JPH0351397B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a novel human T hybrid cell line and its production method. More specifically, human T that produces human B cell growth factor (BCGF) or human B cell differentiation factor (BCDF) and can be cultured permanently.
This invention relates to a cell hybridization cell (human T cell hybridoma) and its production method. Lymphocytes included in the human immune system are broadly classified into T cells, ie, thymus-derived cells, and B cells, ie, bone marrow-derived cells. The above B cells are known to secrete antibodies. On the other hand, the T cells secrete various humoral immune factors (lymphokines), such as factors that suppress the antibody secretion of the B cells and factors that cause division of other T cells (interleukin 2), It plays a central role in regulating the immune response. Therefore, these T cell-derived humoral immune factors,
In particular, obtaining large amounts of homogeneous humoral immune factors will greatly contribute to the elucidation of immune responses. However, it is difficult to obtain large amounts of these T cell-derived humoral immune factors from the human body, and it is also possible to extract humoral immune factors by stimulating human T cells in vitro. However, it has been difficult to collect large amounts of the above factors, and it has been almost impossible to obtain homogeneous factors. Therefore, various attempts have been made to apply cell fusion technology to fuse human T cells and extract these humoral immune factors in large quantities and in a homogeneous state. For example, Okada M. et al, Proc. Natl. Acad.
According to Sci., OSA78, 7717-7721 (1981):
A T-cross that secretes T-cell growth factor (TCGF or IL-2) in the cross between a hypoxanthine-guanine-phosphoribosyl-transferase (HGPRT)-deficient human leukemic T cell line and normal human T cells. It is stated that it contains cells. However, there is no specific description in the above literature regarding factors that induce proliferation or differentiation of B cells (BCGF or BCDF) and T hybrid cells that secrete these factors, and in particular regarding B cell growth factor (BCGF). Until then, even its existence was uncertain. The present inventor has obtained the above-mentioned HGPRT-deficient human leukemic T.
We have conducted various studies on hybrid cells between cell lines and normal human T cells. As a result, they discovered that these hybrid cells included hybrid cells that secreted BCGF and hybrid cells that secreted BCDF, and that these hybrid cells could be subcultured stably for a long period of time in vitro. It has been found that by culturing these hybrid cells, it is possible to produce large amounts of BCGF or BCDF in a homogeneous state. The present invention has been completed based on these new findings. That is, the present invention provides hybrid cells between HGPRT-deficient human leukemic T cells ATCC CRL-8081 and normal human T cells, which are human cells that produce human B cell growth factor (BCGF) or human B cell differentiation factor (BCDF). This invention relates to T hybrid cells and methods for producing these hybrid cells. As used herein, human B cell growth factor (BCGF) is a factor that promotes the proliferation of human B cells, and human B cell differentiation factor (BCDF) is
A factor that causes human B cells to differentiate into antibody-producing cells. According to the present invention, both BCGF and BCDF are produced in large quantities in a homogeneous state, so that research in this field that was previously impossible, such as human T.
This will greatly benefit the elucidation of interactions between cells and human B cells. Also, BCGF and
BCDF is also useful as a therapeutic agent for immunodeficiency caused by decreased T cell function (insufficiency). Furthermore, by utilizing these factors, it becomes possible to obtain large quantities of desired human antibody-producing cells in vitro, and these can be used, for example, as the hitherto desired raw material for human B cell fusion methods. The human T hybrid cell line capable of producing BCGF or BCDF of the present invention can be obtained by fusing a specific parent cell line derived from a human leukemic T cell line with normal human T cells, and cloning the resulting hybrid cell. You can get it. The above parent cell line is publicly known and was deposited with the American Type Culture Collection (ATCC) on July 30, 1981 by the present inventors.
It is entrusted as "CRL-8081". The parent cell line is derived from a human leukemic T cell line and is characterized by lacking HGPRT. Its cytological and other properties are as follows. (1) Morphological characteristics: The diameter is approximately 2 to 3 times that of normal human peripheral blood T cells, and the shape is almost spherical. The nucleus occupies a large proportion of the cell, and a small amount of protoplasm is observed. A few granules are observed within the protoplasm. Occasionally, a pseudopod-like protrusion may appear. (2) Chromosome number: Parent cells were cultured at 37°C for 3 hours in the presence of colchicine at a concentration of 0.1 μg/ml, and after centrifugation, 0.075 M-KCl
The number of nuclear chromosomes after fixation on a slide glass using a fixative of methanol:ethanol = 3:1 was counted by microscopic observation using a 1000x oil immersion lens. As a result, 100 cells divided. In metaphase, the number of nuclear chromosomes in each parent cell is between 69 and 87, with an average of 78, as shown in Table 1 below.

[Table] (3) T cell-specific antigen expression: Parent cells were adjusted to a concentration of 5 × 10 ⁵ cells, and 100 μ of a monoclonal antibody at an appropriate concentration (0.1 mg/0.5 ml) was added.
Stock solutions of anti-Leu1, anti-Leu2A, and anti-Leu3A antibodies were diluted 100 times, 10 times, and 10 times, respectively, and used as 100μ), and after incubation at 4°C for 30 minutes, the cells were diluted with 5% fetal MEM medium containing bovine serum (FCS) [Osaka University Institute of Technology]
Wash with FITC (fluorescein isothiocyanate)
- Binding - Reacting with rabbit anti-mouse immunoglobulin [Miles-Yeda LTD., Israel],
T cell-specific antigen expression was measured by a direct immunofluorescence antibody method. Each monoclonal antibody was purchased from Becton Dickinson Co., Ltd.
Obtained from [J. Exp. Med., Sunnyvale, Ca]
153310−323 (1981)]. As a result of examining 200 cells, anti-Leu1 and anti-Leu3A antibodies
More than 95% were positive, and less than 1% were positive for anti-Leu2A antibodies. (4) Rosette formation: Rosette formation in antibody complement-sensitized sheep red blood cells (EAC) was measured for 200 cells under a microscope at 400x magnification, and less than 1% were positive. (5) B cell marker expression: Surface immunoglobulin (Ig) analyzed by direct immunofluorescence antibody method using FITC-conjugated mouse anti-human immunoglobulin (Behring werke AG, Marburg) was positive in less than 1%. be. Monoclonal anti-DR antibody (Becton-
Human HLA-DR antigen (DR) analyzed by indirect immunofluorescence antibody method using (Dickinson Co.)
Less than 1% is positive. Mouse splenocytes and myeloma P3U 1 immunized with Epstein-Barr virus-transformed human B cell lines (obtained from Dr. Peter Ralph, CESS, Sloan, Kettering Cancer Institute) and myeloma P3U ₁ (Elbert Einstein College
of Medicine) [G.Kohler and C.Milstein, Nature
256495, 1975] The expression of human B cell-specific antigen (B antigen), analyzed by indirect immunofluorescence antibody method using a monoclonal anti-human B cell antibody, is positive in 1% or less. The above antibody reacts with B cells or their lines, but not with T cells or their lines. (6) HLA phenotype Parent cells were treated with anti-HLA serum (Tokyo Medical and Dental University,
Rabbit complement (Veritas) absorbed by parent cells of the present invention after incubation at 37°C for 30 minutes with (obtained from Professor Sasazuki)
As a result of incubating for 60 minutes and examining the cell viability, the HLA phenotype was A2, A11, B8,
Shows B37 and Bw22. (7) Proliferation: 8-azaguanine (8-AG, 100μM), 10
% fetal calf serum (FCS), containing 5 x 10 ^-5 M2-mercaptoethanol and 1mM glutamine.
Grows well in RPMI1640 medium (Flow Laboratories). (8) Growth conditions: generally at a temperature of 36-38℃ and pH7.2-7.3
It grows well under these conditions. Further, culturing in an incubator containing 5% carbon dioxide gas and 95% air is suitable. (9) Subculture: Subculture is possible without limit. (10) Cryopreservation: Can be easily stored for long periods in liquid nitrogen. (11) 8-azaguanine resistance: Resistant to 8-azaguanine (100μM),
It is killed in a medium containing hypoxanthine, aminopterin and thymidine (HAT medium). This indicates that the parent cell is an HGPRT-deficient strain. (12) Mitodiene reactivity: Concanavalin A (ConA) 10-100μg/ml
By adding 1 to 10% of phytohemagglutinin and phytohemoglutenin (PHA),
Mitotic proliferation is partially suppressed. Potskweed mitogen (PWM) and protein A
(ProA) does not affect the division and proliferation of this cell line at any concentration. These are shown in the attached FIGS. 1 to 4.
Each figure shows 2 x ¹⁰⁴ parental cell lines treated with various concentrations of mitogen (ConA in Figure 1, ConA in Figure 2).
RPMI1640 + 10%FCS with PHA, PWM in Figure 3 and ProA in Figure 4)
Incubate for 60 hours within the culture and add 0.2 μCi during the last 12 hours of culture.
^3H -thymidine ( ^3H -HdR) was added, the cultured cells were collected on a glass fiber strip (Labo Science), and the incorporation of ^3H -TdR into the deoxyribonucleic acid fraction was counted using a liquid scintillation counter. The results are plotted as the average value (SD is 10% or less) of each repeat ^three times.
×10 ^-5 ). The HGPRT-deficient human leukemic T cell line ATCC CRL-8081, which has the above-mentioned properties, is a human leukemic T cell line.
Cells (CCRF-CEM) [J.KAPLAN, TC
SHOPE and WDPETERSON, Jr., J. Exp.
Med.139, 1070-1076, 1974]
This was cultured in RPAI 1640 medium containing 10% FCS and 8-azaguanine (8-AG) added.
It can be obtained by sequentially increasing the amount of 8-AG added. More specifically, for example, first, cells are cultured for one week in a medium supplemented with 2 μM 8-AG, then viable cells are cultured for one week in a medium containing 16 μM 8-AG, and then the 8-AG concentration is increased to 2 μM. The 8-AG resistant cell line is obtained by sequentially culturing in a medium with a doubling of the concentration, and finally survives in a medium with an 8-AG concentration of 100 μM. The parent cell line thus obtained is then
It shows strong growth in a medium containing 100μM 8-AG,
Subculture can be performed in this medium. There are no particular limitations on the normal human T cells that can be fused with the parent cell ATCC CRL-8081, and for example, any of various T cells derived from peripheral blood, bone marrow, lymph nodes, spleen, tonsils, etc. can be used. These T cells are isolated and purified by known separation means, such as physical methods, chemical methods, surface membrane methods, etc. commonly used in this field. The obtained T cells are then treated with various mitogens, such as protein A (ProA),
After stimulation with concanavalin A (ConA), alloantigen, etc., the cells are fused with the above parent cells. Details of the production of the human T cells and stimulated T cells described above are as shown in the Examples described below. HGPRT-deficient human leukemic T cells as parental cells
The fusion reaction between ATCC CRT-8081 and the human T cells described above is basically the same as known cell fusion methods, and is carried out in an appropriate medium in the presence of a fusion promoter. Viruses such as Sendai virus (HVJ) may be used as the fusion promoter, but polyethylene glycol (PEG), which has been developed in recent years, may also be used.
It is preferable to use The PEG preferably has an average molecular weight of about 1000 to 6000, and is suitably present in the medium at a concentration of about 30 to 60 W/V%. Furthermore, as the medium, MEM medium, Dolbecco's modified medium, RPMI 1640 medium, and various other usual media used for this type of cell culture can be used. Furthermore, if desired, an auxiliary agent such as dimethyl sulfoxide may be added to the above cell fusion medium to increase the fusion efficiency. Parent cell ATCC CRL used for the above cell fusion
There is no particular restriction on the ratio of the amount of -8081 to human T cells used, but in general, the number of human T cells can be used about 1 to 10 times the number of parent cells. Preferred cell fusion is carried out, for example, as follows. That is, predetermined amounts of parent cell line and human T cells are thoroughly mixed in an appropriate medium, centrifuged, the supernatant is removed, and an appropriate amount of a PEG solution preheated to 37° C. is added and mixed. This initiates a cell fusion reaction. Thereafter, the desired fused cells are observed to appear by repeating the steps of sequentially adding an appropriate medium, centrifugation, and discarding the supernatant. Isolation of the desired fused cells is carried out by culturing the cells after the cell fusion described above in a normal hybrid selection medium. This selection medium is a medium in which the parent cells cannot proliferate, and only the target hybrid cells can proliferate (human T cells cannot naturally proliferate), and typical examples thereof include hypoxanthine, aminopterin, etc. and a medium containing thymidine (HAT medium). As the HAT medium, for example, 10 to
In MEM or RPMI 1640 medium containing 20% FCS, aminopterin 4 x 10 ^-7 M, hypoxanthine 1 x 10 ^-4 M, thymidine 1.6 x 10 ^-5 M and optionally glycine 3 x 10 ^-6 An example is a medium to which M is added. Cells are cultured in the HAT medium according to the usual limiting dilution method for a period of time sufficient to kill cells other than the desired hybridized cells (unfused cells, etc.), usually about several days to several weeks. As a result, only the target human T cell fused cells are selectively proliferated. The fused cells thus obtained are characterized by their karyotype (number of nuclear chromosomes), cell surface phenotype, mitogen reactivity,
It has new characteristics that are clearly different from the parent cell line, human T cells, in terms of lymphokine production ability, etc. This fused cell can be grown and maintained in the same suitable medium as above, but after selection with HAT medium, it contains hypoxanthine and thymidine.
It is preferable to culture in HT medium for 1 to 2 weeks and then transfer to normal medium. Furthermore, the above-mentioned human T cell hybrid cell lines can be cloned by growing them in a conventional medium according to a conventional method, and thereby each can be separated into a single fused cell line. The human T hybrid cell line having the ability to produce BCGF and the human T hybrid cell line having the ability to produce BCDF of the present invention were discovered from among the large number of hybrid cell clones obtained as described above, and the present invention This was discovered by the inventor for the first time to have the ability to produce BCGF or BCDF. A human T hybrid cell line (i.e., clone No. 77-
A, 94-C, 98-F, etc.) and a human T hybrid cell line with BCDF-producing ability (i.e., clone No. 90-E)
etc.) are as follows. That is, these hybrid cell clones are derived from the HGPRT-deficient parental cell line ATCC CRL, as shown in the Examples below.
This is a hybrid cell line obtained by cell fusion of -8081 and stimulated human peripheral blood lymphocyte T cells.
It has the cytological properties listed in the table. Each measurement method is as follows. (1) T cell-specific antigen expression: (a) Leu antibody Adjust each hybrid clone to a concentration of 5 x 10 ^-5 cells. monoclonal antibody anti-Leu-1,
Stock solutions of anti-Leu2A, anti-Leu3A, and anti-Leu4 (obtained from Dr. R. Evans, Sloan Kettering Cancer Institute, 0.1 mg/0.5 ml concentration) were 100x, 10x, 10x, and 10x, respectively. diluted to
Prepare 100μ monoclonal antibody solution.
After incubating the above hybrid clone with this monoclonal antibody solution at 4°C for 30 minutes, the cells were washed with MEM medium (Osaka University Biken) containing 5% FCS (fetal calf serum), and FITC (fluorescein
isothiocyanate)-conjugated with rabbit anti-mouse immunoglobulin (Miles-Yeda, Ltd., Israel), and T cell-specific antigen expression was measured by indirect immunofluorescence antibody method. (b) OKT antibody As a monoclonal antibody, OKT-monoclonal antibody (Ortho Pharm.Corp.USA) was used, and OKT3, OKT4, and OKT8 antibodies were each diluted 10 times and made into a 100 μ solution, 5×
10 ⁵ cells were treated and measured by indirect immunofluorescence in the same manner as for the Leu antibody described above. (2) Macrophage antibody Human macrophage tumor cell line U937 cells
BALB/C mice were immunized and this BALB/C
Spleen B cells and myeloma cells obtained from mice
P ₃ U ₁ was fused with polyethylene glycol to obtain a mouse B hybridoma that produced a monoclonal antibody against human macrophages. (G.Kohler and C.Milstein, Nature,
256495, 1975). This antibody specifically binds to U937 and normal human macrophages, but
It does not react with normal human T cells, B cells, human T cell lines, or human B cell lines. Using this antibody, human macrophage antigen (Mφ) was similarly measured by indirect fluorescence antibody method. (3) B cell and macrophage antigen monoclonal anti-human DR antibody (Becton-
Dickinson Co.) was diluted 10 times to 100μ, reacted with 5×10 ⁵ cells, and human HLA-DR antigen (DR) was similarly measured by indirect immunofluorescence antibody method. Table 2 shows parent cell ATCC for comparison.
CRL-8081 (CEM- ^AGR ), human histiocytic lymphoma cell line (U937, obtained from Dr. Taniyama of the National Institute for Preoperative Research), human peripheral blood T cells (PBL-T), and human peripheral blood B cells (PBL-B). Other than using
The results of tests conducted in the same manner as in (1) to (3) above are also listed.

【table】

[Table] Furthermore, clone No. 77-A, 94-C,
98-F etc. are homogeneous as shown in the test examples below.
Characterized by secreting BCGF.
In addition, clone No. 90-E of the present invention etc. are homogeneous.
Characterized by secreting BCDF.
These things are clear from FIG. That is, as is clear from FIG. 5, BCGF produced by these hybridomas does not have interleukin-2 activity or BCGF activity. On the other hand, BCDR is associated with interleukin-2 activity and
Does not have BCGF activity. The hybrid cell clones of the present invention having the above characteristics can be subcultured stably over a long period of time, so by culturing and proliferating them, a large amount of BCGF or BCDF can be dissolved in the culture supernatant. can be manufactured. Moreover, you can get
BCGF or BCDF is obtained from a single clone and is homogeneous. Hereinafter, in order to explain the present invention in detail, examples of isolation and preparation of human T cells, cell fusion of T cells and parent cells, selection of the obtained fused cells, cloning, and test examples of the obtained BCGF or BCDF producing clones will be given below. . Example 1 Isolation and preparation of human T cells 50 ml of blood collected from a healthy adult with heparin
Lymphocyte specific gravity separation liquid, “Fuicol Pack”
(Pharmacia Japan Co., Ltd.) to isolate 5 x 10 ⁷ peripheral blood lymphocytes. Isolation of T cells from the lymphocytes was performed by rosetting with neuraminidase-treated sheep red blood cells (SRBC) [T. HIRANO, T. KURITANI,
T. KISHIMOTO and T. YAMAMURA, J.
Immunol., 119, 1235-1241 (1977)]. The thus obtained peripheral blood T cells (1×10 ⁶ cells/ml) were diluted to a concentration of 10μ.
Stimulated T cells were prepared by stimulation with g/ml ProA (manufactured by Pharmacia) for 72 hours. Example 2 Parental cell-human T cell fusion Parental cells (having the characteristics of ATCC number CRL-8081, hereinafter referred to as "CEM- ^AGR ") were incubated in culture medium (RPMI 1640 + 10%
FCS + 100 μM 8-AG) was exchanged to maintain active growth. 1×10 ⁷ pieces of the above CEM-AG ^R and the above Example 1
2 x 10 ⁷ of each human T cell obtained in the above were used for cell fusion. That is, each of the above-mentioned cells was washed three times with FCS-free MEM medium pre-warmed at 37°C, then transferred to a 50 ml conical tube and mixed well.
It was centrifuged at 1000 rpm for 10 minutes. The cell pellet obtained by removing the supernatant was gently shaken, and then 0.3% of 45% PEG-6000 (Cotsholite) heated to 37°C was placed on top of the cell pellet.
Poured ml. After shaking well for 30 seconds, the mixture was left standing at 37° C. for 6 minutes in a carbon dioxide incubator containing 5% carbon dioxide gas and 95% air. A total of 12 ml of FCS-free MEM (preheated to 37°C) was added to this at a rate of 2 ml per minute. After quickly adding 25 ml of MEM, the mixture was centrifuged at 800 rpm for 10 minutes, and the supernatant was removed. Gently add 50 ml of 20% FCS-containing RPMI 1640 medium (prewarmed to 37°C) to a cell concentration of 2 x 10 ⁵ cells/ml, and place this in a 2 cm diameter microculture plate (Linbro). 50 each well
1 ml was dispensed into each individual. After 24 hours, half of the supernatant was discarded and added to HAT medium (hypoxanthine (Sigma)).
1×10 ^-4 M, aminopterin (Sigma) 4×
1 ml of RPMI 1640 + 20% FCS medium containing 10 ^-7 M and 1.6 x 10 ^-5 M thymidine (Sigma) was added to each well. Repeat the same operation every 2 days until 2~
The cells were cultured at 37°C in a 5% carbon dioxide gas incubator for 4 weeks. The proliferating cell line was then transferred to HT medium without aminopterin (A) (HAT medium without A), and after culturing for an additional week, HAT
The cells were transferred to RPMI 1640 + 10% FCS medium (normal medium) that does not contain the following, and thereafter, cloning was performed according to a conventional method. That is, dilute the hybrid cells in a normal medium to 1 cell/ml, dispense this into a falcon microplate at 0.2 ml/well, discard half of the supernatant every 2 to 3 days, and preheat to 37°C. Add warmed medium,
The cells continued to proliferate. Thus, single cloning of the hybrid cell lines was carried out, and each human T hybrid cell line, clone No. 77-A, 94-C, 98-F and 90E, having the above-mentioned characteristics was obtained. Example 3 Production of human T hybrid cell supernatant Each hybrid cell obtained in Example 2 above was treated with RPMI
The concentration was adjusted to 1 × 10 ⁵ cells/ml using 1640 + 10% FCS medium, and this was incubated at 37°C for 2 days in 2 cm diameter wells of a microculture plate (Linbro).
Below, the cells were cultured in a 5% carbon dioxide incubator. Centrifuge at 3000 rpm for 10 minutes to collect the culture supernatant, which is sterilized with a 0.45μ Millipore filter.
Culture supernatants of each hybrid cell were obtained. Test Example (1) BCGF activity of hybrid cell supernatant (A) BCGF activity against anti-human IgM-stimulated human peripheral blood B cells Human peripheral blood lymphocytes obtained in the same manner as in Example 1 were Rosettes were formed using SRBC in the same manner, non-rosette-forming cells were fractionated by specific gravity centrifugation, and the same procedure was performed. Next, cells that adhere to the non-rosette forming cells are removed by a petri dish method,
Obtain human B cell fraction. On the other hand, rabbit-anti-human obtained by a conventional method
IgM serum (Hirano T. et al, J. Immunol,
119; 1235 (1977)) is affinity purified using a human IgM-binding Sepharose column to obtain an insolubilized antibody bound to Sepharose-4B beads (manufactured by Pharmacia). Containing 5 x 10 ⁴ B cells obtained above, and 10
Rabbit anti-human IgM-conjugated sepharose obtained above was added to RPMI 1640 medium (0.2 ml) containing %FCS.
4B and each culture supernatant obtained in Example 3 above are added. After culturing for 4 days, 1 μCi of ³ H-thymidine (hereinafter referred to as " ³ H-TdR") is added and cultured for 5 hours. Then, the deoxyribonucleic acid fraction of B cells
The uptake of ³ H-TdR was measured using a liquid scintillation counter to examine the division and proliferation of B cells. The results are shown in Figure 5, 1-A. (B) BCGF activity against B cells obtained from PHA-induced B colonies Contains 15% FCS, 5 x 10 ^-5 M 2-mercaptoethanol and 1/1600 PHA-P (phytohemagglutinin-P, manufactured by Difco) Matsukoi 5A medium (manufactured by Flow Laboratories) 2
ml of human B cells obtained in the same manner as in (A) above.
T cells obtained by irradiating 2000 rad of X-rays on ^peripheral blood T cells from the same person as the person from whom the B cells were collected.
After adding 2×10 ⁶ cells and culturing for 3 days, the cells are washed with the above medium. On the other hand, it contains the following ingredients and contains agar 0.3% and 0.5
Prepare a soft agar medium containing %. Γ85% Matsukoi 5A medium Γ15% FCS Γ2mM L-glutamine (manufactured by Flor Laboratory) Γ16μg/ml L-asparagine (manufactured by Takara Kosan Co., Ltd.) Γ8μg/ml L-serine (manufactured by Takarakosan Co., Ltd.) Γ1mM Sodium pyruvate ( Γ0.8% MEM Essential amino acid solution (50 times diluted) (Flow Laboratory) Γ0.4% MEM Non-essential amino acid solution (100 times diluted) (Flow Laboratory) Γ0.6% hydrogen carbonate Sodium (7.5% solution) (manufactured by Flow Laboratory) Γ5×10 ^-5 M 2-Mercaptoethanol Γ100 units/ml Penicillin Γ100 μg/ml Spreptomycin In this soft agar medium, the above cells were
In the presence of 1600PHA-P, two-layer method [Muraguchi, A. et al J. Immunol.125564
(1980)] at 37°C for 7 days, and colony-forming cells were collected. The obtained cells are
with FITC-conjugated rabbit anti-human immunoglobulin (Behring Werke AG, Marburg).
Direct immunofluorescence showed 92% positive results. Additionally, 3.5% of these cells showed SRBC and rosette formation. 2×10 ⁵ of these colony-forming cells were incubated with anti-
Leu1 antibody-producing hybridoma (Dr.R.Evans:
(obtained from Sloan Kettering Cancer Institute, NY)
The cells were treated with 100 μl of the culture supernatant and complement (4-fold diluted neonatal rabbit serum) in a cytotoxic reaction to remove contaminating T cells. 1 × 10 ³ cloned B cells thus obtained,
3 along with 0.2 ml of each culture supernatant obtained in Example 3 above.
After culturing for one day, 0.5 μCi of ³ H-TdR was added, cultured for 6 hours, and the uptake of ³ H-TdR was measured. The results are shown in Figure 5, 1-B. (2) BCDF activity (A) CESS (B cell line transformed with EB virus, Dr. P. Ralph, Sloan Kettering Cancer Institute), which is a cell that can differentiate into IgG producing cells (PFC) in the presence of BCDF. After culturing 1 x 10 ⁴ cells (obtained from J.D. Co., Ltd.) for 48 hours with 0.2 ml of each culture supernatant obtained in Example 3, reverse
Measure the number of IgG producing cells (PFC) by plaque assay. That is, protein A-bound SRBC was added to agarose melted at 46-48°C, and the CESS suspension after the above culture, anti-human IgG antibody (Behring Werke AG, Marburg), and complement (guinea pig serum, diluted 5 times) were added. ), spread on a Petri dish, and keep warm at 37℃ for 4 hours.
Next, measure the number of hemolytic plaques (equivalent to the number of PFC),
Calculate the increase in PFC (ΔPFC/culture) using the formula below. ΔPFC/culture=PFC/culture
(Group to which culture supernatant was added) -PFC/culture (Group to which culture supernatant was not added) The results are shown in FIG. 5, 2-A. (B) Human B cells obtained in the same manner as (1)-(A) above
10 ⁶ pieces, 0.0025% Staphylococcus aureus Cowan (Calbiochem−
Behring Corp., La Jolla, Ca.)
The B cells are stimulated by culturing in 1 ml of RPMI 1640+10% FCS medium for 3 days, and then the stimulated cells are washed with the medium. The increase in the number of PFCs is measured in the same manner as in (2)-(A) above, except that 2×10 ⁵ of these stimulated cells are used. The results are shown in FIG. 5, 2-B. (3) T cell growth factor (IL-2) activity IL-2 activity of each culture supernatant was measured using IL-2-dependent human cytotoxic T cells [Okada, M. etal, Proc. Natl.Acad.Sci.USA
78, 7717-7721 (1981)]. Specifically, 3 x ¹⁰ 3 IL-2-dependent human cytotoxic T cells were cultured for 24 hours in the presence of each culture supernatant obtained in Example 3, and then 0.5 μCi of ³ H-TdR was added, and 6 After culturing for an hour, the amount of ³ H-TdR taken up was counted and IL-2 activity was measured. The results are shown in FIG. 5. The results of Test Examples (1) to (3) above are shown as the average ±SD of the same test repeated 2 to 3 times. In the figure, nd means not measured. In test examples (1) and (3), the horizontal axis indicates the
³ H-TdR counts (cpm x 10 ³ ) are calculated as ΔPFC/culture (counts x 10 ² ) in test example (2).
are shown respectively. As a control, a group (C) in which no culture supernatant was added, CEM obtained in the same manner as in Example 3.
-AG ^R or 24-A [Okada, M. et al, Pvoc.
Natl. Acad. Sci. USA 78, 7717-7721 (1981)]
The culture supernatant addition group and the PHA-stimulated T cell factor (PHA-Sup) addition group are shown together. still,
For PHA-Sup, human T cells obtained in the same manner as in Example 1 were cultured for 48 hours in the presence of PHA (1/1000), and then the culture supernatant was added to Sephadex G-100.
(Pharmacia), molecular weight
A collection of 15,000 to 20,000 fractions was used.
1 PHA-Sup obtained from ¹⁰⁶ human T cells
Shown as a unit. From Figure 5, the BCGF activity of each culture supernatant of 77-A, 94-C, and 98-F and the culture supernatant of 90-E are shown.
BCDF activities are evident and appear to be distinct from IL-2 activity. (4) 77-A and 94 in the same manner as in Test Example (1)-(B) above.
-C culture supernatant was tested for synergistic effect of BCGF activity. The results are shown in Figure 6. In the figure, the horizontal axis shows the dilution factor of each culture supernatant, and the vertical axis shows the uptake of ³ H-TdR (cpm×10 ³ ). Each mark in the figure is as follows. △; CEM-AG ^R undiluted culture supernatant addition group □; PHA-Sup 10 unit addition group 〇; 77-A culture supernatant addition group ▲; 94-C culture supernatant addition group ■; 2-fold dilution of 94- C culture supernatant at each dilution factor of 77
-Group with mixed A culture added The results are the average ± S of the same test repeated three times.
Shown as D. The figure shows the additive effect of the BCGF activity of the 77-A and 94-C culture supernatants. That is, there are at least two types of BCGF, and their synergistic effects cause B cells to proliferate significantly. This result shows that untransformed human B
This provides a powerful weapon for long-term culture cloning of cell lines. Experimental Example 1 Stability test of T hybrid cells Using the following human leukemic T cells, an 8-azaguanine-resistant strain was created as follows: JUR: Sloan-Kettering Institute Research, New York, USA (Sloan −Kettering Institute
Dr. Peter for Cacer Research)
Obtained from Peter Ralph, MOLT-4: Biochem.Biophys.Res.Commun.
51, 529 (1973), HSB-2: Cancer Research
Research), 28, 1121 (1968), HPB-ALL: Int.J. Cancer, 21, 166 (1978). The above T cells were first treated with RPMI 1640 + 10% FCS containing 2 μM 8-azaguanine (8-AG).
Suspend the cells in a medium at a concentration of 1 x ¹⁰ cells/ml, take 10 ml of the cells into a culture bottle (manufactured by Corning),
With the bottle lying on its side, 5% carbon dioxide and 95
The cells were cultured for one week in an incubator at 37° C. under aeration conditions of % air. Thereafter, viable cells were taken out, suspended in the same medium containing 8-azaguanine at twice the above concentration, that is, 4 μM, at a concentration of 1×10 ⁶ cells/ml, and similarly cultured for one week. As mentioned above, the concentration of 8-azaguanine added to the medium was increased by about 2 times (2→4→8→16
→ 32 → 50 → 75 → 100 μM), cells surviving in each medium were sequentially cultured, and finally cells surviving in a medium containing 100 μM of 8-azaguanine were obtained. Thus, after about 8 weeks, the desired 8
-Azaguanine resistant strain was obtained. Next, in the same manner as in Example 2, the above 8-azaguanine-resistant human leukemic T cells and Example 1
The cells were fused with ProA-stimulated peripheral blood T cells obtained according to the method of . Two weeks after fusion, the number of wells containing hybridized cells that were still producing were counted. The results are shown in the table below and compared with the results obtained using the parent cell CEM- ^AGR in the present invention.

[Table] From the above results, it can be seen that the 8-azaguanine-resistant strain of human leukemic T cells does not necessarily fuse with normal T cells to give stable hybrid cells.

[Brief explanation of drawings]

Figures 1-4 are graphs showing the mitogen reactivity of the parental cell lines. Figure 5 shows the BCGF activity (1), BCDF activity (2) and IL-2 activity (3) of the hybrid cells of the present invention.
This is a graph showing. FIG. 6 is a graph showing the synergistic effect of BCGF activity when the culture supernatant of the hybrid cell clone No. 77-A of the present invention and the culture supernatant of 94-C were mixed.

Claims (1)

[Claims] 1. HGPRT-deficient human leukemic T cells ATCC
A human T hybrid cell line, which is a hybrid cell between CRL-8081 and normal human T cells, and is characterized by having the ability to produce human B cell growth factor or human B cell differentiation factor. 2 HGPRT-deficient human leukemic T cell ATCC
Fuse CRL-8081 with normal human T cells, selectively proliferate the resulting hybrid cells in a selective medium,
A method for producing a human T hybrid cell capable of producing a B cell growth factor or a B cell differentiation factor, which comprises cloning this to obtain a hybrid cell line capable of producing a B cell growth factor or a B cell differentiation factor. .