Biomedical subjects

T R Malek

Publications and source records attributed to T R Malek.

At least 37 records · Page 2Linked to original sources

PubMed1998

The structure and function of gamma c-dependent cytokines and receptors: regulation of T lymphocyte development and homeostasis.

Five cytokines, IL-2, IL-4, IL-7, IL-9, and IL-15, form one group that is characterized by utilizing the common gamma chain (gamma c) as a receptor subunit. Examination of the phenotype of various cytokine or cytokine receptor "knockout" mice demonstrates that these cytokines are critical for normal lymphocyte development and subsequent functional activity of the peripheral immune compartment. This review summarizes the structural and functional properties of each of these five cytokines and their receptors, including the known redundant pathways for each cytokine or receptor. The contribution of these cytokines and receptors will then be considered in detail with respect to regulation of T lymphocyte development and homeostasis of the peripheral T cell compartment.

Animals↗

PubMed1997

Regulation of Fas-dependent activation-induced T cell apoptosis by cAMP signaling: a potential role for transcription factor NF-kappa B.

TCR-mediated activation of T cell hybridomas induces programmed cell death by a Fas-dependent pathway. We now show that costimulation of 2B4 cells, in the absence or presence of transgenic Bcl-2, with anti-CD3 epsilon and forskolin, an activator of cAMP signaling, resulted in antagonism of Fas-dependent activation-induced cell death that was always accompanied by selective downregulation of the nuclear levels of NF-kappa B p65-p50 (RelA-p50) transcription factor. Forskolin not only inhibited activation-induced cell death and NF-kappa B activation, but also suppressed expression of Fas and Fas ligand (Fas-L). Furthermore, NF-kappa B p65 antisense oligonucleotide down-regulated nuclear levels of NF-kappa B, inhibited cell surface expression of Fas-L and apoptosis of 2B4. Collectively, these finding demonstrate a potential role of NF-kappa B in the regulation of activation-induced apoptosis in T lymphocytes.

Antibodies↗

PubMed1997

Distribution of Ly-6C on lymphocyte subsets: I. Influence of allotype on T lymphocyte expression.

The expression patterns of the Ly-6C Ag were examined on splenic and thymic lymphocyte subsets of Ly-6.1 and Ly-6.2 strains of mice using the rat mAb 15.1. Ly-6C is expressed on subsets of CD4+ and CD8+ splenocytes, and a portion of NK cells. Within the splenic and lymph node CD4+ T cell compartment, Ly-6C expression is restricted to Ly-6.2 strains of mice, and is present on a subset of naive cells. Ly-6C is expressed on the majority of peripheral CD8+ T cells in both Ly-6.1 and Ly-6.2 strains, and is found primarily on the Ag-experienced subset. In the thymus, Ly-6C is present on subpopulations of CD4- CD8+, CD4- CD8-, and CD4+ CD8- cells. Ly-6C+ CD4- CD8+ thymocytes show a mature phenotype, while Ly-6C+ CD4- CD8- and Ly-6C+ CD4+ CD8- thymocytes appear to be part of the recently described NK1.1+ alphabeta TCR+ population. On account of the marked differences in Ly-6C expression on peripheral CD4+ T cells from Ly-6.1 and Ly-6.2 strains of mice, additional experiments were undertaken to assess Ly-6C expression in parental and Ly-6.1 x Ly-6.2 F1 mice. Neither phenotype dominated in the F1 offspring, with frequencies of Ly-6C+ CD4+ splenocytes falling in the intermediate range. Further experiments compared the staining patterns of the rat anti-pan Ly-6C (Ly-6.1 and Ly-6.2) Ab with a mouse anti-Ly-6.2 allotype specific Ab, with emphasis on both Ly-6.2 and Ly-6.1 x Ly-6.2 F1 mice. The results demonstrate the presence of lymphocytes that express the pan form of Ly-6C but not the form recognized by the alloantibody. This latter finding suggests the presence of more than one form of the Ly-6C Ag.

Alleles↗

PubMed1997

The common gamma-chain of cytokine receptors regulates intrathymic T cell development at multiple stages.

Signaling through the common gamma chain (gamma c), a subunit of the receptors for IL-2, -4, -7, -9, and -15, is critical for lymphocyte development, with the IL-7/IL-7R representing one important interaction. To investigate the stages of intrathymic T cell development that are dependent on gamma c and to determine whether gamma c controls T cell development solely as a component of the IL-7R, intrathymic T cell development was compared in IL-7R alpha-deficient mice and anti-gamma c-treated chimeric mice reconstituted with bone marrow and purified pro-T cells. In the presence of anti-gamma c, each of four phenotypically distinguishable stages of CD4- CD8- thymocytes failed to reconstitute T cell development, suggesting that each of these subsets of pro-T cells required gamma c for their differentiation and/or growth. Reconstitution of anti-gamma c-treated chimeric mice with bone marrow from IL-7R alpha-deficient mice indicated that IL-7R only partially contributed to intrathymic T cell development. Furthermore, when compared with IL-7R-deficient mice, anti-gamma c chimeric and gamma c-deficient mice exhibited a distinct phenotypic pattern of pro-T cell development. Collectively, these results indicate that several gamma c-sharing cytokines may contribute to T cell development in the thymus and suggest that one of these cytokines may be novel.

Animals↗

PubMed1996

Interleukin-7 receptor alpha is essential for the development of gamma delta + T cells, but not natural killer cells.

Mice that lack a functional gamma c subunit of the receptors for interleukin (IL)-2, IL-4, IL-7, IL-9, and IL-15 display profound defects in lymphoid development. The IL-7/IL-7R system represents a critical interaction for conventional T and B cell development. In this report, the role of IL-7R alpha in the development of lymphoid lineages other than conventional T and B cells was examined. We demonstrate that gamma delta + T cells were absent in IL-7R alpha-deficient mice, whereas the development and function of natural killer cells were normal. Thus, IL-7R alpha function is required for the development of gamma delta + T cells but not natural killer cells.

Animals↗

PubMed1996

Generation of primary tumor-specific CTL in vitro to immunogenic and poorly immunogenic mouse tumors.

This study investigated the generation of primary tumor-specific CTL activity in vitro to several mouse tumors. We report that the development of optimal primary tumor-specific CTL to the P815 mastocytoma, the EL4 thymoma, and the Lewis lung carcinoma is dependent on tumor Ags, on enhancement of T cell costimulation by B7.1, and on exogenous T helper activity in the form of IL-2 and IL-4. A relatively low concentration of IL-2 and IL-4 was required to limit the induction of lymphokine-activated killer cells. In the case of P815, the CTL were directed toward molecularly defined tumor rejection Ags. These primary cultures yielded long term T cell lines that were heterogeneous in fine tumor Ag specificity and in cytokine production.

Animals↗

PubMed1995

The IL-2 receptor gamma c chain does not function as a subunit shared by the IL-4 and IL-13 receptors. Implication for the structure of the IL-4 receptor.

The IL-2 receptor (IL-2R) gamma c subunit is also a component of the receptors for IL-4, IL-7, IL-9, and IL-15. The IL-4R and IL-13R appear to share a common subunit, and gamma c was proposed to be this shared subunit. In this study, we have assessed the relative contribution of gamma c to the mouse IL-4R and IL-13R. The MC/9 mast cell line constitutively expresses gamma c and proliferates to IL-4 and IL-13, but only the response to IL-4 was blocked by anti-gamma c mAbs. After transfection of the IL-4- and IL-13-responsive gamma c-negative B9 plasmacytoma with full length (m gamma) or cytoplasmic-tailless gamma c cDNA (m gamma t), only the proliferative response to IL-4 was affected by the surface expression of these gamma c molecules. The inability of m gamma or m gamma t expression to affect IL-13-induced proliferation by B9 indicates that gamma c does not obviously contribute to the IL-13R and does not function as the shared subunit of the IL-4R and IL-13R. This study suggests that there are two distinct IL-4R, one of which is independent of gamma c.

Animals↗

PubMed1995

Blockade of T- and B-lymphocyte development by antibody to the gamma c subunit of the receptors for interleukins 2, 4, and 7.

Cytokines are important regulators of hematopoesis. Mutations in gamma c, which is a subunit shared by the receptors for interleukin (IL) 2, IL-4, and IL-7, have been causally associated with human X chromosome-linked severe combined immunodeficiency disease. This finding indicates a mandatory role for cytokine receptor signaling at one or more stages of lymphocyte development. To evaluate the cellular level at which gamma c is critical for lymphopoiesis, the effect of monoclonal antibodies to gamma c on the capacity of syngeneic bone marrow cells to reconstitute the hematopoietic compartment of lethally irradiated recipient mice was examined. We show that monoclonal antibody to gamma c blocked lymphocyte development at or before the appearance of pro-B cells and prior to or at the seeding of the thymus by precursor cells while erythromyeloid cell development was normal. These results suggest that one level of lymphocyte development that requires gamma c is a point in hematopoietic cell differentiation near the divergence of lymphopoiesis and erythromyelopoesis.

Animals↗

PubMed1995

Expression and function of the gamma c subunit of the IL-2, IL-4, and IL-7 receptors. Distinct interaction of gamma c in the IL-4 receptor.

IL-2R, IL-4R, and IL-7R share a common subunit referred to as gamma c and the IL-13R has been proposed to contain gamma c as a subunit. In this report we have used two novel mAbs (3E12 and 4G3) to distinct epitopes of mouse gamma c to determine its lymphoid cell distribution and to examine whether gamma c uses similar epitopes to interact with different cytokines and cytokine receptors. FACS analysis revealed that gamma c is expressed in most lymphocytes, myeloid cells, embryonic thymocytes, and lymphoid cell lines. Results from radiolabeled ligand binding studies, biochemical analysis of ligand-receptor cross-linked complexes, and cytokine bioassays indicate that the epitope defined by mAb 4G3 closely defines the IL-7 binding region of gamma c and overlaps the IL-2 binding region of gamma c. These studies also indicate that gamma c interacts with IL-4 in the context of the IL-4R in a manner that is distinct from its role in the IL-2R and IL-7R and suggest that the 3E12 epitope defines a region of gamma c that intimately interacts with the IL-4R. The B9 plasmacytoma, which proliferates in response to IL-4 and IL-13, was shown to not express gamma c. Thus, at least in some circumstances, gamma c is dispensable for signaling via the IL-4R and is not a required subunit of the IL-13R.

Animals↗

PubMed1995

Biochemical identity and characterization of the mouse interleukin-2 receptor beta and gamma c subunits.

Although the mouse IL-2 receptor (IL-2R) beta and gamma c subunits have been identified by molecular cloning, the biochemical identity of these subunits has not yet been established. In the present study, the mouse IL-2R was biochemically characterized from cell lines expressing normal and aberrant IL-2R. Using novel monoclonal antibodies specific for the beta or gamma c subunits, we established that the M(r) of the beta chain is 90,000-100,000 and that of the gamma c subunit is 75,000-80,000. Analysis of transfected EL4 cells that expressed alpha, gamma c, and truncated beta subunits or mutant EL4 cells, which selectively lacked cell surface gamma c, revealed that no other material migrated to a position on SDS-PAGE characteristic of IL-2/IL-2R beta and IL-2/IL-2R gamma c cross-linked complexes, respectively. Thus, the beta and gamma c subunits appear to be the sole IL-2R constituents of these IL-2 cross-linked complexes. The IL-2/IL-2R gamma c, but not the IL-2/IL-2R beta, complex exhibited enhanced mobility after SDS-polyacrylamide gel electrophoresis under nonreducing conditions, suggesting a more compact structure for gamma c as a result of intrachain disulfide bonds. The primary posttranslational modification of the mouse beta and gamma c subunits is N-linked glycosylation. These biochemical studies reconcile past uncertainties concerning the subunit composition of the mouse IL-2R and are consistent with a model of the IL-2R containing only three subunits.

Amidohydrolases↗

PubMed1995

Pleiotropic effects of Bcl-2 on transcription factors in T cells: potential role of NF-kappa B p50-p50 for the anti-apoptotic function of Bcl-2.

Bcl-2 functions to repress apoptosis by regulation of genes which encode proteins required for programmed cell death and by interference with peroxidative damage. We investigated the interrelationship between expression of bcl-2 and regulation of transcription factor DNA binding activities in the 2B4 T cell hybridoma and IL-2-dependent CTLL T cell line. Over-expression of bcl-2 in 2B4 resulted in enhanced basal levels of activator protein (AP)-1, octamer binding factor (Oct)-1, lymphoid enhancer binding factor (LEF)-1, RelA-p50 and NF-kappa B p50-p50 DNA binding activities. After apoptotic signaling, down-regulation of AP-1, NF-AT and Oct-1 binding activities was observed in control 2B4 and CTLL, whereas suboptimal, but higher, levels of these transcription factors were found in bcl-2-transfected cells, potentially promoting cell survival. Furthermore, after apoptotic signaling, expression of bcl-2 led to differential changes of NF-kappa B levels, resulting in a decrease in RelA-p50 and an increase in NF-kappa B p50-p50, altering the ratio of these DNA binding activities such that now p50-p50 markedly predominated in both 2B4-Bcl-2 and CTLL-Bcl-2. Apoptotic signaling in the presence or absence of Bcl-2 resulted in induction of the RelB-p50 heterodimer in 2B4. The changes in NF-kappa B/Rel levels raise the possibility that this family of transcription factors may play an important role in the regulation of apoptosis.

Animals↗

PubMed1994

Formation of high affinity IL-2 receptors is dependent on a nonligand binding region of the alpha subunit.

The IL-2R is composed of three subunits, alpha, beta, and gamma, each of which individually contributes to binding IL-2 in the high affinity IL-2R. The molecular mechanism by which the high affinity IL-2R assembles has not been established. Previous studies have shown that human IL-2R subunits form high affinity hybrid IL-2R when mixed with appropriate mouse IL-2R subunits, but the efficiency of this process is not known. In this study, we produced stable cell lines that expressed hybrid human/mouse IL-2R, comprised of heterologous alpha- and beta-chains, to determine the contribution of individual receptor subunits in stabilizing the high affinity IL-2R. Quantitative ligand binding studies and FACS analysis were performed for EL4J cells that expressed hybrid mouse/human IL-2R, and these data were compared with those of cells that expressed homologous mouse IL-2R subunits. EL4J cells that expressed human beta/mouse alpha formed high affinity IL-2R, comparable with cells that express homologous mouse alpha and beta subunits. In contrast, EL4J cells that expressed homologous mouse IL-2R contained an approximately 10-fold higher level of high affinity IL-2R than did EL4J cells that expressed heterologous human alpha/mouse beta IL-2R. This difference was not accounted for by lower levels of human alpha-chains in these cell lines or lower expression of the mouse IL-2R beta or gamma subunits. These results suggest that amino acid sequences within the alpha subunit, distinct from the IL-2 binding site, are important in the assembly and stabilization of the high affinity IL-2R.

Animals↗

PubMed1994

Regulation of nuclear factor-kappa B and activator protein-1 activities after stimulation of T cells via glycosylphosphatidylinositol-anchored Ly-6A/E.

Cross-linking of glycosylphosphatidylinositol-anchored proteins, including mouse Ly-6A/E, leads to IL-2 secretion and T cell activation, whereas engagement of Ly-6A/E uniquely inhibits IL-2 production induced via TCR. However, little is known concerning the molecular mechanism by which glycosylphosphatidylinositol-anchored proteins regulate IL-2 expression. In this study, we have examined the ability of an anti-Ly-6A/E mAb to regulate transcription factors controlling IL-2 expression. Stimulation of EL4J(Ly-6E).A4 cells with anti-CD3 epsilon or anti-Ly6A/E mAbs induced nuclear factor (NF)-kappa B p65-p50 (RelA/p50) and AP-1 (Fos/Jun) binding activities and increased nuclear factor of activated T cells (NF-AT) activity, whereas octamer-binding factor and NF-Y levels were stable. Cyclic AMP response element binding protein and T cell-specific factor-1 (alpha) activities were selectively enhanced by anti-CD3 epsilon, but not by anti-Ly6A/E, which suggests that signaling via the TCR and Ly-6 were not identical. Costimulation of these cells with both mAbs produced substantially reduced levels of AP-1, NF-AT, and, especially, NF-kappa B p65-p50 whereas cyclic AMP response element binding protein and T cell-specific factor-1(alpha) were induced to a level seen after stimulation by anti-CD3 epsilon. The inducibility of the IL-2 enhancer in vivo and the contribution of individual transcription factors for this induction were assessed with use of reporter chloramphenicol acetyltransferase constructs containing the IL-2 enhancer or oligomerized binding sites for transcription factors. These experiments also demonstrated a key role for NF-kappa B and AP-1 in the transcriptional regulation of the IL-2 gene by TCR- and Ly6A/E-mediated signaling. By using the 2B4.11 T cell hybridoma and a mutated variant, were revealed a crucial role for the zeta-chain in Ly6A/E-mediated activation of NF-kappa B.

Antibodies, Monoclonal↗

PubMed1994

Multiple glycosylphosphatidylinositol-anchored Ly-6 molecules and transmembrane Ly-6E mediate inhibition of IL-2 production.

Cross-linking of Ly-6 molecules on T lymphocytes leads to IL-2 production, whereas costimulation of T cells via Ly-6A/E and the TCR inhibits IL-2 secretion. This study was initiated to determine whether there are unique structural requirements at the level of the Ly-6 molecule for its capacity to activate or block IL-2 production. Functional studies in which transfected EL-4J cells that expressed various Ly-6 proteins or chimeric Ly-6 molecules were used have demonstrated that direct activation of IL-2 secretion or inhibition of anti-CD3-induced IL-2 production occurred after mAb binding to Ly-6A/E, Ly-6C, and Ly-6G and was independent of whether the mAbs bound to amino- or carboxyl-terminal epitopes of Ly-6. The blockade of IL-2 production by costimulation with anti-CD3 and anti-Ly-6 was detected even when the addition of either mAb was temporally delayed for up to 17 h in a reciprocal fashion. Stimulation of mouse Ly-6 proteins expressed in Jurkat cells antagonized PMA/OKT3-induced IL-2 production, thus revealing that the Ly-6 inhibitory pathway is operative in human cells. EL-4J cells were also transfected with a chimeric construct in which the glycosylphosphatidylinositol anchor of Ly-6E was replaced by the transmembrane and a portion of the intracytoplasmic tail of H-2Db. Anti-Ly-6A/E also blocked anti-CD3-induced IL-2 production for these cells, although anti-Ly-6A/E failed to directly induce IL-2 secretion. Thus, anti-Ly-6A/E blockade of IL-2 production is independent of the glycosylphosphatidylinositol anchor of Ly-6E. This finding suggests that there may be aspects of signaling via Ly-6 that are solely dependent on the extracellular amino acid sequence of this protein.

Animals↗

PubMed1994

Regulation of T lymphocyte function by glycosylphosphatidylinositol (GPI)-anchored proteins.

Monoclonal antibody binding to GPI-anchored proteins, e.g. Thy-1 and Ly-6, on the surface of T lymphocytes usually leads to stimulation of interleukin-2 production. This phenomenon is dependent upon expression of the zeta-chain of the T cell receptor complex and requires the GPI anchor. Recent studies suggest that this activation may proceed through a common pathway resulting in tyrosine phosphorylation of intracellular substrates and association of various GPI-anchored proteins to src-family tyrosine kinases. Several models are discussed to explain the signaling capabilities of GPI-anchored proteins. In contrast, under some experimental conditions antibody binding to selected GPI-anchored proteins, i.e. Ly-6A/E and sgp60 (CD48), leads to inhibition of T cell activation. Furthermore, induction of Ly-6A/E expression on CD4 effector T cells correlates with a decreased capacity to secrete IL-2. These latter results suggest that Ly-6A/E may also function to down-regulate an immune response.

Animals↗

PubMed1993

Subsets of activated CD4 T lymphocytes refractory for secretion of IL-2 are distinguished by expression of Ly-6A/E in BALB/c mice.

Ly-6A/E, a cell surface glycosylphosphatidylinositol-anchored protein that modulates T cell activation, is expressed on developing and mature T cells and is tightly regulated in a haplotype- and subset-specific manner. We examined whether Ly-6A/E(low)CD4+ and Ly-6A/E(high)CD4+ T cells comprised functional subsets. Peripheral CD4+ T cells were primed in vitro with Con A in the presence or absence of IL-4 or IFN-gamma, sorted for Ly-6A/E expression, and restimulated to induce lymphokine production. Regardless of priming conditions, IL-2 production by Ly-6A/E(high)CD4+ effector T cells was markedly reduced (mean = 83%) compared to the Ly-6A/E(low)CD4+ subset. The Ly-6A/E(high)CD4+ subset also produced less IFN-gamma than Ly-6A/E(low)CD4+ cells and little IL-4 when primed without exogenous IL-4. In contrast, Ly-6A/E(high)CD4+ cells primed with exogenous IL-4 produced ample IL-4 and IFN-gamma. Interestingly, the difference in IL-2 production between Ly-6A/E(low) and Ly-6A/E(high)CD4+ subsets was not due to a failure of the Ly-6A/E(low)CD4+ cells to become primed because substantially greater amounts of IL-2 and IL-4 were produced by the Con A-pretreated Ly-6A/E(low)CD4+ subset in comparison to unprimed Ly-6A/E(low)CD4+ cells. Taken together these data suggest Ly-6A/E marks a subset of CD4+ effector T cells that differs from Ly-6A/E(low)CD4+ cells by a greatly reduced capacity to produce IL-2 but not IL-4.

Animals↗

PubMed1993

Selective expression of Ly-6G on myeloid lineage cells in mouse bone marrow. RB6-8C5 mAb to granulocyte-differentiation antigen (Gr-1) detects members of the Ly-6 family.

Mouse Ly-6 proteins are characterized by lineage-restricted patterns of expression on lymphoid cells. A mAb (1A8) was produced to Ly-6G, a newly described member of the Ly-6 locus. Based on selective reactivity to cloned Ly-6 gene products expressed in EL4J cells, 1A8 was determined to be specific for Ly-6G. Furthermore, mAb to other Ly-6 specificities did not bind to Ly-6G-transfected EL4J cells, indicating that Ly-6G is distinct from other serologically defined Ly-6 specificities. FACS analysis using 1A8 demonstrated that Ly-6G was expressed in bone marrow but not substantially on other lymphoid tissues, including activated T and B cells. In the bone marrow, Ly-6G expression was primarily restricted to the cells with more forward angle light scatter, which are mostly granulocytes. The RB6-8C5 mAb, previously described to detect a myeloid-restricted Ag (Gr-1) on more differentiated granulocytes, also reacted with Ly-6G- and Ly-6C-transfected EL4J cells. Both 1A8 and RB6-8C5 selectively precipitate a M(r) 21 to 25 kDa, glycosylphosphatidylinositol-anchored protein. Collectively, these data indicate that the Gr-1 Ag is a member of the Ly-6 family and further link expression of individual Ly-6 genes with distinct lineages in mouse bone marrow cells.

Animals↗

PubMed1993

Characterization of two novel Ly-6 genes. Protein sequence and potential structural similarity to alpha-bungarotoxin and other neurotoxins.

Genomic clones cross-hybridizing with Ly-6A.2 cDNA were isolated and characterized for functional Ly-6-related genes. Two new Ly-6 genes, designated Ly-6F.1 and Ly-6G.1, were found to have high nucleotide homology (> or = 70%) and the characteristic four exon gene organization of Ly-6A/E and Ly-6C. By a PCR-based assay, Ly-6G.1 mRNA was readily found in bone marrow, whereas Ly-6F.1 mRNA was not detected in lymphoid tissues. Thus, Ly-6G.1 represents an additional Ly-6 gene with apparent selective expression in hematopoietic cells distinct from Ly-6A/E and Ly-6C. Using the available deduced protein sequence data for mature Ly-6 proteins, searches of the database uncovered an evolutionary relationship of Ly-6 proteins with neurotoxins isolated from snake venoms. The protein sequence conservation between the two groups was selective for, but not limited to, residues in neurotoxins that have been found to be important for their tertiary structures. From this relationship, we propose a neurotoxin-like structure for Ly-6 and Ly-6-related proteins, such as CD59.

Amino Acid Sequence↗