Methods and materials for the generation of regulatory t cells

Abstract

Methods are disclosed for the generation of immunosuppressive regulatory T cells. The methods can include contacting a population of CD4+CD25− T cells with a T cell receptor (TCR)/CD3 activator, a TCR co-stimulator activator, and rapamycin. Kits for the generation of immunosuppressive regulatory T cells, methods of use, and cell populations are also disclosed.

1 . A method of generating immunosuppressive regulatory T cells from a sample,
 the method comprising:   providing an initial sample containing a population of CD4+CD25− T cells; and   contacting the population with a T cell receptor (TCR)/CD3 activator, a TCR co-stimulator activator, and rapamycin to generate a final sample comprising immunosuppressive regulatory T cells.   
     
     
         2 . The method of  claim 1 , further comprising purifying the population prior to the contacting step. 
     
     
         3 . The method of  claim 2 , wherein the purifying step comprises isolating CD4+ T cells. 
     
     
         4 . The method of  claim 1 , wherein the generated immunosuppressive regulatory T cells are CD4+CD25+. 
     
     
         5 . The method of  claim 1 , wherein the generated immunosuppressive regulatory T cells are CD4+CD25+FOXP3−. 
     
     
         6 . The method of  claim 1 , wherein the population of CD4+CD25− T cells comprise CD4+CD25−CD8+ T cells. 
     
     
         7 . The method of  claim 1 , wherein the sample is blood. 
     
     
         8 . The method of  claim 1 , wherein the sample is obtained from leukopheresis products, bone marrow, lymph tissue, thymus tissue, spleen tissue, or umbilical cord tissue. 
     
     
         9 . The method of  claim 1 , wherein the final sample comprises at least about 50% CD4+CD25+ cells. 
     
     
         10 . The method of  claim 1 , wherein the population is contacted with the rapamycin before the T cell receptor (TCR)/CD3 activator and TCR co-stimulator activator. 
     
     
         11 . The method of  claim 1 , wherein the population is contacted with the rapamycin concurrently with the T cell receptor (TCR)/CD3 activator and TCR co-stimulator activator. 
     
     
         12 . The method of  claim 1 , wherein the rapamycin is added to the population at a concentration of about 0.01 μM to about 10 μM. 
     
     
         13 . The method of  claim 1 , wherein the rapamycin is added to the population at a concentration of about 0.5 μm to about 2 μm. 
     
     
         14 . The method of  claim 1 , wherein the rapamycin is added to the population at a concentration of about 1 μM. 
     
     
         15 . The method of  claim 1 , wherein the contacting step further comprises contacting the population with at least one cytokine or growth factor. 
     
     
         16 . The method of  claim 1 , wherein the contacting step further comprises contacting the population with IL-2, IL-4, TGF-β, IL10, or combinations thereof. 
     
     
         17 . The method of  claim 1 , wherein the T cell receptor (TCR)/CD3 activator is an antibody or a ligand for TCR/CD3. 
     
     
         18 . The method of  claim 1 , wherein the T cell receptor (TCR)/CD3 activator is a TCR/CD3 antibody. 
     
     
         19 . The method of  claim 1 , wherein the T cell receptor (TCR)/CD3 activator is a ligand for TCR/CD3. 
     
     
         20 . The method of  claim 1 , wherein the TCR co-stimulator activator is an antibody. 
     
     
         21 . The method of  claim 1 , wherein the TCR co-stimulator activator is a monoclonal antibody. 
     
     
         22 . The method of  claim 1 , wherein the TCR co-stimulator activator is a CD28 antibody, CD137 (4-1BB) antibody, GITR antibody, B7-1/2 antibody, CD5 antibody, ICOS antibody, OX40 antibody, or CD40 antibody. 
     
     
         23 . The method of  claim 1 , wherein the TCR co-stimulator activator is a CD28 antibody. 
     
     
         24 . The method of  claim 1 , wherein the TCR co-stimulator activator is a ligand for CD28, CD137 (4-1BB), GITR, B7-1/2, CD5, ICOS, OX40 or CD40. 
     
     
         25 . The method of  claim 1 , wherein: the T cell receptor (TCR)/CD3 activator is immobilized on a solid phase; and the TCR co-stimulator activator is immobilized on a solid phase. 
     
     
         26 . The method of  claim 25 , wherein the T cell receptor (TCR)/CD3 activator and the TCR co-stimulator activator are immobilized on the same solid phase. 
     
     
         27 . The method of  claim 25 , wherein the T cell receptor (TCR)/CD3 activator and the TCR co-stimulator activator are immobilized on different solid phases. 
     
     
         28 . The method of  claim 1 , wherein:
 the T cell receptor (TCR)/CD3 activator is immobilized on beads; and   the TCR co-stimulator activator is immobilized on beads.   
     
     
         29 . The method of  claim 25 , wherein the solid phase comprises glass, silica, latex, polymeric materials, plastic, tissue culture plastic, dextran, cellulose, polyethylene glycol, iron, or combinations thereof. 
     
     
         30 . The method of  claim 25 , wherein the solid phase comprises particles, beads, bottles, tubes, strips plates or wells, sheets, fibres, capillaries, needles, combs, pipette tips, microarrays, chips, filters, or membranes. 
     
     
         31 . The method of  claim 25 , wherein the solid phase comprises spherical beads. 
     
     
         32 . The method of  claim 25 , wherein the solid phase comprises monodisperse spherical beads. 
     
     
         33 . The method of  claim 25 , wherein the solid phase comprises spherical beads having diameters of about 1 um to about 10 um. 
     
     
         34 . The method of  claim 25 , wherein the solid phase comprises spherical beads having diameters of about 1 um to about 5 um. 
     
     
         35 . The method of  claim 25 , wherein the solid phase comprises spherical beads having diameters of about 1 um, about 2.8 um, or about 4.5 um. 
     
     
         36 . The method of  claim 25 , wherein the solid phase comprises magnetic beads, superparamagnetic beads, or magnetizable beads. 
     
     
         37 . A kit for the generation of immunosuppressive regulatory T cells, the kit comprising: a T cell receptor (TCR)/CD3 activator, a TCR co-stimulator activator, and rapamycin. 
     
     
         38 . The kit of  claim 37 , wherein:
 the T cell receptor (TCR)/CD3 activator is immobilized on a solid phase; and   the TCR co-stimulator activator is immobilized on a solid phase.   
     
     
         39 . A method of treating a mammal, the method comprising:
 providing a first mammal;   obtaining CD4+CD25− T cells from the first mammal;   expanding/activating the T cells ex vivo by contacting the T cells with a T cell receptor (TCR)/CD3 activator, a TCR co-stimulator activator, and rapamycin; and   administering the expanded/activated T cells to a second mammal to be treated.