Methods for the isolation and expansion of cord blood derived T regulatory cells
View Patent ↗The present invention encompasses methods, and kits for the isolation and expansion of T regulatory cells having the CD45RA + phenotype, including such cells from human umbilical cord blood.
1. A method of isolating a regulatory T cell (T reg cell) from a population of phenotypically CD45RA + blood cells and culture activating said T reg cell to generate a suppressor cell line, wherein said suppressor cell line suppresses T cell proliferation in a mixed lymphocyte reaction by a factor greater than about 95% at a ratio of 1:32 (Treg: T cell), said method comprising:
a) isolating a population of mononuclear cells from a human umbilical cord blood sample;
b) contacting said population of mononuclear cells with an antibody that specifically binds CD25 under conditions suitable for formation of a mononuclear cell-antibody complex;
c) substantially separating said mononuclear cell-antibody complex from said population of mononuclear cells, thereby isolating a population of CD25 + cells, wherein said CD25 + cells are CD45RA + ;
d) expanding said isolated population of CD25 + cells in the presence of anti-CD3/CD28 antibody coated beads at a 3:1 bead to cell ratio;
e) activating said expanded population of CD25+ cells in the presence of IL-2;
f) substantially separating said activated population of CD25 + cells; thereby generating a suppressor cell line that uniformly express CD25, intracellular CTLA4, CD27 and CD62L.
2. The method of claim 1 , wherein said population comprises an enhanced population of phenotypically CD45RA + blood cells, wherein said blood cells are isolated from umbilical cord blood.
3. The method of claim 1 , wherein said T reg cell expresses the FoxP3 antigen.
4. The method of claim 1 , wherein said antibody in step b is selected from the group consisting of an isolated antibody, a biological sample comprising an antibody, an antibody bound to a physical support and a cell-bound antibody.
5. The method of claim 4 , wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a biologically active fragment of an antibody, and combinations thereof.
6. The method of claim 5 , wherein said biologically active fragment is selected from the group consisting of an Fab fragment, a F(ab′) 2 fragment, a Fv fragment, and an scFv fragment.
7. The method of claim 4 , wherein said physical support is selected from the group consisting of a microbead, a magnetic bead, an absorption column and an adsorption membrane.
8. The method of claim 1 , wherein said mononuclear cell-antibody complex is substantially separated from said population of mononuclear cells by a method selected from the group consisting of fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS).
9. The method of claim 1 , wherein steps b) and c) are repeated.
10. A method of isolating a regulatory T (T reg ) precursor cell and culture activating said precursor cell to generate a suppressor cell line, wherein said suppressor cell line suppresses T cell proliferation in a mixed lymphocyte reaction by a factor greater than about 95% at a ratio of 1:32 (Treg: T cell), said method comprising:
a) isolating a population of mononuclear cells from a human umbilical cord blood sample;
b) contacting said population of mononuclear cells with an antibody that specifically binds CD25 under conditions suitable for formation of a mononuclear cell-antibody complex; and
c) substantially separating said mononuclear cell-antibody complex from said population of mononuclear cells, thereby isolating a population of CD25 + cells, wherein said CD25 + cells are CD45RA + ;
d) expanding said isolated population of CD25 + cells in the presence of anti-CD3/CD28 antibody coated beads at a 3:1 bead to cell ratio;
e) activating said expanded population of CD25+ cells in the presence of IL-2;
f) substantially separating said activated population of CD25 + cells; thereby generate a suppressor cell line that uniformly express CD25, intracellular CTLA4, CD27 and CD62L.
11. The method of claim 10 , wherein said method produces a population of suppressor T cells increased in number by about 100-fold from the original isolated T cell population.
12. The method of claim 11 , wherein said population comprises an enhanced population of phenotypically CD3 + blood cells, wherein said blood cells are isolated from umbilical cord blood.
13. The method of claim 11 , wherein said T reg cell expresses the FoxP3 antigen.
14. The method of claim 11 , wherein said antibody in step b is selected from the group consisting of an isolated antibody, a biological sample comprising an antibody, an antibody bound to a physical support and a cell-bound antibody.
15. The method of claim 14 , wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a synthetic antibody, a biologically active fragment of an antibody, and combinations thereof.
16. The method of claim 15 , wherein said biologically active fragment is selected from the group consisting of an Fab fragment, a F(ab′) 2 fragment, a Fv fragment, and an scFv fragment.
17. The method of claim 14 , wherein said physical support is selected from the group consisting of a microbead, a magnetic bead, an absorption column and an adsorption membrane.
18. The method of claim 11 , wherein said mononuclear cell-antibody complex is substantially separated from said population of mononuclear cells by a method selected from the group consisting of fluorescence activated cell sorting (FACS) and magnetic activated cell sorting (MACS).
19. The method of claim 11 , wherein steps b) and c) are repeated.
20. The method of claim 1 , wherein said method produces a population of suppressor T cells increased in number by about 100-fold from the original isolated cell population.