Graft reaction in tissue culture: II. Quantification of the lytic action on mouse fibroblasts by rat lymphocytes sensitized on mouse embryo monolayers

A method for assaying the lytic action of large pyroninophilic cells (LPC) in cultures of rat lymphocytes on target fibroblasts in mouse embryo monolayers has been described. The basis of the assay was the labelling of fibroblasts with ⁵¹Cr. The rate of ⁵¹Cr release to the medium was found to be proportional to the number of LPC and expressed precisely the actual number of lysed fibroblasts. The lytic activity in LPC suspension was expressed by the lysis index which is the number of LPC per fibroblast lysed at 50 per cent lysis. A suspension of LPC collected 5–6 days after exposure of rat lymphocytes to the mouse monolayers showed a lysis index ranging from 0.7 to 1.3 after 16–48 hours of incubation. The lytic power of a culture was expressed by a value which was obtained by dividing the total number of LPC in a culture by the index. This determined the total number of fibroblasts lysed if the whole culture content were distributed among plates of test monolayer in such a way as to produce 50 per cent lysis in all the plates. Cultures started with 25 × 10⁶ to 30 × 10⁶ lymphocytes produced on the 5th and 6th day a lytic power of as high as 6 × 10⁶ to 12 × 10⁶ fibroblasts. The study has indicated that rats injected with horse serum and boosted 2 days prior to culturing were completely unable to produce graft reaction cultures. Lymphoid cells from non-boosted rats did generate LPC with lytic ability. A quantitative study of the specificity of the lytic reaction has indicated that LPC originated on C57BL/6 monolayers lysed much more strongly monolayers isologous to originator than C3H monolayers, and vice versa.     

Quantitative studies on phytohaemagglutinin-induced cytotoxicity by human lymphocytes against homologous cells in tissue culture

The cytotoxic effect of normal human blood lymphocytes on Chang cells in tissue culture was investigated. Cell damage was measured by release of ⁵¹Cr from pre-labelled tissue culture `target' cells. This method was sensitive and rendered highly reproducible results. Released ⁵¹Cr was not re-utilized.

About 25 per cent of the ⁵¹Cr was spontaneously released from labelled Chang cells when incubated for 24 hours at 37°. Lymphocytes at a lymphocyte/Chang cell ratio of 25:1 led to a slight increase of this release. When phytohaemagglutinin was also present, about 50–60 per cent of the isotope appeared in the medium. Under these conditions target cells were significantly damaged within 1 hour. At a lymphocyte/Chang cell ratio of only 1:1, weak cytotoxic effects were also noted after 24 hours of incubation. The results of dose—response experiments suggested that a considerable proportion of the lymphocytes participated in the reaction. Individual variation of the cytotoxic effect of lymphocytes from different donors suggested that it could be related to the degree of histoincompatibility between lymphocytes and Chang cells. Under the present conditions contaminating erythrocytes or granulocytes did not interfere with the cytotoxic action of the lymphocytes.

     

Quantitative titration, kinetic behaviour, and inhibition of cytotoxic mouse isoantisera

A precise method has been used to investigate the kinetics and inhibition of isoimmune cytolysis of mouse lymphocytes. Target cells were labelled with ⁵¹Cr and lysis was estimated by measurement of the radioisotope released in a non-sedimentable form.

Kinetic studies under conditions of limited complement or limited antiserum reveal a close similarity with rabbit anti-sheep haemolysis, and suggest that at least some of the molecular events causing lysis are the same in both cases. With the antisera studied, C′ probably requires a bimolecular antibody complex in order to bind to a cell, but the overall order of reaction, with respect to antiserum, may not be two. The observed variations in lytic response are believed to be due to the effects of antigen density and the presence in antisera of antibodies made of different kinds of immunoglobulins having the same or different specificities.

Inhibition of lysis by syngeneic unlabelled lymphocytes is proposed as a standard in a quantitative assay for histocompatibility antigens.

     

Graft reaction in tissue culture: III. Effect of phytohaemagglutinin

Rat lymphocytes cultured on mouse embryo fibroblast monolayers transformed and differentiated to large pyroninophilic cells (LPC) that lysed the mouse fibroblasts. Exposure to phytohaemagglutinin (PHA), induced the development of a different type of culture culminating in a population of medium-size lymphoid cells. The sensitization of rat lymphocytes to the mouse cell antigens was blocked by adding PHA. The resulting cells were unable to lyse mouse fibroblasts. PHA, added at any time up to 72 hours after plating, also blocked sensitization; added to a population of mature LPC, it clumped the cells and markedly decreased the lytic process.     

Assessment of the mixed leucocyte reaction by target cell death: a study of leukaemic cells

In one way mixed cultures between normal and acute leukaemic cells, cytotoxic effect was assessed by the subsequent amount of isotope released from ⁵¹Cr labelled allogeneic target cells (Chang liver cells). Observations were made during the acute phases and, where possible, in remission. All cells used in these cultures were of known HL–A phenotype.

Normal lymphocytes exhibited a variable response to leukaemic cells. In many cultures the increased cytotoxicity was greater than that found after mixed cultures of normal cells. This was not related to the number of circulating blast cells nor directly to HL–A differences between cells.

     

Early detection of potentially lethal events in T cell-mediated cytolysis

The short-term kinetics of the interaction between mouse cytolytic T lymphocytes (CTL) and ⁵¹Cr-labeled target cells was investigated. It was found that addition of EDTA to mixtures of CTL and target cells instantaneously blocked de novo lytic interactions, but did not inhibit the release of ⁵¹Cr from already damaged target cells. Using this information, a modified cytolytic assay was developed. By applying this assay to highly active CTL populations generated in secondary mixed leukocyte cultures, it was possible to detect appreciable target cell damage as early as 30 seconds after exposure to CTL. Quantitative studies demonstrated linear relationships between cytolysis and time and between rate of cytolysis and cell number under these assay conditions.     

E Rosette Formation by Human T Lymphocytes: A Spontaneous Cell Mediated Cytotoxic Phencmenon

The nature and significance of spontaneous association between unsensitized human T lymphocytes and sheep erythrocytes has Seen studied in relation to a possible cytotoxic reaction. Human lymphocytes and 51Cr labelled sheep erythrocytes when mixed in ratios of 5 or 10:l released 70–100% of 51Cr into the supernatants. This suggests that E rosette formation may be the first step in a spontaneous T cell mediated cytotoxic reaction to sheep erythrocytes.     

Studies of allograft immunity in mice: I. Induction, development and in vitro assay of cellular immunity

Cellular immunity induced by tumour allografts in inbred mice was studied with the help of an in vitro assay system measuring the cytotoxic effect of sensitized lymphocytes on ⁵¹Cr-labelled target cells. It is shown that lymphoid cells from spleen, lymph nodes and blood of the allograft recipients reach a peak of cytotoxic activity on days 10–11 after immunization. Incubated with labelled target cells at a ratio of 100:1, the sensitized lymphocytes caused the specific release of up to 70 per cent of the radioactivity within 1 hour. A second population of target cells added to the same cell suspension was destroyed at a slightly accelerated rate, suggesting stimulation of the effector cells by the first interaction. The cytotoxic activity of circulating lymphocytes was found to reach a plateau between days 20 and 60 after immunization, while the activity of spleen cells dropped to low levels in the same time period. The specificity of target cell destruction by sensitized lymphocytes is demonstrated by the lack of lytic activity for syngeneic target cells, and by the selective destruction of target cells carrying a tumour specific antigen. Tumour cells, lymphocytes and embryonic fibroblasts of the donor strain are shown to differ considerably in their sensitivity to lysis by immune lymphocytes.     

Purification and properties of a lymphocyte inhibition factor from human serum

Human lymphocytes, activated by PHA or PPD, damage homologous cells in tissue culture, as shown by release of radioactivity from target cells labelled with ⁵¹Cr-chromate. Cytotoxic effects of supernatants from activated lymphocytes are difficult to demonstrate by this method. However, such supernatants inhibit the incorporation of radioactive amino acids into homologous and heterologous target cells.

In this investigation the cytopathic effects of supernatants from human lymphocytes, stimulated with PHA or PPD, and that of the stimulated lymphocytes themselves were quantitatively assessed by four different methods. Established cell lines of human (Chang and HeLa cells) or mouse origin (L-cells) were used as target cells. Cytotoxic effects of supernatants were demonstrated in a micro modification of the cell counting assay. Supernatants also inhibited incorporation of ¹⁴C-leucine or ¹⁴C-thymidine into target cells. However, when assayed by release of ⁵¹Cr, this cytotoxic effect was usually weak or absent. In contrast, stimulated lymphocytes showed strong cytotoxicity in the chromium assay.

The results suggest that stimulated human lymphocytes release factors which are not primarily cytolytic but which inhibit target cell metabolism and proliferation, eventually leading to cell death.

     

Susceptibility of adherent versus suspension target cells derived from adherent tissue culture lines to cell-mediated cytotoxicity in rapid 51Cr-release assays

Preparation of target cells from tissue culture lines which grow adherent to tissue culture vessels is often desirable for tests of cell-mediated cytotoxicity (CMC). In the present study we used cells derived from adherent tissue culture lines to compare the merits of suspension vs. adherent target cells in short-term ⁵¹Cr-release assays. Cytotoxic activity of murine spleen cells sensitized in vitro against allogeneic spleen cells or syngeneic sarcoma cells was tested with fibroblast or sarcoma target cells. In parallel tests, aliquots of tissue culture lines were detached and used as either suspension or adherent target cells in CMC assays, matching the concentrations of suspension and adherent target cells. In both allogeneic and syngeneic combinations adherent target cells released less ⁵¹Cr spontaneously and were more susceptible to CMC than their suspension counterparts.     

Quantitative estimation of cytotoxic activity of immune lymphocytes using 51Cr-labelled peritoneal macrophages as target cells.

Peritoneal macrophages labelled with 51Cr in suspension and cultivated for 48 hr on glass pretreated with poly-L-lysine were used as target cells for determination of the cytotoxic effect of immune lymphocytes. 51Cr release from such target cell from different strains of mice is 15.5 +/- 0.8% of the total target cell radioactivity after 20 hr incubation with normal allogeneic lymphocytes. The use of 2% sodium dodecylsulphate ensures 100 percent solubilisation of labelled target cells growing on the glass surface and permits the cytotoxic effect to be determined by both 51Cr release and measuring the label retained by the intact cells. The two methods proved to be accurate, reproducible and in accord with each other as well as with the method of direct cell counting. Determination of released 51Cr enables the cytotoxic effect of lymphocytes to be measured after 4 hr incubation with the macrophage monolayer. SaI and Mc11 ascitic sarcomas display different sensitivities to the cytotoxic effect of immune lymphocytes and require different optimum conditions for its development.     

Quantitation of a whole blood assay for human natural killer cell activity

A method is described for the measurement of human natural killer cell activity using heparinized whole blood in a ⁵¹chromium release assay. Fractionation-reconstitution experiments showed that the cytotoxic activity was abolished by removal of the Fc receptor bearing lymphocytes, but not by the elimination of monocytes and granulocytes. Autologous or pooled plasma was not found to possess inherent cytolytic activity. By analogy to an enzyme kinetic reaction, the results were expressed as kinetic lytic units (KLU) which were defined as the maximum number of target cells that could be lysed per unit time per milliliter of whole blood.The buffy coat cytotoxicity (BCC) assay is quick, easy to perform, and suitable for screening and monitoring of natural cytotoxicity. Since this methodology preserves the actual concentration of natural killer cells, it may represent a truer reflection of in vivo events.     

Lysis of pulmonary fibroblasts by lymphokine (IL-2)-activated killer cells—a mechanism affecting the human lung microenvironment?

In this study we investigated whether IL-2-activated killer cells may bind and exert lytic activity against non-transformed lung fibroblasts. We demonstrated that human lymphokine-activated killer (LAK) cells generated in vitro following incubation with recombinant IL-2 of either peripheral blood mononuclear cells (PB-LAK) or lymphocytes obtained from bronchoalveolar lavage (BAL-LAK), but not resting cells, can lyse normal lung fibroblasts obtained from transbronchial lung biopsies in a 4-h ⁵¹Cr release assay. Both autologous and allogeneic fibroblasts were consistently lysed by LAK cells, thus suggesting that the phenomenon we observed is not MHC-restricted. Since fibroblasts can bind IL-2 through specific receptors, we evaluated whether long-term culture with rIL-2 could modulate the susceptibility to lysis of target cells. Our data showed that autologous fibroblasts were more resistant to lysis than allogeneic fibroblasts when they were cultured with rIL-2. Since LAK cells have been demonstrated to release a series of different immunomodulatory cytokines, we evaluated the effect of short-term incubation of fibroblasts with different factors, including IL-1, IL-2, IL-3, IL-4, IL-6, tumour necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ), on the binding and the lysis mediated by LAK cells. These cytokines were not directly cytotoxic on fibroblasts. Only IFN-γ was found to have a significant protective effect against the lysis. Our data support the concept that a self-directed cytotoxicity against pulmonary fibroblasts is generated during lymphocyte activation with rIL-2.     

The role of macrophages in the cytotoxic killing of tumour cells in vitro: I. Primary immunization of lymphocytes in vitro for target cell killing and the mechanism of lymphocyte-macrophage cooperation

Lymph node and spleen cells from normal mice were cultured for 3 days with polyoma virus-induced tumour, Ehrlich's ascites tumour or leukaemia L 1210 cells. This resulted in in vitro immunization of the lymphocytes, which were then transferred to irradiated target cells labelled with ⁵¹Cr. Normal, i.e. non-immune thioglycollate-stimulated peritoneal macrophages were also added to some tubes. Non-immune macrophages mixed with immunized lymphocytes showed a significantly increased ability to destroy tumour cells as compared with macrophages in the absence of immunized lymphocytes. The immunized lymphocytes were almost entirely inactive alone. When the number of macrophages was kept constant the cytotoxicity was dependent on the number of viable immunized lymphocytes placed on the target cells. Immunized lymphocytes, in the presence of macrophages, only exhibited strong killing of the target cells against which they had been immunized; some lysis of `bystander' cells was, however, seen provided specific target cells were present. Macrophage monolayers exposed to immunized lymphocytes upon contact with specific antigen became `armed' and showed a significant cytotoxicity for specific target cells. When immunized lymphocytes and normal macrophages were treated with actinomycin D and puromycin, cytotoxicity was inhibited in the immunized lymphocytes but not in the macrophages. The possible mechanism of normal macrophage cooperation with immunized lymphocytes in the cytotoxic killing reaction is discussed. Results presented in this paper favour the view that immunologically specific cytophilic factor (presumptive cytophilic antibody) is involved in the macrophage-mediated cytotoxicity in the system studied.     

Serotonin Modulated CA++ Dependent K+ Channels in Alloimmune Effector Cell Lytic Function

Abstract

Potassium channel activity has been implicated in the lytic function of cloned murine effector T lymphocytes (5) and human NK cells (12) as well as in the initiation of the injury process in tumor cells (23). In the present studies, the effects of various K⁺ channel blockers on the cytolytic function of in vivo derived alloimmune lymphocytes towards P815 tumor cells were evaluated. The classical K⁺ channel blocker 4-aminopyridine (4-AP), the naturally occurring monoamine serotonin (5-hydroxytryptamine, 5-HT) and its agonist, quipazine, as well as the Ca⁺⁺ dependent K⁺ channel blocker quinidine were chosen for investigation based on their known ion channel gating properties. These agents, when present in the assay medium, inhibited in a dose dependent manner the lysis of P815 tumor cells as measured by specific ⁵¹Cr release. Preincubation of effector lymphocytes with the various K⁺ channel blockers resulted in greater inhibition of lysis than did the preincubation of target cells. The 5-HT agonist quipazine was of particular interest in that it inhibited the lytic process with equal effectiveness when continuously present in the assay medium or when the effector cells alone were preincubated. Quinidine was used to investigate whether Ca⁺⁺ dependent K⁺ channels were the predominant ion channel involved in the lytic process. When present during the lytic assay, quinidine was similar to quipazine in terms of their dose range at which they inhibited the lytic process. These results indicate that 5-HT sensitive Ca⁺⁺ dependent K⁺ channels are likely to be involved in the delivery of lytic signal (s) by immune effector lymphocytes and suggests that neuroenocrine products may modulate the functional activity of in vivo derived lymphocytes.     

The interaction of normal lymphocytes and cells from lymphoid cell lines: VI. Line-directed cytotoxic specificity of lymphocytes activated by autochthonous or allogeneic LCL cells

Human peripheral blood lymphocytes activated by contact with X-irradiated cells from lymphoblastoid cell lines (LCL cells) acquire cytotoxic capacity directed against LCL cells as demonstrated by release of ⁵¹Cr from the labelled target. In a large series of cross-over experiments it was possible to demonstrate an element of specificity in the cytotoxic phase in the sense that lymphocytes activated by irradiated cells from line `A' tended to kill target cells of line `A' more efficiently than those of an unrelated line. This `line-directed' specificity was not absolute and in some experiments could not be demonstrated at all. Several factors could be identified which tend to obscure line-directed specificity. Both specific and non-specific cytotoxicity as observed in this in vitro system are probably relevant to the immunological defence of the intact organism against proliferating aberrant lymphoid cells.     

The cytotoxic activity of lymphocytes from human lymph in vitro

The in vitro cytotoxicity and DNA synthesis of thoracic duct and blood lymphocytes from four patients have been studied on the 1st day of drainage. Three patients were being drained as a pretreatment for kidney transplantation and one had myasthenia gravis. In one patient lymphocytes were obtained from a lymphatic fistula in the groin and from the blood 5 weeks after drainage began. Lysis of tissue culture cells (Chang cells) in the presence of PHA or antiserum to target cell antigens was quantitated by [⁵¹Cr]chromate release.

Lymphocytes from lymph were at best poorly cytotoxic to antibody-treated target cells under conditions where purified blood lymphocytes from the same donors had normal lytic activity. PHA-induced cytotoxicity by lymph-borne lymphocytes was noted but was considerably weaker than that of blood lymphocytes. In contrast, incorporation of [¹⁴C]thymidine into DNA of thoracic duct lymphocytes after stimulation with PHA was about 60% of that of the patients' blood lymphocytes. The DNA synthesis of thoracic duct lymphocytes induced by PPD or allogeneic lymphocytes was as good as that of blood lymphocytes. The mitotic response to PHA by lymphocytes from the lymph was reduced after two weeks drainage.

It is assumed that the number of effector cells and/or supporting cells in antibody-induced cytotoxicity in thoracic duct lymph is too small to induce target cell lysis under the present experimental conditions. Moreover, our data indicate that PHA-induced and antibody-mediated cytotoxicity are at least partly mediated by different lymphocyte subpopulations.

     

Quantitative assay of the lytic action of immune lymphoid cells of 51Cr-labelled allogeneic target cells in vitro; inhibition by isoantibody and by drugs

The in vitro cytotoxic effect of spleen cells of mice immunized by tumour allografts was studied by measuring target cell inactivation as a function of release of radioactive label (⁵¹Cr) or loss of cloning efficiency. When sensitized lymphoid cells were incubated with target cells at a ratio of 100:1, up to 90 per cent of the incorporated label was released within 6–9 hours, while the number of clone-forming cells was reduced by up to 99 per cent in the same time period. Isoantiserum from the graft recipients, as well as its 19S and 7S fractions, protected target cells against the toxic effect of the spleen cells, but a lipoprotein antigen isolated from the tumour cells failed to inhibit the cytotoxic reaction. Target cell lysis as measured by specific release of ⁵¹Cr was partially inhibited by actinomycin-D and by cycloheximide at concentrations which effectively blocked DNA-dependent RNA and protein synthesis.     

Specific antisera against recirculating and non-recirculating lymphocytes in the rat

Heterologous anti-lymphocyte sera were prepared by injecting suspensions of recirculating or non-recirculating lymphocytes into rabbits. Recirculating lymphocytes were obtained from a thoracic duct fistula, and non-recirculating lymphocytes were obtained from the blood of rats in which thoracic duct lymph had been drained away for 3 days. The cytotoxic activity of the sera was assayed by measuring the isotope release from target cells labelled with ⁵¹Cr. Antibodies specific for recirculating or non-recirculating lymphocytes could be demonstrated with the aid of cell adsorption techniques.

Cell-specific sera were used to estimate the proportion of recirculating and non-recirculating lymphocytes in lymphocyte suspensions obtained from thymus lymph nodes, blood and bone marrow. All thymocytes and most of the lymph node lymphocytes appeared to have antigenic properties in common with recirculating lymphocytes, whereas about 20% of the blood lymphocytes and most of the bone marrow lymphocytes belonged to the non-recirculating lymphocyte antigenic type.

     

E Rosette Formation by Human T Lymphocytes: A Spontaneous Cell Mediated Cytotoxic Phencmenon

The nature and significance of spontaneous association between unsensitized human T lymphocytes and sheep erythrocytes has Seen studied in relation to a possible cytotoxic reaction. Human lymphocytes and 51Cr labelled sheep erythrocytes when mixed in ratios of 5 or 10:l released 70-100% of 51Cr into the supernatants. This suggests that E rosette formation may be the first step in a spontaneous T cell mediated cytotoxic reaction to sheep erythrocytes.