Background:

Allogeneic haematopoietic stem cell transplantation (HCT) is a curative option for haematopoietic diseases. However, a major limitation to the success of HCT is graft-versus-host disease (GvHD). The use of T regulatory cells (Tregs) as prophylaxis or treatment for GvHD has now emerged with a number of active clinical trials. Whilst many studies use Tregs from the original HCT donor, the future of this cell therapy could be third-party Tregs. Cord Blood (CB), with its established banks, represents a potential source of third-party cells. However, isolating a high purity Treg cells from cryopreserved CB is difficult without employing flow sorting. Here we investigated the feasibility of using reversible streptamer technology-based selections to obtain clinical grade Tregs from cryopreserved CB units.

Results:

: A streptamer-based Treg selection was developed with both single-step, CD25 only positive cell selection and two-step, CD4 then CD25 positive selection from cryopreserved CB units, to yield a method that can be readily adapted to full good manufacturing practice (GMP). The best purity was achieved with a two-step streptamer isolation method, giving median purities of 89% Tregs of total cells. This method took advantage of the reversible nature of the streptamer technology allowing for successive positive selections for CD4 + and then CD25 + cells from cryopreserved CB units in an enclosed bag system. Isolated Tregs subsequently demonstrated 300-fold culture expansion using anti-CD3/28 beads. The expanded cells contained high proportions of cells with a Treg phenotype (both by flow cytometry and epigenetics) and demonstrated suppressive function.

Conclusions:

: Using streptamer selection, highly enriched Tregs could be isolated from cryopreserved CB. The method could be performed in an enclosed bag system utilizing readily available clinical processing materials. Moreover, Tregs selected in this manner could be expanded in culture to make a clinically relevant dose from a cryopreserved CB unit. Thus, streptamers represent a viable alternative to column based magnetic bead or fluorescent activated cell sorting (FACS)-based methods for selection of clinical grade Tregs from banked CB units.