(1) Seminar on "Receptor-mediated Modulation of Lymphocyte Growth and Activation"
This meeting was held January 11-13 in Kona, Hawaii. There were ten U.S. participants and seven Japanese participants at this seminar which focused upon the discrimination of self from non-self in the immune system, and the relevance to tumor rejection, transplantation, and autoimmunity.
I. Receptor-ligand interactions in lymphocyte activation
The first session of this meeting dealt with receptor-ligand interactions in lymphocyte activation. Dr. Takashi Saitoh described T-cell development in CD3 zeta knockout mice. Initial studies were described which utilized zeta mutants to reconstitute zeta negative 2B4 cells. Signalling of transfected cells with different stimuli including antigenic ligand and antibody cross-linking of the T cell receptor (Tcr) revealed different requirements for components of the zeta molecule. Mice in which zeta gene was knocked out by homologous recombination showed a profound defect in maturation of Tcr!
!!expressing T cells in thymus and in the periphery. Signalling defects in T cells of mature phenotype were also observed. Carl June described the structure and function of the CTLA4 molecule. CTLA4 serves as a co-receptor for the B7/BB1 molecule on activated B cells. CTLA4 is expressed at an extremely low level in resting T cells, and its expression is increased after Tcr-mediated activation of T cells. Anti-CD28 stimulation also induced expression of CTLA4. The difference in signal transduction through Tcr and CD28 were discussed.
Ko Okumura described the involvement of LFA in cell interactions and activation. He first described the interaction of soluble CD2 with CD48 and CD58 (LFA3). He further described in vivo studies in which treatment with anti-ICAM1 plus anti-LFA1 resulted in prolonged cardiac allograft survival in the rat.
Kazuo Sugamura described the functional significance of the IL2 receptor!!
!chain in signal transduction. Transfection into non-lymphoid cells demonstrated the role of a third component of the IL2 receptor in addition to the previously desrcibed!!!and!!!chains. This third component, the!!!chain, contributes to both IL2 binding affinity and to signal transduction. The!!!chain of the IL2 receptor appears not to be central for signal transduction; the!!!chain is critical; and the!!!gene is required for some but not other gene activation events in response to IL2. The structure and function of the Fas antigen in mediating apoptosis was described by Shigekazu Nagata. The Fas product is a member of the NGF/TNF receptor family. Cells transfected with human Fas are rendered susceptible to induction of apoptotic death and response to anti-fas antibody. The nature of signal transduction through the Fas molecule and its physiologic role remain unknown.
John Cambier discussed the B lymphocyte antigen receptor complex and the interaction of receptor subunits with cytoplasmic enzymes involved in signal propagation. The!!
!and!!!subunits of the Ig receptor complex interact with Ig heavy chains. In turn, the!!!and!!!molecules interact with a number of additional intracellular proteins including kinases. The functional state of the Ig receptor complex may be mediated by the association and phosphorylation of the various components of this complex. Peter Cresswell described the mechanisms of class II MHC-restricted antigens processing. Transfection experiments involving class II!!
!and!!!chains with or without the invariant (I) chain demonstrated that the I chain acts as chaperone directing class II molecules to endosomes. A second role of the I chain is to prevent class II association with peptide. II. Mechanisms of self-nonself discrimination
Jon Sprent described tolerance mechanisms in CD8 T cells. Using adoptive transfer techniques and analysis of tolerance in both thymus and peripheral T cells, it was shown that the class I tolerance of CD8 T cells was more complete in lymph nodes than in thymus.
Marcia Blackman described multiple mechanisms of T cell tolerance for CD4 T cell specific for Mls superantigen. Using a Tcr transgeneic model, it was found that both clonal deletion and anergy induction play a role in tolerance to this antigen. Evidence for anergy was found exclusively in the periphery and not in thymus, indicating that this is a peripheral mechanism of T cell tolerance.
The role of co-stimulator signals in T cell activation and anergy was discussed by Ronald Schwartz. In a model of Th1 T cell clones, it was shown that a co-stimulatory signal, in addition to Tcr signaling is necessary for T cell activation. Tcr signaling in the absence of co-stimulus results in anergy which is reflected in a lack of IL2 production and a failure to develop responsiveness to IL4. The interaction of T cell CD28 with B7 expressed on antigen presenting cells appears to represent an important co-stimulatory pathway. Analysis of the molecular basis for anergy has indicated that the AP-1 signal for IL2 production appears to be a major locus for unresponsiveness.
Yasunobu Yoshikai descibed the increased expression of!
!!T cells reactive to heat shock protein in host defense to microbial infection. In a mouse model of Listeria, antibody to!!!resulted in increased susceptibility to infection, suggesting a protective role for!!!-expressing cells in host resistance. The role of immune responsiveness in mouse mammary tumor virus (MMTV) infection was discussed by Richard Hodes. It was demonstrated that infectious and tumorigenic MMTV share the property of superantigenicity. The expressed ORF products of distinct MMTV have unique specificities for T cells expressing particular V?? products and are capable of mediating both expansion of mature peripheral T cells and long-term deletion of T cells expressing the relevant V!!
! product. Experiments using MHC class II transgeneic mice indicated a critical role for the E!!!transgene product mediating both V!!!specific T cell deletion and host susceptibility to MMTV infection. It thus appears that the superantigenic stimulation of host T cells by MMTV ORF products plays an important role in facilitating in vivo infection.III. Self-nonself discrimination in tumor immunity, transplantation, and autoinumunity.
The heterogeneity of graft rejection pathways and tolerance induction was discussed by Hiromi Fujiwara. Tolerance induction was studied for both MHC class I and class II antigens. Treatment modalities included the pre-treatment of recipients with fractionated or nonfractionated donor lymphoid cells, with or without depletion of host T cell sub-populations, or the use of the immunosuppressive drug FK506. Different requirements were shown for induction of class I- and class II-specific tolerance. In addition, different class I-different combinations also showed differences in the relative importance of CD4 or CD8 host cells in response to the graft. In vitro proliferation and CTL responses were at times dissociated in tolerance states. Collectively, these data indicate the complexity of response pathways to MHC alloantigens, and the corresponding complexity of tolerance mechanisms.
Jeff Bluestone discussed the mechanisms of tolerance to pancreatic islet xenograft. CTLA4-Ig was shown to block xeno- and allo-antigen responses in vitro. In addition, the survival of human islet cells in the mouse was facilitated by treatment with CTLA4-Ig in vivo. Graft survival was accompanied by specific tolerance to re-grafting up to 60 days but not thereafter, despite the fact that the original graft continued to survive in vitro, there was no evidence of corresponding tolerance as measure by MLR or IL2 production. Antibody to human B7 but not to mouse B7 also blocked the rejection of human islet cells by the mouse host, indicating that there may be direct presentation of xeno antigen involved in graft rejection. The combined use of antibody to ICAM1 and CTLA4-Ig produced even more marked graft survival in vivo.
Toshiyuki Hamaoka described a model system in which TGF!!
!-mediated immunosuppression leads to tumor escape from normal rejection mechanisms. In this model it appears that TGF!!!produced by tumor cells themselves led to suppressed IL2 production by the host in response to tumor antigen. After removal from contact with tumor cells, T cells are capable of recovery from this effect. These findings point to a potentially important site of immune response suppression in tumor bearing animals which might be a target for therapeutic intervention. The role of the interaction between T cell CD28 and presenting cell B7 in the activation of CD8 positive T cells was discussed by James Allison. It was shown that in the in vitro CTL response to class I antigen, B7-CD28 interaction plays a critical role. This occurred in a model in which no exogenous CD4 help is needed and appears to reflect IL2 production by CD8 T cells. In an in vivo tumor model, it was shown that transfection of a B7 negative UV-induce melanoma converted a progressor tumor into one which was susceptible to immune host rejection. Exposure to the B7 positive tumor also induced immunity against the B7 negative tumor line. These data provide striking evidence for a role of co-stimulator signal in host response to tumor antigens and suggest a strategy for immunization against non-stimulatory tumors based on introduction of a co-stimulator signal.
The use of peptides as immunotherapeutics was discussed by Gary Fathman. Two strategies for peptide-induced unresponsiveness were discussed. In the first of these, peptides corresponding to TCR sequences were used to pre-immunize mice. Peptides corresponding to V!!
!8.2 were used to pre-immunize mice prior to there subsequent challenge with sperm whale myoglobin (Mg). The response to Mg normally is predominated by use of V!!!8.2 Tcr. This pre-immunization protocol resulted in a specific and profound decreased responsiveness of both CD4 and CD8 T cells both to antigenic challenge in vivo and Tcr crosslinking in vitro. The second approach involved immunization with an antigeneic peptide in IFA. This approach also led to specific unresponsiveness to subsequent challenge with protein antigen. Pretreatment with a MBP peptide protected animals from subsequent development of autoimmune EAE. Peptide treatment also worked to successfully treat disease after onset of clinical signs. These strategies have therapeutic implications as approaches to the treatment and or prevention of autoimmune diseases. In general discussion following these presentations, the therapeutic implications of understanding basic cellular and molecular mechanisms of host response were discussed. As detailed above, the understanding of basic immune responses has now reached a critical point at which preclinical and clinical applications are apparent for tumor immunity, transplantation, and autoimmunity. Techniques of gene transfection and the use of monoclonal antibodies and specific peptides are examples of current biotechnology which are now poised for clinical application. This meeting represented a unique forum at which the latest advances in basic immunobiology were integrated and discussed in the context of clinical application.
(2) Seminar on "Signal Transduction from Plasma Membrane to the Nucleus "
Maui, Hawaii, January 25-27, 1993
The meeting was organized jointly by Tadatsugu Taniguchi, Osaka University and by Benoit de Crombrugghe, The University of Texas, M. D. Anderson Cancer Center. The central theme of the meeting was Signal Transduction, from Membrane to Nucleus.
The meeting was subdivided into five Sessions.
Session I. Functions of Signaling Molecules I.
Tadatsugu Taniguchi discussed his work on the IL-2 receptor and signal transduction triggered by interactions between IL-2 and its receptor complex. He first described structure-functional studies with three distinct IL-2 binding receptor components, and then the interactions between one of these receptor components and a lymphocyte specific protein tyrosine kinase, p56 Ilk. Other tyrosine kinases appear to be involved as well in the signaling that is triggered by IL-2 which leads to the induction of c-fos and c-jun.
It appears that the complex IL-2 receptor which is not a tyrosine kinase itself, interacts with one or more tyrosine kinases to activate cellular signaling pathways.
Martin Schwartz discussed his work on mechanism by which integrins have a synergistic role in signal transduction together with cytokine-receptor interactions. PDGF triggers a protein kinase C-dependent increase in intracellular pH when cells are adherent to fibronectin but not when they are in suspension. Specific integrins also regulate the activity of a calcium channel and, thereby, control intracellular calcium concentrations. The interactions between the extracellular matrix and specific integrins can, therefore, modulate the signaling activity of cytokines but also by themselves produce significant changes in intracellular components.
Tadashi Yamamoto discussed his work with several members of the tyrosine kinase family and the mechanisms by which they influence nuclear events and progression through the cell cycle.
Yukiko Goto discussed her work on the structure-function characterization of a MAP kinase activator which is itself a kinase and functions as a MAP kinase kinase (MAPKK). In mammalian fibroblasts a similar MAPKK is activated in response to growth factor stimulation. MAPKK has therefore, a key role in relaying growth stimulatory signals between the membrane and the nucleus.
Session 2. Functions of signaling molecules II.
Kunihiro Matsumoto described his studies on the signaling pathways in yeast that are triggered via a Protein Kinase C homologue (PKC1). His studies have identified a cascade of three successive MAP kinases downstream of PKC1. His results and those of others strongly suggest that a similar pathway exists both in yeast and in mammalian cells.
Shin Yonehara discussed his studies on apoptosis in T cells which is mediated by interaction between the NGF receptor-like molecule Fas and a monoclonal antibody. Fas antigen appears to play a role in the negative selection of autoreactive T cells. Fas mediated cell death is inhibited by the presence of the adenovirus E1b transcription factor. This study provided evidence that Fas-mediated programmed cell death occurs through a signaling pathway. The nature of the signals between the membrane and the nucleus are beginning to be unraveled.
Session 3. Signaling molecules in development and disease.
Gregor Eichele discussed the role of retinoids and their receptors in pattern formation during vertebrate development. He described his discovery of a new retinoic acid metabolite, 9-cis-retinoic acid, which is the ligand of the retinoid-X-receptors. He further discussed the involvement of all- transretinoic acid in pattern formation during limb development and how an early embryonic tissue organizer known as Hansen' s node, is a high-point of retinoic acid production.
John Wozney discussed his work on the identification of the fanilly of bone morphogenetic proteins (BMPs) which are a new class of TGF-!!
!relatives. These cytokines are capable in vivo to reproduce the complete chain of events which leads to endochondral bone formation. They are also able to convert in vitro multipotent mesenchymal cells into chondroblasts and osteoblasts. The mechanisms by which the BMPs trigger intracellular signaling is not understood. The BMPs also play a role during early embryonic development in mesenchymal-epithelial cell interfaces in non-skeletal tissues. Kenichi Yamamura discussed his work on the effect of genetic background on tumorigenesis in transgenic mice harboring a c-myc gene activated by the immunoglobulin enhancer, Transgenic mice in a B6 background developed B cell lymphoma, whereas T cell lymphoma predominated in a C3H background.
Eric Olson discussed his work on the control of myogenesis by muscle-specific helix-loop-helix (HLH) proteins. The activity of these proteins are blocked by the FGF and TGF-!!
!signal transduction pathways. Specific residues have been identified which upon phosphorylation inhibit either DNA binding or the transcriptional activation of these HLH proteins. These studies illustrated the mechanisms by which signals that favor cellular proliferation inhibit differentiation. Session 4. Signal transduction and gene reglation
Benoit de Crombrugghe discussed his work on lineage-specific control mechanisms of type I collagen gene. He described the use of transgenic mice to delineate the minimal sequences in the promoter of one of these collagen genes which confer tissue-specificity in transgenic mice, the spatial and temporal correlation between the activity of this promoter during embryonic development and the presence of extracellular TGF-!!
!1, and possible mechanisms by which TGF-!!!activates this promoter. Matoo Kitagawa described his work together with Tadatsugu Taniguchi on two transcriptional regulation of the type I interferon gene and interferon-inducible gene, IRF-1 and IRF-2. IRF-1 displays properties of a tumor suppressor and is often deleted in patients with leukemia and myelodysplasia. In contrast, IRF-2 has properties of a dominant oncogene since NIH-3T3 cells stable transfected by IRF-2 show increased tumorigenicity, a situation which can be reversed by the concomitant overexpression of IRF-1.
Michael Karin described his work on the control of the c-jun transcriptional activator by phosphorylation of specific residues in the transcriptional activation domain of this protein. He also identified a novel kinase which could be responsible for these phosphorylation events. His work showed the complexity of regulatory events in the cell that are triggered by growth factors.
Session 5. Nuclear factors.
Naoko Imamoto discussed her work on the identification of a heat-shock cognate protein (hsc 70) which is involved in the transport from the cytosol to the nucleus Antibodies to hsc 70, when injected in the cytoplasm of tissue culture cells, inhibit nuclear import of several nuclear proteins. She also discussed the establishment of a permeabilized cell-free system to study transport of nucleophilic proteins into the nucleus and demonstrated a critical role for hsc 70 in this system.
Eva Lee described her work with mice carrying a germline mutation in the retinoblastoma (Rb) gene. Mice heterozygous for the Rb mutation develop normally but are prone to occurrence of brain tumors not retinoblastoma. Mice homozygous for the Rb mutation die during embryonic development with multiple abnormalities.
In further work she discussed the isolation of a new protein which interacts with pRb, RbAp48. This protein shows homologies with a yeast protein implicated in the negative regulation of the RAS-adenyl cyclase pathway. Her work suggests that pRb could inhibit cellular growth by acting at several levels in the pathways of growth stimulation.
Yoichi Taya discussed his work on the isolation of new proteins which interact with the retinoblastoma gene product (pRb). He also described a low molecular weight inhibitor of cdc2/cdk2 knases isolated from streptomyces which could help control the activity of these kinases. In further work he described the synthesis of phosphopeptides containing phosphate groups on Ser, Thr and Tyr residues and the use of these phosphopeptides to study phosphorylations sites phosphorylated by odc2 kinase.
Overall conclusion.
During this seminar the participants had in-depth discussions of critical molecules of specific signal transduction pathways. The talks and discussions illustrated the considerable progress which has been made recently in defining molecules which have an active regulatory role in some of the pathways which control cellular proliferation and cellular differentiation. Despite obvious complexities very substantial and exciting progress was reported in a number of areas and it is likely that progress will continue at an accelerated pace. Discussions centered around the nature of the molecules interacting with either membrane receptors or tyrosine kinases located at proximity of the membrane, nuclear transcription factors, molecules which control events of the cell cycle, and proteins or enzymes which alter the activities of these proteins. All participants felt that the seminar had been very informative and highly stimulating.
(3) Seminar on "Transcription Factors in Lymphocyte Development and Malignancy"
Silverado Country Club, February 15-16, 1993
Organizers: Stanley J. Korsmeyer, Washington University
School of Medicine,
Takehiko Sasazuki, Kyushu University
Sesssion I. Transcription Factors in Lymphoid Development and Neoplasia
Dr. Hisashi Harada, Ph.D. Institute for Molecular & Cellular Biology, Osaka University led off the discussion with a presentation of the IRF1 and IRF2 genes in transcription and oncogenesis. It has long been noted that interferon can function as a negative regulator of growth. Interferon genes have been noted to have a conserved motif that regulates their interaction with IRF1, a transcriptional activator, and IRF2, a transcriptional repressor. IRF1 has a relatively short protein half-life of 20 minutes whereas IRF2 is long at greater than 6 hours. IRF2 is rather constitutively expressed throughout the cell cycle although IRF1 shows accumulation in G0 and G1 and decrease in S phase. IRF2 was noted to increase colony formation in NIH 3T3 cells. However, IRF1 had an anti-oncogene capacity in 3T3. Of interest, IRF1 mapped to 5q31.1, the area implicated within myelodysplastic syndrome (MDS). FISH analysis confirmed that IRF1 was in the minimal area of deletion in MDS. In fact 5 cases were identified in which both alleles had been lost. Another case was noted which had a rearrangement breakpoint within exon 1 of IRF1. Thus, IRF1 is an extremely attractive candidate for the gene deleted on chromosome 5 in the genesis of MDS.
Dr. Mark Kamps, University of California, San Diego discussed the t(1;19) translocation and its E2A-Pbx-1, fusion protein product. This translocation of pre-B ALL results in a p85 as well as p77 E2A-Pbx product. The translocation eliminates the helix-loop-helix portion of E2A fusing its activation domain with the homeobox domain region from Pbx. Pbx-1 is expressed in all lineages except B and T lymphoid cells. Of interest is that 90% of pre-B ALL with the t(1;19), express p85 and p77 and have a better prognosis than the 10% of pro-B phenotype that lack the p85 or p77 proteins. Dr. Kamps has utilized a murine leukemia virus based retro-viral vector to overexpress p77 within 3T3 cells. Moreover, this model has been used within bone marrow and transplantation studies have revealed development of acute myeloblastic leukemia complete with chloromas. Roughly half of these cases are factor dependent and others independent of IL-3. The E2A-Pbx product has also been shown to block differentiation in a bone marrow culture system with increase in blasts of myeloid type.
Shizuo Akira, M.D. of the Institute for Molecular & Cellular Biology, Osaka University detailed further elegant work on NF-IL6, a transcription factor involved in inflammation, immune response & acute phase reaction. The IL-6 recognition element is present in many acute phase response genes and is bound by the NF-IL-6 protein which has homology to CEBP and possesses a basic DNA binding domain as well as a leucine zipper. NF-IL6 has been noted to be phosphorylated and is a target for MAP kinase. Moreover, oncogenic Ras will activate NF-IL6 increasing phosphorylation on threonine 235. Phosphorylation of NF-IL6 occurs in PC12 cells stimulated with NGF and it will then bind to the c-fos gene.
Takeshi Watanabc, M.D., Medical Institute of Bioregulation, Kyushu University presented studies on protein phosphorylation and B cell activation. Dr. Watanabe screened a human cDNA library of a B cell with an E1A probe. He isolated a gene encoding a 75 kD protein in hematopoietic cells. The N-terminal domain has a nuclear localization signal and a 37 amino acid repeat, replicated 4 times. The carboxy terminal domain has an E1A homologous acidic stretch and!!
!-helix as well as an SH3 domain. He named this protein HS1 and found that it was rapidly phosphorylated after activation. SH3 domain appears to keep HS I within the cytosol as its deletion results in a nuclear translocation. That normal movement with activation of the immunoglobulin receptor is related to phosphorylation of HS1. There is a feeling that the p56 lyn molecule may interact with HS1 and be responsible for its phosphorylation. Jeff Leiden, M.D., Ph.D. of the University of Chicago presented his work on the ets-related transcription factor Elf-1 and its interactions with the retinoblastoma gene product. Dr. Leiden has noticed that many of the ets family members are expressed in T cells including ets-1, fli-1, erg-3 and elf-1. He has noted potential ets binding sites in a wide variety of genes expressed within T cells including the IL-2 enhancer. Of interest, all elf sites are next to AP-1 sites. Elimination of the ets binding site and the IL-2 promoter abolishes its activation by PMA in ionomycin. Elf-1 is comprised of an acidic domain, an!!
!-helix, basic domain and a serine threonine rich domain. Of interest elf-1 has a classic Rb binding motif similar to that found in E1A, T antigen or the E7 product of HPV. Elf-1 product will bind to Rb in vitro and E7 peptide will compete with it. Mutation of the classic binding site for elf- I prevents that interaction. Elf-1 was transfected into cos cells and anti-elf antibodies co-precipitated an underphosphorylated form of Rb. With T cell activation the elf- I Rb association is lost upon phosphorylation of Rb. Thus, Dr. Leiden has generated both in vivo as well as in vitro evidence for this exciting association of an ets oncogene product with Rb. Session II concemed the NF!!
!B/Rel Family. Dr. Warner Greene of the Gladstone Institute Virology & Immunology, San Francisco began the discussion with his presentation of work on NF-I!!!B. I!!!B is a cytosolic tether of 37 kD size that complexes with both p65 and p50 forms of NF!!!B. Phosphorylation of I!!!b with activation results in translocation of p50 and p65 to the nucleus. There appears to be a family of I!!!Bs. A nuclear localization sequence within p65 is required for the interaction of I!!!B. Of note, Dr. Greene has also noted that free radical scavengers decrease the activity of NF!!!B. It has been noted that the tax protein will interact with a subset of Rel family members including a p100 and p49 molecule that have the Rel homology domain with or without an ankyrin domain. Differential associations of Tax relate to its localization. Tax + p100 is found in the cytoplasm, Tax + p105 is nuclear. Thus a dynamic situation of differential translocation and redox regulation is evolving for this family of genes. Dr. Riccardo Dalla-Favera of New York University Hospital followed with a discussion of the role of NF!!
!B/rel genes in lymphomagenesis. He has noted that roughly 7% of B cell non-Hodgkins's lymphomas have a deletion or translocation at chromosome segment 10q24. He cloned out an interchromosomal translocation with immunoglobulin C!!!locus and discovered Lyt-10, a molecule now renamed as NF!!!B2. Whole length molecule has both a rel homology domain and an ankyrin domain. Chromosomal breakpoint ends up shaving off the ankyrin domain leaving the active rel DNA binding portion. Additional breakpoints hit within the ankyrin coding domain even if they do not translocate C!!!. Expression of these products are constitutively present within the nucleus and bind DNA. Session III Transcription Factors in T Cell ALL was initiated on February 16 by Ilan Kirsch, M. D. of the Bethesda Naval Hospital. Dr. Kirsch discussed the involvement of SCL/Tal-1 in hematopoietic development and malignancy. Dr. Kirsch has observed that GATA-1 will activate the promoter region of SCL. Overexpression of SCL in murine erythroleukemia cells has been noted to drive differentiation of those cells. A study of the incidence of SCL rearrangement found that 13 of 40 T cell ALLs had activated SCL. Of that fully 11 of 13 are an intra-chromosonral rearrangement that deletes approximately 80 kilobases. This juxtaposes a gene Sil with SCL. Of note, Sil has a enkaryotic topo-1 consensus motif within it.
Richard Baer, Ph.D. of the University of Texas Southwestern Medical Center followed with a discussion of the TAL1 and TAL2 helix-loop helix proteins. TAL1 is synonymous with SCL. Richard found an evolutionarily conserved homolog TAL2 at 9p32 that is also involved in approximately 2% of T cell ALL. Moreover he has noted that the large number of T cell ALLS that do not have rearrangements of the helix-loop-helix implicated genes actually express them. Thus, the presence of one of these basic HLH proteins may be more ubiquitous in T cell ALL than appreciated by translocation and rearrangement. E47, one of the ubiquitous bHLH proteins will interact with TAL1. Also he has noted that anti-E2A will co-precipitate a pp42 and p41 form of TAL1 in vivo. He has used a casting procedure to identify a consensus recognition sequence for TAL1 which is CAXXTG. Of interest, TAL1 does not bind DNA by itself, but must complex with either E47, E12 or E22. Furthermore, TAL1 has an N-terminal region that functions as a transactivator.
Stanley J. Korsmeyer, M. D. of Washington University School of Medicine followed with a presentation on Transgenic mice which redirect either Ttg-1 or HOX11 to the thymus. These transgenics use the lck proximal promoter to overexpress in early developing thymocytes two putative oncogenes isolated from chromosomal breakpoints. Ttg-1 transgenics have relatively normal pre-malignant phenotype but with a mean age of 9 months develop very aggressive monoclonal T cell acute lymphoblastic lymphoma/leukemia. Of interest overexpression of this LIM domain protein results in a select set of T ALLs that are immature CD8+ cells. In contrast the HOX11 animals in pre-malignant thymus demonstrate cell cycle acceleration and accelerated cell death which results in a maturational arrest between double negative and double positive thymocytes. From that backdrop of increased death and decreased numbers of thymocytes emerges very aggressive T cell acute lymphoblastic lymphoma/leukemia. This provides a model for understanding the interrelationship of cell cycle progression, apoptosis and genomic instability.
The final session of the meeting on February 16 concerned the regulation of T cells.
Co-organizer Dr. Takehiko Sasazuki of the Medical Institute of Bioregulation at Kyushu University initiated this session with a discussion of T cell repertoire selection. A HLA-DR transgene proved to be expressed only in thymus and not the periphery, and had variable expression in dendritic cells and B cells within several transgenic lines. A peptide, MCC, of amino acids 81 to 103 was recognized by 2B4 in the context of DR!!
!. Lines 24 and 30 deleted V!!!5 and V!!!11 in the context of the appropriate superantigen. However only line 24 will demonstrate positive selection of T cell receptors while strain 30 will not. Strain 30 also expresses DR in the thymic epithelium, however it does not express in dendritic cells or B cells. Thus bone marrow derived cells appear to be necessary for positive selection within this Class II MHC model. Dr. Motoharu Seiki of the Cancer Institute at Kanazawa University presented evidence for crosstalk between HTLV-1 Tax and its effects upon immediately early responses of fos and jun. He noted that with a metallothioneine driven Tax construct in Jurkatt cells that induction of fos, jun, egr-1 and egr-2 would follow. A c-fos promoter reporter was also stimulated by Tax expression. This appears to be mediated through a cArG box found in fos, egr-1 and egr-2. The thesis is that p67 SRF interacts with the cArG box and that it then interacts with Tax to activate the transcription of these genes.
The conference was completed with a talk by Shigekazu Nagata, Ph. D. from Osaka Bioscience Institute concerning the Fas-gene. Dr. Nagata has mapped Fas to human chromosome segment 10q23. I and to chromosome 19 in the mouse. Deletional analysis of this receptor reveals that 100 amino acid cyioplasmic domain has the effector function. This molecule is homologous to the TNFR1 and TNFR2 molecules. He found that deletions at aa 296 as well as loss of the N-terminus destroyed function of the Fas antigen receptor. He presented data that overexpression of Bcl-2 would partially block Fas effect for transient period of time but that these cells would still die after 12 hours.
SEMINAR AGENDA AND PARTICIPANTS
(1) "Self-non-self discrimination in the immune system"
Hawaii, January 11-13, 1993
AGENDA
!-mediated immune suppression as a model of tumor escape mechanisms