WO2006081619A1

WO2006081619A1 - Improving survival and proliferation of cd4+ and cd25+ t cells

Info

Publication number: WO2006081619A1
Application number: PCT/AU2006/000132
Authority: WO
Inventors: Bruce Milne Hall; Suzanne Jean Hodgkinson
Original assignee: Newsouth Innovations Pty Limited
Priority date: 2005-02-02
Filing date: 2006-02-02
Publication date: 2006-08-10

Abstract

The invention relates to a method of growing CD4+,CD25+ T cells in vitro, comprising culturing CD4+,CD25+ T cells under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4+,CD25+ T cells. The invention also relates to methods of increasing tolerance in a subject by administering CD4+, CD25+ T cells grown in vitro.

Description

IMPROVING SURVIVAL AND PROLIFERATION OF CD4+ AND CD25+ T CELLS

The invention relates to a method for growing CD4⁺, CD25⁺ T cells , to methods for increasing immune tolerance in a subj ect, and to methods for assessing whether a subj ect comprises CD4⁺, CD25⁺ T cells that have been activated with a specific antigen .

BACKGROUND OF THE INVENTION

The immune system provides a mechanism to protect the body against infection by foreign entities such as infectious organisms or foreign antigens . Under normal conditions , the immune system is capable of recognising and eliciting an immune response against foreign entities , while largely- ignoring host tissue . The ability of the immune system to ignore the host' s tissue is known as immune tolerance . Immune tolerance also refers to a state where the immune system is adapted to ignore antigens such as transplanted foreign tissues , infected tissues , allergenic substances or malignant tissues .

It would be advantageous to be able to induce tolerance in a subj ect as the ability to induce tolerance is needed in, for example, the treatment of subj ects with autoimmune or allergic disease , or to prevent rej ection following tissue transplantation . Autoimmune disease occurs when T-cells recognise and react to "self" molecules , that is , molecules produced by the cells of the host . This occurs when specific self molecules interact with proteins on the surface of T-cells such that the T-cells recognise the molecule as foreign and consequently elicit an immune response against the self molecule . In tissue transplantation, non-self maj or histocompatibility antigen present on the foreign tissue contacts the surface of T- cells , resulting in T-cell activation against the foreign antigen . This activation ultimately results in allograft or xenograft rej ection by the immune system. The ability to suppress the immune response to "self" molecules in a person having an autoimmune disease by inducing tolerance to the self molecules, or to suppress the immune response to transplant tissue by inducing tolerance to antigens present on the transplant tissue, would be highly desirable .

Present methods for preventing allograft rej ection, or for treating autoimmune disease , typically cause a general immunosuppression that is not specific for a specific antigen or antigens . As a result , the subj ect is rendered susceptible to infection from pathogenic and opportunistic organisms , and may be at an increased risk of malignancy . The more specific immunosuppressive drugs such as cyclosporin A, steroids , azathioprine , anti-T-cell antibodies , rapamycin, mycophenolate mofetil, desoxyspergualine and FK506, typically have undesirable side-effects, and typically require that the subj ect be administered the drugs for life or at least extended periods of time, thereby placing the subj ect at considerable risk of infection, cancer, and/or other conditions due to long term effects of the treatment . For example, long term use of high doses of steroids can result in weight gain, osteoporosis , fluid retention and thinning of skin . Long term use of azathioprine may lead to an increased risk of skin cancer . Prolonged use of mycophenolate mofetil can cause stomach upsets , bleeding and increased bruising and cancer . Cyclosporin A may cause tremor, excessive hair growth, headaches , and increase the risk of tumours . It would be advantageous to provide alternative methods for preventing allograft rejection, and for treating auto-immune disease .

SUMMARY OF THE INVENTION

CD4⁺, CD25⁺ T cells (or CD4⁺, CD25⁺ lymphocytes ) are a subpopulation of CD4⁺ T cells . CD4⁺, CD25⁺ T cells suppress the activity of T cells that elicit an immune response to an antigen . CD4⁺, CD25⁺ T cells activated to an antigen are capable of imparting to cells of the immune system, including CD4⁺, CD25^" T cells , tolerance to the specific antigen to which the activated CD4⁺, CD25⁺ T cells have been activated. Accordingly, immune tolerance to an antigen may be induced or increased in a subj ect by administering CD4⁺, CD25⁺ T cells that have been activated to the antigen . It would therefore be advantageous to be able to grow in vitro CD4⁺, CD25⁺ T cells .

The inventors have found that when naϊve CD4⁺, CD25⁺ T cells are activated by contacting the CD4⁺, CD25⁺ T cells with an antigen in the presence of cytokines capable of supporting activation of CD4⁺, CD25⁺ T cells , and are cultured in vitro, the cells typically undergo proliferation for a short period of time (typically 24 hrs to 1 week) , after which they are less capable of proliferation . This can significantly limit the amount of growth of activated CD4⁺, CD25⁺ T cells that can be obtained .

In a first aspect, the invention provides a method of growing CD4⁺, CD25⁺ T cells in vitro, comprising culturing

CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T cells .

Typically, the effect of nitric oxide production is inhibited by culturing the CD4⁺, CD25⁺ T cells in the presence of at least one nitric oxide inhibitor .

In one embodiment, the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one factor capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and under conditions which inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T cells . The at least one factor capable of supporting activation, survival and/or proliferation of the CD4⁺, CD25⁺ T cells is typically a cytokine .

In one embodiment , the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one antigen to activate the CD4⁺, CD25⁺ T cells . The CD4⁺, CD25⁺ T cells may be cultured in the presence of at least one antigen prior to, or simultaneously with, culturing the CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide on the CD4⁺, CD25⁺ T cells . The CD4⁺, CD25⁺ T cells may be cultured in the presence of at least one antigen simultaneously with culturing the CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide on the CD4⁺, CD25⁺ T cells . Typically, the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one antigen and a nitric oxide inhibitor . The CD4⁺, CD25⁺ T cells may be cultured in the presence of at least one antigen, at least one nitric oxide inhibitor, and at least one factor capable of supporting activation, survival and/or proliferation of the CD4⁺, CD25⁺ T cells .

The inventors have found that by culturing CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide on the CD4⁺, CD25⁺ T cells , for example, in the presence of a nitric oxide inhibitor, growth of CD4⁺, CD25⁺ T cells in vitro can be enhanced . Without wishing to be bound by theory, the inventors believe that upon activation of CD4⁺, CD25⁺ T cells , these cells produce nitric oxide . The inventors further believe that nitric oxide production by CD4⁺, CD25⁺ T cells contributes to the death, or at least a reduction in proliferation, of these cells in culture .

In a second aspect, the invention provides a method of growing CD4⁺, CD25⁺ T cells in vitro, comprising culturing CD4⁺, CD25⁺ T cells in the presence of at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells, and at least one nitric oxide inhibitor .

In one embodiment, the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one antigen . The CD4⁺, CD25⁺ T cells may be cultured in the presence of at least one antigen prior to, or simultaneously with, culturing the CD4⁺, CD25⁺ T cells with the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells and at least one nitric oxide inhibitor .

In one embodiment , the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells is at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , IGF-β and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

Typically, IL-12 is IL-12p70.

In one embodiment , the at least one factor capable of supporting activation, survival and/or proliferation of

CD4⁺, CD25⁺ T cells is IL-2 , a biologically active fragment thereof, or a functionally equivalent molecule thereof .

In yet another embodiment, the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells is one or more of IL-4 , IL-5 , IL-IO , IL- 12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β or IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

In a third aspect , the invention provides a method of growing CD4⁺, CD25⁺ T cells in vitro , comprising culturing naϊve CD4⁺, CD25⁺ T cells in the presence of at least one antigen and IL-2 , a biologically active fragment thereof or functionally equivalent molecule thereof, and a nitric oxide inhibitor .

In one embodiment , the method comprises :

(a) culturing naive CD4⁺, CD25⁺ T cells in vitro in the presence of at least one antigen, IL-2 , a biologically active fragment thereof, or a functionally equivalent molecule thereof, and a nitric oxide inhibitor; (b) thereafter culturing the CD4⁺, CD25⁺ T cells in the presence of IL-2 , a biologically active fragment thereof, or a functionally equivalent molecule thereof , in the absence of a nitric oxide inhibitor .

In another embodiment , the method comprises :

(a) culturing naive CD4⁺, CD25⁺ T cells in vitro in the presence of at least one antigen, IL-2 , a biologically active fragment thereof, or a functionally equivalent molecule thereof , and a nitric oxide inhibitor; and

(b) thereafter culturing the CD4⁺CD25⁺ T cells in the presence of at least one cytokine selected from the group consisting of IL-2, IL-4 , IL-5, IL-IO, IL-12, IL-13, IL-15 , IL-18 , IL-23 , TGF-β and IFN-γ, a biologically active fragment thereof, or functionally equivalent molecule thereof .

In a fourth aspect, the invention provides a method of growing CD4+, CD25+ T cells in vitro, comprising culturing activated CD4⁺, CD25⁺ T cells with the antigen to which the CD4⁺, CD25⁺ T cells have been activated, in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5, IL-IO, IL-12 , IL-13, IL-15 , IL-18 ,

IL-23 , TGF-β and IFN-γ, a biologically active fragment thereof, or functionally equivalent molecule thereof, and a nitric oxide inhibitor .

In one embodiment, the method comprises :

(a) obtaining from a subj ect a sample of CD4⁺, CD25⁺ T cells that have been activated to a specific antigen in the subj ect; and (b) culturing the activated CD4⁺, CD25⁺ T cells in the presence of the specific antigen and one or more cytokines selected from the group consisting of IL- 2 , IL-4 , IL-5 , IL-IO , IFN-γ, IL-12 , IL-13 , IL-15 , IL-23 , TGF-β and IL-18 , a biologically active fragment thereof, or a functionally equivalent molecule thereof , and a nitric oxide inhibitor .

The nitric oxide inhibitor may be any agent or substance which inhibits the effect of nitric oxide on the CD4⁺, CD25⁺ T cells . The nitric oxide inhibitor may operate by any mechanism which inhibits the effect of nitric oxide on the CD4⁺, CD25⁺ T cells . The nitric oxide inhibitor may inhibit the effect of nitric oxide by inhibiting nitric oxide production during culturing of the cells . For example , the nitric oxide inhibitor may inhibit nitric oxide production by inhibiting enzymes which produce nitric oxide in the CD4⁺, CD25⁺ T cell . The nitric oxide inhibitor may inhibit nitric oxide production by blocking expression of enzymes which produce nitric oxide in the CD4⁺, CD25⁺ T cell . The nitric oxide inhibitor may inhibit enzymes which produce nitric oxide in the CD4⁺, CD25⁺ T cell by inhibiting the activity of those enzymes .

In one embodiment , the nitric oxide inhibitor is an inhibitor of nitric oxide synthase inhibitor . Typically, the nitric oxide synthase inhibitor is an iNOS inhibitor . Examples of suitable iNOS inhibitors include L-NIL (N6- ( l- Iminoethyl ) -L-lysine ) , L-NAME (N-nitro-L-arginine-methyl ester) , aminoguanidine, GDIPS , FeTPPS , N- ( 3- aminomethyl) benzyl ) acetamidine dihydrochloride . Typically, the iNOS inhibitor is L-NIL . - ci - In another embodiment, the nitric oxide inhibitor inhibits nitric oxide production by blocking nitric oxide synthase expression in the CD4⁺, CD25⁺ T cells . Examples of molecules which may block nitric oxide synthase expression include antisense DNA or RNA molecules , iRNA and siRNA molecules which are capable of hybridising to nucleic acid encoding nitric oxide synthase, typically iNOS .

The nitric oxide inhibitor may be a nitric oxide antagonist, that is a compound that reduces or blocks the interaction between nitric oxide and parts of the cells that are capable of interacting with nitric oxide . For example , the nitric oxide antagonist may sequester nitric oxide in the cell so that access of the nitric oxide to parts of the cell capable of interacting with nitric oxide is reduced or eliminated . The nitric oxide antagonist may convert the nitric oxide to an inactive molecule . The nitric oxide antagonist may bind nitric oxide to thereby prevent interaction between nitric oxide and the CD4⁺, CD25⁺ T cells .

In those embodiments of the present invention where CD4⁺, CD25⁺ T cells are cultured in the presence of at least one antigen, the CD4⁺, CD25⁺ T cells may be cultured in the presence of the at least one antigen in any manner which presents the at least one antigen to the T cells in a form which will permit the T cells to recognise the antigen . Typically, the antigen is an antigen located on the surface of an antigen presenting cell . Typically, the antigen is associated with a class II maj or histocompatibility molecule on the surface of the antigen presenting cell . The antigen presenting cell may be any cell that expresses an antigen presenting molecule (typically class II MHC) and, typically, ligands required to facilitate activation of the CD4⁺, CD25⁺ T cells . Examples of ligands include ICAMl , ICAM2 , LFA3 , ligands for CD28 and CTLA or any activation ligands . Examples of antigen presenting cells include dendritic cells , phagocytes , B-lymphocytes , Langerhans cells or unfractionated lymphocytes in which the proliferation of the stimulator cells is impaired (for example, by irradiation or mitomycin C treatment) .

Typically, the CD4⁺, CD25⁺ T cells are cultured by incubating the cells in a medium containing at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and the nitric oxide inhibitor . The at least one factor capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25^~ T cells may be added to the medium from an exogenous source . The at least one factor capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25^~ T cells may be purified protein, recombinant or otherwise . Alternatively, the CD4⁺, CD25⁺ T cells may be incubated in the presence of cells which express the at least one factor capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25^~ T cells . The medium may comprise growth factors , nutrients and/or buffers . The nitric oxide inhibitor is typically added from an exogenous source .

The CD4⁺, CD25⁺ T cells may in addition be incubated with an antibody which reduces proliferation of CD4⁺, CD25^~ T cells . Proliferation of CD4⁺, CD25^" T cells may be reduced, for example , by incubating the T cells in the presence of one or more antibodies selected from the group consisting of anti-CD3 , anti-CD45RB/RO or any other antibody which specifically binds to CD4⁺, CD25^~ T cells . The CD4⁺, CD25⁺ T cells may be incubated with the antibody prior to, with, or subsequent to culturing the CD4⁺, CD25⁺ T cells .

The antigen may be any antigen . When the CD4⁺, CD25⁺ T cells consist of or include activated CD4⁺, CD25⁺ T cells , the antigen may be an antigen to which the activated CD4⁺, CD25⁺ T cells have been activated .

In one embodiment, the antigen is an autoantigen of a subj ect and the subj ect has an autoimmune disease or condition .

In another embodiment , the antigen is an alloantigen, for example, an antigen of allograft tissue following, or prior to, an allograft to a subj ect , or in other words , a transplant of tissue to the subj ect from a member of the same species .

In yet another embodiment, the antigen is a xenoantigen, for example, an antigen of xenograft tissue following, or prior to, a xenograft to a subj ect, or in other words , a transplant of tissue to the subj ect from a species different to that of the subj ect .

In some embodiments , the antigen is an allergen or part thereof .

In some embodiments , the antigen is a self antigen .

In a fifth aspect , the invention provides a method of increasing tolerance in a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of CD4⁺, CD25⁺ T cells grown in vitro by the method of the first, second, third or fourth aspects of the invention .

In a sixth aspect , the invention provides a method for increasing tolerance in a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of CD4⁺, CD25⁺ T cells grown in vitro by the method of the first , second, third or fourth aspects of the invention .

In one embodiment of the fifth or sixth aspects of the present invention, tolerance may be further increased by reducing the number of CD4⁺ T cells , typically the number of CD4⁺, CD25^~ T cells , in the subj ect prior to administering the CD4⁺, CD25⁺ T cells . The CD4⁺ T cells may be reduced in number by any methods known in the art . In some embodiments , the number of CD4⁺, CD25^~ T cells is reduced by reducing all lymphocytes , including CD4⁺ T cells , for example , by irradiation in various forms including whole body irradiation or total lymphoid irradiation . In some embodiments , the number of CD4⁺ T cells is reduced by administering to the subj ect antibodies which bind to CD4⁺ T cells , typically to

CD4⁺, CD25^~ T cells . Suitable antibodies include one or more antibodies selected from the group consisting of anti-CD3 , anti-CD4 , anti-CD45RB/RO , anti-lymphocyte globulin or anti-thymocyte globulin . In embodiments where the CD4⁺ T cells are reduced by administering antibodies that bind to CD4⁺ T cells , the antibodies may be subsequently removed or inactivated . Methods for removal or inactivation of antibodies include antiidiotype antibodies , soluble CD4 ligand, or antibodies against the treating antibody or any other technique that removes or neutralizes the treating antibody .

In one embodiment , the CD4⁺, CD25⁺ T cells cultured in the presence of at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and the nitric oxide inhibitor, and are then further cultured in the presence of at least one factor selected from the group consisting of IL-2 , IL-4 , IL-5 ,

IFN-γ, IL-12p70 , IL-IO , IL-13 , IL-15 , IL-18 , IL-23 and TGF- β , or a biologically active fragment thereof, or a functionally equivalent molecule thereof , in vitro, prior to administration to the subj ect . Typically, the CD4⁺, CD25⁺ T cells are further cultured in the presence of at least one factor selected from IL-2 , IL-5, IFN-γ, IL-13, IL-23 and IL-12 , a biologically active fragment thereof, or a functionally equivalent molecule thereof , more typically IFN-γ or IL-2 , or a biologically active fragment thereof, or a functionally equivalent molecule thereof .

In one embodiment , the method comprises the further step of administering to the subj ect an effective amount of at least one factor selected from the group consisting of IL- 2 , IL-4 , IL-5 , IFN-γ, IL-12p70 , IL-10 , IL-13 , IL-15 , IL-18 IL-23 and TGF-β, or a biologically active fragment thereof, or a functionally equivalent molecule, thereof , prior to, simultaneously with, or subsequent to, administering the CD4⁺, CD25⁺ T cells . Typically, IL-2 , IFN-γ, and/or IL- 12p70 , a biologically active fragment thereof, or functionally equivalent molecule thereof , is administered prior to, simultaneously with, or subsequent to, administering the CD4⁺, CD25⁺ T cells .

The effective amount of CD4⁺, CD25⁺ T cells may be any amount of T cells which increases tolerance in the subj ect .

In a seventh aspect, the present invention provides a composition comprising CD4⁺, CD25⁺ T cells activated to an antigen and/or pharmaceutically acceptable carrier wherein the CD4⁺, CD25⁺ T cells have been cultured in vitro by the method of the first , second, third or fourth aspects of the invention .

In an eighth aspect , the invention provides a method for treating or preventing in a subj ect in need thereof a disease resulting from an immune response to an antigen, the method comprising the step of administering to the subj ect a therapeutically effective amount of CD4⁺, CD25⁺ T cells activated to the antigen grown in vitro by the method of the first, second, third or fourth aspect of the invention .

The disease may be any disease resulting from an immune response to the antigen . In one embodiment, the diseases associated with an immune response to an autoantigen include autoimmune diseases . Examples of the types of autoimmune disease that may be prevented or treated using the method of the present invention include , for example, type 1 insulin dependent diabetes mellitis , inflammatory bowel syndrome including ulcerative colitis and Crohn' s disease, thrombotic thrombocytopenic purpura, Sj ogren' s syndrome, encephalitis , acute encaphaliomyelitis , Guillain Barre Syndrome, chronic inflammatory demyelination polyneuropathy, idiopathic pulmonary fibrosis/alveolitis , asthma, uveitis , iritis , optic neuritis , rheumatic fever, Reiter' s syndrome, psoriasis , psoriatic arthritis , multiple sclerosis , progressive systemic sclerosis , primary biliary cirrhosis , pemphigus , pemphigoid, necrotising vasculitis, myasthenia gravis, polymyositis, sarcoidosis , granulomatosis , vasculitis , pernicious anemia, CNS inflammatory disorder, autoimmune haemolytic anaemia, Hashitomo' s thyroiditis , Graves disease , habitual spontaneous abortions , Raynaud' s syndrome, dermatomyositis , chronic active hepatitis , celiac disease, autoimmune complications of AIDS , atrophic gastritis , ankylosing spondylitis , Addison' s disease, atopic dermatitis, glomerulonephritis including membranous nephropathy, focal sclerosing glomerulonephritis and minimal change nephropathy, systemic lupus erythematosis , scleroderma, rheumatoid arthritis , and j uvenile arthritis .

In another embodiment, the disease is the result of an immune response to a non-self antigen in contact with the subject . This may be the case following, for example, transplantation of tissue to the subj ect where the transplanted tissue undergoes rej ection by the immune system of the subj ect . Typically, the transplanted tissue is allograft or xenograft tissue .

Alternatively, the disease may be the result of an immune response to an allergen in contact with the subj ect . Examples of diseases resulting from an allergen include asthma, eczema, atopic dermatitis , anaphylaxis , hayfever, allergic conj unctivitis , contact dermatitis , food allergy, and drug or other chemical allergy, venom allergy, allergy to a fungus or other microorganism, or any other allergen . The invention also contemplates a kit for use with the methods of the invention . A kit for increasing tolerance , or for treating disease, may comprise one or more factors , such as cytokines , capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and a nitric oxide inhibitor . The factors may be selected from the group consisting of IL-2, IL-4 , IL-5, IL-IO, IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , IFN-γ and TGF-β, biologically active fragments thereof, or functionally equivalent molecules thereof . The nitric oxide inhibitor may be any of the nitric oxide inhibitors mentioned above .

The kit may further comprise at least one antigen in a form suitable for contacting CD4⁺, CD25⁺ T cells . For example, the kit may comprise at least one antigen on the surface of an antigen presenting cell . It will be appreciated by persons skilled in the art that the relevant part of the antigen may be incorporated into an appropriate MHC molecule on the surface of an antigen presenting cell .

In a ninth aspect , the invention provides a method of inducing tolerance in a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of at least one factor capable of supporting activation, survival and/or proliferation of CD4+, CD25⁺ T cells , and a nitric oxide inhibitor . Typically the factor is a cytokine capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , a biologically active fragment thereof, or a functionally equivalent molecule thereof . Typically the at least one factor is selected from the group consisting of IL-2, IL-4, IL-5, IL-IO, IL-12, IL-13, IL-15, IL-I 8,

IL-23 , IFN-γ and TGF-β a biologically active fragment thereof, or a functionally equivalent molecule thereof .

It is envisaged that administration of at least one factor selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , IFN-γ and TGF-β, and a nitric oxide inhibitor, will result in enhanced proliferation of CD4⁺, CD25⁺ T cells in vivo and will thereby increase tolerance in the subj ect .

The nitric oxide inhibitor may be administered simultaneously with, or subsequent to, administration of the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells . Suitably, the nitric oxide inhibitor is administered simultaneously with the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells .

The disease may be any disease resulting from an immune response to one or more antigens . In one embodiment, the disease is associated with an immune response to an autoantigen, for example an autoimmune disease . Examples of the types of autoimmune disease that may be prevented or treated using the method of the present invention include, for example , type 1 insulin dependent diabetes mellitis , inflammatory bowel syndrome including ulcerative colitis and Crohn' s disease, thrombotic thrombocytopenic purpura, Sj ogren' s syndrome, encephalitis , acute encaphaliomyelitis , Guillain Barre Syndrome, chronic inflammatory demyelination polyneuropathy, idiopathic pulmonary fibrosis/alveolitis , asthma, uveitis , iritis , optic neuritis , rheumatic fever, Reiter' s syndrome, psoriasis arthritis , multiple sclerosis , progressive systemic sclerosis , primary biliary cirrhosis , pemphigus , pemphigoid, necrotising vasculitis , myasthenia gravis , polymyositis , sarcoidosis , granulomatosis , vasculitis , pernicious anemia, CNS inflammatory disorder, autoimmune haemolytic anaemia, Hashitomo' s thyroiditis , Graves disease, habitual spontaneous abortions , Raynaud' s syndrome, dermatomyositis , chronic active hepatitis , celiac disease, autoimmune complications of AIDS, atrophic gastritis , ankylosing spondylitis , Addison' s disease, atopic dermatitis , eczema, glomerulonephritis including membranous nephropathy, focal sclerosing glomerulonephritis and minimal change nephropathy, systemic lupus erythematosis, scleroderma, rheumatoid arthritis, and j uvenile arthritis .

In another embodiment , the disease is the result of an immune response to a non-self antigen in contact with the subj ect . This may be the case following, for example, transplantation of tissue to the subj ect where the transplanted tissue undergoes rej ection by the immune system of the subj ect . Typically, the transplanted tissue is allograft or xenograft tissue .

Alternatively, the disease may be the result of an immune response to an allergen in contact with the subj ect . Examples of diseases resulting from an allergen include asthma, eczema, atopic dermatitis , anaphylaxis , hayfever, allergic conj unctivitis , contact dermatitis , food allergy, drug or other chemical allergy, venom allergy, allergy to microorganisms such as fungus or mites , or any other allergy to an allergen or part thereof . In a tenth aspect , the invention provides a kit when used with the method of the ninth aspect, the kit comprising at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and one or more nitric oxide inhibitors . Typically, the factor is a cytokine capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , a biologically active fragment thereof, or a functionally equivalent molecule thereof . Typically, the factor is one or more cytokines selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-

23 , TGF-β and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

Typically, the nitric oxide inhibitor is any one or more of those nitric oxide inhibitors mentioned above .

In an eleventh aspect , the invention provides a method of assessing whether a subj ect comprises CD4⁺, CD25⁺ T cells that have been activated to a specific antigen, comprising :

(a ) obtaining from the subj ect a sample of lymphocytes comprising CD4⁺, CD25⁺ T cells ; (b) incubating at least one portion of the sample of lymphocytes so as to promote distinction of CDA⁺, CD25⁺ T cells have been activated with the specific antigen from those that have not been activated to the specific antigen; (c) thereafter determining whether activated CD4⁺, CD25⁺ T cells activated to the specific antigen are present in the sample . In one embodiment, the at least one portion of the sample of lymphocytes is incubated in the presence of the specific antigen .

The inventors have found that CD4⁺, CD25⁺ T cells activated to an antigen produce nitric oxide, and that the production of nitric oxide may therefore be used as an indicator to detect whether CD4⁺, CD25⁺ T cells activated to a specific antigen are present in the sample .

In one embodiment, the at least one portion of the sample of lymphocytes is incubated in the presence of the specific antigen and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-12 , IL-13 , IL-18 , IL-23 and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof , and the presence of CD4⁺, CD25⁺ T cells activated to the specific antigen is determined by detecting nitric oxide production by the CD4⁺, CD25⁺ T cells .

In another embodiment, wherein the sample comprises CD4⁺, CD25⁺ T cells and CD4⁺, CD25^" T cells , the at least one portion of the sample is incubated in the presence of the specific antigen , a nitric oxide antagonist , and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-12 , IL-18 , IL-23 and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof , and the presence of CD4⁺, CD25⁺ T cells activated to the specific antigen is determined by detecting a decrease in proliferation of CD4⁺, CD25^" or an increase in proliferation of CD4⁺, CD25⁺ T cells .

In another embodiment, the at least one portion of the sample is incubated in the presence of the specific antigen, a nitric oxide antagonist and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-12 , IL-13 , IL-18 , IL-23 and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof, and the presence of CD4⁺, CD25⁺ T cells activated to the specific antigen is determined by detecting an increase in proliferation of the CD4⁺, CD25⁺ T cells .

The nitric oxide inhibitor may be any of the nitric oxide inhibitors as listed above .

The subj ect may be any subj ect which produces CD4⁺, CD25⁺ T cells . The subj ect may be a mammal . The mammal may be a human or non-human animal, such as rodent, non-human primate, cattle , pig, sheep, camel, goat, dog, cat or horse . Typically, the subj ect is a human .

BRIEF DESCRIPTION OF THE FIGURES

Figure IA is a graph of proliferation of naϊve unfractionated lymphocytes (dashed line and filled in circles ) , naϊve CD4⁺ T cell/lymphocytes (hard line, filled in circles ) , naϊve CD4⁺, CD25⁺ T cells (open circles thin line ) , and naϊve CD4⁺, CD25^" T cells (open circles thick line ) . Proliferation assayed at days 2 , 3 , 4 , 5 and 6 following contacting the lymphocytes with an alloantigen .

Figure IB is a graph showing a comparison of proliferation of naϊve CD4⁺ T cell lymphocytes , naϊve CD4⁺, CD25⁺ T cells and naϊve CD4⁺, CD25^" T cells in response to self antigen (black) , PVG antigen (donor antigen) (light grey) and Lewis antigen (third party antigen) (dark grey) .

Figure 1C is a graph of the proliferation (on y axis ) of serial dilutions ( shown on x axis ) of populations of naive CD4⁺ T cell lymphocytes (triangles ) , naϊve CD4⁺, CD25⁺ T cells ( squares ) and naϊve CD4⁺, CD25^~ T cells ( circles ) following contacting the lymphocytes with alloantigen .

Figure ID is a graph showing the effect on proliferation of mixing separate naϊve CD4⁺, CD25⁺ T cells with separate CD4⁺, CD25^" T cells over 6 days following contacting the lymphocytes with alloantigen . Mixtures are as indicated .

Figure IE are graphs showing proliferation of CD4⁺ T cells/lymphocytes , CD4⁺, CD25⁺ T cells and CD4⁺, CD25^~ T cells from DA rats tolerant to a PVG cardiac allograft following contacting the lymphocytes with self antigen (black) , donor antigen (PVG antigen - light grey) or third party antigen (Lewis - dark grey) .

Figure IF are graphs showing the results of a limiting dilution assay of unfractionated lymphocytes , fractionated CD4⁺ T cells/lymphocytes and fractionated CD4⁺, CD25^~ T cells from DA rats tolerant to a PVG cardiac allograft at day 4 following contacting the lymphocytes with self antigen (open circles ) , donor antigen (dashed line) or third party antigen (thick line) .

Figure 2A is a graph showing proliferation of naϊve CD4⁺, CD25⁺ T cells following contact with self antigen (white) or donor antigen (black) and incubation in the presence of cytokines as indicated . Figure 2B is a graph showing proliferation at day 3 of activated CD4⁺, CD25⁺ T cells against self antigen (black) or donor antigen (grey) in the presence of TGF-β (D) , IFN- γ (E) , IL-12 (p70) ( F) , IL-5 (G) or IL-IO (H) .

Figure 3A is a graph showing rej ection time of heart allografts in rats following whole-body irradiation and administration of various doses (as indicated at A to D) of naϊve CD4⁺ T cells/lymphocytes .

Figure 3B is a graph showing heart graft function up to 50 days post-transplantation in rats following administration of A, 5xlO⁶ naive CD4⁺ T cell lymphocytes (closed triangles ) ; B, 20 x 10⁶ naϊve CD4⁺ T cells (closed circles ) ; C, 5xlO⁶ naϊve CD4⁺, CD25^~ T cells (open triangles ) ; D, 0.5 x 10^s naϊve CD4⁺, CD25⁺ T cells plus 5 x 10^δ naϊve CD4⁺ T cells (open squares ) ; E , 5 x 10⁶ naϊve CD4⁺, CD25⁺ T cells plus 5 x 10⁶ naϊve CD4⁺ T cells (closed squares ) .

Figure 4A are graphs showing the effect of IL-2 (upper) and IL-4 ( lower) on proliferation of naϊve CD4⁺, CD25⁺ T cells alone (black) , in contact with self antigen ( cross- hatch) or in contact with alloantigen ( shaded) .

Figure 4B is a graph showing the effect from day 3 to day 6 on proliferation of naϊve CD4⁺, CD25⁺ T cells cultured with alloantigen and no cytokines (dark shading) , IL-2 (mid-shading) or IL-4 ( light shading) .

Figure 4C shows the results of semi-quantitative RT-PCR using primers to cytokines or cytokine receptors (as indicated) on mRNA isolated from naϊve CD4⁺, CD25⁺ T cells cultured in the presence of self or alloantigen and IL-2 or IL-4 as indicated .

Figure 4D shows the results of semi-quantitative RT-PCR using primers to cytokines or cytokine receptors (as indicated) on mRNA isolated from CD4⁺, CD25⁺ T cells from DA rats tolerant to PVG allografts in the presence of self antigen ( DA) , donor antigen (PVG) or third party antigen (Lewis ) in the presence of IL-2 or IL-4.

Figure 4E shows the results of semi-quantitative RT-PCR using primers to the IL-5 receptor alpha chain or to GADPH on mRNA isolated from naive CD4⁺, CD25⁺ T cells incubated in the presence of either self stimulators or PVG stimulators , alone or with either IL-2 or IL-4.

Figure 4F is a graph showing the results of real-time RT- PCR using primers to the IFN-γ receptor or to GADPH on mRNA isolated from naive CD4⁺, CD25⁺ T cells incubated in the presence of self or alloantigen, and with no cytokine or with either IL-2 or IL-4.

Figure 5A shows the results of semi-quantitative RT-PCR using primers to cytokine mRNA isolated from CD4⁺, CD25⁺ T cells and CD4⁺CD25^" T cells alone or admixed in a 1 : 1 ratio . All populations were from naive DA rats and were incubated in the presence of self or alloantigen .

Figure 5B are graphs showing proliferation of CD4⁺, CD25⁺ T cells , CD4⁺, CD25^" T cells and CD4⁺, CD25^" T cells admixed with CD4⁺, CD25⁺ T cells in a ratio of 1 : 1 following incubation in the presence of self antigen (black) or alloantigen (grey) . The effects of L-NIL, and antibodies to block IL-5, TGF-β or IL-IO were compared to a control antibody Mog-Ig2a .

Figure 6A are graphs showing the survival of heterotopic cardiac allografts transplanted from PVG rats or Lewis rats into irradiated DA rats that were adoptively restore with CD4⁺ T cells from DA rats tolerant to a PVG heart allografts where the cells had been cultured for 3 days with PVG stimulator cells in media supplemented with either IL-4 (dotted line ) or IL-5 (hard line) .

Figure 6B are graphs showing the proliferation of CD4⁺, CD25⁺ T cells and CD4⁺, CD25^" T cells from DA rats tolerant to PVG allografts following incubating the lymphocytes with PVG antigen in the presence or absence of IL-4 (as indicated) , followed by incubating the cells alone (white) , or in the presence of self antigen ( DA) ( dark grey) , donor antigen ( PVG) (black) or third party antigen (Lewis ) (light grey) , and in the presence or absence of IL-4.

Figure 7 is a graph showing proliferation of unfractionated naive lymphocytes from DA rats tolerant to PVG allografts at day 4 following contact with self- antigen or alloantigen in the presence of various cytokines .

Figure 8 is a graph showing proliferation of CD4⁺, CD25⁺ lymphocytes in various cytokines ( as indicated) following preincubation in IL-2 , IL-2 and L-NIL, IL-4 or IL-4 and L- NIL .

Figure 9A is a graph showing the clinical time course of Experimental Allergic Neuritis (EAN) in Lewis rats following administration of lymphocytes from tolerant (recovered) Lewis rats . Closed squares are untreated rats , closed circles are rats treated with CD4⁺, CD25⁺ T cells from rats tolerant to EAN and open circles are rats treated with CD4⁺, CD25^" T cells from rats tolerant to EAN .

Figure 9B is a graph showing weight change over a time course of Experimental Allergic Neuritis (EAN) in Lewis rats following administration of lymphocytes from tolerant (recovered) Lewis rats . Closed squares are untreated rats , closed circles ' are rats treated with CD4⁺, CD25⁺ T cells and open circles are rats treated with CD4⁺, CD25^~ T cells .

Figure 1OA is a graph showing proliferation of various combinations of lymphocytes from naive Lewis rats in response to PNM the immunizing antigen for EAN .

Figure 1OB is a graph showing the proliferation of various combinations of lymphocytes ( as indicated) from Lewis rats tolerant to PNM antigen ( recovered from the EAN 4 weeks after immunisation) in response to PNM antigen .

Figure 11 shows the % survival of PVG cardiac grafts (upper panel ) and Lewis (third party) cardiac grafts (lower panel ) over 50 days in irradiated DA rats which received 20 x 10⁶ CD4⁺ T cells from DA rats tolerant to PVG antigen cultured with IFN-γ in the presence of PVG antigen ( full line, upper graph) (dotted line , lower graph) ; 5 x 10⁶ Naϊve CD4⁺ T cells (thick dashed line, upper and lower graph) ; 20 x 10⁶ Naϊve CD4⁺ T cells (thin dashed line, upper graph) ; 20 x 10⁶ CD4⁺, CD25⁺ T cells from DA rats tolerant to PVG antigen mixed with 5 x 10⁶ Naϊve CD4⁺ T cells (dotted line, upper graph) / irradiated DA rats (no cells ) ( solid line , upper and lower graph) .

Figure 12 shows the effect on proliferation of CD4⁺, CD25⁺ T cells of incubating CD4⁺ T cells (top row) , CD4⁺, CD25^" T cells (middle row) or CD4⁺, CD25⁺ T cells (bottom row) in the presence of autoantigen from DA rats ' (left column of graphs ) or alloantigen ( from PVG rats ) (right column of graphs ) in the presence of IL-2 , IL-12 , or IL-2 and IL-12 as indicated at the bottom of the diagram.

Figure 13 shows proliferation of CD4⁺, CD25⁺ T cells after activation in the presence of IL-2 and PVG antigen for 3 days , followed by incubation in the presence of no supplement (Resp . Stimul) , CHO-K supernatant (CHO-K) , IL-2 (IL-2 ) , IL-12p70 ( IL-12p70 ) or IL-12p40 ( IL-12p40 ) .

Figure 14 shows the results of RT-PCR GAPDH, of IL-2 or IL-12Rβ2 mRNA from CD4⁺, CD25⁺ T cells following culturing with autoantigen ( DA antigen) or alloantigen ( PVG antigen) for 4 days in the presence of IL-2 or IL-4.

Figure 15 shows the effect of administration of IL-5 on severity (A) and weight loss (B) in an experiment disease model of EAN . Open squares = Rats administered IL-5 ; closed circles = rats administered CHO-K supernatant; open circles = no treatment .

Figure 16 shows the percent of demyelinated nerve fibre in peripheral nerves from an experimental rat model of EAN 14 and 21 days after immunisation with PNM . Rats treated with IL-5 at immunisation are represented by grey bars , rats that were untreated are represented by black bars . Figure 17 shows the normalised copy number of rtiRNA following real time RT-PCR from draining lymph nodes (LN) or cauda equina (CE) of rats at 14 days or 21 days post- immunisation with PNM using primers to TCR, IFN-γ, IL-2 , IL-IO or TNF-α (as indicated) . Black bars represent IL-5 treated, open bars represent untreated . Significant is indicated as * p<0.05 and ** p<0.01.

Figure 18 shows the results of RT-PCR analysis of IL-5 , ^• IL-5Rα, IL-4 and IL-13 mRNA expression at day 14 and day 21 in draining lymph nodes (LN) and cauda equine (CE) of rats immunised with DNA and either administered IL-5 (IL- 5 ) or no IL-5 (CTL) .

Figure 19 shows the effect of CD4⁺, CD25⁺ T cells on proliferation of naϊve CD4⁺, CD25^~ T cells in a limiting dilution assay, A: naϊve CD4⁺, CD25^~ T cells were mixed with 1 : 2 serial dilution of naϊve CD4⁺CD25⁺ T cells; B : naϊve CD4⁺, CD25^~ T cells were mixed with 1 : 2 serial dilution of CD4⁺, CD25⁺ T cells which had been cultured in the presence of PVG antigen and IL-2.

Figure 20 shows the effect of CD4⁺, CD25⁺ T cells on proliferation of naϊve CD4⁺, CD25^~ T cells in a limiting dilution assay, A: naϊve CD4⁺, CD25^~ T cells were mixed with 1 : 2 serial dilution of naϊve CD4⁺, CD25⁺ T cells ; B : naϊve CD4⁺, CD25^"" T cells were mixed with 1 : 2 serial dilution of CD4⁺, CD25⁺ T cells which had been cultured in the presence of PVG antigen and IL-4.

DETAILED DESCRIPTION OF THE INVENTION Tolerance is a critically important immunological discriminatory process without which a serious disease state may exist . As described above, there are circumstances when abnormal induction or maintenance of tolerance leads to autoimmune diseases . There are also special circumstances when induction of tolerance would be desirable , for example , following organ transplantation . The re-establishment of tolerance is also desirable in autoimmune diseases and allergic diseases .

As used herein, the term "tolerant" or "tolerance" will be understood by those skilled in the art as meaning a state of immune unresponsiveness or reduced immune responsiveness to an antigen . It will be appreciated by persons skilled in the art that the term "tolerance" as used herein has the same meaning as "immune tolerance" .

For many years , animals of various species such as , for example mice and rats , have been used as models for studying the human immune system as well as the immune system of other mammals . This has been the case because findings in mice and rats , for example , have been directly applicable to models of the immune system of humans and other mammals . Accordingly, results obtained in studies of mice, rats and other mammals are directly applicable to humans and other mammals (Kostakis et al . IRCS Med Sci Libr Compend 1977 , 5 , 280) .

One aspect of the invention provides a method of growing CD4⁺, CD25⁺ T cells in vitro, comprising culturing CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T^' cells . The inventors have found that CD4⁺, CD25⁺ T cells undergo enhanced proliferation when the cells are cultured under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of the CD4⁺, CD25⁺ T cells . Typically, the effects of nitric oxide production are inhibited by culturing the cells in the presence of a nitric oxide inhibitor .

Typically, the cells are cultured in the presence of a factor which is capable of supporting activation, survival, and/or proliferation of CD4⁺, CD25⁺ T cells, and a nitric oxide inhibitor . Typically, the factor is a cytokine which is capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells, a biologically active fragment thereof, or a functionally- equivalent molecule thereof . The cytokine may be any one or more of the following cytokines : IL-2 , IL-4 , IL-5 , IL-

10 , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β, IFN-γ. Typically, the IL-12 is IL-12p70.

As used herein, the term "proliferation" refers to division or growth of cells . Proliferation of CD4⁺, CD25⁺ T cells may be determined by any methods known in the art for measuring proliferation of lymphocyte populations . Examples of suitable methods are described in, for example, Transplantation ( 1999 ) 67 : 605-613.

As used herein, "enhanced proliferation" refers to increased division or cell growth resulting in increased cell numbers . The increased cell numbers may be due to an increased rate of proliferation, to proliferation over a longer period of time, or to an increased rate of proliferation over a longer period of time . Naϊve CD4⁺, CD25⁺ T cells may be used in the method of the first to fourth aspects of the present invention . The term "naive CD4⁺, CD25⁺ T cells" refers to CD4⁺, CD25⁺ T cells which have not been contacted with an antigen and activated . Naϊve CD4⁺, CD25⁺ T cells may be isolated from thymus , bone marrow, peripheral lymphoid tissue or blood .

Naϊve CD4⁺, CD25⁺ T cells , may be isolated from a subj ect whose immune system has not been contacted with the antigen . For example, if the antigen is an antigen from an allograft, naϊve CD4⁺, CD25⁺ T cells may be obtained from the subj ect prior to the subj ect receiving the allogaft .

The subj ect may be any subj ect which produces CD4⁺, CD25⁺ T cells .

In one embodiment, naϊve CD4⁺, CD25⁺ T cells are cultured in the presence of at least one antigen and IL-2 , a biologically active fragment thereof, or functionally equivalent molecule thereof, and a nitric oxide inhibitor . The CD4⁺, CD25⁺ T cells may thereafter be cultured in the presence of IL-2 and/or IL-4 , a biologically active fragment thereof, or a functionally equivalent molecule thereof , and/or one or more cytokines selected from the group consisting of IL-5 , IFN-γ, Il-12p70 , IL-IO , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

Activated CD4⁺, CD25⁺ T cells are CD4⁺, CD25⁺ T cells that have been activated to an antigen . Activated CD4⁺, CD25⁺ T cells recognise, and proliferate in the presence of, the antigen to which they have been activated, but do not recognise other antigens .

Activated CD4⁺, CD25⁺ T cells may be used in the method of the invention . Activated CD4⁺, CD25⁺ T cells are formed when naϊve CD4⁺, CD25⁺ T cells contact an antigen in the presence of cytokines capable of supporting activation of CD4⁺, CD25⁺ T cells , such as IL-2 and/or IL-4. The inventors have found that naϊve CD4⁺, CD25⁺ T cells do not express receptors for IL-5 , IL-12 or IFN-γ. The inventors have further found that when naϊve CD4⁺, CD25⁺ T cells are activated by contacting the naive CD4⁺, CD25⁺ T cells with an antigen and IL-2 , the CD4⁺, CD25⁺ T cells express the IFN-γ receptor and the IL-12 β2 receptor . The inventors have also found that when naϊve CD4⁺, CD25⁺ T cells are activated by contacting the naϊve CD4⁺, CD25⁺ T cells with an antigen and IL-4 , the CD4⁺, CD25⁺ T cells express the IL- 5Rα . Activated CD4⁺, CD25⁺ T cells are capable of conferring tolerance in a subj ect to the antigen to which the CD4⁺, CD25⁺ T cells have been activated .

In one embodiment, CD4⁺, CD25⁺ T cells activated to an antigen are cultured in the presence of the antigen and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-10 , IL-12 , IL-13 , IL-18 , IL-23 , IFN-γ and TGF-β , a biologically active fragment thereof, or a functionally eguivalent molecule thereof , and a nitric oxide inhibitor . In some embodiments , the at least one cytokine is selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-18 , IL-23 , IFN-γ and TGF-β . The isolation and characterisation of a population of T cells in vitro has been described in a number of prior art documents , for example , those shown in US Patent No . 5 , 622 , 853 and International Patent Application No . WO00/20445 ; however, any known procedure for isolating T cells may be used . Briefly, in one optional approach a sample containing T-cells is taken from a mammal . Lymphocytes are then isolated from the sample using the methods for T cell isolation referred to above . For example, lymphocytes may be isolated by Ficoll-Hypaque gradient centrifugation ( Pharmacia, Piscataway, N . J . ) .

Following isolation of lymphocytes , CD4⁺, CD25⁺ T cells are typically isolated from the population of isolated lymphocytes prior to use in the method of the invention .

As used herein, "CD4⁺, CD25⁺ T cell" refers to a T cell that expresses on its surface the cluster of differentiation markers known as a CD4 and CD25. A CD4⁺, CD25⁺ T cell is also known as CD4⁺ and CD25⁺ T cell, or in other words , a CD4 positive and CD25 positive T cell . The CD4⁺, CD25⁺ T cell may also express other markers which may aid in the isolation of CD4⁺, CD25⁺ T cells such as , for example, CD45RO^~, RB^~ . Naive CD4⁺, CD25⁺ lymphocytes may express L- selectin . Typically, the CD4⁺, CD25⁺ T cells are CD4⁺, CD25^+high T cells .

Typically, CD4⁺, CD25⁺ T cells are isolated from a population of lymphocytes by positive enrichment of CD25⁺ T cells using an anti-CD25 monoclonal antibody . For example , CD4⁺, CD25⁺ T cells may be isolated by means of multiparameter flow cytometric analysis using one or more fluorescent labelled anti-CD25 antibodies . This method includes the analysis of both light scatter parameters as well as one or more fluorescence parameters . Other methods of isolation include, for example , magnetic bead based separation as previously described in U . S . Pat . No . 517 , 101. Flow cytometric analysis may be performed, for example, on a FACScanTM flow cytometer or a FACStarTM plus cell sorter (both available from Becton Dickinson Immunocytometry Systems , ^λλBDIS" ) . Data acquisition may be performed with FACScan Research software and FACStar Plus software (Becton Dickenson) . Forward light scatter, orthogonal light scatter and three fluorescence signals are determined for each cell and stored in listmode data files . Each experiment measures approximately 30 , 000 cells , although it will be appreciated that the number of cells may vary greatly depending on the subj ect and available lymphocytes . The analysis of the listmode data files is preferably performed with Paint-A-Gate, TM software (BDIS ) . ( See U . S . Pat . No . 4 , 845 , 653 ) . To increase the orthogonal light scattering resolution, the orthogonal light scattering signals may be transformed by using a polynomial function as described in US Pat . Application ser . No . 517 , 096. For light microscope examination, 10, 000 sorted cells are centrifuged for five minutes at 20Og and resuspended in 100ml RPMI 1640 containing 10% FCS . Cytospin preparations are made on a Shandon cyto-centrifuge ( Southern Products Ltd) . Slides containing sorted cells may be stained with Wright Giemsa stain ( Sigma) .

CD4⁺, CD25⁺ T cells may be fluorescently labelled for identification and/or isolation using a variety of monoclonal antibodies available from BDIS . Antibodies may be fluorescent labelled with one of the following fluorochromes : phycoerythrin ( "PE" ) , fluorescein isothyocyanate ( "FITC") and peridinin chlorophyll complex

("PerCp") . For a description of PE and PerCp, see U . S . Pat . Nos . 4 , 520 , 110 and 4 , 876 , 190 respectively . The monoclonal antibodies which may be used include, for example : anti-CD4 FITC, PE or PerCp; anti-CD25 PE . (All antibodies commercially available from BDIS ) .

As used herein, the term "culturing" refers to the growth and maintenance in a viable state of cells in vitro . The step of culturing may be accomplished by simply incubating the T cells in suitable media . The T cells may be cultured in a culture medium which provides sufficient carbon, nitrogen, oxygen and other nutrients , growth factors , buffers , co-factors and any other substance as reguired to at least maintain the viability of the lymphocytes . For example, CD4⁺, CD25⁺ T cells may be cultured in RPMI or DMEM supplemented with 10% fetal calf- serum ( FCS ) and other supplements such as antimicrobial agents , growth factors , other cytokines ( see, for example, Transplantation ( 1993 ) 55 : 374-379 ) . Examples of suitable medium include medium formulations that are known to those skilled in the art such as , for example, RPMI , IMDM, DMEM, DMEM/F12 , EMEM with or without serum or with reduced serum, and further optionally including antibiotics, lipids , transferrin, insulin, additional nutrient supplements such as amino acids and co-factors as reguired .

Generally, cultured lymphocytes are incubated at 37⁰C in a 5% CO₂ atmosphere .

It will also be understood that the expression "biologically active fragment thereof" in relation to IL- 2 , IL-4 , IL-5 , IL-I O , IL-12 , IL-13 , IL- 15 , IL-18 , IL-23 ,

TGF-β or IFN-γ refers to any fragment of these cytokines which has the ability to support activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells in the presence of a nitric oxide inhibitor .

It is envisaged by the inventors that as cytokines interact with specific receptors to activate signal transduction pathways , any molecules which interact with the same receptor on CD4⁺, CD25⁺ T cells to activate the same signal transduction pathways as a cytokine capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells will also be capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells . Accordingly, the expression ^λλa functionally equivalent molecule thereof" refers to molecules that are not the actual cytokine, or a biologically active fragment thereof, but which are nonetheless ligands for the cytokine receptor, and which are capable of supporting the activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells .

In embodiments where the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , the CD4⁺, CD25⁺ T cells may be cultured in the presence of the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and one or more nitric oxide inhibitors , in any manner . Typically, the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells is an isolated polypeptide that is added exogenously to the medium in which the CD4⁺, CD25⁺ T cells are cultured, either as part of a culturing medium or as a purified polypeptide . Alternatively, the at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells may be the product of heterologous gene expression in cells that are co-cultured with the lymphocytes .

Typically, the CD4⁺, CD25⁺ T cells are in addition cultured in the presence of at least one antigen . The CD4⁺, CD25⁺ T cells may be cultured in the presence of at least one antigen in any manner which presents the at least one antigen to the CD4⁺, CD25^~ T cell in a form which will permit the T cell to recognise the antigen . As used herein, a reference to contacting a CD4⁺, CD25⁺ T cell with an antigen refers to contacting a CD4⁺, CD25⁺ T cell with an antigen in a manner which permits the T cell to recognise the antigen . In other words , the CD4⁺, CD25⁺ T cells are contacted with the an antigen in a manner which would permit activation of the T cells .

Typically, the antigen is on the surface of a stimulator cell such as , for example , an antigen presenting cell . Typically, the antigen is presented to the CD4⁺, CD25⁺ T cells associated with a maj or histocompatability (MHC) molecule (typically class II ) on the surface of an antigen presenting cell . As defined herein, a "stimulator cell" is a cell which is capable of presenting an antigen to a lymphocyte in a manner in which the lymphocyte can recognise the antigen . For example, the stimulator cell may be a tumour cell ( see for example US Pat . No .

5 , 342 , 774 , Knuth et al . ( Proc . Natl . Acad . Sci . USA 86 : 2804-2808 , 1989) and Van Den Eynde et al . ( Int . J . Cancer 44 : 634-640 , 1989 ) or the stimulator cell may be an antigen presenting cell .

An "antigen presenting cell" will be understood by those skilled in the art to be a cell which contributes to the induction of an immune response by presenting antigen to T cells . Antigen presenting cells may be dendritic cells , mononuclear phagocytes , B-lymphocytes , unfractionated lymphocytes or Langerhans cells . The antigen presenting cells may be isolated from, for example , bone marrow, blood, thymus , epidermis , liver or fetal liver . The antigen presenting cells may be unfractionated lymphocytes in which stimulator cells have been impaired by treatment with, for example , irradiation or mitomycin C . The antigen presenting cells may be cells expressing the relevant antigen presenting molecule (eg . Class II MHC) and other ligands that are required to facilitate binding and activation of naive CD4⁺, CD25⁺ T cells . Suitable ligands include ICAMl , ICAM2 , LFA3 , and the ligands for CD28 and CTL-A and other activation ligands or part of the antigen that is presented on self MHC molecules and recognised by T cells activated in an autoimmune response .

The CD4⁺, CD25⁺ T cells may be contacted with the antigen using synthetic antigen presenting systems , such as those described in US Patent No . 6 , 828 , 150 or 6, 787 , 154.

The antigen may be any substance which elicits an immune response in a subj ect that is not tolerant to the antigen . The antigen may or may not be derived from the subj ect . The antigen may be an autoantigen, which will be understood by those skilled in the art as referring to an antigen that can elicit a reaction in persons with a propensity to allergy . The antigen may be an alloantigen, which will be understood by those skilled in the art as referring to an antigen derived from a subj ect of the same species . The antigen may be a xenoantigen, which will be understood by those skilled in the art as referring to an antigen derived from a subj ect of a different species . The antigen may be an allergen .

A typical alloantigen may, for example, be donor transplant cells or tissue from another human . A typical xenoantigen may be transplant cells or tissue from a non- human animal such as , for example, a pig . Donor transplant cells or tissue from humans or non-human animals may include kidney, liver, heart, lung, skin, pancreas, cornea, lens , bone marrow, muscle, connective tissue, vascular tissue , gastrointestinal tissue, nervous tissue, bone, valves , stem cells , cells , such as stem cells , transfected with an agent such as a therapeutic agent .

If a subj ect is exposed to an antigen , antigen presenting cells with the antigen already presented on the surface of the cell may be isolated from the subj ect . For example, antigen presenting cells isolated from, for example, the spleen of a subj ect suffering from an autoimmune disease will have the autoantigen presented on the surface of the cell . In the case of tissue transplantation, antigen presenting cells isolated from the tissue of a transplant donor will have the alloantigen presented on the surface of such cells . For example, the antigen presenting cells may be frozen or stored spleen or lymph node cells from the cadaver of a donor, or peripheral blood cells from a living donor . Alternatively, empty MHC molecules of antigen presenting cells isolated from the subj ect may be loaded with antigens as described in U . S . Pat . No .

5 , 731 , 160 whereby empty MHC molecules are loaded with immunogenic exogenous peptides of approximately 8 to 18 amino acids in length .

Antigen presenting cells may be isolated from blood or tissue by methods known in the art . For example , B- lymphocytes can be purified from a mixed population of cells ( e . g . other cell types in peripheral blood or spleen) by standard cell separation techniques . For example , adherent cells can be removed by culturing spleen cells on plastic dishes and recovering the non-adherent cell population . T-lymphocytes can be removed from a mixed population of cells treated with an anti-T cell antibody ( e . g . anti-CD3 ( see for example WO 01/37860 ) , anti-CD2 ) and complement . In one embodiment, resting B- lymphocytes are used as the antigen presenting cell . Resting B-lymphocytes can be isolated by methods based on the small size and density of the B-lymphocytes . Resting lymphoid cells may be isolated by counterflow centrifugal elutriation as described in Tony, H-P, Parker, D . C . ( 1985 ) J. Exp . Med . 161 : 223-241. Using counterflow centrifugal elutriation, a small, resting lymphoid cell population depleted of cells which can activate T cell responses can be obtained as described in US Pat . No . 6 , 312 , 692.

In another embodiment, unfractionated lymphocytes may be used as the antigen presenting cell . Typically, the unfractionated lymphocytes are treated to impair proliferation of stimulator cells . Examples of treatments suitable for impairing proliferation of stimulator cells include irradiation, or treatment with mitomycin C . Typically, the CD4⁺, CD25⁺ T cells are cultured under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T cells by culturing the CD4⁺, CD25⁺ T cells in the presence of a nitric oxide inhibitor . The nitric oxide inhibitor may be any agent or substance which inhibits the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T cells .

In one embodiment, the nitric oxide inhibitor is a nitric oxide synthase inhibitor . The nitric oxide synthase inhibitor may be any compound that inhibits nitric oxide synthase activity . Typically, the nitric oxide synthase inhibitor is an iNOS inhibitor . Suitable iNOS inhibitors include L-NIL, L-NAME, aminoguanidine, GDIPS (SOD mimetic copper [II] [ 3 , 5-dii-sopropylsalisylate acid] _{2 ?} FeTPPS , N- ( 3- aminomethyl) benzyl) acetamidine dihydrochloride .

In another embodiment, the nitric oxide inhibitor removes nitric oxide from the CD4⁺, CD25⁺ T cells .

In yet another embodiment, the nitric oxide inhibitor inhibits nitric oxide production by blocking nitric oxide synthase expression . Examples of molecules which may block nitric oxide synthase production include antisense DNA and RNA molecules , interference RNA molecules , ribozymes . Methods for the production of antisense, iRNA, siRNA and ribozymes are known in the art and are described in, for example, Nature , vol . 431 , no . 7006 , pp . 337-378.

Suitably, once the CD4⁺, CD25⁺ T cells have been cultured under conditions that inhibit the effect of nitric oxide production on CD4⁺, CD25⁺ T cells for a period of time , further proliferation may be stimulated by culturing the

CD4⁺, CD25⁺ T cells in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IFN-γ, IL-12p70 , IL-IO , IL-13 , IL-18 , IL-15 , IL-23 , IFN-γ and TGF-β, a biologically active fragment thereof, or a functionally equivalent molecule thereof , in the presence or absence of a nitric oxide inhibitor . Thus , the method may comprise , after culturing the CD4⁺, CD25⁺ T cells in the presence of a nitric oxide inhibitor, culturing the CD4⁺, CD25⁺ T cells in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-12p70 , IL-10 , IL-13 , IL-15 , IL-23 , IFN-γ and TGF-β, a biologically active fragment thereof, or a functionally equivalent molecule thereof . In some embodiments , the at least one cytokine is selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , IFN-γ and TGF-β .

In another aspect , the invention provides a method of increasing tolerance in a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of CD4⁺, CD25⁺ T cells grown in vitro by culturing under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T cells . The CD4⁺, CD25⁺ T cells grown in vitro may be administered to a subj ect in need thereof to increase the subj ect' s tolerance to one or more antigens . In one embodiment , the invention further provides a method for increasing immune tolerance in a subj ect in need thereof which comprises administering to the subj ect an effective amount of CD4⁺, CD25⁺ T cells activated to an antigen grown in vitro by culturing the CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide production on survival and/or proliferation the CD4⁺, CD25⁺ T cells . Typically, the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one factor capable of supporting activation, survival and/or proliferation of the CD4⁺, CD25⁺ T cells , and a nitric oxide inhibitor . Typically, the factor capable of supporting activation, survival and/or proliferation of the CD4⁺, CD25⁺ T cells is a cytokine . Typically, the CD4⁺, CD25⁺ T cells are also cultured in the presence of the antigen . The CD4⁺CD25⁺ T cells are typically cultured in the presence of the antigen under conditions that permit contacting of the CD4⁺, CD25⁺ T cells with the antigen . As used herein, the expression "increasing tolerance" means an increase in tolerance to one or more antigens relative to the tolerance to the one or more antigens prior to application of the method of the invention .

As discussed above, culturing the CD4⁺, CD25⁺ T cells in the presence of at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells , and a nitric oxide inhibitor, results in enhanced proliferation of the CD4⁺, CD25⁺ T cells .

Upon culturing the CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide production on survival and/or proliferation the CD4⁺, CD25⁺ T cells , the T cells may then be administered to the subj ect .

Typically, the T cells are cultured for a further period in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof , prior to administering to the subj ect . In some embodiments , the at least one cytokine is selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β and IFN-γ.

The CD4⁺, CD25⁺ T cells are typically administered by parenteral administration . Preparations for parenteral administration include suspensions in sterile aqueous carriers . Aqueous carriers for suspensions may include saline and buffered media . Parenteral vehicles include any solution which is capable of maintaining the activity and viability of the T cells , and may include, for example, cell culture medium, sodium chloride solution, Ringer ' s dextrose, dextrose and sodium chloride, lactated Ringer ' s intravenous vehicles include fluid and nutrient replenishers , electrolyte replenishers ( such as those based on Ringer ' s dextrose ) , and the like . Preservatives and other additives may also be present such as , for example, anti-microbials , anti-oxidants , chelating agents , growth factors and inert gases and the like .

The CD4⁺, CD25⁺ T cells can be administered, parenterally by inj ection or by gradual perfusion over time independently or together . Administration may be intravenously, intra- arterial, intraperitoneally, intramuscularly, intracavity, intraarticular or transdermally . Typically, administration is intravenously .

In another aspect, the invention provides a method for treating or preventing in a subj ect in need thereof a disease resulting from an immune response to an antigen comprising administering to the subj ect a therapeutically effective amount of CD4⁺, CD25⁺ T cells activated to the antigen grown in vitro using the method described above .

For example, the disease may be an autoimmune disease, or host-versus-graft disease resulting from allograft or xenograft rej ection, or an allergic reaction .

In one embodiment, the disease is an autoimmune disease . As used herein, "autoimmune disease" refers to a disease resulting from an immune response to an autoantigen . Autoimmune disease may include , but is not intended to be limited to, these particular types of autoimmune diseases : type 1 insulin dependent diabetes mellitis , inflammatory bowel syndrome including ulcerative colitis and Crohn' s disease, thrombotic thrombocytopenic purpura, Sj ogren' s syndrome , encephalitis , acute encaphaliomyelitis , Guillain Barre Syndrome , chronic inflammatory demyelination polyneuropathy, idiopathic pulmonary fibrosis/alveolitis , asthma , uveitis , iritis , optic neuritis , rheumatic fever, Reiter' s syndrome, psoriasis arthritis , multiple sclerosis , progressive systemic sclerosis , primary biliary cirrhosis , pemphigus , pemphigoid, necrotising vasculitis , myasthenia gravis , polymyositis , sarcoidosis , granulomatosis , vasculitis , pernicious anemia, CNS inflammatory disorder, autoimmune haemolytic anaemia, Hashitomo' s thyroiditis , Graves disease, habitual spontaneous abortions , Raynaud' s syndrome, dermatomyositis , chronic active hepatitis , celiac disease, autoimmune complications of AIDS , atrophic gastritis , ankylosing spondylitis , Addison' s disease, atopic dermatitis , eczema, contact dermatitis , allergy to food, drugs or venoms , glomerulonephritis including membranous nephropathy, focal sclerosing glomerulonephritis and minimal change nephropathy, systemic lupus erythematosis , scleroderma, rheumatoid arthritis , j uvenile arthritis , psoriasis and psoriatic arthritis .

In another embodiment , the disease is a host-versus-graft disease resulting from allograft rej ection . The term "allograft rej ection" will be understood by those skilled in the art as referring to an immune response to an antigen ( s ) of a graft or transplanted tissue in a subj ect wherein the graft or tissue is obtained from a different member of the same species as the subj ect .

Allograft rej ection includes rej ection of all types of allograft and may include for example, allografts of cornea, heart , lung, kidney, liver, pancreas , pancreatic islets , brain, bone , intestine, cells , including stem cells and hematopoietic cells . Cells may include cells such as stem cells transfected with an agent, therapeutic or otherwise .

In yet another embodiment, the disease is a host-versus- graft disease resulting from xenograft rej ection .

The term "xenograft rej ection" will be understood by those skilled in the art as referring to an immune response to an antigen ( s ) of a graft or tissue transplant in a subj ect wherein the tissue is obtained from a member of a different species from the subj ect .

Xenograft rej ection includes rej ection of all types of xenograft and may include for example, xenografts of cornea, heart , lung, kidney, liver, pancreas , pancreatic islets , brain, bone , intestine, skin cells , including stem cells and hematopoietic cells from, for example, rodent, non-human primate, human, cattle, pig, sheep, camel , goat or horse . The cells may include cells , such as stem cells , that have been transfected with an agent, therapeutic or otherwise .

In a further embodiment, the disease is an allergy . The term "allergy" will be understood by those skilled in the art to refer to a type I hypersensitivity that is associated with a Th2 response mediated by CD4⁺ cells and occurs following contact with an allergen and which results in the triggering of IgE-sensitised mast cells by the allergen . The allergy may be an allergy to any allergen and includes , for example, asthma, eczema, atopic dermatitis , anaphylaxis , hayfever, allergic conjunctivitis , contact dermatitis, food, drug or other chemical , and venom allergy .

Generally, the terms "treat" , "treating" , "treatment" and the like are used herein to mean affecting a subj ect, tissue or cell to obtain a desired pharmacologic and/or physiologic effect . The effect may be prophylactic for example , completely or partially inducing immune tolerance to a specific antigen, in a subj ect or completely or partially preventing signs or symptoms of disease resulting from an immune response to an autoantigen, an alloantigen, a xenoantigen, an allergen or other foreign antigen, and/or may be therapeutic in terms of a partial or complete cure of disease .

The disease may be treated by administering to the subj ect a therapeutically effective amount of a composition comprising the CD4⁺, CD5⁺ T cells that have been activated to the antigen and grown in vitro according to the methods described above and a pharmaceutically acceptable carrier . As used herein, the term "therapeutically effective amount" is meant as an amount of a composition effective to yield a desired therapeutic response . For example, sufficient to prevent or treat disease such as those mentioned above .

The specific therapeutically effective amount will, obviously, vary with such factors as the particular condition being treated, the physical condition of the subj ect , the type of mammal being treated, the duration of the treatment , the nature of concurrent therapy (if any) , and the specific formulations employed and the relative constituent cell populations of the subj ects immune system.

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable suspending agent , medium or vehicle for delivering a compound to a subj ect . Pharmaceutically acceptable carriers include aqueous solutions , non-toxic excipients , including salts , preservatives , buffers and the like, as described, for instance, in Remington ' s Pharmaceutical Sciences , 15th ed . Easton : Mack Publishing Co . , 1405-1412 , 1461-1487 ( 1975 ) and The National Formulary XIV . , 14th ed . Washington :

American Pharmaceutical Association ( 1975 ) . The pH and exact concentration of the various components of the pharmaceutical composition are adj usted to maintain cell viability and activity according to routine skills in the art . See Goodman and Gilman ' s The Pharmacological Basis for Therapeutics ( 7th ed . ) .

The compositions of the invention may be administered parenterally in formulations containing conventional nontoxic pharmaceutically acceptable carriers , adjuvants , and vehicles .

The compositions are typically prepared and administered in dose units . For treatment of a subj ect, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subj ect , different daily doses can be used . Under certain circumstances , however, higher or lower daily doses may be appropriate . The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals .

The compositions according to the invention are typically administered systemically in a therapeutically effective dose . Amounts effective for this use will , of course, depend on the severity of the side effects and the weight and general state of the subj ect . Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects . Various considerations are described, eg . , in Langer, Science, 249 : 1527 , ( 1990 ) .

The compositions comprising CD4⁺, CD25⁺ T cells may be in the form of a sterile inj ectable suspension . This suspension may be formulated according to known methods using those agents suitable for suspending and administering cell suspensions which have been mentioned above . Among the acceptable vehicles and solvents that may be employed to suspend cells are cell culture medium, Ringer ' s solution, and isotonic sodium chloride solution .

Dosage levels of the compositions comprising CD4⁺, CD25⁺ T cells are typically of the order of about 5xlO⁶ to about 5xlO⁹ cells per kilogram body weight, with a typical dosage range between about 5xlO⁶ to about 5xlO⁸ cells per kilogram body weight per day ( from about 3xlO⁸ cells to about 3XlO¹¹ cells per patient per day) . The amount of cells that may be combined with the carrier materials to produce a single dosage will vary depending upon the host treated and the particular mode of administration . For example , a formulation intended for administration to humans may contain about 5xlO⁸ to 5xlO¹⁰ cells with an appropriate and convenient amount of carrier material which may vary from about 5 to 95 percent of the total composition . Dosage unit forms will generally contain between from about 5xlO⁸ to 10⁹ cells .

It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the cells , the age, body weight , general health, sex, diet, time of administration, drug combination and the severity of the particular disease undergoing therapy .

Also contemplated for use with the method of the invention are kits . As used herein, the term "kit" refers to a group of components that are capable of being used together in the methods of the invention . For example, the kit may be used to prepare cells for inducing tolerance in accordance with the invention, or to administer CD4⁺, CD25⁺ T cells to a subj ect, or to administer antibody and/or cytokines to a subj ect . A kit may include, for example, one or more of IL-2 , IL-4 , IL-5,

IL-IO, IL-12 , IL-13 , IL-15, IL-18 , IL-23 , TGF-β and/or IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof, isolated as a protein or in a medium such as, for example, a medium suitable for the culturing of lymphocytes , together with one or more nitric oxide inhibitor . The kit may further include instructions for applying the method of the invention using the components of the kit .

The present invention further provides methods for assessing whether a subj ect comprises CD4⁺, CD25⁺ T cells that have been activated by a specific antigen . As activated CD4⁺, CD25⁺ T cells are typically capable of imparting tolerance to the specific antigen with which they are activated, there is provided a method for determining whether a subj ect is tolerant, or at least capable of becoming tolerant, to a specific antigen .

In one embodiment of the seventh aspect, there is provided a method comprises the steps of :

(a) obtaining from the subj ect a sample comprising CD4⁺ lymphocytes or CD4⁺, CD25⁺ T cells ;

(b) contacting at least one portion of the sample with the specific antigen;

(c) incubating the at least one portion of the sample in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5, IL-IO , IL- 12 , IL-13, IL-15, IL-18 , IL-23 and IFN- γ, or a biologically active fragment thereof, or a functionally equivalent molecule thereof, and in the presence or absence of a nitric oxide inhibitor; (d) thereafter determining the proliferation of

CD4⁺, CD25⁺ T cells in the presence of a nitric oxide inhibitor, whereby increased proliferation of

CD4⁺, CD25⁺ T cells in the presence of the inhibitor relative to proliferation of CD4⁺, CD25⁺ T cells in the absence of the inhibitor indicates that the subj ect is tolerant to the specific antigen .

In some embodiments , in step ( c) the cytokine is selected from the group consisting of IL-5 , IL-12 and IFN-γ.

The inhibitor may be any inhibitor which inhibits the biological effect of nitric oxide . In one embodiment, the agent is a nitric oxide inhibitor . The nitric oxide inhibitor may be any compound that inhibits nitric oxide production . Typically, the nitric oxide inhibitor is an iNOS inhibitor . Suitable iNOS inhibitors include L-NIL, L-NAME , aminoguanidine, GDIPS ( SOD mimetic copper [II ] [3 , 5- dii-sopropylsalisylate acid] _{2 /} FeTPPS , N- ( 3- aminomethyl) benzyl ) acetamidine dihydrochloride .

The invention will now be further described by way of reference only to the following non-limiting examples . It should be understood, however, that the examples following are illustrative only, and should not be taken in any way as a restriction on the generality of the invention described above . In particular, while the invention is described in detail in relation to autoimmune disease, tissue rej ection and allergic reactions , it will be clearly understood that the findings herein are not limited to treatment of autoimmune disease , tissue rej ection and allergic reactions .

EXAMPLES Example 1

These studies compared proliferation of CD4⁺ T cells to CD4⁺, CD25⁺ T cells or CD4⁺, CD25^~ T cell subsets or combinations of these CD4⁺ T cell subsets in Mixed Lymphocyte Cultures (MLC) . Cells were prepared from lymph nodes and spleen of either naϊve DA rats or DA rats that are tolerant to a fully allogenic PVG heterotopic heart graft . Tolerance was defined as non-rej ection of the graft after 75 days in the absence of maintenance immunosuppression . Responses to self (DA) , and to fully MHC incompatible specific donor (PVG) and fully MHC incompatible third party (Lewis ) were compared . DA, PVG, Lewis share no MHC antigens and are fully allogeneic to each other . The nature of this tolerance has previously been described and can be induced in DA rats with PVG heterotopic cardiac allografts by a variety of therapies in the first 2 weeks post transplant including cyclosporine, as well as either anti-CD4 or anti-CD3 monoclonal antibody therapy (J . Exp Med . 1990, 171 : 141 , Transplantation, 1993 , 55 : 459 , Transplantation 1997 , 64 : 1559 ) . The MLC proliferative response of unfractionated peripheral lymphocytes to specific donor stimulation is generally comparable to that to third party (Transplantation, 1993 , 55 , 380-385 ) .

Materials and Methods

Animals and procedures . DA (RTl^a) , PVG (RT1^C) , Lewis (RTlI) _ancj sprague Dawley rats were bred and maintained as previously described (J . Immunol . 1998 , 161 ; 5146) . Operative procedures including heterotopic heart grafts, irradiation and preparation of lymph node and spleen cells have been previously described (J . Exp . Med 1978 , 148 ; 878 ) . Heterotopic heart graft survival was monitored daily by palpation for loss of contraction and swelling associated with rej ection . Rej ection was defined as total loss of contraction equivalent to that when there is total loss of electrocardiographic activity . Some grafts destined to become tolerant have transient swelling and reduced contractility, but recover . This is consistent with an acute inflammatory response that may be required to induce the specific tolerance mediating CD4⁺, CD25⁺ T cells .

Preparation of peripheral lymphoid cells .

Single cell suspensions from spleen and lymph node cells (LNC) were prepared, as described (34 ) and RBC lysed by a buffer of 0.83% NH₄Cl , 0.1%KHCO₃ and 1OmM EDTA at pH 7.2. Cells were resuspended in PBS/2% BSA (MultiGel, Biosciences , Castle Hill , NSW, Australia) .

Subsets were identified by mAb and indirect immunofluorescence staining, and analysis on a FACScan, as described (35) . Monoclonal antibodies used were R7.2 (TCR-α,β), G4.18 (CD3 ) , W3/25 (CD4 ) , MRCOx8 (CD8 ) , MRCOx39 (CD25 , IL-2R alpha chain) , L316 (CD122 IL-2R beta chain) , (Pharmingen/ Becton Dickenson, San Diego, CA) . Subsets of T cells were enriched by a combination of an indirect panning technique to deplete CD8⁺ T cells and B cells , as described ( 9) and Magnetic bead separation techniques and a MACS column, as described by the manufacturer, (Miltenyi , Bergisch Gadenbach, Germany) . Briefly, cells were incubated at 4°C with optimised concentrations of MRCOxδ (anti-CD8 monoclonal antibody) and MRC 0x33 (anti-CD45RA monoclonal that binds to B cells and other leucocytes except it does not bind T cells ) , washed three times with PBS/2%BSA then resuspended at 2xlO⁷ cells/ml . These cells were incubated for 30 min at 4°C on Petri dishes (Greiner Labortechnik , Frickenhausen,

Germany) coated with rabbit anti-rat Ig and anti-mouse Ig

(DAKO A/s . Glostrup, Denmark) . This supernatant was concentrated in 85μl of PBS and incubated for 4⁰C for 15 minutes with 13μl of goat anti-mouse Ig micro-beads (Miltenyi ) per 10⁶ cells . After washing, cells were eluted on a CS MACS column (Milentyi) to obtain 97-99% enrichment for CD4⁺ T cells . The enriched CD4⁺ T cells were then incubated at 4⁰C for 20 min with PE conj ugated MRCOx39 (anti-CD25 monoclonal antibody) , then washed twice before incubation for 15 min at 4⁰C with δμl/10⁶ cells of mouse anti-PE mAb microbeads (Miltenyi) . Cells were then eluted through a LS MACS column (Miltenyi) and were either resuspended in media with 20% Lewis rat serum for use in MLC or in PBS/2%BSA for inj ection to rats . The cells were 96-99% CD4⁺ and the depleted population had <1% CD4⁺, CD25^+high T cells . The enriched population was 85-95% CD4⁺, CD25^{+ high} T cells . For those experienced in the art, it is known that enriched CD4⁺, CD25⁺ T cells populations refers to the CD4⁺, CD25^{+ high} T cells , as separation techniques preferentially enrich this population of CD4⁺, CD25⁺ T cells .

In some experiments CD4⁺, CD25⁺ T cells were directly enriched by incubation of unfractionated lymphoid cells at 4°C for 20 min with PE conjugated MRCOx39, then washed twice before incubation for 15 min at 4⁰C with 8μl/10⁶ cells of mouse anti-PE mAb microbeads (Miltenyi ) . Cells were then eluted through a LS MACS column (Miltenyi) and were either resuspended in media with 20% Lewis rat serum for use in MLC or in PBS/2%BSA for inj ection to rats .

Mixed lymphocyte cultures (MLC) Stimulator cells were from thymus of rats given 8.5 gray whole body irradiation 24 hours before . This population of stimulator cells is depleted of mature lymphocytes and is enriched for antigen presenting cells . Enriched antigen presenting cells are preferred as functional lymphoid cells will be stimulated and may produce cytokines that will activate responder cells or may produce background stimulation . Stimulator cells from whole body irradiated donors have the peripheral and thymic lymphoid cells destroyed in vivo within 24 hours . An alternate method that could be used to enrich dendritic cells could be with monoclonal antibody selection and Magnetic bead separation . The stimulator cells can also be irradiated and left over night to allow peripheral lymphocytes to die of the effects of irradiation, leaving an antigen presenting cell enriched population . 10⁴ of these stimulator cells were as effective as 2xlO⁵ in vitro irradiated spleen cells . The normal ratio of responder to stimulator cells is 1 : 1 to 2 : 1 when peripheral lymphoid cells are used as stimulators but when there is enrichment of antigen presenting cells by depletion of T and B lymphocytes then responders to stimulators cells may be 10-100 : 1. Microcultures in ϋ-bottom microtiter plates (Linbro, Flow

Labs , VA) had 2 x 10 stimulators cells and either 2 x 10 or 1 x 10⁵ responder cells/well in a total volume of 200 μl . Usually there are 4-6 replicate wells set up for each experimental sample . Cell culture medium used was RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 100 ng/ml penicillin, 100 U/ml streptomycin (Glaxo, Boronia,

Victoria, Australia) , 2 mM L-glutamine, 5x10^" M 2- mercaptoethanol ( Sigma Chemicals , St . Louis , MO) , and 20%

Lewis rat serum. 20% Lewis rat serum produced low background stimulation . Autologous or same species serum results in a very low background stimulation . This low background is due to elimination of the response to heterologous proteins in products such as fetal calf serum that are not used in the media . In other experiments , serial dilutions of T cells subsets were cultured with 2 x 10 stimulators cells . Different ratios of mixtures of CD4⁺ T cell subsets were also

4 cultured with 2 x 10 stimulators cells in limiting dilution assays . Cells were cultured at 37⁰C in humidified air containing

5% CO₂ and at various time points , usually at 3 , 4 , 5 and 6 days the cultures were pulsed with 0.5 μCi H-TdR (Amersham, Arlington Heights , IL) 16 hr prior to harvesting with a Titretek Cell Harvester (Flow Lab, Ayrshire, Scotland) . Proliferation was assayed by adding liquid scintillation fluid before counting on a beta counter ( 1450 Microbeta Plus, Beckman Instruments , Palo Alto, CA) . Other wells were harvested at 24 and 48 hours to extract mRNA for RT-PCR analysis of cytokine mRNA induction .

Results

Figure IA

The results of measurement of the proliferation of naive unfractionated lymphocytes , and the enriched populations of CD4⁺, CD25⁺ T cells, CD4⁺ T cells or CD4⁺, CD25^" T cells are shown in Figure IA. As can be seen from Figure IA, the response at 4 and 5 days of CD4⁺ T cells is less that that after depletion of CD4⁺, CD25⁺ T cells as seen with the enriched CD4⁺, CD25^~ T cells . The response of CD4⁺, CD25⁺ T cells is much smaller than with either unfractionated CD4⁺ T cells or CD4⁺, CD25^~ T cells . This kinetic study demonstrates that the proliferation at day 4 and 5 is most useful , to demonstrate proliferative responses for unfractionated CD4⁺ T cells and CD4⁺, CD25^" T cells . The response of naϊve CD4⁺, CD25⁺ T cells peaks earlier at day 3 and 4 and wanes by day 5 and 6, where as the response of unfractionated CD4⁺ T cells and of naϊve CD4⁺, CD25^" T cells does not appear until day 4 and peaks at days 5 or 6 before waning .

Figure IB

The results of comparison of proliferation to self, and two allogeneic stimulator strains of naϊve CD4⁺ T cells, naϊve CD4⁺, CD25⁺ T cells and naϊve CD4⁺, CD25^~ T cells are shown in Figure IB . As can be seen from Figure IB , the proliferative response at day 4 to syngeneic DA stimulators is low in all cases . The responses to PVG and Lewis are similar in each subtype of cells . As described above , the response of CD4⁺, CD25⁺ T cells is less than the other populations and CD4⁺, CD25^~ T cells have a greater response than unfractionated CD4⁺ T cells .

Figure 1C Figure 1C shows the result of serial dilution of naϊve CD4⁺, CD4⁺, CD25⁺ or CD4⁺, CD25^~ T cells . As can be seen from Figure 1C , at all dilutions the response by CD4⁺, CD25^~ T cells is greater that the equivalent number of unfractionated CD4⁺ T cells . The unfractionated CD4⁺ T cells are a mixed population with approximately 5% naϊve CD4⁺, CD25⁺ T cells and 95% CD4⁺, CD25^~ T cells . Thus the greater proliferation of CD4⁺, CD25^~ T cells is not simply due to an effect of enrichment (ie due to the loss of the 5% CD4⁺, CD25⁺ T cells) . It is consistent with an active effect of the minority population of naϊve CD4⁺, CD25⁺ T cells inhibiting the maj or population of CD4⁺, CD25^" T cells .

Figure ID

The effect of admixing separated naϊve CD4⁺, CD25⁺ T cells with separated naϊye CD4⁺, CD25^~ T cells was examined and the result is shown in Figure ID . As can be seen from Figure ID, naϊve CD4⁺, CD25⁺ T cells have an active suppressor effect on naϊve CD4⁺, CD25^" T cells . In these experiments 1 : 10 mixes of CD4⁺, CD25⁺ with CD4⁺, CD25^" T cells resulted in proliferative responses similar to the mixed population in CD4⁺ T cells where the ratio is 1 : 10- 1 : 20. Increasing the ratio to 1 : 1 resulted in near total suppression of the proliferative responses . This is consistent with the demonstrated non-specific inhibitory effect of naϊve CD4⁺, CD25⁺ T cells on immune responses in vitro .

Figure IE

Figure IE is a graph showing the proliferation of CD4⁺ T cells , CD4⁺, CD25⁺ T cells or CD4⁺,CD25^" T cells from animals tolerant to PVG antigen following contact with PVG antigen . The response at 4 and 5 days of CD4⁺ T cells is similar to the tolerated strain ( PVG) as it is to third party Lewis . After depletion of CD4⁺, CD25⁺ T cells , as seen with the enriched CD4⁺, CD25^~ T cells, the proliferative response to the tolerated strain is less than to the third party strain . The response of CD4⁺, CD25⁺ T cells is much smaller than with either unfractionated CD4⁺ or CD4⁺, CD25^~ T cells . These CD4⁺, CD25⁺T cells do not respond to specific donor ( PVG) in that their response is similar to the response to self ( DA) . These CD4⁺, CD25⁺ T cells from tolerant animals retain their response to third party, which is greater than to either specific donor or self .

The response to self ( DA) is less in all cultures than that to fully allogeneic stimulators , PVG or Lewis . The response to PVG and Lewis which are MHC unrelated strains is similar for unfractionated CD4⁺ T cells , but is less to PVG than to Lewis for both CD4⁺, CD25⁺ T cells and CD4⁺, CD25^~ T cells .

These results suggested that the CD4⁺, CD25⁺ T cells from tolerant animals die in standard culture conditions . Thus they do not inhibit the CD4⁺, CD25^" T cells in the mixed population of unfractionated CD4⁺ T cells , thereby their removal does not lead to an enhanced response of enriched CD4⁺, CD25^~ T cells . Alone CD4⁺, CD25⁺ T cells do not respond to donor alloantigen, again consistent with them not surviving, and their possible dependence on cytokines for growth .

Figure IF

Figure IF shows serial dilution of CD4⁺, CD4⁺, CD25⁺ T cells or CD4⁺, CD25^~ T cells from DA rats tolerant to PVG heart allografts . As can be seen from Figure IF, at all dilutions the response to specific donor ( PVG) by

CD4⁺, CD25^~ T cells is less that the response to third party (Lewis ) and no greater than that to self ( DA) . In contrast, with unfractionated peripheral lymphocytes or with CD4⁺ T cells the response to specific donor ( PVG) is similar to third party (Lewis ) and greater than to self ( DA) . At all dilutions the response of CD4⁺, CD25^" T cells to third party (Lewis ) or to self ( DA) is much greater than the equivalent number of unfractionated CD4⁺ T cells .

This study demonstrates that the response to PVG by CD4⁺, CD25^" T cells compared to Lewis is reduced and is different to that observed with naϊve CD4⁺, CD25^" T cells where there was a similar increase to both PVG and Lewis above that observed with unfractionated CD4⁺ T cells . Further naϊve CD4⁺, CD25^~ T cells response to PVG is always greater than to self .

This study suggested that the response of unfractionated CD4⁺ T cells from tolerant animals is due to a diminished response of the CD4⁺, CD25^~T cells to PVG being unchecked by CD4⁺, CD25⁺ T cells in the absence of cytokines capable of stimulating activation of CD4⁺, CD25⁺ T cells or of prolonging survival or stimulating proliferation of activated CD4⁺, CD25⁺ T cells . In contrast CD4⁺, CD25⁺ T cells from animals tolerant to PVG retain the capacity to inhibit the response to third party Lewis and their removal allowed an enhanced response of CD4⁺, CD25^~ T cells to Lewis compared to unfractionated CD4⁺ T cells .

These studies demonstrate that there is a difference in the CD4⁺, CD25⁺, T cells in tolerant animals compared to naϊve . This is consistent with the CD4⁺, CD25⁺ T cell from tolerant host dying in culture without critical growth factors, and that it thereby cannot suppress in this assay. Alternately the activation of CD4⁺, CD25^~ T cells results in a change of cytokine milieu that destroys rather than promotes the function of specific CD4⁺, CD25⁺ T cells that are maintaining tolerance . That is , new activation and release of early T cell activation cytokines destroys the specific tolerance mediating CD4⁺, CD25⁺ T cells .

Conclusions

In mixed lymphocyte cultures (MLC) , the proliferative response of CD4⁺, CD25⁺ T cells : a) to MHC incompatible alloantigens can be reproducibly assayed with the defined culture conditions that eliminate any background. b) from naϊve animals is only to MHC incompatible stimulators , not to self . c) from naϊve animals inhibits the proliferation of naϊve CD4⁺, CD25^" T cells . d) from tolerant animals is only to third party and not to specific donor or self . e) from tolerant animals cannot inhibit the response of tolerant CD4⁺, CD25^~ T cells to specific donor but can inhibit the response to third party , f) From naϊve animals has a non-alloantigen specific suppressor effect, in distinction from those from tolerant animals that retain the capacity to suppress response to third party alloantigen but not to specific donor alloantigens in the absence of cytokines capable of stimulating activation of CD4⁺, CD24⁺ T cells or of prolonging survival or stimulating proliferation of activated CD4⁺CD25⁺ T cells .

Example 2

The following experiments examined the effects of various cytokines on the proliferative response in MLC of CD4⁺, CD25⁺ T cells , and the response of naϊve and tolerant enriched CD25⁺, CD4⁺ T cells to individual cytokines was compared .

Materials and Methods

Cytokines were produced as described, and include IL-2 and one unit was defined as that required to induce 50% of maximal proliferation of the IL-2 dependent CTLL line . The cloning , production and assaying of these cytokines has been described and used standard methods for transfection into CHO-Kl cells (Transplantation Proc . (1999) 31 , 1574-5 , 1999 and 31 , 1572 , 1999) and included IL-4 , IL-5 , IFN-γ, IL-IO , IL-12 (p70 ) , IL-12 (p40 ) , IL-13. Human TGF-β was purchased from Sigma . Cytokine was added - to each relevant well as a 50μl aliquot of CHO-K transfected cell line supernatant . These supernatants have 5000-50 , 000 units per ml . Thus the relevant cytokines were at a final concentration of 1000-12 , 000 units per ml . Controls had supernatant from a non transfected CHO-kl cell line added to the medium.

Reverse transcription - polymerase chain reaction (RT- PCR) : The methods for mRNA extraction, cDNA synthesis and semi-quantitative PCR have been described ( 31, 35 ) . All samples were standardized by quantitation of cDNA by spectroscopy and by PCR of the house-keeping gene GAPDH . The primers and optimal cycle conditions for the cytokines IL-2 , IL-4 , IL-5 , IL-IO, IFN-γ, TNF-α, have been described (Transplantation 1998 ; 1145-1142 ) . Primers for IFN-γ receptor and IL-5-receptor alpha chain were designed and validated . Reaction and amplification conditions were optimised for each primer set using a PCR machine (Corbett Research, Sydney, NSW, Australia) . PCR product was then analysed by electrophoresis on 6% polyacrylamide gels and stained with ethidium bromide . The specificity of the RT- PCR products were verified by Southern transfer and hybridisation with dideoxygenin ( DIG) 3 ' -end labelled oligonucleotide probes . Hybridised probe was detected using the DIG luminescent detection kit (Boehringer Mannheim) . The methods for the semi-quantitative technique RT-PCR used were as previously described (Transplantation 1997 , 64 ; 1559-1567 ) . Starting cDNA samples were assayed at neat, 10 , 100 and 1000 fold dilutions and reactions terminated at a cycle in the optimum range for each product . For most samples there were duplicate samples assayed at each dilution . Levels of πiRNA expression were compared using the lowest dilution at which the PCR product was detected . Negative controls without cDNA, and positive controls of cDNA from ConA stimulated rat lymphocytes , were included in all experiments . Results

Enriched CD4⁺, CD25⁺ T cells were cultured in MLC against self ( DA) , specific donor (PVG) and third party (Lewis ) stimulators . Cultures were assayed for proliferation at 3- 4 days as described above .

Figure 2A

Figure 2A illustrates the response of naive DA cells to either DA antigen or PVG antigen in the presence of cytokines as indicated . As can be seen from Figure 2A, only IL-2 and IL-4 resulted in enhanced proliferation of naive CD4⁺, CD25⁺ T cells . These cytokines enhance proliferation to self, and both allogeneic stimulators , to a similar degree . This is consistent with poly-clonal activation by either IL-2 , or IL-4. Addition of IL-5 , IL-IO , IL-13 , IFN-γ did not enhance proliferation of naϊve CD4⁺, CD25⁺ T cells . Note the background response to self is at approx 100 cpm, equivalent to counts obtained with distilled water . That is with the methods used, (as described in Figure 1 ) there was no non-specific proliferation to extraneous factors like media, or stimulator cells . The background counts are equivalent to having distilled water assayed .

Figure 2B

Figure 2B is a graph showing the proliferation at day 3 of activated CD4⁺, CD25⁺ T cells against self antigen (black) or donor antigen (grey) in the presence of TGF-β (D) , IFN-γ (E) , IL-12p70 ( F) , IL-5 (G) , or IL-10 (H) . Figure 2B shows that the response of activated CD4⁺, CD25⁺ T cells is different to that of naϊve CD4⁺, CD25⁺ T cells in several aspects . First as described in Figure IE, activated CD4⁺, CD25⁺ T cells do not proliferate to specific donor in the absence of specific cytokines and therefore differ from naive CD4⁺, CD25⁺ T cells . Activated CD4⁺, CD25⁺ T cells proliferate to third party Lewis in a manner similar to naϊve CD4⁺, CD25⁺ T cells . Both activated and naϊve CD4⁺, CD25⁺ T cells do not proliferate to self (DA) .

The addition of IL-2 or IL-4 to cultures resulted in polyclonal activation, like that observed with naϊve CD4⁺, CD25⁺ T cells , in that there was a marked enhanced proliferative response of naϊve CD4⁺, CD25⁺ T cells to self ( DA) , specific donor (PVG) and third party (Lewis ) . The response to other cytokines identified a different pattern for activated CD4⁺, CD25⁺ T cells to the specific donor PVG for some cytokines but not others . Activated CD4⁺, CD25⁺ T cells response to both self ( DA) and third party Lewis stimulators were similar to that of naϊve cells for all cytokines .

Thus these experiments demonstrated a different response to PVG (donor or specific antigen) , that was most consistent with IFN-γ and IL-5. IL-12 (p7 Q ) and TGF-β also showed an effect . Control cultures and those with cytokines that did not induce proliferation, had responses at background, approx . 100 cpm.

These experiments demonstrated that CD4⁺, CD25⁺ T cells activated to a specific antigen require either IL-5 , IL-12 or IFN-γ to grow and survive .

Conclusions Cytokines have different effects on naive and tolerant

CD4⁺, CD25⁺ T cells . a) IL-2 and IL-4 markedly enhance proliferation of CD4⁺, CD25⁺ T cells from naϊve or tolerant cell donors to self, specific donor and third party. b) IL-5 and IFN-γ only enhances proliferation of tolerant CD4⁺, CD25⁺ T cells to specific donor and not to self or third party. They have no effect on naϊve CD4⁺, CD25⁺ T cells proliferation to self or to alloantigens .

Example 3

These studies examine the ability of CD4⁺, CD25⁺ T cells to transfer tolerance to a host .

Materials and Methods

Adoptive Transfer assays . These were conducted as described (J. Exp . Med 1978 , 148 ; 878-889 and Transplantation 1993, 55; 374-379) . Briefly, DA rats were irradiated with 7.5-8.5gray from a ⁶⁰Co source, then grafted with heterotopic adult heart from a PVG donor that had also been irradiated (Figure 3 A) . This source of irradiation required treatment over 15-30 minutes , as described (J. Exp . Med 1978 , 148 ; 878-889) . In the later experiments the irradiation source was a linear accelerator and whole body irradiation was delivered in 2-3 minutes . These irradiated hosts were given a heterotopic heart graft and also were restored within twenty-four hours of irradiation with CD4⁺T cells , either unfractionated or enriched for CD4⁺, CD25^~ T cells or CD4⁺, CD25⁺ T cells . Heart graft rejection is monitored initially by daily palpation and if there is doubt by ECG monitoring . Rej ection is scored when there is loss of all palpable contraction that is equivalent to total loss of electrocardiograph activity .

Results

Figures 3A and 3B show the capacity of naive CD4⁺ T cells to effect rej ection response on adoptive transfer to irradiated hosts .

For the studies reported in Figure 3B a semi-quantitative scale was used where a score of 4+ indicated robust contraction with normal auxiliary heart graft rate . 3+ indicated minor slowing and or reduced contractility. 2+ indicated obvious slowing and swelling of the graft but clear palpation of beat . 1+ indicated marked slowing and poorly palpable beat , usually associated with markedly reduced amplitude of the ECG . 0 indicated no palpable contraction and equivalent to no detectable ECG activity .

Figure 3A

Figure 3A shows a comparison of rej ection time in adoptive irradiated DA rats restored with different doses of naϊve CD4⁺ cells . Data combined from all laboratories and sites of Prof B . Hall . Group A, n=7 ; Group B, n=17 ; Group C, n=8 β ; Group D, n=6. To alter the balance of cells in a host, adoptive transfer experiments were performed where the DA hosts own lymphocytes were destroyed by near lethal whole body irradiation ( 700-850 rads) . In this model, heterotopic PVG heart grafts are not rej ected by the whole body irradiated hosts , whilst in non irradiated hosts rej ection occurs in 6-9 days which is first set rej ection time . Restoration of the irradiated host with an enriched population of CD4⁺ T cells restores a rej ection response , but not to the normal first set time . There is no dose response with these cells, in that 5 million CD4⁺ T cells are as effective as 20 or 100 million CD4⁺ T cells . The rej ection time on average is >12 days with these cells .

Table 1. Rej ection of PVG heterotopic cardiac allografts in irradiated DA recipients restored with unfractionated CD4⁺ T cells and/or subpopulations of CD4⁺ T cells

* Compared to 5xlO CD4⁺T cells ( see row 3 )

The effects of enriched naϊve CD4⁺ T cells , CD4⁺, CD25⁺ T cells or CD4⁺, CD25^~ T cells in the adoptive DA host with a PVG allograft were compared and the results are shown in Table 1. As in Figure 3A, unfractionated naive CD4⁺ T cells did not restore first-set rej ection time but restored rej ection to a median of 11 days . In this model the irradiation source was different to that in Figure 1. CD4⁺, CD25⁺ T cells had no capacity to effect rej ection, and no grafts rej ected . CD4⁺, CD25^" T cells restored rej ection to near first-set tempo . Admixing naϊve CD4⁺, CD25⁺ T cells with naive CD4⁺ T cells in a ratio of 1 : 1 prevented restoration of rejection, and the grafts appeared to function without a significant rej ection episode for over 100 days . CD4⁺, CD25⁺ T cells expanded by culture for three days with donor antigen (PVG) and IL-2 to expand the numbers suppressed naϊve CD4⁺ T cells . This was done as described in methods for mixed lymphocyte culture , but in bulk in 50 ml tissue culture flasks . These results demonstrate that naϊve CD4⁺, CD25⁺ T cells can be cultured with donor antigen and expanded in vitro to maintain suppressor function and not acquire the capacity to effect rej ection .

Figure 3B .

The effects of varying the ratios of naϊve CD25⁺, CD4⁺ T cells to naϊve CD25^~, CD4⁺ T cells in the restorative inoculum on rejection times in irradiated adoptive hosts was examined and a graph of the results in shown in Figure 3B .

Figure 3B illustrates heart graft function up to 50 days post-transplantation in rats following administration of groups were given; A, 5xlO⁶ naϊve CD4⁺ T cells (closed triangles ) (n=9) ; B, 2OxIO⁶ naive CD4⁺ T cells (closed circles ) (n=4 ) ; C, 5xlO⁶ naϊve CD4⁺, CD25^', T cells (open triangles ) (n=4 ) ; D, 0.5xl0⁶ naϊve CD4⁺, CD25⁺ T cells plus

5xlO⁶ naϊve CD4⁺ T cells (open squares ) (n=9 ) ; E , 5xlO⁶ naϊve CD4⁺, CD25⁺ T cells plus 5xlO⁶ naϊve CD4⁺ T cells

( closed squares ) (n=9 ) .

On this scale rats give 20 million CD4⁺ T cells effected rej ection most rapidly and those given CD25^", CD4⁺ T cells had a similar rapidity of rej ection . Those given 5 million CD4⁺ T cells alone rej ected less rapidly than those restored with CD25^", CD4⁺ T cells . Mixing 0.5 million CD4⁺, CD25⁺ T cells with 5 million CD4⁺ T cells further slowed the rejection processes , in that grafts were not totally rej ected but had poor function long term. In these animals the ratio of CD25⁺, CD4⁺ T cells : CD25^", CD4⁺ T cells was 1 : 5-10 compared to 1 : 20-40 in those given unfractionated cells .

Mixing 5 million CD4⁺, CD25⁺ T cells with 5 million CD4⁺ T cells totally suppressed rej ection with all grafts surviving indefinitely with very good function .

These studies demonstrate that the naϊve CD4⁺, CD25⁺ T cells can suppress naϊve CD4⁺, CD25^" T cells if present in a ratio approaching 1 : 1.

Further it shows that expansion of CD4⁺, CD25⁺ T cells by culture with IL-2 may allow production of sufficient self suppressor T cells to achieve such ratios , if the host is depleted of CD4⁺, CD25^~ T cells or the CD4⁺, CD25^~ T cells function is impaired . Culture of naϊve CD4⁺, CD25⁺ T cells with donor antigen does not convert these cells into ones that can effect rej ection . Thus culture of naϊve CD4⁺, CD25⁺ T cells may expand the number of suppressor

CD4⁺/CD25⁺ T cells . Treatment of unfractionated CD4⁺ T cells in vitro with IL-4 , does not enhance the tolerance effect of that CD4⁺, CD25⁺ T cells, indicating enriched CD4⁺, CD25⁺ T cells must be used. (Data not shown)

Conclusions

Altering the balance of CD4⁺, CD25⁺ T cells to CD4⁺, CD25^~ T cells in vivo can lead to tolerance, manifest by experiments with naive cells used to restore rejection in irradiated hosts ;

a) Naive unfractionated CD4⁺ T cells do not show a dose response effect, suggesting there is a homeostatic balance of the 1-10% CD4⁺, CD25⁺ T cells against the >90% CD4⁺, CD25^~ T cells .

b) Removal of the 1-10% CD4⁺, CD25⁺ T cells , allows the CD4⁺, CD25^~ T cells to effect rapid rejection, confirming that the naive CD4⁺, CD25⁺ T cells in their natural mixed population in naϊve CD4⁺ T cells function to moderate rej ection .

c) CD4⁺, CD25⁺ T cells alone do not effect rejection and can inhibit rej ection when the ratio of naϊve CD4⁺, CD25⁺ T cells to naive CD4⁺, CD25^" T cells is increased, with near total suppression of rejection when the ratio is 1 : 1.

d) Naϊve CD4⁺, CD25⁺ T cells when expanded in MLC with specific donor antigen and IL-2 retain the capacity to suppress at 1 : 1 ratio and do not revert to cells that effect rej ection . Example 4

This study examines proliferation of CD4⁺, CD25⁺ T cells in response to antigen and culturing in IL-2 or IL-4.

Figure 4A

Figure 4A shows the results of culture of naϊve CD4⁺, CD25⁺ T cells from DA rats with no stimulator cells ( first column) , self stimulator cells (middle column) , or allogeneic PVG stimulator cells . Top panel of Figure 4A shows the effect in the presence or absence of IL-2 , which increased proliferation in all assays , with greater effect at day 5 than day 4. The proliferation to self and allogeneic stimulator cells , was similar, and that with no stimulators much less . The bottom panel of Figure 4A shows cell proliferation in the presence or absence of IL-4 which also enhanced all responses . The response to allogeneic was greater than that to self or those with no stimulators . This suggests the IL-4 effect is more on specific antigen stimulation rather than to self-antigens .

Figure 4B

Figure 4B illustrates a time course of proliferation of naϊve CD4⁺, CD25⁺ T cells from DA rats cultured with allogeneic PVG stimulator cells alone, or in the presence of IL-2 or IL-4. Cells cultured with no cytokine supplementation had peak proliferation at day 3 as described in Figure IA. No proliferation was detectable at day 5 and day 6 with counts at background (equal to distilled water counts <150 ccpm) . IL-2 induced marked proliferation at all days with a peak at day 5. IL-4 enhanced proliferation on all days and this proliferation peaked at day 4. Profiles of the phenotype of these cells after 3-4 days in culture shows there is an emergence of a double positive population that expresses both CD8 and CD4 ( 10-35% of cells) and continue to express CD25. The original cells were <1% CD4⁺, CD8⁺ cells , and <2% CD8⁺ T cells .

Figure 4C mRNA expression of cytokines and cytokine receptors by CD4⁺, CD25⁺ T cells cultured with IL-2 and IL-4 were assayed by semi-quantitative RT-PCR, and the results are shown in Figure 4C .

Two experiments are shown in which naive CD4⁺, CD25⁺ T cells were cultured in MLC with IL-2 , IL-4 or controls with CHO- kl cell supernatants . Culture was for 3 days before mRNA was extracted . Semi-quantitative RT-PCR was performed as described (Transplantation, 1997 , 64 , 1559-1567 ) . The samples are serial dilutions of cDNA from right to left . The results show that there was no induction of IL-2 by any treatment, but abundant mRNA for IL-4 , IL-5, IL-IO, IL-13 , IFN-γ and inducible nitric oxide synthetase . Those cells cultured with IL-2 expressed IL-12Rβl that was not expressed in those cultured with IL-4. With IL-2 there was no IL-5R expression but in other experiments this receptor is induced by culture with IL-4. Figure 4D

Figure 4D illustrates cytokine and cytokine receptor induction in cultured tolerant CD4⁺, CD25⁺ T cells . This compares tolerant cells stimulated by specific donor, third party stimulators or self stimulators . Cultures contained no cytokine supplement (CTL top three lines ) or were supplemented with IL-2 , IL-4 or CHO-k supernatant . &+ CTLS were cDNA from ConA activated T cells was used as a control for the assay itself (-&+ controls ) . mRNA was assayed using semi-quantitative RT-PCR with serial dilutions of cDNA from right to left .

There was no induction of IL-2 mRNA in any CD4⁺, CD25⁺ T cells preparation . The most consistent and abundant mRNA was for IFN-γ and iNOS that was detected in all assays and tended to be enhanced after culture of CD4⁺, CD25⁺ T cells with either IL-2 or IL-4. IL-12Rβ2 was induced in CD4⁺, CD25⁺ T cells cultured with IL-2 and to a lesser extent with IL-4 , and was not observed in CD4⁺, CD25⁺ T cells cultured with no cytokines . This suggested that the IL-12p70 enhancement of IFN-γ, and in turn induction of iNOS , which inturn produces the cell toxic molecule nitric oxide, may explain some of the suppressor effects of activated CD4⁺, CD25⁺ T cells . IFN-γ mRNA was induced in cells cultured with IL-4.

mRNA for IL-4 and IL-5 was only mainly induced in CD4⁺, CD25⁺ T cells exposed to IL-2 or IL-4 and in neither control culture with no cytokines or CHO-k supernatant . IL-13 mRNA was detected in all samples with some induction with IL-2 and IL-4 , but this may in part reflect differences in starting cDNA as per GAPDH levels . mRNA for IL-IO and TGF-β were present in all cultures and were not discriminatory . mRNA for IL-5Rα was induced in CD4⁺, CD25⁺ T cells exposed to IL-4 as indicated by very faint bands . Samples assayed at day 4 show a more definite expression of IL-5Rα mRNA in cells cultured with IL-4 mRNA . See Figure 4E .

This suggests a different population of T suppressor cells may be induced with IL-2 and with IL-4 , the IL-2 inducing cells that might respond to ThI cytokines and IL-4 ones that might respond to Th2 cytokines such as IL-5.

Figure 4E

Figure 4E shows the result of RT-PCR analysis of IL-5Rα mRNA expression in the presence of antigen and cytokines as indicated . As can be seen from Figure 4E , IL-5Rα chain is induced in CD4⁺, CD25⁺ T cells cultured with IL-4 and not with IL-2 or no cytokine . Naϊve CD4⁺, CD25⁺ T cells from DA rats were cultured with either self DA stimulators or PVG stimulators for 4 days . IL-5Rα mRNA was assayed in cDNA collected from cells . Cells were culture with nil cytokine , IL-2 or IL-4. IL-5Rα mRNA was only detected in cells cultured in IL-4 with greater levels in those culture with alloantigen than those cultured with self antigen . Controls were uncultured CD4⁺, CD25⁺ T cells , cDNA from ConA activated lymphocytes and a known cDNA which had IL-5ROC. Figure 4F .

Figure 4F shows the result of real time RT-PCR for IFN-γ receptor (IFNGR) on CD4⁺, CD25⁺ T cells contacted with antigen in the presence of IL-2.

This study clearly demonstrated that there was induction of IFN-γ receptor on CD4⁺, CD25⁺ T cells cultured with IL-2 and alloantigeneic PVG stimulators and to a lesser degree with self DA stimulators , and not in cells cultured with no cytokines .

Taken together, the studies in Figures 4A to 4F show that CD4⁺, CD25⁺ T cells exposed and activated with ThI responses (ie IL-2) and alloantigen, developed receptors that would allow them to respond to ThI cytokines such as IFN-γ and IL-12. On the other hand, CD4⁺, CD25⁺ T cells exposed and activated by Th2 cells ( ie IL-4 ) in the presence of alloantigen develop receptors for Th2 cytokines such as IL-5. They also express the ThI cytokine IFN-γ mRNA.

These results are consistent with the inventors ' observation that activated CD4⁺, CD25⁺ T cells from rats with tolerance to PVG proliferate to PVG but not self or third party when either IFN-γ or IL-5 are added to the culture, but not if there is no cytokines .

The inventors propose that under normal circumstances of an immune response, with early activation there would be both IL-2 and IL-4 resulting in activation of both types of CD4⁺, CD25⁺ T cells . The inventors propose to call these two types of activated CD4⁺, CD25⁺ T cells TsI cells and Ts2 cells . TsI cells are CD4⁺, CD25⁺ T cells activated by IL-2 and acquire the IFN-γ and IL-12p70 receptor and Ts2 cells are CD4⁺, CD25⁺ T cells activated by IL-4 that acquire the IL-5Rα receptor and also express IFN-γ. Ts refers to suppressor T cells of the CD4⁺, CD25⁺ T cell phenotype .

Conclusions

IL-2 and IL-4 have different effects on CD4⁺, CD25⁺ T cells in culture, including; a) Whilst both stimulate proliferation to self and alloantigen, the response to IL-4 peaks and tapers earlier than that with IL-2. b) Culture with IL-2 leads to development of a CD4⁺, CD8⁺ double positive CD25⁺ T cells . This development of double positive cells is probably de-differentiation of CD4⁺, CD25⁺ T cells to re-express CD8. c) Neither culture with IL-2 or IL-4 induces detectable expression of IL-2 mRNA, but both cells cultured with either IL-2 or IL-4 have mRNA for IL-4 , IL-5, IL- 12p40, iNOS . Those cells cultured with IL-2 expressing less IFN-γ and typically more IL-5 than those cells cultured with IL-4. Those cells cultured with IL-4 express more IFN-γ and typically less IL-5 and iNOS mRNA than those cells cultured with IL-2. There appears to be no detectable specific induction of IL-IO , IL-13 and TGF-β. d) There is late de novo cytokine receptor mRNA expression, with IFN-γR appearing when cultured with IL-2 and antigen . IL-5R appears with IL-4. IL-12Rβ2 appears with both stimuli but possibly more with IL-2 than IL-4. e) The pattern of cytokine mRNA expression with tolerant cells cultured with IL-2 or IL-4 is similar to that with naive CD4⁺, CD25⁺ T -cells, consistent with this being polyclonal activation of CD4⁺, CD25⁺ T cells , not activation of alloantigen specific CD4⁺, CD25⁺ T cells , f) That IL-2 induces an IFN-γ/IL-12p70 responsive suppressor cell, described herein as TsI . g) That IL-4 induces an IL-5 responsive suppressor cell , described herein as Ts2.

Example 5

This study examines the effect of naive CD4⁺, CD25⁺ T cells on CD4⁺, CD25^~ T cells .

Figure 5A

Figure 5A shows the result of RT-PCR on T cells cultured in the presence of self or alloantigen in the presence of cytokines as indicated . In this assay duplicate samples at maximum concentration of cDNA were examined as there is limited mRNA extracted from these cultures . CD4⁺, CD25⁺ T cells alone cultured with self (DA) stimulators , have minimal cytokine induction . With PVG stimulators there is no IL-2 mRNA but some IFN-γ, IL-4 , IL-IO and

TGF-β expression . In contrast the CD4⁺, CD25^~ T cells have marked induction of all cytokine mRNA tested when stimulated by PVG, and this profile is similar to that observed when stimulated with DA . With self-stimulators there is no IL-5 mRNA induction, however . It should be noted that the CD4⁺, CD25^" T cells have a marked proliferative response to self, known as the autologous MLC, and thus marked induction of cytokine mRNA in the CD4⁺, CD25^" T cells responding to self is consistent with a marked autologous proliferative response . In the mixing experiment of CD4⁺, CD25^~ T cells and naive

CD4⁺, CD25⁺ T cells at a 1 : 1 ratio (combined in Figure 5A) there is total suppression of proliferation as described before . In these cultures there is marked reduction in expression of IL-2 , IL-4 , IL-5 mRNA in the allogeneic response compared to that with the CD4⁺, CD25^~ T cells . A small reduction on IFN-γ, IL-IO, TGF-β mRNA and no effect on iNOS mRNA levels compared to those observed with CD4⁺, CD25^~ T cells alone against PVG . CTL are assays with cDNA from ConA stimulated T cells .

This shows the suppression of MLC by the CD4⁺, CD25⁺ T cells is associated with IFN-γ and iNOS induction, suggesting NO production may mediate this effect of suppression . This possibility was examined by adding an iNOS inhibitor L-NIL to MLC .

Figure 5B

Figure 5B is a series of graphs showing the effect of blocking IL-5 , TGF-beta, IL-IO and iNOS on the inhibitory effects of CD4⁺, CD25⁺ T cells inhibition of CD4⁺ , CD25^" T on MLC proliferation .

This experiment examined whether monoclonal antibodies to IL-5 , TGF-β and IL-10 could block the suppression of CD4⁺, CD25⁺ T cells on CD4⁺, CD25^" T cells proliferation when cultured in a ratio of 1 : 1. As can be seen in the bottom panel of Figure 5B, the controls (CTN) are totally- suppressed and enhanced proliferation occurs to fully allogeneic donor with anti-IL-5 , TGF-β and anti-IL-10 but not with the control monoclonal antibody, anti-Mog Ig2a . None restored proliferation to that of CD4⁺, CD25^~ T cells alone (see top right panel) and the effect was not non specific as none of these antibodies had an effect on the proliferation of CD4⁺, CD25^~, T cells alone or on CD4⁺, CD25⁺

T cells alone, except for anti-TGF-beta which did enhance CD4⁺, CD25⁺ T cells proliferation . L-NiI which blocks iNOS and thereby blocks NO production enhanced both CD4⁺, CD25^~ T cells alone proliferation and partially blocked the inhibition of CD4⁺, CD25⁺ T cells in the mixed culture . These results suggest that IL-5, IL-IO and TGF-beta have a functional effect in the suppressive effects of CD4⁺, CD25⁺ T cells on CD4⁺ CD25^" T cells in MLC . This suggests that the non-specific suppression of CD4⁺, CD25⁺ T cells may also be in part mediated through the effects of IL-5 and IFN-γ.

Conclusions

The suppressive effect of naive CD4⁺, CD25⁺ T cells on naϊve CD4⁺, CD25^" T cells in MLC is associated with : a) Naive CD4⁺, CD25⁺ T cells when admixed 1 : 1 with naϊve CD4⁺, CD25^" T cells suppress the MLC proliferation as seen in Figure ID . b) A marked reduction in induction of IL-2 , IL-4, IL-5 mRNA expression and a smaller reduction in induction of IFN-γ and TGF-β mRNA expression, but no reduction in iNOS induction when compared with the induction in CD4⁺, CD25^~ T cells . The relative preservation of IFN-γ, and iNOS induction suggests nitric oxide production may be preserved and may contribute to the suppression of proliferation observed . c) Blocking of iNOS with L-NIL prevents the suppression mediated by naϊve CD4⁺, CD25⁺ T cells . d) Blocking with anti-IL-5 or anti-TGF-β or anti-IL-10 monoclonal antibodies partially blocks suppression suggesting IL-5 , IL-IO and TGF-β have an effect in mediating the suppression by naive CD4⁺, CD25⁺ T cells

Example 6

The effect of IL-4 or IL-5 on survival in culture of CD4⁺ T cells from DA rats with tolerance to PVG allografts examined . The survival of tolerance mediating cells was assayed by their ability to adoptively transfer specific tolerance to PVG cardiac allografts and to retain the capacity to effect rej ection of third party Lewis allografts .

These studies used CD4⁺ T cells from spleen and lymph nodes of DA rats tolerant to a PVG cardiac allograft that has survived over 75 days . Graft acceptance and tolerance developed after a short course of immunosuppression at the time of transplantation, either 10 days of cyclosporine treatment, or anti-CD4 or anti-CD3 monoclonal antibody therapy, as described (Transplantation, 1997 , 64 , 1559- 1567 and 1993 , 55 ; 459-468 ) . The ability of these cells to transfer tolerance was examined in the irradiated adoptive host that is described above in Figure 3A and 3B .

In early studies we had demonstrated that fresh tolerant cells transfer tolerance to specific donor ( PVG) but rej ect third party grafts . Admixing tolerant cells with naive CD4⁺ T cells in a ratio of 4 : 1 , ie 20 million tolerant CD4⁺ T cells with 5 million naive CD4⁺ T cells is used to show the tolerant cells can suppress the naive cells from effecting rej ection . Previous studies and controls for these studies showed that the culture of tolerant CD4⁺ T cells with specific donor stimulators in a mixed lymphocyte culture for 3 days , resulted in their loss of capacity to transfer tolerance . Culture of naϊve cells under similar conditions resulted in cells that effected rej ection .

When tolerant CD4⁺ T cells were cultured with specific donor stimulators and with a cytokine rich media of ConA supernatant, then they retained their capacity to transfer tolerance to specific donor but retained the capacity to effect third party graft rej ection as described (Transplantation 1993, 55, 374-379) . This baseline data was reproduced as controls to the experiments and was as per the previous described results .

Figure 6A

CD4⁺ T cells from DA rats tolerant to a PVG heart allograft were cultured with either IL-4 or IL-5 alone in MLC with PVG antigen and no ConA supernatant . The cells were subsequently administered to irradiated DA rats which had received either a graft from PVG rats or Lewis rats . The % survival of the grafts is shown in Figure 6A. As can be seen from Figure 6A, this experiment demonstrated that IL- 5 alone sustained the tolerance transferring specific suppressor CD4⁺ T cells . The same cells retained the capacity to effect third party graft rej ection, demonstrating that the IL-5 alone did not induce tolerance mediating cells . Neither IL-2 (Transplantation 1993 , 55 , 374-379) nor IL-4 ( see data above) alone maintain these suppressor CD4⁺ T cells in culture with specific antigen, while a Con A supernatant with all these cytokines maintains the specific suppres sor function of

CD4⁺ T cells .

Taken together these experiments with culture of CD4⁺ T cells from rats with allograft tolerance demonstrated that IL-5 alone can substitute for the ConA supernatant . Either IL-2 or IL-4 alone is insufficient to maintain the tolerance maintaining cells .

Figure 6B

Figure βB illustrates the results of examination of the effect of culture in MLC with IL-4 and specific donor antigen on the capacity of tolerant CD4⁺, CD25⁺ T cells and CD4⁺, CD25^~ T cells to proliferate in vitro when exposed to different stimulator cells .

CD4⁺, CD25⁺ T cells from DA rats tolerant to a PVG cardiac allograft were cultured against PVG ( specific allogeneic) stimulator cells in a primary MLC with or without IL-4 for 3 days . These cells were subsequently washed, rested for 24 hours and then cultured in a secondary MLC alone or against DA ( syngeneic) , PVG ( specific allogeneic) or Lewis (3rd party allogeneic) stimulator cells . Proliferation was assessed at day 2-3 of secondary culture .

The tolerant CD4⁺, CD25⁺ T cells have significantly reduced proliferation against specific donor PVG whether or not IL-4 is added to the primary MLC . The CD4⁺, CD25^~ T cells had a similar response to donor and third party stimulators . These data are consistent with the findings in the adoptive transfer assay where IL-4 did not maintain the suppressor effect of unfractionated CD4⁺ T cells , suggesting the specifically tolerant CD4⁺, CD25⁺ T cells die in culture if IL-4 is the only cytokine available . Also that the CD4⁺, CD25^~ T cells cultured with IL-4 survive and maintain alloreactivity to specific donor and third party . These results are consistent with the adoptive transfer studies described in Figure 6A where tolerant cells cultured with IL-4 do not maintain their ability to transfer specific tolerance . This study also demonstrates that the culture of specific CD4⁺, CD25^" T cells from tolerant animals cannot be maintained by IL-4 and IL-4 does not reinduce tolerance . That is activated CD4⁺, CD25⁺ T cells from tolerant animals are not maintained or expanded by the IL-4 and alloantigen .

Conclusion

The Th2 cytokine IL-5 but not IL-4 , promotes the survival of specific CD4⁺, CD25⁺ T cells tolerance mediating suppressor cells in culture and allows them to retain the ability to transfer tolerance . a) Survival of the alloantigen specific tolerance mediating cells in CD4⁺ T cells in MLC can be supported by IL-5 , Con A sup . IL-4 supplement alone does not promote survival of the tolerance mediating CD4⁺ T cells . b) The failure of tolerant CD4⁺, CD25⁺ T cells to proliferate to specific donor in MLC is not restored by pre-culture with specific donor alloantigen and IL-4.

Example 7 This study examined the effect of various cytokines on proliferation of activated CD4⁺, CD25⁺ T cells in MLC .

Figure 7 Figure 7 examines the effect of individual cytokines on proliferation in MLC of unfractionated lymphocytes from DA rats with tolerance to PVG heart allografts .

In this experiment the effects of various cytokines on proliferation of unfractionated lymphocytes from DA rats with tolerance to a PVG allograft were examined in MLC . The proposal was that key cytokines would promote survival of the activated CD4⁺, CD25⁺ T cells that maintain tolerance and these would inhibit the proliferative response of unfractionated cells in MLC .

This figure shows IL-2 and IL-4 markedly enhance proliferation of unfractionated lymphocytes to self DA and to specific-donor PVG . Both IL-5 and IFN-γ inhibit responses to PVG but not self . Other cytokines had a similar effect, including IL-IO , IL-12p40 homodimer, and IL-12p70.

This data suggest the test for the tolerant state may be detected by adding IL-5, IL-12 or IFN-γ. These cytokines may inhibit proliferation of unfractionated tolerant lymphocytes by promoting the survival and function of the donor antigen specific activated CD4⁺, CD25⁺ T cells so they suppress the proliferative response in MLC . This effect may be detected by decreased proliferation or by enhanced nitric oxide production or by other means that detect active function of the activated CD4⁺, CD25⁺ T cells .

Conclusion Addition of cytokines that promote the survival of activated CD4⁺, CD25⁺ T cells from tolerant animals reduces the proliferation of unfractionated peripheral lymphocytes to specific donor antigen in MLC, in particular IL-5 , IFN-γ and IL-12.

Example 8

This study examined the effect of IL-2 , IL-4 and a nitric oxide inhibitor on proliferation of CD4⁺, CD25⁺ T cells .

Figure 8

Figure 8 illustrates the effect of cytokines and iNOS inhibitors on culture of naϊve CD4⁺, CD25⁺ T cells stimulated by self and with either IL-2 or IL-4.

This experiment investigated whether naϊve CD4⁺, CD25⁺ T cells cultured with IL-2 or IL-4 would die unless stimulated with a second cytokine that would be required to promote ongoing growth, and whether the induction of iNOS in this process would produce NO that would inhibit further growth .

Naϊve CD4⁺, CD25⁺ T cells from Sprague-Dawley rats were cultured with either IL-2 or IL-4 in the presence or absence of L-NIL in bulk cultures for 3 days (as per the methods with autologous rat serum and usual media as described above) . These cells were then placed in micro wells and subj ected to a further 3 days culture with a variety of individual cytokines (as indicated in Figure 8 ) for 3 more days of culture before proliferation was assayed . Naϊve CD4⁺, CD25⁺ T cells grown with IL-2 died without a cytokine supplement, as shown in first two bars of first panel of Figure 8. However, in the presence of L-NIL these cells survive and grow without further cytokines, first two bars of second panel of Figure 8. Re-exposure to IL-2 and to a lesser extent IL-4 allowed further proliferation of the CD4⁺, CD25⁺ T cells culture with IL-2. L-NIL enhanced proliferation of IL-2 exposed cells to further IL-2.

For naϊve CD4⁺, CD25⁺ T cells cultured with IL-4 (see third panel) , they again died without further cytokines, but did grow if cultured again with either IL-2 or IL-4 , with a much lesser effect with IL-4. The addition of L-NIL had no effect on the survival of IL-4 stimulated cells (see fourth panel) .

These results showed that inhibition of i-NOS can enhance growth of CD4⁺, C25⁺ T cells cultured with IL-2.

Conclusions

The proliferation of naϊve CD4⁺, CD25⁺ T cells induced by

IL-2 is enhanced by blocking iNOS . a) Naϊve CD4⁺, CD25⁺ T cells cultures with IL-2 but not IL-4 have enhanced growth if iNOS is blocked . b) Naϊve CD4⁺, CD25⁺ T cells cultured with self antigen and IL-2 and IL-4 , have some growth induced by IL-12 and TGF-β. There is no additive effect of IFN-γ or IL- 5 as in this setting specific antigen is not available and full expression of the receptors for these cytokines may has not occurred . c) Culture of naϊve CD4⁺, CD25⁺ T cells with specific antigen and IL-2, may lead to a state where blocking iNOS and other inhibitors of activated naϊve

CD4⁺, CD25⁺ T cells combined with IFN-γ and/or other cytokine (not IL-2 , IL-4 or IL-5 ) may lead to prolonged growth to allow clonal expansion of TsI cells and the generation of specific CD4⁺, CD25⁺ TsI cells and/or CD4⁺, CD8⁺, CD25⁺ T cells that can mediate, promote or restore tolerance . d) Culture of naive CD4⁺, CD25⁺ T cells with specific antigen and IL-4 , may lead to a state where these cells' growth and expansion can be facilitated by cytokines such as IL-5 and/or other cytokine (not IL- 2 , IL-4 or IFN-γ), and may be inhibited by cytokines , such as IL-4. Substitution of IL-4 at days 4-6 in culture with IL-5 and/or other cytokine (not IL-2 , IL-4 or IL-5) may facilitate growth of specific Ts2 cells and the generation of specific CD4⁺, CD25⁺ TsI cells and/or CD4⁺, CD8⁺, CD25⁺ T cells that can mediate, promote or restore tolerance . e) Polyclonally activated CD4⁺, CD25⁺ T cells, ie . those not responding to a specific antigen, will not grow further when cultured with either IL-5 or IFN-γ, thereby allowing growth of antigen specific CD4⁺, CD25⁺ T cells when these polyclonally activated populations are grown with a specific antigen and either IL-5 or IFN-γ. f) Initial culture with specific antigen and IL-2 and/or IL-4 may lead to polyclonal activation of CD4⁺, CD25⁺ T cells that recognise the specific antigen .

Example 9 This study examined the ability to treat EAN in rats by administering activated CD4⁺, CD25⁺ T cells .

Methods

Methods; EAN was induced in 10-15 week old female Lewis rats by immunization with bovine peripheral nerve myelin in Freund' s complete adjuvant, as described (J . Neurol . Sci . 1994 , 123 : 162-172 ) . The animals were monitored for disease activity daily by weighing and clinical observation and scoring of paralysis using a semiquantitative score . The score used was ; 5+ death or total paralysis requiring euthanasia, 4+ paralysis of all limbs , 3+ Total hind limb paralysis , and weak forearms , 2+ weak hind limbs , 1+ weak tails , 0 normal .

Results

Figure 9

In Figures 9A and 9B, the effect of adoptive transfer of activated CD4⁺, CD25⁺ T cells or CD4⁺, CD25^~ T cells on the clinical course of Experimental Allergic Neuritis (EAN) in Lewis rats was examined .

As proof of concept , the effect of CD4⁺, CD25⁺ T cells from Lewis rats that had recovered from EAN on the development of EAN in naϊve rats was examined by adoptive transfer of these cells to naϊve Lewis rats at the time of their immunization with PNM in Freund' s complete adj uvant . The effect of these tolerant CD4⁺, CD25⁺ T cells was compared to the effect of CD4⁺, CD25^~ T cells from these tolerant animals . In this model rats that recover, appear normal and are resistant to re-induction of EAN by re-immunization with immunogenic antigen . That is they are considered tolerant after their original disease, thus rats that had j ust recovered from EAN were considered a good source of activated tolerant CD4⁺, CD25⁺ T cells . Lewis rats that had recovered from EAN at 30 days post immunization were thus used to prepare CD4⁺, CD25^~ T cells and CD25⁺, CD4⁺ T cells . All groups of Lewis rats (n=5-12 ) were immunized with peripheral nerve myelin (PNM) and Freund' s adj uvant . One group was given 5 million CD25⁺, CD4⁺ T cells ivi and another group was given 5 million CD4⁺, CD25^~ T cells ivi . The control group was not given any cells at the time of immunization . Those given activated CD4⁺, CD25⁺ T cells from tolerant rats had a much milder clinical course with a maximum disease score j ust over one, compared to controls whose diseases peaked at 2.5+ around 15-16 days post immunization . Rats given CD4⁺, CD25^~ T cells from tolerant animals developed more severe disease peaking at 3+ and with an earlier onset and slower recovery .

In another experiment naive CD4⁺CD25⁺ T cells were given on day 0 of immunization . These had no effect on the clinical course of EAN confirming this was an effect of activated CD4⁺, CD25⁺ T cells from a tolerant animal .

Weight loss was much less in those given activated CD4⁺, CD25⁺ T cells from a tolerant animal and slightly greater in those given CD25^~, CD4⁺ T cells from a tolerant animal than controls given no cells . ( Figure 9B) As adult rats have 500-1000 million peripheral lymphocytes , we suggest that 5 million CD4⁺, CD25⁺ T cells would not have had a significant impact of the CD25⁺, CD4⁺ T cell : CD4⁺, CD25^~ T cell ratio in these naive hosts . This is further supported by the finding that giving 5 million naϊve CD4⁺, CD25⁺ T cells had no effect on the course of EAN . Thus the effect was most likely due specifically to the specifically activated CD4⁺, CD25⁺ T suppressor cell effect , not the well described non-specific effect of naive CD4⁺, CD25⁺ T cells .

This suggests a finite number of activated CD4⁺, CD25⁺ T cells may significantly alter the disease course and reestablish tolerance, during an acute disease process .

Conclusions

Specific activated CD4⁺, CD25⁺ T cells from animals that have recovered from acute autoimmune diseases can ameliorate the severity of autoimmune disease .

We propose that autologous CD4⁺, CD25⁺ T cells may be obtained from diseased animals in remission and further activated to transfer back to maintain or re-establish tolerance .

Example 10

Cell subsets and cultures were as per the mixed lymphocyte cultures . Antigen presenting cells with PNM were prepared by pre-culturing for 1 hours at 37 degrees with PNM and the autologous stimulator cell preparation . These cells were then washed . Culture conditions and measurement of proliferation was as described for MLC . Re suits

These studies demonstrate distinct patterns of response of CD4⁺, CD25⁺ T cells to an autoantigen before and during exposure to the autoantigen, which in this case was PNM in the EAN model .

First the response of naϊve CD4⁺ T cells , CD4⁺, CD25⁺ T cells and CD4⁺, CD25^" T cells , showed that the CD4⁺, CD25^" T cells had a much greater response than the CD4⁺ T cells , and that the response of CD4⁺, CD25^" T cells could be inhibited by naϊve CD4⁺, CD25⁺ T cells . Naϊve CD4⁺, CD25⁺ T cells alone had little or no response to PNM . This was similar to the ability of naive CD4⁺, CD25⁺ T cells to inhibit naϊve CD4⁺, CD25^" T cells in MLC . ( Figure 10A)

Second in animals that are j ust recovering there is an active suppressor phenotype, where the CD4⁺, CD25⁺ T cells do not greatly respond to specific antigen and cannot fully inhibit the response of CD4⁺, CD25^" T cells to the specific antigen . We would predict that these cells may respond to IL-5 , IFN-γ and possibly IL-12. ( Figure 10B)

Figure 1OA.

Figure 1OA illustrates proliferation of lymphocytes from naϊve Lewis rats when stimulated in culture with self- antigen presenting cells with and without PNM . Proliferation at day 4 vs day 5.

The proliferation of naϊve CD4⁺, CD25⁺ T cells and CD4⁺, CD25^" T cells to that of naϊve unfractionated CD4⁺ T cells was examined . This response is similar to that observed with naive cells response to alloantigens in MLC . At day 4 the response of unfractionated CD4⁺ T cells was similar to self and self plus PNM . That of CD4⁺, CD25^~ T cells was greater than unfractionated CD4⁺ T cells , consistent with removal of the non-specific suppression by naive CD4⁺, CD25⁺ T cells . Again there was no difference in the response to self alone and self plus PNM . CD4⁺, CD25⁺ T cells had minimal proliferation alone, and when admixed with CD4⁺, CD25^" T cells in a normal ratio, suppressed the response to that of unfractionated CD4⁺ T cells . With a ratio of 1 : 1 naϊve CD4⁺, CD25⁺ T cells : naϊve CD4⁺, CD25^" T cells there was near total suppression .

The results on day 5 had an essentially similar pattern but there was greater proliferation than on day 4. Again admixing the two populations at 1 : 1 ratio markedly suppressed the response to both self alone and self with PNM .

Figure 1OB

Figure 1OB shows the result of a study in which T cell subsets were prepared from Lewis rats that had recovered from EAN, 30 days after immunization . Again culture stimulated with self antigen presenting cells alone or primed with PNM were set up and proliferation assayed at days 4 and 5. The response of unfractionated CD4⁺ T cells and CD4⁺, CD25^" T cells was greater to PNM than to self- antigen presenting cells , consistent with a specific sensitisation . The CD4⁺, CD25⁺ T cells alone had a nonspecific proliferation above that normally seen with naϊve CD4⁺, CD25⁺ T cells . This may be due to polyclonal activation of the CD4⁺, CD25⁺ T cells . The maj or difference was that the suppression with CD25⁺, CD4⁺ T cells at 1 : 1 against PNM was incomplete, whilst that to self was more complete . Our interpretation of the results of this assay is that there are two responses, that to self-antigens and that to PNM. The data is consistent with partial suppression of the anti-self response but failure to suppress that anti- PNM response . These data are similar to that observed in transplant tolerance in that the activated CD4⁺, CD25⁺ T cells which have alloantigen specificity do not suppress the response to the specific antigen, but retain their capacity to non-specifically inhibit a third party response .

Conclusions

That CD4⁺, CD25⁺ T cells from naive animals behave in autoimmune responses like they behave with alloimmune responses . a) That in naive animals there is no specific response to autoantigen, in that they are the same as that to self stimulators . b) That in naϊve animals removal of CD4⁺, CD25⁺ T cells enhances the response to self stimulators . That is naϊve CD4⁺, CD25^~ T cells proliferation is greater than that of unfractionated CD4⁺ T cells . c) Further naϊve CD4⁺, CD25^" T cells proliferation can be suppressed by admixing with CD4⁺, CD25⁺ T cells, especially at a 1 : 1 ratio . d) That in animals recovering from acute episode of autoimmunity, there is an enhanced response to specific antigen compared to self antigens, for CD4⁺ T cells and CD4⁺, CD25^~ T cells , but no specific response by CD4⁺, CD25⁺ T cells to the autoantigen . These CD4⁺, CD25⁺ T cells do not fully suppress the CD4⁺, CD25^~ T cells proliferation, and are more effective against the self response than against the specific alloantigen . That is the activated specific CD4⁺, CD25⁺ T cells do not suppress in vitro .

Example 11

This example illustrates the ability of CD4⁺, CD25⁺ T cells cultured in vitro in the presence of donor antigen and IL- 2 or IL-4 to suppress donor heart graft rej ection .

TsI cells ( activated CD4⁺, CD25⁺ T cells produced by alloantigen stimulation in the presence of IL-2 , expressing IFNGR and IL-12Rβ2 ) and Ts2 cells (activated CD4⁺, CD25⁺ T cells produced by alloantigen stimulation in the presence of IL-4 , expressing IL-5Rα and IFN-γ) were produced by 3 day culture of naϊve CD4⁺, CD25⁺ T cells from DA rats in the presence of antigen presenting cells from PVG rats and >100 units/ml of IL-2 or IL-4 respectively . These cells were then tested for their capacity to prevent PVG or third party cardiac allograft rej ection in irradiated DA rats restored with 5xlO⁶ naϊve CD4⁺ T cells . The results of the experiment are shown in Table 2. As can be seen from Table 2 , naϊve CD4⁺, CD25⁺ T cells in a ratio of 1 : 10 with naϊve CD4⁺ T cells did not suppress rej ection of hearts from PVG donor strains . Similarly, CD4⁺, CD25⁺ T cells contacted with PVG antigen in the presence of IL-2 or IL-4 and mixed in a ratio of 1 : 10 with CD4⁺ T cells did not suppress rej ection of hearts from Lewis donor strains . In contrast , CD4⁺, CD25⁺ T cells contacted with PVG antigen and incubated in the presence of IL-2 or IL-4 and mixed in a ratio of 1 : 10 with naive CD4⁺ T cells did suppress rej ection of hearts from PVG donor strains . Thus , both TsI and Ts2 cells at a ratio of 1 : 10 (TSl or TS2 T cells to CD4⁺ T cells) prevented rej ection of PVG but not Lewis allografts , demonstrating specificity of induction of suppression and enhanced suppression as naϊve CD4⁺CD25⁺ T cells at ratio of 1 : 10 did not suppress . , Naϊve CD4⁺CD25⁺ T cells at a ratio of 1 : 1 suppressed both PVG and Lewis rej ection . Thus a short culture with specific alloantigen and either IL-2 or IL-4 selected for and expanded specific suppressor CD4⁺CD25⁺ T cells leading to a 10-fold increase in suppressor capacity.

Table 2

Adoptive transfer* animals restored with Heart donor Animals with severe rejection* -

CD4⁺,CD25⁺ cells Naive CD4⁺ Number / Total Median Days post-transplant⁸ Significance rat strain

No. Culture with cells Day (number of animals) (P)*

- 5xlO⁶ PVG 12/12 12 10(3), 12(4), 13, 14(2), 18, 20 -

- 5xlO⁶ Lewis 3/3 10 9, 10(2) -

5xlO⁶ 5x10⁶ PVG 3/9 >100 16(3), >100(6) p=0.0006

0.5x10⁶ - 5xlO⁶ PVG 8/9 14 8, 13(3), 14(4), >100 NSD

0.5x10⁶ IL-2 and PVG cells 5xlO⁶ PVG 1/6 >100 12, >100(5) p=0.0066

0.5xl0⁶ IL-2 and PVG cells 5x10⁶ Lewis 5/5 11 9, 11(4) NSD O O

0.5x10⁶ IL-4 and PVG cells 5xlO⁶ PVG 2/6 >50 10, 14, >50 (4) p=0.0351

0.5xl0⁶ IL-4 and PVG cells 5xlO^δ Lewis 4/6 19(13-24) 8, 9, 13, 24, >50 (2) NSD

* In the adoptive transfer assay, the recipient and donor are irradiated and a heart graft performed one day later. The irradiated recipients do not reject their graft but rejection can be restored with naive CD4⁺ T cells.

I Severe rejection refers to rejection associated with major swelling, loss of contraction and slowing of beat, equivalent to major graft dysfunction. Clinically this severity 5 of rej ection would be incompatible with life.

§ Number of days post-transplantation at which severe rejection occurred for those animals which underwent rejection. Animals that did not have a severe rejection episode had excellent heart graft function for >50-100 days.

J Compared to animals reconstituted with 5xlO⁶ naive CD4⁺ T cells of the same heart donor strain. NSD; not significantly different.

Example 12

These experiments examined whether CD4⁺, CD25⁺ T cells from DA rats activated to antigen from PVG rats and cultured in the presence of PVG cells and IFN-γ were capable of suppressing rej ection of a PVG cardiac graft in DA rats .

CD4⁺ T cells from DA rats tolerant to a cardiac allograft from PVG rats were cultured in mixed lymphocyte culture with PVG alloantigen (as described in Example 1 ) and >100 units/ml of IFN-γ. After three days the T cells were adoptively transferred to irradiated DA rats grafted with either specific donor PVG cardiac allografts (grafts from PVG rats) , or third party Lewis allografts (grafts from Lewis rats ) . Each irradiated rat was then restored with 5xlO⁶ naive CD4⁺T cells . The survival of grafts was then monitored over a 50 day period . The results of the experiment are shown in Figure 11. The upper graph of Figure 10 shows the survival when T cells were adoptively transferred to irradiated DA rats grafted with PVG cardiac allografts . The lower graph of Figure 11 shows the survival when T cells were adoptively transferred to irradiated DA rats grafted with third party Lewis cardiac allografts .

As can be seen from Figure 11 , the only grafts which demonstrated significant survival over the 50 day period were grafts in rats which received no cells ( see Fig 10 upper and lower graphs, solid line) , or PVG grafts (but not Lewis grafts ) in rats which received CD4⁺ T cells cultured in the presence of PVG antigen and IFN-γ ( see Fig . 10 , upper graph, dotted line closed circles ) . The CD4⁺ T cells cultured with IFN-γ when admixed with 5xlO⁶ naive CD4⁺ T cells suppressed allograft rej ection, indicating that they retained suppressor capacity .

These results indicate that the cultured CD4+ T cells continued to suppress PVG allograft rej ection but did not suppress Lewis graft rej ection . Naϊve cells alone effected rej ection, whereas irradiation delayed rej ection .

Thus , these results demonstrate that IFN-γ can preserve survival of activated CD4+, CD25+ T cells in a CD4⁺ T cell population from tolerant animals .

Example 13

Figure 12 illustrates an experiment that was conducted to compare the proliferation of unfractionated CD4⁺ T cells , a CD4⁺CD25^~ T cell subset and a CD4⁺CD25⁺ T cell subset from naϊve DA rats after culturing the T cells for 4 days in the presence of autoantigen ( DA antigen ) or alloantigen (PVG antigen) in media supplemented with IL-2 or IL-12p70 or both IL-2 and Il-12p70. The effect of the antigen and cytokine combination on cell proliferation is shown in Figure 12. Proliferation of unfractionated CD4⁺ T cells is shown in the top row of graphs , proliferation of the CD4⁺CD25^~ T cell subset is shown in the middle row of graphs , and proliferation of the CD4⁺CD25⁺ T cell subset is shown in the lower row of graphs . The left hand column of graphs show proliferation in response to autoantigen, and the right hand column of graphs show proliferation in response to alloantigen (PVG) .

As can be seen from Fig . 12 , proliferation was not enhanced by IL-12p70 alone when compared to control nil cytokines . IL-2 induced marked proliferation of all subsets , and addition of IL-12p70 enhanced this proliferation in the CD4⁺ and the CD4⁺CD25⁺ populations but not in the CD4⁺CD25^" T cells .

These results indicate that Il-12p70 enhances proliferation of CD4⁺, CD25⁺ T cells that have been activated with antigen and IL-2 (TsI cells ) . In parallel experiments , culturing of cells with IL-4 and IL-12p70 did not enhance proliferation above that with IL- 4 clone .

Example 14

This experiment examined the effect of IL-2 and IL-12p70 on growth of TsI cells (CD4⁺, CD25⁺ T cells activated by culturing in the presence of antigen and IL-2 ) .

TsI cells were prepared by culturing naϊve CD4⁺CD25⁺ T cells in the presence of IL-2 and alloantigen ( PVG antigen) for 3 days . Cells were then washed and place in fresh media with alloantigen and either no supplement, CHO-K supernatant, IL-2 , IL-12p70 or IL-12p40 > 100 units/ml . Cell proliferation was then measured as described above . The results of cell proliferation are shown in Figure 13.

As can be seen from Figure 13 , cultures with control CHO-K supernatant or IL-12p40 exhibited proliferation similar to those with no supplement . Culturing in the presence of

IL-12p70 induced significant extra proliferation compared to controls , as did culturing in the presence of IL-2. These experiments indicate that TsI cells are responsive to IL-12p70.

Example 15

Proliferation of unfractionated CD4⁺ T cells , a CD4⁺CD25⁺ T cell subset and a CD4⁺CD25⁺ T cell subset from naϊve DA rats was compared after culture for 4 days against auto- antigen ( DA antigen) or alloantigen (PVG antigen) when media was supplemented with no cytokine, IL-4 or IL-12p70 or both IL-12 and IL-12p70. Proliferation was not enhanced by IL-12p70 alone when compared to control nil cytokines . IL-4 induced marked proliferation of all subsets , and addition of IL-12p70 did not enhance proliferation of any of the subpopulations . This showed IL-12p70 did not enhance proliferation of Ts2 cells .

Example 16

RT-PCR was used to analyse the expression of IL-2 and IL- 12β2 receptor mRNA in CD4⁺, CD25⁺ T cells from naϊve DA rats following culturing with alloantigen (cells from PVG rats ) or autoantigen (cells from DA rats ) and IL-2 or IL-4 > 100 units/ml . The results of the RT-PCR analysis are shown in Figure 14.

Figure 14 illustrates RT-PCR of GAPDH ( control ) , IL-2 or

IL-12β2 receptor mRNA following culture will alloantigen (PVG) or autoantigen (DA) for 4 days with no cytokine (upper panels ) ; IL-2 , (middle panel ) ; or IL-4 ( lower panel ) . As can be seen from Figure 14 , strong bands were observed for IL-12β2 receptor when cells were cultured with IL-2 and alloantigen . A feint band was observed for IL- 12β2 receptor following alloantigen stimulation alone . This result suggests preferential up regulation of the IL-

12 receptor on CD4⁺ CD25⁺ T cells activated by contacting with alloantigen in the presence of IL-2.

Example 17

EAN was induced in 10-15 week old female Lewis rats by immunization with bovine peripheral nerve myelin ( PNM) in Freund' s complete adj uvant, as described in J. Neurol . Sci . 1994 , 123 : 162-172. The animals were divided into three groups : (a) those immunised with PNM and Freund' s adjuvant only (control) ; (b) those immunised with PNM and Freund' s adjuvant and administered CHO cell supernatant ( control ) ; and (c) those immunised with PNM and Freund' s adj uvant and administered IL-5 ( 5000 units/day daily intraperitoneal inj ection from the day of onset of clinical for 10 days ) .

All groups of Lewis rats (n=5-12 ) were immunised with peripheral nerve myelin ( PNM) and Freund' s adjuvant as described above .

The animals were monitored for disease activity daily by weighing and clinical observation and scoring of paralysis using a semi-quantitative score . The score used was ; 4+ paralysis of all limbs , 3+ Total hind limb paralysis , and weak forearms , 2+ weak hind limbs , 1+ weak tails , 0 normal .

Referring to Figure 15A, the effect of administration of IL-5 on the clinical course of Experimental Allergic Neuritis (EAN) in Lewis rats was examined .

As can be seen from Figure 15A, those rats administered IL-5 had a milder clinical course with a maximum disease score just over one, compared to controls whose diseases peaked at 2.5+ around 15-16 days post immunization . Figure 15B illustrates weight loss over the course of the disease . Weight loss was less in those rats administered IL-5 when compared to the untreated control of following treatment with CHO cell supernatant .

The percent demyelination was also investigated in rats immunised with PMN with or without IL-5. The effect of treatment with IL-5 on demyelination at day 14 and 21 is shown in Figure 16. As can be seen from Figure 16 , treatment with IL-5 may reduce the demyelination normally observed in the EAN model .

The above data suggests that administration of IL-5 may be effective in reducing the severity of EAN by inducing ■ tolerance to PMN .

Example 18

To determine the expression of cytokines in animals immunised with PNM and treated with IL-5 as described in Example 16, real time PCR analysis of mRNA from draining lymph nodes (LN) and cauda equina (CE) was performed at day 14 and 21 post immunisation with PNM . The results of the real-time PCR is shown in Figure 17. Open bars represent mRNA levels of control rats that were not treated with IL-5 , while closed bars represent mRNA levels of rats treated with IL-5. Cytokines that were measured are as indicated at the top of the diagram.

As can be seen from Figure 17 , in the lymph node draining the site of immunization, there was a marked increase in IFN-γ expression but not IL-2 in IL-5 treated rats ( closed bars ) at day 21 compared to controls ( open bars ) , whereas IL-IO was reduced at day 14. In the cauda equina, there was marked reduction in IL-2 and IFN-γ, indicating reduced ThI cell infiltration . TCR-α was less in both the lymph nodes and cauda equina at day 21 but not day 14. TCR-α, IL- 10 and TNF-α results are expressed as copy number/100000 GAPDH copies . IL-2 and IFN-γ results are expressed as copy number/100 TCR-α copies . Data was combined of duplicate assays from 5 samples , expressed as mean±SD . Statistical significant differences *p<0.05 , ** p<0.005.

Example 19

Expression of IL-5 , IL-5 receptor, IL-4 and IL-13 in draining lymph nodes and cauda equine in rats that had been immunised with PNM and were either untreated (CTL) or treated with IL-5 as described in Example 16 were examined using semi-quantitative RT-PCR . The results of semiquantitative RT-PCR are shown in Figure 18.

Referring to Figure 18 , semi-quantitative RT-PCR of cytokine mRNA showed IL-5 treated animals have increased IL-5 and IL-5Rα in the lymph node draining the site of immunization . There was similar expression of IL-4 but reduced IL-13 in IL-5 treated compared to control (CTL) . In the cauda equina, IL-5 and IL-5Rα was still detected, even though TCR-α copies were reduced ( refer to figure 3 ) . Each box shown serial cDNA dilutions with neat on right and 1 : 10 dilution on the left . Control GAPDH expression was similar in all samples . Results from 5 samples per group with one representative sample shown . Example 20

The effect of naϊve and activated CD4⁺CD25⁺ T cells on proliferation of naϊve CD4⁺CD25^~ T cells in MLC was tested in a limiting dilution assay with a 1 : 2 serial dilutions of CD4⁺CD25⁺ T cells were mixed with a constant number of naϊve ( 10⁵) CD4⁺CD25^~ T cells . The results of the limiting dilution study are shown in Figure 19.

Figure 19 (A) shows the ability of fresh naϊve CD4⁺CD25⁺ T cells to partially suppress responses to PVG antigen and Lewis antigen, at a ratio of 1 : 1. The ability of the naϊve T cells to suppress response to PVG antigen is significant diminished at a ratio of 1 : 8 (naϊve CD4⁺, CD25⁺ : CD4⁺, CD25^~ T cells ) .

Figure 19 (B) shows Ts 1 cells (CD4⁺, CD25⁺ T cells activated to antigen (in this case PVG antigen in the presence of IL-2 ) selectively fully suppress responses to PVG antigen to a ratio of 1 : 16 but only suppress to Lewis antigen (third party) at a ratio of 1 : 4 then lose suppression .

Referring to Figure 2OA, Figure 2OA shows that fresh naϊve CD4⁺CD25⁺ T cells partially suppress responses to PVG antigen and Lewis antigen, at a ratio of 1 : 1 but loose significant inhibition at a ratio of 1 : 8. Figure 20 (B) shows that Ts2 cells (CD4+, CD25+ T cells activated to (in this case PVG antigen) in the presence of IL-4 ) partially suppress both responses to PVG and Lewis antigen and do so in ratios similar to that observed with fresh naϊve cells . In other studies we have shown the MLC mainly induces ThI responses with induction of IL-2 and IFN-γ. The only Th2 cytokine induced is IL-4 with no IL-5 or IL-10 unless cells are exposed to extra IL-4 , when IL-2 and IFN-γ induction is significantly suppressed an IL-4 and IL-5 expression enhanced .

Example 21

This experiment was conducted to determine the affect of incubating CD4⁺, CD25⁺ T cells in the presence of IL-23 and IL-13 following activation of naive CD4⁺, CD25⁺ T cells in the presence of IL-2 or IL-4.

Naϊve CD4⁺, CD25⁺ T cells from naϊve DA rats were incubated with stimulator cells from PVG rats in the presence of either IL-2 ( group A) or IL-4 (group B) for 3 to 4 days as described above . Following 3 to 4 days , the cells were washed and the culture medium replaced with culture medium containing either 100 units /ml IL-12p70 (positive control ) , IL-23 , IFN-γ, IL-10 or IL-12p70 and IFN-γ for group A, and 100 units/ml IL-13 or IL-13 and IL-5 for group B . Negative control was CHO cell supernatant .

CD4⁺, CD25⁺ T cells incubated in the presence of IL-12p70 , IL-23 and IL-13 alone exhibited further proliferation . CD4⁺, CD25⁺ T cells incubated in the presence of IL-13 and IL-5 , or IL-12p70 and IFN-γ also showed enhanced proliferation .

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i . e . to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention .

Claims

- Ill -CLAIMS :

1. A method of growing CD4⁺, CD25⁺ T cells in vitro, comprising culturing CD4⁺, CD25⁺ T cells under conditions that inhibit the effect of nitric oxide production on the survival and/or proliferation of CD4⁺, CD25⁺ T cells .

2. The method of claim 1 , wherein the effect of nitric oxide production is inhibited by culturing the CD4⁺, CD25⁺ T cells in the presence of at least one nitric oxide inhibitor .

3. The method of claim 1 or 2 , wherein the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells .

4. The method of claim 3 , wherein the at least one factor capable of supporting activation, survival and/or proliferation of the CD4⁺ _f CD25⁺ T cells is a cytokine .

5. A method of growing CD4⁺, CD25⁺ T cells in vitro, comprising culturing CD4⁺ _f CD25⁺ T cells in the presence of at least one factor capable of supporting activation, and/or survival or proliferation of CD4⁺, CD25⁺ T cells , and a nitric oxide inhibitor .

6. The method of claim 1 to 5 , wherein the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one antigen .

7. The method of any one of claims 3 to 6 , wherein the at least one factor capable of supporting activation, and/or survival or proliferation of CD4⁺, CD25⁺ T cells , is at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

8. The method of any one of claims 3 to 6 , wherein the at least one factor capable of supporting activation, and/or survival or proliferation of CD4⁺, CD25⁺ T cells is IL-2 , or a biologically active fragment thereof, or a functionally equivalent molecule thereof .

9. The method of any one of claims 3 to 6 , wherein the at least one factor capable of supporting activation, and/or survival or proliferation of CD4⁺, CD25⁺ T cells is at least one cytokine selected from the group consisting of IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 and

IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

10. A method of growing CD4⁺, CD25⁺ T cells in vitro, comprising culturing naϊve CD4⁺, CD25⁺ T cells with one or more antigens in the presence of IL-2 , a biologically active fragment thereof or functionally equivalent molecule thereof, and a nitric oxide inhibitor .

11. A method of growing CD4⁺, CD25⁺ T cells in vitro , comprising culturing activated CD4⁺, CD25⁺ T cells with the antigen to which the activated CD4⁺, CD25⁺ T cells have been activated, in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-10 , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β and IFN-γ, or a biologically active fragment thereof, or a functionally equivalent molecule thereof, and a nitric oxide inhibitor .

12. A method of growing CD4⁺, CD25⁺ T cells in vitro , comprising the step of :

(a) culturing activated CD4⁺, CD25⁺ T cells with the antigen to which the activated CD4⁺, CD25⁺ T cells have been activated, in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , TGF-β and IFN- γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof, and a nitric oxide inhibitor; and (b) thereafter culturing the CD4⁺, CD25⁺ T cells in the absence of a nitric oxide inhibitor .

13. The method of claim 12 , wherein the CD4⁺, CD25⁺ T cells are cultured in step (b) in the presence of a factor capable of supporting activation, survival and/or proliferation of CD4⁺, CD25⁺ T cells .

14. The method of claim 13 , wherein the factor is IL-2 , a biologically active fragment thereof, or a functionally equivalent molecule thereof .

15. The method of claim 13 , wherein the factor is at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5, IL-IO , IL-12 , IL-13, IL-15, IL-18 , IL- 23 , TGF-β and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof .

16. The method of claim 11 , wherein the CD4⁺, CD25⁺ T cells have been activated to the specific antigen in a subj ect from which the CD4⁺, CD25⁺ T cells have been taken .

17. The method of any one of claims 2 to 14 , wherein the nitric oxide inhibitor inhibits nitric oxide production during culturing of the CD4⁺, CD25⁺ T cells .

18. The method of claim 17 wherein the inhibitor of nitric oxide production is an iNOS inhibitor .

19. The method of claim 18 wherein the iNOS inhibitor is selected from the group consisting of L-NIL, L-NAME, aminoguanidine , GDIPS, FeTPPS , N- ( 3- aminomethyl ) benzyl) acetamidine dihydrochloride .

20. The method of any one of claims 2 to 14 wherein the nitric oxide inhibitor is a compound which removes nitric oxide from the medium in which the CD4⁺, CD25⁺ T cells are cultured .

21. The method of any one of claims 2 to 14 wherein the nitric oxide inhibitor inhibits nitric oxide production by blocking nitric oxide synthase expression in the CD4+, CD25+ T cells .

22. The method of any preceding claim further comprising the step of incubating the CP4⁺, CD25⁺ T cells with an antibody which reduces proliferation of CD4⁺, CD25^" T cells .

23. The method of claim 22 wherein the antibody is selected from the group consisting of anti-CD3 , anti- CD45RB/RO or an antibody which specifically binds to CD4⁺, CD25^~ T cells .

24. The method of any one of claims 6 or 10 to 16, wherein the at least one antigen is an autoantigen .

25. The method of any one of claims 6 or 10 to 16 , wherein the at least one antigen is an alloantigen .

26. The method of any one of claims 6 or 10 to 16 , wherein the at least one antigen is a xenoantigen .

27. The method of any one of claims 6 or 10 to 16 , wherein the at least one antigen is an allergen .

28. The method of claim 6 or 10 to 16 wherein the at least one antigen is a self antigen .

29. A method of increasing tolerance in a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of CD4⁺, CD25⁺ T cells grown in vitro in accordance with the method as claimed in any one of claims 1 to 28.

30. The method of claim 29 wherein the CD4⁺, CD25⁺ T cells are cultured in the presence of at least one factor capable of supporting activation, and/or survival or proliferation of CD4⁺, CD25⁺ T cells , and a nitric oxide inhibitor .

31. The method of claim 29 or 30 , further comprising reducing the number of CD4⁺ T cells in the subj ect prior to administering the CD4⁺CD25⁺ T cells .

32. The method of claim 31 wherein the number of CD4⁺ T cells are reduced by administering to the subj ect antibodies which bind to CD4⁺ T cells .

33. The method of 32 , wherein the antibodies are selected from the group consisting of anti-CD3, anti-CD4 , anti-

CD45RB/RO , anti-lymphocyte globulin or anti-thymocyte globulin .

34. The method of any one of claims 29 to 33 , wherein the CD4⁺, CD25⁺ T cells are further cultured prior to administration to the subj ect in the presence of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IFN-γ, IL-12p70 , IL-IO , IL-13 , IL-15 , IL-18 , IL- 23 and TGF-β in vitro .

35. The method of any one of claims 29 to 34 comprising the further step of administering to the subj ect an effective amount of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IFN-γ, IL-12p70 , IL-IO , IL-13 , IL-15 , IL-18 , IL-23 and TGF-β, prior to, simultaneously with, or subsequent to, administering the CD4⁺, CD25⁺ T cells .

36. A method for treating or preventing in a subj ect in need thereof a disease resulting from an immune response to an antigen, the method comprising the step of administering to the subj ect a therapeutically effective amount of CD4⁺, CD25⁺ T cells activated to the antigen grown in vitro in accordance with a method as claimed in any one of claims 1 to 28.

37. The method of claim 36 wherein the disease is a disease resulting from an immune response to the antigen .

38. The method of claim 36 wherein the antigen is an autoantigen .

39. The method of claim 36 wherein the antigen is an alloantigen or a xenoantigen .

40. The method of claim 36 wherein the antigen is an allergen in contact with the subj ect .

41. A kit for use with the methods of any one of claims 1-40 , comprising one or more cytokines selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL-12 , IL-13 , IL-15, IL-18 , IL-23 , TGF-β and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof, and a nitric oxide inhibitor .

42. A method of inducing tolerance in a subj ect in need thereof, the method comprising administering to the subj ect an effective amount of at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-10 , IL-12 , IL-13 , IL-15 , IL-18 , IL-23 , IFN-γ and TGF-β, a biologically active fragment thereof, or a functionally equivalent molecules thereof, and an effective amount of a nitric oxide inhibitor .

43. The method of claim 42 wherein the nitric oxide inhibitor is administered simultaneously with, or subsequent to, administration of the at least one cytokine .

44. A kit when used with the method of claim 42 or 43 , comprising at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-12 , IL-13 , IL-15, IL-

18 , IL-23 , TGF-β and IFN-γ, a biologically active fragments thereof, or a functionally equivalent molecules thereof, and one or more nitric oxide inhibitor .

45. A method of assessing whether a subj ect comprises CD4⁺, CD25⁺ T cells that have been activated to a specific antigen, comprising :

(d) obtaining from the subj ect a sample of lymphocytes comprising CD4 4., CD254. T cells; '

(e) incubating at least one portion of the sample of lymphocytes so as to promote distinction of CD4⁺, CD25⁺ T cells that have been activated to the specific antigen from those that have not been activated to the specific antigen;

( f) thereafter determining whether' CD4⁺, CD25⁺ T cells activated to a specific antigen are present in the sample .

46. The method of claim 45, wherein the sample of lymphocytes is incubated with a test antigen .

47. The method of claim 45, wherein the test antigen is the specific antigen .

48. The method of claim 45 , wherein the at least a portion of the sample of lymphocytes is incubated in the presence of the specific antigen and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5, IL-12 , IL-13 , IL-15 , IL-18 , IL-23 and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof and the presence of CD4⁺, CD25⁺ T cells activated to a specific antigen in the sample is determined by detecting nitric oxide production by

CD4⁺, CD25⁺ T cells .

49. The method of claim 45 , wherein the sample of lymphocytes comprises CD4⁺, CD25⁺ T cells and CD4⁺, CD25^~ T cells , and the at least one portion of the sample is incubated in the presence of the specific antigen, a nitric oxide inhibitor and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-IO , IL- 12 , IL-13 , IL-15 , IL-18 , IL-23 and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof, and the presence of CD4⁺, CD25⁺ T cells activated to a specific antigen is determined by detecting a decrease in proliferation of CD4⁺, CD25^~ T cells , or an increase in proliferation of CD4⁺, CD25⁺ T cells .

50. The method of claim 45, wherein the at least one portion of the sample of lymphocytes is incubated in the presence of specific antigen, a nitric oxide inhibitor, and at least one cytokine selected from the group consisting of IL-2 , IL-4 , IL-5 , IL-10 , IL-12 , IL-13 , IL- 15 , IL-18 , IL-23 and IFN-γ, a biologically active fragment thereof, or a functionally equivalent molecule thereof, and the presence of CD4⁺, CD25⁺ T cells activated to a specific antigen is determined by detecting an increase in proliferation of CD4⁺, CD25⁺ T cells .

51. A composition comprising CD4⁺, CD25⁺ T cells and/or pharmaceutically acceptable carrier wherein the CD4⁺, CD25⁺ T cells have been cultured in accordance with a method as claimed in any one of claims 1 to 28.

52. The method of any one of claims 29 to 51 , wherein the subj ect is a mammal .

53. The method of any one of claims 29 to 52 wherein the subj ect is a human .