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Biomedical subjects

J Marbrook

Publications and source records attributed to J Marbrook.

At least 37 records · Page 2Linked to original sources
PubMed1985

The use of hybridoma cells as a murine model to study tumor immunity.

The secretion of antibody by hybridoma cells allows the growth of individual cells to be measured by a hemolytic plaque assay. Similarly, plaque reduction assays were sensitive indicators of tumor immunity. A series of strains of hybridoma have been isolated from an original isolate which is sensitive to cytotoxic T cells and NK cells. In the derivation of a spleen-seeking variant, the cells appear to have lost immunogenic but not antigenic characteristics. This model lends itself to the quantitative study of immune constraints on tumor growth in vivo. Combined with the selection of tumor variants, the model is of wide application to many fields of tumor biology, particularly where sensitive measurement of tumor cell number and viability is crucial.

Animals↗
PubMed1985

The analysis of an anti-fluorescein response at limit dilution: the effect of lymphokine preparations on the apparent specificity.

The specificity of cytotoxic T cells in an anti-fluorescein response has been analysed at limit dilution. According to the ability to discriminate between fluoresceinated and non-fluoresceinated targets, the apparent specificity of CTL depends on the quality and quantity of the lymphokine preparations used to support the response. An acid-labile component of the factor preparations contributes to the generation of non-specific CTL. No cytotoxicity against NK-sensitive target cells was detected in the 'non-specific' component of the responses.

Acids↗
PubMed1984

The adaptation of the plaque reduction assay for measuring specific cytotoxic cells in limiting dilution cultures.

The use of the original haemolytic plaque reduction technique to measure cytotoxic T lymphocytes (CTL) has been developed further as a rapid screening assay, particularly suitable for limiting dilution analyses. Using hybridoma cells as targets, the cytotoxicity has been measured by the loss of haemolytic plaque formation and by the reduction of the amount of haemolytic monoclonal antibody secreted from viable target cells into the assay supernatants. The assessment of large numbers of cytotoxic samples has been greatly facilitated by quantitating the amount of haemoglobin released in the assay with an automated microELISA multiscanner and by scoring visually using a modification of the spot test. Using these new techniques, relatively high frequency estimates of cytotoxic cell precursors in an allogeneic response (1 in 462 spleen cells) and an anti-fluorescein response (1 in 3970 spleen cells) were obtained.

Animals↗
PubMed1984

T-cell development in the absence of a thymus: the number, the phenotype, and the functional capacity of T lymphocytes in nude mice.

A small but definite proportion of T-lymphocyte-like cells have been reported in nu/nu (nude) mouse spleen despite the congenital absence of a thymus in these animals. We have determined the number and the characteristics of such cells using flow cytometry. The level of T-like cells increased with age. In 4-month-old nu/nu CBA spleen, 14% of all cells expressed some Thy 1 antigen. However, only 4% expressed mature T-cell levels, and only the 2% with the highest Thy 1 also showed a normal distribution of Ly 1 and Ly 2 antigens. These T-like cells were slightly larger than normal nondividing T lymphocytes. We have assessed the total functional capacity of T-like cells in nu/nu CBA spleen using a high-cloning-efficiency limit-dilution culture system. Almost all precursor cells capable of forming clones when stimulated with concanavalin A in the presence of irradiated spleen cells and growth factors, and almost all precursors of those clones that were cytolytic in a lectin-mediated tumor-cell-lysis assay, were within this 2% subpopulation of nu/nu spleen cells with mature T-cell markers. Increased levels of purified interleukin 2 failed to induce further precursor function, indicating that maturation of pre-T cells was not obtained. However the nu/nu spleen cells bearing mature T-cell markers displayed only 10-30% of the cloning efficiency of normal splenic T cells. The majority of nu/nu spleen T-like cells, even within this phenotypically "normal" subset, appeared to be nonfunctional. We conclude that the absence of a thymus leads to qualitative, as well as quantitative, deficiencies in the T-cell population, and various interpretations are discussed.

Animals↗
PubMed1984

Regulation of cytotoxic lymphocyte precursors: I. Interactions between concanavalin A and T cell growth factors.

The induction of cytotoxic T lymphocytes by concanavalin A has been analyzed under conditions of limit dilution. The dose-response curves deviate from linearity in a way that has been interpreted as revealing successive zones of suppression as the cell concentration was increased. The magnitude of suppression was influenced by both the concentration of concanavalin A and the amount of T cell growth factors added to the culture. These regulatory events involve the cytotoxic T cell clones produced by (CBA X DBA)F1 spleen cells which are detected by DBA mastocytoma (P815) targets at a maximum detectable frequency of 1 in 2000 cells. Similar multiphase dose-response data were also obtained with syngeneic and allogeneic combinations with the same target cell. It is suggested that the successive zones of suppression and activation are a consequence of the relative frequencies of CTL-P, suppressive and helper cells, and the ease with which the cells are activated in limit dilution cultures. The experimental approach illustrates how CTL production can be manipulated to study the balance of signals required to control effector cell production.

Aging↗
PubMed1983

The analysis of an anti-fluorescein cytotoxic response.

The detailed kinetics of the appearance of clones of anti-fluorescein cytotoxic T lymphocytes (CTL) in a primary limiting dilution response have been examined. The addition of factors from the supernatants of concanavalin A-stimulated rat spleen cell cultures was necessary to obtain linear dose-response curves and strong primary responses. In a standard primary response the maximum detectable number of specific clones was found, on day 5, to be derived from precursors having a frequency of 1 in 10 000 spleen cells. When limiting dilution cultures were restimulated on day 3, responses on day 5 were unaffected but new clones appeared on day 7 with a frequency of 1 in 1000. The emergence of the day-7 response depends on the addition of factor(s) on day 3. It is proposed that the early low frequency precursors detected on day 5 and the high frequency precursors detected on days 7-9 are derived from two stages in the differentiation lineage of CTL precursors. Restimulation of limiting dilution cultures involves not only the continued clonal expansion of primary responses but also the emergence of new "clones" of specific CTL.

Animals↗
PubMed1983

Antibody-induced cell-cycle synchrony.

Synergism between specific antibodies and cytotoxic drugs in killing tumor cells is an established phenomenon. When cells grown in a suspension culture are treated with antibody, a subsequent increase in susceptibility to cytosine arabinoside has been shown to be due to the induction of cell-cycle synchrony. This observation may lead to a novel way of investigating cell-cycle-dependent processes and also an aspect of tumor-antibody interactions that is not generally recognized.

Animals↗
PubMed1983

The enhancement of cultured spontaneous cytotoxic T cells from normal and athymic (nu/nu) mice.

Subsets of natural killer cells may be characterised by the target cell profile, cell surface markers and the effect of growth factors on the production of effector cells. The subset which appears in primary cultures (spontaneous or natural T killer cells) is enhanced by the supernatants of Con A-stimulated rat spleen cultures. The enhancement can be attributed to an increased number of clones rather than an expansion of clone size in 5-day cultures. With semi-purified IL-2 preparations, clones of spontaneous cytotoxic cells are also detectable in nu/nu spleen cultures. These results suggest such spontaneous cytotoxicity is a general phenomenon within cytotoxic responses.

Animals↗
PubMed1983

Spontaneous cytotoxic T cells in murine spleen-cell cultures. I. Some characteristics of effector and precursor cells.

Cytotoxic T cells that arise spontaneously in culture (SCTL) have been characterized using a sensitive plaque reduction (PR) assay. Although SCTL effector cells are highly susceptible to anti-Thy-1 antibody plus complement treatment, they are derived from precursor cells having very low levels of Thy-1 antigen. SCTL are barely detectable in cultures of thymus cells, bone marrow cells, and spleen cells from athymic nu/nu and very old mice. Since SCTL have some similarities to both natural killer cells and normal cytotoxic T cells, SCTL may represent a stage in the T-cell lineage that links these two forms of cytotoxicity.

Aging↗
PubMed1983

Spontaneous cytotoxic T cells in murine spleen-cell cultures. II. Distinguishing between spontaneous cytotoxic T cells and NK cells according to kinetics and target selectivity.

Cytotoxic T cells that arise spontaneously in cultures (SCTL) appear to be distinct from natural killer (NK) cells. The specificity of these two types of cytotoxic cell populations has been compared by direct cytotoxicity and cold target inhibition tests. The SCTL population consists of an array of cytotoxic cells each of which is specific for a series of target cells. The NK cells had a more limited range of target selectivity although at least two types of NK effector cells were detected on the basis of specificity measurements.

Animals↗
PubMed1982

The haemolytic plaque reduction technique to measure cytotoxicity with hybridoma cells as a target.

The use of hybridoma cells as targets to measure cell-mediated cytotoxicity has been established. The ability of target hybridoma cells to form haemolytic plaques has been used as an indicator of target viability and therefore, cytotoxicity has been detected by plaque reduction (PR). Allogeneic responses, spontaneous cytotoxic responses and anti-hapten responses have been measured by the PR assay. Compared with the standard [51Cr]chromate release assay, small numbers of target cells (400 or less) can be used in the PR assay and this results in a greatly enhanced sensitivity in detecting small amounts of cytotoxicity. The ability to construct a range of hybridoma targets with the appropriate cell surface determinants presents a new approach, of general applicability, to the sensitive detection of cytotoxic lymphocytes.

Animals↗
PubMed1982

The segregation of specific clones of cytotoxic lymphocytes in an in vitro primary response against influenza virus.

CBA spleen cells have been stimulated in vitro with A/Jap influenza virus-infected CBA spleen cells to generate a 'primary' cytotoxic lymphocyte (CL) response. The culture conditions were devised to allow the segregation of individual clones of CL and cytotoxicity measured by the lysis of infected or non-infected L-929 cells. The specificity was assessed by splitting clones and measuring the ability of the clones to discriminate between pairs of targets. Influenza A/FMI and A/Jap strains were used. Subsets of clones were detected which could lyze either A/Jap-infected or A/FMI-infected target cells. In addition CL clones were found which lyzed uninfected L-929 cells and a fourth category were clones which could not discriminate between A/FMI-and A/Jap-infected targets.

Animals↗
PubMed1981

The measurement of cytotoxicity by the reduction of hybridoma plaque-forming cells.

Hybridoma cells secreting antibody against heterologous erythrocytes have been used as targets to measure cytotoxic T cell activity. The ability of the target cells to form hemolytic plaques has been used as an index of target viability. It has been possible to devise a sensitive plaque reduction method for measuring cytotoxicity which lends itself to screening small numbers of cytotoxic cells.

Animals↗
PubMed1981

Influenza A2 inhibits murine in vitro antibody synthesis.

A2 influenza inhibited in a dose-dependent fashion the in vitro antibody-forming cell (AFC) response of CBA mouse spleen cells to sheep erythrocytes. The greatest amount of inhibition occurred if virus was added to the splenocytes on the day the cells were placed in culture. Inoculation of spleen cells with influenza on day 3 of culture or immediately before assay on day 4 had no effect on the AFC response. Experiments indicated that the inhibition could be transferred by adding viable, infected cells to uninfected spleen cultures. The origin of the cells that caused the suppression of AFC was not resolved; however, the data suggest that infected B or T cells were not solely responsible for the inhibition. Although infectious virus was most effective, inactivated influenza preparations also suppressed the AFC response. The possible in vivo significance of influenza's inhibitory effect on antibody synthesis is discussed.

Animals↗
PubMed1979

Biochemical and biological characterization of lymphocyte regulatory molecules. I. Purification of a class of murine lymphokines.

Murine spleen cells activated by concanavalin A (Con A) in culture produce a class of lymphokine molecules which possess biological activity in a number of lymphocyte response assays. Lymphokines with a mol wt of 30,000, as estimated from gel filtration studies, can be resolved into two components which differ by charge, with isoelectric point (pI) values of 4.3 and 4.9, respectively. Both components stimulate (a) the growth of established T-cell lines in culture, (b) the proliferation of thymocytes in the presence of Con A under culture conditions where Con A alone is nonmitogenic, (c) the induction of antibody responses to heterologous erythrocyte antigens in athymic (nude) spleen cultures, (d) the generation of cytotoxic T lymphocytes (CTL) in thymocyte cultures, and (e) the generation of CTL in nude spleen cultures. In each of these culture systems we suggest that the assays are detecting a single class of lymphokine which acts directly on activated T cells. Nonactivated T cells must be stimulated by either antigen or mitogen before becoming responsive to lymphokine, but do not require antigen or mitogen for continued growth with lymphokine. The two molecular species, separable by isoelectric focusing are referred to as the T-cell growth factor (TCGF). A lymphokine, similar in size (30,000 daltons) to TCGF but heterogeneous in charge (pI 3.0--4.0), stimulates immune responses to erythrocyte antigens in T-cell-depleted spleen cultures but has no stimulatory activity in the other lymphocyte assay systems described. The data have been interpreted as showing the two molecular forms of murine TCGF (pI 4.3 and 4.9) are responsible for many of the lymphokine activities described elsewhere as thymocyte mitogenic factor, nonspecific T-cell-replacing factor and killer helper factor or costimulator. The other lymphokine, separable from TCGF by charge, appears to have true T-cell-replacing activity.

Animals↗
PubMed1979

The clonal analysis of cytotoxic lymphocytes against 2,4,6-trinitrophenyl (TNP)-modified cells.

The frequency and specificity of clones of cytotoxic lymphocytes (CL) produced when (CBA X C57BL)F1 spleen cell populations were cultured with 2,4,6-trinitrophenyl (TNP)-modified syngeneic F1 cells, was examined. The frequency of clones which lysed F1-TNP targets was 1/3.3 X 10(4) spleen cells, and the frequency of clones which lysed the modified parental cells, CBA-TNP and C57BL-TNP was 1/6.7 X 10(4) and 1/2.9 X 10(5) spleen cells, respectively. Using a clonal analysis of the specificity of the CL, it was shown that the majority of the clones of CL which lysed the two modified parental tarets, were specific for one or the other of the targets. Activity against modified allogeneic DBA/2-TNP targets was also detected. The activity against DBA/2-TNP targets was due mainly to clones of CL which were specific for DBA/2-TNP targets. Only a minor part of the activity was due to clones which were cross-reactive for both F1-TNP and DBA/2-TNP target cells.

Animals↗
PubMed1979

Some limits to post-antigen generation of diversity: failure to detect variants in clones of hapten-specific antibody-forming cells (AFC) developing in culture from direct AFC-progenitor B cells.

A search was made for variants in clones of hapten-specific antibody-forming cells (AFC) arising by stimulation of mature B cells with either thymus-independent hapten-POL (polymerized bacterial flagellin) conjugates, or the polyclonal activator lipopolysaccharides. Enriched, hapten-binding B cells or unfractionated spleen cells were cultivated for 3-4 days at limiting dilution in the presence of thymus filler cells, and the AFC in each microculture well were then assayed for plaque formation on various hapten-sheep red cell monolayers. No variants were found from (4-hydroxy-3-iodo-5-nitrophenyl) acetyl (NIP) to 2,4-dinitrophenyl specificity, nor from fluorescein (FLU) to NIP specificity. No variants were found in avidity for FLU hapten. All 374 clones examined, including clones of up to 300 AFC, appeared to be homogeneous in antibody specificity and plaque morphology under our conditions. These results differ from published findings using erythrocytes as antigens. Reasons for this discrepancy are discussed, including differences in sensitivity differences in immunological similarity between the test antigens, and in the particular B cell subsets involved.

Animals↗