(1) Seminar on "Analysis of Host's Immune Responses Involved in Anti-Tumor Immune Resistence"
The U.S.-Japan Cooperative Cancer Research Program in the Cancer Immunology Area was held at Kyoto Kaikan, Kyoto, Japan on November 11- 13, 1985.
I. The Immunobiology and Molecular Biology of Tumor Antigens
The four presentations in this area dealt with abnormal gene expression by malignant cells and with the biochemical isolation of tumor antigenes. Dr. Kuzumaki employed temperature-sensitive src gene mutants of Rous sarcoma virus and somatic cell hybrids to study the src gene product. His findings suggested that the src gene-coordinated tumor associated cell surface antigen of RSV tumors is antigenically related to the extra cellular domain of EGF receptors or their associated tyrosine kinase-modified molecules. Dr. Greene described the expression of the neu oncogene obtained from a neuroblastoma induced in the offspring of pregnant rats injected with ethylnitrosourea. This oncogene has been cloned and demonstrated to encode a 185 kd glycosylated phosphoprotein that is expressed on the surface of transformed cells. Transfection of the cloned oncogene is capable of transforming murine NIH 3T3 cells. Monoclonal antibodies have been generated to different epitopes on the p185 protein. It has been demonstrated that the interaction of these antibodies with cell surface molecules leads to the modulation of cell surface molecules and a reversal of the malignant growth phenotype of the neu-expressing cells. In vivo treatment has been shown to be effective as an immunotherapeutic modality in the treatment of neu-transformed cells. Dr. Yodoi described the abnormal expression of the IL 2 receptor gene on human adult T cell leukemia (ATL) cells. In contrast to the behavior of normal cells, antibodies to the IL 2 receptor failed to down-regulate IL 2 receptor expression on ATL cells or on rat cells transformed with the human T cell leukemia virus I (HTLV-I). To date, no evidence of abnormal gene rearrangement has been found, and it is hypothesized that a soluble factor derived from ATL cells induces constitutive IL 2 receptor expression. Dr. Ishii compared methods for the extraction of solubilized tumor antigens from murine colon tumor cells. It was found that n-butanol extracts of tumor specific transplantation antigens were potent immunogens, capable of protecting immunized hosts from subsequent challenge with specific colon tumor cells.
II. The Biology of Host-Tumor Interaction
A. Immune Response Mechanisms
Both cellular and molecular biologic techniques were employed in several studies of basic immune response mechanisms. Dr. Germain described a molecular genetic analysis of T cell recognition and activation. Employing techniques of gene cloning, "exon shuffling", and transfection, Dr. Germain demonstrated the preferential association of cis-inherited class II A!!
!and A!!
!gene products on the cell surface. These studies mapped the determinants critical in!!!-!!!chain associations as well as in the determination of serologic and T cell-determined epitopes on la molecules. Dr. Vitetta described the biologic activities of the lymphokine B cell stimulating factor-1 (BSF-1). It was demonstrated that this factor, purified to homogeneity, functioned both' as an activation factor exerting its effect upon resting B cells, and as a differentiation factor acting to selectively drive the differentiation of IgG1 antibody forming cells. The action of this factor at a genetic level was described.
Dr. Asano presented the results of experiments which characterized the adaption differentiation of cell-recognizing T cell receptor structures. Monoclonal antibodies were generated which detected apparently I-region encoded determinants on T cells. It was demonstrated through the use of chimeric animals that the-expression of these determinants was related to the host maturation environment of T cells. These antibodies were capable of functionally inhibiting the MHC-restricted action of T helper cells as well as the proliferative response of autoreactive T cells. It was therefore concluded that these monoclonal antibodies detect structures on the surface of T cells which are related to MHC restriction specificities and perhaps to the T cell receptor structure itself.
Dr. Hodes employed transgenic mice to analyze the T cell repertoire for xenogeneic histocompatability gene products. A cloned miniature swine class I MHC gene was success-fully transfected into the genome of a B 10 mouse. The heritable line derived from this progenitor mouse was used to characterize cytotoxic T cell responses against the expressed xenogeneic histocompatability gene product. Evidence was presented suggesting that the foreign transplantation antigen may be, at least in part, recognized in the context of self (murine) MHC products. In addition, a striking and unanticipated cross reactivity between T cell recognition of the xenogeneic gene product and T cell recognition of mutant H-2Kb products was described and the potential significance of this observation discussed.
Analysis of Anti-Tumor Effector Mechanisms
The regulation of tumor cell growth by natural killer (NK) cells was discussed by Dr. Minato. Cloned lines of NK cells were analyzed for their cell surface phenotype and were found to be Thyl+, L3T4-, Lyt2-, ASGM1+ and IL-2 receptor+. This phenotype applied even to NK clones derived from nude mice. Growth of these lines was IL-2 dependent. More-over, it was demonstrated that the!!
!,!!!and!!
!chains of the T cell receptor were rearranged and expressed in cloned NK cell lines from both normal and nude mice. It remains un-known whether the conventional T cell receptor is employed by these NK cells in their functional cytotoxicity. Dr. Segal described a model for retargeting of cytotoxic cells for tumor killing using cross-linked antibody heteroaggregates. Antibody heteroaggregates were made by disulfide linking an antibody against an effector cell receptor structure to an antibody specific for a target cell determinant. ADCC effector cells were targeted by linking antibodies to the Fc!!
!R, whereas cytotoxic T cells were targeted using antibodies directed at the T cell receptor-associated T3 structure. In each case, successful retargeting of ADCC or CTL effectors to antigens, including human tumor specific antigens was successfully achieved. This approach was discussed for its potential in tumor immunotherapy. In vivo studies were presented by Dr. Fujiwara which analyzed the mechanism of Lytl+2- cell-mediated tumor growth inhibition. Using diffusion chambers in vivo, it was determined that these T cells interact with target tumor in the presence of adherent cells, and subsequently release lymphokine(s) which then activate macrophage-like effector cells. It was emphasized that this represents only one potential pathway for antitumor cell cytotoxicity in vivo.
In vivo treatment with anti-Lyt antibodies was employed by Dr. Nakayama to analyze the relevant effector cells involved in skin graft or tumor cell rejection. The results of Dr. Nakayama's studies indicated that Lyt2+3+ positive cells were involved in syngeneic or semisyngeneic tumor rejection as well as in some (but not other) genetic combinations studied for skin graft survival. The timing of effect in these studies suggested that they may have identified effector cells mediating tumor or graft rejection.
Dr. Kuribayashi analyzed tumor eradication mechanisms employing a series TCGF-independent T cell lines. Long term cultured T cell lines were effective in inhibiting tumor growth in vivo, both in Winn assays and in systemic transfer experiments. Analysis of sublines suggested that cytotoxic subsets may be directly involved in tumor rejection, whereas non cytotoxic cells may help the maintenance of activity and proliferation in these cytotoxic T cells.
The cellular effector mechanisms involved in eradication of established tumors were studied in experiments presented by Dr. Greenberg. An adoptive chemoimmunotherapy model was presented for treatment of FBL3 murine leukemia. In combination with cyclophosphamide, specifically immune and H-2 restricted T cells were effective in preventing growth of FBL3. Using Thyl congenic animals and adoptive transfer, it was demonstrated that long lived resistence to tumor in recipient hosts was maintained by adoptively transferred cells, even when these cells persisted at extremely low numbers in the host. Lytl+2-and not Lytl-2+ cells were effective in this protection, and no evidence for CTL generation was detected under these conditions.
Dr. Uede studied antitumor effector mechanisms of tumor infiltrating lymphoid cells. Mononuclear cells isolated from regressing tumors were found to be mostly Lyt2+ T cells. Moreover, factors secreted from tumor infiltrating cells were found which were important to the generation of cytotoxic T cells. This cytotoxic cell generating factor was produced by lymphoid cells of tumor-immune animals challenged with tumor. It was suggested that such factors may play an important role in the host response to tumor.
The study of effector T cells mediating skin graft rejection was utilized by Dr. Singer. In studies of rejection of class I or class II different skin grafts, it was found that class I rejection was mediated by Lyt2+ T cells whereas class II different grafts were rejected only by L3T4+ cells. Correlation with in vitro studies and the behavior of multiple class I mutant skin grafts suggested that graft rejection was mediated by helper cells and not by cytotoxic T lymphocytes.
Systemic immunodeficiency (which is clearly related to tumor susceptibility), was studied by Dr. Shearer, employing the murine graft versus host (GVH) model. The effects of class I or class II-induced GVH upon cytotoxic T cells responses was examined. It was suggested that under certain circumstances, GVH may lead to a selective deficiency in L3T4 T cells. The parallel of this model to the human AIDS pathology was discussed.
III. Preclinical Approaches to Tumor Immunotherapy
In this final session, a series of presentations described application of basic immune principles to preclinical tumor immunotherapy. Dr. Hashimoto utilized a continuous infusion of IL-2 in combination with the adoptive transfer of lymphokine-activated killer cells (LAK). Whereas neither LAK nor IL-2 alone significantly prolonged the survival of B6 mice bearing the syngeneic EL4 lymphoma cells, the combination of both modalities resulted in cured mice or in significant prolongation of survival.
Dr. Hamuro reported that cloned murine IL-2, as well as human recombinant IL-2, was effective in the therapy of various syngeneic tumors in mice. In vitro studies of CTL differentiation revealed that, in addition to IL-2, other lymphokines were potent in the support or amplification of CTL generation. These factors included a T cell differentiation potentiating factor which synergized with IL-2 in the induction of high affinity IL-2 receptors.
Dr. Vitetta reported on recent progress in the use of immunotoxins. Covalent linkage of the ricin A chain to targeting antibodies has provided successful immunotherapy in the BCL1 murine model system. Recent improvements in strategies for the use of immunotoxins were described. In addition, recent results employing ricin conjugated anti T3 antibodies have been aimed at T cell depletion in human bone marrow. Clinical studies employing such depletion in the prevention of GVH during bone marrow transplantation are about to begin.
The biologic response modifier properties of DNA were described by Dr. Tokunaga. Initially discovered as the active moiety in extracts of BCG, intratumor injections of certain DNA preparations were found to have therapeutic affect.
A novel haptenic muramyl dipeptide derivative was described by Dr. Hamaoka in a tumor immunotherapy model. Animals were preimmunized with BCG, and challenged with tumor. Established tumors were then treated with the BCG-related MDP hapten. Such treatment resulted in significant tumor regression and survival. The potential application to clinical tumor systems was discussed.
(2) Seminar on "Lymphoid Markers and Their Genes" was held at Sapporo, Japan, January 31- February'3, 1986.
The meeting dealt with cell surface markers, lymphocyte differentiation and cancer. The sessions were focused on 5 separate areas: 1. The T cell receptor and accessory molecules on T lymphocytes. 2. IL-2 and IL-2 receptor gene expression and function. 3. B-lymphocyte surface markers. 4. Studies of B-lymphocyte differentiation. and 5. Oncogenes and transformation. Each section will be discussed separately.
1. The T cell receptor and accessory molecules:
Dr. Cox Terhorst of Harvard Medical School (Boston) discussed the early events of T cell activation in the context of the identification and characterization of the cell surface molecules involved. Antigen induced stimulation and subsequent activation of T cells are initiated through interactions with the T cell antigen receptor. Several lines of evidence were presented which indicated the intimate association between the T cell antigen receptor and surface marker T3, forming the so-called T3/T cell receptor complex. The evidence included the following: l. by immunoprecipitations with either anti-T3 monoclonal antibodies, or with anti-T cell receptor antibodies five polypeptide chains were been detected. These were two disulfide bridged variable glycoproteins (!!
!and!!!chains) and three invariable structures the T3-!!!,!!
!and!!
!chains with mw 25kD, 20kD, 20kD respectively. 2. mutants of a T leukemic cell line which were selected for the loss of the T3 complex from their surface by treatment with an anti-T3 antibody and complement concomitantly lost expression of the clonotypic heterodimer. 3. monoclonal antibodies directed at either the T cell receptor and chains or at the T3 chains affect T cell functions in an identical fashion. Thus, it was concluded that the complex formed between the T cell receptor and the T3 molecules is functionally as well as structurally central to the immune response. Dr. Dan Littman of the University of California (San Francisco) discussed the significance of the finding that the T4 and T8 glycoproteins are expressed on the surface of functionally distinct subpopulations of T lymphocytes. T4+ T cells were described as primarily helper cells that are restricted to interact with class 11 histocompatibility molecules; T8+ cells were described as cytotoxic or suppressor cells which interact with class I MHC molecules. Antibodies directed against either T4 or T8 block activation and effector function of the T cells. The isolation and characterization of the genes which encode T4 and T8 was described. The gene isolation was achieved through a combination of gene transfer and subtractive hyibridization. Both genes are members of the immunoglobulin gene superfamily and have N-terminal domains homologous to light chain variable regions. In addition, the T4 gene contains a short region homologous to J regions of immunoglobulin and T cell receptor genes. Full-length cDNA's for both genes were introduced into retroviral expression vectors which have allowed expression of the glycoproteins in a variety of infected cell lines and also in transgenic mice. Fragments containing up to 4.5 kb of upstream T8 sequence are not appropriately expressed; they found high level expression of T8 mRNA in spleen, but little expression in the thymus. They also isolated and sequenced the murine counterpart of T4, L3T4. The protein sequence homology between the mouse and human genes is 52% for the external domain, but 29/32 residues of the cytoplasmic tail are conserved, suggesting that this region of the molecule plays an important role in transducing transmembrane signals. Both L3T4 and T4 RNA's are expressed in mouse and human brain, respectively, but appear to be processed differently from the RNA forms found in the T cells. Dr. Littman also discussed the role of T4 and AIDs, a disease characterized by the depletion of the T4+ subset of T lymphocytes, comprising the bulk of helper cells. There is mounting evidence that the in vivo and in vitro tropism of the virus for T4+ cells is the result of a direct interaction between the viral envelope glycoprotein and the T4 glycoprotein on the target cells. Antibodies against several epitopes of T4 were found to be effective in blocking infection; cell variants which turn off expression of T4 are not susceptible to infection; and there is evidence for direct binding of the viral gp 110 glycoprotein to T4. A dramatic decrease in the level of surface T4 after infection was not due to a decrease in T4 mRNA levels, and was therefore suggested to be due to interaction of newly-synthesized T4 with the envelope glycoprotein.
While most of the dramatic clinical manifestations of AIDS are directly related to the depletion of helper T cells, the AIDS virus has been found in brain and in peripheral nerves. Dr. Littman found that the T4 gene is expressed not only in lymphocytes and monocytes, but also in normal human brain. While T cells express a 3.5 kb mRNA, cells within the brain express both the 3.5 kb and a 2,0 kb message. There is no evidence for the expression of the T cell specific TB and T3 mRNA'S in the brain. The homologous murine gene, L3T4, is also expressed in mouse brain, as a 2.5 kb mRNA, compared to a 3.5 kb mRNA expressed in the mouse thymus and spleen. These results were strongly suggested to imply that the means of infection of cells of the CNS is also via the T4 glycoprotein, which serves as receptor on all susceptible tissues.
2. Characterizations of the expression of the IL-2 and IL-2 Receptor Genes.
Dr. Takashi Fujita of the Institute for Molecular and Cellular Biology of Osaka University discussed regulation of interleukin-2 (IL-2) expression. IL-2 is a lymphokine which plays a crucial role in the immune system, especially for the growth control of T lymphocytes. Expression of this lymphokine is restricted to activated, T-4 positive T lymphocytes. Dr. Fujita reported the presence of unique DNA sequences in the 5'-flanking region of the human IL-2 gene which control specific expression of the gene in T cells. He also indicated that the sequences contain element(s) which may be defined as a regulatory enhancer: the DNA sequence appears to function independent of its orientation and activate a heterologous promoter in induction dependent, T cell specific manner. The element was also found to contain regions homologous to other T cell tropic genes.
Dr. Gerald Crabtree of Stanford University (Palo Alto) described the activation of the IL-2 gene by retroviral insertion. Evidence collected over the past years has indicated that normal T-lymphocyte proloiferation occurs by an autocrine mechanism in which the lymphocyte both makes and responds to Interleukin 2 or T-cell growth factor. Such an autocrine mechanism underlying normal T-cell proloiferation suggests that a similar mechanism may occasionally account for malignant T-cell growth. Dr. Crabtree reported that the IL-2 gene of the Gibbon Ape cell line, MLA-144, lies between two integration sites of the Gibbon Ape Leukemia Virus. A complete LTR is present about 1kb upstream of the IL-2 promoter while a copy of the entire virus lacking a 3.25 kb fragment is present within the 3' non-translated region of the IL-2 gene. This second integration is situated so that transcripts arising within the IL-2 gene terminate within the viral LTR, producing a transcript elongated by inclusion of 250 bp of viral sequence. The 5' integration is situated so that any transcript arising within the viral LTR will be directed away from the IL-2 gene. S 1 nuclease mapping demonstrated that the 1st exon of the IL-2 gene is properly initiated despite the viral insertion. Transcripts of the IL-2 gene in MLA 144 cells constitutively expressing IL-2 arise predominantly, if not entirely from the inserted allele since Northern analysis indicated that the predominant mRNA is about 200 Lp longer than the IL-2 mRNA of normal Gibbon lymphocytes. A role for the constitutive production of IL-2 in driving the proliferation of the MLA-144 cell line is suggested by previous experiments in which dexamethasone was used to inhibit IL-2 production. In sensitive clones of MLA-144 cells dexamethasone produced a 40% reduction in the rate of proliferation which could be overcome by IL-2. Dr. Crabtree proposed that the viral insertion contributes to the proliferation of the cell line but probably an additional factor was involved in transformation.
Dr. Masanori Hatakeyama of Osaka University presented studies on the expression of human IL-2 receptor gene in murine T cells and L cells. The IL-2 receptor exists in two forms having different (high and low) affinities to the ligand and the IL-2 specific signal seems to be delivered via the high-affinity one. A cDNA that encodes the Tac (IL-2 receptor) antigen was cloned from ATL cells. Transfection of the cDNA into non-T cells resulted in the expression of only a low affinity IL-2 receptor. This observation raised a question whether or not the cloned cDNA for Tac antigen actually encodes the functional, high affinity IL-2 receptor. To clarify this problem, this group first obtained Tax antigen cDNA from a human PBL cDNA library. This cDNA was inserted into an expression vector and then transfected into mouse T Lymphoma line EL4 and into L929 fibroblasts. They found that the EL4 transformants expressed high affinity as well as low affinity receptor for human IL-2. In contrast, the L929 transformant expressed only a low affinity receptor. The growth of the EL4 transformants was inhibited by human, but not mouse, IL-2. The results presented demonstrated that the cloned cDNA does encode functional IL-2 receptor, and that the affinity of IL-2 receptor is variably modified by post-translational events. They also concluded that IL-2/receptor interaction mediates reverse signal transduction in EL4.
A second detailed study of IL-2 receptor function was presented by Dr. Akira Shimizu of Kyoto University. His laboratory cloned the cDNAs of the human and mouse IL-2 receptor genes and by nucleotide sequence comparison found several evolutionally conserved regions localized to particular region such as the cytoplasmic portion (which is too short to have any enzymatic activity) and the transmembrane portion. They speculated that these regions were thought to be important for function and regulation of the IL-2 receptor. To test this possibility they used a human cDNA clone subcloned into an expression vector which contained an SV40 promotor to do a series of cDNA tranfection experiments. The cDNA drived human IL-2 receptors expressed on IL-2 dependent murine T cell-line, CTLL-2, were shown to be functionally active, by blocking the endogenous mouse IL-2 receptors with monoclonal antibodies. The IL-2 response of the human IL-2 receptor expressing CTLL-2 cells (CT/hR cells) could not blocked by anti-mouse IL-2 receptor antibody alone nor anti-human IL-2 receptor antibody alone. Addition of both anti-mouse and anti-human IL-2 receptor antibodies, completely abolished the IL-2 response of CT/hR cells, providing proof that the cDNA clone encodes the IL-2 receptor. On the other hand, cDNA derived IL-2 receptors expressed on non-lymphoid cells were functionally inactive. They could not mediate the growth signal, and consisted of low affinity species. The reason for this dysfunction of the IL-2 receptors on non-lymphoid cells was proposed to be due to the lack of additional protein(s) expressed specifically on lymphoid cells and necessary for IL-2 receptor function.
Dr. Kazuo Sugamura of Kyoto University discussed the relationship between HTLV-1 and the IL-2 receptor. HTLV-I/ATLV has been considered to be the etiological agent of ATL. However, in spite of extensive molecular characterization of HTLV-I, the mechanism of HTLV-I-induced malignant T cell transformation is still unknown. Cytological studies revealed that all ATL leukemia cells and HTLV-I-transformed T cells have a unique surface phenotype with receptors for IL-2. The expression of IL-2 receptors in these HTLV-I-carrying cells was suggested to be attributable to the IL-2 receptor-inducing ability of HTLV-I. However, there was no direct evidence that IL-2 receptors are involved in the mechanism of T cell transformation induced by HTLV-I. HTLV-I-carrying, IL-2-dependent cell lines are known to become transformed into cells that can proliferate independently of IL-2; the growth of these transformed cell lines was demonstrated to be inhibited by IL-2. This growth inhibitory effect was mediated by the high-affinity IL-2 receptors. Dr. Sugamura also discussed the functional difference of IL-2 receptors between IL-2-dependent and IL-2-independent T cells infected with HTLV-I.
3. B-lymphocyte Surface Markers.
Dr. Hitoshi Kikutani of Osaka University presented studies which were aimed at defining the B-lymphocyte activation pathway by monoclonal antibodies. Antigen and T cell dependent maturation of B lymphocytes can be dissected to activation, proliferation and differentiation steps. Each step is regulated by several B cell stimulatory factors including BSF1, B cell growth factors and B cell differentiation factor (BCDF or BSF2). Some of these factors such as BSF1 and BCDE have been purified to homogeneity. However, to elucidate the regulatory mechanisms by these molecules, identification and isolation of B cells at each muturation step has been initiated. They have begun to identify B cell subpopulations at each activation stage utilizing monoclonal antibodies, anti-Ba and anti-L30 which recognize activated and resting B cells, respectively, and anti-isotype antibodies. Multi-parameter FACS analysis showed that tonsillar B cells could be separated into at least four subpopulations;
Ba-/L30+/IgD+, Ba+/L30+/IgD+, Ba+/L30-/IgD- and Ba-/L30+/IgD-.
Ba-/L30+/IgD+ cells could be switched to Ba+/L30+/IgD+ and further to
Ba+/L30-/LgD- by various mitogenic stimulations. Ba-/L30+/IgD+, Ba+/L30+/IgD+ and
Ba+/L30-/IgD- cells turned out to be resting, early activated and late activated B cells, respectively, as defined by cell volume, expression of transferrin receptor and proliferative response to BCGF. Purified BCDF-induced Ig secretion preferentially in Ba+/L30-/IgD-cells, suggesting that there B cells were in mature stage. Furthermore, Ba-/L30+/IgD- B cells, which mostly expressed IgG or IgA, might be a kind of memory cell since these cells were in resting stage with respect to the expression of Ba and L30 antigens. The subpopulations of cells identified and isolated in this study should be useful for the analysis of activation of resting B cells and memory B cells.
Dr. Kikuchi of Sapporo Medical College discussed B cell differentiation antigens with respect to B cell malignancies. They generated a battery of monoclonal antibodies (Mab) that distinguish different antigen molecules selectively expressed in human B cells including expression of L27, L26, L29, L30 and L10 (IL-2 receptor, Tac). L27 and L26 were expressed in most surface Ig-positive B cells and thus should pan-b cell specificity. L30 appeared to be expressed on small resting B cells but not on large activated B cells. In contrast to L30, L29 was not expressed on resting B cells, but was expressed after activation by Staph. A protein plus IL-2. Lymphoid tissues were studied immunohistologically with anti-L26, anti-L30 and anti-L29. Anti-L-30-labeled small B cells located in the mantle zone of the lymphoid follicles, but did not stain large blastic B cells within the germinal centers. In contrast, anti-L29 labeled large B cells in the germinal center, but not B cells in the mantle zone. Anti-L26 Stained B cells in both germinal center and mantle zone. These B cell antigens were all lost from B cells when they differentiate into plasma cells. Two color FACS analysis and immunoperoxidase staining of lymphoid tissues indicated that IL-2R was expressed on a small fraction of splecic B cells in the marginal zone, and on few of B cells in the lymph nodes. Dr. Kikuchi also presented studies on the utilization of these MABS for the classification and diagnosis of human B cell malignancies. Clear differences were found between the expression of L30 and L29 on non-Hodgkin's lymphoma. L30 was expressed on small lymphocytic (SL) and diffuse small cleaved (DSC), but not on diffuse large (DL). In contrast, L29 was expressed on DL, but not on SL and DSC. Lymphoma cells of four out of 6 cases of DSC, had IL-2R.
Dr. Toru Abo of Tohoku University (Sendai) discussed surface antigens and cytokines of NK cells. There has been a concensus that a large granular lymphocyte (LGL) population with natural killar (NK), function is non-adherent and non-phagocytic. However, a significant proportion of the non-adherent cells purified by the two step depletion of a adherent cells with plastic surface and nylon wool columns engulfed Sta. aureus. These cells were morphologically identified as LGL. Two-color immunofluorescence tests demonstrated that Leu11+, Leu11+ 7- and Leu11+ 7+ LGL phagocytosed Ste. aureus. Among the particles they tested, only gram (+) bacteria were preferentially phagocytosed, whereas gram (-) bacteria, other large-sized microbes (e.g., Baker's yeast and Candida albicans), latex, silica and carbonyl iron were not. When gram (+) bacteria were incubated with non-adherent cells for 18 hrs, significant amounts of interleukin 1(IL1)-like factors (or IL1 itself) as well as interferon (IFN) were detected in the supernatants. On the other hand, this incubation did not induce interleukin 2 (IL2). The phagocytosis was suggested to be an important stimulus to produce IL1-like factors from LGL. Dr. Abo concluded that his data identified an additional unique characteristic similar to, but not identical with, a myelomonocytic nature for Leul 11+ LGL.
Dr. Shin Yonehara of the Tokyo Metropolitan Institute of Medical Science discussed the receptor system of interferon-alpha and tumor necrosis factor. To obtain monoclonal antibody to the cell surface receptor molecules for human interferon-a monoclonal anti-idiotype antibodies for anti-IFN were obtained. Immune splenocytes of Balb/c mouse, immunized with polyclonal mouse anti-human IFN-!!
!Ig6, were fused with NS-1 myeloma line. Of 2000 individual hybridoma clones, two secreted monoclonal antibody, termed CAB-11 IgN-!!!.The binding of CAB-11 IgG on human B lymphoblastoid Daudi cells was measured by means of indirect immunofluorescence on a FACS. CAB-11 IgG bound to the surface of Daudi cells, and the binding was completely inhibited by IFN-!!!but not by IFN-!!!CAB-11 IgG specifically precipitated a protein with MU 95K from radioiodinated cell surface proteins. The MW of the precipitated protein was coincided with the MW of IFN-!!!2 receptor, calculated from the MW of the cross-linked complex of radioactive IFN-!!!2 and the receptor molecules for IFN-!!!. Dr. Yonehara also reported an analysis of redioactive tumor necrosis factor (TNF)-binding to various human cells with different sensitivities to the cytolytic activity of TNF. Presence of the high affinity binding sites was shown to be indispensable for the sensitivity to TNF, while the amounts of high affinity binding sites were not correlated with the sensitivity to the cytolytic activity of TNF. Radioactive TNF was cross-linked to the high affinity binding sites on human cells. When cross-linked materials were analyzed by SDS-PAGE, radioactive bands of MW 98K and 140K were observed. The 98K band was observed in all cells with high affinity binding sites, while the 140K band was observed only in cells sensitive to the cytolytic activity of TNF. He suggested that the cytolytic activity of TNF may be mediated by another subunit of TNF receptor complex from the TNF-binding molecule. Dr. Yonehara also obtained monoclonal antibody for human cell surface that directly killed human cells just like TNF. The cytolytic activity of the purified monoclonal antibody (termed anti-Fas IgM) was enhanced by the treatment with IFN-g, actinomycin D and mitomycin C, just like TNF activity was enhanced. FACS analysis showed that the amounts of Fas antigen increased significantly by the treatment with IFN-!!
!. Binding of anti-Fas IgM to cell surface was not inhibited by TNF, but binding molecules to TNF were shown to be co-internalized with Fas antigen by the treatment of Anti-Fas IgM. Dr. Yonehara suggested that Fas antigen may be a subunit of TNF which mediates the cytolytic activity of TNF receptor complex to mediate the cytolytic activity of TNF. Dr. Yoshiyuki Hashimoto of Tohoku University (Sendai) discussed a novel cell surface antigen related to cell growth. The murine monoclonal antibody (MOAb) B3 raised against a rat bladder cancer cell line and the MoAb HBJ127 and HBJ98 raised against a human bladder cancer cell line recognize homologous gp 125 antigens predominantly present on proliferating cells such as those in basal layers of skin and esophagus of the corresponding species. All malignant and nonmalignant cultured cell lines express this antigen. With regard to lymphocytes, the expression of the antigen was found to be marginal in peripheral lymphocytes, have a negative correlation with the maturation of the lymphocytes, and was augmented on activation with lectins or alloantigens. Upon activation, the antigen expression in peripheral lymphocytes was readily manifested at the G0 to G1a transition and augmented progressively with the progression of cell cycle. The appearance of the gp 125 antigen was suggested to precede the appearance of the Tac antigen (IL 2 receptor) and the onset of cellular DNA synthesis but is later than myc gene expression. Addition of the MoAb to tumor cells inhibited nuclei acid synthesis or proliferation of the cells in vitro in a dose-dependent fashion. The inhibitory effect of the MoAb is reversible as indicated from the finding that removal of the MoAb results in the recovery of the inhibited cell growth. The MoAb did not cause down regulation of the cell-surface gp 125 antigen and did not arrest the cell cycle of tumor cells in a certain phase. Simultaneous addition of Con A and the MoAb to lymphocytes inhibited the cell cycle at G1a and inhibited the appearance of the gpl25 antigen. By contrast, addition of the MoAb to lymphocytes after 24 hr-Con A stimulation did not modulate the gp 125 antigen, although it inhibited the cellular DNA synthesis strongly. Dr. Hashimoto concluded that the cell surface gp 125 component detected in both rat and human systems may play a requisite role for proliferation of both normal and neoplastic cells.
4. B-lymphocyte Differentiation
Dr. Frederick Alt of Columbia University (New York) discussed the control of gene expression and gene rearrangement during lymphocyte differentiation. The genes which encode the variable region of antigen receptor chains are assembled from multiple germline DNA elements. Control of the recombinational events involved in V gene assembly appears to be directly associated with lymphocyte differentiation. With respect to the assembly of genes which encode the variable region of immunoglobulin heavy chains (VH-D-JH), pre-B cell lines preferentially rearrange the most J [H]-proximal (3') V[H] gene segments. Studies of the in vivo V[H] repertoire revealed a fetal repertoire which, relative to the repertoire of the adult splenic lymphocytes, is highly skewed toward J [H]-proximal V[H] segments. A V[H] repertoire reminiscent of that of the normal fetus appeared to be maintained in autoantibodies and in the splenic lymphocytes of an adult mouse with a severe combined immunodeficiency disorder.
Evidence was presented which suggested that a common recombinase performs all of the many variable region gene assembly events seen in B and T cells, and that the tissue-and stage-specificity of these joining events as well as the phenomenon of allelic exclusion is mediated by regulating the accessibility of the involved gene segments. This evidence included the demonstration that the normal specificity of recombinase-mediated joining events can be altered by manipulating the chromatin configuration and transcriptional activity of the involved segments; in particular, introduced lambda light chain V gene segments and T-cell receptor V gene segments were transcriptionally targeted to join at a high rate in pre-B cells which only rearrange their endogenous V[H] gene segments. The general importance of accessibility in recombinase control was also indicated by analyses of heavy chain class switching in pre-B cells; specific class switch events also appeared to be targeted by the accessibility of the involved constant region genes.
Dr. Owen Witte of the University of California (LOS Angeles) discussed the topic of lymphoid cultures and transformation by Myc and Ras oncogenes. In vitro culture conditions for the long term growth of B lineage cells from bone marrow and fetal liver have been developed in his laboratory. Lymphoid cells from these cultures can repopulate the SCID mouse. Bone marrow culture cells (BMC) can develop into cells capable of secreting IgM, IgA and multiple IgG subclasses. These BMC reconstituted mice can mount immune responses against T-dependent (TNP-Ficoll) antigens. Fetal liver cutures (FLC) can only develop to secrete IgM and IgG3 subclass efficiently and respond to T-independent antigen only. These long term culture conditions can be exploited to demonstrate the transforming effects of the myc oncogene for lymphoid cells in a totally in vitro system. Viral myc constructs were found to be complementary for transformation with a viral ras vector. The combined oncogenes gave rise to early B lineage transformants with DJ (incomplete) heavy chain gene rearrangements.
Dr. Tadamitsu Kishimoto of Osaka University discussed the structure and function of human B cell differentiation factor (BCDF OR BCF-2). Several B cell stimulatory factors are involved in the proliferation and differentiation of B cells into antibody secreting cells. These factors are divided into three categories, i.e., i) factor(s) responsible for the activation of resting B cells (BSF-1), ii) factor(s) required for the continuous proliferation of activated B cells (BCGF) and iii) factors for the final differentiation into Ig-secreting cells (BCDF). Dr Kishimoto's laboratory purified human BCDF to homogeneity and studied its physiochemical and immunological properties. A human T cell clone, which secreted more than 100-fold as much as BCDF compared to that from mitogen-stimulated T cells, was established and the culture supernatant was employed for the isolation of BCDF. BCDF was purified by sequential chromatographies, i.e. gel filtration on ACA34, chromatofocusing on Mono P column and reversed-phase HPLC. Approximately 10 ug of purified BCDF had the M.W. of 21 Kd and the P.1. of 5. 1. A partial amino acid sequence from N-terminal to the 1 6th amino acid residue was determined. On the basis of the determined amino acid sequence, the synthetic peptide column could absorb BCDF activity from PHA-sup but IL-2 and BSF-1 activities were not absorbed. Patients with the myxoma, a benign intratrial heart tumor, often show hyper-gamma-globulinemia and autoantibody formation, such as ANF and RF. The culture supematant of myxoma cells showed high BCDF activity, which could be absorbed by the anti-peptide column, suggesting that BCDF may be able to induce hyper-gamma-globulinemia and autoantibody production in vivo. The purified BCDF could induce Ig-secretion in B cell lines as well as in activated B cells at the concentration of 3pM. Nuclear run off experiments showed that BCDF could induce an increase in the transcription of mRNA specific for secretory type heavy chains.
Dr. Phil Tucker of the University of Texas Health Science Center (Dallas) discussed allelically excluded expression of IgM and IgG1 by a B cell line without DNA rearrangement of constant region genes. They have subcloned from BCL1 an IgD and IgM positive lymphoma, variant cell genes that express both IgM and IgG1. Analysis by metabolic and cell surface labelling demonstrated that both mu and gamma I heavy chains bear the parental variable region. All varients express more IgM than IgG1 (~10:1) the lines express similar rations of the corresponding heavy chain mRNAs. A single variable region rearrangement was observed by Southern blotting representing two identically rearranged alleles as confirmed by cytogenetic examination. No DNA rearrangements were associated with Cµ, C!!
!1, or any of the intervening DNA. However, one allele had an unusual rearrangement just 3' to VDJ which appeared to eliminate (perhaps delete) all CH genes. This data coupled with quantification of JH/C!!!1 and JH/C!!!3 ratio between parental and variant DNAs suggested that both u and gl are derived from the nonrearranged (with respect to CH ) chromosome. This interpretation was strengthened by nuclear RNA "sandwich" hybridization that demonstrated transcripts containing both µ and!!!1. Nuclear run-on transcription showed two major differences in the hybridization pattern. First, an increased loading of polymerases between Cµ and C!!!1 within a region containing an unusual inverted repeat posulated to play a role in u transcriptional terminator was observed in the µ+, !!!1+ variants. Second, apparent "sterile" transcripts could be detected in the parental line which appeared to initiate upstream of C!!!3 and C!!!1. Their presence correlated with hypomethylation patterns of the correspond genes. Dr. Tuckers' observation may have general implications for isotgre switch "Committment" and for regulation of transcriptional termination. Dr. Takeshi Watanabe of Kyusyu University (Fukuoka) described a novel method for elucidating the function of trans-acting nuclear proteins responsible for induction heavy chain gene expression. A complete human G1 heavy chain gene was transferred into mouse cells by protoplast fusion. The gene was strongly expressed in mouse myelomas but not in fibroblast (L) cells. Nuclear extracts from myeloma cells were injected via erythrocyte ghosts into L cell transformants containing a single integrated construct copy. This injection procedure triggerred accurate transcription of the integrated heavy chain gene. Injection of nuclear extracts from non-lymphoid cells had no effect on heavy chain transcription. Dr. Watanabe concluded that positive regulatory trans-acting factors are involved in the activation of immunoglobulin heavy chain gene expression through its enhancers; he also concluded that these factors are only present in B lineage cells.
5. Oncogenes and Maligninancy
Dr. Witte described the relationship between the Ab1 oncongene and murine and human leukemias. The viral abl oncogene is a chimeric protein with the structure NH2-gag-abl-COOH with tyrosine kinase activity. He described an abl-related protein in human chronic myelogenous leukemia (called P210) with the structure NH2-bcr-abl-COOH. This protein also has tysosine kinase activity. Dr. Wittes' group found that this protein can be expressed in both myeloid and lymphoid cell lineages, although the level of expression can vary considerably among different cell types. In addition this group prepared monoclonal antibodies that recognized viable, c-abl and bcr-abl. These anti-bodies were used to develop a sensitive Western immunoblot technique. Examination of over 50 clinical samples showed a nearly 100% correlation between P210 expression and CML. Progress towards molecular cloning of complete copies of the 8kb chimeric bcr-abl, c-abl (7kb) and c-bcr (4.5 and 6.5 kb) mRNAs using enriched mRNA preparations and internal oligo priming was reported.
Dr. Alt described differential expression of Myc family genes during mammalian development. Evidence was presented which indicated that the myc-family of cellular oncogenes contains three well-defined members c-myc, N-myc and L-myc, as well as multiple additional members. Despite many similarities in structure and function of myc-family genes, high level expression of the N- and L-myc genes was found to be very restricted with respect to tissue and developmental stage while that of c-myc was more generalized. The unique patterns of myc-family gene expression generally predicted the types of tumors in which they were expressed; activated n-myc expression is a property of a variety of tumors which derive from primitive normal cell precursors including a set of childhood embryonic tumors which may have a genetic component to their etiology. The highest levels of N- and L-myc expression were found in developing neural tissues; however, the N-myc gene appeared to have a role in the early stages of multiple differentiation pathways. This property was clearly evidenced by the differential patterns of myc gene expression in developing B-lymphoid cells; both N- and C-myc are expressed during the pre-B stages while only c-myc is expressed at the B cell and later stages of the pathway. Dr. Alt suggested that differential myc-family gene expression could play a role in normal differentiation.
Finally, Dr. Noriyuki Sato (Sapporo Medical College) described a new sensitive recipient cell line for DNA transfection. Transfection experiments using cellular DNAs of malignant tumors have provided an opportunity to investigate cellular transforming genes and to analyze the transforming process of cells as well as the products of these genes. These assays have often employed 3T3 mouse cells, mainly NIH 3T3. However, of all human tumor DNA tested, some 80% are ineffective in 3T3 transformation assays. Thus, there was a demand to establish sensitive DNA recipient systems. Dr. Sato developed a new DNA transfection recipient cell, designating WFB that was derived from WKA rat fetus. This new cell line shows a strict growth inhibition by cell contact and no piling-up of cells on confluent monolayer cultures; these properties were maintain even after in vitro passage for more than 200 generations. The karyotype of WFB was that of normal Zn + XY. Although 3T3 cells demonstrated a background growth in semi-solid agar, there was completely no growth of WFB in agar. Once WFB was transformed, it demonstrated and maintained an easily observable transformed phenotype. This was deemed beneficial because they could obtain only transformed cells after transfection, and, in particular, it became possible to analyze tumor specific antigens which could be closely associated with cell transformation. Furthermore, WFB was more sensitive than BALB 3T3 for oncogene transfer; at least for 6.6 kb EJ ras oncogene. Dr. Sato suggested that WFB would be very useful for oncogene detection experiments and for the analysis of transformation-related antigens on cells.
SUMMARY REPORT
By Stephen Oroszlan
The United States- Japan Cooperative Cancer Research Program, Seminar on "Regulation of Expression and Function of Transformation-related Genes" was held February 17-19, 1986 at the Sheraton Makaha Hotel, Makaha, Hawaii, USA. This meeting was the 2nd joint meeting of the 3rd five year agreement of the U.S.- Japan CCRP, Cellular and Molecular Biology sub-area. Dr. Stephen Oroszlan (USA) and Dr. Yoji Ikawa (Japan) were the organizers. Other invited participants were from the USA: Dr. Roger Davis, Dr. Ales-sandra Eva, Dr. George Pavlakis, Dr. Neal Rosen, Dr. Peter Vogt, Dr. Michael Dean, Dr. Takis Papas, Dr. David Stern, and Dr. Owen Witte, and from Japan: Dr. Yoji Ikawa, Dr. Toshiaki Katada, Dr. Makoto Noda, Dr. Kumao Toyoshima, Dr. Yoshito Kaziro, Dr. Toshikazu Nakamura, Dr. Toshitada Takemori, and Dr. Mitsuaki Yoshida. The following five scientific sessions were held: Session I: src and other oncogenes coding for tyrosine kinase (Co-Chairmen: P. Vogt and K. Toyoshima); Session II: abl and other oncogenes (Co-Chairmen: O. Witte and Takemori); Session 111: Genes for growth factors and their receptors (Co-Chairmen: T. Papas and Y. Kaziro); Session IV: G-proteins and ras gene products (Co-Chairmen: Y. Ikawa and R. Davis); Session V: X-genes of BLV/HTLV-I family-Expression and function (Co-Chairmen: M. Yoshida and S. Oroszlan).
Most of the known oncogenes have been classified into four distinct groups according to sequence homology and function of their protein products. K. Toyoshima presented an overview of the src family which is the largest group comprising of about half of the known oncogenes. He discussed v-erb B and c-erb B which is divided into four domains: a glycosylated outer domain, a membrane anchorage domain, a kinase domain and an erythroblastosis inducing C-terminal domain. It shows substantial amino acid sequence homology to the EGF receptor. In addition, exon and intron sequences are completely conserved between c-erb B-2 and EGF receptor. Similarly c-src in both chicken and human as well as c-fes and yes are spliced at the same places and they are all different from c-erb B-2 and c-erb B-2 which again are the same to each other. He proposed a new way of classifying the protein kinase family into subgroups based on exon-intron structure. N. Rosen studied the pp60-src kinase activity in a large number of human tumors and cell lines and found both quantitative and qualitative changes which may play a role in transformation. Another rapidly transforming avian retrovirus, the acute leukemia virus S 1 3 was discussed by P. Vogt. The sea (for sarcoma-erythroblastosis-anemia) oncogene and erb proteins show similarities in that both are tyrosine kinases, membrane anchored glycosylated proteins and transform erythroid cells but they do not show nucleic acid homology. While erb B induces only erythroblastosis, sea is active in reticuloytes also. The sea transformation-specific protein is linked to the N-terminal domain of the viral env protein gp 37. The met oncogene which shows sequence homology to sea and is also a tyrosine kinase was discussed by M. Dean: sea may be the chicken equivalent of met. The met oncogene which was activated in vitro by chemical carcinogen in the osteosarcoma cell line, HOS via a genetic rearrangement is a truncated form of the met-protoncogene product. Dean and colleagues discovered that met is tightly linked to the recessive genetic disease cystic fibrosis caused by a single defective gene, located on chromosome 7.
Another tyrosine kinase gene the abl oncogene was discussed by O. Witte and Takemori. The abl oncogene is capable of inducing a variety of hematopoietic tumors in mice and is involved in the progression of human chronic myelogenous leukemia. Witte described two different modes of activation of c-abl which are viral transduction and chromosomal translocation. Both generate similar chimeric products with structural alterations of the NH2-terminal region. Takemori presented interesting data on mouse bone marrow cells transformed in vitro by A-MuLV treated with mutagen, 5-Azacytidine. He obtained several lymphoid cell lines showing temperature sensitive phenotypic changes in surface properties. S. Oroszlan presented data from his laboratory showing the significance of myristylaton in virus assembly and in transformation by abl and src.
Studies with human hematopoietic primary tumor DNAs using transfection assays in NIH/3T3 cells described by A. Eva lead to the discovery of a new transforming gene derived from human diffuse B-cell lymphoma and designated dbl. A protein product of 66K which is phosphorylated at a serine was identified.
The regulation of intracellular events by extracellular signals such as growth factors often involves reversible phosphorylation of intracellular proteins. The first steps in signal transduction often are the phosphorylation of growth factor receptor and activation of its kinase activity. The binding of epidermal growth factor (EGF) to its receptor initiates transmembrane signaling resulting in activation of tyrosine kinase activity of the receptor and the stimulation of cellular growth. The regulation of the EGF. receptor by platelet derived growth factor was discussed by R. Davis. PDGF causes a decrease in the affinity of EGF receptor, a decrease in Vmax of EGF tyrosine kinase and synergistic stimulation of EGF induced mitogenesis. The action of PDGF on the EGF receptor appears to be mediated by the specific phrosphorylation of the receptor at threonine-654 by protein kinase-C and at two additional sites either by protein kinase-C or some unknown kinases. D. Stern described the neu oncogene which was activated in rat neuro/glioblastomas induced by transplacental mutagenesis with ENU. It is also amplified in a number of known tumors. Neu is related to c-er B which encodes the EGF receptor. The protein product of both neu-protooncogene and neu oncogene is a 185 Kda transmembrane protein and is probably a growth factor receptor. Neu is most likely activated by mutations. Monoclonal antibodies to p185 eliminated the protein from transformed cells. This provides an extremely important and exciting model system for the immunotherapy of tumors. T. Nakamura reported on the purification and characterization of hepatocyte growth factor (HGF) from rat platelets. HGF which is released from platelets by thrombin treatment is also found in human platelets. HGF is one of the major device of controlling hepatocyte growth may be expected to show up in retrovirus vectors and be oncogenic.
An example of activation of protroncogenes by chromosomal translocation was described by T. Papas who discussed studies on the cellular ets genes and their implication in human leukemia. Translocation of human ets-1 gene from chromosome 11 to 4 as well as the translocation of ets-2 from chromosome 21 to 8 in acute myeloid leukemias were found to be associated with altered transcriptional expression of ets gene.
Another intensively studied class of oncogenes are the members of the ras family. The products of the ras genes have been shown to be fatty acylated and located at the inner plasma membrane. They acts as GTP binding proteins, "coupling factors" or transducers in the growth hormone receptor signal-transduction systems. Y. Kaziro reviewed the function of GTP binding proteins in transmembrane signaling and described studies with a ras-homolog cloned from Shizosaccharomyces pombe. His studies uncovered a new function of ras protoncogene in yeast by showing that it was essential for mating. Kaziro has also cloned and sequenced the a subunits of Gs, Gi and Go proteins and discussed their homologies with transducin and the ras proteins. I. Katada discussed the role and mechanism of action of guanine mucleotide-binding regulatory proteins (Ns and Ni) in receptor-adenylase cyclase coupling. Ns is involved in the activation while Ni in the inhibition of adenylate cyclase. M. Noda described an interesting system to study the mechanism of action of v-ras. He found that in the rat pheochromocytoma cell line, PC12, the Ki-v-ras and Ha-v-ras mimic the activities of nerve growth factor by cessation of cell division and expression of a number of properties which resemble those of differentiated neurons. Noda also described studies with flat revertants isolated from K-v-ras transformed NIH/3T3 cells after treatment with quabain. He found that in these revertants which retain high levels of v-ras protein the amount of tropomyosin is restored to normal level. Fibronectin which is also reduced in transformed phenotypes is apparently overexpressed in the revertants. The similarities and diversities among the transformation pathways mediated by the various oncogenes may be reflected from the patterns of susceptibilities or resistance to retransformation. He found that v-mos, v-fes and v-sis were capable of readily transforming the revertants which were however resistant to retransformation by v-ras, v-fes and v-src.
Human T-cell leukemia virus type I (HTLV-I) has been shown to play a causative role in adult T-cell leukemia. HTLV-I and bovine leukemia virus (BLV) are highly related in nucleic acid and amino acid sequences are similar in gene organization and show immunological cross sections. These viruses do not contain a typical oncogene sequence derived from cellular DNA, but they contain a unique sequence called px region. The last session of this meeting dealt with gene expression in HTLV/BLV and focused on the function of the px gene and its products. In an introductory presentation S. Oroszlan discussed the translational control of gene expression in retroviruses including suppression of termination codons resulting in gag-pol in-frame readthrough as known for type C-viruses, and frameshifts as expected to occur in HTLV/BLV.
M. Yoshida reported on HTLV-I pX gene for which he had earlier identified four open reading frames. He described experiments which now prove that the previously isolated p40x protein translated from reading frame IV is a DNA binding protein and is the transcriptional transactivating factor responsible for the specific activation of HTLV-LTR. He has also identified two new proteins designated p27x and p21x derived from open reading frame III. They were found to be phosphorylated and localized in the nucleus. The exact function of these pX products is not yet known. Y. Ikawa reported that the functional homology of HTLV-I p40x is found in BLV. He also identified a smaller protein in BLV which is translated from the same message as p38x but in a different reading frame. Again the function of this smaller pX product remains to be seen. G. Pavlakis expressed the transcriptional activator proteins of HTLV-1, HTLV-II and BLV in eukaryotic vectors and showed that they all can activate the HTLV-I promoter indicating that they are functionally related. These activators were shown to interact with a short sequence within U3 region of the LTR which has the characteristics of a conditional enhancer. Pavlakis studied the HTLV-III/LAV activator which unlike that of HTLV-I requires sequences within the R-region of LTR for appropriate function. He pointed out the possibility that mechanisms other than transcriptional regulation are involved in the activation of HTLV-III/LAV by its transactivator protein coded by the short open reading frame which overlaps the 3' end of the pol gene.
SEMlNAR AGENDA AND PARTICIPANTS
Analysis of host's immune responses involved in anti-tumor immune resistance
AGENDA