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Improved method for cloning human B-cell lines
CELLULAR
IMMUNOLOGY
Improved
72, 2 19-230 ( 1982)
Method for Cloning Human B-Cell Lines’
SUSANHocH,*** PETERH. SCHUR,* ANDJERROLD SCHWABER~'3 Departments of *Rheumatology and *Immunology, Brigham and Womenk Hospital, tDivision Immunology, ChildrenS Hospital Medical Center; and Departments qf *Medicine, TPediatrics, and YPathology, Harvard Medical School, Boston, Massachusetts 02115
of
Received April 13. 1982; accepted June 27, 1982
Difficulties in the successfulcloning of B-cell lines have prevented widespread establishment of specific antibody-forming human B-cell lines. We have improved on standard methods of cloning by culturing human lymphoid cells suspended in agarose with nonproliferating human fetal lung fibroblasts. Using this method, cloning efficiencies up to 20% were observed, from as few as 50 plated lymphoid cells. These results are significantly better than the efficiencies of up to 1% using 5 to 10 X lo3 cells with traditional soft agar methods. The number of colonies observed increased in proportion to the number of fibroblasts plated with the lymphoid cells. A threshold number of fibroblasts was found. Microscopically, close association between lymphoid cells and fibroblasts was seen. A growth-enhancing factor appears to be produced by fibroblasts suspended in agarose. This cloning method is applicable to both well-established and newly established lymphoid cell lines and should lx useful for growing and cloning B cells.
INTRODUCTION Recent developments in production of monoclonal antibodies of rodent origin have renewed interest in the possibilities of producing human monoclonal antibodies (1). Difficulties with interspecies hybrid cells, and the absence of a reliable human myeloma cell line comparable to the MPC 11 derivative mouse myeloma cell line, have led to investigations of using Epstein-Barr virus (EBV)-transformed human B-cell lines for production of human antibodies (2). EBV infection of human B cells in vitro transforms them into lymphoid cell lines which produce and secrete immunoglobulins (3). Specific antibody-producing B-cell lines have been isolated following successive cloning of early cultures of human B cells (2, 4) or by antigen preselection, EBV transformation, and cloning (5-8). Present methods for cloning human B-cell lines have low efficiencies, no greater than I%, requiring the introduction of large numbers of cells, representing approximations to derivation of a true clone (9). We report here an adaptation of the method of Kirk et al. (lo), in which we used normal human fetal lung fibroblasts suspended in agarose, to culture human B-cell lines, with an approximately IO-fold increase in cloning efficiency over the best previous method for cloning. ’ Supported in part by grantsfrom the USPHS (AI16731, AM1 1414, AM05577), March of Dimes Birth Defects Foundation, The Lupus Foundation of America, and the New England Peabody Home Foundation. ’ S.H. is a Postdoctoral Fellow of the Arthritis Foundation. ’ J.S. is a Scholar of the Leukemia Society of America. 219 0008-8749/82/ 1402I9- 12$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights al reproduction in any form reserved.
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METHODS Cells Midpassage level human fetal lung fibroblasts were grown in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 pg/ml streptomycin, and 2 mM L-glutamine (Microbiological Associates, Walkersville, Md.). Cells were grown to confluence and detached with trypsinEDTA for cloning. The lymphoid cells used in these studies included well-established EBV-transformed cell lines, newly established (l-3 months in culture) B-cell lines, and peripheral blood lymphocytes from normals incubated with EBV for 2 hr and directly cloned. The well-established cell lines included 007, a B-cell line from normal adult blood (11); SMI 4, a B-cell line established from healthy cord blood (12); LAZ 267 ( 13) and LAZ 166 ( 14), both derived from blood of patients with agammaglobulinemia grown for 2-3 months (6- 12 passagesafter establishment) and stored frozen in liquid nitrogen until thawed for use in these experiments. Newly established Bcell lines were derived from blood of patients with systemic lupus erythematosus, and cloned 3-4 weeks after establishment without freezing. Freshly transformed B cells were peripheral blood leukocytes from a healthy adult male, incubated with EBV for 2 hr as previously described (15), and then cloned without prior culture. EBV was obtained from the supernatant of the marmoset cell line B 95-8 as described previously ( 15). This concentrated supernatant produces EBV-determined nuclear antigen (EBNA) expression in 14% of peripheral blood lymphocytes after 48 hr.
Cloning All studies used the same medium for cloning: RPM1 1640 supplemented with 20% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin, and 50 pg/ml streptomycin. Various concentrations of agarose (SeaPlaque, FMC Corp., Rockland, Maine) were used. Two cloning methods were compared. For both methods studied, cultures were observed weekly through an inverted microscope for colony formation. Colonies were defined as clusters of more than 25 lymphoid cells which maintained continued growth. Method 1. Lymphoid cells were cultured by the soft agar method described by Steinitz and Klein (9). Feeder layers of confluent human fetal lung fibroblasts were prepared in 24-well tissue culture plates. Five-tenths milliliter of cloning media containing 0.45% agarose was layered in each well of a 24-well tissue culture plate (Costar, Cambridge, Mass.) and allowed to solidify. The lymphoid cells at concentrations ranging from 1 to 10,000 thousand cells per well were suspended in 0.37% agarose and layered onto the base agarose layer. After solidification, the cultures were incubated, fed weekly with cloning media, and observed for colony formation. Method 2. B-cell lines were cloned by a modification of the method of Kirk et al. for cloning human prostatic carcinoma cell lines using nonproliferating fibroblasts embedded in agar ( 10). Five-tenths milliliter of cloning media containing 0.45% agarosewas layered in each well of a 24 well tissue culture plate and allowed to harden. Confluent fibroblasts were trypsinized, washed in medium to remove trypsin, and resuspended to the desired concentration. The lymphoid cells were
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counted and the concentration was adjusted. The fibroblasts and the lymphoid cells were mixed together in cloning medium containing 0.3% agarose and layered onto the base agarose layer. The top agarose layer was allowed to harden and 0.2 ml cloning media added weekly to keep it from drying out and observed for colonies. To determine whether cell to cell association was necessaryfor this cloning method to work, a triple-layer cloning method was used. The base layer consisted of 0.45% agarose in cloning media as previously described. The middle layer contained the suspended fibroblasts and consisted of 0.3% agarose in a total volume of 0.375 ml. This layer was allowed to harden and then a top layer containing the lymphoid cells alone in 0.3% agarose, total volume 0.375 ml, was added. This was compared to plates in which the same concentrations of lymphoid cells and fibroblasts were mixed in 0.3% agarose in a total volume of 0.750 ml. Photography Colonies were examined and photographed using a Leitz Diavert microscope equipped with a Vario-Orthomat camera, 35 mm. Panatonic X film was used. RESULTS Cloning Eficiencies A comparison of these two cloning methods is presented in Table 1. Method 1, the soft agar method of Steinitz and Klein (9) represents an improvement over prior methods. One thousand to five thousand lymphoid cells were plated per well of a 24-well culture dish, with the bottom layer of agarose inhibiting contact between the lymphoid cells and the confluent fibroblasts. With this method, we achieved an initial cloning efficiency of three colonies per 100,000 lymphoid cells or 0.003% with the well-established B-cell line 007. Recloning of a 007 clone, 007 Clone 4, increased the cloning efficiency to approximately l%, comparable to the efficiency reported by Steinitz and Klein for lymphoma cell lines (9), suggesting selection for cells best able to grow suspended in soft agarose. The cell line SMI 4 plated at 1000 lymphoid cells per well yielded two colonies per 6000 cells plated or 0.03%. Similar efficiencies could be achieved with recently established human B-cell lines derived from patients with common varied or X-linked agammaglobulinemia although a higher concentration of cells per well appeared to be necessary. For example, the cell line LAZ 166 formed no colonies when plated at 500 and 1000 cells per well using the Steinitz or soft agar method but at 10,000 cells per well, six colonies per 100,000 cells plated were found yielding a cloning efficiency of 0.006%. In contrast, our modification of the method of Kirk et al. (10) (Method 2) using fibroblasts suspended in agaroseyielded cloning efficiencies more than 1O-fold higher than Method 1. Moreover, smaller numbers of lymphoid cells could be plated to help ensure the clonal nature of resulting colonies. For example, plating 500 lymphoid cells from the cell line LAZ 166 with 250,000 fibroblasts per well yielded an average of three colonies per well after 2 weeks in culture or 0.6%. For the previously cloned cell line, 007 Clone 4, a mean cloning efficiency of 20% was achieved after plating 50 lymphoid cells with 125,000 fibroblasts per well. Similar increases in cloning efficiencies were found with LAZ 267 and SMI 4 (Table 1). This represents
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HOCH, SCHUR, AND SCHWABER TABLE I
Comparison of Soft Agar Method and Suspended Fibroblast Method for Cloning Human B-Cell Lines
Method Soft agar
Cell line 007 007 c4 LA2 166
LAZ 267 SMI 4 Suspended fibroblasts
007 C4 LAZ 166 LAZ 267 SMI 4 New cell lines A.M. E.S. C.S. P3Ul mouse myeloma line
No. of lymphoid cells/well 5,000 500 500 l,OC@ 10,000 500 l,ooo 500 1,000 50 500 1,000 500 1,000 100 500 50 100 500 50 100 500 500 50 100
No. of colonies/ cells plated
Maximum cloning efficiency (8)
3/100,000 41500 0 0 6/ 100,000 0 0 0
0.003 0.8 0 0 0.006 0 0 0 0.03
2/6,C@CI IO/50 31500 51500 10/1,000 2/100 51500
20 0.6 0.6 1.0 1.0 2.0 1.0
0 0 31500 0 0 61500 21500 20150 31/100
0 0 0.6 0 0 1.2 0.4 40 31
6/1,ooO
a considerable improvement over other methods used to clone these B-cell lines (see Table 1). That these colonies were clones was tested by immunofluorescence. Eight colonies of SMI 4 tested each expresseda homogeneous single or double (1 clone IgD + IgM) immunoglobulin heavy-chain isotype. In Fig. 1, the effect of increasing numbers of fibroblasts on colony formation at 2 weeks of culture is depicted for cell line 007 Clone 4. The lymphoid cells were plated at a concentration of 50 cells per well. For this cell line, a threshold effect of nonproliferating fibroblasts on number of colonies formed by the lymphoid cells was clearly seen between 3 1,000 and 62,000 fibroblasts per well. In most experiments, a plateau was observed at a concentration above 250,000 fibroblasts per well. In some, but not all experiments, fewer clones were observed at concentrations of 1,OOO,OOO fibroblasts per well, the maximum concentration of fibroblasts studied. We were interested in seeing if this method could be applicable for cloning newly established lymphoid cell lines. Cell lines derived from EBV transformation of peripheral lymphocytes 1 month previously were cultured with varying concentrations of fibroblasts. Initial studies using 50 and 100 lymphoid cells per well from these newly established cell lines gave no colony formation. However, at concentrations of 500 lymphoid cells/well, using Method 2, colonies were observed. Figure 2 pre-
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2-
0
3.9
7.0
156
31
62
# FIEROBLASTS/WELL
125
250
500
1000
x lo3
FIG. 1. Using the suspended fibroblast method for cloning human B-cell lines, the number of lymphoid colonies observed increases with the concentration of suspended fibroblasts plated per well, as demonstrated here for the well-established normal B-cell line 007 Clone 4, plated at 50 lymphoid cells/well.
sents the data for two different cell lines after 3 weeks in culture with fibroblasts in agarose. Here again, a threshold effect on colony formation seemsevident with the threshold somewhat higher, around 200,000 fibroblasts per well. Using a concentration of 500,000 fibroblasts per well and 500 lymphoid cells, cell lines A.M. and ES. yield a cloning efficiency of approximately three colonies per 500 cells or 0.6%. Finally, we have utilized this method to see if newly isolated peripheral blood lymphocytes (PBL) obtained by Ficoll-metrizoate density gradients (16) could be EBV infected and cloned immediately rather than grown first as a mass culture and then cloned 1 month or more later. PBL cloned at a density of 1 X 1O3mononuclear cells per well or 100 B cells per well did not give rise to any colonies when plated with 500,000 fibroblasts/well. However, when PBL were plated at a concentration of 1 X IO4mononuclear cells per well or 1000 B cells per well with the same number
0) Y6 6 d0 “4
r I’ I’ I
t ‘t --
0
--
17
--
--
3.9 7.8 158 31 # FIBROBLASTS/WELL
I
82
125 x lo3
I
250
1
500
I
loco
FIG. 2. A similar concentration-dependent increase in lymphoid cloning efficiency is present for newly established B-cell lines A.M. and ES. plated at 500 lymphoid cells per well.
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FIG. 3. Microscopic studies of colony formation using the suspended fibroblast method. In a two lymphoid cells and a fibroblast are closely associated. In b and c clusters of lymphoid cells are seen around the original fibroblast nidus. A mature colony is present (d) with a clump of fibroblasts still discernable.
of fibroblasts, two to five colonies were observed after 1 month in culture. In this way, a cloned cell line could be established directly from PBL. These cells appear to grow initially less rapidly than cells grown in mass culture and then cloned later, probably paralleling the initial lag of growth seen in mass culture.
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FIG. 3. (Continued)
A4icroscopic Studies We observed microscopically that the primary event in this cloning method iIP-
peared to be the close association between a lymphoid cell and a fibroblast or clur w
of fibroblasts (Fig. 3a). This was followed by the gradual formation of a cluster of 1Ylaphoid cells immediately adjacent to the fibroblast. Over 2 to 3 weeks, an enlargi w
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FIG. 3. (Continued)
clone of lymphoid cells was clearly visible, often developing around the original fibroblast (Figs. 3b, c). In Fig. 3d, a mature colony is presented still associated with the original clump of fibroblasts. The morphology of the lymphoid cells is clearly distinguishable from that of the fibroblasts. Subculture of these colonies yielded cell lines that were morphologically indistinguishable from the parental lymphoid cells.
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FIG. 3. (Continued)
In comparing whether or not direct cellular contact was necessaryfor the (sbserved enhanced cloning efficiency, we plated duplicate tissue culture dishes with fil 3roblasts and lymphoid cells mixed together in the agarose as well as suspended in separate layers. The presence of the nonproliferating fibroblasts suspended in the agarose increased the cloning efficiency of 007 twofold over that observed using a feeder
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layer of confluent fibroblasts with agarose over them. However, maximum colony formation occurred only when the fibroblasts and lymphoid cells were mixed together in the agarose. DISCUSSION Difficulties with established techniques for cloning human B-cell lines have hindered use of these cells to generate human monoclonal antibodies. While a few specific antibody-producing cell lines have been described (2, 4-8), lack of an adequate method for cloning antigen-specific B cells has precluded extensive production of such cell lines. Several of the described lines have been clearly heterogeneous in regard to their antibody production perhaps because of difficulty in successful cloning (2,4-8). In comparing several established methods for the cloning of human B-cell lines, we were initially unable to improve on the I % cloning efficiency reported by Stein&z and Klein (9) for well-established lymphoma lines cloned at 10,000 cells per well. They had reported three different EBV-positive lymphoma cell lines in which 60 to 120 colonies were observed when 10,000 cells were plated for a cloning efficiency of 1%. However, when 1000 cells were plated, only one of three lines gave rise to any colonies at all. By adapting the method of Kirk et al. (10) to culture of human B-cell lines, we report a method which increases the cloning efficiency of B-cell lines approximately lo-fold. Furthermore, this method allows for the cloning of small numbers of cells from well-established cell lines ensuring the derivation of a true clone, and can be used for a first approximation to cloning newly established lines and freshly transformed cells. We have previously attempted to clone lymphoid cells by limiting dilution with or without feeder layers of fibroblasts or human thymocytes (17). None of these methods was routinely successful. The method of Steinitz and Klein (9) was the first method which routinely gave rise to colonies provided sufficient numbers of cells were plated. We have not been able to achieve greater than 1% cloning efficiency with this method, and that with a limited number of cell lines. Most cell lines of interest had cloning efficiencies significantly less than 1%. Some cell lines could not be cloned by this method. Using the method of fibroblasts suspended in the agarose together with small numbers of lymphoid cells, enhancement of cloning efficiency was observed with cloning efficiencies ranging from 0.4 to 20%. The 20% cloning efficiency was achieved with a well-established cell line, 007 C4, previously cloned by the soft agar method suggestingselection for the ability to grow well in soft agar. In addition, this improved efficiency was achieved using small numbers of lymphoid cells, usually 1OO/perwell for well-established lymphoid cell lines and 500 to 1000 per well for newly established cell lines. Using lower numbers of lymphoid cells helps to ensure that colonies derived from this method are indeed clonal. Since the number of antigen-specific B cells for any antigen has been estimated to range from 1 in 10,000 to 1 in 1,OOO,OOO by some (18), this method will allow more efficient cloning of specific antibodyproducing cell lines when combined with preselection techniques. The presence of nonproliferating fibroblasts in soft agar in proximity to lymphoid cells appeared to be the factor that allowed for the increased cloning efficiency with
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this method. Microscopically, we observed that the primary event in colony formation was the close association between a lymphoid cell and a fibroblast or clump of fibroblasts. Subsequently, lymphoid colonies arose by the division of this initial lymphoid cell and retained their association with the original fibroblast. In cultures where fibroblasts and lymphoid cells were not in direct contact, but the fibroblasts were suspended in agarose,the cloning efficiency was still enhanced over that of the Steinitz method. These observations suggest that a growth factor is derived from nonproliferating fibroblasts suspended in agarose, confirming the findings of Kirk et al. (10). Our data indicate that this growth enhancement, mediated by nonproliferating fibroblasts, is not specific for prostatic carcinoma cells but may be stimulatory to a wide range of cells, including B lymphocytes. The growth enhancement factor is not specific for human cells in that we have used this method to clone P3U 1, a mouse myeloma cell line (Table 1). The linear relationship observed between the number of fibroblasts added and the number of lymphoid colonies arising suggestsa dose-response relationship. We consistently observed a threshold level of fibroblasts. It may be that a critical number of fibroblasts is needed either to ensure that one of a small number of lymphoid cells will be close to a fibroblast or to produce a desired concentration of a growth factor; or the growth stimulating factor may be short lived. Our improvement over the 1% cloning efficiency reported by Steinitz and Klein (9) using a feeder layer of confluent fibroblasts suggeststhat the growth-enhancing factor is not produced by anchored, confluent, fibroblasts. Whether or not attachment, nonproliferation, or confluency is critical in determining whether or not fibroblasts enhance growth of other cells is not clear. Our data demonstrate that fibroblasts suspended in agarose produce growth enhancement. Under conditions of close association between fibroblasts and lymphocytes as when they are mixed together in the agarose, this enhancement is magnified. To date, we have only used fibroblasts derived from fetal lung. Whether or not human fibroblasts from other sources or of nonhuman origin can stimulate colony formation by lymphoid cells remains to be determined. This method for cloning EBV-transformed human B lymphocytes with increased efficiency coupled with appropriate preselection of antigen-specific B cells should allow for isolation of specific antibody-producing B-cell lines. ACKNOWLEDGMENTS We are grateful to Dr. Herbert Lazarus for many helpful discussions and to Mr. John Young, Ernst Leitz, Inc., for loan of equipment.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Olsson, L., and Kaplan, H. S., Proc. Nat. Acad. Sri. USA 77, 5429, 1980. Zurawski, V. R., Haber, E., and Black, P. H., Science 199, 1439, 1978. Fahey, J. L., Finegold, I., Rabson, A. S., and Manaker, R. A., Science 152, 1259, 1966. Hirano, T., Teraoka, O., Teranish, T., Tsoyugushi, I., Todha, H., and Oikawa, A., Microhiol. Immunol. 24, 879, 1980. Steinitz, M., and Klein, G., Nature (London) 269, 420, 1977. Steinitz, M., Seppala, I., Eichmann, K., and Klein. G., Immunobiology 156, 41, 1979. Steinitz, M., Koskimies, S., Klein, G., and Makela O., J. C/in. Lab. Immunol. 2, 1, 1979. Steinitz, M., Izak, G., Cohen S., Ehrenfeld M., and Flechner I., Nature (London) 287, 443. 1980.
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9. Steinitz, M., and Klein, G., Eur. J. Cancer 13, 1269, 1977. 10. Kirk, D., &lay, M. F., and Kaign, M. E., Cancer Rex 41, 1100, 1981. 11. Schwaber, J., and Rosen, F. S., J. Ch. Immunoi. 2, 30, 1982. 12. Evans, J., Steel, M., and Arthur E., Cell 3, 153, 1974. 13. Schwaber, J., Klein, G., Emberg, I., Rosen, A., Lazarus, H., and Rosen, F. S., J. Immunol. 124, 2191, 1980. 14. Schwaber, J., Lazarus, H., and Rosen., F. S., Proc. Nat. Acad. Sci. USA 75, 242 1, 1978. 15. Robinson, J., and Miller, G., J. Virul. 15, 1065, 1975. 16. Boyum, A., &and. J. Clin. Lab. Invest. 21 (Suppl. 87), 9, 1968. 17. Lemhardt, W., Andersson, J., Coutinho, A., and Melchers, F., Exp. Cell. Rex 111, 309, 1978. 18. Makinodan, T., and Albright, J. F., Prog. Allergy 10, 1, 1967.
IMMUNOLOGY
Improved
72, 2 19-230 ( 1982)
Method for Cloning Human B-Cell Lines’
SUSANHocH,*** PETERH. SCHUR,* ANDJERROLD SCHWABER~'3 Departments of *Rheumatology and *Immunology, Brigham and Womenk Hospital, tDivision Immunology, ChildrenS Hospital Medical Center; and Departments qf *Medicine, TPediatrics, and YPathology, Harvard Medical School, Boston, Massachusetts 02115
of
Received April 13. 1982; accepted June 27, 1982
Difficulties in the successfulcloning of B-cell lines have prevented widespread establishment of specific antibody-forming human B-cell lines. We have improved on standard methods of cloning by culturing human lymphoid cells suspended in agarose with nonproliferating human fetal lung fibroblasts. Using this method, cloning efficiencies up to 20% were observed, from as few as 50 plated lymphoid cells. These results are significantly better than the efficiencies of up to 1% using 5 to 10 X lo3 cells with traditional soft agar methods. The number of colonies observed increased in proportion to the number of fibroblasts plated with the lymphoid cells. A threshold number of fibroblasts was found. Microscopically, close association between lymphoid cells and fibroblasts was seen. A growth-enhancing factor appears to be produced by fibroblasts suspended in agarose. This cloning method is applicable to both well-established and newly established lymphoid cell lines and should lx useful for growing and cloning B cells.
INTRODUCTION Recent developments in production of monoclonal antibodies of rodent origin have renewed interest in the possibilities of producing human monoclonal antibodies (1). Difficulties with interspecies hybrid cells, and the absence of a reliable human myeloma cell line comparable to the MPC 11 derivative mouse myeloma cell line, have led to investigations of using Epstein-Barr virus (EBV)-transformed human B-cell lines for production of human antibodies (2). EBV infection of human B cells in vitro transforms them into lymphoid cell lines which produce and secrete immunoglobulins (3). Specific antibody-producing B-cell lines have been isolated following successive cloning of early cultures of human B cells (2, 4) or by antigen preselection, EBV transformation, and cloning (5-8). Present methods for cloning human B-cell lines have low efficiencies, no greater than I%, requiring the introduction of large numbers of cells, representing approximations to derivation of a true clone (9). We report here an adaptation of the method of Kirk et al. (lo), in which we used normal human fetal lung fibroblasts suspended in agarose, to culture human B-cell lines, with an approximately IO-fold increase in cloning efficiency over the best previous method for cloning. ’ Supported in part by grantsfrom the USPHS (AI16731, AM1 1414, AM05577), March of Dimes Birth Defects Foundation, The Lupus Foundation of America, and the New England Peabody Home Foundation. ’ S.H. is a Postdoctoral Fellow of the Arthritis Foundation. ’ J.S. is a Scholar of the Leukemia Society of America. 219 0008-8749/82/ 1402I9- 12$02.00/O Copyright 0 1982 by Academic Press, Inc. All rights al reproduction in any form reserved.
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METHODS Cells Midpassage level human fetal lung fibroblasts were grown in Dulbecco’s modified essential medium (DMEM) supplemented with 10% fetal calf serum, 50 units/ml penicillin, 50 pg/ml streptomycin, and 2 mM L-glutamine (Microbiological Associates, Walkersville, Md.). Cells were grown to confluence and detached with trypsinEDTA for cloning. The lymphoid cells used in these studies included well-established EBV-transformed cell lines, newly established (l-3 months in culture) B-cell lines, and peripheral blood lymphocytes from normals incubated with EBV for 2 hr and directly cloned. The well-established cell lines included 007, a B-cell line from normal adult blood (11); SMI 4, a B-cell line established from healthy cord blood (12); LAZ 267 ( 13) and LAZ 166 ( 14), both derived from blood of patients with agammaglobulinemia grown for 2-3 months (6- 12 passagesafter establishment) and stored frozen in liquid nitrogen until thawed for use in these experiments. Newly established Bcell lines were derived from blood of patients with systemic lupus erythematosus, and cloned 3-4 weeks after establishment without freezing. Freshly transformed B cells were peripheral blood leukocytes from a healthy adult male, incubated with EBV for 2 hr as previously described (15), and then cloned without prior culture. EBV was obtained from the supernatant of the marmoset cell line B 95-8 as described previously ( 15). This concentrated supernatant produces EBV-determined nuclear antigen (EBNA) expression in 14% of peripheral blood lymphocytes after 48 hr.
Cloning All studies used the same medium for cloning: RPM1 1640 supplemented with 20% fetal calf serum, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 units/ml penicillin, and 50 pg/ml streptomycin. Various concentrations of agarose (SeaPlaque, FMC Corp., Rockland, Maine) were used. Two cloning methods were compared. For both methods studied, cultures were observed weekly through an inverted microscope for colony formation. Colonies were defined as clusters of more than 25 lymphoid cells which maintained continued growth. Method 1. Lymphoid cells were cultured by the soft agar method described by Steinitz and Klein (9). Feeder layers of confluent human fetal lung fibroblasts were prepared in 24-well tissue culture plates. Five-tenths milliliter of cloning media containing 0.45% agarose was layered in each well of a 24-well tissue culture plate (Costar, Cambridge, Mass.) and allowed to solidify. The lymphoid cells at concentrations ranging from 1 to 10,000 thousand cells per well were suspended in 0.37% agarose and layered onto the base agarose layer. After solidification, the cultures were incubated, fed weekly with cloning media, and observed for colony formation. Method 2. B-cell lines were cloned by a modification of the method of Kirk et al. for cloning human prostatic carcinoma cell lines using nonproliferating fibroblasts embedded in agar ( 10). Five-tenths milliliter of cloning media containing 0.45% agarosewas layered in each well of a 24 well tissue culture plate and allowed to harden. Confluent fibroblasts were trypsinized, washed in medium to remove trypsin, and resuspended to the desired concentration. The lymphoid cells were
METHOD FOR CLONING HUMAN B-CELL LINES
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counted and the concentration was adjusted. The fibroblasts and the lymphoid cells were mixed together in cloning medium containing 0.3% agarose and layered onto the base agarose layer. The top agarose layer was allowed to harden and 0.2 ml cloning media added weekly to keep it from drying out and observed for colonies. To determine whether cell to cell association was necessaryfor this cloning method to work, a triple-layer cloning method was used. The base layer consisted of 0.45% agarose in cloning media as previously described. The middle layer contained the suspended fibroblasts and consisted of 0.3% agarose in a total volume of 0.375 ml. This layer was allowed to harden and then a top layer containing the lymphoid cells alone in 0.3% agarose, total volume 0.375 ml, was added. This was compared to plates in which the same concentrations of lymphoid cells and fibroblasts were mixed in 0.3% agarose in a total volume of 0.750 ml. Photography Colonies were examined and photographed using a Leitz Diavert microscope equipped with a Vario-Orthomat camera, 35 mm. Panatonic X film was used. RESULTS Cloning Eficiencies A comparison of these two cloning methods is presented in Table 1. Method 1, the soft agar method of Steinitz and Klein (9) represents an improvement over prior methods. One thousand to five thousand lymphoid cells were plated per well of a 24-well culture dish, with the bottom layer of agarose inhibiting contact between the lymphoid cells and the confluent fibroblasts. With this method, we achieved an initial cloning efficiency of three colonies per 100,000 lymphoid cells or 0.003% with the well-established B-cell line 007. Recloning of a 007 clone, 007 Clone 4, increased the cloning efficiency to approximately l%, comparable to the efficiency reported by Steinitz and Klein for lymphoma cell lines (9), suggesting selection for cells best able to grow suspended in soft agarose. The cell line SMI 4 plated at 1000 lymphoid cells per well yielded two colonies per 6000 cells plated or 0.03%. Similar efficiencies could be achieved with recently established human B-cell lines derived from patients with common varied or X-linked agammaglobulinemia although a higher concentration of cells per well appeared to be necessary. For example, the cell line LAZ 166 formed no colonies when plated at 500 and 1000 cells per well using the Steinitz or soft agar method but at 10,000 cells per well, six colonies per 100,000 cells plated were found yielding a cloning efficiency of 0.006%. In contrast, our modification of the method of Kirk et al. (10) (Method 2) using fibroblasts suspended in agaroseyielded cloning efficiencies more than 1O-fold higher than Method 1. Moreover, smaller numbers of lymphoid cells could be plated to help ensure the clonal nature of resulting colonies. For example, plating 500 lymphoid cells from the cell line LAZ 166 with 250,000 fibroblasts per well yielded an average of three colonies per well after 2 weeks in culture or 0.6%. For the previously cloned cell line, 007 Clone 4, a mean cloning efficiency of 20% was achieved after plating 50 lymphoid cells with 125,000 fibroblasts per well. Similar increases in cloning efficiencies were found with LAZ 267 and SMI 4 (Table 1). This represents
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HOCH, SCHUR, AND SCHWABER TABLE I
Comparison of Soft Agar Method and Suspended Fibroblast Method for Cloning Human B-Cell Lines
Method Soft agar
Cell line 007 007 c4 LA2 166
LAZ 267 SMI 4 Suspended fibroblasts
007 C4 LAZ 166 LAZ 267 SMI 4 New cell lines A.M. E.S. C.S. P3Ul mouse myeloma line
No. of lymphoid cells/well 5,000 500 500 l,OC@ 10,000 500 l,ooo 500 1,000 50 500 1,000 500 1,000 100 500 50 100 500 50 100 500 500 50 100
No. of colonies/ cells plated
Maximum cloning efficiency (8)
3/100,000 41500 0 0 6/ 100,000 0 0 0
0.003 0.8 0 0 0.006 0 0 0 0.03
2/6,C@CI IO/50 31500 51500 10/1,000 2/100 51500
20 0.6 0.6 1.0 1.0 2.0 1.0
0 0 31500 0 0 61500 21500 20150 31/100
0 0 0.6 0 0 1.2 0.4 40 31
6/1,ooO
a considerable improvement over other methods used to clone these B-cell lines (see Table 1). That these colonies were clones was tested by immunofluorescence. Eight colonies of SMI 4 tested each expresseda homogeneous single or double (1 clone IgD + IgM) immunoglobulin heavy-chain isotype. In Fig. 1, the effect of increasing numbers of fibroblasts on colony formation at 2 weeks of culture is depicted for cell line 007 Clone 4. The lymphoid cells were plated at a concentration of 50 cells per well. For this cell line, a threshold effect of nonproliferating fibroblasts on number of colonies formed by the lymphoid cells was clearly seen between 3 1,000 and 62,000 fibroblasts per well. In most experiments, a plateau was observed at a concentration above 250,000 fibroblasts per well. In some, but not all experiments, fewer clones were observed at concentrations of 1,OOO,OOO fibroblasts per well, the maximum concentration of fibroblasts studied. We were interested in seeing if this method could be applicable for cloning newly established lymphoid cell lines. Cell lines derived from EBV transformation of peripheral lymphocytes 1 month previously were cultured with varying concentrations of fibroblasts. Initial studies using 50 and 100 lymphoid cells per well from these newly established cell lines gave no colony formation. However, at concentrations of 500 lymphoid cells/well, using Method 2, colonies were observed. Figure 2 pre-
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FIG. 1. Using the suspended fibroblast method for cloning human B-cell lines, the number of lymphoid colonies observed increases with the concentration of suspended fibroblasts plated per well, as demonstrated here for the well-established normal B-cell line 007 Clone 4, plated at 50 lymphoid cells/well.
sents the data for two different cell lines after 3 weeks in culture with fibroblasts in agarose. Here again, a threshold effect on colony formation seemsevident with the threshold somewhat higher, around 200,000 fibroblasts per well. Using a concentration of 500,000 fibroblasts per well and 500 lymphoid cells, cell lines A.M. and ES. yield a cloning efficiency of approximately three colonies per 500 cells or 0.6%. Finally, we have utilized this method to see if newly isolated peripheral blood lymphocytes (PBL) obtained by Ficoll-metrizoate density gradients (16) could be EBV infected and cloned immediately rather than grown first as a mass culture and then cloned 1 month or more later. PBL cloned at a density of 1 X 1O3mononuclear cells per well or 100 B cells per well did not give rise to any colonies when plated with 500,000 fibroblasts/well. However, when PBL were plated at a concentration of 1 X IO4mononuclear cells per well or 1000 B cells per well with the same number
0) Y6 6 d0 “4
r I’ I’ I
t ‘t --
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FIG. 2. A similar concentration-dependent increase in lymphoid cloning efficiency is present for newly established B-cell lines A.M. and ES. plated at 500 lymphoid cells per well.
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FIG. 3. Microscopic studies of colony formation using the suspended fibroblast method. In a two lymphoid cells and a fibroblast are closely associated. In b and c clusters of lymphoid cells are seen around the original fibroblast nidus. A mature colony is present (d) with a clump of fibroblasts still discernable.
of fibroblasts, two to five colonies were observed after 1 month in culture. In this way, a cloned cell line could be established directly from PBL. These cells appear to grow initially less rapidly than cells grown in mass culture and then cloned later, probably paralleling the initial lag of growth seen in mass culture.
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FIG. 3. (Continued)
A4icroscopic Studies We observed microscopically that the primary event in this cloning method iIP-
peared to be the close association between a lymphoid cell and a fibroblast or clur w
of fibroblasts (Fig. 3a). This was followed by the gradual formation of a cluster of 1Ylaphoid cells immediately adjacent to the fibroblast. Over 2 to 3 weeks, an enlargi w
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FIG. 3. (Continued)
clone of lymphoid cells was clearly visible, often developing around the original fibroblast (Figs. 3b, c). In Fig. 3d, a mature colony is presented still associated with the original clump of fibroblasts. The morphology of the lymphoid cells is clearly distinguishable from that of the fibroblasts. Subculture of these colonies yielded cell lines that were morphologically indistinguishable from the parental lymphoid cells.
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FIG. 3. (Continued)
In comparing whether or not direct cellular contact was necessaryfor the (sbserved enhanced cloning efficiency, we plated duplicate tissue culture dishes with fil 3roblasts and lymphoid cells mixed together in the agarose as well as suspended in separate layers. The presence of the nonproliferating fibroblasts suspended in the agarose increased the cloning efficiency of 007 twofold over that observed using a feeder
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layer of confluent fibroblasts with agarose over them. However, maximum colony formation occurred only when the fibroblasts and lymphoid cells were mixed together in the agarose. DISCUSSION Difficulties with established techniques for cloning human B-cell lines have hindered use of these cells to generate human monoclonal antibodies. While a few specific antibody-producing cell lines have been described (2, 4-8), lack of an adequate method for cloning antigen-specific B cells has precluded extensive production of such cell lines. Several of the described lines have been clearly heterogeneous in regard to their antibody production perhaps because of difficulty in successful cloning (2,4-8). In comparing several established methods for the cloning of human B-cell lines, we were initially unable to improve on the I % cloning efficiency reported by Stein&z and Klein (9) for well-established lymphoma lines cloned at 10,000 cells per well. They had reported three different EBV-positive lymphoma cell lines in which 60 to 120 colonies were observed when 10,000 cells were plated for a cloning efficiency of 1%. However, when 1000 cells were plated, only one of three lines gave rise to any colonies at all. By adapting the method of Kirk et al. (10) to culture of human B-cell lines, we report a method which increases the cloning efficiency of B-cell lines approximately lo-fold. Furthermore, this method allows for the cloning of small numbers of cells from well-established cell lines ensuring the derivation of a true clone, and can be used for a first approximation to cloning newly established lines and freshly transformed cells. We have previously attempted to clone lymphoid cells by limiting dilution with or without feeder layers of fibroblasts or human thymocytes (17). None of these methods was routinely successful. The method of Steinitz and Klein (9) was the first method which routinely gave rise to colonies provided sufficient numbers of cells were plated. We have not been able to achieve greater than 1% cloning efficiency with this method, and that with a limited number of cell lines. Most cell lines of interest had cloning efficiencies significantly less than 1%. Some cell lines could not be cloned by this method. Using the method of fibroblasts suspended in the agarose together with small numbers of lymphoid cells, enhancement of cloning efficiency was observed with cloning efficiencies ranging from 0.4 to 20%. The 20% cloning efficiency was achieved with a well-established cell line, 007 C4, previously cloned by the soft agar method suggestingselection for the ability to grow well in soft agar. In addition, this improved efficiency was achieved using small numbers of lymphoid cells, usually 1OO/perwell for well-established lymphoid cell lines and 500 to 1000 per well for newly established cell lines. Using lower numbers of lymphoid cells helps to ensure that colonies derived from this method are indeed clonal. Since the number of antigen-specific B cells for any antigen has been estimated to range from 1 in 10,000 to 1 in 1,OOO,OOO by some (18), this method will allow more efficient cloning of specific antibodyproducing cell lines when combined with preselection techniques. The presence of nonproliferating fibroblasts in soft agar in proximity to lymphoid cells appeared to be the factor that allowed for the increased cloning efficiency with
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this method. Microscopically, we observed that the primary event in colony formation was the close association between a lymphoid cell and a fibroblast or clump of fibroblasts. Subsequently, lymphoid colonies arose by the division of this initial lymphoid cell and retained their association with the original fibroblast. In cultures where fibroblasts and lymphoid cells were not in direct contact, but the fibroblasts were suspended in agarose,the cloning efficiency was still enhanced over that of the Steinitz method. These observations suggest that a growth factor is derived from nonproliferating fibroblasts suspended in agarose, confirming the findings of Kirk et al. (10). Our data indicate that this growth enhancement, mediated by nonproliferating fibroblasts, is not specific for prostatic carcinoma cells but may be stimulatory to a wide range of cells, including B lymphocytes. The growth enhancement factor is not specific for human cells in that we have used this method to clone P3U 1, a mouse myeloma cell line (Table 1). The linear relationship observed between the number of fibroblasts added and the number of lymphoid colonies arising suggestsa dose-response relationship. We consistently observed a threshold level of fibroblasts. It may be that a critical number of fibroblasts is needed either to ensure that one of a small number of lymphoid cells will be close to a fibroblast or to produce a desired concentration of a growth factor; or the growth stimulating factor may be short lived. Our improvement over the 1% cloning efficiency reported by Steinitz and Klein (9) using a feeder layer of confluent fibroblasts suggeststhat the growth-enhancing factor is not produced by anchored, confluent, fibroblasts. Whether or not attachment, nonproliferation, or confluency is critical in determining whether or not fibroblasts enhance growth of other cells is not clear. Our data demonstrate that fibroblasts suspended in agarose produce growth enhancement. Under conditions of close association between fibroblasts and lymphocytes as when they are mixed together in the agarose, this enhancement is magnified. To date, we have only used fibroblasts derived from fetal lung. Whether or not human fibroblasts from other sources or of nonhuman origin can stimulate colony formation by lymphoid cells remains to be determined. This method for cloning EBV-transformed human B lymphocytes with increased efficiency coupled with appropriate preselection of antigen-specific B cells should allow for isolation of specific antibody-producing B-cell lines. ACKNOWLEDGMENTS We are grateful to Dr. Herbert Lazarus for many helpful discussions and to Mr. John Young, Ernst Leitz, Inc., for loan of equipment.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.
Olsson, L., and Kaplan, H. S., Proc. Nat. Acad. Sri. USA 77, 5429, 1980. Zurawski, V. R., Haber, E., and Black, P. H., Science 199, 1439, 1978. Fahey, J. L., Finegold, I., Rabson, A. S., and Manaker, R. A., Science 152, 1259, 1966. Hirano, T., Teraoka, O., Teranish, T., Tsoyugushi, I., Todha, H., and Oikawa, A., Microhiol. Immunol. 24, 879, 1980. Steinitz, M., and Klein, G., Nature (London) 269, 420, 1977. Steinitz, M., Seppala, I., Eichmann, K., and Klein. G., Immunobiology 156, 41, 1979. Steinitz, M., Koskimies, S., Klein, G., and Makela O., J. C/in. Lab. Immunol. 2, 1, 1979. Steinitz, M., Izak, G., Cohen S., Ehrenfeld M., and Flechner I., Nature (London) 287, 443. 1980.
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9. Steinitz, M., and Klein, G., Eur. J. Cancer 13, 1269, 1977. 10. Kirk, D., &lay, M. F., and Kaign, M. E., Cancer Rex 41, 1100, 1981. 11. Schwaber, J., and Rosen, F. S., J. Ch. Immunoi. 2, 30, 1982. 12. Evans, J., Steel, M., and Arthur E., Cell 3, 153, 1974. 13. Schwaber, J., Klein, G., Emberg, I., Rosen, A., Lazarus, H., and Rosen, F. S., J. Immunol. 124, 2191, 1980. 14. Schwaber, J., Lazarus, H., and Rosen., F. S., Proc. Nat. Acad. Sci. USA 75, 242 1, 1978. 15. Robinson, J., and Miller, G., J. Virul. 15, 1065, 1975. 16. Boyum, A., &and. J. Clin. Lab. Invest. 21 (Suppl. 87), 9, 1968. 17. Lemhardt, W., Andersson, J., Coutinho, A., and Melchers, F., Exp. Cell. Rex 111, 309, 1978. 18. Makinodan, T., and Albright, J. F., Prog. Allergy 10, 1, 1967.