Isolation, propagation and characterization of cord blood derived CD4+ CD25+ regulatory T cells

Regulatory T cells (Treg) have recently come to the fore in studies of immune regulation, particularly in autoimmune disease and cancer. While there appear to be several distinct subsets of T cells with regulatory function, a population described as natural Treg and characterized by expression of the transcription factor FOXP3 has attracted particular interest. These cells can be enriched using the surface markers CD4 and CD25, and cord blood is a convenient source of CD25+ Treg. We present detailed protocols for the enrichment of Treg from cord blood using CD25 and a magnetic bead procedure, yielding populations > 80% positive for CD25 and 50–65% FOXP3 positive. This enrichment can be followed by a second magnetic bead or a flow sorting step, yielding > 95% CD25 and > 65% FOXP3 positive populations. Protocols are presented for propagation of these cells in culture (yielding > 80% FOXP3 positive cells) and for their phenotypic and functional characterization.