Infusion of host derived ex vivo expanded regulatory T cells (eTregs) is an attractive therapeutic strategy for promotion of transplant tolerance. We report herein that human eTregs cultivated for in vivo therapy abundantly express the death molecules T-cell Immunoglobulin and Mucin domain-3 protein (TIM-3) and program death 1(PD-1), are highly potent yet fragile. We studied the functional and phenotypic features of sorted peripheral CD4⁺CD25^hiCD127^lo Tregs expanded in the presence of anti-CD3 and anti-CD28 coated micro beads and IL-2 for 14 days. Notably, >95% of eTregs generated using protocols under investigation for Treg cellular therapy are TIM-3⁺ of which ∼60% co-expressed PD-1, both molecules known to contribute to effector T cell apoptosis, exhaustion or dysfunction. Contrastingly, only ∼10% of nTregs in peripheral blood express TIM-3. Compared to their nTregs, expanded TIM-3⁺ Tregs are more potent suppressors of in vitro Teff proliferation and robustly express CD25, CTLA-4, CD39, CD45RO, IL-10, but are programmed for apoptotic cell death. Exposure to the TIM-3 ligand galectin-9 in a dose and time dependent manner or absence of a continuous proliferative micro environment led to eTreg death; though these cells could be rescued by TCR stimulation and/or IL-2. Thus these studies indicate that the highly potent eTregs are terminally differentiated Tregs expressing death molecules TIM-3 and PD-1 and may be short lived in vivo. As use of eTregs is a promising therapeutic strategy our findings provide further insight into novel properties and fate of expanded human Tregs instructive for advancement of Treg cellular therapeutic approaches.

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