ClGn. exp. Immunol. (1976) 25, 347-351.
BRIEF COMMUNICATION
Cytotoxic activity of human lymphocyte plasma membranes JANEZ FERLUGA, GEORGE J. FRIOU* & ANTHONY C. ALLISON Division of Cell Pathology Clinical Research Centre, Harrow, Middlesex
(Received 12 March 1976) SUMMARY
Plasma membrane fractions isolated from normal human peripheral blood lymphocytes have considerable cytolytic activity towards a cultured human lymphoblast cell line and mouse mastocytoma cells. Other subcellular fractions, including lysosomes, have low cytolytic activity. It is suggested that this cytotoxic potential in lymphocyte plasma membranes is normally latent but can be activated by prolonged and intimate contact with target cell plasma membranes.
INTRODUCTION Certain cells from lymphoid organs have the capacity to kill the cells with which they come into contact. This process is thought to be important in tumour immunity and autoimmunity. In experimental animals such killing by T lymphocytes can be immunologically specific (Cerottini & Brunner, 1974), although when normal lymph node cells are incubated with tumour cells some non-specific killing is observed (Behelak & Richter, 1975; Herberman et al., 1975). This non-specific killing can be augmented by phytohaemagglutinin (PHA), which binds lymphocytes and target cells together (Asherson, Ferluga & Janossy, 1973; Waterfield, Waterfield & Moller, 1975). In human peripheral blood lymphocytes (PBL) several distinct types of cytotoxicity can be observed. Normal human lymphocytes incubated with tumour cells in the presence of PHA kill tumour cells within a few hours, before the lymphocytes are transformed; however, human PBL transformed by PHA have greater cytotoxic activity (Holm & Perlmann, 1967a, b). Human PBL stimulated by mixed lymphocyte reactions also acquire increased cytolytic capacity, which has a substantial non-specific component (Holm & Perlmann, 1967b; Steel et al., 1974), so that lymphocytes with histocompatibility antigens other than those of the reacting cell types are killed. These results suggest that normal PBL have cytotoxic potential which can be manifested under appropriate conditions not always involving recognition of specific antigen of target cells. The mechanism by which lymphocytes kill target cells is still largely unknown. If the effector cells are killed, or their motility is inhibited before they come into contact with target cells, the cytotoxic activity is abolished, and other evidence shows that contact of effector and target cells is required (Cerottini & Brunner, 1974). However, if the effector cells are selectively lysed by antibody and complement soon after contact is established, the lytic reaction goes to completion (Martz & Benacerraf, 1973; Sanderson & Taylor, 1975). The simplest interpretation of the observations is that once close apposition of effector cell and target cell membranes is established the effector cell membranes are themselves able to lyse the target cells. We have presented evidence that purified plasma membrane fractions from mouse lymph node cells have considerable cytotoxic activity when tested against mouse tumour cells (Ferluga & Allison, 1975). Because of the general interest of this system, and the possibility that it opens up a new means of characterizing the mechanism of cell-mediated cytolysis, we have undertaken comparable studies of human PBL. * Present address: Department of Medicine, Clinical Immunology and Rheumatic Disease Section, University ofSouthern California School of Medicine, Los Angeles, California, U.S.A. Correspondence: Dr A. C. Allison, Division ofCell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.
347
J. Ferluga, G. J. Friou Cg A. C. Allison
348
MATERIALS AND METHODS Lymphocytes. Human peripheral blood lymphocytes were from buffy coat residues left after platelet removal from normal human blood. Buffy coats from approximately 7 litres of blood were used in each experiment. Leucocytes were purified by sedimentation of red blood cells with 1% dextran solution (T 500, Pharmacia), followed by Ficoll-Triosil density-gradient centrifugation (Boyum, 1968). Mononuclear and polymorphonuclear phagocytes were removed by filtration through nylonwool columns (Greaves & Brown, 1974), reducing these cells from 14% to 1%. Phagocytes were counted using acridine orange staining of lysosomes (Allison & Young, 1969). Subcellular fractions. Subcellular fractions were obtained by breaking the cells (1-2 x 109) by passage through a cellrupturing pump (Wright, Edwards & Jones, 1974), followed by differential centrifugation (Crumpton & Snary, 1974). Pellets were obtained by centrifugation at 300 g for 15 min (nuclear), at 4000 g for 15 min (large granular) and at 20,000 g for 30 min (microsomal). The microsomal pellet was resuspended in 10 mm Tris-HCI-0 15 M-NaCl, pH 7-4 (Tris-saline), and made up to 43% sucrose (w/w), overlaid with 39, 36 and 20% (w/w) sucrose, and centrifuged at 75,000 g for 12 hr in a swinging-bucket rotor. Fractions collected at interfaces between sucrose layers were diluted with tris-saline, sedimented at 70,000 g for 1 hr and resuspended again in Tris-saline. Pellets and sucrose-gradient fractions were assayed for protein (Lowry; standard, bovine serum albumin) and markers for subcellular components. These were: for plasma membrane, 5'-nucleotidase (Persijn, van der Slik & Bon, 1969); for lysosomal membranes, 8i-glucuronidase (Talalay, Fishman & Huggins, 1946); for endoplasmic reticulum, glucose-6-phosphatase (Hers, Beaufays & de Duve, 1953) and for mitochondria, cytochrome oxidase (Cooperstein & Lazarow, 1951). Cytotoxicity assay. In the cytotoxicity assays, subcellular fractions suspended in 0 05 ml tris-saline were mixed in plastic tubes with 3 x 104 s Cr-labelled human CLA-4 lymphoblastoid cells or P-815 mouse mastocytoma cells (Cerottini & Brunner, 1974), suspended in 0 1 ml RPMI 1640 medium with 2-5% foetal bovine serum, and the mixtures incubated at 37°C for the times indicated. After addition of 1 ml of medium the cells were centrifuged and 0 5 ml of supernatant taken for counting of radioactivity. The per cent specific lysis produced by a membrane fraction was calculated as follows: Cr released in presence- Cr released in the absence of the fraction Cr released by freezing and thawing- Cr released in the absence of the fraction The relationship between percentage lysis and protein concentration was plotted for each fraction and the relative cytolytic activity was expressed as the reciprocal as follows: 100 ,ug protein required to produce 20% specific lysis The CLA-4 cells were kindly provided by Dr N. R. Ling. Experiment :i
I
U~~~~~A
0
80~~~~~
tn40
^
w
/
40~~~~~~~~ 0
_ 20 0
20
50I
~ 01~ ~~~~000
~ OE
A010 0
Protein
0
001 on 0
010
(tig/150 kLI
FIG. 1. Per cent lysis (5 1Cr release) of mastocytoma cells in 6 hr and of CLA-4 cells in 15 hr by subcellular fractions from human lymphocytes. The fractions are marked as follows: 1 (e) sucrose density fraction 20/36%; 2 (m) 36/39%; 3 (A,) 39/43%; 4 (o) large granular and 5 (o) nuclear pellets. For each fraction the per cent lysis is expressed in relation to the concentration of protein added.
Cytotoxicity ofplasma membranes
349
RESULTS Observations on the cytotoxicity of different fractions of normal human PBL are summarized in Fig. 1. Sucrose-gradient fractions had considerable dose-dependent lytic activity towards human (CLA-4) and mouse (P-815) cells. On the other hand, nuclear and large-granular fractions did not show detectable cytolytic activity, even in much higher concentrations. 5-O
cytolytic
activity25
2-5 0 40
5' - Nucleotidase
20 _
0n /3-Glucuronidose
4
0
Cytochrome
2.5
RH
Glucose - 6phosphatose
3 Fraction no.
FIG. 2. Relationship between cytolytic and enzymatic activities of subcellular fractions from human lymphocytes. Cytolytic activity was obtained from the cytotoxicity assay on CLA4 cells (solid columns) in 15 hr and on mastocytoma cells in 6 hr (open columns) and 15 hr (hatched columns). * Less than 20% lysis was obtained although protein concentrations were above 84 pg/150 p1. ** Only 32 pg of protein was added which did not produce 20% lysis. Enzyme activities are expressed as pmoles of product liberated per minute per pg of protein except for the cytochrome oxidase which is presented as a decrease in absorbance (550 mpu), resulting from the oxidation of the reduced cytochrome c, in optical density units/mg protein/minute. * * * The microsomal fraction was estimated instead of the sucrose density gradient fractions. The subcellular fractions are numbered as in Fig. 1.
To obtain information about the subcellular fraction with which cytolytic activity is associated, various markers were employed. As shown in Fig. 2, cytolytic activity paralleled the activity of the plasma membrane marker, 5'-nucleotidase, in various fractions, but was poorly or negatively correlated with glucose-6-phosphatase (endoplasmic reticulum), fl-glucuronidase (lysosomal) and cytochrome oxidase (mitochondrial) activities.
350
7. Ferluga, G. J. Friou and A. C. Allison
DISCUSSION This investigation has been concerned with the cytolytic potential of normal human PBL plasma membranes. The activity described does not appear to be due to monocytes, since depletion of these cells to 1% did not significantly reduce it. In fractionated PBL cytolytic activity was closely correlated with the presence of the plasma membrane marker, 5'-nucleotidase. No cytolytic activity was demonstrable in the large-granule fraction which was enriched in the lysosomal enzyme marker, which does not support the suggestion (Hibbs, 1974) that lysosomes play a role in cytotoxicity. Although it is widely thought that normal lymphocytes are not cytotoxic, evidence from several sources shows that under certain conditions they can exert cytotoxic effects. This is most obvious when normal human PBL are incubated with tumour cells in the presence of PHA (Holm & Perlmann, 1967a). Even in the absence of PHA, normal lymphocytes can lyse tumour cells (Peter et al., 1975; Hersey et al., 1975). The nonspecificity of human T-lymphocyte killing has also been observed by Steel et al. (1974) who used the same human target cells as we did in the present study. The aforementioned studies have been carried out using the chromium release technique. With the microcytotoxicity technique similar results have been obtained. Indeed non-specific or non-selective killing of human tumour cells by lymphocytes from normal persons has been a major problem in human tumour immunology (Bean et al., 1975). These results are consistent with our finding of cytolytic potential in normal human PBL membranes, expressed against both human and mouse target cells. For this cytotoxicity to be effective, certain requirements can be envisaged: intimate and prolonged contact of effector cell and target cell plasma membranes; and inability of the target cells to repair damage inflicted by the lymphocyte membranes. Other aspects of the problem, such as the effects of prior specific or non-specific stimulation of the human PBL and the binding of plasma membranes to target cells are under investigation. The skilful assistance of Miss Luisa C. Baptista and Mr G. R. Mirick is gratefully acknowledged. J. Ferluga and Luisa C. Baptista are supported by the Cancer Research Campaign.
REFERENCES ALLISON, A.C. & YOUNG, M.R. (1969) Vital staining and GREAVES, M.F. & BROWN, G. (1974) Purification of human T and B lymphocytes. ]. Immunol. 112, 420. fluorescence microscopy of lysosomes. Lysosomes in Biology and Pathology (ed. by J. T. Dingle and H. B. Fell), HERBERMAN, R.B., NuNN, M.E., HOLDEN, H.T. & LAVRIN, D.H. (1975) Natural cytotoxic reactivity of mouse vol. 2, p. 600. North-Holland Publishing Co., Amsterdam. lymphoid cells against syngeneic and allogeneic tumors. ASHERSON, G.L., FERLUGA, J. & JANossy, G. (1973) NonII. Characterisation of effector cell. Int. J. Cancer, 16, specific cytotoxicity by T cells activated with plant 230. mitogens in vitro and the requirement for plant agents during the killing reaction. Clin. exp. Immunol. 15, 573. HERS, H.G., BEAUFAYS, H. & DE DUVE, C. (1953) L'analyse simultanee des hexoses, des trioses et de leurs esters BEAN, M.A., BLOOM, B.R., HERBERMAN, R.B., OLD, L.J., phosphores. Biochim. biophys. Acta (Amst.), 11, 416. OETTGEN, H.F., KLEIN, G. & TERRY, W.D. (1975) Cellmediated cytotoxicity for bladder carcinoma: evaluation HERSEY, P., EDWARDS, A., EDWARDS, J., ADAMS, E., MILTON, G.W. & NELSON, D.S. (1975) Specificity of cell-mediated of a workshop. Cancer Res. 35, 2902. cytotoxicity against human melanoma lines: evidence for BEHELAK, Y. & RICHTER, M. (1975) Immunocompetent 'non-specific' killing by activated T cells. Int. . Cancer, cells in man. III. The killer cell activity of normal circu16, 173. lating lymphocytes. Clin. Immunol. Immunopathol. 4, 286. BOYUM, A. (1968) Separation of leukocytes from blood and HIBBS, J.B., JR (1974) Heterocytolysis by macrophagcs activated by BCG; lysosome exocytosis into tumour bone marrow. Scand.]. clin. Lab. Invest. 21, (supplement cells. Science, 184, 468. 97). CEROTTINI, J.C. & BRUNNER, K.T. (1974) Cell-mediated HOLM, G. & PERLMANN, P. (1967a) Quantitative studies on phytohaemagglutinin-induced cytotoxicity by human cytotoxicity, allograft rejection, and tumour immunity. lymphocytes against homologous cells in tissue culture. Advanc. Immunol. 18, 67. Immunology, 12, 525. COOPERSTEIN, S.J. & LAZAROW, A. (1951) A microspectrophotometric method for the determination of cytochrome HOLM, G. & PERLMANN, P. (1967b) Cytotoxic potential of stimulated human lymphocytes. J. exp. Med. 125, 721. oxidase. J. biol. Chem. 187, 665. CRUMPTON, M.J. & SNARY, D. (1974) Preparation and MARTZ, E. & BENACERRAF, B. (1973) An effector-cell independent step in target cell lysis by sensitized mouse properties of lymphocyte plasma membranes. Contemporlymphocytes. J. Immunol. 111, 1538. ary Topics in Molecular Immunology (ed. by G. L. Ada), PERSIJN, J.P., VAN DER SLIK, W. & BON, A.W.M. (1969) A vol. 3, p. 27. Plenum Press, New York. new method for the determination of serum nucleotidase. FERLUGA, J. & ALLISON, A.C. (1975) Cytotoxicity of isolated III. Inhibition of alkaline phosphatase. Z. klin. Chem. u. plasma membranes from lymph node cells. Nature (Lond.), klin. Biochem. 7, 493. 255, 708.
Cytotoxicity ofplasma membranes PETER, H.H., KALDEN, J.R., SEELAND, P., DIEHL, V. & ECKERT, G. (1975) Humoral and cellular immune reactions in vitro against allogeneic and autologous human melanoma cells. Clin. exp. Immunol. 20, 193. SANDERSON, C.J. & TAYLOR, G.A. (1975) The kinetics of 1 Cr release from target cells in cell mediated cytotoxicity and the relationship to the kinetics of killing. Cell Tissue Kinet. 8, 23. STEEL, C.M., HARDY, D.A., LING, N.R. & LAUDER, I.J. (1974) The interaction of normal lymphocytes and cells from lymphoid cell lines. VI. Line-directed cytotoxic specificity of lymphocytes activated by autochthonous
351
or allogeneic LCL cells. Immunology, 26, 1013. TALALAY, P., FISHMAN, W.M. & HUGGINS, C. (1964) Chromogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. i. biol. Chem. 166, 757. WATERFIELD, J.D., WATERFIELD, E.M. & MOLLER, G. (1975) Lymphocyte mediated cytotoxicity against tumor cells. I. Con A activated cytotoxic effector cells exhibit immunclogical specificity. Cell. Immunol. 17, 392. WRIGHT, B.M., EDWARDS, A.J. & JoNEs, V.E. (1974) Use of a cell rupturing pump for the preparation of thymocyte subcellular fractions. J. immunol. AMeth. 4, 281.
BRIEF COMMUNICATION
Cytotoxic activity of human lymphocyte plasma membranes JANEZ FERLUGA, GEORGE J. FRIOU* & ANTHONY C. ALLISON Division of Cell Pathology Clinical Research Centre, Harrow, Middlesex
(Received 12 March 1976) SUMMARY
Plasma membrane fractions isolated from normal human peripheral blood lymphocytes have considerable cytolytic activity towards a cultured human lymphoblast cell line and mouse mastocytoma cells. Other subcellular fractions, including lysosomes, have low cytolytic activity. It is suggested that this cytotoxic potential in lymphocyte plasma membranes is normally latent but can be activated by prolonged and intimate contact with target cell plasma membranes.
INTRODUCTION Certain cells from lymphoid organs have the capacity to kill the cells with which they come into contact. This process is thought to be important in tumour immunity and autoimmunity. In experimental animals such killing by T lymphocytes can be immunologically specific (Cerottini & Brunner, 1974), although when normal lymph node cells are incubated with tumour cells some non-specific killing is observed (Behelak & Richter, 1975; Herberman et al., 1975). This non-specific killing can be augmented by phytohaemagglutinin (PHA), which binds lymphocytes and target cells together (Asherson, Ferluga & Janossy, 1973; Waterfield, Waterfield & Moller, 1975). In human peripheral blood lymphocytes (PBL) several distinct types of cytotoxicity can be observed. Normal human lymphocytes incubated with tumour cells in the presence of PHA kill tumour cells within a few hours, before the lymphocytes are transformed; however, human PBL transformed by PHA have greater cytotoxic activity (Holm & Perlmann, 1967a, b). Human PBL stimulated by mixed lymphocyte reactions also acquire increased cytolytic capacity, which has a substantial non-specific component (Holm & Perlmann, 1967b; Steel et al., 1974), so that lymphocytes with histocompatibility antigens other than those of the reacting cell types are killed. These results suggest that normal PBL have cytotoxic potential which can be manifested under appropriate conditions not always involving recognition of specific antigen of target cells. The mechanism by which lymphocytes kill target cells is still largely unknown. If the effector cells are killed, or their motility is inhibited before they come into contact with target cells, the cytotoxic activity is abolished, and other evidence shows that contact of effector and target cells is required (Cerottini & Brunner, 1974). However, if the effector cells are selectively lysed by antibody and complement soon after contact is established, the lytic reaction goes to completion (Martz & Benacerraf, 1973; Sanderson & Taylor, 1975). The simplest interpretation of the observations is that once close apposition of effector cell and target cell membranes is established the effector cell membranes are themselves able to lyse the target cells. We have presented evidence that purified plasma membrane fractions from mouse lymph node cells have considerable cytotoxic activity when tested against mouse tumour cells (Ferluga & Allison, 1975). Because of the general interest of this system, and the possibility that it opens up a new means of characterizing the mechanism of cell-mediated cytolysis, we have undertaken comparable studies of human PBL. * Present address: Department of Medicine, Clinical Immunology and Rheumatic Disease Section, University ofSouthern California School of Medicine, Los Angeles, California, U.S.A. Correspondence: Dr A. C. Allison, Division ofCell Pathology, Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ.
347
J. Ferluga, G. J. Friou Cg A. C. Allison
348
MATERIALS AND METHODS Lymphocytes. Human peripheral blood lymphocytes were from buffy coat residues left after platelet removal from normal human blood. Buffy coats from approximately 7 litres of blood were used in each experiment. Leucocytes were purified by sedimentation of red blood cells with 1% dextran solution (T 500, Pharmacia), followed by Ficoll-Triosil density-gradient centrifugation (Boyum, 1968). Mononuclear and polymorphonuclear phagocytes were removed by filtration through nylonwool columns (Greaves & Brown, 1974), reducing these cells from 14% to 1%. Phagocytes were counted using acridine orange staining of lysosomes (Allison & Young, 1969). Subcellular fractions. Subcellular fractions were obtained by breaking the cells (1-2 x 109) by passage through a cellrupturing pump (Wright, Edwards & Jones, 1974), followed by differential centrifugation (Crumpton & Snary, 1974). Pellets were obtained by centrifugation at 300 g for 15 min (nuclear), at 4000 g for 15 min (large granular) and at 20,000 g for 30 min (microsomal). The microsomal pellet was resuspended in 10 mm Tris-HCI-0 15 M-NaCl, pH 7-4 (Tris-saline), and made up to 43% sucrose (w/w), overlaid with 39, 36 and 20% (w/w) sucrose, and centrifuged at 75,000 g for 12 hr in a swinging-bucket rotor. Fractions collected at interfaces between sucrose layers were diluted with tris-saline, sedimented at 70,000 g for 1 hr and resuspended again in Tris-saline. Pellets and sucrose-gradient fractions were assayed for protein (Lowry; standard, bovine serum albumin) and markers for subcellular components. These were: for plasma membrane, 5'-nucleotidase (Persijn, van der Slik & Bon, 1969); for lysosomal membranes, 8i-glucuronidase (Talalay, Fishman & Huggins, 1946); for endoplasmic reticulum, glucose-6-phosphatase (Hers, Beaufays & de Duve, 1953) and for mitochondria, cytochrome oxidase (Cooperstein & Lazarow, 1951). Cytotoxicity assay. In the cytotoxicity assays, subcellular fractions suspended in 0 05 ml tris-saline were mixed in plastic tubes with 3 x 104 s Cr-labelled human CLA-4 lymphoblastoid cells or P-815 mouse mastocytoma cells (Cerottini & Brunner, 1974), suspended in 0 1 ml RPMI 1640 medium with 2-5% foetal bovine serum, and the mixtures incubated at 37°C for the times indicated. After addition of 1 ml of medium the cells were centrifuged and 0 5 ml of supernatant taken for counting of radioactivity. The per cent specific lysis produced by a membrane fraction was calculated as follows: Cr released in presence- Cr released in the absence of the fraction Cr released by freezing and thawing- Cr released in the absence of the fraction The relationship between percentage lysis and protein concentration was plotted for each fraction and the relative cytolytic activity was expressed as the reciprocal as follows: 100 ,ug protein required to produce 20% specific lysis The CLA-4 cells were kindly provided by Dr N. R. Ling. Experiment :i
I
U~~~~~A
0
80~~~~~
tn40
^
w
/
40~~~~~~~~ 0
_ 20 0
20
50I
~ 01~ ~~~~000
~ OE
A010 0
Protein
0
001 on 0
010
(tig/150 kLI
FIG. 1. Per cent lysis (5 1Cr release) of mastocytoma cells in 6 hr and of CLA-4 cells in 15 hr by subcellular fractions from human lymphocytes. The fractions are marked as follows: 1 (e) sucrose density fraction 20/36%; 2 (m) 36/39%; 3 (A,) 39/43%; 4 (o) large granular and 5 (o) nuclear pellets. For each fraction the per cent lysis is expressed in relation to the concentration of protein added.
Cytotoxicity ofplasma membranes
349
RESULTS Observations on the cytotoxicity of different fractions of normal human PBL are summarized in Fig. 1. Sucrose-gradient fractions had considerable dose-dependent lytic activity towards human (CLA-4) and mouse (P-815) cells. On the other hand, nuclear and large-granular fractions did not show detectable cytolytic activity, even in much higher concentrations. 5-O
cytolytic
activity25
2-5 0 40
5' - Nucleotidase
20 _
0n /3-Glucuronidose
4
0
Cytochrome
2.5
RH
Glucose - 6phosphatose
3 Fraction no.
FIG. 2. Relationship between cytolytic and enzymatic activities of subcellular fractions from human lymphocytes. Cytolytic activity was obtained from the cytotoxicity assay on CLA4 cells (solid columns) in 15 hr and on mastocytoma cells in 6 hr (open columns) and 15 hr (hatched columns). * Less than 20% lysis was obtained although protein concentrations were above 84 pg/150 p1. ** Only 32 pg of protein was added which did not produce 20% lysis. Enzyme activities are expressed as pmoles of product liberated per minute per pg of protein except for the cytochrome oxidase which is presented as a decrease in absorbance (550 mpu), resulting from the oxidation of the reduced cytochrome c, in optical density units/mg protein/minute. * * * The microsomal fraction was estimated instead of the sucrose density gradient fractions. The subcellular fractions are numbered as in Fig. 1.
To obtain information about the subcellular fraction with which cytolytic activity is associated, various markers were employed. As shown in Fig. 2, cytolytic activity paralleled the activity of the plasma membrane marker, 5'-nucleotidase, in various fractions, but was poorly or negatively correlated with glucose-6-phosphatase (endoplasmic reticulum), fl-glucuronidase (lysosomal) and cytochrome oxidase (mitochondrial) activities.
350
7. Ferluga, G. J. Friou and A. C. Allison
DISCUSSION This investigation has been concerned with the cytolytic potential of normal human PBL plasma membranes. The activity described does not appear to be due to monocytes, since depletion of these cells to 1% did not significantly reduce it. In fractionated PBL cytolytic activity was closely correlated with the presence of the plasma membrane marker, 5'-nucleotidase. No cytolytic activity was demonstrable in the large-granule fraction which was enriched in the lysosomal enzyme marker, which does not support the suggestion (Hibbs, 1974) that lysosomes play a role in cytotoxicity. Although it is widely thought that normal lymphocytes are not cytotoxic, evidence from several sources shows that under certain conditions they can exert cytotoxic effects. This is most obvious when normal human PBL are incubated with tumour cells in the presence of PHA (Holm & Perlmann, 1967a). Even in the absence of PHA, normal lymphocytes can lyse tumour cells (Peter et al., 1975; Hersey et al., 1975). The nonspecificity of human T-lymphocyte killing has also been observed by Steel et al. (1974) who used the same human target cells as we did in the present study. The aforementioned studies have been carried out using the chromium release technique. With the microcytotoxicity technique similar results have been obtained. Indeed non-specific or non-selective killing of human tumour cells by lymphocytes from normal persons has been a major problem in human tumour immunology (Bean et al., 1975). These results are consistent with our finding of cytolytic potential in normal human PBL membranes, expressed against both human and mouse target cells. For this cytotoxicity to be effective, certain requirements can be envisaged: intimate and prolonged contact of effector cell and target cell plasma membranes; and inability of the target cells to repair damage inflicted by the lymphocyte membranes. Other aspects of the problem, such as the effects of prior specific or non-specific stimulation of the human PBL and the binding of plasma membranes to target cells are under investigation. The skilful assistance of Miss Luisa C. Baptista and Mr G. R. Mirick is gratefully acknowledged. J. Ferluga and Luisa C. Baptista are supported by the Cancer Research Campaign.
REFERENCES ALLISON, A.C. & YOUNG, M.R. (1969) Vital staining and GREAVES, M.F. & BROWN, G. (1974) Purification of human T and B lymphocytes. ]. Immunol. 112, 420. fluorescence microscopy of lysosomes. Lysosomes in Biology and Pathology (ed. by J. T. Dingle and H. B. Fell), HERBERMAN, R.B., NuNN, M.E., HOLDEN, H.T. & LAVRIN, D.H. (1975) Natural cytotoxic reactivity of mouse vol. 2, p. 600. North-Holland Publishing Co., Amsterdam. lymphoid cells against syngeneic and allogeneic tumors. ASHERSON, G.L., FERLUGA, J. & JANossy, G. (1973) NonII. Characterisation of effector cell. Int. J. Cancer, 16, specific cytotoxicity by T cells activated with plant 230. mitogens in vitro and the requirement for plant agents during the killing reaction. Clin. exp. Immunol. 15, 573. HERS, H.G., BEAUFAYS, H. & DE DUVE, C. (1953) L'analyse simultanee des hexoses, des trioses et de leurs esters BEAN, M.A., BLOOM, B.R., HERBERMAN, R.B., OLD, L.J., phosphores. Biochim. biophys. Acta (Amst.), 11, 416. OETTGEN, H.F., KLEIN, G. & TERRY, W.D. (1975) Cellmediated cytotoxicity for bladder carcinoma: evaluation HERSEY, P., EDWARDS, A., EDWARDS, J., ADAMS, E., MILTON, G.W. & NELSON, D.S. (1975) Specificity of cell-mediated of a workshop. Cancer Res. 35, 2902. cytotoxicity against human melanoma lines: evidence for BEHELAK, Y. & RICHTER, M. (1975) Immunocompetent 'non-specific' killing by activated T cells. Int. . Cancer, cells in man. III. The killer cell activity of normal circu16, 173. lating lymphocytes. Clin. Immunol. Immunopathol. 4, 286. BOYUM, A. (1968) Separation of leukocytes from blood and HIBBS, J.B., JR (1974) Heterocytolysis by macrophagcs activated by BCG; lysosome exocytosis into tumour bone marrow. Scand.]. clin. Lab. Invest. 21, (supplement cells. Science, 184, 468. 97). CEROTTINI, J.C. & BRUNNER, K.T. (1974) Cell-mediated HOLM, G. & PERLMANN, P. (1967a) Quantitative studies on phytohaemagglutinin-induced cytotoxicity by human cytotoxicity, allograft rejection, and tumour immunity. lymphocytes against homologous cells in tissue culture. Advanc. Immunol. 18, 67. Immunology, 12, 525. COOPERSTEIN, S.J. & LAZAROW, A. (1951) A microspectrophotometric method for the determination of cytochrome HOLM, G. & PERLMANN, P. (1967b) Cytotoxic potential of stimulated human lymphocytes. J. exp. Med. 125, 721. oxidase. J. biol. Chem. 187, 665. CRUMPTON, M.J. & SNARY, D. (1974) Preparation and MARTZ, E. & BENACERRAF, B. (1973) An effector-cell independent step in target cell lysis by sensitized mouse properties of lymphocyte plasma membranes. Contemporlymphocytes. J. Immunol. 111, 1538. ary Topics in Molecular Immunology (ed. by G. L. Ada), PERSIJN, J.P., VAN DER SLIK, W. & BON, A.W.M. (1969) A vol. 3, p. 27. Plenum Press, New York. new method for the determination of serum nucleotidase. FERLUGA, J. & ALLISON, A.C. (1975) Cytotoxicity of isolated III. Inhibition of alkaline phosphatase. Z. klin. Chem. u. plasma membranes from lymph node cells. Nature (Lond.), klin. Biochem. 7, 493. 255, 708.
Cytotoxicity ofplasma membranes PETER, H.H., KALDEN, J.R., SEELAND, P., DIEHL, V. & ECKERT, G. (1975) Humoral and cellular immune reactions in vitro against allogeneic and autologous human melanoma cells. Clin. exp. Immunol. 20, 193. SANDERSON, C.J. & TAYLOR, G.A. (1975) The kinetics of 1 Cr release from target cells in cell mediated cytotoxicity and the relationship to the kinetics of killing. Cell Tissue Kinet. 8, 23. STEEL, C.M., HARDY, D.A., LING, N.R. & LAUDER, I.J. (1974) The interaction of normal lymphocytes and cells from lymphoid cell lines. VI. Line-directed cytotoxic specificity of lymphocytes activated by autochthonous
351
or allogeneic LCL cells. Immunology, 26, 1013. TALALAY, P., FISHMAN, W.M. & HUGGINS, C. (1964) Chromogenic substrates. II. Phenolphthalein glucuronic acid as substrate for the assay of glucuronidase activity. i. biol. Chem. 166, 757. WATERFIELD, J.D., WATERFIELD, E.M. & MOLLER, G. (1975) Lymphocyte mediated cytotoxicity against tumor cells. I. Con A activated cytotoxic effector cells exhibit immunclogical specificity. Cell. Immunol. 17, 392. WRIGHT, B.M., EDWARDS, A.J. & JoNEs, V.E. (1974) Use of a cell rupturing pump for the preparation of thymocyte subcellular fractions. J. immunol. AMeth. 4, 281.
