Production of human — human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization of peripheral blood lymphocytes

Immunology Leiters, 15 (1987) 89-93 Elsevier IML 00879

Production of human-human hybridomas secreting antibody to sheep erythrocytes after in vitro immunization of peripheral blood lymphocytes M a s a z u m i Terashima, Shigetoshi S h i m a d a , H i r o t s u g u K o m a t s u a n d Toshiaki Osawa Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan (Received 30 December 1986) (Revision received and accepted 20 February 1987)

1. Summary This report describes the formation of human hybridomas after in vitro immunization of peripheral blood lymphocytes (PBL) with an antigen and fusion of the stimulated lymphocytes with a HAT-sensitive human myeloma cell line, RPMI 8226. PBL were stimulated in vitro with sheep erythrocytes (SRBC) plus fresh human serum. PBL of some donors produced anti-SRBC antibody when they were cultured at 2x106 cells per well in a 24-well plate with the antigen plus fresh human serum for 7 days. Although lymphocytes of some donors were "low-responders" under the above conditions, they responded to SRBC when they were cultured with not only the antigen plus fresh human serum but also with the culture supernatant obtained after phytohemagglutinin (PHA) stimulation of a mixture of PBL from two donors (MLCP H A sup). The cells sensitized by this procedure were fused with RPMI 8226 cells. Hybrids secreting IgM or IgG anti-SRBC antibodies were obtained.

Key words: Sensitization in vitro; Sheep red blood cell; Humanhuman B cell hybridoma; Human monoclonal antibody

Correspondence to: Toshiaki Osawa, Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.

Additionally the ratio of total IgG-producing hybridomas to IgM-producing ones was higher when the MLC-PHA sup was used at the time of the in vitro immunization.

2. Introduction During the past several years, a number of reports have appeared describing the construction of human-human B cell hybridomas [1, 2]. To ensure wide application of the method, the human B cell hybridoma technique must be efficiently worked out with peripheral blood lyrnphocytes (PBL), and successful in vitro sensitization with an antigen [3-14] prior to fusion is essential. In most of the studies on human B cell hybridomas, however, B cells of donors who had previously been immunized with certain antigens were used [2, 15, 16]. Only two reports dealt with studies which used PBL primarily immunized in vitro [17, 18]. One of the difficulties appears to reside in the difference in antigen-specific response among donors of PBL. In this paper, we describe the requirements for the successful i n vitro immunization of human PBL with sheep red blood ceils (SRBC) irrespective of the sensitivity of donors to the antigen, and the construction of anti-SRBC antibody-producing human B-B hybridomas.

0165-2478 / 87 / $ 3.50 © 1987 Elsevier Science Publishers BN. (Biomedical Division)

89

3. Materials and Methods

3.1. Mutant myeloma cell line A clone of the human myeloma cell line R P M I 8226, designated JMS-3, which had been rendered sensitive to a medium containing hypoxanthine, aminopterin and thymidine (HAT medium) was kindly provided by Dr. T. Kasahara of Jichi Medical School, Japan. This cell line was maintained in RPM1 1640 medium containing 20% fetal calf serum (FCS) and 10 #g/ml 6-thioguanine. No immunoglobulin heavy or light chains were detected in culture medium of the cell line. 3.2. In vitro immunization The medium used for cell cultures for in vitro immunization of human peripheral blood B cells was R P M I 1640 supplemented with 2 m M glutamine, 1 mM sodium pyruvate, 0.05 m M 2-mercaptoethanol, 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and 10% fresh human serum. The serum used was prepared from fresh autologous or allogeneic serum exhaustively absorbed with SRBC. PBL were isolated by Ficoll-Urografin density gradient centrifugation [19]. 2 x 10 6 PBL in 1.5 ml of the medium were plated in a 24-well tissue culture plate (Falcon No. 3047), and exposed to SRBC (0.013% final concentration) with or without PHAactivated mixed lymphocyte culture supernatant ( M L C - P H A sup). The culture was maintained for 5 to 7 days in 5% CO2 at 37 °C. At the end of the immunization period, the culture was harvested and, after enumeration of anti-SRBC plaqueforming cells (PFC), the cells were fused with JMS-3 cells. 3.3. Preparation of MLC-PHA sup Equal numbers of normal h u m a n peripheral blood lymphocytes (PBL) from two donors were mixed and the mixture was cultured at 37 °C at a total cell density of 2 x 106 cells/ml in R P M I 1640 supplemented with 5% fetal calf serum (FCS), 2 mM glutamine, 1 mM sodium pyruvate, 0.05 m M 2-mercaptoethanol, and P H A (Wellcome Research Laboratories, Beckenham, England) at a final concentration of 2/~g/ml. After 2 days of incubation, the cells were washed 90

four times with R P M I 1640, and then resuspended in the same medium without P H A at the same cell density. The culture was harvested after additional incubation for 3 days, and the cells were removed by centrifugation at 4 0 0 × g for 10 min. The supernatant was passed through a membrane filter (0.22-#m) and stored at - 2 0 ° C until use.

3.4. Cell fusion In vitro immunized PBL and human myeloma JM3-3 cells were washed three times with R P M I 1640, and equal numbers of these two kinds of cells were mixed in a 15-ml tube (Corning No. 25310, Corning Glass Works, Corning, N.Y., U.S.A.). After centrifugation at 150xg, cells were fused essentially as described by K6hler and Milstein [20], by using 45% (w/v) polyethyleneglycol (M.W. 4000; Merck & Co., Rahway, N.J., U.S.A.), in R P M I 1640 containing 5 /~g/ml poly-L-arginine. The fused cells were washed and cultured in a 96-well microtiter plate at 5 X 1 0 4 cells/well. The culture medium was R P M I 1640 medium supplemented with 20% FCS, 2 mM glutamine, 1 mM sodium pyruvate, 5×10 5 M 2-mercaptoethanol, 1 x l 0 -4 M hypoxanthine, 4×10 _7 M aminopterin and 1.6x10 -~ M thymidine. In most instances cell growth was apparent 16 to 20 days after the fusion. When cell growth was apparent in the wells, the culture supernatants were collected and assayed for Ig secretion and for anti-SRBC antibody by the methods described below. 3.5. Ig secretion Goat anti-human IgM or IgG (Jackson I m m u n o Research Laboratories, Inc., Avondale, Pa., U.S.A.) was absorbed onto flexible polyvinyl plates at a concentration of 20 #g/ml for 18 h at 4 °C. The excess antibody was removed and 1% bovine serum albumin (BSA) in 10 mM sodium phosphate buffer, p H 7.4, containing 0.15 M NaC1 (PBS) was added to each well, and the plates were incubated for another 18 h at 4°C. The plates were then washed with PBS and 50 #1 of the hybridoma culture supernatant was added to each well. After 1.5-h incubation at room temperature, the plates were washed five times with PBS-0.05% Tween 20, and then 50 #1 of the same buffer containing 100,000 cpm of

125I-labeled rabbit anti-human F(ab')2 antibody (Amersham International plc., Buckinghamshire, England) was added to each well. After standing for 1.5 h, the plates were again washed and bound radioactivity in each well was counted in a g a m m a counter. Background counts were determined by using 20% FCS in R P M I 1640 instead of the culture supernatants and were found to be below 70 cpm.

Table 1 Effect of various sera on anti-SRBC antibody production? Serum

FCS Human (fresh) (pooled AB) a

3.6. Specific anti-SRBC antibody Flexible polyvinyl plates were coated with 1% BSA in PBS for 18 h at 4°C. The BSA solution was then removed, and 50/zl of hybridoma culture supernatant and either 50/zl of 10% SRBC in 1°70 BSA solution or the BSA solution alone were added to each well. After 1.5 h incubation at 4 °C, the plates were washed five times by centrifugation and 50/zl of lZSl-labeled rabbit anti-human F(ab')2 antibody was added to each well. After 1.5 h, the plates were again washed and each well was dried and the radioactivity bound to SRBC was counted. Specific binding was determined by subtracting the background count without SRBC from the count with SRBC for each sample. Background counts were found to be below 100 cpm.

4. Results and Discussion 4.1. Serum requirements of in vitro immunization To determine whether fresh h u m a n serum was critical for the in vitro immunization of human peripheral B cells, pooled AB serum or FCS was used instead of fresh h u m a n serum. The use of pooled AB serum or FCS did not affect the viability of immunized PBL. The culture in the presence of fresh h u m a n serum gave rise to higher antiSRBC responses than those observed in the culture in FCS, and pooled AB serum failed to support antigen-dependent generation of PFC (Table 1). In the immunization procedure described by Cavagnaro and Osband [17], autologous serum, but not allogeneic serum, was reported to be essential for the optimal response. However, the use of autologous serum did not seem to be an essential requirement in our system.

PFC/106 cultured PBL SRBC ( - )

SRBC ( + )

1+ 1 15 _+ 11 0

34+ 15 279 _+ 139 1 _+ 1

PBL (2× 106/1.5 ml) were cultured with or without SRBC for 7 days and harvested, and anti-SRBC plaque-forming cells were counted. Each value is the mean of triplicate cultures with the standard error of the mean.

4.2. Dependency of anti-SRBC antibody produc-

tion on the donor of PBL Table 2 shows that PBL from different donors can differ in their response to SRBC and production of anti-SRBC antibody under the optimal conditions in the presence of fresh human serum. 4.3. The effect of MLC-PHA sup on anti-SRBC

antibody production Since it is known that the supernatants of PHAactivated PBL or mixed lymphocyte cultures contain various kinds of lymphokines including B-cell growth factor (BCGF), B-cell differentiation factor (BCDF), interleukin-2 (IL-2), interferon-3, (IFN30, we expected that the addition of the M L C - P H A sup would enhance anti-SRBC antibody production, even if the d o n o r s were low-responders to SRBC. As shown in Table 3, the addition of the Table 2 Anti-SRBC antibody production by PBL from different doors a. PBL

Donor 1 Donor 2

Serum

autologous allogeneic autologous allogeneic

PFC/106 cultured PBL SRBC ( - )

SRBC ( + )

34 _+ 1 36 + 1 2+ 1 3_+ 1

471 _ 68 140 _ 29 4+ 2 2_+ 1

a PBL (2× 106/1.5 ml) were cultured with or without SRBC for 7 days and harvested, and anti-SRBC plaque-forming cells were counted. Each value is the mean of triplicate cultures with the standard error of the mean.

91

Table 3 Effect of M L C - P H A sup on anti-SRBC antibody production a PBL donor No.

PFC/106 PBL SRBC ( - )

SRBC ( + )

M L C - P H A sup plus SRBC

1 2 3

1 0 0

27_+ 13 29_+ 9 1_+ 1

339+ 144 203_+ 40 281_+ 86

a PBL (2× 106/1.5 ml) were cultured with or without SRBC for 7 days and harvested, and anti-SRBC plaque-forming cells were counted. Each value is the mean of triplicate cultures with the standard error of the mean.

MLC-PHA sup at the concentration of 10°70 to the cultures greatly augmented the anti-SRBC response even in the case of PBL from a low-responder. Although it has recently been revealed by other investigators that IL-2 and/or IFN-3, can stimulate B cells and promote Ig secretion in the case of polyclonal activation with mitogens or in secondary immunization with antigens [21-24], neither of them had any effect on the in vitro immunization of human B cells under our antigen-specific conditions (data not shown). Furthermore, it was found that the MLC-PHA sup used in our experiments contained BCGF, BCDF, and IFN-3, (52 U/ml) but not detectable IL-2 (data not shown). 4.4. Duration of in vitro immunization for fusion In vitro stimulated cells were harvested and used for fusion after various periods of time in culture with pokeweed mitogen (GIBCO, Grand Island, N.Y., U.S.A.), antigen, or MLC-PHA sup plus antigen. The fusion ratio of PBL to JMS-3 used was

1:1. The plating after fusion was at a cell density of 5 × 104 cells per well. Although stimulation in the absence of antigen yielded no hybrids secreting anti-SRBC antibody, it yielded growing hybridoma cells that secreted Ig (Table 4). Stimulation by antigen alone yielded hybrids showing growth in 97°7o of the wells and 41070 of them secreted Ig. Then, the total hybrid yield after fusion with JMS-3 was very high (0.005-0.0107o). As shown in Table 4, stimulation with MLCP H A sup and antigen for 5 days gave more wells with growing cells and Ig secretion than those observed after stimulation for 7 days. More IgGproducing hybridomas were seen when the MLCP H A sup was used at the time of the in vitro immunization. Hybrids in 1 of 30 wells (307o) in each of experiments No. 2 and No. 3 produced anti-SRBC antibody. SJ-l, the hybrids obtained in experiment No. 3, secreted IgG class anti-SRBC antibody, and SJ-2 obtained in experiment No. 2 secreted IgM class anti-SRBC antibody (Fig. 1). ;

i

SJ-1 SJ-2 JMS-3

Bound

I

!

5

10

la51

(cpm

x

10 "a )

Fig. 1. Binding of the supernatants of anti-SRBC antibodyproducing hybridomas (S J-1 and S J-2) and parent myeloma JMS-3 to sheep erythrocytes. Experimental details are described in Materials and Methods.

Table 4 S u m m a r y of experiments on in vitro immunization of h u m a n peripheral lymphocytes. Exp. No.

Antigen

Stimulator

Immunization time (day)

Viability (07o)

Hybrids (070 well)

Ig-producing hybrids (07o)

1 2 3 4

SRBC SRBC SRBC

PWM M L C - P H A sup M L C - P H A sup

3 7 5 7

79 88 94 87

88 97 75 70

60 (lgM 47070, lgG 53%) a 41 (IgM 69070, IgG 31070) 27 (IgM 38070, IgG 62070) 10

a Percentages of IgM-producing hybrids and IgG-producing hybrids regardless of the specificity of Ig produced.

92

The results presented in this paper show that production of antigen-specific antibodies against SRBC can be induced with our simplified experimental system, utilizing fresh h u m a n serum and an antigen. However, if the donor of the PBL is a nonresponder to the antigen, the addition of M L C - P H A sup is required. It is expected that this simple system will be a useful method for obtaining B cells sensitized in vitro to various antigens as a source of custommade h u m a n hybridomas secreting monoclonal antibody. The application of this method to other antigens is now under study in our laboratory.

Acknowledgement This investigation was supported by a research grant from the Japan Health Sciences Foundation.

References [1] Olsson, L. and Kaplan, H. S. (1980) Proc. Natl. Acad. Sci. USA 77, 5429. [2] Croce, C. M., Linnerbach, A., Hall, W., Steplewski, Z. and Koprowski, H. (1980) Nature (London) 288, 488. [3] Luzzati, A. L., Taussig, M. J., Meo, T. and Pernis, B. (1976) J. Exp. Med. 144, 573. [4] Delfraissy, J.-F., Galanaud, P., Dormont, J. and Wallon, C. (1977) J. Immunol. 118, 630. [5] Dosch, H. M. and Gelfand, E. W. (1979) Immunol. Rev. 45, 244.

[6] Hoffmann, M. K. (1980) Proc. Natl. Acad. Sci. USA 77, 1139. [7] Morimoto, C., Reinherz, E. L. and Schlossman, S. E (1981) J. Immunol. 127, 69. [8] Nonaka, M., Zuraw, B. L., O'Hair, C. H. and Katz, D. H. (1981) J. Exp. Med. 153, 1574. [9] van Tol, M. J., Zijlstra, J., Thomas, C. M. G., Zegers, B. J. M. and Ballieux, R. E. (1984) J. Immunol. 133, 1902. [10] Vazguez, A., Balavoine, J. F., Delfraissy, J. E, Wakasugi, H. and Galanand, P. (1984) Cell. Immunol. 86, 287. [11] Brenner, M. K., Newton, C., North, M., Weyman, C. and Farrant, J. (1983) Immunology 50, 377. [12] Cupps, T. R., Goldsmith, P. K., Volkman, D. J., Gerin, J. L., Purcell, R. H. and Fauci, A. S. (1984) Cell. Immunol. 86, 145. [13] Ten, P. L. J., Booth, R. J., Prestidge, R. L., Watson, J. D., Dower, S. K. and Gillis, S. (1985) J. Immunol. 135, 2128. [14] Komatsu, H., Shimada, S., Terashima, M. and Osawa, T. (1986) Int. Arch. Allergy Appl. Immun. 80, 431. [15] Chiorazzi, N., Wasserman, R. L. and Kunkel, H. G. (1982) J. Exp. Med. 156, 930. [16] Osband, M., Cavagnaro, J. and Kupchick, H. Z. (1981) Blood 60 suppl. 1., 81a (abstract). [17] Cavagnaro, J. and Osband, M. (1983) Hybridoma 2, 128. [18] Strike, L. E., Devens, B. H. and Lundak, R. L. (1984) J. Immunol. 132, 1798. [19] Kawaguchi, T., Matsumoto, I. and Osawa, T. (1974) J. Biol. Chem. 259, 2786. [20] K6hler, G. and Milstein, C. (1975) Nature 256, 495. [21] Zubler, R. H., Lowenthal, J. W., Erard, E, Hashimoto, N., Devos, R. and MacDonald, H. R. (1984) J. Exp. Med. 160, 1170. [22] Ralph, R., Jeong, G., Welte, K., Mertelsmann, R., Rabin, H., Henderson, L. E., Souza, L. M., Boone, T. C. and Robbi, R. J. (1984) J. Immunol. 133, 2442. [23] Mayer, L., Crow, M. K. and Thompson, C. (1985) J. Immunol. 135, 3272. [24] Bich-Thuy, L. T., Lane, H. C. and Fauci, A. S. (1985) Cell. Immunol. 94, 353.

93