Biomedical subjects

G F Burns

Publications and source records attributed to G F Burns.

At least 73 records · Page 4Linked to original sources

PubMed1985

TLiSA1, a human T lineage-specific activation antigen involved in the differentiation of cytotoxic T lymphocytes and anomalous killer cells from their precursors.

The characteristics of a novel T lineage-specific activation antigen, termed TLiSA1, are described. The antigen was detected with a mouse monoclonal antibody, LeoA1, that was raised against activated human T cells generated in mixed lymphocyte culture (MLC). The antigen became strongly expressed on T cells 48-72 h after stimulation with phytohemagglutinin, and retained expression on MLC-activated T cells after 10 d of culture. The antigen was absent from a range of human T, B, myeloid, fibroblast, and tumour cell lines, but was present on the surface of the interleukin 2 (IL-2)-dependent gibbon cell line MLA-144. Analysis of the antigen by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitates obtained from activated human T cells demonstrated a broad band in the region of 70 kD, whereas precipitates obtained from MLA-144 revealed a single narrow band of 95 kD. The molecule was expressed with a maximum density of 66,000 copies per cell on the surface of MLC-activated T cell blasts, as assessed by Scatchard analysis. TLiSA1 was distinguished from the IL-2 receptor bound by the anti-Tac monoclonal antibody by demonstrating that the antigens did not comodulate or coprecipitate, and by constructing an IL-2-independent human T X T hybrid that expressed the TLiSA1 but not the Tac antigen. MLC with B lymphoblasts was used to generate cytotoxic T lymphocytes (CTL) specific for the stimulating cell, and anomalous killer (AK) cells able to kill melanoma target cells. The presence of LeoA1 or F(ab')2 fragments of the antibody from the beginning of coculture did not affect proliferation in these cultures, but did inhibit the induction of both CTL and AK cells from their precursors. This inhibition of differentiation by LeoA1 was confirmed under conditions of limiting dilution, where it was shown that the antibody reduced the frequency of CTL produced, and greatly (fourfold) reduced the frequency of AK cells generated from their precursors. We discuss the possibility that human CTL may express a differentiation factor receptor that is distinct from the receptor for IL-2.

Adult↗

PubMed1985

The isolation of natural killer (NK)-resistant variants of the K562 cell line by mutagenesis and selection with antibodies which inhibit NK cell-mediated lysis.

Mechanisms involved in the lysis of tumor cells by natural killer (NK) cells were investigated by using mutagenized K562 targets resistant to the effects of NK cells. K562 cells were treated with the mutagen methyl methanesulfonate (MMS) and, to select for resistant mutants, rabbit anti-idiotypic (anti-id) antibodies were used. This anti-id was raised to a monoclonal antibody 9.1C3 which itself blocked lysis by NK cells by binding to the effector cells; the anti-id inhibited killing by binding to the K562 targets, presumably to a cell surface protein relevant to a secondary event in the NK lytic pathway. MMS-derived mutants showed a heterogeneity of staining with the anti-id, allowing the antibody to be used with flow cytometry to select a population of K562 cells relatively negative in antigen expression. The degree of reactivity of K562 cultures with the anti-id antiserum and the resistance to lysis by NK cells were inversely related. Cultures of NK-resistant K562 cells with low expression of the anti-id structure were cloned by limiting dilution: 96 clones were analyzed and one subclone, C9/2, which was six-to sevenfold less sensitive to lysis than the parental K562 cell line, was used in further studies by cold target inhibition and single cell binding assays. The increased resistance to lysis of C9/2 was not due to a reduced expression of target recognition structures, and resistance could not be overcome by prolonging the time allowed for lysis to 18 hr nor by adding exogenous recombinant leukocyte interferon. Killing of the NK-resistant variant was inhibited by mannose-6-phosphate but not by the monoclonal antibody against which the anti-id antibody was raised. It is therefore suggested that the structure on the K562 cells recognized by the anti-id antibodies is a novel secondary receptor which is important in the later stages of the NK cell cytolytic cascade.

Antibodies, Monoclonal↗

PubMed1985

In vitro generation of human activated lymphocyte killer cells. II. N-acetyl-D-galactosamine inhibits a distinct subpopulation of human activated lymphocyte killer cells generated in mixed lymphocyte culture.

A range of monosaccharides was tested for its ability to inhibit the generation of cytotoxic cells during mixed lymphocyte culture. The most discriminatory effect was produced by N-acetyl-D-galactosamine (NADG). The presence of this sugar at the initiation of the coculture significantly inhibited in a dose-dependent manner the induction of a subset of nonspecific activated lymphocyte (ALK) cells preferentially able to lyse the K562 target cell (natural killer, NK-like cells) but had no effect on the generation of either specific cytotoxic T lymphocytes or another separate subset of ALK cells mediating lysis of an NK-insensitive melanoma cell line. The addition of conditioned medium containing interleukin 2 and interferon (IFN) at the start of culture reversed the inhibitory effect of the sugar. Under conditions of limiting dilution, the frequency of NK-like precursors ranged from 1/50 to 1/1200 with different mononuclear cells (MNC) and in all cases the presence of NADG from Day 0 of culture selectively decreased the frequency of these precursors. At the concentrations used NADG had no effect on NK-like cell cytolysis once generated. The addition of recombinant gamma-IFN did not abrogate the inhibitory effect of NADG and in MLC of some individuals decreased the frequencies of ALK cell precursors. These data provide further evidence for the heterogeneity of ALK cells and indicate that what is usually referred to as NK-like cell activity in in vitro culture is mediated by a subpopulation of MNC which are activated and induced to differentiate along a pathway independent of that of other ALK subsets.

Acetylglucosamine↗

PubMed1985

Lymphocytes from patients with hairy cell leukaemia enable the distinction of two separate subpopulations of activated lymphocyte killer cells generated in mixed lymphocyte culture.

Blood from patients with hairy cell leukaemia (HCL) was used to investigate subpopulations of the non-specific activated lymphocyte killer (ALK) cells generated in mixed lymphocyte culture (MLC). Mononuclear cells from four of five patients with HCL had decreased natural killer (NK) cell activity and, of these, all showed a decrease in ALK cell activity against K-562 target cells (NK like cells), but three had normal levels of ALK cells able to kill melanoma cells. One patient who had received splenectomy had normal NK cell activity, and in MLC his cells generated normal levels of both subpopulations of ALK cells. The results support the hypothesis of separate subsets of ALK cells: NK like cells with NK cell precursors and which preferentially lyse the K-562 target cell and a second separate population which arises independent of NK cell precursors and which has strong cytolytic activity against an NK insensitive melanoma cell line.

Cell Line↗

PubMed1985

Antibodies to the T3 antigen complex on human T cells render thymocytes unresponsive to mitogen.

Human thymocytes differed from peripheral blood T cells, in that they did not proliferate in response to mouse monoclonal antibodies specific for the T3 antigen complex (UCHT1 and OKT3), even in the presence of T-cell growth factor (IL-2) or with monocytes plus IL-2. The failure to respond was not the result of inhibition by cortical thymocytes, since OKT6-negative cells also did not respond when tested under conditions of limiting dilution. Of more importance, these antibodies rendered human thymocytes unresponsive to the lectin phytohaemagglutinin when added before, during, or immediately after addition of the lectin, an effect which was much more profound than the decrease in mitogenesis caused with blood mononuclear cells. These results illustrate a clear difference between medullary thymocytes and peripheral T cells in their ability to respond to signals transduced through the T3 antigen complex.

Adolescent↗

PubMed1985

Identification of a structure on human melanoma cells recognized by CTL exhibiting anomalous killer cell function.

The structures involved in the recognition of melanoma cells by nonspecific cytotoxic T lymphocytes (CTL) activated in mixed lymphocyte culture were investigated with monoclonal antibodies (MAb) which blocked this anomalous killer (AK) function. Of over 2000 MAb raised against melanoma cells, only three inhibited killing; one of these, an IgMk termed Leo Me13, was investigated in detail. In antibody-binding studies using a large range of cultured tumor cells, it was shown that Leo Me13 was relatively specific for melanoma cells. Of more importance, Leo Me13 inhibited conjugate formation between AK cells and melanoma target cells by 60 to 80% and caused an eight- to 10-fold reduction in killing. The MAb did not immunoprecipitate protein from melanoma cells surface-labeled with 125I, and thin-layer chromatography followed by immunoblotting of the separated glycolipids from melanoma cells indicated that the epitope was on acidic glycolipids migrating between GM1 and GD1a; moreover, treatment of melanoma cells with neuraminidase resulted in complete loss of binding of Leo Me13 but not of other anti-melanoma antibodies which did not inhibit AK cell-mediated lysis. Other melanoma-reactive MAb of the same isotype as Leo Me13 did not block killing of melanoma cells, but one documented antibody, R24, an IgG3 with specificity for the ganglioside GD3, was found to inhibit this function. These data suggest that the AK cells recognize and bind to melanoma cells by a secondary "lectin-type" receptor for a carbohydrate moiety.

Animals↗

PubMed1985

Rapid changes in surface antigen expression by blood monocytes cultured in suspension or adherent to plastic.

Cell surface antigens used as markers of in vivo differentiation may not be stable on monocytes maintained under different conditions of in vitro culture. Monocytes were isolated from blood by centrifugation over Percoll or by adherence to plastic dishes, and the cells cultured in suspension or as adherent monolayers. Initially, monocytes obtained by both methods were similar in size, morphology, and surface antigen expression detected with the antimonocyte monoclonal antibodies OKM1, FMC17, PHM2, and PHM3. After culture, cells maintained in suspension were predominantly small, whereas those adherent to plastic rapidly increased in size; however, cytochemical staining for nonspecific esterase and acid phosphatase showed increased enzymic activity by monocytes in both systems, possibly reflecting increased cell maturation. The most striking difference was a substantial loss of FMC17 antigen by most monocytes within four hours in suspension culture, as compared with a qualitative and quantitative increase in expression by plastic adherent cells within two hours. These changes occurred even if the cells were first reacted with lipopolysaccharide. Monocytes taken from suspension culture and allowed to adhere to plastic rapidly synthesized the antigen, a process inhibited by cycloheximide, and conversely, cells removed from plastic progressively displayed decreased FMC17 antigen expression when transferred to suspension culture. No functional role in adherence or phagocytosis has been found for the FMC17 antigen. The results suggest that antigen expression may depend as much on the physical state of the cells as on apparent activation or maturation events.

Acid Phosphatase↗

PubMed1984

Transfer of human nasal papilloma into nude mice.

We transferred tumor tissue from two inverted schneiderian nasal papillomas to hypothymic nude mice. Tissue from one tumor, which later underwent malignant change, was transferred three times. Forty days lapsed before growth was evident, with a subsequent period of rapid growth. Histologic appearances of the primary tumor and xenografts were similar. Although the data are derived from only two cases, our findings suggest that the capacity of these tumors to grow in nude mice may be an index of their malignant potential.

Adolescent↗

PubMed1984

High frequency of precursors of anomalous killer cells in human peripheral blood: evidence for T-cell regulation.

The frequency of precursors (P) of the anomalous killer (AK) cells able to kill a melanoma target cell line without prior sensitization was determined by limiting dilution analysis. The frequencies obtained from the peripheral blood mononuclear cells of six healthy individuals ranged from 1/250 to 1/750, which was considerably higher than those of alloreactive cytolytic T-lymphocyte (CTL) precursors induced in the same cultures (range 1/900 to 1/7500). The presence of phytohemagglutinin (PHA) inhibited the appearance of both CTL and AK in bulk cocultures, and in limiting dilution analysis the presence of the lectin resulted in multiphasic cell dose-response curves rather than linear single hit responses for both types of precursor cells. The results suggest that AK-P are under the same type of regulation as are CTL-P.

Cell Differentiation↗

PubMed1984

Epitope diversity of monoclonal antibodies revealed by cross-species reactivity.

The epitope specificity of two monoclonal antibodies (MAb) which have the same functional activity has been studied. These two independently raised rat IgG2b MAb, NIMP-R10 and M1/70 (Springer et al., 1979), blocked the complement (C) receptor on mouse macrophages. Both MAb showed essentially the same binding pattern with mouse cells, binding to the same extent mouse eosinophils, macrophages, neutrophils, a small proportion of spleen and bone marrow cells, but not thymocytes. That both MAb were apparently recognizing the same epitope was suggested from experiments in which MAb M1/70 inhibited the binding of MAb NIMP-R10. In addition, both MAb showed identity at the molecular level, precipitating the same molecules from the surface of mouse cells. However, NIMP-R10 and M1/70 could be shown to recognize different epitopes when they were tested on human cells. Thus, NIMP-R10 was found to bind to neutrophils and to large granular lymphocytes with natural killer cell activity but not to eosinophils or monocytes, while M1/70 bound to all of these cell types. It is suggested that inter-species testing may have general application in the analysis of antibody specificity.

Animals↗

PubMed1984

A mature myeloid antigen identified on human natural killer cells.

A rat monoclonal antibody (MAb), NIMP-R10, selected for binding to mouse granulocytes was found to identify all mature human peripheral blood neutrophils and a subpopulation of mononuclear cells. By cell sorting experiments the mononuclear cells labelled with NIMP-R10 were shown to be predominantly large granular lymphocytes encompassing most of the natural killer cell activity of peripheral blood cells. NIMP-R10 blocks the complement receptor on mouse myeloid cells, but the MAb was found to have no such biological activity when human cells were examined.

Animals↗

PubMed1984

Activated lymphocyte killer cells derived from melanoma tissue or peripheral blood.

Lymphoid cells infiltrating metastatic melanomas were grown directly from cell suspensions of tumour tissue by the addition of T cell growth factor. Lymphoid cells grew out at the expense of tumour cells in six of seven freshly excised tumours, and cells from two cultures were expanded for in vitro testing of cytolytic function against different target cells. Early in culture the tumour derived lymphocytes killed fresh autologous melanoma cells and, particularly later in culture, were highly and non-specifically cytolytic for cultured melanoma and non-melanoma cells. Cultured peripheral blood lymphocytes from patients with melanoma, and from normal subjects, were cytolytic to the same degree as tumour derived lymphocytes, and also resembled cells grown from tumour tissue in possessing acid phosphatase activity which was resistant to tartrate. Cultured lymphoblasts from both tumour and peripheral blood had a T cell phenotype when analysed with monoclonal antibodies. An in vitro co-culture system was employed to study the kinetics and the precursors of these non-specific killer cells among blood mononuclear cells. Blood mononuclear cells cultured with irradiated B lymphoblasts led to the generation of non-specific cytolytic cells, referred to as activated lymphocyte killer (ALK) cells, after 7-10 days of culture and the progenitors of these ALK cells were demonstrated to be distinct from those of specific cytolytic T cells.

Cells, Cultured↗

PubMed1984

A novel antigenic cell surface protein associated with T200 is involved in the post-activation stage of human NK cell-mediated lysis.

A monoclonal antibody, 9.1C3, was used to investigate the mechanism of natural killer (NK) cell-mediated lysis. In addition to blocking NK cell function, the antibody blocked antibody-dependent cellular cytotoxicity against the K562 target cell at the effector cell level. The stage at which 9.1C3 antibody inhibited cytolysis was established with a Ca++ pulse technique, whereby it was shown that the antibody inhibited killing at a discrete step after the Ca++-dependent programming for lysis. The 9.1C3 antigen appeared to be associated with the T200 glycoprotein complex. Thus the 66 and 77 Kd proteins detected by 9.1C3 were also precipitated with a monoclonal antibody to T200, and in sequential immunoprecipitations, 9.1C3 antibody removed these bands from immunoprecipitates with antibody to T200. Also, in co-modulation studies, it was found that antibody to T200 co-capped the 9.1C3 antigen but that capping with 9.1C3 antibody did not induce co-modulation of the T200 antigen. Expression of the 9.1C3 and T200 antigens on different cell types, however, was not identical, and the 9.1C3 antibody did not immunoprecipitate high m.w. proteins in the region of 200 Kd. Functionally, in NK cell killing studies, the antibody to T200 used alone did not block but was synergistic with the 9.1C3 antibody. The differential effect of the enzymes pronase and trypsin on the cell surface expression of the 9.1C3 and T200 antigens reflected the ability of these enzymes to inhibit NK cell killing. These data suggest that the 9.1C3 antigen participates in a late event in the cytolytic pathway.

Antibodies, Monoclonal↗

PubMed1984

Anti-idiotype antibodies to the 9.1C3 blocking antibody used to probe the lethal hit stage of NK cell-mediated cytolysis.

We previously described a monoclonal antibody, 9.1C3, which blocked natural killer (NK) cell-mediated cytolysis by acting on effector cells during a late step in the lethal hit stage. The present work describes the production in rabbits of anti-idiotypic (anti-id) antibodies to the 9.1C3 antibody. In addition to reacting specifically with the 9.1C3 antibody, the anti-id antibodies bound strongly to the K562 target cell. The anti-id antibodies blocked killing of K562 targets by NK, antibody-dependent cellular cytotoxicity, and NK-like cells but did not inhibit killing by cytotoxic T lymphocytes (CTL). Pretreatment of cells and washing before assay indicated that blocking occurred at the target cell level. Of particular interest, single cell assays with Percoll-enriched large granular lymphocytes demonstrated that the antibodies caused no reduction in binding. These data are consistent with a model for NK cell-mediated lysis that involves a secondary target cell receptor independent of the primary NK-target cell interaction. The anti-id antibodies immunoprecipitated cell surface proteins of relative m.w. 79K and 62K unreduced, and 94K and 79K reduced from K562 target cells. The development of anti-id antibodies may be a useful procedure to explore the structure and function of cellular receptors involved in NK cell-mediated cytolysis.

Animals↗

PubMed1984

In vitro generation of human activated lymphocyte killer cells: separate precursors and modes of generation of NK-like cells and "anomalous" killer cells.

The activation of human peripheral blood mononuclear cells (PBM) in culture leads to the generation of nonspecific killer cells. These cells, termed activated lymphocyte killer (ALK) cells, can kill fresh tumor cells and tumor cell lines, in addition to the natural killer (NK) cell sensitive target K562. ALK cells have features in common with both T and NK cells, but their nature and origin are unknown. In the present study, it is shown that ALK cells are in fact heterogeneous and can be generated from both large granular lymphocytes with the same phenotype as NK cells and from T cells. Cell populations enriched for NK cells, when cultured with lymphokines, rapidly acquired a T cell phenotype, enhanced cytolytic activity against K562, and the ability to lyse NK-insensitive target cells such as a melanoma cell line LiBr; these ALK cells were described as NK-like cells. On the other hand, of the cloned cells derived from PBM stimulated with irradiated B lymphoblasts and grown in lymphokines, the major proportion of cytolytic T cells (CTC) able to kill the specific stimulator lymphoblasts were also found to kill LiBr but not K562 cells. These ALK cells, which were derived from the same precursors as CTC, were designated anomalous killer (AK) cells. Consistent with this, the presence of the pan T monoclonal antibody UCHT1 from the beginning of mixed cell cultures inhibited the generation of CTC and of the AK-type of ALK cell, which killed melanoma cells, but not the NK type, which killed K562 targets. By contrast, at the effector cell level, the antibodies UCHT1 and OKT8 only blocked specific killing by CTC but did not block the killing of LiBr or of K562 targets by ALK cells. However, at the effector cell level there was additional evidence for the heterogeneity of ALK cells. Thus, monoclonal antibody 9.1C3, which blocks killing by freshly isolated NK cells, also blocked the killing of K562 targets by NK-like cells, but did not block B lymphoblast killing by CTC or melanoma cell killing by AK cells. It is concluded that after mixed lymphocyte culture, the majority of ALK cells measured by the killing of melanoma target cells arise from the same precursors and are under the same influences as classical CTC (AK cells), whereas cells killing K562 targets are derived from NK cells (NK-like cells). Once generated, the AK cells have a different mechanism of killing from both classical CTC and from NK and NK-like cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗

PubMed1983

A method for in vitro clearance of mycoplasma from human cell lines.

Rapid and reproducible clearance of mycoplasma contaminating human cell lines was achieved using macrophages and antibiotics. Human peripheral blood monocytes were purified by Percoll density gradient centrifugation and allowed to mature into macrophages by 7 days culture in vitro. To the adherent monolayers of macrophages were added the cells to be cleared. Optimal results were obtained with a macrophage to cell concentration of 100:1, together with 200 micrograms/ml of the antibiotics tylosin and lincomycin. The cleared cells were recovered after 7 days of treatment. Monitoring with the Hoechst 33258 stain demonstrated that cells cleared by this method have remained mycoplasma-free for over 6 months. The method is unlikely to cause cell mutation or to introduce mouse viruses and is effective on both adherent and non-adherent cell lines.

Cell Line↗

PubMed1983

Mouse monoclonal antibodies can nonspecifically inhibit the proliferation of human T-cell growth factor-dependent T cells.

A large series of mouse monoclonal antibodies was found to inhibit the proliferation of T-cell growth factor (TCGF)-dependent human T-cell blasts as measured by the incorporation of tritiated thymidine. The specificity of the antibody appeared to be irrelevant for inhibition and two T-cell-specific antibodies did not prevent the absorption of TCGF by treated T cells. It is suggested that the antibodies function by the indirect release of suppressor factors by Fc receptor-bearing TCGF-dependent cells.

Animals↗

PubMed1983

Human natural killer cells, activated lymphocyte killer cells, and monocytes possess similar cytotoxic mechanisms.

The relationship between the killing mechanisms of human natural killer (NK) cells, mitogen- and mixed-lymphocyte-culture-induced activated lymphocyte killer (ALK) cells, and monocytes was investigated with a monoclonal antibody. The IgG2 antibody 9.1C3 was prepared from mice immunized with purified human large granular lymphocytes and selected from clones that inhibited NK cell killing. The 9.1C3 antibody bound to all mononuclear cells but not to granulocytes or K562 cells, and it selectively blocked killing of K562 targets by both NK and ALK cells without affecting the binding of effector to target cells. The antibody blocked killing when present from time zero and it still inhibited partially even when added 1 hr after initiation of the lytic reaction. Killing of Epstein-Barr virus-transformed B lymphoblasts by classical cytotoxic T lymphocytes was not inhibited. Of interest, 9.1C3 did block the killing of K562 target cells by cultured peripheral blood monocytes. Other monoclonal antibodies that bound to monocytes did not block killing, and a nonspecific effect of the antibody on monocytes was excluded. These data suggest that NK cells, ALK cells, and monocytes can kill tumor cell targets by using similar lytic mechanisms.

Animals↗