Gordon F. Burns

Spontaneous reverse haemolytic plaque formation. I. Technical aspects of the protein A assay

Abstract The technical factors affecting the detection and measurement of spontaneous immunoglobulin secretion by circulating normal human lymphocytes with a protein A plaque assay have been investigated. Each of the parameters influencing the numbers of plaques obtained, protein A coupling to indicator red cells, antibody and complement concentrations and mixing conditions are considered and a recommended procedure for optimum plaque formation is suggested.

Surface antigen changes occurring in short-term cultures of activated human T lymphocytes: Analysis by flow cytometry

Abstract Surface antigens of activated and cultured human T cells were studied using peripheral blood lymphocytes activated with conditioned medium from phytohemagglutinin-activated leukocytes and maintained in liquid culture for 2 weeks with conditioned medium containing Interleukin 2. The ensuing cell population was tested for kinetic changes in cell size and for the expression of surface antigens by immunofluorescence staining with a panel of monoclonal antibodies and analysis by flow cytometry. Upon activation, the cell population progressively increased in size to large blasts, with the rapid appearance on all of the large dividing cells of the antigen recognized by OKT9, the transferrin receptor. Cells within the population continued to express the common peripheral T-cell antigens bound by OKT3 and UCHT1, and also the antigen bound by 3A1, but never the antigen bound by OKT6, a thymic cell marker. From the time of activation an increasing proportion of the T cells, up to 80%, expressed the antigen detected with OKIa and FMC4, which recognise nonpolymorphic Ia determinants. This sequence of events was followed by a general decrease in size of the cell population, a process accompanied by further phenotypic changes. The percentage of cells expressing Ia antigens decreased, but most striking was the rapid change in the OKT4:OKT8 ratio of cells within the population, from 60:40 to 40:60. Thereafter the proportions of OKT4 + to OKT8 + cells within the cultures remained relatively stable and it is suggested that these data provide evidence for a possible change in phenotype of cultured human T lymphoblasts, from OKT4 to OKT8.

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.

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.

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.

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.

Colony formation by T-lymphocytes infiltrating human tumours.

Cell suspensions from 10 breast cancers and 4 melanomas were cultured in soft Agar with phytohaemagglutinin-stimulated lymphocyte-conditioned medium (PHA-LCM). Between 3 and 10 days, some of the plated cells formed colonies of greater than 20 cells indentifiable as T-lymphocytes by morphology, cytochemical staining and capacity to form rosettes with sheep erythrocytes. The frequency of colony-forming T-lymphocytes was 0-32 per 2 X 10(5) viable cells plated, and correlated with the degree of lymphocytic infiltration within the tumour. This cloning procedure appeared to select for a subpopulation of T-cells which is well represented within primary tumours. It should prove useful for investigating lymphocyte tumour relationships in vivo.

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.

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.