Method for identifying and obtaining activated T-cells that have a demethylated Foxp3 TSDR region

Abstract

This disclosure provides a method of identifying or separating activated regulatory T cells that have a demethylated Foxp3 TSDR region from a mixture of activated T cells. The mixture is depleted of cells bearing the CD154 molecule (CD40 ligand). The desired cells can be recovered using a Treg marker such as CD25, GITR, CTLA4, CD137, latent TGF-beta (LAP), GARP (LRRC32) or CD121a/b. The Treg cells having a demethylated Foxp3 TSDR region can be used, for example for preparation of pharmaceutical compositions for use in treatment.

1 . A method of identifying activated regulatory T cells that have a demethylated Foxp3 TSDR region in a cell mixture, the method comprising:
 (1) obtaining a cell mixture that includes such cells and other T cells;   (2) contacting the cell mixture with a first molecule that specifically binds CD154;   (3) contacting the cell mixture with a second molecule that specifically binds an additional marker that is different from CD154 and is specifically present on regulatory T cells; and   (4) detecting cells in the mixture that have not bound with the first molecule but have bound the second molecule,   thereby identifying activated regulatory T cells in the cell mixture that have a demethylated Foxp3 TSDR region.   
     
     
         2 . The method of  claim 1 , wherein the additional marker is CD137. 
     
     
         3 . The method of  claim 1 , wherein the additional marker is selected from CD25, GITR, latent TGF beta (LAP), GARP (LRRC32), and CD121a/b. 
     
     
         4 . The method of  claim 1 , wherein the first molecule and the second molecule are each an antibody or an antibody fragment. 
     
     
         5 . The method of  claim 1 , wherein the first molecule and the second molecule are fluorescently labeled, and cells that have not bound the first molecule but have bound the second molecule are detected by flow cytometry. 
     
     
         6 . The method of  claim 1 , further comprising determining demethylation of the Foxp3 TSDR region in the cells detected in step (4). 
     
     
         7 . The method of  claim 1 , further comprising separating cells that have not bound the first molecule but have bound the second molecule from other cells in the cell mixture. 
     
     
         8 . The method of  claim 7 , further comprising administering the separated cells to a subject in need thereof. 
     
     
         9 . A method of producing an enriched cell population of activated regulatory T cells that have a demethylated Foxp3 TSDR region, the method comprising:
 (1) obtaining a cell mixture that includes such cells and other T cells;   (2) combining cells from the cell mixture with a first molecule that specifically binds CD154;   (3) separating and recovering cells that have not bound the first molecule;   (4) combining cells from the cell mixture with a second molecule that specifically binds an additional marker that is different from CD154 and is specifically present on regulatory T cells; and   (5) separating and recovering cells that have bound the second molecule;   wherein the combining the cells with the first molecule is done before, during, or after the combining the cells with the second molecule;   thereby obtaining an isolated population of activated regulatory T cells that have a demethylated Foxp3 TSDR region.   
     
     
         10 . The method of  claim 9 , whereby CD154 positive cells are labeled with a fluorescent label, and separation of CD154 positive cells is performed by flow cytometry. 
     
     
         11 . The method of  claim 9 , comprising combining the cell mixture with both the first molecule and the second molecule; wherein the first molecule and the second molecule are labeled with different fluorescent labels; and
 separating cells from the cell mixture by fluorescence activated cell sorting for cells that have not bound the first molecule but have bound the second molecule.   
     
     
         12 . The method of  claim 9 , whereby CD154 positive cells are labeled with a magnetic microparticle, and separation of CD154 positive cells is performed using a magnetic field. 
     
     
         13 . The method of  claim 9 , comprising combining the cell mixture with both the first molecule and the second molecule; wherein the first molecule is labeled with a magnetic microparticle; and
 separating cells from the cell mixture that are not labeled with the microparticle but have bound the second molecule.   
     
     
         14 . The method of  claim 9 , wherein the additional marker is CD137. 
     
     
         15 . The method of  claim 9 , wherein the additional marker is selected from CD25, GITR, latent TGF beta (LAP), GARP (LRRC32) and CD121a/b. 
     
     
         16 . The method of  claim 9 , wherein the molecule binding CD154 is an antibody or an antibody fragment. 
     
     
         17 . The method of  claim 9 , wherein the cell mixture has been cultured in the presence of an antigen, and the isolated population is antigen specific. 
     
     
         18 . The method of  claim 9 , wherein the cell mixture has been cultured in the presence of a pan T cell stimulator (such as CD3/CD28 antibody), and the isolated population as a whole is not specific for any particular antigen. 
     
     
         19 . The method of  claim 9 , further comprising determining whether activated regulatory T cells obtained according to the method have a demethylated Foxp3 TSDR region. 
     
     
         20 . The method of  claim 9 , further comprising administering the isolated cells to a subject in need thereof.