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Generation of human T-cell hybrids with the characteristics of human peripheral blood T-lymphocytes
Immunology Letters, 6 (1983) 203 207 Elsevier BiomedicalPress
GENERATION
OF HUMAN
OF HUMAN
T-CELL HYBRIDS
PERIPHERAL
BLOOD
WITH
THE CHARACTERISTICS
T-LYMPHOCYTES
G. G A L L A G H E R and W. H. STIMSON* Dept. Bioscience and Biotechnology, Biochemistry Division, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow G40NR, U.K.
(Received 8 February 1983) (Accepted 11 February 1983)
1. Summary Using a selection system based on the two irreversible biochemical inhibitors, actinomycin-D and emetine hydrochloride, we have constructed human T-lymphocyte hybrids between the human T-cell line, M o l t - 4 F , and mitogen-activated human lymphocytes. The cells were identified as true hybrids by their karyotype, differences in supernatant activities, responses to mitogen and membrane characteristics. These hybrid cells are stable in culture and express functions found in mature human T-lymphocytes.
2. Introduction Human T-lymphocytes are recognized to be a heterologous population of cells, in terms of surface antigens and cell function. This causes great problems when attempting to dissect the complex interactions which take place between cells during every immune response. It would, therefore, be an advantage to be able to work with homogeneous populations when studying the functions of these very important cells. Two approaches exist to obtain clones of human T-cells. The first relies on supplementing the growth medium with lnterleukin 2 (IL2), the T-cell growth factor. This method has allowed the propagation of
human T-cell clones with distinct helper, suppressor and cytotoxic functions [1-3]. The clones, however, remain totally dependent on IL2 and large numbers of cells are difficult to generate and maintain. The second technique stems from the work of Kohler and Milstein [4], who adapted somatic cell fusion to the study of the immune system. Their method allows the generation of clones with large populations by 'immortalizing' the function under study within a hybrid cell generated from a myeloma or lymphoblastic leukaemia cell and an activated Bor T-cell. Many successes have been reported in the murine system. Using enzyme-deficient parent celllines sensitive to aminopterin, T-cell hybrids have been described which express functional properties of the mature partner cell [5,6]. Initial work with human cells also relied on enzyme-deficient parent cells for both B- and T-lymphocytes [7,8"1. However, the irreversible inihibitor method of Wright [9] has been adapted successfully to the construction of human B- and T-cell hybrids [10,11]. We report here the use of a variation of this method to generate human T-cell hybrids, which express functions characteristic of human T-cells, between the human T-cell line, Molt-4F, and mitogen-activated human peripheral blood T-cells.
3. Materials and methods *To whom correspondenceshould be addressed. Key words: human T-cell hybrids Molt-4F- T-lymphocyte
0165-2478/83/0000 0000/$3.00 © 1983ElsevierScience Publishers B.V.
3.1. Cells The human T-cell line, Molt-4F, was obtained 203
from Flow Laboratories (Irvine, U.K.). The line is regularly maintained in HAMS F10 culture medium (Flow), supplemented with 20 mM Hepes, 2 mM glutamine, 5 mM NaHCO3, 50 tzg/ml gentamycin sulphate and 10% (v/v) foetal calf serum (FCS). Heparinized human peripheral venous blood was obtained from healthy male adults. The mononuclear cells were prepared by centrifugation of the plasma layer over Ficoll-Isopaque (300 g, 25 min, 4 ° C) and the cells obtained from the interphase were washed twice before use. These were adjusted to 5 X 105/ml in medium with 10% autologous plasma (no FCS) and the population of activated T-cells highly enriched by incubation with 5 #g/ml phytohaemagglutinin (PHA-P, Difco) for 72 h in 25 cm 3 flasks (Costar). The cells were harvested by incubating with 100 mM N-acetyl-D-galactosamine (Sigma) for 2 h at 37 ° C, washed twice and used for fusion, a small aliquot being set aside for control purposes.
3.2. Fusion procedure The protocol was adapted from that of Kobayashi et al. [11]. Briefly, Molt-4F cells were adjusted to 106/ml and incubated with 0.25 #g/ml actinomycin-D and 5 X 10-5 M emetine hydrochloride (Sigma) for 2 h at 37 ° C. These concentrations were seen to be optimal following a thorough investigation of the minimum lethal dose of the two compounds. After washing 4 times, a small aliquot was set aside and the remainder used for fusion. The cells were mixed in the ratio: PHA-stimulated cells:Molt-4F 10:1, then pelleted and gently resuspended in a polyethelene glycol (PEG) solution containing: 46% (w/v) PEG 1550 (Serva), 5/~g/ml polyL-arginine, (mol. wt. = 40,000 (Sigma) [12]) and 15% (v/v) dimethylsulphoxide (DMSO). The solution was prewarmed to 37 ° C and the cells were exposed to 0.5 ml for 45 s. Using prewarmed medium, the PEG was diluted to 20 ml over 5 min and the mixture centrifuged (150 g, 10 min). The pellet was gently resuspended in 5 ml of medium containing 10% FCS and incubated for 2 h at 37 ° C. Finally, the cell concentration was brought to 5 X 105/ml in complete medium and plated (200 ~zl per well) into 96-well, flat-bottomed plates (Costar). No feeder cells or conditioned media were employed. As controls, each plate was seeded with 8 wells of each: (a) drug-treated Molt-4F; (b) PHA-stimulated 204
cells; (c) (a) + (b) mixed but not fused. The cells were fed by 50% medium exchange every day for one week, and thereafter twice a week. Macroscopic cell growth was observed after 4-5 weeks, although small colonies were often evident at 3 weeks. No growth was seen in any of the control wells at any time. 3.3. Mitogen response of hybrid cells The ability of the hybrid cells to bind and respond to various mitogens was measured. Cells (105/200 ~zl) of each type were incubated in microtitre plates (triplicate cultures) each with: (a) no mitogen (control); (b) 5 ~zg/ml PHA; or (c) 10 tzg/ml concanavalin A (Con A). These were cultured for 72 h at 37 ° C and the mitogenic response estimated following the addition of 1 #Ci[3H]thymidine (Radiochemicals Centre, Amersham) for 4 h. The results are expressed as a Stimulation Index (SI) and a Mitogen Response Ratio (MRR) calculated as follows: SI-
Mean cpm of mitogen-stimulated cultures Mean cpm of control cultures (no mitogen)
MRR
SI of hybrid cells =
SI of parental cell line
The M R R allows a comparison of the mitogen response of the hybrid cell with that of the parent cell line. The patterns of mitogen-induced cellular aggregation (at 24 h) in every well were also compared using inverted-stage phase-contrast microscopy and taken as an indication of mitogen binding. 3.4. Assessment of supernatant activity Supernatants from the hybrid cells and the parental Molt-4F were gathered after incubating 5 X 105 cells/ml for 24 h with 10% FCS. The supernatant were harvested, adjusted to pH 7.4, filter sterilized (0.22 #m pore size) and tested for their ability to affect the PHA-induced proliferation of human lymphocytes (5 #g/ml PHA). Supernatants were tested at 50%, 25% and 5% (v/v), in triplicate. Control cultures received no supernatant and were incubated with and without mitogen. Human lymphocytes were prepared as above
(3.1), brought to 106/ml in 10% autologous plasma and plated onto 100 ~zl cultures. The test supernatant was added and the final culture volume was brought to 200 #1 with medium containing 10% FCS. The cells were incubated for 72 h at 37 ° C and the Sis were calculated and compared as in 3.3. 3.5. Assessment of SRBC receptors Fresh sheep red blood cells (SRBC) were treated with a 2% (w/v) solution of 2-aminoethylisothiouronium bromide hydrobromide (AET), pH 8.0, for 15 min, 37 ° C. The washed A E T - S R B C were mixed in complete medium with an equal volume of the cells being tested (106/ml), centrifuged (150g, 10 min) and left at 4 ° C for 1 h. Thereafter, the pellet was gently resuspended and the degree of rosette formation assessed. Cells bearing ~>5 SRBC were regarded as positive. 3.6. Karyotype analysis Standard methods were used in the karyotype analysis. Briefly, Colcemid-induced metaphase spreads were prepared by incubating exponentiallygrowing ceils with 0.01 # g / m l Colcemid (Sigma) for 4 h. The cells were swollen in 0.075 M potassium chloride (15 min, 37 ° C), fixed in methanol-glacial acetic acid (3:1), dropped onto cold, wet slides, allowed to dry, and stained with Geimsa. Suitable spreads were photographed to enable accurate counting. 10-15 spreads of each type were counted [13]. 3.7. Surface antigen analysis A standard, double-antibody technique was employed. The monoclonal antibodies OKT8 (Ortho) and Hu.Lyt. 1 (New England Nuclear) were used at their optimum concentrations (1 # g / m l protein, 4 ° C, 45 rain). These antibodies were visualized on the surface of the cells by staining with a 1:20 dilution of a fluorescein-linked rabbit anti-mouse IgG (Miles) followed by examination on a Wild microscope equipped with a HBO-200 mercury-vapour light source.
4. Results and discussion
PHA-activated human lymphocytes were success-
fully fused with drug-pretreated Molt-4F cells, resulting in good growth of hybrid cells in 5 out of 144 wells 4 weeks after fusion. No growth was seen in any of the control wells. Once the cells had become approximately 50% confluent, the cultures were expanded into 1 ml of complete medium in 25 cm 3 flasks, which were then balanced on their corners [14]. Two of the cultures grew rapidly and were designated 2E6M and 2CllM. The supernatant from 2C11 M, unstimulated by mitogen or antigen, was capable of significantly inhibiting the PHA-induced proliferation of normal human peripheral lymphocytes (Fig. 1). When it was introduced at a concentration of 25% (v/v), the response was suppressed by 55% while at the 50% level thymidine uptake was completely inhibited. The 2E6M and Molt-4F supernatants did not possess this activity and none of the supernatants were cytotoxic (as assessed by trypan blue exclusion at 72 h), or exhibited any mitogenic activity (data not shown). It is not yet known whether this inhibition reflects some general, non-specific suppression, or a specific effect on the PHA-induced response. Further immunological and physiological characterizations of this supernatant have been initiated.
OP~ I ~1041
HPBL
I
MOLt -4F
2E6M
L 2c~M
CCL~-rVpE
Fig. 1. Effect of hybrid cell supernatants on the PHA-induced proliferation of normal human peripheral blood lymphocytes (HPBL). Results are expressed as the mean + S.D. of 3 experiments performed in triplicate. (a) without and (b) with 5 ~tg/ml PHA. Cell supernatant was added at: (m) 5%; (A) 25%; and (~) 50% (v/v) to 200 I.d cultures. 205
Both the 2E6M and 2C11M hybrid cells responded to the mitogens, P H A and Con A, 2E6M dramatically so (Table 1). The Molt-4F cells showed a slight growth inhibition in the presence of PHA, while both of the hybrids (2E6M and 2C11 M) were stimulated to double and quadruple their respective growth rates in its presence. The 2E6M cells displayed an exceedingly high transformation rate in the presence of Con A and this cell may provide a vehicle for the study of blastogenesis in T-cells. However, unlike Molt-4F or 2E6M, 2C11M did not appear to respond either positively or negatively to the addition of Con A, behaving as if no mitogen had been added. Support for these results was shown by the patterns of aggregation obtained for the cells. While Molt-4F showed a small degree of aggregation with the two mitogens, 2E6M and 2C11M behaved quite differently (Table 2). 2E6M aggregated intensely in the presence of Con A and hardly at all with PHA while 2C11M did not aggregate at all with Con A
Table 1 Effects of mitogens on hybrid cells
Molt-4F 2E6M 2CIIM
Stimulation index a,b
Mitogen response ratio a
PHA
Con A
PHA
0.889 2.056 4.813
2.626 414.1 1.005
1.0 2.313 5.414
Con A 1.0 157.7 0.383
4
a As defined in text (3.3). b Mean of 3 experiments, carried out in triplicate.
and only moderately with PHA. This appears to indicate that the surface characteristics are different in the 3 cell types, at least with respect to the carbohydrate moieties displayed. The hybrid cell, 2E6M, also expressed the SRBC receptor on its surface (20%), while 2C11M did not, reflecting a further degree of similarity of 2E6M with normal T-cells. In our hands Molt-4F did not form rosettes with AET-treated SRBC while normal peripheral human lymphocytes did (40%). Karyotypic analysis of the cells showed that the hybrids 2E6M and 2C11M each had greater numbers of chromosomes than Molt-4F (95, 91 and 88, respectively). This small difference was not unexpected since Molt-4F is nearly tetraploid, but it was significant. A summary of the properties of the cells is layed out in Table 2. Microscopic examination of the cells has shown differences in their respective morphologies. Although all of the cell types grow in suspension, both Molt-4F and 2C11M grow as discrete cells without any visible marks or irregularities of shape. However, 2E6M grows in groups of 5-10 cells adhering to each other, and the cells appear very granular. Studies into the antigens displayed on the surface of the cells have shown that neither our Molt-4F nor the hybrid cells carry any antigens which would react with the monoclonal antibodies Hu.Lytl or OKT8; further studies are continuing. Other workers have reported that rigorous cloning is essential for the maintainance of functional human T-cell hybrids [15], that feeder systems are desirable for initial growth [11], or that they may be
Table 2 S u m m a r y of properties of hybrid cells
Karyotype a SRBC-rosette Aggregation (a) P H A (b) Con A MRR b (a) P H A (b) Con A Supernatant action d
M olt-4F
2E6M
2C11 M
HPBL
88 + 1.4
94+3.6
91 5:0.8
46
20% +/+++
++
++ ++
1.0 1.0
2.3 157.7
5.4 0.38 Yes
nd c nd
Mean 5: S.D. of 10-15 observations. b As defined in text (3.3). c n d = not determined. d As defined in text (3.4). a
206
40%
+ +
unstable and quickly die [16]. We have not encountered any of these problems, perhaps because there was only one focus of growth in each well and therefore the cells are clones, or because the culture conditions were such that only the stable cells survived the first few weeks after fusion and were subsequently selected. It was decided that the mitogen-stimulated cells themselves would act as feeders for the critical initial period after fusion and that the addition of other drug-treated cells as feeders [11] would not be required. Further successful experiments have borne this out. 2E6M and 2C11M have been in continuous culture now for over 8 months and show no signs of losing any of the properties reported here. The use of irreversible inhibitors as selection tools should open up the field of human-human cell hybridization by allowing many (if not all) cell-lines to be used as parents for the construction of hybrids with suitable partners, and work into this area is proceeding in our laboratories. Further studies into the effects of different parent cells on the fusion efficiency are also being carried out. We believe that it may be desirable to choose the parent cell carefully in relation to the partner cell and the function that one wishes one's hybrids to express. In conclusion, we believe that we have successfully constructed functional human T-lymphocyte hybrid cells and that the use of such cells will be of great use as investigative tools in human immunology.
Acknowledgements We would like to thank Dr. E. Boyd for preparing the karyotype analysis and Drs. A. Boylston. M.
Feldman and S. Metcalf for helpful discussion. This work was supported by a grant and a training award from the Medical Research Council.
References [1] Fischer, A., Zanders, E. D., Beverley, P. C. L. and Feldman, M. (1982) in: The l_.ymphokines, Vol. 5, Monoclonal T-cells and Their Products (Feldman, M. and Schreier, M. H. eds.) pp. 465-491, Academic Press. [2] Lamb, J. and Feldman, M. (1982) Nature (London) 300, 456-458. [3] Wallace, L. E., Rickinson, A. B., Rowee, M. and Epstein, M. A. (1982) Nature (London) 297,413~115. [4] Kohler, G. and Milstein, J. A. (1978) Nature (London) 256, 495497. [5] Goldsby, R. A., Osbourne, B. A., Simpson, E., Herzenberg, L. A. (1978) Nature (London) 267,707 708. [6] Kontianen, S., Simpson, E., Bohrer, E., Beverley, P. C. L., Herzenberg, L. A., Fitzpatrick, W. C., Vogt, P., Torano, A., McKenzie, 1. F. C. and Feldman, M. (1978) Nature (London) 274, 477 480. [7] Olsson, L. and Kaplan, H. S. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5429-5431. [8] Grillot-Courvalin, C., Brouet, J.-C., Berger, R. and Bernheim, A. (1981) Nature (London) 292, 844-845. [9] Wright, W. E. (1978) Exp. Cell. Res. 112, 395~107. [10] Clark, S. A., Stimson, W. H., Williamson, A. R. and Dick, H. M. (1981) J. Supramol. Struct. Cell. Biochem. Suppl. 5, 10th Ann. ICN-UCLA Symp., Abstr. 100. [11] Kobayashi, Y., Asada, M., Higuchi, M. and Osawa, T. (1982) J. Immunol. 128, 2714-2718. [12] Wantanabe, T. and Ohara, J. (1981) Nature (London) 290, 58 60. [13] Boyd, E., personal communication. [14] Metcalf, S., personal communication. [15] lrigoyen, O., Rizzolo, P. R., Thomas, Y., Rogozinski, L. and Chess, L. (1981) J. Exp. Med. 154, 1827-1837. [16] Greene, W. C., Fleisher, T. A., Nelson, D. L. and Waldman, T. A. (1982) J. Immunol. 129, 1986-1992.
207
GENERATION
OF HUMAN
OF HUMAN
T-CELL HYBRIDS
PERIPHERAL
BLOOD
WITH
THE CHARACTERISTICS
T-LYMPHOCYTES
G. G A L L A G H E R and W. H. STIMSON* Dept. Bioscience and Biotechnology, Biochemistry Division, University of Strathclyde, The Todd Centre, 31 Taylor Street, Glasgow G40NR, U.K.
(Received 8 February 1983) (Accepted 11 February 1983)
1. Summary Using a selection system based on the two irreversible biochemical inhibitors, actinomycin-D and emetine hydrochloride, we have constructed human T-lymphocyte hybrids between the human T-cell line, M o l t - 4 F , and mitogen-activated human lymphocytes. The cells were identified as true hybrids by their karyotype, differences in supernatant activities, responses to mitogen and membrane characteristics. These hybrid cells are stable in culture and express functions found in mature human T-lymphocytes.
2. Introduction Human T-lymphocytes are recognized to be a heterologous population of cells, in terms of surface antigens and cell function. This causes great problems when attempting to dissect the complex interactions which take place between cells during every immune response. It would, therefore, be an advantage to be able to work with homogeneous populations when studying the functions of these very important cells. Two approaches exist to obtain clones of human T-cells. The first relies on supplementing the growth medium with lnterleukin 2 (IL2), the T-cell growth factor. This method has allowed the propagation of
human T-cell clones with distinct helper, suppressor and cytotoxic functions [1-3]. The clones, however, remain totally dependent on IL2 and large numbers of cells are difficult to generate and maintain. The second technique stems from the work of Kohler and Milstein [4], who adapted somatic cell fusion to the study of the immune system. Their method allows the generation of clones with large populations by 'immortalizing' the function under study within a hybrid cell generated from a myeloma or lymphoblastic leukaemia cell and an activated Bor T-cell. Many successes have been reported in the murine system. Using enzyme-deficient parent celllines sensitive to aminopterin, T-cell hybrids have been described which express functional properties of the mature partner cell [5,6]. Initial work with human cells also relied on enzyme-deficient parent cells for both B- and T-lymphocytes [7,8"1. However, the irreversible inihibitor method of Wright [9] has been adapted successfully to the construction of human B- and T-cell hybrids [10,11]. We report here the use of a variation of this method to generate human T-cell hybrids, which express functions characteristic of human T-cells, between the human T-cell line, Molt-4F, and mitogen-activated human peripheral blood T-cells.
3. Materials and methods *To whom correspondenceshould be addressed. Key words: human T-cell hybrids Molt-4F- T-lymphocyte
0165-2478/83/0000 0000/$3.00 © 1983ElsevierScience Publishers B.V.
3.1. Cells The human T-cell line, Molt-4F, was obtained 203
from Flow Laboratories (Irvine, U.K.). The line is regularly maintained in HAMS F10 culture medium (Flow), supplemented with 20 mM Hepes, 2 mM glutamine, 5 mM NaHCO3, 50 tzg/ml gentamycin sulphate and 10% (v/v) foetal calf serum (FCS). Heparinized human peripheral venous blood was obtained from healthy male adults. The mononuclear cells were prepared by centrifugation of the plasma layer over Ficoll-Isopaque (300 g, 25 min, 4 ° C) and the cells obtained from the interphase were washed twice before use. These were adjusted to 5 X 105/ml in medium with 10% autologous plasma (no FCS) and the population of activated T-cells highly enriched by incubation with 5 #g/ml phytohaemagglutinin (PHA-P, Difco) for 72 h in 25 cm 3 flasks (Costar). The cells were harvested by incubating with 100 mM N-acetyl-D-galactosamine (Sigma) for 2 h at 37 ° C, washed twice and used for fusion, a small aliquot being set aside for control purposes.
3.2. Fusion procedure The protocol was adapted from that of Kobayashi et al. [11]. Briefly, Molt-4F cells were adjusted to 106/ml and incubated with 0.25 #g/ml actinomycin-D and 5 X 10-5 M emetine hydrochloride (Sigma) for 2 h at 37 ° C. These concentrations were seen to be optimal following a thorough investigation of the minimum lethal dose of the two compounds. After washing 4 times, a small aliquot was set aside and the remainder used for fusion. The cells were mixed in the ratio: PHA-stimulated cells:Molt-4F 10:1, then pelleted and gently resuspended in a polyethelene glycol (PEG) solution containing: 46% (w/v) PEG 1550 (Serva), 5/~g/ml polyL-arginine, (mol. wt. = 40,000 (Sigma) [12]) and 15% (v/v) dimethylsulphoxide (DMSO). The solution was prewarmed to 37 ° C and the cells were exposed to 0.5 ml for 45 s. Using prewarmed medium, the PEG was diluted to 20 ml over 5 min and the mixture centrifuged (150 g, 10 min). The pellet was gently resuspended in 5 ml of medium containing 10% FCS and incubated for 2 h at 37 ° C. Finally, the cell concentration was brought to 5 X 105/ml in complete medium and plated (200 ~zl per well) into 96-well, flat-bottomed plates (Costar). No feeder cells or conditioned media were employed. As controls, each plate was seeded with 8 wells of each: (a) drug-treated Molt-4F; (b) PHA-stimulated 204
cells; (c) (a) + (b) mixed but not fused. The cells were fed by 50% medium exchange every day for one week, and thereafter twice a week. Macroscopic cell growth was observed after 4-5 weeks, although small colonies were often evident at 3 weeks. No growth was seen in any of the control wells at any time. 3.3. Mitogen response of hybrid cells The ability of the hybrid cells to bind and respond to various mitogens was measured. Cells (105/200 ~zl) of each type were incubated in microtitre plates (triplicate cultures) each with: (a) no mitogen (control); (b) 5 ~zg/ml PHA; or (c) 10 tzg/ml concanavalin A (Con A). These were cultured for 72 h at 37 ° C and the mitogenic response estimated following the addition of 1 #Ci[3H]thymidine (Radiochemicals Centre, Amersham) for 4 h. The results are expressed as a Stimulation Index (SI) and a Mitogen Response Ratio (MRR) calculated as follows: SI-
Mean cpm of mitogen-stimulated cultures Mean cpm of control cultures (no mitogen)
MRR
SI of hybrid cells =
SI of parental cell line
The M R R allows a comparison of the mitogen response of the hybrid cell with that of the parent cell line. The patterns of mitogen-induced cellular aggregation (at 24 h) in every well were also compared using inverted-stage phase-contrast microscopy and taken as an indication of mitogen binding. 3.4. Assessment of supernatant activity Supernatants from the hybrid cells and the parental Molt-4F were gathered after incubating 5 X 105 cells/ml for 24 h with 10% FCS. The supernatant were harvested, adjusted to pH 7.4, filter sterilized (0.22 #m pore size) and tested for their ability to affect the PHA-induced proliferation of human lymphocytes (5 #g/ml PHA). Supernatants were tested at 50%, 25% and 5% (v/v), in triplicate. Control cultures received no supernatant and were incubated with and without mitogen. Human lymphocytes were prepared as above
(3.1), brought to 106/ml in 10% autologous plasma and plated onto 100 ~zl cultures. The test supernatant was added and the final culture volume was brought to 200 #1 with medium containing 10% FCS. The cells were incubated for 72 h at 37 ° C and the Sis were calculated and compared as in 3.3. 3.5. Assessment of SRBC receptors Fresh sheep red blood cells (SRBC) were treated with a 2% (w/v) solution of 2-aminoethylisothiouronium bromide hydrobromide (AET), pH 8.0, for 15 min, 37 ° C. The washed A E T - S R B C were mixed in complete medium with an equal volume of the cells being tested (106/ml), centrifuged (150g, 10 min) and left at 4 ° C for 1 h. Thereafter, the pellet was gently resuspended and the degree of rosette formation assessed. Cells bearing ~>5 SRBC were regarded as positive. 3.6. Karyotype analysis Standard methods were used in the karyotype analysis. Briefly, Colcemid-induced metaphase spreads were prepared by incubating exponentiallygrowing ceils with 0.01 # g / m l Colcemid (Sigma) for 4 h. The cells were swollen in 0.075 M potassium chloride (15 min, 37 ° C), fixed in methanol-glacial acetic acid (3:1), dropped onto cold, wet slides, allowed to dry, and stained with Geimsa. Suitable spreads were photographed to enable accurate counting. 10-15 spreads of each type were counted [13]. 3.7. Surface antigen analysis A standard, double-antibody technique was employed. The monoclonal antibodies OKT8 (Ortho) and Hu.Lyt. 1 (New England Nuclear) were used at their optimum concentrations (1 # g / m l protein, 4 ° C, 45 rain). These antibodies were visualized on the surface of the cells by staining with a 1:20 dilution of a fluorescein-linked rabbit anti-mouse IgG (Miles) followed by examination on a Wild microscope equipped with a HBO-200 mercury-vapour light source.
4. Results and discussion
PHA-activated human lymphocytes were success-
fully fused with drug-pretreated Molt-4F cells, resulting in good growth of hybrid cells in 5 out of 144 wells 4 weeks after fusion. No growth was seen in any of the control wells. Once the cells had become approximately 50% confluent, the cultures were expanded into 1 ml of complete medium in 25 cm 3 flasks, which were then balanced on their corners [14]. Two of the cultures grew rapidly and were designated 2E6M and 2CllM. The supernatant from 2C11 M, unstimulated by mitogen or antigen, was capable of significantly inhibiting the PHA-induced proliferation of normal human peripheral lymphocytes (Fig. 1). When it was introduced at a concentration of 25% (v/v), the response was suppressed by 55% while at the 50% level thymidine uptake was completely inhibited. The 2E6M and Molt-4F supernatants did not possess this activity and none of the supernatants were cytotoxic (as assessed by trypan blue exclusion at 72 h), or exhibited any mitogenic activity (data not shown). It is not yet known whether this inhibition reflects some general, non-specific suppression, or a specific effect on the PHA-induced response. Further immunological and physiological characterizations of this supernatant have been initiated.
OP~ I ~1041
HPBL
I
MOLt -4F
2E6M
L 2c~M
CCL~-rVpE
Fig. 1. Effect of hybrid cell supernatants on the PHA-induced proliferation of normal human peripheral blood lymphocytes (HPBL). Results are expressed as the mean + S.D. of 3 experiments performed in triplicate. (a) without and (b) with 5 ~tg/ml PHA. Cell supernatant was added at: (m) 5%; (A) 25%; and (~) 50% (v/v) to 200 I.d cultures. 205
Both the 2E6M and 2C11M hybrid cells responded to the mitogens, P H A and Con A, 2E6M dramatically so (Table 1). The Molt-4F cells showed a slight growth inhibition in the presence of PHA, while both of the hybrids (2E6M and 2C11 M) were stimulated to double and quadruple their respective growth rates in its presence. The 2E6M cells displayed an exceedingly high transformation rate in the presence of Con A and this cell may provide a vehicle for the study of blastogenesis in T-cells. However, unlike Molt-4F or 2E6M, 2C11M did not appear to respond either positively or negatively to the addition of Con A, behaving as if no mitogen had been added. Support for these results was shown by the patterns of aggregation obtained for the cells. While Molt-4F showed a small degree of aggregation with the two mitogens, 2E6M and 2C11M behaved quite differently (Table 2). 2E6M aggregated intensely in the presence of Con A and hardly at all with PHA while 2C11M did not aggregate at all with Con A
Table 1 Effects of mitogens on hybrid cells
Molt-4F 2E6M 2CIIM
Stimulation index a,b
Mitogen response ratio a
PHA
Con A
PHA
0.889 2.056 4.813
2.626 414.1 1.005
1.0 2.313 5.414
Con A 1.0 157.7 0.383
4
a As defined in text (3.3). b Mean of 3 experiments, carried out in triplicate.
and only moderately with PHA. This appears to indicate that the surface characteristics are different in the 3 cell types, at least with respect to the carbohydrate moieties displayed. The hybrid cell, 2E6M, also expressed the SRBC receptor on its surface (20%), while 2C11M did not, reflecting a further degree of similarity of 2E6M with normal T-cells. In our hands Molt-4F did not form rosettes with AET-treated SRBC while normal peripheral human lymphocytes did (40%). Karyotypic analysis of the cells showed that the hybrids 2E6M and 2C11M each had greater numbers of chromosomes than Molt-4F (95, 91 and 88, respectively). This small difference was not unexpected since Molt-4F is nearly tetraploid, but it was significant. A summary of the properties of the cells is layed out in Table 2. Microscopic examination of the cells has shown differences in their respective morphologies. Although all of the cell types grow in suspension, both Molt-4F and 2C11M grow as discrete cells without any visible marks or irregularities of shape. However, 2E6M grows in groups of 5-10 cells adhering to each other, and the cells appear very granular. Studies into the antigens displayed on the surface of the cells have shown that neither our Molt-4F nor the hybrid cells carry any antigens which would react with the monoclonal antibodies Hu.Lytl or OKT8; further studies are continuing. Other workers have reported that rigorous cloning is essential for the maintainance of functional human T-cell hybrids [15], that feeder systems are desirable for initial growth [11], or that they may be
Table 2 S u m m a r y of properties of hybrid cells
Karyotype a SRBC-rosette Aggregation (a) P H A (b) Con A MRR b (a) P H A (b) Con A Supernatant action d
M olt-4F
2E6M
2C11 M
HPBL
88 + 1.4
94+3.6
91 5:0.8
46
20% +/+++
++
++ ++
1.0 1.0
2.3 157.7
5.4 0.38 Yes
nd c nd
Mean 5: S.D. of 10-15 observations. b As defined in text (3.3). c n d = not determined. d As defined in text (3.4). a
206
40%
+ +
unstable and quickly die [16]. We have not encountered any of these problems, perhaps because there was only one focus of growth in each well and therefore the cells are clones, or because the culture conditions were such that only the stable cells survived the first few weeks after fusion and were subsequently selected. It was decided that the mitogen-stimulated cells themselves would act as feeders for the critical initial period after fusion and that the addition of other drug-treated cells as feeders [11] would not be required. Further successful experiments have borne this out. 2E6M and 2C11M have been in continuous culture now for over 8 months and show no signs of losing any of the properties reported here. The use of irreversible inhibitors as selection tools should open up the field of human-human cell hybridization by allowing many (if not all) cell-lines to be used as parents for the construction of hybrids with suitable partners, and work into this area is proceeding in our laboratories. Further studies into the effects of different parent cells on the fusion efficiency are also being carried out. We believe that it may be desirable to choose the parent cell carefully in relation to the partner cell and the function that one wishes one's hybrids to express. In conclusion, we believe that we have successfully constructed functional human T-lymphocyte hybrid cells and that the use of such cells will be of great use as investigative tools in human immunology.
Acknowledgements We would like to thank Dr. E. Boyd for preparing the karyotype analysis and Drs. A. Boylston. M.
Feldman and S. Metcalf for helpful discussion. This work was supported by a grant and a training award from the Medical Research Council.
References [1] Fischer, A., Zanders, E. D., Beverley, P. C. L. and Feldman, M. (1982) in: The l_.ymphokines, Vol. 5, Monoclonal T-cells and Their Products (Feldman, M. and Schreier, M. H. eds.) pp. 465-491, Academic Press. [2] Lamb, J. and Feldman, M. (1982) Nature (London) 300, 456-458. [3] Wallace, L. E., Rickinson, A. B., Rowee, M. and Epstein, M. A. (1982) Nature (London) 297,413~115. [4] Kohler, G. and Milstein, J. A. (1978) Nature (London) 256, 495497. [5] Goldsby, R. A., Osbourne, B. A., Simpson, E., Herzenberg, L. A. (1978) Nature (London) 267,707 708. [6] Kontianen, S., Simpson, E., Bohrer, E., Beverley, P. C. L., Herzenberg, L. A., Fitzpatrick, W. C., Vogt, P., Torano, A., McKenzie, 1. F. C. and Feldman, M. (1978) Nature (London) 274, 477 480. [7] Olsson, L. and Kaplan, H. S. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5429-5431. [8] Grillot-Courvalin, C., Brouet, J.-C., Berger, R. and Bernheim, A. (1981) Nature (London) 292, 844-845. [9] Wright, W. E. (1978) Exp. Cell. Res. 112, 395~107. [10] Clark, S. A., Stimson, W. H., Williamson, A. R. and Dick, H. M. (1981) J. Supramol. Struct. Cell. Biochem. Suppl. 5, 10th Ann. ICN-UCLA Symp., Abstr. 100. [11] Kobayashi, Y., Asada, M., Higuchi, M. and Osawa, T. (1982) J. Immunol. 128, 2714-2718. [12] Wantanabe, T. and Ohara, J. (1981) Nature (London) 290, 58 60. [13] Boyd, E., personal communication. [14] Metcalf, S., personal communication. [15] lrigoyen, O., Rizzolo, P. R., Thomas, Y., Rogozinski, L. and Chess, L. (1981) J. Exp. Med. 154, 1827-1837. [16] Greene, W. C., Fleisher, T. A., Nelson, D. L. and Waldman, T. A. (1982) J. Immunol. 129, 1986-1992.
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