Method and Kit for Rapid Isolation of Human Foxp3+ Treg Cells

Abstract

The present invention relates to methods for isolating human forkhead box P3 (Foxp3+) CD4+ regulatory T cells (herein referred to as Foxp3+ Treg cells) from a sample containing (i) peripheral blood mononuclear cells (PBMCs), (ii) a lymphocyte containing fluid, or (iii) a lymphocyte containing tissue, a kit for isolating human Foxp3+ Treg cells, and the use of anti-CD49d antibody for the isolation of human Foxp3+ Treg cells.

1 - 15 . (canceled) 
     
     
         16 . A method for isolating human Foxp3+ regulatory T cells comprising
 (a) treating a sample containing (i) peripheral blood mononuclear cells, (ii) a lymphocyte containing fluid, or (iii) a lymphocyte containing issue, with an anti-CD49d antibody and   (i) an anti-CD25 antibody or   (ii) an anti-CD127 antibody; and   (b) separating Foxp3+ Treg cells.   
     
     
         17 . The method of  claim 16 , wherein steps (a) and (b) are carried out simultaneously. 
     
     
         18 . The method of  claim 16 , wherein step (a) comprises treating the sample with an anti-CD25 antibody and an anti-CD127 antibody. 
     
     
         19 . The method of  claim 16 , wherein step (a) further comprises separating non-CD4+ T cells from the sample. 
     
     
         20 . The method of  claim 19 , wherein one or more antibodies are used to specifically deplete non-CD4+ T cells from the sample. 
     
     
         21 . The method of  claim 16 , wherein at least one of the antibodies used in step (a) is labeled or immobilized. 
     
     
         22 . The method of  claim 16 , wherein step (b) is carried out by depleting the sample of
 (i) CD49d+ cells via the anti-CD49d antibody;   (ii) CD127+ cells via the anti-CD127 antibody; or   (iii) CD49d+CD127+ cells via the anti-CD49d or anti-CD127 antibody.   
     
     
         23 . The method of  claim 16 , wherein step (b) is carried out using centrifugation, cell elutriation, magnetic separation, fluorescence activated cell sorting, immunological separation, adhesion, complement lysis or flow cytometry. 
     
     
         24 . The method of  claim 16 , wherein step (b) is carried out using magnetic cell separation, fluorescence activated cell sorting, or a column-based immunological separation. 
     
     
         25 . The method of  claim 16 , wherein an anti-CD45RO antibody is used as an additional antibody in step (a), and wherein the isolated Foxp3+ Treg cells are CD45RA+ T cells. 
     
     
         26 . The method of  claim 16 , wherein an anti-CD45RA antibody is used as an additional antibody in step (a), and wherein the isolated Foxp3+ Treg cells are CD45RO+ T cells. 
     
     
         27 . A method for isolating human Foxp3+ regulatory T cells comprising
 (a) treating a sample containing (i) peripheral blood mononuclear cells, (ii) a lymphocyte containing fluid, or (iii) a lymphocyte containing issue, with an anti-CD49d antibody, an anti-CD25 antibody, and an anti-CD127 antibody; and   (b) separating Foxp3+ Treg cells.   
     
     
         28 . The method of  claim 27 , wherein steps (a) and (b) are carried out simultaneously. 
     
     
         29 . The method of  claim 27 , wherein step (a) further comprises separating non-CD4+ T cells from the sample. 
     
     
         30 . The method according of  claim 27 , wherein one or more antibodies are used to specifically deplete non-CD4+ T cells from the sample. 
     
     
         31 . The method of  claim 27 , wherein at least one of the antibodies used in step (a) is labeled or immobilized. 
     
     
         32 . The method of  claim 27 , wherein step (b) is carried out by depleting the sample of
 (i) CD49d+ cells via the anti-CD49d antibody;   (ii) CD127+ cells via the anti-CD127 antibody; or   (iii) CD49d+CD127+ cells via the anti-CD49d and anti-CD127 antibodies.   
     
     
         33 . The method of  claim 27 , wherein step (b) is carried out using centrifugation, cell elutriation, magnetic separation, fluorescence activated cell sorting, immunological separation, adhesion, complement lysis or flow cytometry. 
     
     
         34 . The method of  claim 27 , wherein step (b) is carried out using magnetic cell separation, fluorescence activated cell sorting, or a column-based immunological separation. 
     
     
         35 . The method of  claim 27 , wherein an anti-CD45RO antibody is used as an additional antibody in step (a), and wherein the isolated Foxp3+ Treg cells are CD45RA+ T cells. 
     
     
         36 . The method of  claim 27 , wherein an anti-CD45RA antibody is used as an additional antibody in step (a), and wherein the isolated Foxp3+ Treg cells are CD45RO+ T cells. 
     
     
         37 . A kit for isolating human Foxp3+ Treg cells, comprising an anti-CD49d antibody and an anti-CD25 antibody or an anti-CD49d antibody and an anti-CD127 antibody. 
     
     
         38 . The kit of  claim 37 , wherein the kit comprises an anti-CD49d antibody, an anti-CD25 antibody, and an anti-CD127 antibody.