GB2108528A - Process for preparing sub-culturable lymphokine-producing human T cell hybridomas - Google Patents

Abstract

A process for preparing human T cell hybridomas which are subculturable and produce lymphokines comprising the steps of 1) treating a human acute leukemia cell with a protein and/or RNA synthesis inhibitor; 2) independently activating a human T cell with a mitogen or antigen; 3) fusing the thus-treated human acute leukemia cell with the thus-activated human T cell in the presence of a fusion accelerator; and 4) isolating the thus-formed hybridoma.

Description

SPECIFICATION Process for producing subculturable lymphokine-producing human T cell hybridomas The present invention relates to suboulturable lymphokine-producing human T cell hybridomas and a process for the preparation thereof.

More particularly, it relates to subculturable lymphokine-producing human T cell hybridomas obtained by the cell fusion of a protein synthesis inhibitor and/or an RNA synthesis inhibitor-treated human acute leukemia cells and mitogen- or antigen-activated human T cells in the presence of a fusion accelerator, and a process for the preparation thereof.

The phenomemon of cell fusion was discovered by Y. Okada using Sendai virus (HVJ) (Y. Okada, Biken J., 1:103 (1958)). Since the discovery of Sendai virus mediated cell fusion, this method has been greatly used in the developing of the field of somatic cell genetics.

In 1975, Kohler and Milstein suceeded in utilizing the cell fusion technique in the field of immunology. That is, it was first reported in G. Kohler and C. Milstein, Nature, 256: 495 (1975) that the fusion of spleen cells obtained from immunized mice with HAT (hy poxanthine-aminopterin-thymidine)-sensitive murine myeloma cells results in the formation of hybridomas capable of permanently producing a monocional antigen-specific antibody.

Lymphocytes contained in human and animal immune systems are divided broadly into cells from thymus (T cells) and cells from bone marrow (B cells).

B cells are antibody-secreting cells. The cell fusion reported by G. Kohler et al. is between mouse-derived B cells and HAT-sensitive murine myeloma cells. On the other hand, T cells are formed by differentiation and maturation of stem cells from the bone marrow in the thymus. Further, T cells circulate in the blood flow through peripheral organs such as lymph nodes and the spleen.

T cells play a significant role in controlling the imune response of a living body. It is well known that the immune response-controlling function of T cells is promoted by a soluble mediator generally called a lymphokine which is released by T cells (H. G. Kunkel and F. J.

Dixon, Advances in Immunology, 29: 56 (1980), Academic Press).

Various attempts have heretofore been made to cure various diseases, such as cancer, allergy, and infectious diseases, by controlling the immune response of a living body.

Lymphokine which is specific to various immune cells may be used as a more effective immunotherapeutic agent and is expected to be utilized widely in the medical field as a clinical diagnostic. (Bernstein, I. D., D. E.

Thor, B. Bar and H. J. Rapp: Science 172 729 (1971) and Piessens, W. F. and W. H.

Churchill: J. Immunol. 114 293 (1975)) Thus, lymphokine is a medically very important substance.

In accordance with conventional methods, however, it is impossible to prepare a large amount of lymphokine, and furthermore the purity of the lymphokine that has been conventionally prepared in the past is low. Thus the utilization of lymphokine in the medical field has been retarded seriously.

Lympholines are non-antibody protein factor groups which are produced by lymphocytes due to, for example, an antigen-specific stimulus or mitogen stimulus. Further, lymphokines are produced mainly by T cells. Typical lymphokines and their actions are shown below: 1. Lymphokines acting on macrophages (1) Migration inhibitory factor (MIF) Action: prevents the migration of macrophages in vitro (2) Macrophage activating factor (MAF) Action: stimulates phagocytosis, the bactericidal action, etc. of macrophages (3) Monocyte-macrophage chemotactic factor (MCF) Action: causes chemotaxis of monocyte macrophages 2.Lymphokines acting on polymorphonuclear leucocytes (1) Leucocyte-migration inhibitory factor (LIF) Action: prevents the migration of polymorphonuclear leucocytes in vitro (2) Chemotactic factor Action: causes chemotaxis of neutrophil, eosinphil and basophil leucocytes 3. Lymphokines acting on lymphocytes (1) Interleukin II (IL-II) Action: stimulates the division and proliferation of T cells activated by an antigen or mitogen 4.Lymphokines acting on other cells (1) Lymphotoxin (LT) Action: damages and peels apart L cells and HeLa cells in vitro (2) y-lnterferon (IFN-y) Action: interferes with virus pathogenicity (3) Colony stimulating factor (CSF) Action: acts on bone marrow lymphocyte precursor cells (GFU-C), accelerating their differentiation and proliferation into granulocytes or macrophages The activity of the above-described lymphokines is measured in vitro. It is reported, however, that there are lymphokines whose activity, as exhibited in vitro, is recognized to correspond to that as exhibited in vivo.For example, MlFs are presumed to inhibit migration of macrophages. (Takeo Kuroyanagi et al: "Lymphokine" Shin Meneki Kagaku Sosho vol. 6 p. 33 Igakushoin Co., Ltd., Tokyo (1979)) In addition, teleangiectatic edema, caused by a chemical mediator in the field of, e.g., a tuberculin reaction, a large accumulation of macrophages is observed. This is because the macrophages, which migrate and collect as a result of MCF derived from sensitized T cells, are further fixed by MIF. This demonstrates the correlation between MCF and MIF with living body immunological defenses and thus permits efficient treatment of foreign substances by effective accumulation and activation of macrophages.

Lymphokines which can be expected to be used as medicines in the future include MAF, lymphotoxin, interleukin II, IFN-y, and CSF, as well as MCF and MIF.

Typical methods of preparing lymphokines are: (1) a method of cultivating peripheral blood lymphocytes sensitized by an antigen together with the antigen (D. J. Cameron and W. H. Churchill: J. Clin. Invest. 63 977 (1979)); (2) a method of cultivating peripheral blood lymphocytes or spleen cells together with a mitogen (Weiser, W. Y., Greineder, D.

K., Remold, H. G. et al: J. Immunol., 126 1958 (1981)); and (3) a method of establishing an antigen-specific T cell clone by the use of T cell growth factor (IL-II) and cultivating the clone (Green, J. A., S. R. Cooperband and S. Kibrick: Science 1641415 (1969)).

Methods (1) and (2) above need a large amount of blood and enable one to prepare only a limited amount of lymphokine. Therefore, it is difficult to prepare a large amount of pure lympholine according to methods (1) and (2). In accordance with method (3), specific lymphokines can be prepared in the presence of IL-II. However, method (3) suffers from disadvantages in that the production of lymphokines from T cells is poor and it is difficult and expensive to obtain human IL-II.

The above-described problems are encountered when using the conventional methods because lymphokine-producing T cells cannot be subcultivated and their growth is poor even if they are cultivated in the presence of a growth factor, such as IL-Il. Thus, it is very difficult at the present time to produce a sufficient amount of iymphokine for clinical use.

As a means of solving the above-described problems using the cell hybridization technique, T cell hybridomas for mice have already been established (Taniguchi, M., Saito, T. and Tada, T.: Nature 278 555-558 (1979)). Therefore, it is now possible to analyze lymphokines produced by these cells.

However, such lymphokines produced by murine T cell hybridomas cannot be used for human clinical purposes. From the viewpoint of human immunology and necessity of clinical application, it has been desired to establish lymphokine-producng human T cell hybridomas which are. subculturable.

It is reasonable to expect that the same method as used for the murine T cell fusion can be applied to the fusion of human T cells.

Indeed, there is a method where lymphokineproducing T cells (not subculturable) and HAT (hypoxanthine-aminopterin-thymidine)-sensitive T line tumor cells (subculturable) have been fused in the presence of fusion accelerator. Thereafter, only those cells which could grow on a HAT medium were screened and cloned to obtain the desired lymphokine-producing T cell hybridomas (see G. Catherine et al., Nature, 292: 842 (1981)).

However, a very complicated and difficult process is required for providing HAT-sensitivity to human T line tumor cells.

SUMMARY OF THE INVENTION Therefore, an object of the present invention is to provide a process for preparing human T cell hybridomas which are subcultural and produce iymphokines.

As a result of extensive investigations to develop a method of preparing subcultural lymphokine-producing human T cell hybridomas, it has been found that such human T cell hybridomas are obtainable by the cell fusion of a protein synthesis inhibitor- or a combination of protein synthesis inhibitor and an RNA synthesis inhibitor-treated human acute leukemia cells with mitogen- or antigenactivated human T cells in the presence of a fusion accelerator.

Therefore, the present invention provides a process for preparing a subculturable lymphokine-producing human T cell hybridoma which comprises treating a human acute leukemia cell with a protein synthesis inhibitor or a combination of a protein synthesis inhibitor and an RNA synthesis inhibitor, while independently activating a human T cell with a mitogen or antigen, fusing the thus-treated human acute leukemia cell with the thusactivated human T cell in the presence of a fusion accelerator, and then isolating the thusformed hybridoma.

DETAILED DESCRIPTION OF THE INVEN nON The process of the present invention generally comprises the following steps (A), (B), (C), (D) and (E).

Step A Lymphocytes separated from human peripheral blood and the spleen or thymus which have been aseptically excised by an operation are activated with a mitogen or antigen. Thereafter, the mitogen or antigen bound to the cells is removed so that there is as low a level of mitogen or antigen present as possible.

Any antigen or mitogen can be used in the present invention as long as it is a substance capable of inducing the transformation of human T cells, and it is selected appropriately depending on the type of lymphokine desired.

Examples of such mitogens include phytohemagglutinin-P (PHA-P) and concanavalin A (Con A). Examples of useful antiqens include PPD (purified protein derivative, i.e., purified tuberculin protein), bacterial toxoid, viral antigens and SK-SD (streptokinase-streptodornase).

Step B Human acute leukemia cells are treated with a protein synthesis inhibitor or a combination of a protein synthesis inhibitor and an RNA synthesis inhibitor. Thereafter, the inhibitor contained in the culture medium is removed by centrifugation.

Human acture leukemia cells which can be used in the present invention include T cell line tumor cells, such as CEM (ATCC No. CCL119), TALL (The Pharmaceutical Department of Tokyo University, Tokyo, Japan) and MOLT-4 (The Pharmaceutical Department of Tokyo University, Tokyo, Japan).

As protein synthesis inhibitors, known protein synthesis inhibitors for eukaryotic cells which can inhibit protein synthesis irreversibly can be used. Emetine hydrochloric acid salt and pactamycin are preferred protein inhibitors for use in the present invention. The protein synthesis inhibitors can be used alone or in combination with RNA synthesis inhibitors. Examples of such RNA synthesis inhibitors include a-amanitin, adriamycin and the like. A preferred RNA synthesis inhibitor for use in the present invention is actinomycin D.

The treatment of human acute leukemia cells with the protein or RNA synthesis inhibitors is performed under conditions such that both division and growth of the cells are completely prevented. For example, when CEM (2 X 106 cells/ml) is treated at 37"C for 2 hours with emetine hydrochloric acid salt alone, the concentration of the salt is from 10-4 to 10-5 5 M, and when the salt is used in combination with actinomycin D, the concentrations of the salt and actinomycin D are preferably 10-4 to 10-5 M and 0.05 to 2.0 yg/ml, respectively.

Step CThe above-prepared lymphokine-producing human T cells and human acute leukemia cells whose growth has been inhibited by a protein synthesis inhibitor or a combination of a protein synthesis inhibitor and an RNA synthesis inhibitor are fused in the presence of a suitable fusion accelerator.

The ratio of number of cells of human T cells to human acute leukemia cells in the fusion step is from 1:1 to 20:1 and preferably from 2:1 to 15:1.

Fusion accelerators which can be used in the present invention include polyethylene glycol (PEG), polyvinyl alcohol, and viruses having a cell-fusion ability, particularly paramyxovirus to which Sandai virus (HVJ) belongs and its inactivated product. Generally, PEG having a molecular weight of from 1,000 to 4,000 is used.

Step D Living cells of the above-obtained fused cells are concentrated to 105 to 2 X 106 cells/ml and incubated on a 96 well culture plate containing a nutrient medium with a feeder layer added thereto.

As feeder layers, human cells whose growth has been inhibited by antibiotics such as mitomycin C or by irradiation with X rays are used.

Any nutrient medium can be used as long as it is a medium on which acute leukemia cells can grow. For example, a medium prepared by adding 10% fetal calf serum (FCS), 5 X10-5 M 2-mercaptoethanol, and 2 mM glutamine to RPMI 1 640 (Nissui Seiyaku Co., Ltd.) is suitable to use.

One week after cultivation, the human acute leukemia cells and the feeder layer treated with the inhibitor completely die and the fused cells remain, i.e., continue to grow.

In order to confirm the formation of fused cells, known techniques are employed for (1) an analysis of cell surface antigens and (2) analysis of cell karyotypes.

Step E The cultivation supernatant of a well in which the fused cells have grown is analyzed to confirm production of the desired lymphokine.

The thus-prepared fused cells can be subcultivated for a long period of time while maintaining the lymphokine activity. Furthermore, cloning enables one to obtain a subline efficiently producing lymphokine.

Lymphokines can be produced by cultivating the subline either in vitro or in vivo. This cultivation can be performed by known techniques. Examples of in vitro cultivation methods include a stirring cultivation method and a stationary cultivation method using a Petri dish and a Roux flask.

Examples of in vivo cultivation methods include a method in which nude mice, nude rats, or animals other than humans, to which human tumor cells can be transplanted, are inoculated with hybridomas to form a solid tumor or an ascite tumor. After a suitable growth period, the ascites and/or blood is collected, and a method, as described in U.S.

Patent 4,276,282, in which a hamster whose immune response has been weakened, is inoculated with the hybridomas which then multiply in the hamster.

Subculturable lymphokine-producing human T cell hybridomas are useful for the mass production of lymphokines and, furthermore, since the desired lymphokine-producing cells are obtainable in a large amount, they can be utilized as an extraction source for lymphokine-production related genes (e.g., messenger RNA (mRNA)) contained therein. It is also possible that after the preparation of complement DNA (cDNA) from the extracted mRNA by the use of a reverse transcriptase, the lymphokines can be prepared using microorganisms (e.g., bacteria, yeasts, actinomycetes, and fungi) according to conventional genetic recombination techniques.

The following examples are given to illustrate the invention in greater detail.

EXAMPLE 1 (1) Preparation of Lymphokine-Producing Human T Cell Hybridomas Human peripheral blood lymphocytes (PBL) (HLA-A2, -Aw24, -B7, -Bw35, -Cw3, -Cw7) (106 cells/ml) were activated with 5 yg/ml PHA-P inan RPMI-1640 medium (Nissui Seiyaku Co., Ltd.) containing 10% fetal calf serum, 5 X 10-5 M 2-mercaptoethanol, and 2 mM glutamine (hereinafter referred to merely as an "RPMI medium") for 2 days. Thereafter, PHA-P bound to the cells was removed so that there was as low a level of PHA-P present as possible.

10-5 Memetine hydrochloric acid salt was added to human acute leukemia cells, CEM (HLA-A1, -Aw30, -B8, -B40) (2 x 106 cells/ml) which had been grown on an RPMI 1 640 medium (containing 10% newborn calf serum). After treatment at 37"C for 2 hours, the emetine hydrochloric acid salt contained in the medium was removed by centrifugation.

The above-prepared PBL and CEM were mixed in a ratio of 10: 1, and the mixture was then centrifuged to obtain cell pellets.

Cell fusion was performed at 37"C for 45 seconds by the addition of 0.5 ml of 46% polyethylene glycol (PEG-1540 (Wako Chemical Co., Ltd)), 15% dimethylsulfoxide (Wako Chemical Co., Ltd.), and 5 ,ug/ml poly-Larginine (MW: 60,000, Sigma, St. Louis, MO). Next, 10 ml of MEM medium containing 25 mM N-2-hydroxyethylpiperazine-N'-2- ethanesuifonic acid (HEPES, Wako Chemical Co., Ltd., Tokyo, Japan) buffer was added slowly, and the resulting mixture was centrifugated and resuspended with an RPMI medium to a cell density of 2 X 105 cells/ml.

After the cell fusion, the number of living cells was concentrated to 2 x 105 cells/ml and cultivated on a 96-well culture plate containing 0.2 ml of RPMI medium per well, the RPMI medium containing as a feeder layer CEM (8 x 104 cells/ml) which had been treated with 25 ,ug/ml of micromycin C at 37"C for 30 minutes. For one week after the start of cultivation, the medium was changed daily in order to moderate the influences exerted by substances released from CEM.

Lyphokine activity was measured by the method as described below in (2) using hybridomas contained in a well in which their proliferation was observed.

Emetine-treated CEM and mitomycin-treated CEM used as controls completely died within one week.

(2) Cultivation of Hybridomas and Lymphotoxin Activity Hybridomas as obtained in (1) above were cultivated in RPMI medium for one day in the presence of 20,us/ ml PHA-P. Measurement of lymphotoxin activity in the supernatant with L-P3 (subline of L cells) as a target cell showed that two hybridomas (E-10, F-8) had activity. This activity was maintained for more than three months. The lymphotoxin activity was measured using the method described by Y. Kobayashi, J. Immunology, 122: 791 (1979). That is, a specimen (25 yI) and 25,ul of actinomycin D (4 yg/ml) was added to 50 l of L-P3 (3 x 104 cells) which had been formed in advance on a microplate, and were cultivated at 37"C for 20-24 hours.Thereafer, the cells were fixed with glutaraldehyde and dyed, and cells which were morphologically normal were counted.

(3) Characteristics of Hybridomas E-10 and F-8 (A) Analysis of Cell Surface Antigen (1) It is known, by an analysis using a fluorescence activated cell sorter, that CEM does not react with monoclonal antibody OKT3, but that human T cells react with OKT3. Utilizing these findings, the OKT3 reactivity of human T cell hybridomas (E-10 and F-8) was examined by the two-step binding assay and immunofluorescence test using 125 protein A. As a result, it was found that CEM had no reactivity, whereas E-10 and F-8 had reactivities which were, respectively, nearly equal to that of PBL-T cell and about 50% of PBL-T cell.

(2) The HLA antigen for E-10 and F-8 was examined by the two-step binding assay using '251-protein A. It was found that PBL was HLA A2 and -Bw35 positive, CEM was HLA-AI and -B6 positive, but that both E-10 and F-8 were HLA-AI, -A2, and -B8 positive.

(B) Karyotype Analysis The number of chromosomes in CEM, E-10 and F-8 was measured in 50 to 80 metaphase nuclei. CEM contained 84.3 + 0.9 (mean value + S.D.) (median of 94) chromosomes.

F-8 contained 91.5 * 1.7 (median of 93) chromosomes. Thus, an increase in the number of chromosomes of about 10 was observed in both E-10 and F-8.

EXAMPLE 2 Lymphokine-producing hybridoma cell line E-10 (106 cells/ml) as obtained in Example 1 was cultured on RPMI medium for one day.

The culture supernatant was then diluted four tines. The MIF activity of the four timediluted solution was measured by the Harrington method described in J.T. Harrington, Jr.

et al., J. Immunology, 110: 752 (1973). The macrophage migration inhibition rate was found to be 29.9% for E-10. No activity was observed for the culture supernatant of CEM which had been cultured under the same conditions. The culture supernatant as obtained by culturing PBL (107 cells/ml) on MEM medium containing HEPES buffer containing Con A (10 ILg/mI) for one day showed a macrophage migration inhibitory rate of 25.2%. E-10 maintained MIF activity for more than 6 months.

EXAMPLE 3 5 X 10-5 M emetine hydrochloric acid salt and 0.1,ug/ml actinomycin D was added to CEM at 37"C for 2 hours. The thus-treated CEM was fused with PBL which had been prepared in the same manner as in Example 1 and was cultured in the same manner as in Example 1. Emetine hydrochloric acid salt and actinomycin D-treated CEM completely died, and only hybrid cells were observed to multiply.

The thus-obtained hybridoma cell line was cultivated in the same manner as described in Example 1-(2) and the lymphotoxin activity of the resulting supernatant was measured. It was observed that two hybridoma cell lines (C5, D-9) had LT activity.

EXAMPLE 4 Lymphokine-producing hybridoma cell line E-10 as obtained in Example 1 was subcloned by limited dilution method (0.5 cell/well) in an RPMI-1640 medium containing as a feeder layer CEM (2 x 105 cell/ml) which had been treated with mitomycin C as in Example 1 and also containing 20% of concanavalin Aactivated PBL supernatant to obtain subline El 0-20.

The E10-20 cells thus obtained were cultivated on an RPMI medium (106 cells/ml) for one day and the resulting supernatant was precipitated by ammonium sulfate (50-100% saturation) and dialyzed against sodium phosphate buffer containing 0.15 M NaCI (PBS, pH 7.2) to obtain a test sample. The MIF activity of the sample was measured in the same manner as in Example 1. The macrophage migration inhibition rate was found to be 29.9%.

Further, the MAF activity of the sample was measured according to a process described below which is a modification of H. W. Murray et al method (J. Exp. Med. 1531690 (1981)), i.e., a process in which human macrophage-like cell line U937 (The Pharmaceutical Department of Tokyo University, Tokyo, Japan) is reacted with MAF and release of 2- (super oxide anion) is detected. That is, substantially the same procedure as Murray et al method was repeated except that 100 it1 of human macrophage-like cell line U937 (5 x 105 cells/ml) was incubated for 48 hours in the presence or absence of the test sample.

As a result, 30% of the total U937 cells revealed to release 2- in the cells, which demonstrates that the test sample contained MAF.

In the same manner as above, CEM cell line was cultivated and the culture supernatant was precipitated with ammonium sulfate (50-100% saturation) and dialyzed against PBS to obtain another test sample. The MIF activity of this ample was measured in the same manner as above and revealed to be 9.9%. Also, the MAF activity was measured in the same manner as above and 21 % of the total U937 cells were shown to release 02 in the cells.

While the invention has been described in detail and with reference to a specific embodi ment thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope thereof.

Claims (21)

1. A process for preparing a subculturable lymphokine-producing human T cell hybridoma which comprises the steps of 1) treating a human acute leukemia cell with a protein synthesis inhibitor or a combination of a protein synthesis inhibitor and an RNA synthesis inhibitor so that the growth of the human acute leukemia cell is completely prevented; 2) independently activating a human T cell with a mitogen or antigen so that blast transformation of the human T cell occurs and the desired lymphokine is produced; 3) fusing the thus-treated human acute leukemia cell with the thus-activated human T cell in the presence of a fusion accelerator; and 4) isolating the thus-formed hybridoma.

2. The process as claimed in Claim 1, wherein said protein synthesis inhibitor is one capable of inhibiting the protein synthesis of eukaryotic cells.

3. The process as claimed in Claim 1, wherein said protein synthesis inhibitor is emetine or its derivative or hydrochloric acid salt thereof and/or pactamycin.

4. The process as claimed in Claim 1, wherein said RNA synthesis inhibitor is at least one member selected from the group consisting of actinomycin D, a-amanitin and adriamycin.

5. The process as claimed in Claim 1, wherein said protein synthesis inhibitor is emetine or its derivative or hydrochloric acid and said RNA synthesis inhibitor is actinomycin D.

6. The process as claimed in Claim 1, wherein said human acute leukemia cell line is T line tumor cells.

7. The process as claimed in Claim 6, wherein said human acute leukemia cell is OEM, TALL or MOLT-4 cell line.

8. The process as claimed in Claim 1, wherein said mitogen is phytohemagglutinin-P or concanavalin A.

9. The process as claimed in Claim 1, 6 or 7 wherein 106 to 107 cells/ml of said human acute leukemia cell line is treated with 10-4 to 10-5 M of said protein synthesis inhibitor.

10. The process as claimed in Claim 1 or 5, wherein 106 to 107 cells/ml of said acute leukemia cell line is treated with 10-4 to 10-5 M of said protein synthesis inhibitor and 0.05 to 0.2,ug/ml of acrinomycin D.

11. The process as claimed in Claim 1, wherein said cell fusion accelerator is a polyethylene glycol having a molecular weight of 1,000 to 4,000.

1 2. The process as claimed in Claim 1, 10 or 11, wherein upon cell fusion, said antigen- or mitogen-treated human T cell is mixed with said human acute leukemia cell line in a cell number proportion of 1:1 to 20:1.

13. The process as claimed in Claim 1, 10 or 11, wherein upon cell fusion, said antigen- or mitogen-treated human T cell is mixed with said human acute leukemia cell line in a cell number proportion of 2:1 to 15:1.

14. The process as claimed in Claim 1, 11 or 13, whrein after cell fusion the resulting hybridoma cells are cultivated in a medium which permits non-protein and/or RNA synthesis inhibitor-treated human acute leukemia cell line to proliferate, thereby allowing for the isolation of the hybridoma cell.

1 5. The process as claimed in Claim 1, wherein said cell fusion accelerator is Sendai virus (HVJ) or its inactivated product.

16. A process as claimed in Claim 1, wherein the lymphokine produced by the thus-formed hybridoma is migration inhibitory factor (MIF).

1 7. The process as claimed in Claim 1, wherein the lymphokine produced by the thus-formed hybridoma is lymphotoxin (LT).

1 8. The process as claimed in Claim 1, wherein the thus-formed hybridoma is then subcultured in vitro or in vivo.

19. The process as claimed in Claim 1, wherein the resulting hybridoma is a cell line selected from the group consisting of E-10, F8, C-S and D-9.

20. The process as claimed in Claim 19, wherein the hybridoma is a cell line E-10.

21. A process for preparing a subculturable lymphokine-producing human T cell hybridoma, substantially as hereinbefore described with reference to any of Examples 1 to 4.