Method for ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune mediated diseases
a technology ex-vivo expansion, which is applied in the field of ex-vivo expansion of regulatory t cells with enhanced suppressive function for clinical application in immune-mediated diseases
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example 1
[0022]Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from 9 persons with SLE, 7 persons with RA, 7 persons with asthma, 10 persons with MS, and 7 persons with CD as well as from 11 persons with no autoimmune disease.
[0023]CD4+ Tregs were isolated and purified from the peripheral blood mononuclear cells (“PBMCs”) of the samples. The PBMCs were isolated from blood samples by density gradient centrifiguation with Ficoll Hypaque (Amersham). The CD4+CD25+ Tregs were purified from PBMCs by autoMACS using the human CD4+CD25+ regulatory T cell isolation kit (Miltenyi Biotec, Auburn, Calif.) according to the manufacturer's instructions. Briefly, CD4+ T cells were first negatively isolated from PBMCs by depleting non CD4 cells with the mixture of monoclonal antibodies against human CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCRy / 8 and CD235a. Human CD4+CD25+ Tregs were then positively isolated with anti-human CD25 antibody-conjugated microbeads from the enriched CD...
example 2
[0024]Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from 9 persons with SLE, 7 persons with RA, 7 persons with asthma, 8 persons with MS, and 7 persons with CD as well as from 25 persons with no autoimmune disease.
[0025]CD4+ Tregs were purified from the PBMCs of the samples. Purification was carried out using as set forth in Example 1. The purity of purified CD4+ Tregs at day 0 was assessed using intracellular Foxp3 staining and analyzed by flow cytometry and the results are displayed on the graph in FIG. 2. Approximately 40 to 75% of the purified cells expressed Foxp3.
example 3
[0026]Approximately 5 to 10 peripheral blood samples of 50 cc each were collected from persons with SLE (FIG. 3A), RA (FIG. 3C), asthma (FIG. 3B), MS (FIG. 3D), CD (FIG. 3E), and UC (FIG. 3F). CD4+ Tregs were purified from the PBMCs of the samples. Purification was carried out using as set forth in Example 1.
[0027]The CD4+Fox3+ Tregs were cultured in X-VIVO 15™ media (Cambrex Bio-Whitaker, East Rutherford, N.J.) supplemented with 10% pooled human AB serum (Cambrex) in the presence of 1000 IU / Ml of human rIL-2 (Proleukin). In addition, anti-CD3 / ant-CD28 coated beads (Dynal, Oslo, Norway) were added at a 1:3 cell to bead ratio. After 3 weeks of culture at 37° C. in an incubator, the expanded Tregs reached over 100 to 1,000 fold expansion, or 1 billion cells, as shown in the graphs in FIG. 3. In FIG. 4 is a graph depicting that an average of 40 to 50% of the ex-vivo expanded cells expressed Foxp3 by intracellular staining at 2 weeks.
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