Use of apoptotic cells ex vivo to generate regulatory T Cells

1 . A method of generating T cells with regulatory activity (T regs) comprising incubating leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).

2 . The method according to claim 1 wherein the incubation is for a time and under conditions sufficient to generate T regs.

3 . The method according to claim 1 further comprising the step of selecting leukocytes expressing CD4 to obtain CD4+ cells.

4 . The method according to claim 3 wherein the incubation is at about a 1:10 to about a 10:1 ratio CD4+:ACs.

5 . The method according to claim 4 wherein the incubation is at about a 2:1 to about a 1:2 ratio CD4+:ACs.

6 . The method according to claim 1 wherein the ACs are obtained by an apoptosis-inducing treatment.

7 . The method according to claim 6 wherein the apoptosis-inducing treatment is an ECP procedure that employs a photoactivatable compound together with light of a wavelength that activates the photoactivable compound.

8 . The method according to claim 7 wherein the photoactivable compound is a psoralen and the light is UVA.

9 . The method according to claim 8 wherein the psoralen is 8-MOP.

10 . The method according to claim 1 wherein the incubation further comprises adding factors that further enhance generation or function of the T regs.

11 . The method according to claim 10 wherein the factors are hormones, proteins, drugs or antibodies.

12 . The method according to claim 11 wherein the factors are selected from at least one of TGFβ, αMSH, anti-CD46, IL-10, vitamin D 3 , dexamethasone, rapamycin and IL-2.

13 . The method according to claim 12 wherein the factor is IL-10.

14 . The method according to claim 13 wherein the IL-10 is present at a concentration of about 1 ng/ml to about 100 ng/ml.

15 . The method according to claim 14 wherein the IL-10 is present at a concentration of about 20 ng/ml.

16 . The method according to claim 1 wherein the incubation is for about 1 to about 14 days.

17 . The method according to claim 16 wherein the incubation is for about 8 days.

18 . The method according to claim 1 further comprising adding an antigen to the incubation to generate Tregs which regulate immune response to the antigen.

19 . The method according to claim 18 wherein the antigen is an alloantigen.

20 . A composition comprising a population of T cells with regulatory activity (T regs) obtained by incubating leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).

21 . The composition according to claim 20 wherein the incubation is for a time and under conditions sufficient to generate T regs.

22 . The composition according to claim 20 further comprising the step of selecting leukocytes expressing CD4 to obtain CD4+ cells.

23 . The composition according to claim 22 wherein the incubation is at about a 1:10 to about a 10:1 ratio CD4+:ACs.

24 . The composition according to claim 23 wherein the incubation is at about a 2:1 to about a 1:2 ratio CD4+:ACs.

25 . The composition according to claim 20 wherein the ACs are obtained by an apoptosis-inducing treatment.

26 . The composition according to claim 25 wherein the apoptosis-inducing treatment is an ECP procedure that employs a photoactivable compound together with light of a wavelength that activates the photoactivable compound.

27 . The composition according to claim 26 wherein, the photoactivable compound is a psoralen and the light is UVA.

28 . The composition according to claim 27 wherein the psoralen is 8-MOP.

29 . The composition according to claim 20 wherein the incubation further comprises adding factors that further enhance generation or function of the T regs.

30 . The composition according to claim 29 wherein the factors are hormones, proteins, drugs or antibodies.

31 . The composition according to claim 30 wherein the incubation further comprises adding factors selected from at least one of TGFβ, αMSH, anti-CD46, IL-10, vitamin D 3 , dexamethasone, rapamycin and IL-2.

32 . The composition according to claim 31 wherein the factor is IL-10.

33 . The composition according to claim 32 wherein the IL-10 is present at a concentration of about 1 ng/ml to about 100 ng/ml.

34 . The composition according to claim 33 wherein the IL-10 is present at a concentration of about 20 ng/ml.

35 . The composition according to claim 20 wherein the incubation is for about 1 to about 14 days.

36 . The composition according to claim 35 wherein the incubation is for about 8 days.

37 . The composition according to claim 20 further comprising adding an antigen to the incubation to generate Tregs which regulate immune response to the antigen.

38 . The composition according to claim 37 wherein the antigen is an alloantigen.

39 . A method of treating autoimmune disorder or ameliorating one or more symptoms thereof, comprising administering to a patient in need thereof an effective amount of a composition comprising a population of T cells with regulatory activity (T regs) obtained by incubating autologous leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).

40 . The method according to claim 39 wherein the apoptosis-inducing treatment is an ECP procedure that employs a photoactivable compound together with light of a wavelength that activates the photoactivable compound.

41 . The method according to claim 40 wherein the photoactivable compound is a psoralen and the light is UVA.

42 . The method according to claim 41 wherein the psoralen is 8-MOP.

43 . The method according to claim 40 wherein the incubation further comprises adding factors that further enhance generation or function of the T regs.

44 . The method according to claim 43 wherein the factors are hormones, proteins, drugs or antibodies.

45 . The method according to claim 44 wherein the factors are selected from at least one of TGFβ, αMSH, anti-CD46, IL-10, vitamin D 3 , dexamethasone, rapamycin and IL-2.

46 . The method according to claim 45 wherein the factor is IL-10.

47 . The method according to claim 46 wherein the IL-10 is present at a concentration of about 1 ng/ml to about 100 ng/ml.

48 . The method according to claim 47 wherein the IL-10 is present at a concentration of about 20 ng/ml.

49 . The method according to claim 40 wherein the incubation is for about 1 to about 14 days.

50 . The method according to claim 49 wherein the incubation is for about 8 days.

51 . The method according to claim 40 further comprising adding an antigen to the incubation to generate Tregs which regulate immune response to the antigen.

52 . The method according to claim 51 wherein the antigen is an alloantigen.

53 . The method according to claim 40 wherein the autoimmune disorder is at least one of acute transverse myelitis, alopecia areata, Alzheimer's disease, amyotrophic lateral sclerosis, ankylosing spondylitis, antiphospholipid syndrome, atherosclerosis, autoimmune Addison's disease, autoimmune hemolytic anemia, Behcet's disease, bullous pemphigoid, cardiomyopathy, celiac sprue-dermatitis, Cerebellar Spinocerebellar Disorders, spinocerebellar degenerations (spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations), chronic alcoholism, alcohol-induced hepatitis, autoimmune hepatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory bowel disease, chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, cold agglutinin disease, CResT syndrome, Creutzfeldt-Jakob disease, Crohn's disease, Dejerine-Thomas atrophy, Dementia pugilistica, diabetes mellitus, Diffuse Lewy body disease, discoid lupus, disorders of the basal ganglia, disseminated intravascular coagulation, Down's Syndrome in middle age, drug-induced movement disorders, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, graft versus host disease, Graves' disease, Guillain-Barré syndrome, Hallerrorden-Spatz disease, Hashimoto's thyroiditis, Huntington's Chorea senile chorea, idiopathic pulmonary fibrosis, idiopathic thrombocytopenia purpura (ITP), IgA nephropathy, infantile or juvenile spinal muscular atrophy, insulin dependent diabetes, juvenile arthritis, Kawasaki's pathology, lesions of the corticospinal system, Leukemias, Hodgkin's lymphoma, non-Hodgkin's lymphoma, lichen planus, Ménière's disease, mixed connective tissue disease, multiple sclerosis, multiple systems degenerations (Mencel, Dejerine-Thomas, Shy-Drager, Machado-Joseph), myasthenia gravis, neurogenic muscular, Parkinson's disease, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychondritis, polyglandular syndromes, polymyalgia rheumatica, polymyositis dermatomyositis, primary agammaglobulinemia, primary biliary cirrhosis, Progressive supranuclear palsy, psoriasis, Raynaud's phenomenon, Reiter's syndrome, rheumatic fever, rheumatoid arthritis, sarcoidosis, scleroderma, Senile Dementia of Lewy body type, Sjögren's syndrome, stiff-man syndrome, Subacute sclerosing panencephalitis, systemic disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, mitochondrial multi-system disorder), systemic lupus erythematosus (SLE), Takayasu arteritis, temporal arteritis/giant cell arteritis, thyroidosis, ulcerative colitis, uveitis, vasculitis, vitiligo, Wegener's granulomatosis, Wernicke-Korsakoff syndrome.

54 . A method of treating atopic disease or ameliorating one or more symptoms thereof comprising administering to a patient in need thereof an effective amount of a composition comprising a population of T cells with regulatory activity (T regs) obtained by incubating autologous leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).

55 . The method according to claim 54 wherein the atopic disorder is at least one of chronic inflammatory pathologies and vascular inflammatory pathologies, including chronic inflammatory pathologies such as sarcoidosis, chronic inflammatory bowel disease, ulcerative colitis, and Crohn's pathology and vascular inflammatory pathologies, such as, but not limited to, disseminated intravascular coagulation, atherosclerosis, and Kawasaki's pathology.

56 . A method comprising administering to a transplant recipient an effective amount of a composition comprising a population of T cells with regulatory activity (T regs) obtained by incubating autologous leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).

57 . The method according to claim 56 wherein the T regs are administered to the transplant recipient according to a schedule selected from the group consisting of two days, one week prior to the transplantation; three days, one week prior to harvesting said transplant; two days a week for two weeks prior to the transplantation; and three days a week for three weeks prior to the transplantation.

58 . A method comprising administering to a GVHD patient an effective amount of a composition comprising a population of T cells with regulatory activity (T regs) obtained by incubating autologous leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).

59 . The method according to claim 58 wherein the T regs are administered to the transplant recipient according to a schedule selected from the group consisting of two days, one week prior to the transplantation; three days, one week prior to harvesting said transplant; two days a week for two weeks prior to the transplantation; and three days a week for three weeks prior to the transplantation.

60 . A method of treating patient with a disorder or the predisposition for a disorder comprising testing the patient to determine whether the patient has a disorder, and administering to a patient in need thereof an effective amount of a composition comprising a population of T cells with regulatory activity (T regs) obtained by incubating autologous leukocytes with autologous apoptotic peripheral blood mononuclear cells (ACs).