John Marbrook

Spontaneous clones of cytotoxic T cells in culture. I. Characteristics of the response.

Abstract A culture system has been developed which allows segregation of individual clones of cytotoxic lymphocytes (CLs). When CBA, DBA, or (CBA × DBA)F1 spleen cells from adult mice were cultured, clones of CLs able to lyse P815 target cells were generated in the absence of stimulating cells. The effector cells are sensitive to anti-Thy-1 serum and include cells with anti-self reactivity. The maximum number of CL clones was detected on Day 4, while the largest size of clones occurred 1 or 2 days later. In contrast to stimulated cultures, there was a nonlinear relationship between the number of clones and the concentration of spleen cells in the culture. The generation of spontaneous cytotoxicity is a characteristic of adult spleens and does not develop until mice are 4 weeks old.

Spontaneous clones of cytotoxic T cells in culture: II. Specificity of the response☆

Abstract When normal (CBA × DBA)F 1 spleen cells are cultured for 4 days in polyacrylamide vessels, clones of cytotoxic lymphocytes (CLs) are generated. The specificity of these apparently spontaneous CL clones has been investigated by assaying cells from individual clones against pairs of different target cells. CL clones were found to discriminate between the two parental strain splenic blasts, between splenic blasts and syngeneic tumour cells, and between two F 1 splenic blasts induced with different mitogens (LPS and PHA). The CL clones generated spontaneously in culture also discriminate between semisyngeneic targets [DBA blasts and (CBA × DBA)F 1 blasts]. Significant cross-reactivity however, was detected when CL clones were assayed against normal P815 targets and TNP-modified P815 targets.

Effect of interleukin 2 on fetal thymocytes in organ cultures: Generation of lymphokine-activated killer cells

Cells with cytolytic activity can be detected in mouse fetal thymic lobes cultured in the presence of interleukin 2 for 6 days. The lymphokine-activated killer cells from 14-day fetal thymic lobes are relatively resistant to treatment with anti-Ly-2 antibody and complement (CD8-) but sensitive to anti-Thy-1 and complement treatment (Thy-1+). They display major histocompatibility complex-unrestricted killing, lysing both syngeneic and allogeneic tumor cells, but will not lyse human xenogenic target cells. Low levels of cytotoxic activity can be detected in thymic lobes from Day 12-13 embryos and this activity increases with embryonic age. While the events which lead to the inhibition of normal maturation of fetal thymocytes by inclusion of IL-2 in fetal thymus organ cultures are unknown, the appearance of cytotoxic cells raises the question of whether they are involved in the normal intrathymic cell death process.

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.

The immunological status of mice during the generation of cyclophosphamide-induced tolerance

Abstract Tolerance to sheep erythrocytes (SRBC) has been induced by a combination of high doses of antigen and treatment with cyclophosphamide (CY). The influence of CY alone or in combination with SRBC has been investigated by using the treated animals as recipients for normal spleen cells. CY treatment appears to produce a mouse which is severely depleted of B cells. The injection of large doses of SRBC together with CY, in a schedule which induces tolerance, generates an environment which suppresses the production of antibody-forming cells by passively transferred normal spleen cells. However, transfer of cells from tolerant mice to irradiated mice failed to demonstrate the presence of suppressor cells.

T cell receptor polymorphisms in Caucasians and Polynesians

The purpose of this study was to find genetic polymorphisms that might be useful in studies of Polynesian‐Caucasian racial admixture and Polynesian disease susceptibility. The allele frequencies of six T cell receptor locus RFLP were measured in 73 Caucasians and two Polynesian ethnic groups comprising 86 Maoris and 95 Samoans. The RFLP studied were (locus/enzyme/probe): Cα/Taq1/Y14, Vα/Taq1/Y14, Cβ/BglII/Y35, Cγ/PvuII/HGP02, Vβ7/Bam HI/Vβ7.4 and VP8/Bam HI/Vβ8.1. Racial differences in allele frequency were present with all six RFLP (P < 0.001). The allele frequencies of the Vα/Taq1/Y14 and the Vp7/Bam HI/7.4 RFLP were similar in the two Polynesian groups, both of which differed from the Caucasians. The 1.4 kb allele of the Vα/Taq1/Y14 RFLP and the 8.0 kb allele of the Vβ7/Bam HI/7.4 RFLP were present in low frequency in both Polynesian groups compared to the Caucasian group, consistent with a gene flow effect. These alleles may be useful in studies of Caucasian–Polynesian racial admixture.

Development of thymocytes in organ culture: Migrant cells with natural killer cell characteristics

During organ culture of foetal thymic lobes, up to 5% of the thymic cells migrate out of the lobes and a majority of these have phenotypic characteristics of thymocytes or macrophages. Interleukin‐2 (IL‐2) increases the number of these migrant cells, and cytotoxic cells can be detected which display natural killer cell surface markers. In contrast, although cytotoxic activity can be detected in cells from adult thymic fragments cultured with IL‐2, the cytotoxic activity is not detected with natural killer sensitive target cells. The appearance of natural killer‐like cells during the culture of foetal thymic lobes suggests that they are involved in differentiation events which occur at this time in the thymus.

Hierarchy of cytotoxic T cell clones: II. Intraclonal triggering of lysis by hapten-specific CTL

Cells from clones of anti-hapten murine cytotoxic T lymphocytes (CTL) can act as both target and effector cells, but will not lyse members of the same clone. The effect of haptenation on the cytolytic activity of anti-fluorescein (FL) and anti-trinitrophenol (TNP) CTL clones was examined. Treatment of anti-FL clones with fluorescein isothiocyanate or anti-TNP clones with trinitrobenzene sulphonic acid induces these clones to kill in an antigen-independent fashion. Targets killed by the haptenated CTL included syngeneic and allogeneic B lymphocyte blast cells, P815, YAC-1 and in one case human GM 4072 tumor cells. The importance of CD8 and T cell receptor (TCR) occupancy is demonstrated by the ability to block autotriggering by antibody directed against Ly 2 and the TCR. The results demonstrate that effects other than antigen recognition of the target play a role in the final outcome of effector-target cell interactions and provide a mechanism which could lead to autodestruction and immunosuppression particularly in some types of viral infection.

Hierarchy of cytotoxic T cell clones. I: Reversal of resistance to lysis and killing between anti-hapten cytotoxic T cells

Cells from clones of anti-hapten cytotoxic T lymphocytes (CTL) can act as both effector cells and, when treated with the specific hapten, as target cells. Individual clones can kill haptenated cells only from other clones that are less efficient killers. Clones specific for both fluorescein and trinitrophenol could be ordered in a single hierarchy in which resistance to lysis correlated with lytic efficiency. When the killing efficiency was reduced with phorbol myristate acetate (PMA) or the colchicine analogue, Colcemid, the degree of resistance to lysis was also reduced. The use of PMA-treated fluoresceinated targets greatly enhanced intraclonal killing and similarly lead to a repositioning of clones within the hierarchy of normal cells. By the haptenation of appropriate clones, efficient CTL could kill cells from other clones in a direction apparently opposite to recognition. The results demonstrate that effects other than antigen recognition of the target cell may result in variations in the nature of T cell immune responses.

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.