Clinical Scale Enrichment and Expansion of Highly Pure CD25+Foxp3+ Regulatory T Cells.

Abstract 3718

CD25+Foxp3+ regulatory T cell (Treg) bear great potential to prevent or treat a variety of immune mediated diseases, including autoimmunity, organ rejection or GvHD. Currently Treg for clinical application can be separated by magnetic cell separation via the CliniMACS® Plus Instrument using CD25 enrichment plus/minus prior depletion of CD8 or CD19 positive cells. With this technology Treg can be enriched to a mean purity of about 50% and first clinical trials for prevention of GvHD show no adverse effects at all. Despite these promising results, concerns have been raised whether in the setting of organ transplantation or autoimmunity higher Treg purities and/or the in vitro expansion of Treg without loss of Foxp3+ expression are required.

Therefore, we have optimized the parameters for CD25 enrichment via CliniMACS to achieve higher purity of the isolated Treg. The purity of Treg could be increased by about 20–30% resulting in an average purity of 70–80% of Foxp3+ Treg. We have also developed a protocol for the in vitro expansion of CliniMACS isolated Treg using CD3/CD28 coated MACSiBead™Particles. In the presence of Rapamycin CliniMACS isolated Treg could be expanded about 20 fold with a single round of stimulation. Importantly Foxp3+ expression was not affected by the expansion but remained constant at about 70–80%. Similarly the expression of effector cytokines by expanded Treg was greatly suppressed by Rapamycin.

These data show that Treg for clinical application can efficiently be isolated with high purity via CliniMACS and subsequently be expanded in vitro without loss of Foxp3 expression.