REPORTS ON SEMINARS

(1) Seminar on "Teansmembrane Signal Teansduction and Cell Proliferation"
The workshop on "Transmembrane Signal Transduction and Cell Proliferation" was held January 10-12, 1994, at the Maui Prince Hotel, Maui, Hawaii. Fifteen leading scientists from the United States and Japan participated. Seminar-style presentations were organized in five separate sessions focusing on the control of cellular responses by nuclear factors, the signaling pathways utilized by cytokine receptors, the elements that underlie signal transduction more generally, and the control of apoptosis. Informal discussions were held throughout the meeting in an attempt to establish common points of reference among the very different systems under study. The major themes discussed during the meeting are summarized below.
Notable progress continues in the analysis of transcription factors that participate in the process of transformation. Dr. Robert Eisenman reviewed the current understanding of the myc family of such factors. An important contributor to the pathogenesis of hematologic malignancies, c-myc has been shown by Dr. Eisenman and colleagues to pair with a second protein called max. The myc/max heterodimer behaves as an activator of transcription, and the interaction of myc with max can be competitively antagonized by a second protein called mad. Levels of mad/max heterodimers were found to increase with differentiation in model systems involving a myeloid cell line or cultured keratinocytes. These observations suggest that the mad-encoded protein may ordinarily serve to interdict responses stimulated by myc. In an attempt to define the set of genes that respond to myc/max regulation, Dr. Eisenman has implemented a screening strategy using specific immunoprecipitation of nuclear complexes to purify DNA associated with myc/max heterodimers.
The theme of contrasting functions in transcriptional regulation was revisited by Dr. Tetsu Akiyama, who summarized recent results obtained in studying the WT-1 locus, which encodes a zinc-finger nuclear factor. Although the WT-1 locus was originally defined as a tumor-suppressor (wherein loss of both alleles heightens the susceptibility to transformation in developing renal cells), a truncated product, resulting from translation of an alternatively spliced transcript, appears to behave as a tumor-inducer. This transcript is represented at high levels in circulating blasts from patients with acute myelocytic or lymphocytic leukemia, but is relatively poorly expressed in normal leukocytes, or in cells from patients with more indolent malignancies, notably chronic myelogenous leukemia. Interestingly, in human clinical samples, levels of WT-1 transcripts may provide a prognostic index for survival. Dr. Akiyama noted that the time of action of WT-1 in suppressing cell proliferation is relatively early, but the mechanism remains under investigation.
Dr. Hisashi Harada has also studied two transcription factors with opposing activities on the same cellular behaviors, the IRF-1 and IRF-2 genes first defined through analysis of the regulation of interferon synthesis. Here IRF-1 activates, while IRF-2 represses, gene expression. Interestingly, inappropriate IRF-2 expression in NIH3T3 fibroblasts yields a transformed phenotype that can be reverted by simultaneous transfection with IRF-1 expression constructs. IRF-1 was also found to antagonize the transforming effects of myc and fos, providing another approach to the mechanism of action of these potent oncogenes. Dr. Harada also reviewed very recent results, obtained in studying mice lacking the IRF-1 gene or IRF-2 genes, which support the antagonistic nature of these two factors.
Discussion of interferon responses provided an appropriate bridge to Session 2 of the workshop, in which the responses to specific cytokines were assessed. Dr. James Ihle presented information on the involvement of the JAK kinases in responses to both Type I and Type II interferons, and in other cytokine signaling systems. He proposed a general model for JAK kinase activation, deduced from study of the interleukin-3 (IL-3) receptor, in which JAK kinases pair directly with the IL-3 receptor!!!chain, and thereby become capable of phosphorylating the p91 STAT proteins. The assignment of specific combinations of JAK and STAT proteins to individual cytokine responses was surveyed.
Dr. Toshio Hirano then outlined the signal transduction pathway emanating from the IL-6 receptor. Here as well, JAK kinase activation leads to phosphorylation of STAT proteins on tyrosine residues, and thereby promotes transcription of a variety of genes. A complex of perhaps four STAT-like proteins was found associated with a typical IL-6-responsive promoter following IL-6 stimulation.
Finally, Dr. Tadatsugu Taniguchi provided new information on the structures involved in signaling from the IL-2 receptor. He noted that formation of heterodimers between!!!and!!!chains of the receptor is required for signaling in the BAF pro-B cell line, and that signaling can be antagonized by introducing expression constructs encoding a C-terminally truncated!!!chain. Prior studies have established that two rather different pathways are activated by the IL-2 receptor!!!chain, and Dr. Taniguchi reported some success in assigning changes in gene activation to these different responses. In particular, he noted that one pathway, stimulated via the serine-rich domain of the!!!chain, augments expression of the Bcl-2 oncogene and hence prevents apoptosis. Together these studies proved quite illuminating regarding the ways in which cytokines can act to control cell growth and differentiation.
Session 3 began with analysis of a receptor-type protein tyrosine kinase, that encoded by the c-kit gene. Dr. Shinichi Nishikawa showed using transgenic mice that the promoter for the gene encoding the c-kit-ligand is active in hair follicles and is stimulated by physical manipulation of the follicle. Dr. Nishikawa has also established precisely when c-kit-ligand and IL-7 are required during B cell development, and has developed an in vitro culture system for B cell progenitors using entirely defined components. This system promises to be extremely useful in studying those factors that control normal and malignant B cell proliferation.
Elaborating on the theme of lymphocyte development, Dr. Roger Perlmutter described efforts that led to the identification of the lck gene as a pivotal regulator of thymocyte maturation. The lck gene is a proto-oncogene encoding a non-receptor protein tyrosine kinase that controls, in part, the transition from immature to mature thymocyte stages in response to expression of the T cell receptor!!!chain. The lck-encoded kinase is also an important regulator of proliferation in mature T cells stimulated via the antigen receptor. Interestingly, while the latter process appears to depend upon p21ras activation, the former is relatively ras-independent.
This discussion of the functions of ras was extended by Dr. Frank McCormick who summarized the current understanding of the regulation and function of the more than 20 ras-like small GTP-binding proteins. Regulation in each case is accomplished through a set of exchange factors that promote GTP binding, and GTPase-activating proteins (GAPs) that stimulate the intrinsic GTPase activity of the ras-family proteins, thereby permitting reestablishment of the baseline, unstimulated condition. Dr. McCormick noted that in many cases signals are delivered from transmembrane protein tyrosine kinases to downstream serine/threonine kinases by both ras-dependent and ras-independent mechanisms. Detailed analysis of the interaction of p21ras with the raf-encoded serine threonine kinase indicates that stable association of these proteins induces an as yet uncharacterized (and apparently covalent) modification of the raf kinase. It is this modified form of the kinase that acts to stimulate the MAP kinases via an intermediate called MEK.
Discussion of the raf/MEK/MAP kinase signaling pathway was the central theme in Session 4 of the workshop. Dr. Eisuke Nishida dissected pathways involved in MAP kinase activation using Xenopus oocytes. She reported that MEK, a pivotal serine/threonine kinase, can be activated directly by both the raf- and mos-encoded kinases, as well as by another kinase that is the equivalent of that encoded by STE11 in yeast. An anti-MEK antibody that neutralizes kinase activity was found to block progesterone-induced oocyte maturation and germinal vesicle breakdown. Similarly, Dr. Gary Johnson emphasized the essential role of raf/MEK/MAPK activation in signaling from serpentine receptors that utilize heterotrimeric G proteins as their most proximal signaling element. However, he also noted the enormous heterogeneity in MAP kinase signaling pathways, mentioning, for example, 5 putative homologues of STE11 that he and his colleagues have identified in mammalian cells. These results helped to reinforce statements made by Dr. Jean Wang at the beginning of the session, who pointed out that signaling responses to the same agent are often cell context-dependent. In her system, two sublines of 3T3 cells were generated that exhibit opposite responses (growth arrest versus transformation) to the BCR-ABL transforming gene. These cells also differ with respect to responses to phorbol esters.
The consensus that emerged from this discussion was that there exists enormous flexibility in response patterns to defined stimuli in cells; hence the general pathways now routinely drawn for signal transduction from growth factor receptors will prove to employ numerous variant connection schemes in specialized cell types. Session 5 was intended to permit discussion of events in cell behavior that are influenced by the signal transduction pathways previously outlined. As a bridge towards this discussion, Dr. Ann Marie Pendergast presented an analysis of the mechanisms underlying hematopoietic cell transformation by BCR-ABL. Of special importance is the interaction between the amino terminal region of the BCR protein, which interacts with the SH2 domain of the ABL-encoded kinase fragment. Also, the BCR-ABL protein is able to interact with GRB-2, a linker protein that stimulates guanine nucleotide exchange factors for p21ras. Together these observations support the view that the BCR-ABL fusion protein behaves as an activated growth factor receptor.
An altogether different kind of ceptor, that encoded by the fas gene, was discussed by Dr. Shigekazu Nagata. Stimulation of the fas-encoded receptor results in apoptotic death, rather than growth, of susceptible cells. The lpr mutation, long studied as a cause of primary lymphoproliferative disease in the mouse, results from synthesis of a dysfunctional fas protein, leading to inappropriately prolonged survival of lymphocytes. Dr. Nagata reported on his recent successful effort to define the fas ligand, which is encoded by a gene called gld in mouse. It was generally agreed that an understanding of the mechanisms underlying gld/fas-induced apoptosis could prove crucial in attempting to define targets for intervention in neoplastic disease.
Finally, Dr. Sachiko Tsukita presented an analysis of transformed cells from the perspective of a cell biologist. By purifying adherens junctions, it was possible to define many of their protein components, including radixin and Éø-catenin. Using antisense oligomers, Dr. Tsukita provided persuasive evidence for the importance of ezrin, radixin, and moesin in conferring substrate adherence. The ability to regulate expression of these proteins (or the function of each) appears to contribute to the metastatic properties of tumors, as well as to their ability to grow in a substrate-independent fashion. For example, Éø-catenin expression is specifically lost in some subtypes of breast carcinomas.
The workshop closed with a reiteration of the need to define the features that confer specificity on signaling pathways that, to date, are remarkable for the generic nature of their components. The ongoing discovery of families of closely-related signaling elements suggests that some differences in signal transduction pathways arise from combinatorial assembly of subsets of similar molecules. In other signaling pathways, specificity is apparently conferred by a network of regulatory influences. Overall, the workshop revealed that stunning progress has been made in enumerating the specific proteins that regulate growth in normal and neoplastic cells.

PARTICIPANTS

United States

Dr. Robert N. Eisenman
Fred Hutchinson Cancer Research Center

Dr. James N. Ihle
St. Jude Children's Research Hospital

Dr. Gary L. Johnson
National Jewish Center For Immunology and Respiratory Medicine

Dr. Frank McCormick
Vice President for Research, Onyx Pharmaceuticals

Dr. Ann Marie Pendergast
Duke University

Dr. Roger M. Perlmutter*
University of Washington

Dr. Jean Y. J. Wang
University of California at San Diego

JAPAN

Dr. Tetsu Akiyama
Research Institute for Microbial Disease
Osaka University

Dr. Hisashi Harada
Institute for Molecular and Cellular Biology Osaka University

Dr. Toshio Hirano
Biomedical Research Center
Osaka University Medical School

Dr. Shigekazu Nagata
Osaka Biosciences Institute

Dr. Eisuke Nishida
Institute for Virus Research, Kyoto University

Dr. Shinichi Nishikawa
Kyoto University, Faculty of Mdicine

Dr. Tadatsugu Taniguchi*
Institute for Molecular and Cellular Biology Osaka University

Dr. Sachiko Tsukita
National Institute for Basic Biology
Okazaki National Research Institutes

(*) = Co-organizers



(2) Seminar on "Cell Biology of the Host Antitumor Immune Response"
The workshop on "Cell Biology of the Host Antitumor Immune Response" was held in Rockville, Maryland, January 10–12, 1994, and covered the cell biology of host antitumor immune responses. A number of diverse topics were covered, focusing on the role of specific cytokines as well as T cells in antitumor immunity. In addition, there were a number of interesting discussions of the role of stress-induced proteins both in antigen presentation as well as antigenic targets in tumors. A major topic of discussion, reflecting the increased activity of the tumor immunology field in general, covered the identification of tumor antigens recognized by T cells.
Dr. Mari Kannagi discussed the analysis of cytotoxic T cell responses against HTLV 1 antigens in adult T cell leukemia. ATL was the first human tumor clearly associated with viral infection. Interestingly, in addition to producing a T cell leukemia, HTLV 1 infection is associated with inflammatory and autoimmune diseases, some of which involve the central nervous system. Dr. Kannagi described the culture of cytotoxic T lymphocytes from HTLV 1 patients with tropical spastic paraparesis (TSP). Interestingly, it is much more difficult to generate CD8+ CTL from the PMBC of most ATL patients. Dr. Kannagi found that one of the major CTL target antigens was the HTLV-1 p40 tax protein. A CTL epitope in p40 tax represented by amino acids 11-19 was presented by HLAA2. Leukemic cells from ATL patients that were HLA A2+ could be recognized by these p40 tax plus HLA A2+ CTL.
Dr. Noriyuki Sato reported on the analysis of a novel transformation associated heat shock protein expressed on the cell surface in H ras oncogene transformed rat fibrosarcoma cells. This antigen can be detected by a monoclonal antibody which immunoprecipitated a 70 kDA protein. CD4-8- T cells from the periphery of rats primed with live BCG demonstrated cytotoxicity against the rat fibrosarcoma cells; the cytotoxicity was blocked by the anti-HSP-70 monoclonal antibody as well as by the anti CD3. This cytotoxicity appeared to be able to be conferred by an acid eluted peptide from the rat fibrosarcoma cells, although it was unclear whether this was an HSP-70 peptide or whether the HSP-70 was presenting a particular peptide that represented a target for the double negative T cells.
Dr. Elizabeth Jaffee presented studies on the identification of tumor-specific peptides recognized by T cells elicited by a vaccination with cytokine gene transduced tumor cells. In a number of tumor models, immunization with tumor vaccines engineered to secrete cytokines such as GM-CSF elicits CTL responses that are enhanced relative to nontransduced tumor vaccines. CTL responses correlate with enhanced in vivo systemic immunity. Dr. Jaffee reported on a technique in which peptides eluted from the tumors MHC class I molecules could be repetitively fractionated on reversed phase HPLC columns. These peptides could be added to surrogate target cells under conditions in which they exchange onto empty MHC molecules. This assay provides the opportunity to test HPLC fractions for the existence of active peptides by virtue of their ability to sensitize the surrogate target cells to killing by the antitumor CTL. Dr. Jaffee used this technique to demonstrate a single immunodominant peptide from tumor cells eliciting CTL responses. She described a new mass spectrometry technique to sequence potential bioactive peptides. This strategy utilized a combination of exopeptidase digestion followed by matrix assisted laser disorption mass spectrometry.
Dr. Yutaka Kawakami described two novel human melanoma-specific antigens that he cloned using a technique pioneered by Theirry Boon's group. This technique involves the shotgun transfection of cos cells with cDNA libraries derived from tumor cells. Pools of transfectants were screened for stimulation of melanoma-specific CD8 clones and ultimately rescreened by sib selection. Two cDNAs encoding melanoma antigens, MART1 and GP 100, were recognized by HLA A2 restricted CTL. Both of these antigens represented shared melanoma antigens expressed by the majority of HLA A2+ melanomas. The GP100 antigen is a cell surface protein that had been previously recognized by certain melanoma-specific antibodies. The MART 1 antigen does not show homology to any previously known protein Dr. Kawakami described the plans to utilize these antigens in active immunotherapy protocols for patients with melanoma.
Dr. Suzanne Topalian discussed her identification of shared MHC class II restricted human melanoma antigens recognized by CD4+ T cells. She showed that by initially selecting the CD4+ cells from tumor infiltrating lymphocytes, it was possible to generate T cell lines and clones that recognized MHC class II restricted tumor antigens. This was demonstrated either by induction of cytokine release by the melanomas themselves treated with gamma interferon to induce MHC class II expression or by a representation assay. The representation assay involved the generation of tumor lysates which are fed to EBV transformed T cells from the patients in which the till were derived. This system allows for the identification of shared MHC class II restricted tumor antigens while assuring MHC matches between the antigen presenting cell and the responding T cells. Dr. Topalian reported the identification of a number of shared MHC class II melanoma antigens.
With regard to the regulation of immunological responses to tumors, a number of presentations centered around the role of specific cytokines.
Dr. Michael Lotze presented his studies on the role of interleukin 4 and interleukin 12 in regulating antitumor reactivity. Both cytokines appeared to display antitumor reactivity either when given systemically or when their genes are transduced into tumor cells as a vaccine. Dr. Lotze presented preliminary results from a phase I study of IL4 gene transduced vaccines in which the IL4 gene was introduced into fibroblasts from patients, and these transduced fibroblasts were mixed with the patient's tumor cells and irradiated prior to injection. Results of these clinical trials are pending.
Dr. Hiromi Fujiwara investigated the role of various cytokines and humoral substances in the inhibition or promotion of antitumor immunological responses. His studies indicated that the tumor-bearing state induces abnormal cytokine networks under which the production of antitumor cytokines are negatively regulated at the transcriptional and posttranslational level. He subsequently found that interleukin 12 could reverse these effects and mediated potent antitumor effects through the induction of gamma interferon.
The role of B7 in antitumor immunity was explored by Dr. Suzanne Ostrand-Rosenberg and by Dr. Miyuki Azuma. Dr. Ostrand-Rosenberg demonstrated that tumors transduced to express both MHC class II as well as B7 displayed enhanced immunogenesity and vaccine potency. The presence of B7 alone or MHC class II alone had much reduced vaccine potency. Furthermore, the double transductants were capable of impacting on established tumors in the murine fibersarcoma model.
Dr. Miyuki Azuma presented her experiments on the identification of a novel B7 family member termed B70 or B7-2. This new family member binds both CD28 as well as CTLA4 similar to the original B7. Antibodies against B70 appeared to completely inhibit primary mixed lymphocyte reactions indicating that the B70 member of the B7 family may be the first expressed and be most important for initiation or priming of immunologic responses. B70 was also demonstrated to be involved in induction of killer cell activation and therefore may be important in the generation of antitumor immune responses.
Dr. Promad Srivastava discussed his work in the identification of heat shock proteins important for generating immune responses. GP96, an endoplasmic reticulum localized heat shock protein, seems to be important for a transfer of initially class I restricted peptides into the MHC class I groove. In addition, HSP70 may be important earlier on in the transfer of peptides from the proteosome to the TAP transporters. Dr. Srivastava demonstrated that GP96 and HSP70 extracted from tumors was capable, in the purified form, of immunizing animals against tumors in a tumor-specific fashion.
Other presentations at the meeting covered the molecular biology of IL6 signal transduction, the regulation of substrate-specific chaperonin induction, alterations in signal transduction in T cells from tumor bearing individuals, and the role of certain oncogenes, such as myk, in the modulation of tumor immunogenesity.

PARTICIPANTS

United States

Dr. Elizabeth Jaffee
Department of Oncology
Johns Hopkins University
School of Medicine
720 Rutland Avenue/Ross 364
Baltimore, MD 21205-2196

Dr. Yutaka Kawakami
Surgery Branch
National Cancer Institute
Bldg. 10, Rm. 2B42
Bethesda, MD 20892

Dr. Michael Lotze
Surgical Oncology
Pittsburgh Cancer Institute
497 Scaife Hall
Pittsburgh, PA 15261-0001

Dr. Augusto Ochoa
National Cancer Institute
Frederick Cancer Research and Development Center
Frederick, MD 21702- 1201

Dr. Drew Pardoll
Department of Oncology
Johns Hopkins University
School of Medicine
720 Rutland Avenue/Ross 364
Baltimore, MD 21205-2196

Dr. Suzanne Rosenberg
Department of Biological Sciences
University of Maryland
540 1 Wilkens Avenue
Baltimore, MD 21228

Dr. Promad Srivastava
Fordham University
300 Larkin Hall
Bronx, NY 10458

Dr. Suzanne Topalian
Surgery Branch
National Cancer Institute
Building 10, Rm. 2B47
Bethesda, MD 20892

JAPAN

Dr. Mari Kannagi
Kumamoto University Medical School
Kumamoto

Dr. Nobuyuki Sato
Sapporo Medical College
Sapporo

Dr. Kazuhiro Nagata
Kyoto University
Kyoto

Dr. Miyuki Azuma
Juntendo University
Tokyo

Dr. Kouji Matsushima
Kanazawa University, Cancer Research Institute
Kanazawa

Dr. Hiromi Fujiwara
Osaka University Medical School
Osaka

Dr. Shizuo Akira
Osaka University
Osaka

Dr. Yoshiyuki Kuchino
National Cancer Center Research Institute
Tokyo



(3) Seminar on "Gene Manipulation of Malignant Cells"
The conference "Gene Manipulation of Malignant Cells" was held on March 25-26, 1994, in the Maui Prince Hotel, Maui, Hawaii, and included scientists from the United States and Japan. The meeting was divided into four major thematic sessions including 1) the structure and function of cancer genes, 2) immunologic approaches to genetic therapy of cancer, 3) systemic gene delivery and prodrug approaches to the treatment of cancer, and 4) hematologic malignancies and stem cell biology.
Dr. Takehiko Sasazuki began the presentations by summarizing the state-of-the-art with respect to human colon cancer. He described the role of multiple oncogenes involved in carcinogenesis, including the non-syntenic chromosomal loss that has been noted sequentially in the development of colon cancer. He described studies aimed to knock out the k-ras gene and showed that he was able to achieve rates of homologous recombination between 1-2 x 104 in independent lines. He showed the morphologic changes in the knockout tumors which suggested reversal of the malignant phenotype and suggested that k-ras may be an appropriate target for anticancer therapy. The discussion focused on how that might be successfully achieved.
Dr. Mitsuaki Yoshida described his pioneering studies with HTLV-1 and summarized the role of the virus in human malignancy and in autoimmune diseases. A major part of his presentation focused on the mechanism of action of tax on the HTLV-1 enhancer, and NF-kB. He presented new findings that the Tat protein is capable of binding to the C-terminal domain of NF-kB1 (IkB-) and suggested that its interactions with these different parts of the molecules may provide multiple roles for the tax gene in inducing T cell proliferation. Discussion focused on what other genetic changes may be responsible for HTLV-1-induced leukemia, as well as the role of perinatal transmission versus horizontal transmission later in life.
Dr. Jeffrey Trent described his work on the mechanism of transformation of human melanoma. He described previous studies in his laboratory on defining a gene on chromosome 6 involved in malignant transformation and reversal of the malignant phenotype by overexpression of superoxide dismutase which is found on chromosome 6 but not in the candidate locus. He also described new methodologies using chromosome gene transfer as well as fluorescence in situ hybridization to assess the efficacy of gene transfer and to develop new methods of gene delivery.
Dr. Yoichi Gondo described his system for doing 2-step homologous recombination in which a knockout can be followed by gene replacement. In his studies, he described his ability to delete mutant p53 genes by homologous recombination and subsequently replace these with a LacZ expression vector. He described a new strain of transgenic mouse where this was essentially accomplished and where this animal can now be used to localize p53 expression.He also described the technical difficulties involved in performing such two-step homologous recombination experiments. Animals deficient in p53 showed a variety of different malignant tumor types, including lymphomas, sarcoma, and others.
Dr. Yusuke Nakamura gave a presentation of the genetic changes in the APC gene in colon cancer. His laboratory has played a major role in defining the various mutations in the disease, and he pointed out that a variety of the mutations led to abnormal gene products which could be the target of immune recognition. He also described the relative importance of other genetic changes in colon cancer, including p53 and NF2. The presence of APC mutations in pancreatic cancer also represented new findings in this area. His laboratory has been involved in the characterization in mismatched repair genes, including MSH2, MLH1, and, more recently, PMS1. His laboratory is currently defining the complexity of PMS1 and its possible role in inducing genetic instability in cancers.
Dr. Nobuyuki Tanaka described the possible role of interferon inducible transfection factors in oncogenesis and showed the role of the IRF1 and IRF2 genes and developed retroviral vectors to express these within cells. In cells, IRF2 overexpression results in transformation, and combination of IRF1 and IRF2 leads to the reverse of the malignant of the phenotype. IRF1 knockout mice showed no tumors up to one year after creation of the transgenic strain. He also showed that there is a possible interaction between IRF1 and the ras gene in malignancies. He noted that IRF deletion has been detected in some patients and is localized to chromosome 5.
Dr. Peter Jones described the role of DNA methylation in the generation of malignant phenotypes. He summarized data on the methylase enzymes. He pointed out the high rate of mutation on CpG sequences and their consequential underrepresentation in the human genome. He also described studies showing that CpG islands at various loci, including myoD, can be changed during oncogenesis and affected by agents such as 5-azacytidine. He also described some promising new studies using a CpG island from the Thy1 gene which has been introduced into a retroviral vector in an effort to maintain expression of recombinant genes following transduction into early hematopoietic cells or into other cells. The session on immunologic approaches was begun by Dr. Michael Bevan who summarized the current state-of-the-art with respect to antigen presentation by the immune system and the role of the proteosome in class I presentation. He described current models of positive T cell selection through low affinity interactions of the T cell receptor with MHC in the thymus. He also characterized the role of various residues of peptides presented in the MHC groove and the role of different anchor and presentation domains in generating immunity to specific epitopes. He also described a novel and innovative approach to the generation of T cells in patients who might not have the ability to generate improved immunity against tumor antigens based on an effort to generate improved immunity against tumor antigens by positive thymic selection in transgenic animals.
Dr. Masashi Okada described a series of studies which focused on cytokine gene therapy in the use of SCID-Hu mice to encourage antitumor responses. He showed that the introduction of IL-6 into tumors improved the antitumor effect. Also, he found that IL-6 transgenic mice could support the development of human CTL against human tumors in this animal model and suggested that it may be used to assess antitumor responses as a potential strategy to encourage immunity. Finally, he described studies with the adenoviral vectors to express as IL-6 in the inhibition of SDL3 tumor in this model. The co-administration of these vectors with leukemic cells led to protection against tumor growth.
Dr. Richard Mulligan described his studies of cytokine-mediated gene transfer to improve immune recognition of tumors. He summarized the systematic characterization of different cytokines which are involved in this process, and the findings of his group that the GM-CSF cytokine appears to give the optimal protection in cancer vaccine models. He further went on to describe a genetically modified strain of mouse in which the GM-CSF gene has been deleted by homologous recombination. These animals show a phenotype of pulmonary alveolar proteinosis. He discussed the potential implications of this defect.
Dr. Gary Nabel discussed the role of direct gene transfer in the treatment of human malignancy. The model systems looking at the ability of a foreign histocompatibility gene to induce site-specific immunity in vivo were discussed. In addition, the results of the human gene therapy trial and the mechanisms underlying immune responses in this trial were summarized, as well as other trials. More recent developments and the improvements incorporated into the newer trial were discussed in detail.
Dr. Jeffrey Leiden discussed the potential of systemic drug delivery for the treatment of cancer. He described the role of the muscle cells as potential drug delivery vehicles in vivo. A major portion of the discussion was on the use of adenoviral gene delivery vehicles, and he described exciting new results demonstrating the potential of this technology to be used for erythropoietin responsive anemias. The muscle cells were shown to correctly process this gene product, and in vivo introduction into animals resulted in high sustained levels of protein in vivo, together with increases in hematocrit. He summarized some of the difficulties with adenoviral vectors and the potential for the immune system to recognize and eliminate these vectors. The difference in persistence of expression in newborns vs. adult animals was described, and several new approaches to the modification of adenoviral vectors which could deal with some of these complications were discussed.
Dr. Savio Woo discussed his work using an adenoviral thymine kinase for the treatment of brain cancer. He described results from an animal model system in which injection of this vector into brain tumors in nude mice resulted in tumor regression. Additional studies in immunocompetent animals also showed a positive response, and he described efforts of his group to bring these studies forward for a potential human clinical trial.
The final portion of the meeting dealt with gene therapy approaches to the treatment of leukemia. Dr. Kenzaburo Tani summarized an extended program with Professor Asano which is underway with hematopoietic targets of gene therapy including potential applications to pyruvate kinase abnormalities, AIDS, and other hematologic disorders. He also described his interest in ex vivo models for bone marrow transplantation and cytokine gene transfer approaches.
The final talk of the meeting was by Dr. Irving Weissman who described the efforts of his laboratory to identify the self-renewing hematopoietic stem cells. He described the phenotype of these cells in the murine model as being Thy1lo Lin-, Sca- 1+ and described methods to isolate this cell. He also discussed its relationship to the human cell which is Thy1loCD34+. He summarized their efforts to isolate stem cells in both mice and man and to undertake purging. The efforts are underway for the treatment of myeloma and for chronic myelogenous leukemia.

PARTICIPANTS

United States

Dr. Michael J. Bevan
Howard Hughes Medical Institute
University of Washington, SL-15
Health Sciences Building, J-625
Seattle, WA 98195

Dr. Jeffrey M. Leiden
Professor of Medicine and Pathology
University of Chicago
5841 S. Maryland Avenue
BRH B608 MC 6080
Chicago, IL 60637

Dr. Peter A. Jones
USC/Norris Comprehensive Cancer Center
NOR 743
1441 Eastlake Avenue
Los Angeles, CA 90033

Dr. Richard Mulligan
Whitehead Institute for Biomedical Research
Nine Cambridge Center
Cambridge, MA 02142

Dr. Gary J. Nabel
Associate Investigator
Howard Hughes Medical Institute
Internal Medicine and Biological Chemistry
1150 W. Medical Center Drive, 4510 MSRB I
Ann Arbor, MI 48109-0650

Dr. Jeffrey Trent
National Center for Human Genome Research
National Institutes of Health
Building 49, Room 4A22
Bethesda, MD 20892

Dr. Irving L. Weissman
Professor, Department of Pathology
Stanford University School of Medicine
300 Pasteur Drive
Palo Alto, CA 94305-5324

Dr. Savio L.C. Woo
Cell Biology & Molecular Genetics
Baylor College of Medicine
One Baylor Plaza, Room T721
Texas Medical Center
Houston, TX 77030

JAPAN

Dr. Mitsuaki Yoshida
University of Tokyo

Dr. Kenzaburo Tani
University of Tokyo

Dr. Yusuke Nakamura
Japan Foundation for Cancer Research

Dr. Nobuyuki Tanaka
Osaka University

Dr. Masashi Okada
Kyushu University

Dr. Takehiko Sasazuki
Kyushu University

Dr. Yoichi Gondo
Kyushu University