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Z Dembic

Publications and source records attributed to Z Dembic.

At least 19 recordsLinked to original sources
PubMed2000

Dendritic cells purified from myeloma are primed with tumor-specific antigen (idiotype) and activate CD4+ T cells.

Multiple myelomas produce tumor-specific antigen (TSA) in the form of idiotype (Id) on monoclonal Ig. CD4(+) T cells can recognize Id-peptide on MHC class II molecules and protect against challenges with MOPC315 cells, which are, as common for myelomas, class II-negative. The present study explains these previous results by demonstrating that Id can be transferred from myeloma cells to antigen-presenting cells (APC), which present processed Id-peptide on their class II molecules to Id-specific T cell receptor-transgenic (TCR-TG) CD4(+) T cells. Id-primed tumor APC were heterogeneous, the majority being dendritic cells with class II(+), CD11b(+) CD11c(+) CD40(+) CD80(+) CD86(+) markers. The APC were localized beneath CD31(+) endothelial cells of tumor microvessels, and their frequency declined with tumor progression. The APC could stimulate Id-specific naive TCR-TG, short-term polarized TCR-TG, and cloned CD4(+) T cells to proliferate and produce cytokines in vitro. Furthermore, small MOPC315 tumors established in Id-specific TCR-TG mice contained clusters of activated (CD69(+)CD25(+)) and proliferating (BrdUrd(+)) Id-specific transgenic CD4(+) blasts. The activated Id-specific T cells were located adjacent to Id-primed dendritic cells in the tumor. Thus, a TSA can be transferred in vivo from myeloma, and possibly other types of cancer cells to APC for MHC class II presentation to CD4(+) T cells.

Animals↗
PubMed1998

Targeted disruption of the interferon-gamma receptor 2 gene results in severe immune defects in mice.

To study the role of the interferon- (IFN) gammaR2 chain in IFN-gamma signaling and immune function, IFN-gammaR2-deficient mice have been generated and characterized. Cells derived from IFN-gammaR2 -/- mice are unable to activate either JAK/STAT signaling proteins or gene transcription in response to IFN-gamma. The lack of IFN-gamma responsiveness alters IFN-gamma-induced Ig class switching by B cells from these mice. In vitro cultures of T cells demonstrate that the T cells from the IFN-gammaR2 -/- mice have a defect in Th1 cell differentiation. The IFN-gammaR2 (-/-) mice also produce lower amounts of IFN-gamma in response to antigenic challenge. In addition, IFN-gammaR2 -/- mice are defective in contact hypersensitivity and are highly susceptible to infection by Listeria monocytogenes. These results demonstrate that the IFN-gammaR2 is essential for IFN-gamma-mediated immune responses in vivo.

Animals↗
PubMed1998

Transient overexpression of CD4 enhances allelic exclusion of T-cell receptor (TCR) alpha chains and promotes positive selection of class II-restricted TCR-transgenic thymocytes.

CD4 contributes to antigen recognition of T cells by binding to class II MHC molecules. There is heterogeneity in expression of CD4 coreceptor among CD4+CD8+ thymocytes. We have investigated whether the expression level of coreceptor influences positive selection. Thymocytes of mice expressing transgenic lambda2(315)-Ig-light-chain/I-Ed specific TCR are poorly positively selected because they fail to allelically exclude endogenous TCR alpha chain genes and because there is no skewing towards CD4. Transient overexpression of CD4 during thymocyte development, in mice transgenic for both TCR and CD4, resulted in skewing towards CD4 in the periphery, reduced rearrangement and expression of endogenous alpha-chains, and decreased levels of thymocyte RAG-1 transcripts. Kinetic BrdU labeling experiments showed that single CD4+ thymocytes developed faster, representing the predominant population even in the cortex of the double transgenic thymi. These results demonstrate that increased coreceptor expression can compensate for poorly selectable TCR, supporting avidity and instructional models for positive selection of thymocytes.

Animals↗
PubMed1997

The gene for the ligand binding chain of the human interferon gamma receptor.

In order to characterize the gene encoding the ligand binding (1(st); alpha) chain of the human IFN-gamma receptor, two overlapping cosmid clones were analyzed. The gene spans over 25 kilobases (kb) of the genomic DNA and has seven exons. The extracellular domain is encoded by exons 1 to 5 and by part of exon 6. The transmembrane region is also encoded by exon 6. Exon 7 encodes the intracellular domain and the 3' untranslated portion. The gene was located on chromosome 6q23.1, as determined by in situ hybridization. The 4 kb region upstream (5') of the gene was sequenced and analyzed for promoter activity. No consensus-matching TATA or CAAT boxes in the 5' region were found. Potential binding sites for Sp1, AP-1, AP-2, and CREB nuclear factors were identified. Compatible with the presence of the Sp1/AP-2 sites and the lack of TATA box, S1-nuclease mapping experiments showed multiple transcription initiation sites. Promoter activity of the 5' flanking region was analyzed with two different reporter genes: the Escherichia coli chloramphenicol acetyltransferase and human growth hormone. The smallest 5' region of the gene that still had full promoter activity was 692 base pairs in length. In addition, we found sequences belonging to the oldest family of Alu repeats, 2 - 3 kb upstream of the gene, which could be useful for genetic studies.

Amino Acid Sequence↗
PubMed1996

Mouse macrophages carrying both subunits of the human interferon-gamma (IFN-gamma) receptor respond to human IFN-gamma but do not acquire full protection against viral cytopathic effect.

Studies of hamster-human and mouse-human somatic fibroblast hybrids and transfected mouse fibroblasts have demonstrated that signaling through the human interferon-gamma receptor (hu-IFN-gammaR) requires the formation of a complex consisting of ligand (IFN-gamma), a ligand binding receptor chain (IFN-gammaR1), and a signal transducing receptor chain (IFN-gammaR2). To date, the ability of this receptor complex to transduce the full repertoire of biological signals has been difficult to assess due to the limited number of activities that IFN-gamma can exert on fibroblasts. The current report assesses the ability of hu-IFN-gammaR chains to transduce signals in the absence of background human gene products by expressing hu-IFN-gammaR2 in a transformed macrophage cell line (F10/96) derived from a hu-IFN-gammaR1 transgenic mouse. Our results indicate that F10/96 clones expressing both human receptor proteins bind hu-IFN-gamma with an affinity comparable to that of human cells. Binding of either human or mouse IFN-gamma to its respective receptor elicits classic IFN-gamma responses such as up-regulation of major histocompatibility complex antigens, enhanced expression of IRF-1, and increased production of NO2- radicals, interleukin-6, tumor necrosis factor-alpha, and granulocyte macrophage-colony stimulating factor. However, hu-IFN-gamma could not fully protect the clones from cytopathic effects of encephalomyocarditis virus and vesicular stomatitis virus while mo-IFN-gamma could. These results demonstrate that while co-expression of hu-IFN-gammaR1 and hu-IFN-gammaR2 is necessary and sufficient for most IFN-gamma-induced responses, it is not sufficient to confer a generalized antiviral state. These findings further suggest that additional species-specific accessory factor(s) are necessary for full signaling potential through the IFN-gamma receptor complex. The nature and potential role of such factors in IFN-gammaR signaling is discussed.

Animals↗
PubMed1996

The structure of the gene for the second chain of the human interferon-gamma receptor.

The gene for the second chain of the human interferon-gamma receptor was analyzed from cosmid DNA clones. The gene spans over 33 kilobases of DNA and contains seven exons. The signal peptide is encoded by exons 1 and 2, the extracellular domain by exons 2, 3, 4, 5, and by part of 6. Exon 6 also encodes the whole transmembrane domain and part of the intracellular domain. Exon 7 encodes the remainder of the intracellular domain and contains the 3'-untranslated region. The sequences at the exon/intron boundaries are well conserved with respect to canonical acceptor/donor sites (AG/GT). The 5'-flanking region was sequenced and analyzed for transcription factor binding sites. No TATA or CAAT boxes in the promoter region were identified. Consistent with the lack of a TATA box, analysis of the mRNAs by primer extension showed multiple transcription start sites. Promoter activity of the 5'-flanking region was investigated with a luciferase reporter gene and the cytomegalovirus minimal promoter. Segments of the 5' region with promoter activity were identified.

Antigens, CD↗
PubMed1996

Impaired differentiation of Schwann cells in transgenic mice with increased PMP22 gene dosage.

An intrachromosomal duplication containing the PMP22 gene is associated with the human hereditary peripheral neuropathy Charcot-Marie-Tooth disease type 1A, and PMP22 overexpression as a consequence of increased PMP22 gene dosage has been suggested as causative event in this frequent disorder of peripheral nerves. We have generated transgenic mice that carry additional copies of the pmp22 gene to prove that increased PMP22 gene dosage is sufficient to cause PNS myelin deficiencies. Mice carrying approximately 16 and 30 copies of the pmp22 gene display a severe congenital hypomyelinating neuropathy as characterized by an almost complete lack of myelin and marked slowing of nerve conductions. Affected nerves contain an increased number of nonmyelinating Schwann cells, which do not form onion bulbs but align in association with axons. The mutant Schwann cells are characterized by a premyelination-like state as indicated by the expression of embryonic Schwann cell markers. Furthermore, continued Schwann cell proliferation is observed into adulthood. We hypothesize that Schwann cells are impaired in their differentiation into the myelinating phenotype, leading to a disorder comparable to severe cases of hereditary motor and sensory neuropathies. Our findings, combined with the analysis of heterozygous and homozygous PMP22-deficient mice, indicate that aberrant pmp22 gene copy numbers cause various forms of myelination defects.

Animals↗
PubMed1996

T cells with two Tcrbeta chains and reactivity to both MHC/idiotypic peptide and superantigen.

It is thought that Tcrbeta genes are effectively allelically excluded, while Tcralpha genes are not. We report here that endogenous Tcrbeta genes are expressed on as much as 5-7% of CD4+ T cells in 8-week-old Tcralphabeta-transgenic mice. In this model, the transgenic Tcr recognizes residues 91 - 101 of a lambda2(315) Ig light chain, presented on the I-Ed class II molecule. From such mice, a CD4+ T cell clone was isolated which not only responded to lambda2(315), but also mobilized Ca2+ and proliferated in response to Mls-1a. (Inadvertently we found that the C3H/Tif substrain, in contrast to C3H/HeJ, is Mls-1a positive.) The clone expressed the transgenic Tcr (Valpha1 and Vbeta8.2), and in addition an endogenous Vbeta6 chain, conferring the Mls-1a reactivity. On the population level, 1-2% of Tcr-transgenic lymph node cells displayed Vbeta6, in addition to the transgenic beta-chain. Such dual Tcrbeta expressor cells could be preferentially expanded in vitro by first stimulating with DBA/2 spleen cells and then with lambda2(315)-pulsed BALB/c antigen-presenting cells. In addition to demonstrating that allelic exclusion of Tcrbeta-chain genes is substantial but not complete in this model, the data show that the double beta-chain expressors can have two different specificities, and be signaled through both receptors, by physiological ligands. However, such dual-Tcr T cells appear to have reduced sensitivity to ligands, due to their decreased expression of each receptor. This holds true for both early (Ca2+ mobilization) and late (proliferation) T cells activation parameters. Dual Tcr cells may have a role in the pathogenesis of autoimmune diseases: If naive T cells are first stimulated by (infectious) superantigen, they could later, as activated T cells, respond to self-peptide/MHC on costimulation-deficient cells and cause autoimmunity. As a corollary, dual Tcr Id-specific T cells could, once activated, directly regulate Id+ B cells.

Animals↗
PubMed1996

Genomic organization and promoter analysis of the gene ifngr2 encoding the second chain of the mouse interferon-gamma receptor.

A clone containing the gene ifngr2 for the second chain (IFN-gamma R2) of the mouse interferon gamma receptor complex was isolated from a cosmid library made of 129/Sv mouse genomic DNA. Sequence analysis revealed that the second chain is encoded by 7 exons. The complete gene spans about 17 kb of the genomic DNA. In the 5'-flanking region several transcription initiation sites between 27 and 136 nucleotides upstream from the translation initiation codon were mapped. This region has a high GC content, but no TATA or CAAT box. Potential binding sites were found for transcription factors Sp1, AP-2, NF1, EGR and NF kappa B. Promoter activity was assayed with a series of constructs with firefly luciferase as a reporter gene, under the control of the promoter fragments of various lengths. This region showed promoter activity in transiently transfected Chinese hamster ovary cells.

Animals↗
PubMed1996

The intracellular domain of the second chain of the interferon-gamma receptor is interchangeable between species.

In this report we show that the mouse interferon (IFN)-gamma R1 and IFN-gamma R2 subunits expressed in hamster cells are capable of rendering the cells sensitive to mouse IFN-gamma as measured by induction of class I MHC antigens and the activation of the transcription factor Stat1 alpha. However, these cells showed no antiviral protection in response to IFN-gamma when challenged with vesicular stomatitis virus (VSV) but limited protection when challenged with encephalomyocarditis virus (EMCV). Furthermore, the cytoplasmic domains of the IFN-gamma R2 subunits, like the cytoplasmic domains of the IFN-gamma R1 chains, can be interchanged between species with no loss of biologic activity, demonstrating that the species-specific interaction of the IFN-gamma R1 and IFN-gamma R2 chains involves only the extracellular domains of the two proteins.

Amino Acid Sequence↗
PubMed1995

Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells. Purification, biochemical characterization, and stoichiometry of binding.

Agents that antagonize the functions of interferon gamma (IFN gamma) are potential pharmaceuticals against several immunological and inflammatory disorders. IFN gamma receptor-immunoglobulin G fusion proteins (IFN gamma R-IgG) function as antagonists of endogenous IFN gamma and have longer half-lives in vivo in comparison with soluble IFN gamma receptors (sIFN gamma R), consisting of the extracellular region of the native sequence. A fusion protein comprising the extracellular domain of the human IFN gamma receptor and the hinge, CH2 and CH3 domains of the human IgG3 constant region, was expressed in Chinese hamster ovary cells. The IFN gamma R-IgG3 fusion protein was secreted into the culture medium as a 175-kDa glycoprotein and was purified over Protein G-Sepharose, DEAE-Sepharose, and size exclusion chromatography. IFN gamma R-IgG3 bound IFN gamma in solid phase assays and ligand blots, competed for the binding of radiolabeled IFN gamma to the cell surface receptor of Raji cells, and inhibited the IFN gamma-mediated antiviral activity with an efficiency at least one order of magnitude higher than that of the soluble receptor produced in the same expression system. Two IFN gamma R-IgG3 fusion proteins bound two IFN gamma dimers forming a complex of approximately 380 kDa. In immunodiffusion assays, the IFN gamma R-IgG3 fusion protein did not precipitate IFN gamma. Dissociation of bound IFN gamma from IFN gamma R-IgG3 was 2-fold slower than from the sIFN gamma R produced in insect cells.

Animals↗
PubMed1995

Naive CD4+ T cells confer idiotype-specific tumor resistance in the absence of antibodies.

CD4+ T cells can recognize a processed idiotypic peptide derived from the mouse lambda 2(315) immunoglobulin light chain. The idiotypic peptide is presented on the I-E(d) class II major histocompatibility complex molecule. Mice made transgenic for a lambda 2(315)-specific alpha beta T cell receptor have been demonstrated to be specifically resistant against a tumor challenge with the MOPC315 (alpha,lambda 2(315)) plasmacytoma (Lauritzsen, G. F., Weiss, S., Dembic, Z. and Bogen, B., Proc. Natl. Acad. Sci. USA 1994, 91: 5700). That study, however, did not rule out a role of either anti-Id antibodies or T cells expressing nontransgenic specificities due to expression of endogenous T cell receptor (TcR) alpha chains. Also, the role of different T cell subsets in protection was unclear. To remove these ambiguities, we have now made the transgenic mice homozygous for the scid mutation, known to inhibit both Ig and TcR gene rearrangements. Such transgenic SCID mice lack B cells and antibodies while they still have plenty of CD4+ and CD4-8- cells expressing the transgenic alpha beta T cell receptor. The number of CD8+ T cell is dramatically reduced. Even so, transgenic SCID mice are protected against a challenge with MOPC315 plasmacytoma cells. Therefore, B cells, as well as novel T cell receptor specificities created by rearrangements of endogenous alpha-chain genes, are both dispensable for effective immunosurveillance in our system. Surprisingly, we found that transgenic CD8+ and CD4-8- cells are idiotype-specific and I-E(d) restricted. However, these T cell subsets are not required for resistance because adoptive transfer experiments demonstrated that highly purified transgenic SCID CD4+ cells suffice for tumor protection.

Animals↗
PubMed1995

Interferon-gamma receptor extracellular domain-IgG fusion protein produced in Chinese hamster ovary cells as mixture of glycoforms.

Glycosylation of proteins fulfills important functions and because of its diversity contributes to apparent protein heterogeneity. We investigated the heterogeneity of a fusion protein comprising the extracellular domain of the human interferon-gamma (IFN-gamma) receptor and parts of the human IgG3 constant region, a potential IFN-gamma antagonist. The protein was produced in Chinese hamster ovary (CHO) cells and was secreted into the culture medium as an 175 kD glycoprotein. Glycosylation represented approximately one-third of the apparent molecular mass of the fusion protein, consisted of N- and O-linked carbohydrate moieties, and included sialic acid residues as part of both N- and O-linked oligosaccharides. Fusion protein forms with different apparent molecular masses and charges were separated by ion-exchange chromatography. Preparative electrofocusing revealed a wide spectrum of glycoforms. Glycosylation of the fusion protein and of soluble IFN-gamma receptors, comprising the extracellular domain of the native sequence, expressed in insect and CHO cells did not interfere with affinity of ligand binding.

Amino Acid Sequence↗
PubMed1995

Preferential positive selection of T lymphocytes which express two different TCR alpha chains, an endogenous and a transgenic.

A hallmark of positive selection in T-cell receptor (TCR)-transgenic mice is a strong skewing towards the CD4+ or the CD8+ subset, depending on the class II or I restriction of the TCR, respectively. However, previous experiments in TCR transgenic mice specific for an Ig light chain (lambda 2(315)/I-Ed class II molecule did not fit into this scheme because the authors observed an anomalous skewing towards CD8. In this paper the authors show that endogenous TCR alpha chains are expressed on > 90% of CD4+ and CD8+ cells in this particular transgenic strain, even on a selecting H-2d haplotype. Endogenous TCR alpha chains are first detected when double-positive thymocytes down-regulate either CD4 or CD8. Endogenous V alpha seems to influence generation of T-cell subsets because CD4+ and CD8+ cells express different frequencies of endogenous V alpha 2 and V alpha 8. In the absence of endogenous TCR alpha chains in recombination-deficient TCR-transgenic severe combined immunodeficiency (SCID) mice, a strong skewing towards CD4+ T cells is seen, but such mice are severely T-cell deficient. As an explanation for these results, the authors suggest that the transgenic TCR has a too low affinity for efficient positive selection, therefore, TCR alpha gene rearrangements proceed. Endogenous TCR alpha paired with transgenic TCR beta could bind to class I or class II molecules, enhance positive selection and thereby production of CD4+ or CD8+ cells. Most of the 'mismatched' CD8+ cells are lambda 2(315)-specific and I-Ed class II restricted, and may function as idiotype-specific suppressors of B cells. These results may help explain the origin of dual TCR alpha T cells. Furthermore, the authors suggest that T cells 'mismatched' for co-receptor/TCR MHC-specificity may be enriched among dual TCR alpha T cells.

Animals↗
PubMed1994

Naive idiotype-specific CD4+ T cells and immunosurveillance of B-cell tumors.

The immunosurveillance hypothesis suggests that lymphocytes can recognize tumor-specific antigens expressed by transformed cells and initiate their elimination. Immunosurveillance implies that lymphocytes of naive phenotype can home to a tumor site and become activated by tumor-specific antigens. In this study, we have employed T-cell receptor transgenic mice as a source of naive, tumor-specific T cells. The transgenic, CD4+ T cells recognize a 91- to 101-residue fragment of the lambda 2(315) immunoglobulin light chain presented by I-Ed class II molecules. Such naive, idiotype-specific, CD4+ T cells protected against tumor development of a class II negative plasmacytoma (MOPC315) and a class II positive B lymphoma (F9), which both secrete lambda 2(315) immunoglobulin. Adoptive transfer experiments demonstrated that 2 x 10(6) lymph node cells were sufficient for protection against MOPC315. Depletion of T-cell subsets indicated that transgenic CD4+ cells were indispensable for tumor resistance. However, an additional role of CD8+ T cells is not ruled out. In contrast to the resistance against the secreting MOPC315 and F9 cells, transgenic mice were not protected against B lymphoma cells (F67), which do not secrete lambda 2(315) but express a truncated lambda 2(315) chain intracellularly. The results suggest that lambda 2(315) is processed and presented by host antigen-presenting cells, which in turn activate naive, idiotype-specific T cells.

Animals↗
PubMed1993

Clonal deletion of specific thymocytes by an immunoglobulin idiotype.

We have investigated whether immunoglobulin can induce clonal deletion of thymocytes by employing two strains of transgenic mice. One strain is transgenic for an alpha/beta T cell receptor (TCR) which recognizes a processed idiotypic peptide of the lambda 2(315) light chain variable region, bound to the I-Ed class II major histocompatibility complex molecule. The other mouse strain is transgenic for the lambda 2(315) gene. Double transgenic offspring from a TCR-transgenic female mated with a lambda 2(315) transgenic male exhibit a pronounced clonal deletion of CD4+CD8+ thymocytes. Analysis of neonates from the reciprocal (lambda 2(315)-transgenic female x TCR-transgenic male) cross suggests that the deletion in double transgenic offspring most likely is caused by lambda 2(315) produced within the thymus rather than by maternally derived IgG, lambda 2(315). Nevertheless, IgG, lambda 2(315) can cause deletion of CD4+CD8+ thymocytes when injected in large amounts intraperitoneally into either adult or neonatal TCR-transgenic mice. Deletion is evident 48 and 72 h after injection, but by day 7 the thymus has already regained its normal appearance. A serum concentration of several hundred microgram/ml is required for deletion to be observed. Therefore, the heterogeneous idiotypes of serum Ig are probably each of too low concentration to cause thymocyte deletion in normal animals.

Animals↗