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Biomedical subjects

E R Podack

Publications and source records attributed to E R Podack.

At least 55 records · Page 3Linked to original sources
PubMed1996

CD80 (B7.1) and CD54 (intracellular adhesion molecule-1) induce target cell susceptibility to promiscuous cytotoxic T cell lysis.

Overexpression of B7 and intracellular adhesion molecule-1 (ICAM-1) on syngeneic cells triggered MHC-unrestricted lysis by MHC-restricted CD8+ CTL without TCR/CD3 engagement. Both CD28/B7 and LFA-1/ICAM-1 interactions were required for MHC-nonrestricted cytotoxicity. Cytotoxicity was measured in 4-h and 16- to 18-h cytotoxicity assays. B7-ICAM-1 overexpression triggered perforin- and Fas ligand-mediated lysis, while TNF-alpha was not elicited in MHC-unrestricted cytotoxicity. These results suggest that target cells may elicit MHC-unrestricted lysis from CTL through up-regulation of membrane adherence and costimulatory receptors.

Animals↗
PubMed1996

Perforin, Fas ligand, and tumor necrosis factor are the major cytotoxic molecules used by lymphokine-activated killer cells.

Lymphokine-activated killer (LAK) cells generated from perforin knockout mice possess significantly reduced cytotoxicity against a panel of tumor target cell lines, with some tumor cells being lysed exclusively by the perforin pathway. LAK cells are also capable of Fas ligand-mediated cytotoxicity. LAK cells generated from mice deficient in both perforin and Fas ligand (PKO/gld) were not cytolytic in short term cytotoxicity assays, demonstrating that perforin and Fas ligand are required for acute target cell lysis. However, PKO/gld LAK cells were cytotoxic in long term cytotoxicity assays against TNF-sensitive tumor lines, and this cytotoxicity was completely inhibited by neutralizing TNF Abs. This potent TNF cytotoxicity has not been fully appreciated previously because of the presence of dominant-acting perforin and Fas ligand in acute tumor cell lysis. TNF-based cytotoxicity by PKO/gld LAK was both soluble and membrane bound, and both forms of TNF were constitutively expressed. Thus, LAK cells are armed with at least three cytotoxic molecules: perforin, Fas ligand, and TNF.

Animals↗
PubMed1996

The role of cell-mediated cytotoxicity in acute GVHD after MHC-matched allogeneic bone marrow transplantation in mice.

The role of cell-mediated cytotoxicity in the complex pathophysiology of graft-versus-host disease (GVHD) has remained poorly defined for several decades. We transplanted T cells from Fas-ligand (FasL)-defective and perforin-deficient mutant donor mice into lethally irradiated MHC-matched allogeneic recipient mice to characterize the role of cell-mediated cytotoxicity in GVHD. Although recipients of allogeneic FasL-defective donor T cells underwent severe GVHD-associated cachexia, they exhibited only minimal signs of hepatic and cutaneous GVHD pathology. Recipients of perforin-deficient allogeneic donor T cells developed signs of acute GVHD, but the time of onset was significantly delayed. These findings demonstrate that Fas-mediated anti-recipient cytotoxicity may be critical for the development of hepatic and cutaneous GVHD, but is not required for GVHD-associated cachexia. In addition, perforin-mediated anti-recipient cytotoxicity appears to play an important role in the kinetics of GVHD pathophysiology, but is not required for GVHD-associated tissue damage.

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↗
PubMed1996

Perforin expression in peripheral blood lymphocytes in rejecting and tolerant kidney transplant recipients.

Perforin (P) is a cytolytic molecule expressed in the granules of cytolytic T cells and natural killer cells. Although cytotoxic cells have been implicated in graft rejection, no prospective clinical study has been published that examines the dynamics of perforin expressing cells in peripheral blood lymphocytes of transplanted patients. The cytofluorimetric assay developed in our laboratory previously for the simultaneous detection of intracellular perforin together with cell surface molecules was used for posttransplantation monitoring of patients, for the assessment of the efficiency of immunosuppressive treatment, and for the prediction of acute kidney transplant rejection and the stability of tolerance to long lived kidney transplants. Immunosuppression for the purpose of allotransplantation causes a decline in the number of perforin-expressing cells in peripheral blood. In contrast, in patients with clinical signs of acute rejection, the total number of perforin-expressing lymphocytes was increased in comparison with nonrejecting patients. Analyzing perforin-expressing subsets, rejection crises were accompanied by a relative decrease of perforin expression in the CD4+ subpopulation while increasing in the CD8+ subset. In the CD56+ and CD16+ NK subpopulations changes in perforin expression were mixed. In nonrejecting patients the ratio of perforin expression in CD4+ cells was high compared with CD8+ cells. Intensive therapy of acute rejection episodes with high doses of corticosteroids (methylprednisolonet [Solumedrol] bolus) strongly and significantly decreased the percentage of both, the subpopulations of perforin-positive T cells and the subpopulation of CD56+P+ NK cells. The lowest level of perforin expression, including low frequencies of perforin among CD8+ and CD4+ cells, was found in the group of patients tolerating transplanted kidneys for several years. These changes in perforin protein expression in peripheral blood can be used to discriminate between immunosuppressed patients who are immunologically quiescent and those who undergo transplant rejection. Our results confirm the hypothesis that cytotoxicity mediated by perforin may be an important effector mechanism in the rejection of allografted kidneys.

Biomarkers↗
PubMed1996

Structure and expression of murine CD30 and its role in cytokine production.

Murine CD30 cDNA predicts a protein of 498 amino acids with homology to the TNF receptor family of proteins characterized by repeated cysteine-rich motifs in the extracellular domain. Murine CD30, although homologous to human CD30, has a 90 amino acid gap in an extracellular region that appears to be duplicated in human CD30. Murine CD30 cDNA was shown to be functional through the production of a soluble murine Ig fusion protein (CD30-Ig) that was active in binding to cells that expressed CD30 ligand. CD30-Ig also served as an immunogen for the production of hamster anti-mouse CD30 mAbs, which recognized both CD30 expressed by murine lymphocytes and CD30 expressed by cells transfected with murine CD30 cDNA. CD30 mRNA is highly expressed in the thymus and in activated spleen cells, but not in other tissues tested. In anti-CD3-activated spleen cells, CD30 ligand is expressed primarily by CD4+ T cells, with peak expression at days 1 and 2, whereas CD30 is expressed primarily by CD8+ T cells, with peak expression on days 4 and 5. Stimulation of CD30 by plate-bound anti-CD30 directly signaled for IL-5 but not IFN-gamma production by CD30+ CTL lines. These studies demonstrate that CD30 directs cytokine secretion and suggest that CD30 signaling may be pivotal in the pattern of cytokine production by T cells.

3T3 Cells↗
PubMed1996

Rapid induction of tumor necrosis factor cytotoxicity in naive splenic T cells by simultaneous CD80 (B7.1) and CD54 (ICAM-1) co-stimulation.

The co-expression of B7.1 (CD80) and intercellular adhesion molecule (ICAM)-1 (CD54) on tumor cells can induce tumor immunity and immunological memory. We show here that the non-immunogenic tumor lines Lewis lung carcinoma and B16F10 melanoma, co-transfected with B7.1 and ICAM-1, induced cytotoxic levels of membrane tumor necrosis factor (TNF) on naive syngeneic T cells within 24 h. Membrane TNF expression, primarily on CD4 cells, was responsible for tumor cell lysis by naive spleen cells and could be completely abolished by anti-TNF antiserum. It is suggested that the strong induction of TNF cytotoxicity may be important in the establishment of tumor immunity.

Animals↗
PubMed1996

Bcl-2 protects against Fas-based but not perforin-based T cell-mediated cytolysis.

Fas ligand and perforin are the two key effector mechanisms in T cell-mediated cytotoxicity. These molecules mediate cytolysis of target cells by membrane damage and apoptosis. bcl-2 is known to protect cells against apoptosis induced by many stimuli including growth factor removal. However bcl-2's effect on Fas ligand and perforin-induced lysis has not been studied extensively. We investigated the effect of overexpression of bcl-2 alone, Fas alone or their combined overexpression on lysis of a commonly used target, P815, by perforin-sufficient, Fas ligand-sufficient and perforin-deficient or Fas ligand-deficient, allospecific cytotoxic T lymphocytes (CTL). Wild-type P815 are susceptible to lysis by perforin-sufficient CTL, regardless of the presence or absence (gld) of Fas ligand, but are poorly lysed by perforin-deficient CTL. Fas transfection of P815 makes target cells highly susceptible to lysis by both perforin-sufficient and -deficient CTL, indicating the presence of the Fas ligand-mediated cytotoxicity on both types of CTL. Co-transfection of P815-fas with bcl-2 abolishes their increased susceptibility to Fas-mediated lysis, even in the face of Fas overexpression on the cell membrane. The protective effect of bcl-2 against cell lysis is evident with perforin-deficient CTL as effector cells or when perforin activity is eliminated by the absence of extracellular calcium in perforin-sufficient CTL. bcl-2 overexpression by P815, however, does not protect against CTL lysis by the perforin pathway, regardless of Fas overexpression, as demonstrated by fas ligand mutated gld and wild-type perforin-sufficient CTL. Therefore bcl-2 can protect P815 target cells against Fas-mediated lysis when triggered by the Fas ligand on CTL, but not against perforin-mediated lysis.

Animals↗
PubMed1995

Transgenic control of perforin gene expression. Functional evidence for two separate control regions.

Perforin is a pore-forming effector molecule of CTL and NK cells. To characterize perforin gene expression and its transcriptional control mechanisms in vivo, expression of a cell surface tag, i.e., human CD4, was driven by 5.1 kb of the murine perforin 5' flanking and promoter region in transgenic mice. Six out of seven transgenic lines expressed the perforin-tag hybrid gene at low to intermediate levels, depending on the integration site. Tissues not yet reported to contain perforin-expressing lymphocytes were identified. Transgene expression occurred in all cells that physiologically are able to express perforin, i.e., in T cells and NK cells, and in some T cells that normally may express little or no perforin. At the whole organ level, significant amounts of transgenic mRNA and endogenous perforin mRNA were co-expressed in the lymphoid organs, as well as in the lung, the ileum, the oviduct/uterus, and the bone marrow. At the single cell level, the perforin tag was present on NK cells and on CD8+, as well as on CD4+ T cells. Also targeted were Thy-1.2+ gamma delta T cells, but not Thy1.2- gamma delta T cells, B cells, nor monocytes. During thymic T cell development, transgene expression occurred in double negative (CD4-CD8-) thymocytes and was detected at all subsequent stages, but exceeded the expression levels of the endogenous gene in the thymus. In conclusion, the analyzed perforin 5' flanking and promoter region contains important cis-acting sequences that restrict perforin expression to T cells and NK cells, and therefore provides a unique tool for manipulating T cell and/or NK cell-mediated immune responses in transgenic mice. On the other hand, the normal control of perforin gene expression involves at least one additional negative control mechanism that was not mediated by the transgenic promoter and upstream region. This control restricts perforin gene expression in thymically developing T cells and in most resting peripheral T cells, but can be released upon T cell activation.

Animals↗
PubMed1995

Functional significance of two cytolytic pathways of cytotoxic T lymphocytes.

Cytotoxic lymphocytes are equipped with several effector molecules mediating cytotoxicity. Two pathways have been molecularly defined and functionally characterized through the analysis of deficiency states. It is evident from these results that lysis by granule exocytosis serves primarily to eliminate exogenous and endogenous hazards such as virally infected and transformed cells. In contrast, lysis by induced suicide in the fas pathway is used to maintain homeostasis of rapidly expanding and contracting cell populations including T cells.

Animals↗
PubMed1995

Execution and suicide: cytotoxic lymphocytes enforce Draconian laws through separate molecular pathways.

With the ability to analyze cytotoxicity in animals deficient in effector molecules, the debate over the biological significance of individual effector pathways is finally being settled. For CD8+ cytotoxic T lymphocytes, the two primary cytotoxic pathways are mediated by the Fas ligand and perforin. The dichotomy between the two killing pathways is mirrored in the dichotomy between their biological roles: the primary function of the Fas ligand is the control of normal cell renewal by inducing programmed cell death of actively proliferating cells via the Fas pathway. This type of cell death is part of the normal endogenous homeostatic mechanism responsible for maintaining rapidly changing cell populations such as clonally expanding and contracting T cells. In contrast, the biological function of perforin and associated granule proteins is the killing and elimination of parasitized, non-compliant cells that arise as part of pathophysiological processes and may resist lysis pathways signalled to induce apoptosis. Thus, the main function of perforin and granzymes is the maintenance of immune surveillance against both exogenous and endogenous hazards.

Animals↗
PubMed1995

Construction of a novel bovine papillomavirus vector without detectable transforming activity suitable for gene transfer.

Bovine papillomavirus type 1 (BPV-1)-derived vectors may be useful for gene therapy because of their episomal maintenance at intermediate to high copy number and stable, high-level expression of gene products. To increase the safety of BPV-1 for human trials, the transforming early genes E5, E6, and E7 were deleted and a new vector, B45-Neo, was established and its transforming potential, episomal maintenance, and cDNA expression determined. Deletion of E5, E6, and E7, caused a decrease of the copy number to 80 in 3T3 fibroblasts when B45-Neo was compared to the parent vector that supported more than 1,000 copies per cell. No significant difference in the copy number, which ranged between 13 and 30 per cell, was detected in other cell lines of murine or human origin. The episomal maintenance of B45 and its ability to express cDNA was retained. B45-Neo, in contrast to BMG-Neo, however, was unable to transform NIH-3T3 and C1271 cells in soft agar colony assays. Unlike BMG-Neo, B45-Neo did not cause morphological changes in 3T3 and C1271 cells characteristic for transformation. It is concluded that B45-Neo is an efficient expression vector without detectable transforming activity and may be useful and safe for human gene therapy trials.

3T3 Cells↗
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↗