Background:

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. The major challenge of implementing Treg therapy in the clinic is to obtain sufficient numbers of stable functional Treg. Furthermore, survival and recruitment of expanded Treg to host lymphoid tissues and sites of inflammation is critical for their function.

Methods:

In this study, we expanded flow-sorted cynomolgus Treg >1000-fold after three rounds of stimulation with anti-CD3 mAb-pulsed artificial APCs (L32 cells), rapamycin and IL-2. After each round of expansion, freshly-expanded vs. cryopreserved expanded Treg were compared in their expression of Treg signature molecules and chemokine receptors, apoptosis, cytokine production upon restimulation and ability to suppress T cell proliferation.

Results:

The expanded Treg from each round maintained high levels expression of Treg signature markers, -CD25, CD27, CD39, Foxp3, Helios, CTLA-4, as well as CXCR3, migration molecule 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. Following cryopreservation, thawed-Treg are less viable compared to freshly-expanded Treg, but maintained Treg signature markers and suppressive capacity.

Adoptively-transferred Treg labeled with CFSE (approximately 2×10^7/kg, i.v.) could be detected in vivo after 50 days in peripheral blood, inguinal LN, mesenteric LN and spleen. Moreover, these cells maintained CD25, CCR7, CXCR3 expression and acquired CD62L after infusion in vivo.

Conclusion:

We optimized the protocols to expand NHP polyclonal Treg massively while maintaining their phenotype and suppressive capacity; and to store Treg without compromising their stability and function.

When infused into autologous recipients, these cells were readily detected in peripheral blood as well as lymphoid tissues for more than 50 days.

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