Biomedical subjects

G Dennert

Publications and source records attributed to G Dennert.

At least 37 records · Page 2Linked to original sources

PubMed1994

Acute rejection of marrow grafts in mice. Dependence on and independence of functional TCR in the rejection process.

The question of how irradiated mice acutely reject marrow grafts has remained controversial, and evidence in support of T cell- and natural killer cell-mediated rejection mechanisms has been provided. Here we show in support of previous data that CB17 severe combined immunodeficiency mice acutely reject allogeneic marrow but the specificity of rejection cannot be mapped within the MHC. It is shown that a similar rejection specificity is also expressed in normal CB17 mice and that it is caused by CD3- or CD3+ effector cells that do not utilize TCR. In search of TCR-independent rejection mechanisms in other mouse strains, use is made of TCR transgenic mice expressing a defect in recognizing H-2Dd. It is shown that, although marrow graft rejection is impaired in these mice, pointing to participation of TCR in the rejection process, residual resistance does exist. This resistance maps to the MHC, cannot be shown to involve TCR, and appears to be expressed by NK1+ CD3+ cells. It is concluded that acute marrow graft rejection in normal mice can be mediated by both TCR-mediated and NK cell receptor-dependent effector mechanisms, depending on the particular mouse strains.

Acute Disease↗

PubMed1994

TCR cross-linking induces CTL death via internal action of TNF.

TCR-mediated stimulation can result in either activation or apoptosis, raising the question of what the mechanisms of these opposing effects might be. Here we demonstrate that microinjection of Ab specific for TNF inhibits activation-induced cell death, implicating TNF in anti-CD3-induced apoptosis. Induction of CTL in the presence of antisense TNF oligodeoxynucleotides inhibits TNF secretion and cell death after stimulation with anti-CD3. Incubation of cells with IL-2 for 48 h before stimulation protects from anti-CD3-induced death, whereas the presence of IL-2 during anti-CD3 treatment is without effect.

Animals↗

PubMed1993

Suppression of stress protein GRP78 induction in tumor B/C10ME eliminates resistance to cell mediated cytotoxicity.

Tumor cells undergo self-destruction when incubated with cytotoxic T-cells (CTL) consistent with the observation that suppression of target protein synthesis causes resistance to apoptosis. Resistance to CTL is also induced by stress, suggesting that pathways exist suppressing apoptosis. Here we examine whether stress induced lysis resistance to CTL and tumor necrosis factor alpha involves stress proteins GRP78 and GRP94. We show that inhibition of GRP78 synthesis by transfection of cells with grp78 antisense vector pRSV-78WO leads to inability to induce resistance to CTL or tumor necrosis factor alpha. Resistance induced in untransfected cells is reversible upon stress removal and correlates with GRP78 rephosphorylation, consistent with the notion that phosphorylated GRP78 is nonfunctional. The possibility that GRP78 plays a role in defense against CTL mediated apoptosis is supported by the finding that CTL but not CD4+ cells express a high level of unphosphorylated GRP78.

Animals↗

PubMed1993

The development of autoimmunity in C57BL/6 lpr mice correlates with the disappearance of natural killer type 1-positive cells: evidence for their suppressive action on bone marrow stem cell proliferation, B cell immunoglobulin secretion, and autoimmune symptoms.

F1 hybrid mice are able to acutely reject parental marrow grafts, a phenomenon that is due to natural killer type 1-positive (NK1+) cells. Circumstantial evidence had suggested that the antigenic determinants recognized by these cells are self-antigens, leading to the hypothesis that the physiological role of NK1+ cells is a downregulatory or suppressive function on bone marrow stem cell proliferation and lymphocyte function. In analyzing this hypothesis it is shown here that in young mice there is a temporal correlation between appearance of NK1+ cells in the spleen and the ability to reject allogeneic marrow or to suppress endogenous stem cell proliferation. The reverse situation exists in mice expressing the homozygous lpr gene. Whereas in young mice cells with NK1+ phenotype are demonstrable, these cells disappear with age, i.e., at the time autoimmunity develops. Concomitant with the disappearance of NK1+ cells, the ability to reject marrow grafts and to control endogenous stem cell proliferation also vanishes. The suggestion that the development of autoimmunity is causally related to the disappearance of NK1+ cells is supported by experiments in which NK1+ cells were either eliminated by antibody injection or increased by adoptively transferring cell populations enriched for NK1+ cells into lpr mice. It is shown that removal of cells enhances autoimmunity, whereas injection of NK1+ cells delays the onset of autoimmunity. In vitro assays are presented that demonstrate that suppression of autoantibody-secreting B cells is due to two NK1+ cell populations, one that expresses CD3 and causes specific suppression and one that lacks CD3 and causes nonspecific suppression.

Aging↗

PubMed1992

Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection.

Lethally irradiated mice reject within 24 h certain marrow grafts, a phenomenon called either allogeneic or hybrid resistance. The cells responsible for this rejection (NK1+ CD3+ cells (TNK) express Ag of NK cells as well as the TCR-associated CD3 complex. This raises the question whether TCR participate in the function of these cells during graft rejection. By using flow cytometry it is shown that the majority of TNK cells expresses the TCR-alpha/beta chains and by using adoptive cell transfer assays evidence is presented that it is the TCR-alpha/beta expressing cells that cause rejection. To explore whether any particular TCR chains have to be expressed on these cells, C57L mice were assayed and found to be responders suggesting that the V beta chains deleted in these mice are not obligatory. However, introduction of a specific TCR V beta 5 chain into C57BL/6 mice as a transgene leads to inability to transfer resistance. TNK cells of V beta 5 transgenic mice express the introduced gene suggesting that it is the transgenic TCR that is responsible for the lack of function. In assessing T cell functions in V beta 5 transgenic mice it is shown that although these mice generate CTL specific for H-2d targets there is a deficiency to recognize H-2Dd, i.e., of determinants presumed to be recognized in the acute rejection mechanism. Thus TNK cells and CTL share the inability to recognize H-2Dd epitopes due to expression of the V beta 5 transgene. The notion that TCR on TNK cells play a role in the acute rejection process makes it necessary to postulate a receptor selection mechanism for these cells.

Acute Disease↗

PubMed1992

Evidence for extrathymic development of TNK cells. NK1+ CD3+ cells responsible for acute marrow graft rejection are present in thymus-deficient mice.

The predominant mechanism responsible for acute specific rejection of allogeneic and parental bone marrow by irradiated mice is due to a cell (TNK) that expresses the NK cell surface markers NK1 and ASGM1 as well as TCR. Here we analyze the question as to whether TNK cells require a functional thymus for their development. Using adoptive cell transfer assays, evidence is presented that, as is the case in normal mice, NK1+ CD3+ effector cells are responsible for rejection in thymus-deficient nude mice and that the specificity of rejection is indistinguishable from that of normal mice. To reveal the presence of TNK cells in the spleen of nude mice, double staining for NK1 and CD3 followed by FACS analysis was done. It is shown that NK1+ CD3+ cells are present in the spleens of nude but not euthymic mice, suggesting that the lack of a functional thymus stimulates either Ag expression or the number of TNK cells. In support of this finding, the treatment of irradiated marrow reconstituted mice with cyclosporin A leads to the appearance of TNK cells in the spleen. The relative efficiency of spleen cells from nude and cyclosporin A-treated mice to transfer resistance in adoptive cell transfers was assessed and found to be higher than that of normal spleen, consistent with the higher frequency of these cells in thymus-defective mice. The fate of NK1+ CD3+ cells subsequent to stimulation with an allogeneic marrow graft indicates that these cells proliferate in nude mice without gaining cytolytic activity. In euthymic mice, however, NK1+ CD3+ cells appear transiently but disappear in favor of CD4+ and CD8+ cells that proliferate in response to an allogeneic marrow graft. The CD8+ cells express cytolytic activity with specificity similar to that of the acute rejection mechanism, consistent with the suggestion that TNK cells differentiate into CD8+ killer cells. The reason why TNK cells in nude mice fail to differentiate into CD8+ CTL is explained by the lack of Th cells.

Animals↗

PubMed1992

Clonal development and karyotype evolution during leukemogenesis of BCR/ABL transgenic mice.

The Philadelphia (Ph) translocation is responsible for the generation of the chimeric BCR/ABL oncogene. The Ph chromosome constitutes the earliest detectable chromosome abnormality in chronic myelogenous leukemia and is also found in acute lymphoblastic leukemia. Mice transgenic for a P190 BCR/ABL-producing DNA construct develop lymphoblastic leukemia/lymphoma and provide an opportunity to study early stages of the disease as well as progression. In this study, we have karyotyped the bone marrow of 10 19-day-old BCR/ABL P190 transgenic mice from a line that reproducibly develops leukemia/lymphoma. Leukemic cells from 17 terminally ill transgenic founders and progeny were also karyotyped as well as bone marrow transplant recipients of leukemic donor marrow. Karyotypically visible aberrations were absent from the early stages of BCR/ABL P190-generated leukemia and normal metaphases could be found even in the terminal stages of the disease. A high frequency of aneuploidy was found in advanced leukemia, with a marked preference for the gain of mouse chromosomes 12, 14, or 17. These results point to a primary role for BCR/ABL in leukemogenesis and suggest a destabilizing effect of the BCR/ABL gene on the regulation of cell division.

Aneuploidy↗

PubMed1991

Nicotinamide and 3-aminobenzamide interfere with receptor-mediated transmembrane signaling in murine cytotoxic T cells: independence of Golgi reorientation from calcium mobilization and inositol phosphate generation.

The two competitive inhibitors of ADP-ribosylation, nicotinamide and 3-aminobenzamide, have been reported to interfere with TNF-induced cell apoptosis, and there is evidence that they inhibit killer-induced target cell lysis as well. There are very few drugs known to specifically interfere with target apoptosis induced by killer cells. We therefore sought to explore the effects these inhibitors have on CTL-mediated cell lysis. Here we show that TcR-mediated transmembrane signaling in CTL, measured by Ca2+ mobilization and generation of inositol phosphates, is inhibited by nicotinamide. The possibility that all cell functions are suppressed by the drug is excluded by the finding that constitutive secretion of BLT serine esterase is not inhibited, whereas stimulated secretion of this enzyme is suppressed. We also show that nicotinamide does not interfere with CTL target cell binding or reorientation of the Golgi apparatus toward the target binding site. It is concluded that nicotinamide inhibits transmembrane signaling in CTL and thereby interferes with delivery of the lethal hit to targets.

Animals↗

PubMed1990

Effects of stress on lysability of tumor targets by cytotoxic T cells and tumor necrosis factor.

The effects of stress on four tumor cell lines are analyzed in view of the possibility that stress protects tumor cells against immune attack. We show that stress causes resistance to CTL and TNF in two cell lines. Induction of resistance is time dependent and reversible and not due to failure of killer cells to interact with stressed targets. It is shown that stress induces stress proteins concomitant with induction of resistance to killer cells and TNF. Moreover experiments are presented suggesting that resistance to either immune effector is due to independent mechanisms. The conclusion that stress can induce mechanisms in targets that interfere with the action of TNF as well as with target lysis following a lethal hit by CTL is discussed.

Animals↗

PubMed1990

Induction of tolerance to parental marrow grafts in F1 hybrid mice. Evidence for recognition of self-antigens.

Lethally irradiated (C57BL/6xC3H)F1 mice are able to acutely reject parental C57BL/6 but not C3H marrow grafts, a phenomenon called hybrid resistance (HR). In attempts to inactivate this rejection mechanism we found that parental spleen cells activated with LPS are very potent in inducing tolerance to a subsequent C57BL/6 marrow graft. Tolerance is likely due to elimination of effector cells responsible for graft rejection as adoptive transfer of spleen cells from normal into tolerized mice reconstitutes responsiveness. Evidence is presented that the Ag on LPS-activated spleen cells responsible for induction of unresponsiveness are expressed on both C57BL/6 and (C57BL/6xC3H)F1 cells. This suggests that the HR effector cells recognize autoantigens. In support of this, induction of tolerance to C57BL/6 parental marrow grafts leads to a concomitant dramatic increase in endogenous CFU-spleen after a dose of gamma-irradiation. Moreover, elimination of the cells responsible for HR by injection of anti-ASGM1 antibody results in a similar increase of endogenous CFU-spleen after irradiation. It is concluded that HR is a reflection of autoimmunity, able to limit the proliferation of syngeneic marrow stem cells.

Animals↗

PubMed1990

Evidence for differentiation of NK1+ cells into cytotoxic T cells during acute rejection of allogeneic bone marrow grafts.

The ability of lethally irradiated C57BL/6 mice to acutely reject H-2d bone marrow is due to a lymphocyte population that is NK1+, ASGM1+, CD4-, CD8-, CD3+. Transfer of spleen cells from C57BL/6 mice expressing these antigens into nonresponder 129 mice adoptively transfers the ability to reject H-2d marrow grafts. The specificity of this rejection maps to the H-2D major histocompatibility complex (MHC) region. Transplantation of high doses of H-2d marrow into C57BL/6 overrides the acute rejection mechanism leading to graft survival. During growth of the graft, a cytolytic activity develops that is due to ASGM1+, CD8+ cytolytic T lymphocytes (CTLs) with H-2Ld specificity. The possibility that the ASGM1+, CD8+ CTLs are descendents of the CD3+, NK1+, ASGM1+, CD8- cells responsible for acute rejection is investigated by adoptive cell transfer experiments. We show that beige mice that lack NK1+ cells as well as the ability to acutely reject H-2d marrow fail to generate specific CTLs after transplantation with a high dose of H-2d marrow. Transfer of highly purified NK1+ cells from B6.PL-Ly-2a/Ly-3a (Lyt-2.1) into beige mice together with H-2d marrow leads to generation of Lyt-2.1 CTLs from donor NK1+ cells. These results show that specific CTLs are generated from NK1+ cells during acute marrow graft rejection.

Animals↗

PubMed1990

Differential effects of protein synthesis inhibition on CTL and targets in cell-mediated cytotoxicity.

The reactions that lead to target cell lysis by cytotoxic T cells (CTL) are despite intensive investigations poorly understood. To examine the relative roles effectors and targets play in the lytic reaction, protein synthesis in either CTL or targets was inhibited before assay of lysis. We show, in agreement with previous results, that de novo protein synthesis is not necessary in either effectors or targets during the cytolytic reaction. However, activation of CTL requires protein synthesis. Activated CTL respond to protein synthesis inhibitors with a cycling of activity, a result that is interpreted to be consistent with a stimulus secretion mechanism. Treatment of targets with protein synthesis inhibitors prior to incubation with CTL leads to a very rapid and irreversible loss of lytic susceptibility. It is shown that the decrease in lysability is not due to lack of proper CTL target interaction: MHC class I antigens are expressed on drug-treated targets and these cells serve as cold targets in competitive inhibition experiments. Moreover, drug-treated targets trigger transient Ca2+ mobilization and generation of inositol phosphates in CTL. It is therefore concluded that drug-treated targets are able to trigger CTL function but lack a component that is required for their successful lysis.

Animals↗

PubMed1989

A novel cell type responsible for marrow graft rejection in mice. T cells with NK phenotype cause acute rejection of marrow grafts.

Acute rejection of allogeneic and semiallogeneic marrow grafts has long been considered to be a function of the natural immune system because it shares many features with NK activity in mice. With the use of a recently developed in vivo adoptive transfer assay in which spleen cells are transferred from mice able to reject a particular marrow graft into mice that fail to do so, we show that the cells responsible for induction of marrow graft rejection indeed display the phenotype of NK cells: they lack the T cell Ag CD4 and CD8 but express the NK Ag NK1 and ASGM1. The rejection induced by adoptively transferred cells is exquisitely specific--a feature that points to a specific recognition process by the transferred cells. To elucidate what the recognition structure on these cells may be we found that they express CD3 and most likely the beta-chain of the TCR. Highly purified responder cells with the NK1+, CD3+, CD4-, CD8- phenotype, when transferred into nonresponder recipients, cause specific marrow graft rejection. We conclude that the acute rejection of bone marrow grafts is caused by a cell that expresses NK phenotype but is of T cell lineage. This may suggest the specificity of acute marrow graft rejection is caused by a specific recognition process that involves TCR.

Animals↗

PubMed1989

Natural cytotoxic activity in a cloned natural killer cell line is mediated by tumor necrosis factor.

The interleukin-2-dependent mouse natural killer (NK) cell line NKB61A2 concomitantly exhibits NK and natural cytotoxic (NC) activities. This was determined by the cells' ability to lyse both the NK-sensitive YAC-1 lymphoma and the NC-sensitive WEHI-164 fibrosarcoma cell lines in a 4- and 18-hour 51Cr release assay, respectively. Cell-free supernatant from NKB61A2 cells grown in culture for 48 h had substantial lytic activity against WEHI-164. The mouse mast cell line PT18-A17 and the rat basophilic leukemia cell line RBL-2H3, which both express NC activity, also produced a soluble factor during culture which lysed WEHI-164 cells. This activity was increased in the basophilic/mast cells by crossbridging the surface IgE receptors. Similar results were obtained by triggering the basophilic NC cells with the calcium ionophore ionomycin and the tumor promoter phorbol-12-myristate-13-acetate (PMA). Such triggering of NKB61A2 cells, however, did not significantly increase their NC activity. Interestingly, both ionomycin and PMA had an inhibitory effect on the NK activity of NKB61A2. Recently it has been found that tumor necrosis factor (TNF) is a major mediator of NC activity. To determine if the soluble factor responsible for the NC activity of the NK clone was related to TNF, a rabbit polyclonal antiserum to mouse TNF was tested against the cell-free culture medium of NKB61A2, PT18-A17, RBL-2H3 and murine recombinant TNF (Mu-rTNF). The lytic activity of the culture medium from all these cells and the Mu-rTNF control was abrogated by this antibody. These data suggest that the murine cell line NKB61A2 has both NK and NC activities and that the NC activity is due to a factor immunologically similar to TNF. In addition, the enhancement of NC activity in the NK cell line is apparently under control by a separate pathway, different from that in the basophilic cells.

Antibodies, Monoclonal↗

PubMed1988

Transmembrane chloride flux is required for target cell lysis but not for Golgi reorientation in cloned cytolytic effector cells. Golgi reorientation, N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester serine esterase release, and delivery of the lethal hit are separable events in target cell lysis.

Cell-mediated cytotoxicity can be inhibited by the replacement of chloride with ions that are incapable of passing through chloride channels or by the presence of stilbene disulfonate derivatives known to interfere with chloride flux. We show that the stilbene disulfonate (4,4-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) inhibits lysis of YAC-1 targets by the cloned cell line NKB61A2. Inhibition of lysis occurs on the level of the effector cell inasmuch as preincubation of effectors but not of targets interferes with subsequent lysis. Moreover, inhibition of chloride flux in the target does not interfere with target cell lysis by cytotoxic granules isolated from killer cells. Target cell binding takes place in the presence of DIDS or absence of external chloride, suggesting that events that follow target cell binding require chloride flux. We show that reorientation of the Golgi apparatus, which occurs subsequent to target cell binding in the effector cell, occurs under conditions that interfere with chloride flux. It is therefore suggested that events in the effector cell taking place subsequent to the Golgi apparatus reorientation reaction are inhibited and that delivery of the lethal hit is a stimulus-induced secretory event that requires transmembrane chloride flux. Delivery of the lethal hit is shown to be independent of the release of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT) serine esterase, suggesting that cytolytic components and BLT serine esterase are likely packaged in different vesicles.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1988

Cell-mediated and glucocorticoid-mediated target cell lysis do not appear to share common pathways.

Target cell lysis by cytolyic lymphocytes follows a sequence of events that culminate in osmotic destruction of the target. Although it is clear that killer cell derived components play a crucial role in target cell lysis it is not clear to what extent the target itself is involved in its destruction. Recent observations have pointed to the possibility that glucocorticoid mediated and cell mediated lysis may utilize common pathways of cell lysis. In analyzing this question we found that cell lines that have nonfunctional glucocorticoid receptors like S49-78 and S49-88 are good targets for both NK and thymus-derived killer (TK) cells. Cell lines that are glucocorticoid sensitive such as Q1(4)6 are sensitive to NK-mediated lysis as its derivative HL4-6-3 which contains glucocorticoid receptors but is glucocorticoid resistant. An intriguing exception to this is the glucocorticoid-resistant mutant S49-4RD which is relatively resistant to both NK and TK lysis compared with parent S49. The resistance of S49-4RD to cell-mediated lysis we show here is most likely due to a defect in the target which results in its failure to trigger the cytolytic machinery in the killer cell rather than in its resistance to lysis per se. In support of this we demonstrate that lysis of S49-4RD by cytolytic granules from TK cells is normal. Moreover TK cells lyse S49-4RD as efficiently as its parent in the presence of the lectin Con A. The conclusion that S49-4RD has a defect in its ability to induce killer cells to initiate the cytolytic reaction is also in agreement with the finding that TK-S49-4RD conjugates show inefficient reorientation of the Golgi apparatus in the effector.

Animals↗

PubMed1988

Lysis of a lung carcinoma by poly I:C-induced natural killer cells is independent of the expression of class I histocompatibility antigens.

Cells from the line 1 murine carcinoma express little if any H-2d when grown in normal medium. These cells are susceptible to splenic cell populations with NK activity, stimulated by prior injection of poly I:C, but are not lysed by NK-deficient splenocytes from homozygous beige mice treated with anti-asialo GM1. Incubation of line 1 cells in medium containing DMSO leads to a dramatic stimulation of H-2d expression but no change in lytic susceptibility to splenic NK cells. Transfection of H-2Dp into line 1 leads to a constitutive and DMSO-inducible expression of H-2Dp at functionally significant levels, but this expression appears to have no influence on NK cytolytic susceptibility.

Animals↗

PubMed1988

Asialo-GM1-positive T killer cells are generated in F1 mice injected with parental spleen cells.

(C57BL/6 x DBA/2)F1 mice transplanted with parental C57BL/6 spleen cells become splenic chimeras, show donor antihost cytotoxic T cell activity, and lose their T cell-mediated, humoral, and natural immunity. Injection of anti-asialo-GM1 (ASGM1) into transplanted mice strongly suppresses splenic cytotoxic activity and causes a significant reduction of spleen cells expressing ASGM1, Thy-1, and Lyt-2. In vitro treatment of spleen cells from transplanted mice with antibody and complement shows that the cytotoxic effector cells are ASGM1+, Thy-1+, Lyt-2+, L3T4-, NK1.1-, and H-2d-, hence of donor origin. The cytotoxic effector cells are specific for H-2d targets and lack NK activity. In an attempt to explore whether in vivo elimination of the cytotoxic effector cells has any influence on splenic chimerism or humoral immunity, F1 mice injected with parental splenocytes were treated with anti-ASGM 1. Results show that this treatment eliminates a substantial proportion of cytotoxic effector cells but has no effect on splenic chimerism or restoration of humoral immunity. It therefore appears that cytotoxic effector cells are not primarily responsible for induction of chimerism or suppression of humoral immunity. In support of this injection of parental spleen cells with the nu/nu mutation induces killer cells in F1 mice but fails to induce splenic chimerism or immunosuppression. In contrast, injection of parental spleen cells with the bg/bg mutation generates both splenic chimerism and suppression of humoral immunity although their ability to generate cytotoxic effector cells in F1 hosts is seriously impaired and comparable to the cytotoxic potential of C57BL/6 nu/nu cells. It is concluded that the ASGM1 + cytotoxic T cells are not primarily responsible for splenic chimerism and suppression of humoral immunity and that the two effects are likely caused by parental cells with a different phenotype and function.

Animals↗