An estimation of the frequency of precursor cells which generate cytotoxic lymphocytes.

The cell-mediated immune response has been generated in vitro with a polyacrylamide culture system which allows the segregation of foci (clones?) of cytotoxic lymphocytes. Using the method of limiting dilutions, the frequency of precursor cells in CBA spleen cells able to generate a cytotoxic response against DBA mastocytoma is estimated at 1 per 1,700 cells.

Primary stimulation and measurement of antibody production to sheep red blood cells in vitro.

A method for the primary stimulation and measurement of antibody production to sheep red blood cells in vitro has been described. In this system when mouse spleen cells are incubated with sheep erythrocytes large complement-dependent plaques are formed. The number and size of plaques increases from day 1 to day 3 of incubation with an average of 4.4 plaque areas per 1 x 10(6) cells plated at day 3. There is a linear relationship between the number of spleen cells plated and the number of plaques formed. Plaque formation is inhibited by colchicine, actinomycin D, and rabbit anti-mouse globulin. This system offers a possible means for the direct in vitro measurement of the number of cells in a population susceptible to antigenic stimulation by sheep erythrocytes.

In vitro and in vivo studies of the immune response to sheep erythrocytes using partially purified cell preparations.

The BSA density-gradient technique for separating mouse spleen cells into partially purified populations has been used to compare the responsiveness of such populations to SRBC using in vivo and in vitro techniques. Two major populations were distinguished, one of which responded very well in vivo with an exponential dose response and poorly in vitro (fraction 3), and another which responded in vivo and in vitro with a linear dose response (fraction 2). A light density, radiation-resistant component was identified which markedly stimulated the response of fraction 3 in vitro, and a density gradient profile was obtained for this cell which did not correspond with a macrophage profile. A high density, radiation-sensitive cell was identified which stimulated the response of PFC precursors in lighter regions of the gradient. The activity of this cell could be replaced using thymus cells. A density profile for the PFC precursor cell was obtained by assaying small numbers of spleen cell fractions in the presence of an excess of the two auxiliary cell types.

The frequency of clones of cytotoxic lymphocytes generated by H-2 antigens.

By using a culture system that allows the segregation of individual precursors of cytotoxic lymphocytes, the number of clones generated by cells from different combinations of congenic mice have been measured. It has been found that 0.3% of the total anti-H2d clones are generated by stimulators which differ predominantly at the H-2 locus. The contribution of non-H-2 antigens to anti-H-2 responses is discussed.

Development of fetal thymocytes in organ cultures. Effect of interleukin 2.

Most fetal thymocytes from 14-d mouse embryos are Thy-1+, L3T4-, Ly-2-, and express the receptor for interleukin 2 (IL-2). The development of thymocytes has been followed in fetal thymus organ cultures. When fetal thymus from 14-d embryos were cultured for a 6-d period, thymocytes increased in number 20-40-fold, and 95% became Thy-1+, L3T4+, Ly-2+. The addition of IL-2 to organ cultures of 14-d fetal thymus inhibited, in a dose-dependent manner, cell proliferation and the appearance of Thy-1+, L3T4+, Ly-2+ thymocytes. The addition of IL-2 also resulted in the appearance of a population of cells that were cytotoxic for syngeneic and allogeneic fetal thymocytes and syngeneic tumour targets. While the events that lead to the expression of the IL-2 receptor on 14-d fetal thymocytes are unknown, IL-2 in fetal thymus organ cultures inhibits the normal maturation of fetal thymocytes and raises the question of whether the cytotoxic cells that appear reflect selection through an alternative pathway of development.

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.

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.

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.

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.

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.

A high efficiency method for purification and assay of bee venom phospholipase A2.

Phospholipase A2 (PLA2) is the main allergen of hymenopteran venoms. We describe a highly efficient reverse phase high performance liquid chromatographic method (HPLC) for isolating PLA2 from crude bee venom. This method removes all detectable contaminants such as melittin from PLA2 while preserving the hemolytic action of PLA2. In addition we describe a simple functional assay of PLA2 based on its propensity to cause hemolysis of guinea pig red blood cells. These techniques are particularly well suited to the isolation and assessment of PLA2 of venoms which are available in limited quantities.

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.

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.

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 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.