Combination therapy for a stable and long term engraftment using specific protocols for t/b cell depletion

A method of treating a subject in need of an immature hematopoietic cell transplantation, the method comprising:

• (a) transplanting into a conditioned subject a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein said dose comprises at least about 5×106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained by separating said T cells from said immature hematopoietic cells, by a method selected from the group consisting of:
• (I) a method comprising:
• (i) adding an antibody to the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent;
• (ii) immobilizing said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field;
• (iii) washing said matrix to remove unbound cells;
• and
• (iv) removing said magnetic field to elute bound cells from said matrix;
• (II) based on a product secreted by said T cells;
• and
• (III) based on an expression of at least one surface marker of T cells selected from the group consisting of 4-1BBS, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF-beta (LAP), GARP (LRRC32) and CD121a/b;
• and subsequently
• (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per kilogram body weight, thereby treating the subject.

A method of treating a subject in need of an immature hematopoietic cell transplantation, the method comprising:

• (a) conditioning a subject under a reduced intensity conditioning protocol, wherein said reduced intensity conditioning comprises a total body irradiation (TBI) and a chemotherapeutic agent;
• (b) transplanting into the subject a dose of T cell depleted immature hematopoietic cells, wherein said T cell depleted immature hematopoietic cells comprise less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein said dose comprises at least about 5×106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained by separating said T cells from said immature hematopoietic cells, by a method selected from the group consisting of:
• (I) a method comprising:
• (i) adding an antibody to the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent;
• (ii) immobilizing said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field;
• (iii) washing said matrix to remove unbound cells;
• and
• (iv) removing said magnetic field to elute bound cells from said matrix;
• (II) based on a product secreted by said T cells;
• and
• (III) based on an expression of at least one surface marker of T cells selected from the group consisting of 4-1BB, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF-beta (LAP), GARP (LRRC32) and CD121a/b;
• and subsequently
• (c) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per kilogram body weight, thereby treating the subject.

A method of inducing donor specific tolerance in a subject in need of a non-syngeneic cell or tissue graft, the method comprising:

• (a) transplanting into a subject a dose of T cell depleted immature hematopoietic cells obtained from a non-syngeneic donor, wherein said T cell depleted immature hematopoietic cells comprise less than 5×105 CD3+ T cells per kilogram body weight of the subject, and wherein said dose comprises at least about 5×106 CD34+ cells per kilogram body weight of the subject, and wherein said T cell depleted immature hematopoietic cells are obtained by separating said T cells from said immature hematopoietic cells, by a method selected from the group consisting of:
• (I) a method comprising:
• (i) adding an antibody to the immature hematopoietic cells that specifically binds to a surface marker, wherein said antibody is labeled with a magnetically responsive agent;
• (ii) immobilizing said immature hematopoietic cells specifically bound to said antibody labeled with said magnetically responsive agent in a matrix through a magnetic field;
• (iii) washing said matrix to remove unbound cells;
• and
• (iv) removing said magnetic field to elute bound cells from said matrix;
• (II) based on a product secreted by said T cells;
• and
• (III) based on an expression of at least one surface marker of T cells selected from the group consisting of 4-1BB, FoxP3, CD154, CD4, CD8, CD25, GITR CD137, latent TGF-beta (LAP), GARP (LRRC32) and CD121a/b;
• and subsequently
• (b) administering to the subject a therapeutically effective amount of cyclophosphamide, wherein said therapeutically effective amount comprises 25-200 mg per kilogram body weight, thereby inducing donor specific tolerance in the subject.