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K T Brunner

Publications and source records attributed to K T Brunner.

At least 19 recordsLinked to original sources
PubMed1979

Cell-mediated immunity to antigens associated with murine sarcoma virus-induced tumors: augmentation of cytolytic T lymphocyte activity by successive specific and nonspecific stimulation in vitro.

It has been shown previously that lymphoid cells from mice which have rejected a tumor induced by the Moloney sarcoma virus-leukemia virus (MSV-MLV) complex develop high levels of specific H-2-restricted cytolytic T lymphocyte (CTL) activity after in vitro stimulation with syngeneic, irradiated MLV-induced lymphoma cells in mixed leukocyte-tumor cell cultures (MLTC). Attempts to further increase lytic activity by restimulating long-term MLTC cells with syngeneic, irradiated lymphoma cells have met but with limited success. This report shows that, in contrast to the lack of increased activity observed after specific stimulation with lymphoma cells, nonspecific stimulation of long-term MLTC cells either with supernatants from secondary mixed leukocyte cultures (2 degrees MLC SN) or with supernatants from concanavalin A-stimulated spleen cells leads, on a per cell basis, to a further 5 to 10-fold increase in CTL activity. The stimulatory activity of 2 degrees MLC SN is due to a factor(s) of apparent mol. wt. of 25,000 to 40,000. The activity of the CTL populations formed under these conditions is at least 100-fold higher against syngeneic as compared to allogeneic MLV-induced or unrelated tumor cells.

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PubMed1979

Generation of cytolytic T lymphocytes in vitro. XII. Detection of memory CTL precursors in peripheral blood of alloimmune mice.

The memory cytolytic T lymphocyte (CTL) response of mice was studied in vitro using peripheral blood leucocytes (PBL) of donors previously immunized with allogeneic cells as responding cells, and particulate alloantigen for stimulation. It is shown that alloimmune but not normal PBL respond to particulate antigen and that small numbers of PBL generate high CTL responses provided non-T accessory spleen cells are added to the cultures. The system provides means to analyse the kinetics of the appearance of memory CTL precursors after primary alloimmunization in individual mice.

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PubMed1979

Generation of cytolytic T lymphocytes in vitro. X. Induction of primary and secondary CTL responses by the mitogen sodium periodate.

Short-term (15 min) sodium periodate (NaIO4) treatment of mouse spleen cells previously primed in vivo or in vitro against alloantigens induced the formation of secondary (2 degree) cytolytic T lymphocytes (CTL) specific for the priming antigens. CTL formation was readily demonstrable within 24 hr after treatment. This early CTL response occurred equally well in the presence or absence of cytosine arabinoside (Ara C), indicating that NaIO4 could induce CTL independently of DNA synthesis. Forty-eight hours after periodate treatment, the lytic activity was similar to that observed in parallel cultures stimulated with irradiated allogeneic spleen cells, although the peak activity was reached earlier (day 4) and was somewhat lower than that induced by alloantigen. The addition of irradiated NaIO4-treated unprimed syngeneic spleen cells to cultures of untreated alloimmune spleen cells also led to CTL formation, which suggests an indirect mechanism of activation. In contrast to alloimmune spleen cells, normal spleen cells treated with NaIO4 developed only very low levels of cytotoxicity after 4 days of incubation. However, in the presence of PHA, such cells were capable of lysing syngeneic and allogeneic target cells.

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PubMed1978

Cholesterol is a critical cellular component for T-lymphocyte cytotoxicity.

Preincubation of cytolytic T lymphocytes (CTLs) generated in secondary C57BL/6 anti-DBA/2 mixed leukocyte cultures with an inhibitor of cellular cholesterol synthesis (25-OH-cholesterol) for 24 hr strongly depressed the cytolytic activity as determined in a 3-hr 51Cr assay. The effect of the inhibitor was reversed by the simultaneous addition of cholesterol or of mevalonic acid during the preincubation period (mevalonate is the product of the regulatory enzyme in the sterol synthesis pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (NADP) [mevalonate:NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.34]). Because, under the same culture conditions, inhibition of DNA synthesis had no effect on CTL activity, the experiments suggest that the effect of 25-OH-cholesterol is related to its inhibitory effect on sterol synthesis, resulting in decreased levels of membrane-bound cholesterol, rather than to inhibition of cellular proliferation.

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PubMed1977

Mechanism of target cell lysis of cytolytic T lymphocytes. I. Characterization of specific lymphocyte-target cell conjugates separated by velocity sedimentation.

Differential velocity sedimentation was applied for separating alloimmune T lymphocytes bound to target cells (TC) from free lymphocytes. Maximal size differences between lymphocytes and TC were achieved a) by isolating the fraction of small peritoneal lymphocytes (SPL) from an alloimmune peritoneal cell population, and b) by selecting large tumor cells as TC. Under the conditions used, most of the conjugates formed at room temperature consisted of one SPL bound to one TC, and adequate separation of bound from free SPL could be achieved within less than 5 min. Functional studies of the conjugate-enriched fractions showed that a minimum of 60% of TC-bound SPL were indeed cytolytic. Conjugate-depleted fractions, however, were still lytic, suggesting that not all effector cells formed stable conjugates at room temperature. Transmission and scanning electron microscopy studies revealed that binding between SPL and TC was achieved through interpenetrating membrane projections and was characterized by point and broad zone contacts. When lysis was allowed to proceed, prominent changes of the TC membrane morphology, including loss of microvillous projections, appearance of localized blebs, pseudopod-like projections, and membrane defects were documented.

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PubMed1976

Enzymatic removal of H-2 alloantigens from the surface of P815-(X2) mouse tumor cells.

After treatment with papain (6 mg/ml) for 1 h, P815 tumor cells became resistant to complement-mediated lysis by mouse alloantibodies of different specificities. Immunofluorescence and absorption studies indicated that this resistance was associated with removel of the corresponding antigenic determinants from the cell surface. In contrast, papain-treated tumor cells were fully susceptible to lysis by rabbit antiserum against P815 cells, indicating a) no alteration of the membrane sensitivity to complement damage, and b) a dissociation between structure and/or localization of allo- and xenoantigens. Papain-treated cells were also completely resistant to lysis by cytolytic T lymphocytes (CTL) during the first 60 min after completion of the enzyme treatment. Susceptibility to lysis by either CTL or alloantibody and complement reappeared within a few hours after incubation in culture medium and was virtually normal by 6 h. Treatment of P815 cells with trypsin (2 mg/ml) had no effect on either humoral or cellular lytic activities.

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PubMed1976

Antigenic specificity of the cytolytic T lymphocyte (CTL) response to murine sarcoma virus-induced tumors. I. Preferential reactivity of in vitro generated secondary CTL with syngeneic tumor cells.

Incubation of spleen cells from mice having rejected a Moloney sarcoma virus (MSV)-induced tumor with syngeneic irradiated lymphoma or sarcoma cells bearing MSV-associated antigens in secondary mixed leukocyte-tumor cell cultures (MLTC) resulted in the generation of highly active cytolytic T lymphocytes (CTL) specifically directed against syngeneic target cells bearing MSV-associated antigens. When MSV-immune spleen cells from C57BL/6 (H-2b) and BALB/c(H-2d) mice were compared with respect to their ability to generate CTL in syngeneic secondary MLTC, it was found that both lymphoid cell populations were equally able to mount an anamnestic CTL response to MSV-associated antigens as assessed by a short-term 21Cr release assay. However, quantitative analysis of the activity of both CTL populations on either H-2b or H-2d tumor cells indicated that target cells sharing the same major histocompatibility complex (MHC) as the effector cells were lysed 10- to 100-fold more efficiently than allogeneic target cells. As suggested by the results of inhibition experiments using mixtures of 51Cr-labeled and unlabeled target cells, preferential lysis of syngeneic versus allogeneic tumor cells might be related to the establishment of effective adhesions between the former and CTL. Direct evidence for the role of MHC in determining the antigenic specificity of CTL directed against MSV-associated antigens was provided by results obtained using MSV-immune spleen cells from congenic resistant mice. Furthermore, studies of the response of F1 (H-2b/d) hybrid mice showed that stimulation of immune spleen cells with tumor cells from one parental strain or the other in secondary MLTC resulted in the generation of CTL capable of lysing tumor target cells of the same perental strain as the stimulating cells, but not of the other. The results thus suggested the presence of two sets of CTL precursor cells in F1 MSV-immune spleens, each set responding exclusively to tumor antigens associated with only one of the two parental phenotypes.

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PubMed1975

Generation of cytotoxic T lymphocytes in vitro. IV. Functional activation of memory cells in the absence of DNA synthesis.

Re-exposure of day 14 mixed leukocyte culture (MLC) cells to the original stimulating alloantigens (secondary response) has previously been shown to result in significant proliferation and in rapid reappearance of high levels of cytolytic T-lymphocyte (CTL) activity within the next 4 days. Moreover, evidence has been presented that CTL precursor cells in day 14 MLC populations, while they derived from cells were large at peak of the primary response (day 4) were themselves small lymphocytes which developed into large CTL after restimulation. In this study, inhibition of DNA synthesis by cytosine arabinoside (ARA-C) was used to investigate whether CTL formation could be dissociated from proliferation during the secondary response. It was found that within the first 24 h after restimulation (a) CTL activity increased 6-to-20-fold, (b) 60-70% of the small T lymphocytes became medium- to large-sized cells, and (c) both events were independent of DNA synthesis. By using two successive cell separations by velocity sedimentation at unit gravity, before and after stimulation of day 14 MLC cells for 24 h in the presence or absence of ARA-C, direct evidence was obtained that small CTL precursor cells developed into large CTL, irrespective of DNA synthesis. The presence of ARA-C for periods longer than 24 h inhibited any further increase in CTL activity, in contrast to a parallel increase in lytic activity and cell number from day 1 to day 4 in control restimulated cultures. Taken together with the finding that 90% of the medium- and large-sized lymphoid cells in control restimulated cultures underwent DNA synthesis within 24 h, these results thus suggest that during a secondary MLC response there is initially a differentiation step leading to the formation of CTL which, although it can be clearly dissociated from DNA synthesis, is under normal conditions followed by proliferation of these effector cells.

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PubMed1975

Effect of delayed addition of 2-mercaptoethanol on the generation of mouse cytotoxic T lymphocytes in mixed leukocyte cultures.

The generation of mouse cytolytic T lymphocytes (CTL) in primary or secondary mixed leukocyte cultures (MLC) is greatly enhanced by the addition of 2-mercaptoethanol (2-ME) to the culture medium. This enhancement can be equally demonstrated with either reduced or oxidized 2-ME. Addition of 2-ME to MLC as late as 3 days after the initiation of the culture results in a peak CTL response which is nearly as high as the one in cultures containing 2-ME throughout the 4 day-incubation period. Quantitative analysis of the CTL response in MLC supplemented with 2-ME on day 3 shows a 50-fold increase in lytic activity within 24 h, suggesting that cell differentiation, in addition to proliferation may be affected by this agent.

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PubMed1975

Primary and secondary in vitro generation of cytolytic T lymphocytes in the murine sarcoma virus system.

Cell-mediated cytotoxic responses in vitro to surface antigens associated with murine sarcoma virus (MSV)-induced tumors were investigated using mixed leukocyte-tumor cell cultures (MLTC). The source of responding cells was either spleens from normal C57BL/6 mice (primary MLTC) or spleens of C57BL/6 mice carrying or having rejected a MSV-induced tumor (secondary MLTC). Graffi virus-induced GiL-4 leukemia cells, Rauscher virus-induced RB1-5 leukemia cells, and MSV-induced MSV-B16 sarcoma cells were used as stimulating syngeneic tumor cells and/or target cells. Under appropriate culture conditions, cytolytic T lymphocytes (CTL) were generated in both primary and secondary MLTC. As assessed by a quantitative short-term 51Cr release assay system, CTL activity in secondary MLTC populations was at least 10-fold higher than that in primary MLTC populations, and 100-fold higher than that in spleen cells taken at the peak of the in vivo response of MSV-infected mice. The ability of spleen cells to mount a secondary CTL response in vitro could be observed as early as 5 days after virus injection, increased up to the time of maximum tumor size and persisted long after tumor regression. This suggests the development of increased numbers of CTL progenitors and/or the formation of "memory" CTL in spleens of MSV-injected mice.

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PubMed1975

Generation of cytotoxic T lymphocytes in vitro. VI. Effect of cell density on response in mixed leukocyte cultures.

Reexposure of day 14 murine mixed leukocyte culture (MLC) populations to the original irradiated allogeneic stimulating spleen cells has previously been found to result in the ratpid generation of cytolytic T lymphocytes (CTL) associated with a net increase in cultured cell number. Under the experimental conditions used, day 5 MLC cells appeared unable to respond to the allogeneic stimulus. In order to characterize further the development of the potential for anamnestic reactivity during the course of MLC, C57BL/6 spleen cells were incubated with irradiated (1000 rads) DBA/2 spleen cells (primary MLC) for up to 3 weeks. At various time intervals after the onset of the primary MLC, the surviving cells were collected and reexposed, at varying cell concentrations, to irradiated DBA/2 spleen cells (secondary MLC). At daily intervals thereafter, CTL activity was assessed using a quantitative 51Cr-release assay system. A paradoxic effect of responding cell concentration on generation of CTL activity was observed; relatively greater increase in CTL activity was observed as the concentration of responding cells was decreased over a 100-fold range. This effect was more pronounced with responding cells reexposed to antigen after primary MLC for 20 days, but was observed even with normal cells. The apparent unresponsiveness of day 5 MLC cells to alloantigen restimulation could be overcome by simple dilution of responding cells. Cytotoxic activity at the time of restimulation with antigen seems to be a major factor determining the magnitude of the secondary response. Since intact cells bearing alloantigens are required for the generation of CTL in MLC, residual cytotoxic cells reduce the effective antigenic stimulus by destroying stimulating cells. This effect of concentration of responding cells on generation of CTL in MLC complicates interpretation of experiments investigating the role of "inhibitor" and "helper" cell in cell-mediated immune responses occurring in vitro. Under optimal conditions, the highest CTL activity and the largest increase in total cell number was observed 4 days after restimulation of day 10 MLC cells. On a per cell basis, the lytic activity was up to 4 times greater than that observed at the peak of a primary response, and the number of viable cells recovered was nearly 20 times higher than that at the onset. Such secondary MLC are thus a convenient source of lymphoid cells selected primarily on the basis of proliferation induced by alloantigens.

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PubMed1975

Generation of cytotoxic T lymphocytes in vitro. V. Response of normal and immune spleen cells to subcellular alloantigens.

Subcellular particulate membrane fragments prepared from murine lymphoid or tumor cells were used as a cell-free antigen source in order to stimulate the generation of cytolytic thymus-derived effector cells (CTL) in vitro. When cultivated with normal spleen cells as a source of responding lymphocytes, particulate antigen preparations induced only low CTL activities; in contrast, virtually normal responses were observed when immune spleens were used as a source of responding cells. In both cases, results were compared to those responses obtained with intact irradiated (1000 rads) normal spleen cells as stimulating antigen. The effector cells generated with particulate antigen preparations (obtained either by hypotonic shock or by sonication) were shown to be T cells and were characterized with regard to the kinetics of their response and their dose-activity relationship. It was also shown that the responses observed were specific, both at the level of initiation and at the effector level. The results obtained suggest that there exists a fundamental difference between normal and alloimmune spleen cells in their ability to respond in vitro to stimulation by subcellular antigen preparations.

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PubMed1974

Generation of cytotoxic T lymphocytes in vitro. III. Velocity sedimentation studies of the differentiation and fate of effector cells in long-term mixed leukocyte cultures.

Separation of cells by velocity sedimentation at unit gravity was utilized to investigate the physical properties of cytotoxic thymus-derived lymphocytes (CTL) generated in long-term mixed leukocyte cultures (MLC). In kinetic studies, CTL were found almost exclusively in the large cell fractions at the peak of the response on day 4, whereas the majority of CTL in day 14 MLC had the sedimentation properties of small lymphocytes. Reculture until day 14 of cells fractionated on the basis of size on day 4 indicated that the small CTL were derived exclusively from cells which had been large on day 4. Re-exposure of day 14 MLC cells to the original stimulating alloantigens resulted in significant cell proliferation and rapid regeneration of CTL activity. Cell fractionation experiments demonstrated that the cells in the day 14 MLC population which responded to the secondary allogeneic stimulus were small T lymphocytes, and that these cells rapidly developed into large, highly cytotoxic CTL following stimulation. Moreover, by restimulating on day 14 fractions which were selected on the basis of size on day 4, it was found that the responding small lymphocytes were themselves the progeny of cells which were large at the peak of the response. Since CTL and CTL progenitors showed concomitant changes in physical properties with time, the possibility exists that they belong to the same cell lineage, and hence that CTL can differentiate into cells which are no longer cytotoxic, but capable of mounting an anamnestic response.

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