Generation, cryopreservation, function and in vivo persistence of ex vivo expanded cynomolgus monkey Treg (TRAN1P.927)

Abstract

Abstract Foxp3+ regulatory T cells (Treg) function to promote immune tolerance and prevent immunopathogenesis. Preclinical studies have demonstrated that adoptive transfer of Treg prevents rejection of organ allografts in mice and humanized mouse models. In this study, we optimized a protocol to expand flow-sorted cynomolgus Treg >1000-fold after three rounds of stimulation with anti-CD3 mAb-pulsed artificial APCs, rapamycin and IL-2. The expanded Treg from each round maintained high level expression of Treg signature markers, -CD25, CD27, CD39, Foxp3, Helios, CTLA-4, as well as CXCR3, a homing receptor of Th1-like cells. In contrast to expanded Teff, Treg produced minimal IFN-γ and IL-17 and no IL-2. In addition, the expanded Treg from each round showed high suppressive capacity in vitro. Compared to freshly-expanded Treg, Treg immediately thawed from cryopreserved stocks maintained Treg signature markers and suppressive capacity, but were less viable. However, additional rounds of stimulation/expansion restored maximal viability. Adoptively-transferred autologous Treg labeled with CFSE (approximately 2x107/kg, i.v.) could be detected in vivo 2-3 months post infusion in peripheral blood, inguinal LN, mesenteric LN and spleen. Moreover, these cells maintained CD25, CCR7, CXCR3 expression and acquired CD62L after infusion in vivo.