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In our effort to find diagnostic markers and to develop therapeutic approaches for prostate cancer, we have identified an mAb that is capable of binding to a cell surface antigen specifically expressed on both androgen-dependent and androgen-independent prostate cancer cells. Immunohistological studies revealed that this mAb, called F77, stained 112 of 116 primary and 29 of 34 metastatic human prostate cancer specimens. Although the mAb F77 alone directly promotes prostate cancer cell death, it also mediates complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. In addition, mAb F77 can significantly inhibit androgen-independent PC3 and Du145 tumor growth in nude mice. Antigen characterization revealed that mAb F77 recognizes a very small molecular species with glycolipid properties. F77 antigen is concentrated in the lipid-raft microdomains, which serve as platforms for the assembly of associating protein complexes. Thus, the present study indicates that mAb F77 defines a unique prostate cancer marker and shows promising potential for diagnosis and treatment of prostate cancer, especially for androgen-independent metastatic prostate cancer.

Expression of FOXP3, a potent gene-specific transcriptional repressor, in regulatory T cells is required to suppress autoreactive and alloreactive effector T cell function. Recent studies have shown that FOXP3 is an acetylated protein in a large nuclear complex and FOXP3 actively represses transcription by recruiting enzymatic corepressors, including histone modification enzymes. The mechanism by which extracellular stimuli regulate the FOXP3 complex ensemble is currently unknown. Although TGF-β is known to induce murine FOXP3 + Treg cells, TGF-β in combination with IL-6 attenuates the induction of FOXP3 functional activities. Here we show that TCR stimuli and TGF-β signals modulate the disposition of FOXP3 into different subnuclear compartments, leading to enhanced chromatin binding in human CD4 + CD25 + regulatory T cells. TGF-β treatment increases the level of acetylated FOXP3 on chromatin and site-specific recruitment of FOXP3 on the human IL-2 promoter. However, the proinflammatory cytokine IL-6 down-regulates FOXP3 binding to chromatin in the presence of TGF-β. Moreover, histone deacetylation inhibitor (HDACi) treatment abrogates the down-regulating effects of IL-6 and TGF-β. These studies indicate that HDACi can enhance regulatory T cell function via promoting FOXP3 binding to chromatin even in a proinflammatory cellular microenvironment. Collectively, our data provide a framework of how different signals affect intranuclear redistribution, posttranslational modifications, and chromatin binding patterns of FOXP3.

Ligation between glucocorticoid-induced tumor necrosis factor receptor (GITR) and its ligand (GITRL) provides an undefined signal that renders CD4 + CD25 − effector T cells resistant to the inhibitory effects of CD4 + CD25 + regulatory T cells. To understand the structural basis of GITRL function, we have expressed and purified the extracellular domain of human GITR ligand in Escherichia coli . Chromotography and cross-linking studies indicate that human GITRL (hGITRL) exists as dimers and trimers in solution and also can form a supercluster. To gain insight into the nature of GITRL oligomerization, we determined the crystallographic structures of hGITRL, which revealed a loosely associated open trimer with a deep cavity at the molecular center and a flexible C-terminal tail bent for trimerization. Moreover, a tetramer of trimers (i.e., supercluster) has also been observed in the crystal, consistent with the cross-linking analysis. Deletion of the C-terminal distal three residues disrupts the loosely assembled trimer and favors the formation of a dimer that has compromised receptor binding and signaling activity. Collectively, our studies identify multiple oligomeric species of hGITRL that possess distinct kinetics of ERK activation. The studies address the functional implications and structural models for a process by which hGITRL utilizes multiple oligomerization states to regulate GITR-mediated signaling during T cell costimulation.

The forkhead family protein FOXP3 acts as a repressor of transcription and is both an essential and sufficient regulator of the development and function of regulatory T cells. The molecular mechanism by which FOXP3-mediated transcriptional repression occurs remains unclear. Here, we report that transcriptional repression by FOXP3 involves a histone acetyltransferase–deacetylase complex that includes histone acetyltransferase TIP60 (Tat-interactive protein, 60 kDa) and class II histone deacetylases HDAC7 and HDAC9. The N-terminal 106–190 aa of FOXP3 are required for TIP60–FOXP3, HDAC7–FOXP3 association, as well as for the transcriptional repression of FOXP3 via its forkhead domain. FOXP3 can be acetylated in primary human regulatory T cells, and TIP60 promotes FOXP3 acetylation in vivo . Overexpression of TIP60 but not its histone acetyltransferase-deficient mutant promotes, whereas knockdown of endogenous TIP60 relieved, FOXP3-mediated transcriptional repression. A minimum FOXP3 ensemble containing native TIP60 and HDAC7 is necessary for IL-2 production regulation in T cells. Moreover, FOXP3 association with HDAC9 is antagonized by T cell stimulation and can be restored by the protein deacetylation inhibitor trichostatin A, indicating a complex dynamic aspect of T suppressor cell regulation. These findings identify a previously uncharacterized complex-based mechanism by which FOXP3 actively mediates transcriptional repression.

We investigated the assembly and activation of the epidermal growth factor receptor (EGFR)–p185 c-neu heterodimer by using a sequential immunoprecipitation methodology. Using this approach we detected heterodimers and also higher-ordered oligomeric complexes. Phosphorylated EGFR–p185 c-neu heterodimeric forms were detected in the absence of EGF, but the species became highly phosphorylated after EGF stimulation. To evaluate heterodimer formation and additional transactivation by EGF, we investigated the roles of the four extracellular subdomains of p185 c-neu and the EGFR. Subdomains I–IV of the EGFR dimerized with subdomains I–IV of p185 c-neu , respectively, in a parallel manner. In addition, subdomains I–IV of the EGFR also associated with p185 c-neu subdomains III, IV, I, and II, respectively. A lack of one of the p185 c-neu cysteine-rich domains (subdomains II or IV) resulted in a loss of EGF-induced transactivation. These data suggest that two cysteine-rich domains play defining roles in ligand-dependent transactivation and that both of these cysteine-rich extracellular subdomains as well as non-cysteine-rich extracellular subdomains are involved in ligand-independent interactions with the EGFR. Our studies provide biochemical evidence of the role of the cysteine-rich domains of p185 c-neu in assembly and transactivation of erbB complexes and also indicate that these subdomains might be useful clinical targets.

Conversion of a malignant phenotype into a more normal one can be accomplished either by down-regulation of erbB family surface receptors or by creating inactive erbB heterodimers on the cell surface. In this report, we report the identification and cloning of differentially expressed genes from antibody-treated vs. untreated fibroblasts transformed by oncogenic p185 neu . We repeatedly isolated a 325-bp cDNA fragment that, as determined by Northern analysis, was expressed at higher levels in anti-p185 neu -treated tumor cells but not in cells expressing internalization defective p185 neu receptors. This cDNA fragment was identical in amino acid sequence to the recently cloned mouse Tat binding protein-1 (mTBP1), which has 98.4% homology to the HIV tat-binding protein-1 (TBP1). TBP1 mRNA levels were found to be elevated on inhibition of the oncogenic phenotype of transformed cells expressing erbB family receptors. TBP1 overexpression diminished cell proliferation, reduced the ability of the parental cells to form colonies in vitro , and almost completely inhibited transforming efficiency in athymic mice when stably expressed in human tumor cells containing erbB family receptors. Collectively, these results suggest that the attenuation of erbB receptor signaling seems to be associated with activation/induction or recovery of a functional tumor suppressor-like gene, TBP1 . Disabling erbB tyrosine kinases by antibodies or by trans-inhibition represents an initial step in triggering a TBP1 pathway.

Inhibition of cell growth and transformation can be achieved in transformed glial cells by disabling erbB receptor signaling. However, recent evidence indicates that the induction of apoptosis may underlie successful therapy of human cancers. In these studies, we examined whether disabling oncoproteins of the erbB receptor family would sensitize transformed human glial cells to the induction of genomic damage by γ-irradiation. Radioresistant human glioblastoma cells in which erbB receptor signaling was inhibited exhibited increased growth arrest and apoptosis in response to DNA damage. Apoptosis was observed after radiation in human glioma cells containing either a wild-type or mutated p53 gene product and suggested that both p53-dependent and -independent mechanisms may be responsible for the more radiosensitive phenotype. Because cells exhibiting increased radiation-induced apoptosis were also capable of growth arrest in serum-deprived conditions and in response to DNA damage, apoptotic cell death was not induced simply as a result of impaired growth arrest pathways. Notably, inhibition of erbB signaling was a more potent stimulus for the induction of apoptosis than prolonged serum deprivation. Proximal receptor interactions between erbB receptor members thus influence cell cycle checkpoint pathways activated in response to DNA damage. Disabling erbB receptors may improve the response to γ-irradiation and other cytotoxic therapies, and this approach suggests that present anticancer strategies could be optimized.

Our aim has been to understand the features of erbB receptor homo- and heterodimer assembly to develop approaches to disrupt receptor activation. We have developed a general approach to cause erbB receptor-specific trans inhibition of human neoplasia. The clonal progression of human astrocytomas to a more malignant phenotype often involves the amplification and overexpression of the epidermal growth factor receptor (EGFr) gene. We have selectively targeted the EGFr in human glioblastoma cells with kinase-deficient mutants of the erbB family derived from the ectodomain of the Neu oncogene that are able to form heterodimers with EGFr and inhibit EGFr-dependent phenotypes. In EGFr-positive U87MG human glioblastoma cells, expression of the Neu ectodomain inhibits EGF-, but not platelet-derived growth factor-, induced DNA synthesis; inhibits cell proliferation in the presence of EGF, but not platelet-derived growth factor; inhibits the ability of U87MG to form colonies in soft agar; and inhibits transforming efficiency in athymic mice. These studies establish that EGFr-mediated signal transduction is important in the maintenance of malignant glioma, and that trans receptor inhibition is a novel way to abrogate abnormal growth of these tumors. Neu ectodomains will be useful in determining the manner in which the EGFr contributes to glial tumorigenesis and in the design of pharmaceuticals that disable erbB family oncoproteins. In addition, these studies provide a rationale for the application of the Neu ectodomain in gene therapy approaches to human malignant glioma and, potentially, to other systemic epithelial malignancies expressing erbB family receptors.

The neu protooncogene (also known as c-erbB2, NGL, and HER2) encodes a 185-kDa transmembrane glycoprotein with intrinsic tyrosine kinase activity that resembles the receptor for epidermal growth factor. The p185 gene and protein were originally identified in the brain and are thought to play a critical role in neurogenesis. Aberrant c-erbB2 protein overexpression also occurs in several human adenocarcinomas. A ligand for p185, neu-activating factor (NAF), specifically binds to neu receptor and increases the p185c-neu tyrosine phosphorylation in vitro and in vivo in a dose-dependent manner. We now show that NAF specifically binds to purified p185 expressed in baculovirus. Direct binding analysis showed that NAF binds with high affinity (Kd = 1.3 nM). We have investigated changes in the structure and association state of baculovirus-produced neu holoreceptor that are induced by ligand binding. In this study, we used sucrose gradients to show that purified p185c-neu exists mainly in the monomeric form at low concentrations, whereas at higher concentrations p185c-neu exists as dimers or multimers. At low concentrations, but in the presence of ligand, p185c-neu sediments as a dimeric or multimeric form. Monomer-oligomer interconversion is absolutely ligand dependent at low receptor concentrations. The high molecular weight form of the receptor is enzymatically more active, as a consequence of ligand-driven activation of the receptor kinase. Oncogenic p185neu receptors sediment predominantly as high molecular weight forms and have constitutively active kinases.

We report the three-dimensional structures, at 1.8-A resolution, of the Fv fragment of the anti-hen egg white lysozyme antibody D1.3 in its free and antigen-bound forms. These structures reveal a role for solvent molecules in stabilizing the complex and provide a molecular basis for understanding the thermodynamic forces which drive the association reaction. Four water molecules are buried and others form a hydrogen-bonded network around the interface, bridging antigen and antibody. Comparison of the structures of free and bound Fv fragment of D1.3 reveals that several of the ordered water molecules in the free antibody combining site are retained and that additional water molecules link antigen and antibody upon complex formation. This solvation of the complex should weaken the hydrophobic effect, and the resulting large number of solvent-mediated hydrogen bonds, in conjunction with direct protein-protein interactions, should generate a significant enthalpic component. Furthermore, a stabilization of the relative mobilities of the antibody heavy- and light-chain variable domains and of that of the third complementarity-determining loop of the heavy chain seen in the complex should generate a negative entropic contribution opposing the enthalpic and the hydrophobic (solvent entropy) effects. This structural analysis is consistent with measurements of enthalpy and entropy changes by titration calorimetry, which show that enthalpy drives the antigen-antibody reaction. Thus, the main forces stabilizing the complex arise from antigen-antibody hydrogen bonding, van der Waals interactions, enthalpy of hydration, and conformational stabilization rather than solvent entropy (hydrophobic) effects.

We have produced bcl-2 transgenic mice by using a construct which mimics the t(14;18) translocation in human follicular lymphomas. Although lymphoid tissues from all transgenic mice contained high levels of human Bcl-2 protein, transgene expression was differentially regulated within the B- and T-cell compartments of lines derived from various founder mice. We have characterized the phenotypes of two lines of bcl-2 transgenic mice (line 2 and line 6) in which bcl-2 transgene expression was restricted primarily to the T- or B-cell lineages, respectively. Analysis of line 6 lymphocytes revealed a polyclonal expansion of B cells, and these B cells exhibited prolonged survival in vitro. In line 2 mice, numbers of T cells in the peripheral lymphoid tissues were more moderately elevated despite enhanced T-cell survival in vitro. Line 2 transgenic mice also showed significantly increased proportions of thymocytes with a mature phenotype. Taken together, these findings suggest different roles for bcl-2 in the in vivo regulation of B- and T-cell development and homeostasis.

Interleukin 2 (IL-2) is a lymphokine, produced by T cells upon antigenic or mitogenic stimulation, that is a critical regulator of T-cell proliferation. Although the binding of IL-2 to its receptor has been well characterized, the molecular mechanisms by which IL-2 transmits its signal from the membrane to the interior of the cell are poorly understood. Like most other growth factors, IL-2 causes rapid phosphorylation of proteins within its target cells. Unlike many other growth factors, however, the known subunits of the IL-2 receptor lack tyrosine-specific kinase activity, and little is known about the kinases whose activities are regulated by IL-2. Here we show that IL-2 (but not IL-4) induces rapid phosphorylation of the p72-74 serine/threonine-specific kinase encoded by the c-Raf-1 protooncogene in an IL-2-dependent murine T-cell line, CTLL-2, and that this phosphorylation is associated with increased kinase activity in p72-74 Raf-1-containing immune complexes. The concentration dependence of IL-2-mediated elevations in Raf-1 kinase activity correlated well with IL-2-stimulated proliferation of CTLL-2 cells. Furthermore, much of the IL-2-stimulated phosphorylation of p72-74 Raf-1 occurred on tyrosines. To our knowledge, the Raf-1 kinase represents the first endogenous substrate of an IL-2-regulated tyrosine kinase to be identified.

Previous studies showed that the cell-surface receptor for reovirus serotype 3 (Reo3R) appears at an early stage of oligodendrocyte differentiation and that anti-Reo3R antibodies and Reo3R-binding peptides induce galactocerebroside expression by developing oligodendrocytes. In the present studies, anti-Reo3R antibodies are shown to stimulate additional features of the program of oligodendrocyte development, including the loss of the A2B5 marker and expression of myelin basic protein. In anti-Reo3R antibody-treated cultures, galactocerebroside was expressed by cells having the morphology of immature oligodendrocyte precursors. Reo3R binding did not appear directly to inhibit or stimulate proliferation of glial progenitor cells or to affect their lineage commitment. Cell-surface structures utilized as a receptor by reovirus type 3 appear to play a role in the regulation of the initiation or rate of execution of the oligodendrocyte developmental program.

To study the mechanisms of tolerance in detail, we have constructed transgenic mice expressing a V beta 8.1-D beta 2-J beta 2.3-C beta 2 T-cell receptor (TCR) gene. Since expression of V beta 8.1 is known to correlate with reactivity of CD4+CD8- T cells to minor lymphocyte-stimulating locus 1a (Mls-1a), we expected to induce tolerance in most CD4+CD8- T cells in V beta 8.1 transgenic mice of the Mls-1a allele. In one line of Mls-1b V beta 8.1 transgenic mice, the V beta 8.1 TCR was expressed on greater than 98% of mature T cells and their response to Mls-1a was highly enriched. In Mls-1a V beta 8.1 transgenic mice, CD4+CD8- T cells in these mice were severely reduced among both peripheral T cells and thymocytes. However, the deletion of these cells was not complete, and most of the residual CD4+CD8- mature T cells still expressed normal densities of V beta 8.1 TCR. The residual CD4+CD8- T cells did not respond to Mls-1a but were still able to proliferate in response to other stimuli via the TCR. Interestingly, CD4+CD8- V beta 8.1+ T-cell clones isolated from Mls-1a V beta 8.1 transgenic mice could respond to Mls-1a. We suggest that these types of T cells escape clonal deletion in the thymus.

Viruses utilize normal cell surface structures as attachment sites. Interaction of viral components with these structures may alter target cell growth. In the present study, the expression and function of the cell surface receptor for reovirus type 3 (Reo3R) was studied in neonatal rat optic nerve glial cultures. The Reo3R is expressed by mature oligodendrocytes and astrocytes but not by O-2A progenitor cells. It appears at an early stage of oligodendrocyte development, coincident with the O4 marker but prior to galactocerebroside or myelin basic protein. Anti-Reo3R antibodies stimulate the expression of galactocerebroside by developing oligodendrocytes. Divalent Reo3R-binding peptides are similarly active. Maximal stimulation of galactocerebroside expression occurs with treatment as short as 4 hr, consistent with a receptor-mediated process. Cell surface structures used as an attachment site by reovirus type 3 may also play a role in the regulation of oligodendrocyte differentiation.

The rat neu oncogene encodes a cell surface glycoprotein, p185, that possesses tyrosine kinase activity. The p185 polypeptide exhibits structural similarity to the epidermal growth factor receptor (EGFR) at both the deduced amino acid and nucleic acid level. However, the neu oncogene and the gene encoding the EGFR have been shown to reside on distinct chromosomes. Comparative analysis of the sequences of the normal neu cDNA and of the neu cDNA from neuroblastomas has revealed a single point mutation leading to a valine-to-glutamic acid substitution in the transmembrane anchoring domain. This mutation converts the neu gene to a transforming gene in rodents. In humans, the gene is called ERBB2 (also NGL and HER2), and amplification and over-expression of its products have been detected in certain tumors. The rat embryonal fibroblast cell line (Rat-1) appears to express both EGFR and cellular p185 polypeptides. We have found that EGF stimulates the phosphorylation of p185 in these cells at tyrosine as well as serine and threonine residues in a specific and dose-dependent manner. This activity occurs even though radiolabeled EGF cannot bind to immunopurified p185. The EGF effect is apparently unique since platelet-derived growth factor, insulin, and transforming growth factor beta all fail to phosphorylate p185 at tyrosine. The EGF-induced effect requires interaction of the EGFR and its cognate ligand because cell lines that lack EGFR cannot be shown to phosphorylate p185, even when exposed to large amounts of EGF. Oncogenic rodent p185 and the human p185 homologue ERBB2 that is overexpressed in human breast tumor cells also can be shown to become phosphorylated on tyrosine residues by the action of EGF. Collectively, these data demonstrate that EGF mediates phosphorylation of p185 at tyrosine as well as serine/threonine through cellular kinases by a receptor-specific mechanism.

The mechanism by which viruses bind to and infect specific tissues to cause disease has only recently begun to be understood. The mammalian reoviruses provide an especially attractive model for studying the details of cell surface recognition. The cell and tissue tropism of reovirus is determined by a portion of the viral hemagglutinin termed the neutralization domain. We have reported previously on the generation of both monoclonal and polyclonal anti-idiotypic antibodies that mimic the viral hemagglutinin in the specificity of binding to the reovirus receptor. By using these anti-idiotypic antibodies as specific probes, we have successfully isolated the mammalian reovirus receptor from neuronal and lymphoid cells. In the present study, we report that the reovirus receptor is structurally similar to the mammalian beta-adrenergic receptor. This conclusion is based on the following observations: (i) purified beta-adrenergic receptor is immunoprecipitable by anti-reovirus receptor antibody; (ii) purified reovirus receptor obtained from murine thymoma cells and beta-adrenergic receptor obtained from calf lung exhibit identical molecular masses and isoelectric points; (iii) trypsin digests of purified reovirus and beta-adrenergic receptors display indistinguishable fragment patterns; (iv) purified reovirus receptor binds the beta-antagonist [125I]iodohydroxybenzylpindolol and this binding is blocked by the beta-agonist isoproterenol.

We have investigated the relationship between CD3 expression and the suppressor T-cell function. We have isolated stable clonal cell lines of the F12.23 suppressor T-cell hybridoma that are either CD3+ or CD3-. These lines were subjected to functional assays including inhibition of in vivo hapten-specific delayed-type hypersensitivity responses, in vitro hapten-specific interleukin 2 responses, as well as hapten-specific cytotoxic T-lymphocyte assays. In all assays, the functional suppressor phenotype absolutely correlated with CD3 surface expression. Furthermore, we have immunoprecipitated heterodimeric proteins that share molecular features with some receptor polypeptides previously described. CD3 polypeptides found on the surface of suppressor T cells are phosphorylated after phorbol ester stimulation. Collectively these studies unambiguously define the suppressive supernatant function as a product of CD3+ receptor-bearing T cells.

We present evidence for a role of I-A subregion-encoded determinants in syngeneic tumor immunity. In animals rendered immune to the S1509a fibrosarcoma, daily treatment with microliter quantities of antisera directed against Kk and I-Ak determinants expressed on lymphoid cells of host origin decreased the capacity for immune tumor rejection. Absorption studies revealed that anti-I-Ak antibody activity alone was sufficient for the manifestation of this effect. Furthermore, experiments utilizing F1 hybrids showed that an antiserum that was genetically unable to interact with H-2 determinants expressed on the tumor was equally effective in inhibiting tumor immunity. Suggestive evidence that the activity of this antiserum is related to interference with the generation of effector T cell function was provided by the observation that hyperimmune animals pretreated with an anti-Kk,I-Ak antiserum were no longer capable of adoptively transferring tumor immunity to naive recipients. Thus, it is possible to regulate the secondary immune response to tumor antigens by using antisera with specificity for I-A determinants expressed on cells or possibly on factors of the host lymphoid system.

The neu oncogene encodes a 185-kDa transmembrane glycoprotein tumor antigen, termed p185. We have recently described a monoclonal antibody reactive with a cell surface domain of the p185 molecule. In vivo treatment with this anti-p185 monoclonal antibody was able to significantly inhibit the tumorigenic growth of neu-transformed NIH 3T3 cells implanted into nude mice. Such treatment had no effect on the tumorigenic growth of Ha-ras-transformed NIH 3T3 cells. Furthermore, anti-p185 antibody treatment was able to inhibit the growth of the rat neuroblastoma cells from which the neu oncogene was initially isolated. These results demonstrate that a monoclonal antibody reactive with the extracellular domain of an oncogene-encoded protein can exert a significant antitumor effect; such antibodies may prove useful in the therapy of certain malignancies.

Data are reported on the genetic control and structure of antigen-specific suppressive molecules obtained from azobenzenearsonate (ABA)-specific suppressor T cells (Ts). Ts-derived suppressor factors (TsFs) were previously shown to bear determinants encoded by genes of the I-J subregion of the H-3 major histocompatibility complex and structures encoded by VH (variable region of heavy chains) genes linked to the Igh locus. To further characterize TsF we have made use of a K light (L) chain variable region (VK) genetic marker linked to the locus governing expression of a crossreactive idiotype on anti-ABA antibodies. The experiments evaluated the possible contribution to TsF of structures under control of the VK locus. TsF was prepared in strains of mice with known Igh and VK genes. Mice carrying Igh-1e genes produced TsF that was retained by an immunoadsorbent containing bound anti-idiotype antibodies; the genetic constitution at the VK locus was not relevant. By using a panel of anti-idiotypic antibodies prepared against the antigen-binding site of antibody carrying the idiotype, against determinants outside the site or against VH structures, we determined that idiotypic structures on the TsF are associated with V-region elements associated with the H chain and not with the binding site formed by a VH-BL combination.

By using polyethylene glycol 1540, BW5147 AKR T lymphoma cells were fused with splenocytes from A/J mice treated so as to induce suppressor T cells specific for azobenzenearsonate (ABA). Of 576 microwells originally seeded, 132 demonstrated growing cell clones, 4 of which produced an ABA-binding supernatant factor. When tested in vivo for suppression of delayed-type hypersensitivity to ABA, two of these cell lines, A4 and F12, were shown to produce suppressive supernatant factors. Fluorescence analysis of the F12 cells with appropriate antisera demonstrated this T cell hybrid to be Thy 1.2+, Lyt 1+,2-, and surface immunoglobulin negative, the surface marker phenotype of conventional ABA-specific suppressor T cells. This cloned suppressor cell line, F12, produces a culture supernatant factor that is suppressive at dilutions up to 1:100 and has provided material for genetic and immunochemical analysis.

Cytolytic T lymphocytes (Tc) specific for cells infected with reovirus type 3 were shown to lyse an uninfected B-cell hybridoma line (designated 87.92.6). This hybridoma expresses and secretes an anti-idiotypic antibody that reacts with a monoclonal antibody (termed G-5). G-5 recognizes a domain on the hemagglutinin of the reovirus that is relevant to virus tropism. The Tc cell response was H-2 restricted and could be inhibited by G-5. As shown by limiting dilution analysis, identical clones lysed reovirus-infected and anti-idiotype-bearing target cells. Tc cells induced by a variant of wild-type reovirus type 3 (immunologically selected by resistance to neutralization by G-5) were unable to recognize the anti-idiotype-bearing cells although they lysed reovirus type 3-infected tumor cells. We conclude that Tc cells must bear determinants that bind anti-idiotype molecules and, furthermore, that B cells and cytolytic T cells can share these idiotypic determinants.

Thy-1 is a major cell surface molecule expressed on murine thymocytes and peripheral T cells. Its physiological function is unknown, but in vitro studies suggest that Thy-1 may transmit activation signals to T cells and may play a role in the growth and/or differentiation of thymocytes [Kroczek, R. A., Gunter, K. C., Seligmann, B. & Shevach, E. M. (1986) J. Immunol. 136, 4379-4384; Kroczek, R. A., Gunter, K. C., Germain, R. N. & Shevach, E. M. (1986) Nature (London) 322, 181-184]. However, not all mouse thymocytes are Thy-1+ [Scollay, R., Wilson, A., D'Amico, A., Kelly, K., Egerton, M., Pearse, M., Wu, L. & Shortman, K. (1988) Immunol. Rev. 104, 81-120]. In addition, C3H-gld/gld mice accumulate large numbers of Thy-1- (and Thy-1+) T-cell antigen receptor-positive CD8- CD4- (double negative) T cells in peripheral lymphoid organs. Our previous studies of these Thy-1- and Thy-1+ double negatives suggested that lack of Thy-1 expression correlated with diminished capacity to respond to T-cell stimuli. In this report, we describe a Thy-1- alpha/beta T-cell receptor-positive major histocompatibility complex-specific cytotoxic T-cell clone derived from C3H-gld/gld lymph node-residing cells. The data show that, at least in this system, Thy-1 (and CD8/CD4) expression is not required for growth, cytolytic activity, or expression of functional T-cell receptor complexes in vitro and raise the possibility that Thy-1 expression may not be obligatory in vivo for development of cytotoxic T-lymphocyte precursors in gld mice.

Highly significant suppression of the growth of S1509a and Sa-I syngeneic sarcomas was observed in A/J mice following daily intravenous injections of 2 microliter of anti-I-Jk alloantiserum. This effect persisted as long as the anti-I-Jk serum was administered (day 15), In contrast, a control anti-I-Js serum had no discernible effect on the growth of the S1509a tumor. The inhibitory activity of the anti-I-Jk serum on the growth of the tumor was absorbed specifically by B10-BR spleen cells bearing I-Jk determinants. In other experiments, we established that A/J mice treated with anti-I-Jk serum, according to the protocol described above, are no longer a source of tumor-specific suppressor cells for adoptive transfer into immune tumor-bearing recipient mice. We conclude that anti-I-Jk serum inhibits tumor growth in A/J mice by abolishing tumor-specific suppressor activity.

Antibody molecules are composed of several functional domains, including a variable domain that contacts antigen and a constant domain. The hypervariable regions of antibody molecules play an integral role in determining their specificity. However, the delineation of specific residues most critical in binding is difficult. We have been studying a monoclonal antibody (87.92.6) that binds to the reovirus type 3 receptor on a number of cell types, down-modulates the receptor, and inhibits DNA synthesis in the cells. We have shown that a peptide derived from the second complementarity-determining region of the monoclonal antibody 87.92.6 light-chain variable region can reproduce both of these effects. We were also able to demonstrate specific amino acid residues and structural features involved in producing these effects. The study of antibody structure, coupled with molecular synthetic techniques, can lead to the development of biologically active substances with potential clinical use.

The developmental patterns of rearrangement and expression of the T-cell antigen-receptor genes are precisely regulated during T-cell differentiation and education. The beta- and gamma-subunit RNAs of the T-cell receptor are abundantly expressed in immature thymocytes. In mature thymocytes the alpha- and beta-subunit RNAs are preferentially expressed, whereas there is minimal expression of the gamma RNA. Although aspects of the pattern of known organization and rearrangement of the T-cell receptor gene in the thymus have been studied and the concept of a thymus selection process generally has been accepted, the cellular and molecular basis of thymus education remains obscure. Certain strains of mice with predilections for autoimmunity demonstrate T-cell developmental abnormalities. This is especially true for the lpr/lpr or gld/gld genotypes, in which the major population of peripheral T cells is developmentally disturbed. We have studied the development, expression, and rearrangement of T-cell receptor genes in the C3H/HeJ gld/gld mouse. Our results indicate a high level of expression of the beta and alpha RNAs in C3H/HeJ gld/gld T cells residing in the periphery. In addition, the beta-subunit gene of gld/gld peripheral T cells undergoes more rearrangements than does its normal C3H/HeJ T-cell counterparts. We speculate that this rearrangement pattern and high level of T-cell receptor mRNA reflects an abnormality or deficit in a thymus selection process that permits emigration of T cells with nonfunctionally rearranged T-cell receptor genes to secondary lymphoid organs. However, the normal level of gamma-subunit RNA expression argues that gamma-subunit gene rearrangements are distinct from processes related to alpha- and beta-subunit selection.

The monoclonal anti-idiotypic antibody (mAb2) 87.92.6 directed against the 9B.G5 antibody specific for the virus neutralizing epitope on the mammalian reovirus type 3 hemagglutinin was previously demonstrated to express an internal image of the receptor binding epitope of the reovirus type 3. Furthermore, this mAb2 has autoimmune reactivity to the cell surface receptor of the reovirus. The nucleotide and deduced amino acid sequences of the 87.92.6 mAb2 heavy and light chains are described in this report. The sequence analysis reveals that the same heavy chain variable and joining (VH and JH) gene segments are used by the 87.92.6 anti-idiotypic mAb2 and by the dominant idiotypes of the BALB/c anti-GAT (cGAT) and anti-NP (NPa) responses. [GAT; random polymer that is 60% glutamic acid, 30% alanine, and 10% tyrosine. NP; (4-hydroxy-3-nitrophenyl)-acetyl.] Despite extensive homology at the level of the heavy chain variable regions, the NPa positive BALB/c anti-NP monoclonal antibody 17.2.25 binds neither 9B.G5 nor the cellular receptor for the hemagglutinin. Amino acid sequence comparison between the viral hemagglutinin and the 87.92.6 mAb2 light chain "internal image," reveals an area of significant homology indicating that antigen mimicry by antibodies may be achieved by sharing primary structure.

The neu oncogene (also referred to as c-erbB-2 and HER2) encodes a 185-kDa transmembrane glycoprotein with tyrosine kinase activity termed p185. The p185 glycoprotein is structurally related to the epidermal growth factor receptor. It is thought that p185 is the receptor for an as yet unidentified growth factor. In the present study, RNA blot analyses and immunohistochemical studies were performed on rat tissues obtained from a variety of prenatal and postnatal stages to examine the expression of the neu oncogene and its product, p185, during normal development. Expression of the neu gene was detected in mid-gestation embryos in a variety of tissues including nervous system, connective tissue, and secretory epithelium, but not in lymphoid tissue. In adult animals, secretory epithelial tissues and basal cells of the skin expressed neu. These studies demonstrate that the neu gene is expressed in a tissue- and developmental stage-specific manner. We suggest that the p185 molecule plays an important role in the growth and development of a variety of tissues, and, in particular, in epithelial tissue.