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Biomedical subjects

G Berke

Publications and source records attributed to G Berke.

At least 91 records · Page 5Linked to original sources
PubMed1982

Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells.

Specific binding (conjugation) of cytotoxic T lymphocytes (CTL) to target cells (TC) is the first step in a multistage process ultimately resulting in dissolution of the TC and recycling of the CTL. We examined the position of the microtubule organizing center (MTOC) of immune CTL bound to specific TC. Immunofluorescence labeling of freshly prepared CTL-TC conjugates with tubulin antibodies indicated that the MTOC in essentially all conjugated CTL but not in the conjugated TC were oriented towards the intercellular contact site. This finding was corroborated by electron microscopy examination of CTL-TC conjugates fixed either immediately after conjugation or during the lytic process. Antibody-induced caps of membrane antigens of CTL such as H-2 and Thy 1, did not show a similar relationship to the MTOC. Incubation of CTL-TC conjugates, 10-15 min at room temperature, resulted in an apparent deterioration of the microtubular system of conjugated CTL. It is proposed that the CTL plasma membrane proximal to the MTOC is particularly active in forming stable intercellular contacts, resulting in CTL-TC conjugation, and that subsequent modulation of the microtubular system of the CTL may be related to the cytolytic response and to detachment of the effector cell.

Animals↗
PubMed1981

Cytotoxic T lymphocyte hybridomas that mediate specific tumor-cell lysis in vitro.

Cytotoxic hybridomas were generated by polyethylene glycol-induced fusion of cytotoxic T lymphocytes (CTL) and BW5147 lymphoma cells. The CTL populations used for fusion were obtained from BALB/c (H-2d) mice primed with leukemia EL4 of C57BL/6 (H-2b) and restimulated either in vivo or in vitro. To circumvent possible CTL-mediated nonspecific lysis of BW5147 cells during fusion, the CTL were transiently inactivated by trypsin prior to fusion. Four cytolytically active hybridomas were obtained, cloned, and subcloned. Hybrid clones lysed all H-2b leukemic target cells tested but not lipopolysaccharide- or concavanalin A-stimulated C57BL/6 lymphoblasts or non-H2b target tumor cells. The mechanism of hybridoma-mediated killing of target cells in vitro appears to be similar to that of parental CTL, although some differences have been observed. The hybridomas appear to possess neither natural killing nor antibody-dependent cytolytic activity. Clones of hybrids propagated in culture for over 6 months without the addition of known external stimulus (i.e., independent of cell growth factor and antigen) exhibit specific lytic activity against H-2b tumor cells. Such autonomous hybridomas will provide a tool for studying the mechanism of CTL-mediated lysis and the nature of the CTL receptors.

Animals↗
PubMed1981

T lymphocyte-mediated cytolysis. II. Role of target cell histocompatibility antigens in recognition and lysis.

Although compelling genetic and serologic evidence implicate target cell (TC) MHC antigens in specific cytotoxic T lymphocyte (CTL)-TC interaction leading to lysis, it is not entirely clear whether TC recognition through an MHC determinant(s) is a prerequisite for lysis to occur. In fact the finding that both specific and nonspecific TC are lysed equally well in lectin-dependent cell-mediated cytotoxicity (LDCC) challenges the necessity for TC MHC involvement in the cytolytic process beyond providing the basis for specificity in direct (nonlectin-dependent) CTL-mediated lysis. In the present paper we present evidence suggesting that even in nonspecific LDCC, as well as in nonspecific lymphocyte-mediated lysis of TC oxidized by periodate treatment or by galactose oxidase (ODCC), TC MHC components are required for lytic interactions with cytotoxic effector cells. This conclusion is based on 3 types of experimental evidence: 1) cells displaying reduced amounts of MHC proteins are poor targets in LDCC; 2) removal of H-2 by papain renders murine target cells refractory to lysis in LDCC, even though Con A binding is only slightly reduced; 3) antisera to target cell H-2-coded products block lysis in both LDCC and ODCC, whereas antisera to other cell surface antigens do not. A theory explaining nonspecific effector target interaction leading to lysis based on involvement of CTL receptor(s) and TC MHC components is presented.

Animals↗
PubMed1981

Cell surface glycoproteins of cytotoxic T lymphocytes induced in vivo and in vitro.

CTL-blasts, generated in vitro or obtained from educated spleens in vivo, have previously been shown to display high levels of a unique membrane glycoprotein T145 (ca. 145,000 daltons), inferred to be a common CTL marker and implicated in CTL cytotoxicity. As shown here, however, these suggestions do not hold for all cytotoxic T lymphocytes. We have shown that T145 is not expressed on highly active CTL-induced in vivo by a standard alloimmunization protocol. The finding that several CTL populations are T145 positive, whereas others are T145 negative, may be due to the existence of functional CTL in 2 stages of differentiation or may represent different CTL lineages. In addition, no correlation was found between cytolytic activity and expression of T145. The functional significance of T145 on CTL is discussed.

Animals↗
PubMed1981

Tumor cell destruction by cytotoxic T lymphocytes: the basis of reduced antitumor cell activity in syngeneic hosts.

Tumor overgrowth in spite of an ongoing antitumor immune response may be due to a basic immunologic defect in T cell-mediated responses against the potentially immunogenic tumor cells. To further understand T cell-mediated responses in syngeneic tumor-host systems, we have analyzed the interaction of cytotoxic T lymphocytes (CTL) with syngeneic tumor cells and have compared it with CTL-allogeneic tumor cell interaction. The major conclusions of this study are: 1) Syngeneic and allogeneic CTL lyse target cells through a similar mechanism. 2) The reduced reactivity in the syngeneic system is due to the low content of effector cells capable of binding to and killing tumor cells. 3) The avidity of CTL-syngeneic tumor cell binding is lower than CTL-allogeneic tumor cell binding. We suggest that the latter 2 observations result from a low immunogenicity of tumor cells in the syngeneic host.

Animals↗
PubMed1981

T lymphocyte-mediated cytolysis. I. A common mechanism for target recognition in specific and lectin-dependent cytolysis.

In this and the accompanying paper we examine the nature of the interactions between effector cells and target cells leading to lysis in T cell-mediated cytolysis reactions. In the first paper, we re-examine the role of lectin (Con A) in the process of lectin-dependent cell-mediated cytotoxicity (LDCC). Lectin has generally been thought to act simply as a bridge between the cytotoxic effector cell (EC) and the target cell (TC), thus bypassing the need for receptor-antigen interaction and accounting for the nonspecific character of this lytic reaction. A role for lectin in the activation of cytotoxic function in the EC has also been suggested. In this paper we confirm that LDCC occurs when TC alone are pretreated with lectin, but not when EC alone are pretreated with lectin, unless free lectin is also present in the assay mixture. In addition, we demonstrate that the failure of lectin-pretreated EC to bind to and lyse TC is not caused by agglutination or self-destruction of the EC. Moreover, lectin-pretreated EC are not inherently deficient in the ability to bind or lyse TC, if the latter are pretreated with lectin, or if free lectin is present in the assay. Finally, when EC are used as both effectors and targets in LDCC, lysis is observed only in the direction of the lectin-pretreated partner. From these results we conclude that in LDCC, as in specific CTL cytolysis, CTL-TC recognition proceeds through interaction of an EC surface receptor (distinct from the lectin receptor), and a TC antigen perhaps modified by, but distinct from, the lectin itself. In the accompanying paper we present evidence that this receptor-determinant interaction involves TC MHC antigen.

Animals↗
PubMed1980

Enucleated cytotoxic T lymphocytes bind specifically to target cells in vitro.

We report the isolation of enucleated particles (cytoplasts) from mouse cytotoxic T lymphocytes (CTLs) which, like intact CTLs, can specifically bind to target cells (TCs) in vitro. CTL-enriched populations were enucleated by centrifugation on a Ficoll density gradient at 31 C in the presence of cytochalasin B. The resulting cytoplasts which consist of 99% of enucleated particles retained about 40% of the nucleated cells' protein but less than 1% of their DNA. The presence of cell surface membrane antigens Thy 1.2 and H-2 on the cytoplasts indicated that their surface membranes originated from the plasma membranes of the intact cells. Electron microscopy of cytoplasts revealed two types of particles, one (type a) contained cytoplasmic organelles such as mitochondria, Golgi apparatus, microfilaments, and microvilli, while the second (type b) contained little cytoplasm and no microfilaments or microvilli. The evidence presented suggests that only type a cytoplasts can bind specifically to TCs. The specific binding to TCs by type a and not type b cytoplasts shows that while the CTL nucleus or nucleus-associated structures affected by enucleation are not essential for specific CTL receptor activity, cytoskeletal structures and activities related to these structures must be preserved.

Animals↗
PubMed1979

A rapid method for generating cytotoxic effector cells in vivo.

A method is described for the production of highly cytotoxic effector T lymphocytes. The cells can be raised in vivo in healthy animals in 4--5 days, thus combining the short time period of sensitization obtained in MLC or radiation GVH, without any of the attendant risks of these two methods (infection or loss of cultures or animals). DNA synthesis and blastogenesis are maximal 4 days after sensitization and are insignificant by day 6. 20--30% of the effector cells from conjugates with the sensitizing cell strain on days 4 through 7. Cytotoxicity was specific for cells of the sensitizing strain; blast cells are cytotoxic at the peak of the reaction (day 4) but small lymphocytes are cytotoxic on subsequent days.

Animals↗
PubMed1979

Competitive inhibition of cytotoxic T lymphocyte-target cell conjugation. A direct evaluation of membrane antigens involved in cell-mediated immunity.

A method to determine directly the antigenic similarity of target cells (TCs) involved in cytotoxic T lymphocyte (CTL)-TC interactions is described. When fluorescently labeled TC (FL-TCs) are bound (conjugated) to CTLs in the presence of antigenically similar but unlabeled TCs, the percentage of CTL-TC conjugates containing unlabeled TCs corresponds linearly to the number of unlabeled TCs in the TC population. This assay is specific, permits direct observation of individual CTLs and TCs which interact to form conjugates, can be completed within 10 min, eliminates complications attributable to CTL recycling, and the degree of competition observed fits that theoretically expected. The results of this paper provide the theoretical basis for "cold" (unlabeled) target inhibition of radioactive assays of lymphocyte-mediated cytolysis.

Animals↗
PubMed1978

Is the presence of serologically defined target cell antigens sufficient for binding of cytotoxic T lymphocytes?

Target cell parameters that coincide with specific binding (conjugation) of target cells to cytotoxic T lymphocytes were examined. It was demonstrated that concentrations of formaldehyde (0 to 1.2%) which do not affect the capacity of target cells to exclude dyes and incorporate sodium chromate, or their susceptibility to cellular and humoral immune lysis did not affect target cell conjugation. Treatments with higher formaldehyde concentrations (1.6 to 3.2%) caused similar decreases in all of the above-mentioned target cell parameters. Formaldehyde-treated (over 0.1%) targets that could not cap alloantigens or incorporate macromolecular precursors could still conjugate with cytotoxic T lymphocytes. Quantitative changes of serologically detected antigens alone cannot account for the decrease in binding of formaldehyde (greater than 1.2%)-treated target cells to cytotoxic T lymphocytes, since absorption of lytic alloantibodies by formaldehyde-fixed targets was not affected over the fixative range used (0 to 3.2%). From this study, it appears that certain target cell properties related to membrane structure and function and not the apparent expression of serologically defined antigens alone are essential for binding of targets by cytotoxic T lymphocytes. Additionally, it was shown that target cell susceptibility to immune lysis is retained, as long as their ability to bind to cytotoxic T lymphocytes is not affected.

Animals↗