Search PubMed⌕ Search

Biomedical subjects

G Berke

Publications and source records attributed to G Berke.

At least 55 records · Page 3Linked to original sources
PubMed1990

The lysis of cytotoxic T lymphocytes and their blasts by cytotoxic T lymphocytes.

After binding to their targets, cytotoxic T lymphocytes (CTL) deliver a lethal hit signal, ultimately leading to target cell lysis, and then can recycle to lyse additional targets, without themselves being destroyed. If non-specific secreted lytic mediators are involved in such lysis. CTL survival would not be expected unless the effectors are immune to CTL-mediated lysis. Therefore the lytic susceptibilities of alloimmune peritoneal exudate lymphocytes (PEL), containing up to 50% CTL, and of the cytolytic PEL blasts (PEB), obtained by culturing with interleukin-2 (IL-2), were examined. 51Cr-labelled BALB/c (H-2d) anti-EL4 (H-2b) (d alpha b) PEL were lysed 88%, 78%, and 48% by C3H/eb (H-2k) anti-P815 (H-2d) (k alpha d) PEL, C57BL/6 (H-2b) anti-P815 (b alpha d) PEL and b alpha d PEB, respectively. Similarly, b alpha d PEL were lysed 82% and 21% by d alpha b PEL and PEB, respectively. b alpha d PEB were lysed 82%, 28-39% and 39-51% by k alpha d PEL, b alpha d PEL and b alpha d PEB, respectively, b alpha d PEB were lysed 29-55% by d alpha b PEL. Furthermore, the CTL-containing populations were no less susceptible to lysis than normal lymphocytes. Since the majority (80-90%) of cells in these two types of CTL-containing populations can be directly and specifically lysed by appropriately immunized PEL CTL, we conclude that both the lytic granule and perforin lacking (PEL) and containing (PEB) CTL are not a priori immune to CTL-mediated lysis. These findings are in accord with theories proposing lysis to be induced by receptor-mediated contact between effector CTL and target cells, and challenge those suggesting the involvement of secreted lytic mediators.

Animals↗
PubMed1989

T cell activation: independent induction of killing activity and interleukin 2 secretion in cytolytic hybridomas.

Memory-like cytotoxic T lymphocytes (CTL) hybridomas exhibiting inducible killing activity and IL2 production were used to analyze the anamnestic response of CTL. Four activating agents were examined; anti-Thy-1 monoclonal antibody G7, staphylococcal enterotoxin B, interferon (IFN)-alpha/beta and IFN-gamma. These agents seemed to affect CTL activities in three distinct ways. Anti-Thy-1 monoclonal antibody, like specific antigen, was found to be a potent inducer of specific killing and IL2 production, whereas staphylococcal enterotoxin B induced IL2 production, but not cytolytic activity. On the other hand, IFN-alpha/beta and IFN-gamma effectively stimulated cytotoxicity without inducing IL2 production. The independent triggering of specific killing and IL2 secretion in the monoclonal cytolytic hybridomas suggests that in CTL distinct signals stimulate killing activity and IL2 production. The results also suggest that IFN-alpha/beta and IFN-gamma trigger the cytolytic program through an alternative activation pathway which does not involve the T cell receptor.

Animals↗
PubMed1989

The cytolytic T lymphocyte and its mode of action.

While the binding step of cytolytic T lymphocyte (CTL) target cell interaction resulting in conjugate formation is a well-characterized event, there seems to be more than one mechanism whereby lymphocytes kill the target. In recent years, infliction of complement (C)-like "holes" (I.D. 10-20 nm) on the target cell membrane, believed to be produced by the Ca2+-dependent lytic protein(s) perforin/cytolysin of secretory lytic granule origin has been proposed to be the mechanism of lymphocytotoxicity. More recent evidence, however, suggests that Ca2+-dependent exocytosis of lytic granules (where detectable) is not involved in lymphocyte-mediated cytolysis. Furthermore, neither formation of C-like "holes" in targets exposed to CTL, nor higher-than-background levels of lytic granules, perforin or BLT-esterases, have been detected in highly potent, peritoneal exudate CTL (PEL) derived directly from the animal or in cytocidal PEL-hybridomas. Hence exocytosis of perforin and formation of the above pores may apply to certain effector cells, particularly those grown in vitro in IL-2, but not to in vivo primed CTL such as PEL. On the other hand, work from this laboratory with Ca2+ probes has shown that lysis induced by CTL such as PEL-not involving lytic granules, perforin or formation of the above "holes"-is preceded by a marked prelytic elevation of cytosolic Ca2+ in the target. CTL-induced target cell membrane perturbation--a direct result of receptor-mediated effector-to-target interaction or through a membrane-bound or secreted effector component(s)--may be responsible for triggering the prelytic influx of Ca2+ from external sources, or its mobilization from internal stores in the target. We propose that CTL-induced, persistent elevation of cytosolic Ca2+, above a critical level, rather than formation of 10-20 nm pores, is responsible for the catastrophic prelytic events observed in the target, such as bleb formation, metabolic exhaustion and DNA degradation, ultimately leading to lysis.

Animals↗
PubMed1988

Highly lytic in vivo primed cytolytic T lymphocytes devoid of lytic granules and BLT-esterase activity acquire these constituents in the presence of T cell growth factors upon blast transformation in vitro.

Demonstration of C-like "rings," lytic granules, and the Ca2+-dependent lytic proteins--perforin/cytolysin--thereof, in certain cytocidal lymphocytes has led to the hypothesis of a mechanism of lytic granule-exocytosis and a common terminal lytic step in lymphocyte and C-induced lysis. However, neither cytolytic granules, nor formation of C-like rings during lysis have been detected in mature, highly potent, peritoneal exudate CTL (PEL) derived directly from the site of allograft rejection or in cytocidal hybridomas derived from them (PEL hybridomas). We now report that when stimulated in vitro in the presence of Con A supernatant, as a source of T cell growth factors (TCGF) or rIL-2, small in vivo primed PEL transform into large, dividing cytolytic T cells (PEL blasts) that express the same lytic specificity of the original PEL in short term lytic assays. The PEL blasts, in contrast to PEL, possess massive quantities of lytic granules, and protease (N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester esterase) (BLT-esterase) activity as well as non-specific, cell-mediated cytolytic activity in a long term (4-h) assay. These results suggest that the proposed lytic mechanism involving exocytosis of lytic granules, perforin, and BLT-esterases and the formation of 10 to 20-nm lesions may apply to lysis induced by granule-containing effectors such as large granular lymphocytes and TCGF-dependent CTL lines, such as PEL blasts. However, killing by mature, in vivo primed CTL, such as PEL or their hybridomas, appears to be effected through an alternative, contact-induced, self-destruction process(es) of the target not involving secretory lytic granules or the above lesions. Hence, although the expression of lytic granules and BLT-esterase activities in cytolytic lymphocytes devoid of these components is induced by TCGF, these cellular constituents are not necessary for the expression of CTL-mediated target cell lysis by mature effector cells.

Animals↗
PubMed1987

CTLA-1 and CTLA-3 serine esterase transcripts are detected mostly in cytotoxic T cells, but not only and not always.

We and other investigators previously reported the cloning of CTLA-1 (or CCP-1) and CTLA-3 (or H Factor) serine esterase-related transcripts preferentially expressed in cytolytic T lymphocytes. We extended the survey of the tissue specificity of these molecules. Two main sets of results were obtained. First, both CTLA-1 and CTLA-3 transcripts could be found in the various cytolytic T cells tested, although in widely different amounts, and in some cases just at the threshold of detection. Secondly, these transcripts were not found in most of the other cells tested, including in some natural cytotoxic cells and in activated cytotoxic macrophages; however, they could be detected in mast cells for CTLA-1 and in some noncytotoxic lymphocytes for CTLA-3. Thus, the CTLA-1 and CTLA-3 serine esterase products are most probably not required for macrophage or natural cytotoxicity; their presence cannot be taken as characteristic of cytotoxic T cells; and a discussion about their relevance to T cell-mediated cytotoxicity should take into account their widely different amounts from one cytotoxic T cell to another.

Animals↗
PubMed1987

Effect of oxidation on the conformation of cell surface H-2 antigens detected by monoclonal antibodies.

Periodate treatment of lymphoid cells is known to render them susceptible to nonspecific lysis by cytotoxic T lymphocytes. To detect alterations in cell surface molecules as a result of such treatment, we investigated the binding of antibodies to oxidized and nonoxidized lymphoid cells and found that oxidation selectively decreases the binding of some anti-H-2 and anti-beta 2m antibodies. This effect was not caused by loss of H-2 molecules from the cells, since the rate of exchange of cell surface beta 2m by exogenously added beta 2m was unchanged, and neither H-2 nor beta 2m molecules could be detected in the supernates of oxidized cells. Our findings suggest that oxidation alters the conformation of H-2 antigens in such a way as to reduce antibody binding. Such conformational changes may further affect the recognition of histocompatibility antigens by cytotoxic T cells.

Animals↗
PubMed1987

Are lytic granules and perforin 1 involved in lysis induced by in vivo-primed peritoneal exudate cytolytic T lymphocytes?

We examined whether in vivo-primed, alloimmune, peritoneal exudate CTL obtained directly from the animal kill TC by perforating their membranes with C-like lesions produced by P1 or P2 originating in cytolytic granules of the CTL. In control experiments lytic granules were isolated from the formerly lytic CTLL-2 line as well as from Con A-activated lymphoblasts but not from the in vivo-primed CTL. The use of anti-P1 of CTLL-2 origin supports the above results in that immunoblots were positive with CTLL-2 extracts and isolated granules thereof but negative with extracts and isolated (nonlytic) granules from in vivo primed CTL; immunofluorescence of permeabilized cells gave similar results. We observed typical "ring structures" on EL4 membranes attacked by Ab + C as well as after exposure to lytic granules extracted from the formerly lytic CTLL-2 line; however, no such "structures" were observed after attack by the in vivo-primed CTL. These results are at variance with the recent suggestion of a common lytic mechanism--employed by CTL--of secreted lytic granules containing P1 (and P2) thereof that acts analogously to C9. An alternative mechanism for CTL-induced lysis not involving initial perforation of the TC membrane by 160A "holes" (Poly P1) has been presented elsewhere. It is important to note that our results with in vivo-primed CTL do not preclude involvement of a secreted, pore (channel)-forming agent(s) that is not observed by negative staining EM.

Animals↗
PubMed1986

The mitogenic activity of staphylococcal enterotoxin B (SEB): a monovalent T cell mitogen that stimulates cytolytic T lymphocytes but cannot mediate their lytic interaction.

Staphylococcal enterotoxin B (SEB), a monovalent T cell mitogen and inducer of T suppressor cells, was found to be a potent polyclonal activator of cytolytic T lymphocytes (CTL) effective against concanavalin A (Con A)-treated target cells. In addition to polyclonal stimulation of CTL, SEB could reactivate "memory" CTL, alloimmunized 60 to 90 days earlier, into "secondary" CTL detectable as early as 24 hr after onset of stimulation and specific for the original priming target cells. Optimal cytolytic activity was induced at 0.5 to 10 micrograms/ml SEB; optimal priming time was 3 days, correlating well with the proliferative activity and morphologic transformation of small lymphocytes into large T lymphoblasts. Long-term cultures of splenocytes, stimulated by SEB, continued to express high cytolytic activity. It is noteworthy that although SEB and Con A are comparable CTL inducers, SEB, unlike Con A, is an ineffective mediator of nonspecific, CTL/target cell interactions. To the best of our knowledge this is the first example of a CTL inducer unable to mediate CTL-target interaction and lysis. The latter observations suggests that different receptors are involved in CTL activation and in CTL-target interaction resulting in lysis.

Animals↗
PubMed1986

Maturation of cytolytic T lymphocytes.

We have studied the maturation of cytotoxic T-lymphocytes (CTL) following primary and anamnestic responses in vivo and in vitro. Parameters evaluated included: frequency of effector CTL, specificity of binding to and lysis of target cells, killing and recycling ability of individual CTL, and the avidity of effector-target conjugation. While the frequency of effector CTL in the peritoneal cavity of BALB/c mice immunized against leukemia EL4 of C57BL/6 origin increases from 0 to 35% in 11 days of priming, a paradoxically lower frequency has been observed usually after 2 degrees and repeatedly after 3 degrees immunizations both in the peritoneal cavity and in the spleen. The H-2 haplotype and H-2 sub-loci specificity of CTL is preserved upon repeated immunizations. Likewise, the rate of killing and recycling of individual CTL do not change throughout immunizations, suggesting that the cytolytic activity of individual effector CTL is discrete ("quantal") and not subject to maturation upon repeated immunizations. On the other hand, inhibition of conjugate formation and of lysis by antibodies against target major histocompatibility complex (MHC) or effector Lyt-2 determinants is consistently less effective with 3 degrees CTL, suggesting an increase in avidity of effector/target interaction upon repeated immunizations. A striking increase in apparent avidity has been observed during CTL priming in mixed lymphocyte reaction, as deduced from blocking by target cell MHC antibodies. These results suggest that alloimmune CTL undergo maturation with respect to their ability to interact with the target, and that the composition of the responding population is subject to moderate selective processes driven by repeated antigenic stimuli.

Animals↗
PubMed1986

Identification of cell surface glycoproteins by periodate-alkaline phosphatase hydrazide.

A novel method for the detection of cell surface glycoconjugates has been developed. Cells are subjected to mild surface oxidation of vicinal hydroxyls with sodium periodate. Afterward, cellular proteins are resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and then probed with alkaline phosphatase hydrazide. The technique is sensitive, reproducible, and inexpensive. It obviates the need for radiolabeled NaBH4 and the subsequent processing of polyacrylamide gels for fluorography. Results are easily obtained in a matter of a few hours.

Alkaline Phosphatase↗
PubMed1986

Interaction of periodate-oxidized target cells and cytolytic T lymphocytes: a model system of "polyclonal MHC recognition".

In oxidation-dependent cytotoxicity (ODCC), cytolytic T lymphocytes (CTL) non-specifically recognize, bind to and lyse oxidized target cells (O-TC) but the precise mechanism whereby CTL react with O-TC is far from clear (Berke, G., Immunol. Rev. 1983. 72:5). Here we present evidence that CTL/O-TC interactions are blocked by aldehyde-reactive reagents such as hydroxylamine, adipic acid dihydrazide and thiocarbohydrazide and that preformed CTL/O-TC conjugates dissociate upon reduction with NaBH4, suggesting that active aldehyde groups of O-TC rather than intercellular Schiff bases are involved in the recognition and lysis of O-TC by CTL in ODCC. The aldehydes are bound to trypsin-sensitive, non-H-2 glycoproteins that appear to be different and unique in the three different target cell lines so far examined (EL4, L1210, R1.1). In view of these and previous findings we would like to suggest that in ODCC, active aldehydes react with adjacent major histocompatibility complex and perhaps other cell-surface molecules to create a multitude of modified conformations, responsible for the "polyclonal" (nonspecific) MHC recognition and lysis of O-TC by CTL, as well as for an altered pattern of H-2 antibody binding to O-TC.

Aldehydes↗
PubMed1986

Clonal expansion of T cells: a cytotoxic T-cell response in vivo that involves precursor cell proliferation.

The response of peritoneal exudate lymphocytes to allogeneic tumor cells was used to determine whether the in vivo generation of cytotoxic T cells (CTL) involved the proliferation of precursor cells. Ten days post-injection, both cytotoxic activity and the formation of conjugates between lymphocytes and target cells were shown to be specific for the immunizing tumor alloantigens and to be effected by Ly-2+ cells. A cell-sorting-based procedure was developed to isolate specific conjugates between red-fluorescence-tagged CTL and blue-fluorescence-tagged tumor target cells. When [3H]thymidine was administered during the response, almost all isolated conjugate-forming CTL were 3H-labeled on autoradiography. Thus, the CTL were clearly products of dividing cells, a result that contradicts published data. Reassessment of a previously studied system, which suggested that CTL were not products of cell division, indicated that in that system many of the conjugate-forming cytotoxic cells studied were Ly-2- and nonspecific, and thus perhaps not T cells. We conclude that the clonal selection model is applicable to at least one in vivo T-cell response.

Animals↗