High purity and yield of natural Tregs from cord blood using a single step selection method.

Natural regulatory T cells (Tregs), characterized as CD4 CD25high Foxp3+, have been described as paramount contributors in immuno-regulation and self-tolerance. CD4 and CD25 have been the main markers used for their isolation, resulting in cells with potent suppressive properties. Nevertheless, low purity and yield continue to be an issue when attempting thorough characterizations and/or up scaling to bigger models and for clinical trials. Here we present a single-step methodology optimized for cord blood CD25+ isolation, using magnetic microbeads that achieves a reproducible purity of 89% for CD4 CD25high CD127low. These cells showed a more consistent suppressive effect in mixed lymphocyte cultures. In addition, the proportion of contaminating effector T cells was < 9% whilst the yield of Tregs was doubled compared to the standard protocol. Gating on CD4 CD25high CD127low populations post isolation showed better correlation with suppressive efficacy compared to CD4 CD25+ gate. These data should facilitate the clinical scale-up of this procedure to obtain consistent Tregs for clinical application and research.