Clin. exp. Immunol. (1975) 21, 226-235.
CYTOTOXIC LYMPHOCYTES FROM NORMAL DONORS A FUNCTIONAL MARKER OF HUMAN NON-T LYMPHOCYTES H. F. PROSS* AND M. JONDAL
Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden (Received 16 December 1974) SUM MARY
A phenomenon we have termed spontaneous lymphocyte-mediated cytotoxicity (SLMC) by non-thymus-derived lymphocytes from normal donors has been described. The phenomenon can be demonstrated using human and xenogeneic (mouse) cell lines as the target cell in a microplate 51Cr release assay which is simple and reproducible. In this paper, SLMC against xenogeneic targets has been evaluated as a potential marker of lymphocytotoxic function with respect to: (a) the nature of the target cell; (b) the variability of the cytotoxic function of lymphocytes from different donors, and from the same donor tested on different days; (c) the nature of the effector cell. Using buoyant density centrifugation of iron plus magnetpurified lymphocytes forming rosettes with sheep red blood cells (SRBC) (T cells) or SRBC-rabbit 19S anti-SRBC-mouse complement (complement receptor lymphocytes), it has been demonstrated that the cytotoxic activity lies in the nonT-lymphocyte fraction, and is probably caused by the complement receptorbearing lymphocyte. The potential usefulness of this phenomenon as a functional marker of non-T-lymphocyte cytotoxic ability, and for the assessment of serological factors which may affect this cytotoxicity, has been discussed.
INTRODUCTION The establishment of various identifying markers of human lymphocytes is an important step in the definition of the function of different cell types, as well as in the characterization of primary and secondary immunodeficiencies. At present it is generally agreed that no single marker is sufficient for adequate lymphocyte identification and assessment of lymphocyte immunocompetence, and that this task is best accomplished by using a panel of different tests. The majority of markers currently in use depend on mitogenic stimulation of certain lymphocyte populations, or can be defined as 'structural' markers, reflecting differences between various cell types on the basis of surface characteristics (Jondal, Wigzell & *
Present address and correspondence: Ontario Cancer Foundation, Kingston Clinic, Kingston, Ontario,
Canada, K7L 2V7.
226
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Auiti, 1973). The limitation of using only structural markers in the assessment of human lymphocytes lies in the fact that the presence of a surface marker may not correlate with the function of the cell types which it defines, e.g. the binding of a sheep erythrocyte to a T cell (Jondal, Holm & Wigzell, 1972), does not necessarily indicate that this cell is capable of cellmediated immune reactions. The present communication describes a functional marker for human lymphocytes based on the ability of these cells to lyse certain tumour cells from lines established in vitro. The characteristics of the reaction have been worked out using the DBA/2 mouse, methylcholanthrene-induced mastocytoma, P815X2 (Dunn & Potter, 1957) (xenogeneic cytotoxicity) and the human cell line K562, derived from the cells of a patient with chronic myelocytic leukaemia (Lozzio & Lozzio, 1973) (homologous cytotoxicity). The test is performed using a microplate 51Cr release assay (Wigzell, 1965) and is simple, sensitive and reproducible. Evidence is presented in this communication that the xenogeneic cytotoxicity assay detects the cytotoxic ability of non-thymus-derived lymphocytes, probably the complement receptor-bearing lymphocytes (Bianco, Patrick & Nussenzweig, 1970) and, as such, represents a convenient functional marker for these cells of potential clinical applicability. Extensive parallel experiments upon cytotoxic lymphocytes from normal donors against K562 and other human cell lines indicate that this natural cytotoxicity is also a non-thymusderived lymphocyte-mediated effect (Jondal & Pross, 1974). This latter observation may have great theoretical relevance to the concept of immune surveillance against malignant cells, as well as being of practical relevance with respect to the interpretation of cytotoxic tests of tumour immunity in patients with malignant disease (Takasugi, Mikey & Terasaki, 1973).
MATERIALS AND METHODS
Lymphocyte purification The methods used to purify and identify human lymphocytes have been described in detail previously (Jondal & Klein, 1973). Human lymphocytes were obtained from healthy donors using 5 i.u. of heparin per millilitre of blood. The lymphocytes were isolated from granulocytes and red cells on a Ficoll-Isopaque gradient (Boyum, 1968) followed by a carbonyl iron magnetism purification (Lundgren, Zukoski & Moller, 1968) to remove phagocytic cells. T lymphocyte (E-RFC) and complement receptor lymphocyte (EAC) rosettes were prepared as described below, and separated from non-rosetting cells by buoyant density centrifugation on Ficoll-Isopaque (Jondal, 1974). In the case of E-RFC-enriched preparations, the SRBC were separated from the T cells using 0.83% ammonium chloride (Boyle, 1968). Cell identification T lymphocytes were identified by rosette formation with sheep red blood cells (SRBC) (Jondal et al., 1972). Briefly, 100 yul of 1% SRBC were mixed with 100 jl of cell suspension containing 4 x 106 cells/ml, in foetal calf serum. The mixture was centrifuged to form a pellet, and incubated for 20 min at 37°C followed by 60 min incubation at 4°C. The pellet was very gently resuspended and placed on a glass slide for counting. Lymphocytes bearing receptors for the third component of complement were identified by EAC rosette formation (Bianco
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et al., 1970). In this case, lymphocytes were mixed with sheep red cells coated with rabbit IgM anti-SRBC and A.Sw. mouse serum as a source of complement. The pelleted mixture was incubated for 30 min at 370C, vigorously resuspended, and the rosettes counted in a Burker chamber. A cell was considered to be a rosette-forming cell if there were three or more RBC adherent to the surface. At least 200 lymphocytes were counted in each pre-
paration. Immunoglobulin-positive lymphocytes were detected by direct immunofluorescence using a polyvalent goat anti-human immunoglobulin serum (Jondal & Klein, 1973). The proportion of monocytes in various preparations was evaluated by counting those cells which had ingested rabbit anti-SRBC-mouse complement-coated SRBC after 30 min incubation at 370C. SRBC external to the monocytes were lysed using ammonium chloride (Jondal, 1974).
Cell lines P81 5X2 cells were maintained as stationary suspension cultures in RPM 1 1640 medium with 10% foetal calf serum, 10 mm extra L-glutamine, and antibiotics. The cultures were subcultured three times weekly. Certain other cell lines and their characteristics are described in the legend to Fig. 1. Monolayer cultures were harvested by trypsinization using 0.25% trypsin for 5 min. Cytotoxic assay The medium used was RPM 1 1640, 10 mm extra L-glutamine, 10% foetal calf serum and antibiotics. Approximately 2 x 106 target cells were labelled with 150 YiCi Na25"CrO4 in 0 4 ml of medium for 1 hr at 370C in 500 C02, and washed three times. 104 Target cells were then incubated overnight with various concentrations of lymphocytes in a volume of 150 pi in Linbro conical well microplates. Cultures with 104 target cells in medium only were included for spontaneous release and total counts. The plates were centrifuged before incubation and before harvesting at 200 g for 2 min in an International PR-J centrifuge. This step was later found to be unnecessary if overnight incubations were being performed and if the plates were handled carefully before harvesting. After incubation, supernatant aliquots of 100 1d were carefully removed from each well using an Eppendorff micropipette and counted in a Nukab gamma counter. Aliquots from resuspended control cultures were also taken to measure the total counts possible per 100 Pul. Percentage cellmediated lysis = [(ct/min (test)-ct/min (spontaneous))/(ct/min (total)-ct/min (spontaneous))] x 100%. The results are expressed as the mean of duplicate cultures. Representative standard errors using this technique are shown in Table 1 and Fig. 3. RESULTS Spontaneous lymphocyte-mediated lysis of mouse tumour cell lines Fig. 1 illustrates the phenomenon of spontaneous cytotoxicity of human lymphocytes against mouse cell lines. It can be seen from this figure that significant lysis can be obtained using many different cell lines as the source of target cells, and that the observation of spontaneous lymphocyte-mediated cytotoxicity (SLMC) is apparently independent of: (a) the mouse strain of origin of the tumour; (b) whether or not the tumour was chemically
Cytotoxicity of normal human lymphocytes
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FIG. 1. SLMC against various cell lines in three experiments. Experiment A (solid lines): (o) P815X2 (methylcholanthrene (MCA) induced mastocytoma, suspension culture, DBA/2 strain); (A) MC57M (MCA sarcoma, monolayer C57BI); (0) MC57G (MCA sarcoma, monolayer, C57B1): (A) SEYF-a (polyoma virus-induced sarcoma, monolayer, ABY); (x) YAC (Moloney leukaemia virus (MLV) induced lymphoma, suspension, A strain); (0) YBA (MLV lymphoma, suspension, CBA). Experiment B (individual points, ratio 25:1): ((o) P815X2; (*) YCAB (MLV lymphoma, suspension, ACAxA); (*) WL4 ('spontaneous' lymphoma, suspension, A.Sw); (A) MC57M, harvested by trypsinization prior to use, as in experiment A; (A) MC57M, harvested by scraping. Experiment C (dotted lines): (o) A9 cells (L cell subline), Mycoplasma-free; (N) A9 cells contaminated with Mycoplasma, 95% viable.
or virally induced; (c) whether or not the tumour cells were maintained in monolayer (and therefore trypsinized off prior to performing the test) or in suspension culture. Fig. 1 also shows that cells from a line known to be Mycoplasma-free (A9 cells, LI - - L), were lysed to the same extent as cells contaminated with Mycoplasma (* - - *), ruling out donor sensitization against Mycoplasma as the cause of the effect. From the number of cell lines available, the mastocytoma P815 X2 was selected for further study as a possible marker of human lymphocytotoxic ability. This line was chosen because of its low spontaneous release of 51Cr (1-30% after 16 hr of incubation), the ease with which it can be maintained in suspension culture, and its availability in most institutions studying tumour immunology.
The reproducibility of SLMC by different donors, and on different occasions Fig. 2 shows the extent of SLMC among a number of different healthy donors, and illustrates several points relevant to the use of the test on a regular basis. (a) Lymphocytes from all individuals tested caused significant lysis of the target cells, although there was some
230
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Lymphocyte: target cel Iratio FIG. 2. SLMC against P815X2 in different individuals. Open symbols, blood bank donors. Closed symbols, laboratory donors. Dotted lines, same individual on four different occasions, (-) four different individuals tested at the same time.
variation from donor to donor. (b) The degree of lysis caused by lymphocytes from the same donor was fairly reproducible from day to day, e.g. the four sets of points joined by dashed lines in Fig. 2. (c) Donor lymphocytes causing low cytotoxicity, e.g. the lowest point at the 20: 1 ratio, Fig. 2, also caused low cytotoxicity on repeat testing, in spite of the fact that other donor lymphocytes tested at the same time gave more 'normal' results. As an example, the closed squares in Fig. 2, ratio 20: 1, represent four different donors tested on the same day. Although three of these points cluster around 32-33°/,, the fourth donor's lymphocytes caused only 17% lysis (P0\
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decreased in cytotoxic ability, an observation which we have attributed to the fact that these cells were mixed with SRBC and sedimented three times on Ficoll-Isopaque to ensure that as many T cells as possible were removed. To further rule out the role of the T cell in this test, purified T-cell preparations were made by complement receptor lymphocyte sedimentation (EAC- preparations), as in Fig. 3. In this case, the T cells remain at the interface of the Ficoll-Isopaque gradient, and lysis of the SRBC by ammonium chloride is not necessary if only the non-rosetted T cells are required. Table 1 (columns E and F) shows the results obtained in two different experiments using EAC rosette-forming cell depletion. In these experiments, as in Fig. 3, the cytotoxic effector cells were completely removed by the removal of complement receptor-bearing lymphocytes. These cells are non-thymus-derived cells which overlap to a large extent, but not completely, with the immunoglobulin-positive B cells (Jondal, 1974). From these data, we have concluded that spontaneous cytotoxicity by human lymphocytes against xenogeneic target cells (P81 5X2) is caused by non-thymus-derived cells, and is probably mediated by complement receptor-bearing lymphocytes. DISCUSSION
The biological significance of SLMC against xenogeneic target cells is not clear, just as it is not clear with other lymphocyte markers such as the ability of human T cells to form rosettes
with sheep erythrocytes. Since the complement receptor lymphocyte also has the Fc receptor for antigen-antibody complexes (Jondal et al., 1972), the cytotoxic effector cell detected in this assay may well be a subpopulation of those cells capable of mediating antibodydependent cell-mediated cytotoxicity (ADCC) (reviewed by Cerottini & Brunner, 1974). If, however, ADCC is the actual mechanism of this phenomenon, IgG antibody against P815 or mouse cell surface antigens must be present in the system, either produced by the lymphocytes or present in the foetal calf serum used in the culture and test medium. Since it is known that human sera contain 'natural antibodies' against mouse antigens (Landy et al., 1960), the presence of minute amounts of IgG anti-mouse antibody is not inconceivable. If this is the case, xenogeneic SLMC would then be a measure of naturally occurring ADCC effector cell activity, and would be a useful assay as such, since it is unnecessary to complicate the SLMC system with the addition of heterologous IgG antibody, as is usually done in the study of ADCC at present. Against this concept, however, are the results from a number of experiments (Pross & Jondal, in preparation). Using nylon wool columns (Julius, Simpson & Herzenberg, 1973), it was possible to almost totally deplete the immunoglobulinpositive cells from lymphocyte preparations, while only partially depleting the complement receptor lymphocytes (to about 5000 of their original proportion) (Jondal & Pross, 1974). These lymphocyte preparations still demonstrated significant SLMC although less than 1% immunoglobulin-positive cells were present in the mixture. Further evidence against the involvement of antibody in the system comes from experiments using mouse spleen cells. CBA mouse spleen cells (effector: target cell ratio of 100: 1) were quite capable of lysing antibody-coated P815 cells, but had no effect on uncoated cells, even when cultured with them in the presence of culture supernatant fluid taken from overnight cultures of human lymphocytes, or with different batches of potentially antibody-containing foetal calf serum. Thus, although our investigations are not complete, there is no evidence at present to indicate that antibody-dependent cell-mediated lysis is the mechanism of xenogeneic (or D
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homologous (Jondal & Pross, 1974)) SLMC. It is possible, however, that human complement receptor lymphocytes have receptors for some structure on the surface of mouse cells, and that the close cell-to-cell contact which results is all that is necessary to initiate target cell lysis. The existence of such a receptor has been suggested by the recent demonstration that human B cells form rosettes with mouse erythrocytes (Stathopoulos & Elliott, 1974). Other preliminary data as to the nature and mechanism of xenogeneic SLMC indicate that 'bystander' target cells (chicken RBC) are not affected by reaction, that it is not mediated by supernatant fluid from cultured lymphocytes per se, and that the effect can be inhibited by the presence of 5 mm ethylenediaminetetraacetate, or by metabolic inhibitors such as 5 mm sodium azide and 5 mm sodium fluoride. The usefulness of this test as a marker of lymphocyte function has been indicated by the results of Petranyi et al. (1974a, b), using a similar independently evolved technique in which human lymphocytes spontaneously lyse DBA/2 fibroblast monolayer cells. The results in this test parallel those obtained using P815 (Petranyi, personal communication). The authors found that low activity in their test correlated with the HL-A haplotype 3,7 antigens known to correlate with the diagnosis of multiple sclerosis (Naito et al., 1972; Bertrams, Kuwert & Liedtke, 1972; Jersild et al., 1973), and suggesting an association with some type of cellular dysfunction in this disease. It is data such as this which leads us to believe that SLMC against xenogeneic target cells represents a potentially valuable tool in the assessment of human non-T-lymphocyte cytotoxic function. The reaction may also serve as an easily available and reproducible cell-mediated cytotoxic system which can be used to assess the effects of serum inhibitory and blocking factors in patients with malignant disease. Experiments are currently underway to investigate this possibility. ACKNOWLEDGMENTS
We thank Dr G. Clements for the proven Mycoplasma-free A-9 cells. The work upon which this publication is based was performed persuant to contract number NO1-CB-33870 and contract number NIH-NO1-CB-33859 with the Division of Cancer Biology and Diagnosis, National Cancer Institute, Department of Health, Education and Welfare. H. F. Pross was a Centennial Fellow of the Medical Research Council of Canada. REFERENCES BERTRAMS, J., KUWERT, E. & LIEDTKE, U. (1972) HL-A antigens and multiple sclerosis. Tissue Antigens, 2, 405. BIANCO, C., PATRICK, R. & NusSENZWEIG, V. (1970) A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization. J. exp. Med. 132, 702. BOYLE, W. (1968) An extension of the 51Cr-release assay for the estimation of mouse cytotoxins. Transplantation, 6, 761. BOYUM, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97, p. 77. CEROTTINI, J.-C. & BRUNNER, K.T. (1974) Cell-mediated cytotoxicity, allograft rejection and tumor immunity. Advanc. Immunol. 18, 67. DUNN, T.B. & POTTER, M. (1957) A transplantable mast cell neoplasm in the mouse. J. nat. Cancer Inst. 18, 587. JERSILD, C., SVEJGAARD, A., FoG, T. & AMMITZBOLL, T. (1973) HL-A antigens and diseases. I. Multiple sclerosis. Tissue Antigens, 3, 243.
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JONDAL, M. (1974) Surface markers on human T and B lymphocytes. IV. Distribution of surface markers on resting and blast transformed lymphocytes. Scand. J. Immunol. 3, 739. JONDAL, M., HOLM, G. & WIGZELL, W. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J. exp. Med. 136,207. JONDAL, M. & KLEIN, G. (1973) Surface markers on human B and T lymphocytes. II. Presence of EpsteinBarr virus receptors on B lymphocytes. J. exp. Med. 138, 1365. JONDAL, M. & PRoss, H.F. (1975) Surface markers on human B and T lymphocytes. VI. Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and lectin-induced cytotoxicity against cell lines as functional markers for different lymphocyte subpopulations. Int. J. Cancer. (In press.) JONDAL, M., WIGZELL, H. & AUITI, F. (1973) Human lymphocyte subpopulations: classification according to surface markers and/or functional characteristics. Transplant. Rev. 16, 163. JULIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Europ. J. Immunol. 3, 645. LANDY, M., MICHAEL, J.G., TRAPANI, R.J., ACHINSTEIN, B., WOODS, M.W. & SHEAR, M.J. (1960) Antibodycomplement system in normal serum lethal to mouse tumor cells. Cancer Res., 20, 1279. Lozzio, C.B. & Lozzio, B.B. (1973) Cytotoxicity of a factor isolated from human spleen. J. nat. Cancer Inst. 50, 535. LUNDGREN, G., ZUKOSKI, C.F. & MOLLER, G. (1968) Differential aspects of human granulocytes and lymphocytes on human fibroblasts. Clin. exp. Immunol., 3, 817. NAITO, S., NAMEROW, N., MICKEY, M.R. & TERASAKI, P.I. (1972) Multiple sclerosis: association with HL-A 3. Tissue Antigens, 2, 1. PETRANYI, G., BENCZUR, M., ODONY, C.E., HOLLAN, S.R. & IVANYI, P. (1974a) HL-A 3,7 and lymphocyte cytotoxic activity. Lancet, ii, 736. PETRANYI, G., IVANYI, P. & HOLLAN, S.R. (1974b) Relations of HL-A and Rh systems to immune reactivity. Vox Sang. (Basel), 26, 470. STATHOPOULOS, G. & ELLIOTT, E.V. (1974) Formation of mouse or sheep red-blood-cell rosettes by lymphocytes from normal and leukaemic individuals. Lancet, i, 600. TAKASUGI, M., MIKEY, M.R. & TERASAKI, P. (1973) Reactivity of normal lymphocytes from normal persons on cultured tumor cells. Cancer Res. 33, 2829. WIGZELL, H. (1965) Quantitative titration of mouse H-2 antibodies using Cr' -labelled target cells. Transplantation, 3, 423.
CYTOTOXIC LYMPHOCYTES FROM NORMAL DONORS A FUNCTIONAL MARKER OF HUMAN NON-T LYMPHOCYTES H. F. PROSS* AND M. JONDAL
Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden (Received 16 December 1974) SUM MARY
A phenomenon we have termed spontaneous lymphocyte-mediated cytotoxicity (SLMC) by non-thymus-derived lymphocytes from normal donors has been described. The phenomenon can be demonstrated using human and xenogeneic (mouse) cell lines as the target cell in a microplate 51Cr release assay which is simple and reproducible. In this paper, SLMC against xenogeneic targets has been evaluated as a potential marker of lymphocytotoxic function with respect to: (a) the nature of the target cell; (b) the variability of the cytotoxic function of lymphocytes from different donors, and from the same donor tested on different days; (c) the nature of the effector cell. Using buoyant density centrifugation of iron plus magnetpurified lymphocytes forming rosettes with sheep red blood cells (SRBC) (T cells) or SRBC-rabbit 19S anti-SRBC-mouse complement (complement receptor lymphocytes), it has been demonstrated that the cytotoxic activity lies in the nonT-lymphocyte fraction, and is probably caused by the complement receptorbearing lymphocyte. The potential usefulness of this phenomenon as a functional marker of non-T-lymphocyte cytotoxic ability, and for the assessment of serological factors which may affect this cytotoxicity, has been discussed.
INTRODUCTION The establishment of various identifying markers of human lymphocytes is an important step in the definition of the function of different cell types, as well as in the characterization of primary and secondary immunodeficiencies. At present it is generally agreed that no single marker is sufficient for adequate lymphocyte identification and assessment of lymphocyte immunocompetence, and that this task is best accomplished by using a panel of different tests. The majority of markers currently in use depend on mitogenic stimulation of certain lymphocyte populations, or can be defined as 'structural' markers, reflecting differences between various cell types on the basis of surface characteristics (Jondal, Wigzell & *
Present address and correspondence: Ontario Cancer Foundation, Kingston Clinic, Kingston, Ontario,
Canada, K7L 2V7.
226
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Auiti, 1973). The limitation of using only structural markers in the assessment of human lymphocytes lies in the fact that the presence of a surface marker may not correlate with the function of the cell types which it defines, e.g. the binding of a sheep erythrocyte to a T cell (Jondal, Holm & Wigzell, 1972), does not necessarily indicate that this cell is capable of cellmediated immune reactions. The present communication describes a functional marker for human lymphocytes based on the ability of these cells to lyse certain tumour cells from lines established in vitro. The characteristics of the reaction have been worked out using the DBA/2 mouse, methylcholanthrene-induced mastocytoma, P815X2 (Dunn & Potter, 1957) (xenogeneic cytotoxicity) and the human cell line K562, derived from the cells of a patient with chronic myelocytic leukaemia (Lozzio & Lozzio, 1973) (homologous cytotoxicity). The test is performed using a microplate 51Cr release assay (Wigzell, 1965) and is simple, sensitive and reproducible. Evidence is presented in this communication that the xenogeneic cytotoxicity assay detects the cytotoxic ability of non-thymus-derived lymphocytes, probably the complement receptor-bearing lymphocytes (Bianco, Patrick & Nussenzweig, 1970) and, as such, represents a convenient functional marker for these cells of potential clinical applicability. Extensive parallel experiments upon cytotoxic lymphocytes from normal donors against K562 and other human cell lines indicate that this natural cytotoxicity is also a non-thymusderived lymphocyte-mediated effect (Jondal & Pross, 1974). This latter observation may have great theoretical relevance to the concept of immune surveillance against malignant cells, as well as being of practical relevance with respect to the interpretation of cytotoxic tests of tumour immunity in patients with malignant disease (Takasugi, Mikey & Terasaki, 1973).
MATERIALS AND METHODS
Lymphocyte purification The methods used to purify and identify human lymphocytes have been described in detail previously (Jondal & Klein, 1973). Human lymphocytes were obtained from healthy donors using 5 i.u. of heparin per millilitre of blood. The lymphocytes were isolated from granulocytes and red cells on a Ficoll-Isopaque gradient (Boyum, 1968) followed by a carbonyl iron magnetism purification (Lundgren, Zukoski & Moller, 1968) to remove phagocytic cells. T lymphocyte (E-RFC) and complement receptor lymphocyte (EAC) rosettes were prepared as described below, and separated from non-rosetting cells by buoyant density centrifugation on Ficoll-Isopaque (Jondal, 1974). In the case of E-RFC-enriched preparations, the SRBC were separated from the T cells using 0.83% ammonium chloride (Boyle, 1968). Cell identification T lymphocytes were identified by rosette formation with sheep red blood cells (SRBC) (Jondal et al., 1972). Briefly, 100 yul of 1% SRBC were mixed with 100 jl of cell suspension containing 4 x 106 cells/ml, in foetal calf serum. The mixture was centrifuged to form a pellet, and incubated for 20 min at 37°C followed by 60 min incubation at 4°C. The pellet was very gently resuspended and placed on a glass slide for counting. Lymphocytes bearing receptors for the third component of complement were identified by EAC rosette formation (Bianco
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et al., 1970). In this case, lymphocytes were mixed with sheep red cells coated with rabbit IgM anti-SRBC and A.Sw. mouse serum as a source of complement. The pelleted mixture was incubated for 30 min at 370C, vigorously resuspended, and the rosettes counted in a Burker chamber. A cell was considered to be a rosette-forming cell if there were three or more RBC adherent to the surface. At least 200 lymphocytes were counted in each pre-
paration. Immunoglobulin-positive lymphocytes were detected by direct immunofluorescence using a polyvalent goat anti-human immunoglobulin serum (Jondal & Klein, 1973). The proportion of monocytes in various preparations was evaluated by counting those cells which had ingested rabbit anti-SRBC-mouse complement-coated SRBC after 30 min incubation at 370C. SRBC external to the monocytes were lysed using ammonium chloride (Jondal, 1974).
Cell lines P81 5X2 cells were maintained as stationary suspension cultures in RPM 1 1640 medium with 10% foetal calf serum, 10 mm extra L-glutamine, and antibiotics. The cultures were subcultured three times weekly. Certain other cell lines and their characteristics are described in the legend to Fig. 1. Monolayer cultures were harvested by trypsinization using 0.25% trypsin for 5 min. Cytotoxic assay The medium used was RPM 1 1640, 10 mm extra L-glutamine, 10% foetal calf serum and antibiotics. Approximately 2 x 106 target cells were labelled with 150 YiCi Na25"CrO4 in 0 4 ml of medium for 1 hr at 370C in 500 C02, and washed three times. 104 Target cells were then incubated overnight with various concentrations of lymphocytes in a volume of 150 pi in Linbro conical well microplates. Cultures with 104 target cells in medium only were included for spontaneous release and total counts. The plates were centrifuged before incubation and before harvesting at 200 g for 2 min in an International PR-J centrifuge. This step was later found to be unnecessary if overnight incubations were being performed and if the plates were handled carefully before harvesting. After incubation, supernatant aliquots of 100 1d were carefully removed from each well using an Eppendorff micropipette and counted in a Nukab gamma counter. Aliquots from resuspended control cultures were also taken to measure the total counts possible per 100 Pul. Percentage cellmediated lysis = [(ct/min (test)-ct/min (spontaneous))/(ct/min (total)-ct/min (spontaneous))] x 100%. The results are expressed as the mean of duplicate cultures. Representative standard errors using this technique are shown in Table 1 and Fig. 3. RESULTS Spontaneous lymphocyte-mediated lysis of mouse tumour cell lines Fig. 1 illustrates the phenomenon of spontaneous cytotoxicity of human lymphocytes against mouse cell lines. It can be seen from this figure that significant lysis can be obtained using many different cell lines as the source of target cells, and that the observation of spontaneous lymphocyte-mediated cytotoxicity (SLMC) is apparently independent of: (a) the mouse strain of origin of the tumour; (b) whether or not the tumour was chemically
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FIG. 1. SLMC against various cell lines in three experiments. Experiment A (solid lines): (o) P815X2 (methylcholanthrene (MCA) induced mastocytoma, suspension culture, DBA/2 strain); (A) MC57M (MCA sarcoma, monolayer C57BI); (0) MC57G (MCA sarcoma, monolayer, C57B1): (A) SEYF-a (polyoma virus-induced sarcoma, monolayer, ABY); (x) YAC (Moloney leukaemia virus (MLV) induced lymphoma, suspension, A strain); (0) YBA (MLV lymphoma, suspension, CBA). Experiment B (individual points, ratio 25:1): ((o) P815X2; (*) YCAB (MLV lymphoma, suspension, ACAxA); (*) WL4 ('spontaneous' lymphoma, suspension, A.Sw); (A) MC57M, harvested by trypsinization prior to use, as in experiment A; (A) MC57M, harvested by scraping. Experiment C (dotted lines): (o) A9 cells (L cell subline), Mycoplasma-free; (N) A9 cells contaminated with Mycoplasma, 95% viable.
or virally induced; (c) whether or not the tumour cells were maintained in monolayer (and therefore trypsinized off prior to performing the test) or in suspension culture. Fig. 1 also shows that cells from a line known to be Mycoplasma-free (A9 cells, LI - - L), were lysed to the same extent as cells contaminated with Mycoplasma (* - - *), ruling out donor sensitization against Mycoplasma as the cause of the effect. From the number of cell lines available, the mastocytoma P815 X2 was selected for further study as a possible marker of human lymphocytotoxic ability. This line was chosen because of its low spontaneous release of 51Cr (1-30% after 16 hr of incubation), the ease with which it can be maintained in suspension culture, and its availability in most institutions studying tumour immunology.
The reproducibility of SLMC by different donors, and on different occasions Fig. 2 shows the extent of SLMC among a number of different healthy donors, and illustrates several points relevant to the use of the test on a regular basis. (a) Lymphocytes from all individuals tested caused significant lysis of the target cells, although there was some
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variation from donor to donor. (b) The degree of lysis caused by lymphocytes from the same donor was fairly reproducible from day to day, e.g. the four sets of points joined by dashed lines in Fig. 2. (c) Donor lymphocytes causing low cytotoxicity, e.g. the lowest point at the 20: 1 ratio, Fig. 2, also caused low cytotoxicity on repeat testing, in spite of the fact that other donor lymphocytes tested at the same time gave more 'normal' results. As an example, the closed squares in Fig. 2, ratio 20: 1, represent four different donors tested on the same day. Although three of these points cluster around 32-33°/,, the fourth donor's lymphocytes caused only 17% lysis (P0\
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decreased in cytotoxic ability, an observation which we have attributed to the fact that these cells were mixed with SRBC and sedimented three times on Ficoll-Isopaque to ensure that as many T cells as possible were removed. To further rule out the role of the T cell in this test, purified T-cell preparations were made by complement receptor lymphocyte sedimentation (EAC- preparations), as in Fig. 3. In this case, the T cells remain at the interface of the Ficoll-Isopaque gradient, and lysis of the SRBC by ammonium chloride is not necessary if only the non-rosetted T cells are required. Table 1 (columns E and F) shows the results obtained in two different experiments using EAC rosette-forming cell depletion. In these experiments, as in Fig. 3, the cytotoxic effector cells were completely removed by the removal of complement receptor-bearing lymphocytes. These cells are non-thymus-derived cells which overlap to a large extent, but not completely, with the immunoglobulin-positive B cells (Jondal, 1974). From these data, we have concluded that spontaneous cytotoxicity by human lymphocytes against xenogeneic target cells (P81 5X2) is caused by non-thymus-derived cells, and is probably mediated by complement receptor-bearing lymphocytes. DISCUSSION
The biological significance of SLMC against xenogeneic target cells is not clear, just as it is not clear with other lymphocyte markers such as the ability of human T cells to form rosettes
with sheep erythrocytes. Since the complement receptor lymphocyte also has the Fc receptor for antigen-antibody complexes (Jondal et al., 1972), the cytotoxic effector cell detected in this assay may well be a subpopulation of those cells capable of mediating antibodydependent cell-mediated cytotoxicity (ADCC) (reviewed by Cerottini & Brunner, 1974). If, however, ADCC is the actual mechanism of this phenomenon, IgG antibody against P815 or mouse cell surface antigens must be present in the system, either produced by the lymphocytes or present in the foetal calf serum used in the culture and test medium. Since it is known that human sera contain 'natural antibodies' against mouse antigens (Landy et al., 1960), the presence of minute amounts of IgG anti-mouse antibody is not inconceivable. If this is the case, xenogeneic SLMC would then be a measure of naturally occurring ADCC effector cell activity, and would be a useful assay as such, since it is unnecessary to complicate the SLMC system with the addition of heterologous IgG antibody, as is usually done in the study of ADCC at present. Against this concept, however, are the results from a number of experiments (Pross & Jondal, in preparation). Using nylon wool columns (Julius, Simpson & Herzenberg, 1973), it was possible to almost totally deplete the immunoglobulinpositive cells from lymphocyte preparations, while only partially depleting the complement receptor lymphocytes (to about 5000 of their original proportion) (Jondal & Pross, 1974). These lymphocyte preparations still demonstrated significant SLMC although less than 1% immunoglobulin-positive cells were present in the mixture. Further evidence against the involvement of antibody in the system comes from experiments using mouse spleen cells. CBA mouse spleen cells (effector: target cell ratio of 100: 1) were quite capable of lysing antibody-coated P815 cells, but had no effect on uncoated cells, even when cultured with them in the presence of culture supernatant fluid taken from overnight cultures of human lymphocytes, or with different batches of potentially antibody-containing foetal calf serum. Thus, although our investigations are not complete, there is no evidence at present to indicate that antibody-dependent cell-mediated lysis is the mechanism of xenogeneic (or D
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homologous (Jondal & Pross, 1974)) SLMC. It is possible, however, that human complement receptor lymphocytes have receptors for some structure on the surface of mouse cells, and that the close cell-to-cell contact which results is all that is necessary to initiate target cell lysis. The existence of such a receptor has been suggested by the recent demonstration that human B cells form rosettes with mouse erythrocytes (Stathopoulos & Elliott, 1974). Other preliminary data as to the nature and mechanism of xenogeneic SLMC indicate that 'bystander' target cells (chicken RBC) are not affected by reaction, that it is not mediated by supernatant fluid from cultured lymphocytes per se, and that the effect can be inhibited by the presence of 5 mm ethylenediaminetetraacetate, or by metabolic inhibitors such as 5 mm sodium azide and 5 mm sodium fluoride. The usefulness of this test as a marker of lymphocyte function has been indicated by the results of Petranyi et al. (1974a, b), using a similar independently evolved technique in which human lymphocytes spontaneously lyse DBA/2 fibroblast monolayer cells. The results in this test parallel those obtained using P815 (Petranyi, personal communication). The authors found that low activity in their test correlated with the HL-A haplotype 3,7 antigens known to correlate with the diagnosis of multiple sclerosis (Naito et al., 1972; Bertrams, Kuwert & Liedtke, 1972; Jersild et al., 1973), and suggesting an association with some type of cellular dysfunction in this disease. It is data such as this which leads us to believe that SLMC against xenogeneic target cells represents a potentially valuable tool in the assessment of human non-T-lymphocyte cytotoxic function. The reaction may also serve as an easily available and reproducible cell-mediated cytotoxic system which can be used to assess the effects of serum inhibitory and blocking factors in patients with malignant disease. Experiments are currently underway to investigate this possibility. ACKNOWLEDGMENTS
We thank Dr G. Clements for the proven Mycoplasma-free A-9 cells. The work upon which this publication is based was performed persuant to contract number NO1-CB-33870 and contract number NIH-NO1-CB-33859 with the Division of Cancer Biology and Diagnosis, National Cancer Institute, Department of Health, Education and Welfare. H. F. Pross was a Centennial Fellow of the Medical Research Council of Canada. REFERENCES BERTRAMS, J., KUWERT, E. & LIEDTKE, U. (1972) HL-A antigens and multiple sclerosis. Tissue Antigens, 2, 405. BIANCO, C., PATRICK, R. & NusSENZWEIG, V. (1970) A population of lymphocytes bearing a membrane receptor for antigen-antibody-complement complexes. I. Separation and characterization. J. exp. Med. 132, 702. BOYLE, W. (1968) An extension of the 51Cr-release assay for the estimation of mouse cytotoxins. Transplantation, 6, 761. BOYUM, A. (1968) Separation of leukocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97, p. 77. CEROTTINI, J.-C. & BRUNNER, K.T. (1974) Cell-mediated cytotoxicity, allograft rejection and tumor immunity. Advanc. Immunol. 18, 67. DUNN, T.B. & POTTER, M. (1957) A transplantable mast cell neoplasm in the mouse. J. nat. Cancer Inst. 18, 587. JERSILD, C., SVEJGAARD, A., FoG, T. & AMMITZBOLL, T. (1973) HL-A antigens and diseases. I. Multiple sclerosis. Tissue Antigens, 3, 243.
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JONDAL, M. (1974) Surface markers on human T and B lymphocytes. IV. Distribution of surface markers on resting and blast transformed lymphocytes. Scand. J. Immunol. 3, 739. JONDAL, M., HOLM, G. & WIGZELL, W. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J. exp. Med. 136,207. JONDAL, M. & KLEIN, G. (1973) Surface markers on human B and T lymphocytes. II. Presence of EpsteinBarr virus receptors on B lymphocytes. J. exp. Med. 138, 1365. JONDAL, M. & PRoss, H.F. (1975) Surface markers on human B and T lymphocytes. VI. Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and lectin-induced cytotoxicity against cell lines as functional markers for different lymphocyte subpopulations. Int. J. Cancer. (In press.) JONDAL, M., WIGZELL, H. & AUITI, F. (1973) Human lymphocyte subpopulations: classification according to surface markers and/or functional characteristics. Transplant. Rev. 16, 163. JULIUS, M.H., SIMPSON, E. & HERZENBERG, L.A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Europ. J. Immunol. 3, 645. LANDY, M., MICHAEL, J.G., TRAPANI, R.J., ACHINSTEIN, B., WOODS, M.W. & SHEAR, M.J. (1960) Antibodycomplement system in normal serum lethal to mouse tumor cells. Cancer Res., 20, 1279. Lozzio, C.B. & Lozzio, B.B. (1973) Cytotoxicity of a factor isolated from human spleen. J. nat. Cancer Inst. 50, 535. LUNDGREN, G., ZUKOSKI, C.F. & MOLLER, G. (1968) Differential aspects of human granulocytes and lymphocytes on human fibroblasts. Clin. exp. Immunol., 3, 817. NAITO, S., NAMEROW, N., MICKEY, M.R. & TERASAKI, P.I. (1972) Multiple sclerosis: association with HL-A 3. Tissue Antigens, 2, 1. PETRANYI, G., BENCZUR, M., ODONY, C.E., HOLLAN, S.R. & IVANYI, P. (1974a) HL-A 3,7 and lymphocyte cytotoxic activity. Lancet, ii, 736. PETRANYI, G., IVANYI, P. & HOLLAN, S.R. (1974b) Relations of HL-A and Rh systems to immune reactivity. Vox Sang. (Basel), 26, 470. STATHOPOULOS, G. & ELLIOTT, E.V. (1974) Formation of mouse or sheep red-blood-cell rosettes by lymphocytes from normal and leukaemic individuals. Lancet, i, 600. TAKASUGI, M., MIKEY, M.R. & TERASAKI, P. (1973) Reactivity of normal lymphocytes from normal persons on cultured tumor cells. Cancer Res. 33, 2829. WIGZELL, H. (1965) Quantitative titration of mouse H-2 antibodies using Cr' -labelled target cells. Transplantation, 3, 423.
