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M S Pasternack

Publications and source records attributed to M S Pasternack.

34 records · Page 2Linked to original sources
PubMed1991

Granzyme A binding to target cell proteins. Granzyme A binds to and cleaves nucleolin in vitro.

The physiologic substrates of cytotoxic T lymphocyte granule-associated serine esterases (referred to hereafter as proteases or "granzymes"), and the role of these enzymes in cell-mediated activity remain unclear. We have developed an assay for possible ligands of the trypsin-like dimeric serine protease granzyme A based on Western immunoblotting techniques. This protein-binding assay demonstrates the selective binding of granzyme A to several proteins present in the target cell P815. The binding specificity is preserved when enzyme binding is performed in the presence of excess competing proteins, including such cationic species as lysozyme and RNase. Enzyme binding is inhibited, however, by heat or detergent inactivation of granzyme A. Subcellular fractionation of target cells shows that the nuclear fraction contains most granzyme A binding reactivity, which is recovered in the nuclear salt wash fraction. A protein with Mr = 100,000 and two closely migrating proteins with Mr = 35,000 and 38,000 are the predominant reactive moieties, and the N-terminal sequence of the 100-kDa protein confirmed that this protein was murine nucleolin. Incubation of granzyme A with nucleolin generates a discrete proteolytic cleavage product of Mr = 88,000. Since nucleolin is known to shuttle between nucleus and cytoplasm, the interaction of granzyme A and nucleolin may be important in the process of apoptosis which accompanies cytotoxic T lymphocyte-mediated lysis of target cells.

Amino Acid Sequence↗
PubMed1989

Combined antibody and ganciclovir treatment of murine cytomegalovirus-infected normal and immunosuppressed BALB/c mice.

The efficacy of treatment with ganciclovir (DHPG) and antibody activity-containing ascitic fluid (AF) separately and in combination was studied in normal and immunosuppressed BALB/c mice challenged intraperitoneally with a lethal dose (10(6) PFU) of murine cytomegalovirus (CMV). With combination therapy, lower doses of both DHPG and AF were often as effective as a higher dose of either agent given singly. For instance, the survival rate of murine CMV-challenged immunosuppressed mice was doubled when 4 mg of DHPG per kg and a 1:16 dilution of AF were both administered in contrast to when each was used alone. In both groups of animals, combination therapy was shown to be more effective than either therapy individually, even when initiation of therapy was delayed as long as 48 h. Such an approach holds promise for decreasing the expense associated with antibody use and the dose-related toxicity associated with DHPG use while maintaining or possibly increasing the efficacy of prophylaxis and therapy of serious CMV disease in humans.

Animals↗
PubMed1988

Cytotoxic T-lymphocytes.

Cytotoxic T-lymphocytes have received intensive scrutiny in the past decade, and have been implicated as important effector cells in diverse clinical settings. A variety of technological advances--the establishment of T cell clones, the production of MAb, the production of pure lymphokines through gene cloning, the modification of target cells by gene transfer and synthetic peptides--has led to tremendous strides in our understanding of CTL function and their role in the pathophysiology of a variety of disease states. It is likely that future efforts will generate important applications of such understanding, such as the pharmacologic modulation of CTL activity and proliferation, novel vaccines, and the introduction of adoptive transfer of cloned CTL as a clinically practical therapy.

Animals↗
PubMed1987

Recognition and lysis of target cells by cytotoxic T lymphocytes.

A single cytotoxic T lymphocyte (CTL) is capable of performing the two most fundamental functions of an immune response, recognition and elimination of foreign antigens. It is now clear that in a CTL these two functions are linked via the antigen-specific, heterodimeric receptor. We review here some experimental approaches that justify this conclusion and provide the means for further examination of the mechanisms by which CTLs lyse their target cells. When antireceptor antibodies serving as antigen substitutes are attached to various cells, they trigger the lytic activity of particular CTLs, which results in lysis of the antibody-modified cell. In the process, a novel serine esterase, which is located within cytolytic granules of the CTL, is released. The presence of this enzyme and a complement-like protein, perforin, in granules of a CTL has led to the suggestion that CTLs and complement have similar cytolytic mechanisms. However, the resistance of some CTLs to lysis by other CTLs, but not to lysis by antibody-activated complement, suggests fundamental differences between cytolytic mechanisms of CTLs and complement.

Animals↗
PubMed1985

Activation requirements for antigen- and mitogen-induced interferon-gamma release from cytotoxic T lymphocytes.

We have studied the activation signals that regulate interferon-gamma (IFN-gamma) secretion from murine cytotoxic T lymphocytes (CTL) upon binding mitogen or antigen. CTL clones were found to require at least 1 hr of stimulation with concanavalin A (Con A) in order to produce detectable levels of IFN-gamma. Full activation of IFN-gamma synthesis in CTL clones occurred after stimulation for 2 hr or more, and in those cultures CTL continued to produce high levels of IFN-gamma even after the effects of Con A had been neutralized. Splenic T cells and uncloned long-term CTL lines required a longer period of stimulation than cloned CTL for Con A-induced IFN-gamma secretion. The relationship between IFN-gamma secretion and cytotoxic activity was studied in an antigen-specific system. These studies reveal marked differences in the types of effector responses generated by CTL upon contact with antigen, demonstrating that some antigen-bearing cells promote high levels of IFN-gamma secretion and are poorly lysed by CTL, whereas other cell lines are lysed with high efficiency by CTL but induce low levels of IFN-gamma secretion.

Animals↗
PubMed1984

Immunoprecipitation of cell surface structures of cloned cytotoxic T lymphocytes by clone-specific antisera.

Clones of cytotoxic T lymphocytes (CTL) differ in their specific reactivity with diverse target cell antigens. To learn about the uniqueness of individual CTL clones we injected rats and mice with cloned CTL in an effort to prepare clone-specific antisera and to analyze the CTL surface molecules that were immunoprecipitated by these antisera. Three clones were studied. They were all derived from BALB.B mice and were specific for antigens encoded by the major histocompatibility complex of the H-2d haplotype. Antisera raised in rats against individual clones contained antibodies to lymphocyte function-associated antigen type 1 (LFA-1) and inhibited the cytotoxic activity of all of the clones. In contrast, BALB/c and BALB.K mice injected with individual clones consistently yielded alloantisera that were clone specific in their ability to inhibit CTL-mediated lysis of target cells (P815). In addition, these alloantisera immunoprecipitated from extracts of 125I-radiolabeled CTL a disulfide-bonded dimer consisting of approximately equal to 45-kilodalton subunits. This dimer resembles the putative T-cell antigen-recognition receptor recently identified in several laboratories. The alloantisera also immunoprecipitated CTL surface molecules that were associated with beta 2-microglobulin and that differed in apparent molecular mass (37-38 kilodaltons) in different clones.

Animals↗
PubMed1984

Isolation and partial characterization of concanavalin A receptors on cloned cytotoxic T lymphocytes.

A small set of concanavalin A (Con A)-binding glycoproteins was isolated from the surface membrane of cloned cytotoxic T lymphocytes (CTL) and partly identified using monoclonal antibodies. The binding of Con A by these glycoproteins on the CTL surface results in the secretion of gamma-interferon and in blocking the effector functions of the cells-namely, antigen-specific and lectin-dependent cytotoxicity. The Con A is evidently bound tightly to some surface structures ("Con A-receptors") that are required for the activation and cytotoxic activity of CTL. To isolate and identify these receptors, antibodies to Con A were used. After Con A was allowed to bind to radiolabeled cloned CTL (labeled with 125I or [35S]methionine or 3H-labeled amino acids), the cells were washed thoroughly, lysed in detergents and anti-Con A antibodies were added to bind to the Con A-receptor complexes. The resulting aggregates were adsorbed with protein A-bearing Staphylococci and the receptors were then specifically released from the pelleted bacteria by alpha-methyl-D-mannoside and analyzed by polyacrylamide gel electrophoresis under reducing conditions. Eight to nine labeled components were seen by autoradiography and with the aid of monoclonal antibodies to known T-cell surface molecules, four were identified as T200, lymphocyte function-associated antigen (LFA)-1, alpha- and beta-chains, and (on some clones) Lyt-2. Other components with Mr congruent to 160,000, 120,000, 46,000, 42,000, and 23,000 have not been identified. The procedures described here may have general application in the studies of the functional properties of other cell surface molecules.

Animals↗
PubMed1984

Monoclonal antibodies specific for a murine cytotoxic T-lymphocyte clone.

Two antibody-secreting murine hybridomas, F1G3.1 and F2A11.5, have been established from B10.D2 mice immunized with cells from the murine cytotoxic T-lymphocyte clone G4. The two clones used, G4 and B10, were derived from BALB.B (H-2b) mice and the target antigen specificity of both maps to the Dd region of the murine H-2 complex. However, B10 has a lower affinity for the target cells, as shown by its lower specific killing of blasts and its higher susceptibility to blocking by anti-Lyt-2 monoclonal antibody 53-6.75. The monoclonal antibodies, F1G3.1 and F2A11.5, react only with cells from clone G4. Similarly, they block only the specific cytolysis mediated by G4; no effect on cytotoxicity mediated by B10 or by heterogeneous populations of cytotoxic T lymphocytes was found. F1G3.1, especially, is very active in stimulating G4 to secrete immune interferon; B10 in contrast did not show any induction on treatment with these monoclonal antibodies. The structure of the surface antigen on G4 cells recognized by these monoclonal antibodies was revealed by immunoprecipitation studies of radioiodinated cell surface proteins. A protein dimer could be identified with an apparent molecular size of 80,000 daltons consisting of monomers migrating as 42,000-dalton proteins on reduction. So far, electrophoresis in the presence of NaDodSO4 does not indicate any heterogeneity in the size of the monomers. This molecule can be distinguished from the Lyt-2 complex.

Animals↗
PubMed1984

Release of discrete interferons by cytotoxic T lymphocytes in response to immune and nonimmune stimuli.

We have examined the potential of murine cytotoxic T lymphocytes (CTL) to synthesize both IFN-gamma and IFN-alpha, beta by using three cloned antigen-reactive CTL lines. These lines, which produce IFN-gamma in an antigen-specific manner or after stimulation with mitogen, were also found to produce IFN-alpha, beta upon nonspecific contact with Newcastle disease virus. The requirement for viral integrity in this process has been examined. Virus infectivity was not necessary to stimulate the release of IFN-alpha, beta from CTL, but acid-inactivation of virus abrogated its IFN-inducing property.

Animals↗
PubMed1983

The site of action of N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK) on cloned cytotoxic T lymphocytes.

N-alpha-tosyl-L-lysyl-chloromethyl-ketone (TLCK), an irreversible inhibitor of trypsin-like serine proteases, is a potent, nontoxic inhibitor of cytotoxic T lymphocyte (CTL) activity with half-maximal inhibition of an alloreactive CTL clone occurring at [TLCK] = 30 microM. We have utilized TLCK as an affinity probe for functionally important CTL surface molecules by raising rabbit antibodies specific for the tosyl group and employing them as immunoprecipitating reagents. When 125I-labeled cloned CTL were treated with TLCK, immunoprecipitation with rabbit anti-tosyl antibodies and analysis by polyacrylamide gel electrophoresis revealed a small number of TLCK-binding proteins. Prior alkylation of radiolabeled CTL with iodoacetamide inhibited TLCK binding only slightly, suggesting that TLCK binding did not occur via free sulfhydryl groups. Thymocytes and a second CTL clone both had very similar patterns of TLCK-binding proteins; in contrast the TLCK-binding proteins of B cells differed greatly. Sequential immunoprecipitation experiments identified the predominant CTL TLCK-binding protein as T200. Lymphocyte function-associated antigen-1 also reacted with TLCK but to a lesser extent. The inhibitory role of cell-surface bound TLCK (vs intracellular TLCK) was demonstrated by protection experiments using Concanavalin A, a reversible ligand of the CTL cell surface. These experiments suggest that T200 may be required for cytotoxic activity of CTL.

Amino Acid Chloromethyl Ketones↗
PubMed1982

Inhibition of cytotoxic T lymphocyte activity by concanavalin A.

Several lectins were tested for the ability to interfere with the recognition or lysis of target cells by cytotoxic T lymphocytes (CTL). Of those tested, concanavalin A (Con A) had the greatest effect: at 0.25 to 10 micrograms/ml, it profoundly blocked antigen-specific lysis of target cells by cloned and uncloned CTL in various allo- and syngeneic systems. Separate pretreatment of CTL and target cells showed that the effect of Con A is exercised predominantly on CTL. Various experimental results ruled out several possible explanations for the inhibitory effect of Con A, such as: CTL-CTL self-killing by nonspecific, lectin-dependent cytotoxicity, impairment of lateral mobility of CTL surface components, and cell agglutination, preventing recycling of CTL among target cells. The findings suggest that subagglutinating concentrations of Con A block CTL activity by binding to and interfering with CTL surface structures that have relatively high affinity for Con A and are critically involved in recognition or lysis of target cells.

Animals↗
PubMedDate not supplied

A novel serine esterase expressed by cytotoxic T lymphocytes.

Cytotoxic T (Tc) lymphocytes recognize and lyse target cells and are thought to serve as an important defence against viral infections and possibly against neoplasms. The nature of the receptors responsible for antigen recognition by these cells is becoming clearer, but the molecular mechanisms responsible for their cytolytic activity remain largely unknown. The possibility that proteases are involved in this process has been suggested by the effects of certain inhibitors. Here we demonstrate that clones of murine Tc cells possess considerable trypsin-like esterase activity when assayed by a sensitive colorimetric assay. This activity was blocked completely by two serine esterase inhibitors, diisopropylfluorophosphoridate (DFP) and phenylmethylsulphonyl fluoride (PMSF), but not by N alpha-tosyl lysyl chloromethyl ketone (TLCK). The use of 3H-DFP as an affinity-labelling reagent demonstrated that the esterase activity resides in a protein of relative molecular mass (Mr) 28,000 (28K). A wide variety of other lymphocytes, including those from thymus, spleen and lymph node, established lines of B cells and noncytotoxic T cells, and clones of T helper cells, had about 300-fold less esterase activity than the Tc-cell clones and far smaller amounts of the DFP-reactive 28K protein. However, in thymocytes the esterase activity increased 20-50-fold and the 28K protein became more prominent 4 days after these cells had been stimulated in vitro to generate Tc cells.

Affinity Labels↗
PubMedDate not supplied

Serine esterase in cytolytic T lymphocytes.

The mechanisms that enable cytotoxic T lymphocytes (Tc cells) to destroy target cells are only vaguely understood. However, recent studies have identified in Tc cells and natural killer cells cytoplasmic granules that contain perforin, a cytolytic protein that resembles the ninth component of complement (C9). Antigen-specific lysis of target cells, traditionally ascribed solely to Tc cells, has now also been demonstrated in some T-helper cell (Th cell) lines, referred to here as T helper-killer or Th/c cells. We recently found a novel serine esterase that is present at greatly elevated levels in cloned murine Tc cell lines and one Th/c cell line, but not in two non-cytolytic Th cell lines. These findings suggest that the serine esterase is involved in cytolytic activity and that a variety of effector cells share a common cytolytic mechanism. To explore the role of the serine esterase in this process, we have been studying additional properties of the enzyme in murine T cells. We show here that it is a membrane-associated, disulphide-linked dimer, it has trypsin-like properties but is not a general protease, in density gradient centrifugation it sediments with perforin, it is secreted by Tc cells during their cytolytic attack on target cells, and antiserum to Tc-cell serine esterase reacts with the enzyme in Th/c cells.

Animals↗
PubMedDate not supplied

Cell-mediated immunity in experimental cytomegalovirus infections: a perspective.

Treatment of cytomegalovirus (CMV) infections has assumed increasing importance in the care of immunocompromised patients, particularly allograft recipients and patients with symptomatic infection with the human immunodeficiency virus. The crucial role of T cells in the host defense against CMV may be inferred by the expression of life-threatening CMV disease in these restricted patient subpopulations. However, detailed understanding of the pathogenesis of CMV disease in the compromised host is lacking, and the mechanisms by which T cells confer protection are unknown. Insight into the pathogenesis of CMV in these patients is crucial for the development of rational therapeutic strategies. A considerable body of data has been generated to explore the role of T cell-mediated immunity in a number of model viral infections as well as in experimental CMV infections in both normal and immunocompromised mice. This experience will be critically reviewed to clarify the often conflicting observations regarding a protective or, conversely, a pathogenic role for T cells in producing CMV disease.

Animals↗