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T D Geppert

Publications and source records attributed to T D Geppert.

At least 19 recordsLinked to original sources
PubMed1999

The cytoplasmic and the transmembrane domains are not sufficient for class I MHC signal transduction.

Class I MHC molecules deliver activation signals to T cells. To analyze the role of the cytoplasmic and the transmembrane (TM) domains of class I MHC molecules in T cell activation, Jurkat cells were transfected with genes for truncated class I MHC molecules which had only four intracytoplasmic amino acids and no potential phosphorylation sites or native molecules or both. Cross-linking either the native or the truncated molecules induced IL-2 production even under limiting stimulation conditions of low engagement of the stimulating mAb. Moreover, direct comparison of transfected truncated and native class I MHC molecules expressed on the same cell revealed significant stimulation induced by cross-linking the truncated molecules, despite low expression. In addition, truncated class I MHC molecules were as able to synergize with CD3, CD2, or CD28 initiated IL-2 production as native molecules. In further experiments, hybrid constructs made of the extracellular portion of the murine CD8 alpha chain and of the TM and the intracytoplasmic domains of H-2Kk class I MHC molecule were transfected into Jurkat T cells. The expression of the transfected hybrid molecules was comparable to that of the native HLA-B7 molecules. Cross-linking the intact monomorphic HLA-A,B,C epitope or the polymorphic HLA-B7 epitope induced IL-2 production upon costimulation with PMA. In contrast, cross-linking the hybrid molecules generated neither an increase in intracellular calcium concentration ([Ca2+]i) nor stimulated IL-2 production. By contrast, cross-linking intact murine class I MHC molecules induced [Ca2+]i, signal and IL-2 production in transfected Jurkat cells. The data therefore indicate that unlike many other signaling molecules, signaling via class I MHC molecules does not involve the cytoplasmic and the TM portions of the molecule, but rather class I MHC signal transduction is likely to be mediated by the extracellular domain of the molecule.

Animals↗
PubMed1997

Structural analysis of class I MHC molecules: the cytoplasmic domain is not required for cytoskeletal association, aggregation and internalization.

The role of the cytoplasmic domain in a variety of the functional activities of class I MHC molecules has not been documented. To address this question, Jurkat cells were transfected with genes for either native class I MHC molecules or constructs in which all but four cytoplasmic amino acids were deleted. Antibody-induced aggregation and internalization of class I MHC molecules were examined by flow cytometry, and cytoskeletal association was determined by analysing the detergent-resistant fraction of FITC-labeled mAb to class I molecules. The results indicate that the truncated class I MHC molecules are comparable to native class I MHC molecules in the ability to move in the plane of the membrane and aggregate, to associate with the cytoskeleton and to undergo mAb-induced internalization at 37 degrees C. Thus, the cytoplasmic domain of class I MHC molecules is not required for these functional activities.

Cell Aggregation↗
PubMed1997

Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) is required for lipopolysaccharide stimulation of tumor necrosis factor alpha (TNF-alpha) translation: glucocorticoids inhibit TNF-alpha translation by blocking JNK/SAPK.

The adverse effects of lipopolysaccharide (LPS) are mediated primarily by tumor necrosis factor alpha (TNF-alpha). TNF-alpha production by LPS-stimulated macrophages is regulated at the levels of both transcription and translation. It has previously been shown that several mitogen-activated protein kinases (MAPKs) are activated in response to LPS. We set out to determine which MAPK signaling pathways are activated in our system and which MAPK pathways are required for TNF-alpha gene transcription or TNF-alpha mRNA translation. We confirm activation of the MAPK family members extracellular-signal-regulated kinases 1 and 2 (ERK1 and ERK2), p38, and Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), as well as activation of the immediate upstream MAPK activators MAPK/ERK kinases 1 and 4 (MEK1 and MEK4). We demonstrate that LPS also activates MEK2, MEK3, and MEK6. Furthermore, we demonstrate that dexamethasone, which inhibits the production of cytokines, including TNF-alpha, significantly inhibits LPS induction of JNK/SAPK activity but not that of p38, ERK1 and ERK2, or MEK3, MEK4, or MEK6. Dexamethasone also blocks the sorbitol but not anisomycin stimulation of JNK/SAPK activity. A kinase-defective mutant of SAPKbeta, SAPKbeta K-A, blocked translation of TNF-alpha, as determined by using a TNF-alpha translational reporting system. Finally, overexpression of wild-type SAPKbeta was able to overcome the dexamethasone-induced block of TNF-alpha translation. These data confirm that three MAPK family members and their upstream activators are stimulated by LPS and demonstrate that JNK/SAPK is required for LPS-induced translation of TNF-alpha mRNA. A novel mechanism by which dexamethasone inhibits translation of TNF-alpha is also revealed.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1996

MEK1 and the extracellular signal-regulated kinases are required for the stimulation of IL-2 gene transcription in T cells.

TCR engagement stimulates the activation of the protein kinase Raf-1. Active Raf-1 phosphorylates and activates the mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase kinase 1 (MEK1), which in turn phosphorylates and activates the MAP kinases/extracellular signal regulated kinases, ERK1 and ERK2. Raf-1 activity promotes IL-2 production in activated T lymphocytes. Therefore, we sought to determine whether MEK1 and ERK activities also stimulate IL-2 gene transcription. Expression of constitutively active Raf-1 or MEK1 in Jurkat T cells enhanced the stimulation of IL-2 promoter-driven transcription stimulated by a calcium ionophore and PMA, and together with a calcium ionophore the expression of each protein was sufficient to stimulate NF-AT activity. Expression of MEK1-interfering mutants inhibited the stimulation of IL-2 promoter-driven transcription and blocked the ability of constitutively active Ras and Raf-1 to costimulate NF-AT activity with a calcium ionophore. Expression of the MAP kinase-specific phosphatase, MKP-1, which blocks ERK activation, inhibited IL-2 promoter and NF-AT-driven transcription stimulated by a calcium ionophore and PMA, and in addition, MKP-1 neutralized the transcriptional enhancement caused by active Raf-1 and MEK1 expression. We conclude that the MAP kinase signal transduction pathway consisting of Raf-1, MEK1, and ERK1 and ERK2 functions in the stimulation IL-2 gene transcription in activated T lymphocytes.

Amino Acid Sequence↗
PubMed1995

The MEK kinase activity of the catalytic domain of RAF-1 is regulated independently of Ras binding in T cells.

Deletion of the amino-terminal domain of Raf-1, which contains the Ras-binding region, results in the constitutive activation of the liberated Raf-1 catalytic domain in fibroblast cell lines. We demonstrate that the MEK kinase activity of the isolated Raf-1 catalytic domain, Raf-BXB, is not constitutively active, but is regulated in Jurkat T cells. Raf-BXB is activated by engaging the antigen receptor-CD3 complex, or treating cells with phorbol myristate acetate or okadaic acid. Increasing intracellular cAMP inhibits Raf-1 activation stimulated by phorbol myristate acetate, but not the activation of Raf-BXB. Serine 621, but not serine 499, is essential for Raf-BXB MEK kinase activity. Because Raf-BXB does not bind Ras, the data establishes a Ras-independent signal in directly regulating the activity of the Raf-1 catalytic domain.

Amino Acid Sequence↗
PubMed1994

A requirement for extracellular signal-regulated kinase (ERK) function in the activation of AP-1 by Ha-Ras, phorbol 12-myristate 13-acetate, and serum.

The role of ERK-1 and ERK-2 in wild-type (wt) Ha-Ras, phorbol 12-myristate 13-acetate (PMA), and serum-induced AP-1 activity was studied. Microinjection of ERK-specific substrate peptides inhibited the induction of AP-1 activity by all three stimuli, whereas a control peptide had no effect. By using eukaryotic expression constructs encoding wt ERK-1 and kinase-deficient mutants of ERKs 1 and 2, it was found that ERK-1 and ERK-2 activities are required for AP-1 activation stimulated by either wt Ha-Ras, PMA, or serum. Overexpression of ERK-1 augmented wt Ha-Ras stimulation of AP-1, while having no effect upon PMA or serum stimulation. Overexpression of either kinase-deficient ERK-1 or kinase-deficient ERK-2 partially inhibited AP-1 activation by wt Ha-Ras but had no effect on PMA or serum-induced activation. Coexpression of both interfering mutants abolished AP-1 induction by wt Ha-Ras, PMA, or serum. We conclude that ERKs are necessary components in the pathway leading to the activation of AP-1 stimulated by these agents.

Amino Acid Sequence↗
PubMed1994

Lipopolysaccharide signals activation of tumor necrosis factor biosynthesis through the ras/raf-1/MEK/MAPK pathway.

BACKGROUND: Lipopolysaccharide (LPS) is known to activate macrophages, causing the release of toxic cytokines that may provoke inflammation and shock. One of the most important and best studied of these cytokines is tumor necrosis factor (TNF). Details of the signaling pathway leading to TNF biosynthesis remain unclear. The pathway is branched in the sense that TNF gene transcription and TNF mRNA translation are both strongly stimulated by LPS. Recent evidence has indicated that MAP kinase homologs become phosphorylated in LPS-stimulated cells, suggesting their possible involvement in signal transduction. We sought to test this hypothesis. MATERIALS AND METHODS: Measurements of LPS-induced MEK and ERK2 activity were undertaken in LPS-sensitive and LPS-insensitive cells. Transfection studies, in which dominant inhibitors of ras and raf-1 were used to block signaling to the level of MAP kinase, were carried out in order to judge whether the TNF gene transcription and TNF mRNA translation are modulated through this pathway. RESULTS: In RAW 264.7 mouse macrophages, both ERK2 and MEK1 activity are induced by LPS treatment. In the same cell line, dominant negative inhibitors of ras and raf-1 block LPS-induced activation of the TNF promoter, as well as derepression of the translational blockade normally imposed by the TNF 3'-untranslated region. A constitutively active form of raf-1 (raf-BXB) was found to augment, but not replace, the LPS signal. In LPS-insensitive cells (RAW 264.7 x NIH 3T3 fusion hybrid cells and primary macrophages derived from C3H/HeJ mice), ERK2 activity was found to be refractory to induction by LPS. CONCLUSIONS: The ras/raf-1/MEK/MAPK pathway is chiefly responsible for transduction of the LPS signal to the level of the TNF gene and mRNA. raf and raf-1 lie upstream from (or actually represent) the physical branchpoints of the transcriptional and translation activation signals generated by LPS. The lesions that prevent LPS signaling in macrophages from C3H/HeJ mice, or in RAW 264.7 x NIH 3T3 fusion hybrid cells, occupy a proximal position in the signaling pathway.

3T3 Cells↗
PubMed1993

Regulation and properties of extracellular signal-regulated protein kinases 1 and 2 in vitro.

Extracellular signal-regulated protein kinases (ERK) 1 and 2 and mutants of each were expressed in bacteria with a hexahistidine tag and purified using nickel-chelate chromatography. Basal activity of wild type ERK2 was approximately 2 nmol/min/mg. Self-catalyzed phosphorylation occurred in vitro on the major physiological site of tyrosine phosphorylation in an intramolecular reaction. Rabbit muscle ERK activator activated ERK2 500-1000-fold up to a specific activity (approximately 2 mumol/min/mg) approximating that of ERK1 purified from stimulated cells (Boulton, T.G., Gregory, J.S., and Cobb, M.H. (1991) Biochemistry 30, 278-286). ERK1 could also be activated by the ERK activator to the same extent. Mutants lacking the major site of tyrosine phosphorylation were autophosphorylated at a greatly reduced rate and were no longer highly activated by the ERK kinase. Mutants lacking the major site of threonine phosphorylation were autophosphorylated at the same or an enhanced rate, but the kinase activity of these mutants depended on the residue used to replace the threonine. Replacement by glutamate rendered the kinase capable of being activated by ERK activator, while replacement by alanine did not. Thus, the carboxyl group of glutamate can provide at least some of the features introduced by phosphothreonine in activated ERKs.

Amino Acid Sequence↗
PubMed1993

Raf-1 is required for T cell IL2 production.

Engagement of the T cell receptor/CD3 complex activates the serine/threonine kinase, Raf-1, but the physiologic consequences of its activation have not been determined. The effects of Raf-1 on interleukin 2 (IL2) production in T cells were examined using activated and inhibitory forms of Raf-1. A truncated active form of Raf-1 was expressed constitutively from the metallothionein promoter in a malignant T cell line, Jurkat. Treatment of the cells with zinc and cadmium greatly increased active Raf-1 expression. This increase in Raf-1 expression allowed antibodies to CD3 and to CD28 to stimulate IL2 production in the absence of phorbol myristate acetate (PMA) and enhanced IL2 production stimulated by these antibodies in the presence of PMA. The action of active Raf-1 was to increase IL2 gene transcription as it enhanced transcription of a reporter gene linked to IL2 promoter. Finally, the dominant negative form of Raf-1 inhibited transcription directed by the IL2 promoter that was induced by the mitogen phytohemagglutinin (PHA) and PMA. We conclude that Raf-1 activity is necessary for IL2 gene transcription and secretion. These data indicate a role for Raf-1 in the immune response.

CD28 Antigens↗
PubMed1992

Comparison of the capacity of murine and human class I MHC molecules to stimulate T cell activation.

The mechanism underlying the apparent differences in the capacity of murine and human class I MHC molecules to function as signal transducing structures in T cells was examined. Cross-linking murine class I MHC molecules on splenic T cells did not stimulate an increase in intracellular calcium ([Ca2+]i) and failed to induce proliferation in the presence of IL-2 or PMA. In contrast, modest proliferation was induced by cross-linking class I MHC molecules on murine peripheral blood T cells or human class I MHC molecules on murine transgenic spleen cells, but only when costimulated with PMA. Moreover, cross-linking murine class I MHC molecules or the human HLA-B27 molecule on T cell lines generated from transgenic murine splenic T cells stimulated only modest proliferation in the presence of PMA, but not IL-2. On the other hand, cross-linking murine class I MHC molecules expressed by the human T cell leukemic line, Jurkat, transfected with genes for these molecules, generated a prompt increase in [Ca2+]i, and stimulated IL-2 production in the presence of PMA. The results demonstrate that both murine and human class I MHC molecules have the capacity to function as signal transducing structures, but that murine T cells are much less responsive to this signal.

Amino Acid Sequence↗
PubMed1992

Extracellular signal-regulated kinases in T cells. Anti-CD3 and 4 beta-phorbol 12-myristate 13-acetate-induced phosphorylation and activation.

Extracellular signal-regulated kinases (ERK) 1 and 2 are growth factor- and cytokine-sensitive serine/threonine kinases that are known to phosphorylate microtubule-associated protein 2 and myelin basic protein. The current studies examined whether ERK1 and/or ERK2 was present in T cells and whether they were phosphorylated and activated as a consequence of T cell activation. The data demonstrated that both ERK1 and ERK2 were present in Jurkat cells and peripheral blood T cells. In T cells, ERK2 was more prevalent than ERK1. The concentrations of ERK1 and ERK2 were not altered by stimulating the cells for 16 h with immobilized anti-CD3 mAb or anti-CD3 mAb and phorbol myristate acetate. mAb to CD3 and phorbol myristate acetate stimulated an increase in ERK1 and ERK2 MBP kinase activity. Anti-CD3 mAb triggered an increase their phosphate content which was detectable at 2 min but reached a maximum at 5 min. A portion of the increase in phosphate was caused by an increase in phosphotyrosine. We also examined the rate of ERK2 degradation. ERK2 was stable for up to 36 h, and its degradation was unaffected by the activation state of the cells. The data demonstrate that ERK1 and ERK2 are part of an anti-CD3 mAb-stimulated signal transduction cascade that is downstream of protein kinase C and, therefore, suggest that these kinases play an important role in T cell activation.

Amino Acid Sequence↗
PubMed1992

Extracellular signal-regulated kinases in T cells: characterization of human ERK1 and ERK2 cDNAs.

Extracellular signal-regulated kinases 1 and 2 are growth factor-sensitive serine/threonine kinases. cDNAs for both human kinases were isolated and sequenced. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 1 were 88% and 96% identical, respectively, to the homologous rat sequences. The nucleic acid and deduced protein sequences of human extracellular signal-regulated kinase 2 were 90% and 98% identical, respectively, to the corresponding rat sequences. A human extracellular signal-regulated kinase 2 specific probe was used to demonstrate that the mRNA for this kinase was present in T cells and did not change with activation. The deduced protein sequences of both human kinases were greater than 95% identical to two Xenopus kinase sequences, indicating that these enzymes are highly conserved across species.

Adult↗
PubMed1992

Engagement of class I major histocompatibility complex molecules by cell surface CD8 delivers an activation signal.

Recent evidence has demonstrated that cross-linking class I major histocompatibility complex (MHC) molecules on human T cells with monoclonal antibodies (mAb) triggers T cell activation. The only known natural ligand for MHC class I molecules is CD8. Therefore, the possibility that CD8+ T cells might provide activation signals to other T cells by engaging MHC class I molecules was examined by culturing CD4+ peripheral blood T cells with Chinese hamster ovary cells (CHO) cells that had been transfected with the alpha chain or alpha and beta chains of CD8 and assessing interleukin (IL)-2 production. CD4+ T cells did not secrete IL-2 when cultured alone, with control or CD8+ CHO cells. In contrast, CD4+ T cells produced IL-2 when cultured with CD8+ CHO cells and co-stimulated with phorbol myristate acetate (PMA) or mAb to CD3 or CD28. PMA stimulated substantially less IL-2 when control CHO cells were employed and the mAb to CD3 and CD28 did not stimulate IL-2 production in the presence of control CHO cells. The co-stimulatory activity of CD8+ CHO cells was completely eliminated by mAb to CD8 or MHC class I molecules. The data demonstrate that CD8 can interact with MHC class I molecules expressed on T cells and deliver a costimulatory signal that increases IL-2 production. Thus, engagement of MHC class I molecules by its natural ligand, CD8, provides an activation signal to T cells. Under some circumstances, such interactions may amplify the responses of T cells.

Animals↗
PubMed1992

Modulatory effect of aggregating the CD3 molecular complex on T cell activation.

The role of cross-linking the TCR/CD3 complex in the induction of T cell activation was examined using human peripheral blood T cells and the Jurkat leukemic T cell line. IL-2 production was induced from these cells by pulsing them with mAb to CD3 and costimulating with phorbol myristate acetate (PMA). Cross-linking the anti-CD3 mAb with soluble goat anti-mouse immunoglobulin (GaMIg) markedly inhibited IL-2 production by these cells. Soluble GaMIg did not induce a generalized inhibition of IL-2 production as it was required for responses induced by mAb to class I MHC molecules. In addition, cross-linking anti-CD3 mAb with GaMIg did not inhibit IL-2 production induced by PMA and ionomycin. Inhibition of IL-2 production induced by soluble GaMIg reflected diminished accumulation of mRNA for IL-2. By contrast, immobilized GaMIg was a potent stimulus for IL-2 production by T cells pulsed with anti-CD3 mAb and costimulated with PMA. Cross-linking anti-CD3 with soluble GaMIg induced enhanced aggregation of the ligated molecules, but it did not alter the profile of the change in intracellular calcium induced. To determine whether cross-linking of mAb played a role in inducing IL-2 production as well as in limiting responsiveness, F(ab) fragments were employed. F(ab) fragments of anti-CD3 mAb failed to induce IL-2 production by PMA costimulated Jurkat cells. However, cross-linking of anti-CD3 F(ab)-pulsed Jurkat cells with low concentrations of soluble GaMIg induced IL-2 production in the presence of PMA, whereas higher concentrations suppressed responses. The data indicate that induction of IL-2 production requires aggregation of the TCR/CD3 complex, whereas excessive cross-linking diminishes the induction of IL-2 production. Moreover, the results indicate that various biologic activities of the CD3 molecular complex, including aggregation, signaling capability, and the ability to induce IL-2 gene transcription, are differentially affected by cross-linking.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1991

Association of various T cell-surface molecules with the cytoskeleton. Effect of cross-linking and activation.

The association of various surface molecules with the cytoskeleton in resting peripheral blood T cells was examined by assaying the capacity of detergent to solubilize them. Cytoskeletal association was assessed by staining T cells with a fluorescein-conjugated mAb, resuspending the cells in buffer with or without the nonionic detergent, NP-40, and determining the capacity of the detergent to remove the mAb from the cell surface by using flow microfluorimetry. MAb to CD3, the TCR, and CD45 were completely removed from the cell surface by detergent. In contrast, 7 to 50% of mAb to CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules were resistant to detergent solubilization, demonstrating that a fraction of these molecules was constitutively associated with the cytoskeleton. The effect of cross-linking these molecules with a mAb and a secondary goat anti-mouse Ig was also examined. Cross-linking CD3 or the TCR induced cytoskeletal association of these molecules. In addition, cross-linking increased the fraction of CD2, CD4, CD8, CD11a/CD18, CD44, and class I MHC molecules that was associated with the cytoskeleton. In contrast, cross-linking CD45 did not induce an association with the cytoskeleton. The effect of T cell activation on the cytoskeletal association of these molecules was also examined. Stimulation of T cells with ionomycin and PMA greatly increased the expression of CD2 and CD44 without increasing the number of molecules associated with the cytoskeleton. Stimulation with PMA alone had no effect on the expression of CD2 or CD44, but was found to decrease the percentage of these molecules associated with the cytoskeleton. Stimulation with ionomycin and PMA increased both the expression of class I MHC molecules and the number of molecules associated with the cytoskeleton proportionally. Finally, stimulation with ionomycin and PMA decreased CD3 expression, but increased the number of CD3 molecules associated with the cytoskeleton. The data establish a pattern of cytoskeletal association of T cell-surface molecules that is a characteristic of each individual molecule and can be altered by cross-linking. Moreover, the results indicate that the association of various T cell surface molecules with the cytoskeleton is a dynamic process that varies with the state of activation and or differentiation of the cells.

Adult↗
PubMed1991

Immobilized anti-CD3-induced T cell growth: comparison of the frequency of responding cells within various T cell subsets.

Human T cells can be divided into subsets based on the expression of CD29, CD45RA, CD45RO, LFA-3, or CD11a. It has been suggested that the subset of CD4+ T cells that expresses high densities of CD29, CD11a, CD45RO, and LFA-3 contains "memory" T cells, whereas the subset of cells that expresses CD45RA contains "naive" T cells. In order to obtain a more complete picture of the functional capacities of human naive and memory CD4+ and CD8+ T cell subsets, highly purified T cells were activated with a uniform stimulus and responses were examined in bulk cultures and under limiting dilution conditions. T cell activation was achieved with an immobilized mAb to the CD3 molecular complex, 64.1. In bulk cultures, immobilized 64.1 stimulated a vigorous response. Moreover, the number of cells entering the cell cycle, the magnitude of the [3H]thymidine incorporation, and the growth of the cells over 6 days in culture by naive and memory CD4+ T cells was comparable. To delineate the frequency of responsive cells in each subset more precisely, cells were cultured with immobilized 64.1 at limiting dilution and the precursor frequency of responding cells was assessed by examining wells microscopically for visible growth. Immobilized 64.1 was able to induce some T cells from each subset to grow in the complete absence of AC, when exogenous IL2 was present. The number of responding CD4+ and CD8+ cells was comparable. The percentage of naive cells responding in each population was approximately three times greater than the frequency of memory cells. IL4 could also support the growth of immobilized 64.1-activated CD4+ T cells, but the frequency of responding cells was much lower than that supported by IL2. The vast majority of the IL-4 responsive CD4+ cells resided within the naive cell subset. The data indicate that the response of CD4+ and CD8+ naive and memory T cell subsets to immobilized anti-CD3 depends on the density of responding cells. Naive T cells have an enhanced capacity to grow when cultured in the absence of other T cells or accessory cells. This ability may facilitate their expansion during primary immune responses.

Antibodies, Monoclonal↗