Therapeutic vaccine for treatment of diabetes type 1 in children, application of the cell sorter and the method of multiplying treg cells to produce therapeutic vaccine for treatment of diabetes type 1

Abstract

The gist of the invention consists in the therapeutic vaccine for treatment of diabetes type 1 in children, which contains Treg cells CD3(+)CD4(+)CD25(high)CD127(−). Claimed too is the cell sorter used to produce the vaccine and the method of multiplying Treg cells in vitro.

1 - 6 . (canceled) 
     
     
         7 . A method of producing a population of cells comprising CD3(+)CD4(+)CD25(high)CD127(−) Treg cells, the method comprising:
 sorting a population of cells using a cell sorter to isolate cells having the phenotype CD3 (+)CD4(+)CD25 (high)CD 127(−] doublet(−)lineage(−)dead(−). 
 
     
     
         8 . The method of  claim 7 , wherein the cell sorter comprises a replaceable sample flow line. 
     
     
         9 . The method of  claim 7 , wherein the step of sorting comprises excluding cells that are positive for antigens selected from the group consisting of: CD14, CD16, CD19, CD8, and combinations thereof. 
     
     
         10 . A method of multiplying CD3(+)CD4(+)CD25(high)CD127(−) Treg cells in vitro comprising:
 isolating CD4 +  T lymphocytes using an immunomagnetic method; 
 marking the CD4 +  T lymphocytes with one or more monoclonal antibodies; 
 sorting the CD4+T lymphocytes using a cell sorter to isolate cells having the phenotype CD3(+)CD4(+)CD25(high)CD127(−]doublet(−)lineage(−)dead(−) to generate a population of sorted Treg cells; 
 multiplying the sorted Treg cells in CellGro or X-VIVO medium supplemented with autologous inactivate serum and interleukin-2; and 
 adding artificial antigen-presenting cells to the medium in a 1:1 proportion to the sorted Treg cells. 
 
     
     
         11 . The method of  claim 10 , wherein the step of marking comprises marking with monoclonal antibodies selected from the group consisting of: an anti-CD3 antibody, an anti-CD4 antibody, an anti-CD8 antibody, an anti-CD19 antibody, an anti-CD14 antibody, an anti-CD16 antibody, an anti-CD25 antibody, an anti-CD127 antibody, and combinations thereof. 
     
     
         12 . The method of  claim 10 , wherein the one or more monoclonal antibodies are conjugated to fluorescent dyes. 
     
     
         13 . The method of  claim 10 , wherein the artificial antigen-presenting cells comprise magnetic beads coated with anti-CD3 and anti-CD28 antibodies. 
     
     
         14 . The method of  claim 10 , wherein the CellGro or X-VIVO culture medium is supplemented with at least 1000 U/ml interleukin-2 and approximately 10% autologous inactivated serum. 
     
     
         15 . The method of  claim 10 , wherein the sorted Treg cells comprise a Treg cell population having a purity of at least 97%. 
     
     
         16 . The method of  claim 10 , wherein the sorted Treg cells are propagated for a time that does not exceed 2 weeks. 
     
     
         17 . The method of  claim 10 , wherein the artificial antigen-presenting cells are in the shape of a magnetic sphere. 
     
     
         18 . The method of  claim 10 , wherein the multiplied Treg cells are tested for a FoxP3 expression level above 90%, a positive result in an IFN-gamma inhibition assay, and a negative result in a microbiological assay.