AU621066B2 - Suppressive t lymphocyte cell lines and supernatant derivative thereof - Google Patents

Description

Lr% I International Bureau INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 4: 1 4 ter na l Number: WO 89/01511 C12N 5/00, A61K 35/26 Interitl nal lic4I Date: 23 February 1989 (23.02.89) (21) International Application Number: PCT/US88/02681 (74) Agents: CAMPBELL, Cathryn et al.; Pretty, Schroeder, Brueggemann Clark, 444 South Flower Street, Suite (22) International Filing Date: 5 August 1988 (05.08.88) 2000, Los Angeles, CA 90071 (US).

(31) Priority Application Numbers: 084,448 (81) Designated States: AT (European patent), AU, BE (Eu- 227,843 ropean patent), CH (European patent), DE (European patent), FR (European patent), GB (European (32) Priority Dates: 11 August 1987 (11.08.87) patent), IT (European patent), JP, LU (European pa- 2 August 1988 (02.08.88) tent), NL (European patent), SE (European patent).

(33) Priority Country: US Published With international search report.

(71) Applicant: MEDICAL UNIVERSITY OF SOUTH Before the expiration of the time limit for amending the CAROLINA [US/US]; 171 Ashley Avenue, Charles- claims and to be republished in the event of the receipt ton, SC 29425-2232 of amendments.

(72) Inventors: BROSTOFF, Steven, W. 2608 La Golondrina Street, Carlsbad, CA 92009 ELLERMAN, A. 2. J.P. 2 7 APR 1989 Karen, E. 2 Oliver Court, Charleston, SC 29403

(US).

AUSTRALIAN

-9 MAR 1989 S PATENT OFFICE (54) Title: SUPPRESSIVE T LYMPHOCYTE CELL LINES AND SUPERNATANT DERIVATIVE THEREOF (57) Abstract A method of culturing antigen-specific lines of lymphocytes of the suppressor T class (Ta) and cell lines generated thereby which suppress TH cell activity. Derivatives of the supernatant of the cell cultures non-specifically suppress helper T cell activity.

1 i WO 89/01511 PCT/US88/02681 with 5% heat-inactivated fetal calf serum, and 2 g ml-1 GP-MBP in 80 cm 2 tissue-culture flasks (Nunc). To test for suppression. CsLN-1 wora A6.. I I I k..i "WO 89/01511 PCr/US88/02681 1 SUPPRESSIVE T LYMPHOCYTE CELL LINES AND -SUPERNATANT DERIVATIVE THEREOF BACKGROUND OF THE INVENTION This invention relates generally to the immune system, and more particularly to T lymphocytes which suppress the immune response.

The immune system in higher organisms is responsible for protection of the individual organism from invasion by microorganisms or other substances, termed antigens, which are recognized as foreign. The system performs this function using two general mechanisms: an antibody mediated response and a cell mediated response. Certain circulating cells, termed B lymphocytes or B cells, produce antibodies that specifically recognize and neutralize foreign antigens. In the cell mediated response, lymphocytes termed T cells activate a response which results in the binding and elimination of the antigen.

There are a variety of T cells that interact during this process. T helper cells (Th) induce B cells to make antibody and assist other T cells to mount a cell-mediated response. Cytotoxic T lymphocytes (CTL) recognize and destroy cells containing foreign antigens on their surface.

T suppressor cells (Ts) suppress the function of other cells, whereas T inducer or T effector cells can stimulate other cells to respond to foreign antigen.

T helper and T inducer cells can have the same phenotype 25 and are often referred to as the T helper subset.

Hereinafter, they will be referred to as T h cells. T suppressor cells were once thought to have the same phenotype as CTL's, but more recently T suppressor cells have also been isolated with phenotypes similar to Th cells. For the purposes of this invention, T s will hereinafter refer to cells having suppressor functions rather than a particular phenotype.

A critical aspect of the immune system is its specificity. A response- occasioned by the presence of a particular antigen will generate antibodies and T cells

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i WO 89/01511 PCrUS8/02681 2 capable of binding and neutralizing only that particular antigen while leaving other antigens untouched.

Mlch of the interaction between the cells of the immune system is mediated by the release of soluble factors, i.e., molecules released by one cell that affect other cells. By binding to cell surface receptors on recipient cells, such factors cause a stimulation or suppression of the activity of the recipient cell. A number of factors which perform a wide variety of functions are released by the cells of the immune system during the immune response. Such factors include the various interleukins, colony stimulating factors, and growth factors.

T

s cells appear to exert their effect on the immune response by suppressing the activity of Th cells. At any time, the immune system may be responding to the presence of a number of antigens. Because each response must be independently regulated, most Th cells appear to be specific to a particular antigen or small group of antigens. Similarly, many soluble factors released by T s cells appear to be antigen specific, and affect only those Th cells specific to the antigen in question.

The immune system is highly integrated and regulated such that, under ordinary circumstances, it does not overact to a foreign substance or react inappropriately to a non-foreign or "self" antigen. Such regulation appears to result from both positive and negative feedback and be mediated in part by various T cells and the soluble factors they secrete. For example, suppressor cells affect such regulation both by direct contact with effector cells as well as by release of soluble factors.

Occasionally, the immune system will perform inappropriately with deleterious consequences to the host organism. The immune response may, for example, be unsuitably severe and sustained, as in an allergic reaction. Alternatively, the immune system may recognize WO 89/01511 PCT/US88/02681 3 and attack a component of the host's own body. Autoimmune diseases result when such responses to self antigens are not controlled. Examples of diseases thought to involve autoimmune responses include multiple sclerosis, rheumatoid arthritis, myasthenia gravis, and Type One diabetes.

Various soluble factors that can be used to stimulate the immune system have previously been isolated from the immune system. In addition, a variety Th cells have been established as homogenous lines or clones and grown indefinitely in culture while still maintaining their ability to release soluble factors. In contrast, T suppressor cell lines have proven difficult to grow indefinitely under culture conditions to produce suppressor factors.

A technique for establishing T suppressor cell lines and the ability to generate cell lines that produce suppressor factors would provide an important tool for the treatment and control of immune mediated diseases particularly autoimmune diseases in which the host has lost the ability to suppress the response to its own self antigens. The isolation of suppressive factors would permit direct intervention to regulate the immune response. Because individual suppressive factors are generally antigen specific, the utility of such a factor would be limited to affecting immune responses related to that particular response. There thus exists a long-felt and compelling need for stable T s cell lines and a non-antigen specific suppression factor. The present invention fulfills these needs and provides related advantages as well.

SUMMARY OF THE INVENTION The invention provides an effective method for culturing lines of lymphocytes that can function to suppress the i response of other T cells. The supernatant of such cell lines contains a soluble non-antigen specific derivative ii -i 4 which suppresses the activity of effector Th lymophocytes.

In order to culture suppressor T lymphocytes, a lymphocyte sample is obtained from an animal having elevated levels of suppressor cells.

Such a state may result from an autoimmune condition, particularly a remission episode. Alternatively, such a condition may occur as a result of immunization or other exposure to an antigen so as to cause an immune response. The lymphocytes are then grown in the presence of a Th cell suppressant, such as cyclosporin A along with specific antigen, and then stimulated alternatively by being exposed first to lymphocyte growth factors and then to antigen. After several such cell cycles, the cell lines exhibit little inducer function while continuing to exhibit suppressor activity. Such suppressor activity may be determined by the ability to inhibit Th mediated transfer of disease or by reduced uptake of H-thymidine by Th lymphocytes. The factor mediating such 15 suppressive activity is derived from the supernatant of the cell lines.

According to a first embodiment of this invention, there is provided a method of culturing antigen specific suppressor T cell lines which retain their ability to produce non-antigen specific, soluble suppression factor, said method comprising the steps of: S 0 a. Recovering lymphocytes from animals having an elevated I level of T s cells, as hereinbefore defined, directed at said antigen; S" b. Growing said lymphocytes in the presence of said antigen and factors which inhibit Th cell, as hereinbefore defined, proliferation without affecting T s cell proliferation; and c. Alternately culturing said lymphocytes in a medium containing lymphocyte growth factors and activating said lymphocytes by exposing them to said antigen.

According to a second embodiment of this invention, there is provided a composition characterized by its ability to non-specifically suppress the activity of effector T lymphocytes, derived from the supernatant of cell cultures generated according to the method of the" first embodiment.

According to a third embodiment of this invention, there is provided a method of supplementing a host's T s cell, as hereinbefore defined, population comprising the steps of: /LMM/567Z 4a a. Recovering a sample of said host's lymphocytes; b. Growing said lymphocytes in the presence of a factor which inhibits Th cell, as hereinbefore defined, proliferation without affecting T s cell proliferation; c. Alternately culturing said lymphocytes in a, medium containing lymphocyte growth factors and activating said lymphocytes by exposing them to said antigen, whereby a culture of antigen-specific T s cells is established; and d. Reintroducing said T s cell into said host.

Other features and advantages of the present invention will become apparent from the following, more detailed description which illustrates, by way of example, the principles of the invention.

DETAILED DESCRIPTION OF THE INVENTION Experimental allergic encephalomyelitis (EAE) is an autoimmune disease mediated by T lymphocytes of the Th cell subset. Following sensitization to guinea pig myelin basic i protein (GP-MBP) in complete Freunds adjuvant (CFA), rats develop an autoimmune response to central nervous system (CNS) a myelin basic protein which is manifested clinically as paralysis, and histologically by a perivascular mononuclear cell infiltration of the CNS parenchyma. Lewis rats, which spontaneously recover from active EAE disease, are resistant to re-induction of active EAE by immunization with GP-MBP.

W/567Z ,'Zy T WO 89/01511 PCT/US88/02681 T suppressor cell lines were generated by recovering a lymphocyte sample from lymph nodes, or other lymphocyte source, of animals, preferably rats, immunized one month previously with an antigen, preferably GP-MBP, in a suitable vehicle such as CFA. Alternatively, cells can be recovered from other animals, such as humans, having an autoimmune disease, particularly if such disease is in a period of remission, or from animals which for one reason or another have elevated T s cell levels. To select for T s cells rather than Th cells, the lymphocytes were grown for approximately a week in the presence of the immunizing antigen and an immunosuppressive agent which is believed to inhibit Th cell function, such as cyclosporin A (CsA). The resulting blasts were purified, as on Ficoll-metrizoate gradients, and grown for 1-7 days in the presence of lymphocyte growth factors, for example those in supernatants from Concanavalin A stimulated cells (CAS).

The cell were then maintained in culture by alternate activation with the immunizing antigen and a source of antigen-presenting cells (APC), followed by culture in growth factors such as CAS. The T s cell lines thus generated were specific for the immunizing antigen. A cell culture so produced by immunizing Lewis rats with GP-MBP, termed CSLN-1, was deposited with the ATCC on August 11, 1987, under accession number CRL 9495. An analogous method can be used to generate T s cell lines specific to other antigens.

Initially, the lymphocytes recovered in the sample may include Th cells. In order to determine whether the cultured cell lines have lost their Th cell activity, the cells were injected into naive recipients. For example, CsLN-1 was tested for its ability to passively transfer EAE to naive rats. After five to six cycles of antigenic stimulation, CsLN-1 produced mild clinical signs of disease in each of four rats. The severity and incidence of passive disease produced by CsLN-1 decreased with subsequent cycles of antigenic stimulation until CsLN-1 was

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WO 89/0511 PCT/US88/02681 6 no longer able to produce any clinical signs of EAE.

Activated cells were tested for effector activity at a dose of 5x10 6 cells per recipient as extant EAE Th cell lines transfer paralytic disease at that cell number, and, in fact, are often lethal at that dose. It appears that some EAE Th cells were included in the initial population of CsLN-1. These Th cells were either lost or their activity suppressed when the cells were cycled six to seven times.

Thereafter CsLN-1 was not an EAE-producing Th cell line.

The T s cell lines generated were assayed for suppressive activity by using the adoptive transfer model of EAE (Richert et al., J. Immunol. 122:494 (1979), wnich is incorporated herein by reference). Adoptive transfer of EAE can be achieved with spleen cells taken from GP-MBP sensitized Lewis rats on day eleven or twelve postimmunization and activated in vitro with antigen.

Recipients of 3 to 4x10 7 activated splenocytes develop sever paralytic EAE 4 to 6 days later. Recipients of splenocytes mixed with GP-MBP activated T s cells during the in vitro antigen activation period failed to exhibit clinical evidence of disease after transfer of up to 5x1P 7 cells. Histologic evidence of disease was seen only if the

T

s cells were not antigen-activated cells or if the T s cell line was used before all Th activity had disappeared from the line. The T s cell lines were able to inhibit the transfer of EAE.

The observation of disease inducing activity during the early cycles in the development of CSLN-1 suggests that the Ts cells may be responding to a specific determinant on the Th cells rather than to the antigen. CSLN-1 was tested for its ability to proliferate to irradiated GP-MBP specific T cells. Lymph node and spleen cells from Lewis rats with EAE were cultured with antigen, the activated blasts separated on a density gradient and irradiated. Resting CsLN-1 (passaged for 3-5 days in CAS) were combined with the antigen-activated blasts, in the presence or absence of APC, in a standard proliferation assay. P 2 -activated b!lasts from a P 2 T cell line were used as negative 'l.

I-I--1 WO 89/01511 PCT/US88/02681 7 controls. P 2 is a peripheral nervous system myelin protein. However, it is immunologically different from MBP, having no cross-reactivity with MBP-specific antibodies. CsLN-1 proliferates to GP-MBP-activated lymphoblasts but not to P 2 -activated lymphoblasts. This response demonstrates that T s cells respond to a BP-MBP specific Th receptor. The response occurs in the absence of APC suggesting that there may be a dichotomy between the activation requirements of Th and T s cells.

In order to test the antigen specificity of T s cells, a radioactive nucleotide uptake assay was employed.

Lymphocytes from an animal previously immunized with a particular antigen were cultured in the presence of that antigen, and replicates cultured with other antigens. The cells were then presented with tritiated thymidine 3

H-

TdR), collected and the incorporation of radioactivity measured by scintillation spectroscopy. Cells cultured with the immunizing antigen exhibited significant 3 H-TdR uptake, while those cultured with non-immunizing antigens did not, thus demonstrating the antigen specificity of T s cells.

To determine whether the suppressor effect of the generated T s cell lines was cell dependent or involved a soluble factor, the activity of supernatant from CSLN-1 cell cultures was investigated. When supernatant derivative was added to mixtures of naive cells and GP-MBP activated cells from GP-MBP immunized animals, the passive transfer of EAE was inhibited. Moreover, the 3 H-TdR uptake of Th cells grown in the presence of antigen was reduced.

Both effects indicate that the suppressive activity is present in the supernatant derivative. As used herein, derivative refers not only to the whole supernatant or a portion thereof, but to an extract or composition thereof also.

The non-specific nature of the suppressor factor was tested by the ability of CsLN-1 to inhibit the transfer of '7 WO 89/01511 PCT/US88/02681 8 an autoimmune disease other than EAE. Experimental allergic neuritis (EAN) is a peripheral nervous system disease produced by P 2 protein in a manner similar to the production of EAE by GP-MBP. Similarly, EAN can be transferred from P 2 immunized donors to naive recipients after incubation of lymphocytes from these donors with P 2 protein in culture in a manner analogous to the transfer of EAE using GP-MBP. The presence of activated CsLN-1 during the culture phase prior to the transfer of the P 2 activated cells into the naive recipients suppresses the ability of the transferred cells to produce disease in these recipients. Since CsLN-1 does not respond specifically to P2 lymphoblasts, the suppressive activity must be the result of the non specific factor it releases.

Because inappropriate immune response, particularly autoimmune diseases, may result from an inability of the host's immune system to effectively regulate the response through T s mediated mechanisms, T s cell supplementation can alleviate or suppress the syndrome. For example, a lymphocyte sample may be removed from an animal and cultured as described above so as to generate T s cell cultures by stimulating them with the antigen to which.the host is inappropriately responding. These T s cell lines can then be perfused back into the host, thereby augmenting the suppression effect and reducing Th activity.

The following examples are intended to illustrate but not limit the invention.

EXAMPLE I ISOLATION OF GUINEA PIG MYELIN BASIC PROTEIN GP-MBP was isolated according to the method of Oshiro and Eylar, Archives of Biochemistry and Biophysics 138:392-393 (1970) as modified by Brostoff and Mason, J. of Immunology 133:1938 (1984), both of which are incorporated herein by reference.

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WO 89/01511 PCT/US88/02681 9 Briefly, 100 membrane stripped guinea pig brains (Pel Freez, Rogers, Arkansas) were homogenized in 700 ml methanol for 2 minutes in a Waring commercial blender (model No. 31BL79) (Waring, New Hartford, CT) followed by the addition of 1400 ml of chloroform and additional blending in a Waring commercial blender (Model No. 31 BL79) for 4 minutes. The homogenate was allowed to separate in a separating funnel for two hours. The lower plasma was discarded and the upper phase mixed with celite (Fisher Scientific, Springfield, NJ) until it reached the consistency of a milkshake. The celite mixture was filtered on a Buchner funnel through Whatman #1 filter paper (Whatman Ltd., England) and washed with 200 ml acetone. The celite mixture was removed and suspended in 3.2 1 of distilled water adjusted to pH 1.9 with concentrated hydrochloric acid. After stirring for 18 hours, the material was centrifuged at 10,000 x g for minutes, the residue discarded and the supernatant adjusted to pH 7.0 with concentrated ammonium hydroxide. The solution was stirred for 1 hour and filtered through Whatman No. 1 filter paper. Ammonium sulfate (350g/liter) was added to the filtrate which was then adjusted to pH with ammonium hydroxide, stirred for 1 hour, and centrifuged at 10,000 x g for 15 minutes. The pellet was resuspended in 200 ml distilled water and dialyzed overnight in Spectrapor (Spectrum Medical Industries, Los Angeles, CA) dialysis tubing (MW cutoff 3500) against 4 changes of 2 liters of distilled water. The precipitate after dialysis was removed by centrifugation at 10,000 x g for 15 minutes. Ammonium acetate was added to the supernatant to bring the concentration to 0.3 M before loading onto a column of Cellex P (Bio-Rad Laboratories, Richmond, CA) equilibrated with 0.3 ammonium acetate pH The Cellex P column (9 x 2.5 cm, flow rate ml/min) was washed successively with 0.2 M and 0.5 M ammonium acetate and the basic optical density at 280 nm.

It was dialyzed as above and lyophilized prior to use. All operations were carried out at 4°C unless otherwise noted.

WO 89/1511 PCT/US88/02681 EXAMPLE II GENERATION OF T S CELL LINES To generate T s cell lines, female Lewis rats, (Charles River Laboratories, Raleigh-Durham, NC) 8-12 weeks of age, were immunized in both hind foot pads with 50Ag GP-MBP emulsified in CFA. One month post-immunization, the draining lymph nodes were removed by dissection, teased into a single cell suspension and washed once in Dulbecco's A+B medium containing 0.2% bovine serum albumin. Lymph node cells were plated at 5 x 106 ml" 1 in 6-well clusters (Costar 3406 Cambridge, MA) in RPM1 1640 (Gibco, Grand Island, NY), 1% Lewis rat serum, 200-300 Mg ml" 1 CsA (Sandimmune IV, Sandoz, Inc.), and 50-60 pg ml 1 GP-MBP, at 37C, in 5% CO 2 After 7 days, the activated blasts were purified on Ficoll-metrizoate (d 1.077 g ml- 1 and plated at 2x10 5 ml" in RPMI 1640, 10% heat-inactivated fetal calf serum (Hyclone, Salt Lake City, UT), and 10% CAS (derived from 48 hour Concanavalin in A-activated Lewis rat splenocytes). After 1-7 days in CAS, the cells were restimulated for 3 days at 3x10 5 ml 1 with GP-MBP (50 pg ml" 1) and 1x10 7 ml-1 irradiated (2500 R) Lewis thymocytes in RPM1 1640, 1% Lewis rat serum. Thereafter, cell lines were alternately passaged as above in 10% CAS or with antigen/APC. Complete culture media consisted of RPM1 1640 supplemented with 1mM sodium pyruvate, 2mM glutamine, HEPES buffer, 5 x 10 5 M 2-mercaptoethanol, 100 u ml 1 penicillin, 100Mg ml 1 streptomycin, and 25 pg ml 1 Fungizone (Gibco).

The T cell lineage of one such cell line was confirmed by cell surface marker phenotyping with monoclonal antibodies against rat T cell differentiation antigens.

The cell line, CsLN-1, reacted with MRC OX19, a rat pat T cell (CD5) marker. Virtually 100% of CsLN-1 reacted with 2 different rat CD4 monoclonal antibodies (MRC OX35, and W3/25), whereas only approximately 15-30% of CsLN-1 were CD8 (MRC OX8) reactive. CD8 reactivity was seen only after stimulation with antigen or the lectin Concanavalin A; 1 II L 3 ~1-11 I ;I 1 WO 89/01511 PCT/US88/02681 11 cells grown for 1 week in CAS were not CD8+. The cell line did not express Major Histocompatibility Complex Class II I-A or I-E antigens.

To test for antigen specificity, CsLN-1 was plated in RPM1 1640 with 1% Lewis rat serum at 2x10 4 cell/well along with 1xl0 6 /well irradiated (2500 R) Lewis thymocytes in 96well flat-bottomed trays (Costar) at 0.2 ml final volume.

After 48 hours, cultures were pulsed with 0.5 C of 3Hthymidine 3 H-TdR) and cells were collected 16 hours later.

Incorporation of 3 H-TdR was measured by liquid scintillation spectroscopy. Results are presented in Table I, expressed as cpm of four replicates. Standard PPD and P2 were used at 25 pg ml 1. NT, not tested.

TABLE I Antigen Specificity of the In Vitro Proliferation (cpm) of CsLN-1 Antigen Cycles of Stimulation 8X 11X None 24,439 19,416 448 GP-MBP 193,894 202,413 64,938 PPD 13,374 5,428 NT Concanavalin A 216,951 133,610 66,929

P

2 NT NT 228 To determine whether CsLN-1 cells retained the ability of Th to produce EAE in naive rats to which they were transferred, 2x10 5 ml 1 cells were grown in RPM1 1640, 1% Lewis rat serum, with 1x10 7 ml-1 irradiated (2500 R) Lewis thymocytes and 20-25 gg ml" 1 GP-MBP or 5gg ml 1 Concanavalin A. After 3 days at 37°C, in 5% C0 2 cells were harvested and 5x10 6 activated cells were injected intraperitoneally in each of 4 naive Lewis rats in qfl 1 r WO 89/01511 PC7/US88/02681 ml of phosphate buffered saline (PBS). The appearance of clinical signs of EAE (approximately 4-6 days post transfer) was graded on a three point scale as follows: 1 limp tail; 2 hind leg weakness; 3= hind leg paralysis.

The results are provided in Table 2.

TABLE 2 Ability of Activated CSLN-1 cells to Produce EAE in Lewis Rats as a Function of Cycles of Stimulation Maximum Disease Severity Activation Cycle Stimulus 6x 7x 8x 9x 9x* 11X

GP-MBP

GP-MBP

GP-MBP

GP-MBP

GP-MBP

Concanavalin A Concanavalin A 0 1 2 3 3 1 3 1 3 1 3 1 4 4 4 4 Each rat received 2x10 7 Concanavalin cells A-activated CsLN-1 EXAMPLE III SUPPRESSION OF PASSIVE TRANSFER OF EAE BY CSLN-1 CELLS CsLn-1 cells were activated in vitro for 3 days with GP-MBP and APC as described in Example II. Spleen cell donors were female Lewis rats immunized 11 to 12 days earlier with Ag GP-MBP in CFA. CP-MBP sensitized spleen cells were t j :1-I;I :il I WO 89/0151 PCT/US88/02681 13 with 5% heat-inactivated fetal calf serum, and 2 pg ml-1 GP-MBP in 80 cm 2 tissue-culture flasks (Nunc). To test for suppression, CsLN-1 were added at a ratio of 1 T s cell to 8 spleen cells. Cells were cultured for 3 days at 37°C, in 5% CO 2 Harvested cells were injected i.p. into naive Lewis rats. Recipients of control cells were given 3 to 4x10 7 viable spleen cells; recipients of spleen cells mixed with Ts cells were given 107 more viable cells than matched controls. Clinical signs of disease was graded on the 3 point scale shown in Table 2. Cryostat sections of brain and spinal cord were taken on days 12 to 14 after cell transfer, stained with haematoxylon and eosin, and graded on the following scale: 0, no abnormality; 1, an occasional foci of pervenular leukocytes within the neuraxis; 2, many small foci of perivenular leukocytes within the neuraxis; 3, large and numerous foci of perivenular leukocytes within the neuraxis. Results are presented in Table 3.

TABLE 3 Incidence Mean Severity Mean Histology of EAE max 3.0 max Activated CsLn-1 0/12 0 0.6 (n=7) Resting CsLN-1 1/3 0.33 1.5 (n=3) Controls 15/15 2.7 2.6 (n=7) CsLN-4, a second cell line established by the same procedure, was found to have the following effect: Activated CsLN-4 0/3 0 0 (n=3) WO 89/01511 PCT/US88/02681 14 EXAMPLE IV SPECIFICITY OF PROLIFERATIVE RESPONSES OF CsLN-1 FOR GP-MBP ACTIVATED LYMPHOBLASTS Lymph node and spleen cells were taken 12 days postimmunization from GP-MBP sensitized Lewis rats and were cultured at 5x10 6 ml 1 with 10 gg ml 1 GP-MBP. After 4 days, activated blasts were purified on Ficoll-metrizoate and given 1500 R of gamma-irradiation. The P 2 T cell line was activated in culture at 2x10 5 cells ml 1 with 40 pg ml" 1 bovine P 2 and APC for 3 days. The P 2 activated T cell line cells also received 1500 R. Antigen activated blasts and the P 2 T cells were used at 3x10 6 ml 1 APC were irradiated (2500 R) Lewis thymocytes at 2x10 7 ml 1 Resting CsLN-1 were used at 3.75x10 5 ml 1 Cells were plated in complete media supplemented with 5% Lewis rat serum, in 96-well flat-bottomed trays at 0.2 ml final volume. After 48 hours, wells were pulsed with 0.5 AC of I 3 H-TdR and cells were collected 16 hours later. Results are presented in Table 4, expressed as cpm of four replicates. Standard errors were within 10% of the mean and are not shown. These results demonstrate that CsLN-1 is specific for GP-MBP specific lymphoblasts and not nonspecific lymphoblasts as demonstrated by the lack of incorporation of thymidine in response to the P 2 T cell line.

TABLE 4 SPECIFICITY OF PROLIFERATIVE RESPONSES OF CsLN-1 FOR GP-MBP ACTIVATED LYMPHOBLASTS mean cpm 3 H-thymidine incorporation CsLN-1 APC 135 CsLN-1 GP-MBP Blasts 18,335

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CsLN-1 P 2 T Cell Line 210 WO 89/01511 PCT/US88/02681 EXAMPLE V SUPPRESSION OF TRANSFER OF EAE BY SUPERNATANTS FROM CsLN-1 CsLN-1 was incubated with GP-MBP for 3 days in culture according to the method of Example II. The supernatant from the cell culture was added to a culture of splenocytes from GP-MBP sensitized Lewis rats and incubated with GP-MBP for an additional 3 days in culture. The splenocytes were harvested and injected intraperitoneally into recipients at 3-6x10 7 cells/animal. Controls received cells which were not treated with CsLN-1 supernatants.

Disease severity was graded on a 3 point scale.

As can be seen in Table 5, splenocytes cultured for three days in the presence of MBP alone develop severe EAE.

In contrast, splenocytes cultured in the presence of supernatant from activated CsLN-1 cells show a lower incident and much milder form of disease. Supernatants from an activated P 2 T helper cell line (P 2 -2A) did not show the suppressive activity (data not shown) indicating that the suppressor activity was not due to the use of depleted media or a cell waste product. Changing the supernatant once during the transfer (after 24 hours) further inhibited the transfer of EAE. With the exception of one animal in experiment 2, the rats that did not show evidence of clinical disease after transfer had no histologic evidence of disease either. In the clinically well animal that had histologic evidence of disease, only 2 small foci of infiltrating cells were found in the brain stem.

WO 89/1511 PCT/US88/02681 16 TABLE Effect of Supernatant From Activated CsLN-1 Cells on the Transfer of Clinical Experimental Allergic Encephalomyelitis* Experi- Presence of Incidence Maximum Mean ment Tissue Culture of Clinical Clinical Disease Supernatant EAE Disease Duration From Activated Grade in Days+ CsLN-1 Cells (Mean) 1 None 3/3 4 66% (by volume) 2/4 1 2 None 4/4 4 4.3 66% (changed 1/4 1 after 24 hrs.) *Animals exhibiting clinical signs had histology grades of 2 or 3. Of the animals that showed no clinical signs of EAE, only one animal in experiment 2 had 2 small foci of infiltrating cells in the brain stem (grade 1) and all the rest showed no histologic evidence of disease.

+Of those showing clinical signs of disease.

EXAMPLE VI SUPPRESSION OF TRANSFER OF EAE BY GP-MBP SPECIFIC LYMPHOBLAST ACTIVATED CsLN-1 GP-MBP specific lymphoblast activated CsLN-1 were assayed for suppressive activity by using the passive transfer model of EAE. GP-MBP specific lymphoblasts were prepared as follows: Draining lymph node and spleen cells were taken 11-12 days post-immunization from GP-MBP sensitized Lewis rats and were caltured at 5x10 6 cells/ml with 10 gg/ml GP-MBP. After 4 days, activated blasts were purified on Ficoll-Metrizoate and given 2500 R of gamma irradiation. Resting 12X CsLN-1 (passaged for 7 days in 1 r WO 89/01511 PCT/US88/02681 17 CAS) were combined with the antigen-activated blasts at a Sratio of 1 CsLN-1 cell to 6 GP-MBP blasts. CsLN-1 were put into culture at 2x10 5 /ml along with GP-MBP Th cells at 1.2x10 6 /ml, in the absence of antigen and APC, in complete media supplemented with 2.5% Lewis rat serum. After 3 days in culture, the activated CsLN-1 cells were purified on a Ficoll-Metrizoate gradient.

GP-MBP sensitized lymphocyte donors were female Lewis rats immunized 11 days earlier with 50 gg GP-MBP in CFA.

GP-MBP-sensitized lymph node cells and spleen cells were plated at 3x10 6 /ml in complete media supplemented with FCS, and GP-MBP at 2.5gg/ml in 80 cm 2 tissue-culture flasks. To test for suppression, GP-MBP Th cell-activated CsLN-1 or resting CsLN-1 (7 days in CAS) were added at a ratio of one T s cell to eight GP-MBP-sensitized lymphoid cells. Cells were cultured for 3 days at 37*C in 5% CO 2 Collected cells were injected i.p. into naive Lewis rats.

Recipients of control cells were given 5.7x10 7 viable cells; recipients of cells mixed with T s were given 6.7x10 7 viable cells. Clinical signs of disease were graded on the three-point scale, shown in Table 2.

As shown in Table 6, recipients of lymphocytes mixed with GP-MBP lymphoblast activated Ts cells during the in vitro antigen activation period had no signs of clinical disease. Resting CsLN-1, which were not activated with Th cells before the 3-day culture period with sensitized lymphocytes, did not suppress the passive transfer of disease. This data suggests that the CsLN-1 T s cells are activated by their cognate Th cells to effect their suppressor program.

I

WO 89/01511 PCT/US88/02681 18 TABLE 6 EFFECT OF GP-MBP Th CELL-ACTIVATED Tg CELLS ON THE ADOPTIVE TRANSFER OF EAE Incidence Mean Severity of EAE max Activated CsLN-1 0/5 0 Resting CsLN-1 2/2 2.75 Control 1/1 EXAMPLE VII INHIBITION OF PROLIFERATION OF SPLENOCYTES FROM MBP SENSITIZED LEWIS RATS WITH SUPERNATANT FROM ACTIVATED CsLN-1 Spleen cells were taken from Lewis rats at day eleven post sensitization and incubation with 2 Ag/ml MBP for 48 hours. 0.5 mg 3 H-TdR was added and the cells incubated for an additional 18 hours prior to harvesting. Stimulation index CPM (antigen)/CPM (no antigen). Results are presented in Table 7, which shows the effect of the supernatant on the proliferative response of splenocytes from MBP sensitized rats. The response of these splenocytes to antigen is considerably reduced in the presence of the supernatant.

WO 89/01511 PCT/US88/02681 19 TABLE 7 INHIBITION OF PROLIFERATIVE RESPONSE OF SPLENOCYTES OF MBP SENSITIZED LEWIS RATS SENSITIZED FROM ACTIVATED CsLN-1 CPM S.I.

Splenocytes (no antigen) 2,112 Splenocytes MBP 10,892 5.2 Splenocytes MBP supernatant 3,846 1.8 EXAMPLE VIII INHIBITION OF PROLIFERATION OF A P 2

-SPECIFIC

T-CELL LINE WITH SUPERNATANT FROM ACTIVATED CsLN-1 The non-specific nature of the suppressor factor was shown using a T cell line responsive to the P 2 protein from bovine PNS myelin, a protein with no sequence homology to the guinea pig myelin basic protein used to establish CsLN- 1 or to produce EAE. Using the P 2 -2A cell line, the supernatant of the CsLN-1 cell line could inhibit the antigen specific proliferation of P 2 -2A in a dose related manner, (as shown in Table P 2 -2A is a P 2 protein specific cell line that produced EAN if activated in culture by P 2 protein or a synthetic peptide representing residues 57 through 81 of the P 2 protein sequence, (Brostoff, et al., Faseb Journel, 2:A476, 1988). When half of the culture medium in which the P 2 -2A cells are activated was replaced with tissue culture medium from CsLN-1 cells activated with MBP, an inhibition of the antigen specific proliferation of P 2 -2A cells greater than as measured by tritiated thymidine incorporation, was achieved. Correspondingly less inhibition was achieved with lower proportions of supernatant. Control li l WO 89/01511: PCT/US88/02681 supernatants from activated T helper cell lines specific for either P 2 protein (P 2 -2A) or MBP (TO-1) did not show any inhibition of the proliferative response indicating that the suppression was not due to the use of depleted media or the presence of a cell waste product.

TABLE 8 Inhibition of P 2 -2a Cell Line Proliferation With Supernatants From Activated Cell Lines Antigen Cell Line* Supernatant (Percent) Stimulation Index P2 (24M) 57-81 (2MM) 57-81 (2AM) 57-81 (2MM) 57-81 (2MM) 57-81 (2MM) 57-81 (1IM) 57-81 (1M) CsLN-1 (50%) CsLN-1 (25%) CsLN-1 (12.5%) CsLN-1

P

2 -2A (50%) TO-1 (50%) 29.0 27.0 12.7 22.0 30.0 25.0 26.0 All cell lines were activated with their specific antigen for 3 days and the cell free tissue culture supernatant used to replace the noted proportions of complete RPMI 1640 medium in the proliferation assay.

WO 89/01511 PCT/US88/02681 21 EXAMPLE IX TIME COURSE FOR THE PRODUCTION OF THE SOLUBLE SUPPRESSIVE FACTOR A time-course for the production of the soluble suppressive activity was first performed to assess when production is maximal. The cell-free tissue culture supernatant (TCS) was collected at 0, 24, 48 and 72 hours after the initiation of the activation culture. The collected TCS (50% vol/vol) was then added into a proliferation assay with the P 2 -2A cell line, P 2 peptide (2gM), and APC (as previously described). As shown in Table 9, the production of a soluble suppressive activity by CsLN-1 cells is maximal at 48-72 hours post-activation with antigen. Of special note is the result obtained with the CsLN-B TCS. The CsLN-B TCS was collected from GP-MBP specific lymphoblast activated CsLN-1 cells. Under these conditions, there was also the production of a soluble suppressive activity.

TABLE 9 Time Course for the Production of a CsLN-1 Derived Soluble Suppressive Activity Antigen Time of Collection Stimulation of CsLN-1 tcs Index

P

2 57-81 23.0

P

2 57-81 0 hours 22.0

P

2 57-81 24 hours P2 57-81 48 hours

P

2 57-81 72 hours P2 57-81 CsLN-B 72 hours WO 89/01511 PCT/US88/02681 22 EXAMPLE X CsLN-1 SUPPRESSION OF EAN PRODUCED 3Y CELL LINE P 2 -2A Not only did supernatant from activated CsLn-1 inhibit the proliferation of the P 2 specific cells, the activated CsLN-1 cells themselves inhibited the ability of the P 2 specific T cells to transfer EAN to naive recipients. The P2 specific cell line P--2A produced EAN if activated in culture with either P 2 protein or a synthetic peptide representing residues 57-81 of the P 2 protein sequence. As shown in Table 10, the presence of CsLN-1 in culture during the activation of P 2 -2A markedly inhibited the ability of

P

2 -2A to produce EAN in recipient rats. All rats receiving cell line P 2 -2A alone developed hind leg paralysis.

Although the rats recovered somewhat after the sixth day, in that they had motor control of their hind legs and tails, a residual hind leg weakness was present until they were sacrificed on day 14 after transfer. In contrast, P 2 2A cells cultured in the presence of CsLN-1 produced clinical signs of disease (very mild hind leg weakness) in only one of four recipient rats. This animal recovered after only two days. None of the other animals showed any evidence of clinical disease.

-r WO 89/01511 PCT/US88/02681 23 TABLE Effect of CsLN-1 Cells on Clinical Experimental Allergic Neuritis Produced by Cell Line P 2 -2A* Presence of Incidence of Disease Disease CsLN-1 in Clinical EAN Severity Duration Culture (max=3.0) in Days No 3/3 3.0 6+ Yes 1/4 2.0 2

P

2 -2A (2x10 5 /ml) cells were activated for 3 days in the presence or absence of activated CsLN-1 cells (2x10 5 /ml).

P

2 -2A cells activated in the presence of CsLN-1 were supplemented with 2,g/ml MBP. Each rat received 6x10 7 cells from the culture containing P 2 -2A cells activated in the presence of CsLN-1 or 3x107 P 2 -2A cells activated in the absence of CsLN-1.

EXAMPLE XI SUPPLEMENTATION OF T s CELL LEVELS Individuals having an immune mediated condition in which the immune response to a given antigen is elevated beyond the control of the host's own regulatory system can be treated by T s cell supplementation. In an autoimmune disease such as, for example myasthenia gravis, in which a reaction to acetylcholine receptor causes an autoimmune response, lymphocytes are removed from the patient and a T s cell line specific to acetylcholine receptor is generated using the method described in Example 2. After the requisite number of cycles to establish the suppressor line (which is determined by comparing its stimulation to antigen with its stimulation by a polyclonal lymphocyte activator such as Concanavalin suppressor cells are readministered intraperitoneally to the individual.

Collection of lymphocytes from individuals is performed by WO 89/01511 PCT/US88/02681 24 lymphocytapheresis, for example, whereby 12 liters of whole blood can be processed to produce as much as 5 x 10 10 mononuclear cells in four hours. (Rosenberg, et al., New England J. Med., 313:1486 (1985) which is incorporated herein by reference). Infusion of the T s cell line cells back into the patient would be at doses of between 106 and 1012 cells per daily infusion, which are repeated as necessary until the desirable immune condition is ablated.

Although the invention has been described with reference to the presently preferred embodiment, it should be understood that various modifications can be made by those skilled in the art without departing from the invention.

Accordingly, the invention is limited only by the following claims.

Claims (16)

1. A method of culturing antigen specific suppressor T cell lines which retain their ability to produce non-antigen specific, soluble suppression factor, said method comprising the steps of: a. Recovering lymphocytes from animals having an elevated level of T s cells, as hereinbefore defined, directed at said antigen; b. Growing said lymphocytes in the presence of said antigen and factors whichi inibit Th cell, as hereinbefore defined, proliferation without affecting T s cell proliferation; and c. Alternately culturing said lymphocytes in a medium containing lymphocyte growth factors and activating said lymphocytes by S exposing them to said antigen.

2. The method of claim 1 wherein said elevated level of T Scells results from the presence of an autoimmune disease in said animal. 15 3. The method of claim 2 wherein said autoimmune disease is Th mediated.

4. The method of claim 2 wherein said autoimmune disease is in a period of remission.

5. The method of claim 1 wherein said elevated T cell level 20 results from prior immunization with said antigen.

6. The method of any one of claims 2 to 4 wherein said autoimmune I disease is selacted from the group consisting of myasthenia gravis, lupus erythromatosis, rheumatoid arthritis, multiple sclerosis, Type one diabetes, Crohn's Disease, Guillain Barre Syndrome, acute disseminated o ILMMI567Z VM\,9 f*ll f t 'L WO 89/01511 PCT/US88/02681 encephalomyelitis, acute necrotizing hemorrhagic encephalomyelitis, chronic relapsing idiopathic polyneuritis, and chronic progressive idiopathic polyneuritis.

7. The method of claim 1 wherein said factor which inhibits Th cell proliferation is cyclosporin A.

8. The method of claim 1 wherein said lymphocyte growth factors are derived from the supernatant of cells stimulated with Concanavalin A.

9. The method of claim 1 wherein said animal is a rat. The method of claim 1 wherein said animal is a human.

11. T s cell lines produced by the method of claim 1.

12. A composition characterized by its ability to non- specifically suppress the activity of effector T lymphocytes, derived from the supernatant of cell cultures generated according to the method of claim 1. Cks lnecer 'Aore de~in>ea,

13. A method of supplementing a host's T s cell population comprising the steps of: a. lymphocytes; Recovering a sample of said host's b. Growing said lymphocytes in the presence of -a qr heen i T rolirertin ewithut factor which inhibits Th cell) proliferation without 27 affecting T s cell proliferation; c, Alternately culturing said lymphocytes in a medium containing lymphocyte growth factors and activating said lymphocytes by exposing them to said antigen, whereby a culture of antigen-specific T s cells is established; and d. Reintroducing said T cells into said host.

14. The CsLN-1 T cell line designated ATCC #CRL 9495. A method of supplementing a human host's T cell, as hereinbefore defined, population, which method is substantially as hereinbefore described with reference to Example XI.

16. The method of claim 15 wherein said supplementing of the human host T cell population is for the treatment of an autoimmune disease selected from myasthenia gravis, lupus 1 erythromatosis, rheumatoid arthritis, multiple sclerosis, Type Sone diabetes, Crohn's Disease, Guillain Barre Syndrome, acute disseminated encephalomyelitis, acute necrotizing hemorrhagic :encephalomyelitis, chronic relapsing idiopathic polyneuritis, and chronic progressive idiopathic polyneuritis.

17. A method of culturing specific suppressor T cell lines, which method is substantially as hereinbefore described S with reference to Example II. DATED this TWENTY-EIGHTH day of JUNE 1991 S• Medical University of South Carolina Patent Attorneys for the Applicant SPRUSON FERGUSON /567Z INTERNATIONAL SEARCH REPORT International Atipllcat io PCT/JS 88 /02681 1. CLASSIFICATION OF SUBJECT MATTER (if 3everal classification symocis oddly. indicate all) 3 According to International Patent Classification (IPC) or to bo03 Nitlonfil Classification and IPC IPC(4): C12N 5/00, A61K 35/26 rT-q l 424/Q'3, 424/95, 4351240.2. 435/240.21 11. FIELDS SEARCHED Minimum Documentation Searched 4 Classification System IClassification Symbols U.S. 435/24o..1, 240.2, 240.21, 68, 9A48 424/93-i 95; 5f'4/21, 885,903 Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched 6 Chemical Abstracts (CAS) 1967-19881; Biological Abstracts Data BASE (BIOSIS) 1967-1988 jr APS 1975-1988 (See Attachment for Search Terms). 11l. DOCUMENTS CONSIDERED TO U1 RELEVANT 1 Category *j Citation of ocument, I I with indication, where appropriate. of the relevant paasages Relevant -o Claim No. to P Nature (London, England), Volume 331, 1-9, 21 January 1988, Ellerman et al. il) 13- Suppressor T-lymphocyte cell line for autoimmune encephalomyelitis" p. 265-

267. Entire document. X Journal of Experimental Medicine, ai ,12,14 y (New York, New York,) Volume 159, .1-10 January 1984, Bogen et al, "In- duction of Acetylcholine Receptor-Specific suppression", p.

292-304, Entire document. Y Journal of Immunology (Baltimore, 1-10, Maryland) Volume 133, July_1984, 114 "Ting et al, "Induction of Suppressor T cells by Interleukin 2" p. 261-266, See Discussion Section, p. 264-265. Special categories of cited documents: As later document published after the international filing date document defining the general state of the art which is not or priority date and not in conflict with the application but cosdrdto be of particular relevance ciled to understand the principle or theory underlying the con~ieredinvention earlier document but publishied on or after the international "X document of particular relevance: the claimed invention filing date cannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an inventive step which Is Cited to establish the Publication date of another document of particular relevance; the claimed invention citation or other special reason (as specified) Cannot be considered to involve an inventive step when the document referring to an oral diactosure. use, snibitlon or document is combined with one or more other such doacu. Other means ments. such combination being obvious to a person skilled 11P' document published prior to the International filing date but in the amt later than the priority date claimed 6 document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the Intrnational Ssarcn D ats of Mailing of this International Search Raponrt O ~CT 988 12 DEC 1988 International Searching Authority Signature a$ Authorized 0 leerip I-ISA/US 6hi F4(Kntox Form PCTIISA/21o (scond shieet) (October 1981) *1 ~1 International Application No. PCT/US 8 8 0 2 68 1 111. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) Category Citation ot Document, with indication, where appropriate, of the relevant passages Relevant to Claim No Y x Y Journal of Biological Response Modifiers, (New York, New York), Volume 3, 1984, Rosenberg, "Immunotherapy of Cancer by qystemic Administration of Tymphoid cells plus Interleukin- 2"1, p. 501-511. Entire document. A, 4,728,614 Mrau) 1 March 1988, see entire document Basic and Clinical Immunology, (rOS Atos, California), 1984, Webb et al, "Immunosuppression, Immunopotentiation, and Nnti- inflammatory Drugs", p. 271-287, qee pages 278-279, section C. 13 11, 12 1-10,. 14 Form PCTIlSW2O (extra shoo) (Rev.1 1-87) PCT/US88/0268l ATTNCHMENT TO FORM PCT/ISA/210 Part II. Fields Searched Search Terms: 1. Suppressor T cell 2. Cyclosporin A 3. Guinea Pig Myelin Basic Protein 4. Antigen Immunization 6. Nutoimmune Disease 7. Culture 8. FRuman