address the function of gp96. By conventional homologous recombination techniques, we have obtained a gp96 gene knockout mouse. Homozygous deletion of gp96 gene (gp96-/-) is found to be lethal during the early stage (day 5.5) of embryogenesis. Heterozygous (gp96±) mice survive, are fertile and do not show apparent gross abnormality. The expression of gp96 is more than 50% reduced in gp96± mice in multiple tissues and organs analyzed. We have studied the immunological phenotype of gp96± C57BL/6 mice, and made the following observations. (a) The expression levels of MHC class I and class II on splenocytes is indistinguishable between normal and gp96± mice. (b) The distribution of CD4+ and CD8+ T lymphocytes is identical. (c) Gp96± mice mount efficient adaptive CD4+ and CD8+ T cell responses against minor histocompatibility antigens. The ability of gp96± mice to generate efficient tumor immunity is being analyzed. These data suggest that gp96 is either haplosufficient for its immunological functions, or other HSPs can compensate for the diminished roles of gp96 in the gp96± mice.

Heterogeneous expression of heat shock proteins in human dendritic cells

Shaoli Sun¹, Yong-Jun Liu² and Zihai Li¹*

¹Center for Immunotherapy of Cancer and Infectious Disease, MC 1601, University of Connecticut Health Center, Farmington, CT 06030–1601, USA. ²Dept. of Immunobiology, DNAX Research Institute of Molecular and Cellular Biology, 901 California Avenue, Palo Alto, CA 94304-1104*Correspondence should be addressed to Z.L. (zli@up.uchc.edu)

Dendritic cells (DC) are important in both the initiation and the fate of adaptive immune responses. There are at least two major subtypes of dendritic cells in human, CD11C⁺ type 1 or myeloid DCs (DC1) and IFN-α/β-producing type 2 CD4⁺CD11C^- plasmacytoid DCs (DC2). Recently, several members of the intracellular protein chaperones known as heat shock proteins (HSP) including gp96, hsp70, hsp60 and hsp65 have been found to modulate the functional state of DCs. To understand the possible roles of HSPs in the differential regulation of DC1 and DC2, we have compared the endogenous expression of various HSPs, by a semi-quantitative reverse transcription based polymerase chain reaction (RT-PCR), in both immature and mature DCs of both DC1 and DC2 types. The mRNA level of hsp 90 (α and β), gp96 or grp94, hsp70 (Bip, 70a, 70b, 70c), calreticulin, hsp60, hsp27 are quantified. We have made the following observations. (a) DC2 expresses less Bip and calreticulin than DC1; (b) There are no differences in the HSP expression profile between immature DC1 and mature DC1; (c) Comparing with immature DC2, mature DC2 expresses less abundant HSPs in general, hsp70c, hsp60, hsp27 and Bip in particular; (d) Hsp 90α mRNA is completely absent in all DCs.

Novel Immunotherapy Using Heat Shock Protein Preparations of Leukemic Cells after Syngeneic Bone Marrow Transplantation in Mice

Yasuaki Tamura^1,2, Kazuya Sato², Yoshihiro Torimotoi², Yutaka Kohgo², Noriyuki Sato¹

¹Department of Pathology, Sapporo Medical University School of Medicine, South-1West–17, Chuo-ku, Sapporo,Hokkaido, 060–8556, Japan, Phone: 81-11-611-2111, FAX: 81-11-643-2310, e-mail address: ytamura@sapmed.ac.jp; ²Third department of internal medicine, Asahikawa medical college, Midorigaoka Higashi 4–5, Asahikawa,Hokkaido, 078–8510, Japan

Recently autologous stem cell transplantations (auto SCT) for hematologic malignancies have been carried out safely and prosperously for patients without donor and for elderly patients. The major problem of SCT is post-transplant relapse due to lack of graft versus leukemia effect. In this study, we investigated immunotherapy with leukemic cell-derived heat shock protein-peptide complexes against minimal residual disease of leukemia after syngeneic bone marrow transplantation in mice.

We found immunotherapy with HSP preparations derived from A20 leukemic cells, against mice with preexisting leukemic cells after syngeneic bone marrow transplantation resulted in increased survival. Treatment with HSP preparations derived from normal liver tissue was not effective. Moreover, vaccination of HSP preparations derived from A20 elicited potent specific cytotoxic T cell lymphocyte responses against A20 and led to memory CD4 T cell responses. These observations may suggest that the leukemic cell-derived HSP peptide complexes lead to specific immunity in mice with preexisting leukemic cells after syngeneic bone marrow transplantation.

Tumor prevention and anti-tumor immunity with heat shock protein 70 induced by 15-deoxy &#8710;12,14 prostaglandin J2 in transgenic adenocarcinoma of the mouse prostate cells

Donkena Krishna Vanaja*, Michael E. Grossmann+, Esteban Celis+ and Charles Y.F. Young*.

Dept. of Urology and Biochemistry/Molecular Biology* and Dept. of Immunology+, Mayo Clinic /Foundation, Rochester MN 55905. Phone: 507-284-9247, Fax: 507-284-2384 E-mail: donkena.krishna@mayo.edu

The biological modifier &#15-deoxy 8710;12,14 prostaglandin J2 and related prostaglandins have been reported to have significant growth inhibitory activity with induction of heat shock proteins (Hsps). Tumor derived Hsps have been shown previously to elicit specific immunity to tumors from which they are isolated. In this study, 15-deoxy &#8710;12,14 prostaglandin J2 (15d-PGJ2) induced Hsp70 was purified from transgenic adenocarcinoma mouse prostate cells (TRAMP-C2). It was then tested for its ability to activate specific cytotoxic T lymphocytes (CTLs) and induce protective immunity against prostate cancer in C57BL/6 mice. Treatment of cells with 8.0 μM 15d-PGJ2 for 24 hrs caused significant induction of Hsp70 expression. Using an ADP-agarose column followed by ion-exchange chromatography (Mono Q FPLC system) Hsp70 peptide complexes were purified. Whole cell lysate and the purified fractions from the mono Q column were immunoblotted with Hsp70 antibodies specific for the inducible and constitutive forms. Densitometry of the purified fractions revealed that 92% of the Hsp70 from TRAMP-C2 cells is the inducible form and 8% is the constitutive form. Vaccination of mice with Hsp70 peptide complex isolated from TRAMP-C2 cells elicited tumor specific CTLs and prevented the growth of TRAMP-C2 tumors. These results indicate that the induced heat shock proteins may have promising applications for anti-tumor, T cell immunotherapy. In particular these findings have important implications for the development of novel anti-cancer therapies aimed at promoting an immune response to prostate tumors.

Stability Analysis of Dimeric Heat Shock Protein gp96 by Adenosine Ligand Binding and Size Exclusion Chromatography

Andrew Wasserman, Kerrie Morin, Neal Gordon and James R. Zabrecky

Antigenics, Inc., 34 Commerce Way, Woburn, MA 01801, USA

It has been proposed that the active form of the endoplasmic reticulum resident heat shock protein, gp96, is a homodimer. Size exclusion chromatograph (SEC) together with ligand binding of the adenosine nucleotide analog, N-ethylcarboxamidoadenosine (NECA), was used to assess the stability of the gp96 dimer. SEC analysis showed that 75% or more of gp96 purified from tissue sources exists as a dimer under non-denaturing conditions. Furthermore, NECA ligand binding activity directly correlates with the relative amount of gp96 dimer. SEC and SDS-PAGE results indicate that denaturation of dimeric gp96 follows two separate pathways. One pathway, which involves aggregation to higher molecular weight forms, can be induced by heat, freeze/thaw and chemical denaturants. Incubation of dimeric gp96 at 60°C induces rapid aggregation and complete loss of NECA bind activity. Similar results were seen at 37°C but at a much slower rate, on the order of days. Loss of gp96 dimer was also observed through a mechanism involving cleavage of peptide bonds (possibly proteolytic) in which there is progressive loss of the 96 KDa species and the appearance of an intermediate of ∼80 KDa, which does not form dimers. SEC analysis showed a loss of gp96 dimer to lower MW species with a concomitant reduction of NECA binding activity. These results support the notion that the dimeric form is necessary for the biological properties of gp96. SEC and NECA binding can be useful for assessing gp96 dimer content and, consequently, may be predictive of biological activity in therapeutic applications.

Development of a Sandwich ELISA for the Quantification of gp96

Jenny Weng and James R. Zabrecky

Antigenics, Inc., 34 Commerce Way, Woburn, MA 01801, USA

A double antibody sandwich ELISA was developed for the quantification of the heat shock protein, gp96. The capture antibody is an affinity-purified goat polyclonal antibody raised against a C-terminal 19 amino acid peptide with the human sequence. A rabbit polyclonal antibody raised against intact gp96 is used as the detector. The assay is sensitive in range of 0.75 to 15 ng/mL. A series of specificity and competition studies demonstrated that the assay is specific for human gp96. Immunoblots and proteinase digestion experiments indicated that the assay specifically measures intact gp96 and not any of the major breakdown products. Spike and recovery tests showed that gp96 can be reliably quantified in the presence of crude cell homogenates. A standard curve was established and the assay has proven useful for the quantification of gp96 in a variety of complex sample matrixes.

Psychological stress primes for an increased heat-shock response which is prolonged in atopic subjects

Miriam Wittmann, Silke Meyer, Elisabeth Stephan, Gerhard Schmid-Ott, Petra Kienlin, Alexander Kapp, Thomas Werfel

Department of Dermatology and Allergology, Hannover Medical University, Hannover, Germany ; phone: 49 511 9246 278 fax: 49 511 9246 440 e-mail: Thomas_Werfel@t-online.de

Mental stress is a recognized “provocation” factor in atopic diseases. We compared the heat shock response in peripheral blood leukocytes of subjects suffering from atopic dermatitis to healthy volunteers after exposure to mental stress. Patients were “stressed” for 10 min by performing an unprepared oral speech and mental arithmetics in front of an audience. Blood samples were taken before, immediatly after, one hour and one day after the “stress event”. Cardiovascular parameters (blood pressure, heart rate) and plasma noradrenaline were determined and showed stress-induced increases. The inducible form of HSP70 (HSP72) was determined by intracellular flow cytometric analysis of heat shocked (44°C for 20 min) and untreated cells. In samples taken before the “mental stress event” basal expression of HSP72 was higher in patients suffering from atopic dermatitis but inducibility was lower in these patients. There was a significant increase of HSP72 expression already observable in samples taken immediatly after the “stress event” in heat shocked monocytes (less pronounced in lymphocytes). Atopic subjects showed highest HSP72 expression values in samples taken the day after “stress” (Figure) which represents a prolonged increased HSP72 expression as compared to normal subjects. In vitro studies demonstrated that noradrenaline as well as IL-4 dose dependently could mimic this intracellular HSP72 increase, but not other mediators of the HPA-axis (hydrocortisone, ACTH, adrenaline) nor substance P or IL-6. IL-4 might be a factor responsible for the prolonged heat-shock response upon mental stress in atopic dermatitis subjects. The meaning of this oberservation for the course of the disease as well as the heat shock response in skin infiltrating leukocytes is currently under investigation.

PRESENCE OF ANTIBODY AGAINST THE INDUCIBLE HEAT SHOCK PROTEIN HSP71 IN PATIENTS WITH ACUTE HEAT-INDUCED ILLNESS

TANGCHUN WU^1,2, SHENG CHEN¹, CHENGFENG XIAO¹, CHANGLAI WANG³, QIN PAN⁴, ZIZHENG WANG³, MEIYUN XIE⁴, ZHICHENG MAO³, YANG WU^1,2 AND ROBERT M. TANGUAY²

¹Institute of Occupational Medicine, Tongji Medical College, Huazhong University of Science & Technology, Hangkong Lu 13, Wuhan 430030, China, ²Laboratory of Cell and Developmental Genetics, Department of Medicine, Pav Marchand, Université Laval and CHUQ (CHUL Research Center), Ste-Foy, Québec, Canada, G1K 7P4, ³Nanjing First Hospital, Nanjing Medical University, Nanjing 210004, ⁴Nanjing Wujing Hospital, Nanjing 210003, China.

Antibodies against heat shock or stress proteins (Hsps) have been reported in patients with a number of diseases where they may be involved in the pathogenesis of disease or may be of use for prognosis. Heat-induced diseases such as heat cramps, heat exhaustion or heat stroke are frequent in hot working or living environments. There are still few investigations on the presence and possible significance of autoantibodies against heat shock proteins in heat-induced illnesses. Using an immunoblotting technique with recombinant human Hsps, we have looked for the presence and measured titers of antibodies against Hsp60, Hsp71, and Hsp90a and β in 42 young patients with acute heat-induced illness. We also examined the presence of antibody against Hsp71 in 57 older patients with acute heat-induced illness and the changes in titers of anti-Hsp71 antibodies in 9 patients hospitalized by emergency physicians. In the group of young persons exercising in a hot environment, the occurrence of antibodies against Hsp71 and Hsp90a was significantly higher among individuals with symptoms of heat-induced illness (P < 0.05) than in the matched group of non-affected exercising individuals. Moreover the titers of antibody against Hsp71 were higher in individuals of the severe and mild heat-induced illness groups, the highest titer being found in the most severe cases. A study of a second group of 57 older heat-affected patients exposed to extreme heat gave similar results. Again patients with the more severe heat-induced symptoms showed a significantly higher incidence of antibodies to Hsp71 than controls and the titer of anti-Hsp71 was higher in the severely affected group. Finally in a study of 9 patients, it ws observed that the titer of anti-hsp71 decreased during recovery from sever heat symptoms.

These results suggest that measurement of antibodies to Hsps may be useful to assess how individuals are responding to abnormal stress within their living and working environment and may be used as one of biomarkers to evaluate the susceptibility to heat-induced diseases.

(Funded by the NNSF of China and the MRC of Canada).

Hsp90 and PA28 coordinate the processing of MHC I peptide precursors

Taketoshi Yamano^1,2, Naoki Shimbara³, Noriaki Tanaka², Keiji Tanaka⁴, Katsuyuki Yui¹ and Heiichiro Udono¹

^1,2Dept. Med. Zool. & Immunol., Nagasaki Univ. Sch. of Med., ²1st Dept. Surg., Okayama Univ. Med. Sch., ³Dept. Biomed. R&D, Sumitomo Electric Ind., ⁴Tokyo Metropolitan Inst. Med. Sci.

We have previously demonstrated that cytosolic HSP90 and HSP70 as well as ER resident HSP gp96 associate with MHC class I ligand (tumor antigen peptide, pRL1a) and its precursors. Also, TAP-translocated peptides have been shown to bind gp96 in the ER. These evidences indicate that HSPs are involved in MHC class I antigen processing, however, functional evidences have been missing. Here, we demonstrate that HSP90 as well as IFN-γ inducible proteasome activator, PA28, take part in the production of MHC I ligands in concert with 20S proteasome in vivo.

As model antigen peptides, OVA-derived peptide OVA248-269, OVA248-264, OVA257-269, all of which encompass Kb-restriced OVA257-264, were used. These peptides were osmotically introduced into EL4 cells, and the processing was evaluated by OVA257-264 specific CTLs. Excess amount of hsp90 or recombinant PA28α enhanced the processing of C- but not N-terminal extended peptides. In contrast, hsp90 specific inhibitor, geldanamycin and a deletion mutant of PA28α lacking C-terminal five amino acids (which acts as dominant-negative form, unpublished results) inhibited the processing of C- but not N-terminal extended peptides. The enhanced processing by HSP90 and PA28α was completely abrogated by prior treatment of EL4 with proteasome specific inhibitor, lactacystin. The contribution of these two molecules on the processing was equal, but mutually exclusive. However, in the presence of IFNγ, the inhibitory effect with geldanamycin was completely abrogated, which was in absolute contrast to the profound inhibition of dominant-negative form of PA28α.

Figure 1