Ex vivo expansion of human CD4+ CD25high regulatory T cells from transplant recipients permits functional analysis of small blood samples.

Regulatory T cell (Treg) function is associated with immune tolerance and could serve as a biomarker for optimization of immunosuppressive regimens. This is hampered by the limited number of Treg in the blood circulation; functional Treg analysis requires large volumes of blood or is dependent on indirect analysis. A more attractive strategy is the ex vivo expansion of Treg, provided the original T cell pool remains unaltered. Here, we show that it is possible to ex vivo expand Treg from limited amounts of blood, preserving the original TCR Vbeta repertoire and suppressive capacity. The protocol proved successful in selected renal transplant recipients and leukopenic patients. Sampling of 10-20 ml blood sufficed; Treg numbers increased over 100-fold during the 2-3 week expansion period, easily reaching cell numbers required for functional analysis (>10(6) cells). This protocol will facilitate the monitoring of Treg function in patients in order to address the role of Treg in transplantation tolerance.