Infusion of ex-vivo expanded natural regulatory T cells (Tregs) is emerging as a promising cellular therapy for the induction of allograft tolerance. However, the lack of a clear understanding of the in-vivo quantitative and functional dynamics of Tregs has limited their translation to the clinic. Here we have used a primate model to infuse escalating doses of CFSE-labeled, autologous CD4+CD25+CD127-/lowFoxP3+ Tregs to allow us to estimate the accessible, peripheral Treg pool, and to longitudinally evaluate the infused cells in blood, bone marrow (BM) and lymph nodes.

CFSE-labeled Tregs, infused at a dose of 2.0×10⁷ /kg, constituted 14.3±3.9% of the peripheral Treg pool when measured 30 minutes after infusion. This allowed us to make what we believe is the first documented calculation of the accessible peripheral Treg pool in a primate model, which we found to be quite large: 1.14±0.33×10⁸ Tregs/kg body weight (> 5-50-times the number of Tregs that have previously been infused into transplant recipients). Post infusion, the frequency of infused Tregs in the peripheral blood fell quickly, with no labeled cells remaining by Day 16. Infused Tregs trafficked to the BM and lymph nodes and were detectable there on Days 2 and 6, but no longer detectable in the BM at Day 13.

Surprisingly, infused Tregs quickly underwent phenotypic alterations after infusion. Thus, within 3 days of infusion, they began to exhibit a loss of both CD25 and FoxP3 and a gain of CD127 expression, suggestive of a concomitant loss of suppressive function. By Day 10, only 13-25% of infused CD4+CFSE+ cells were CD25+FoxP3+ compared to 70-75% on the day of infusion.

In preliminary studies, the presence of sirolimus extended both the persistence and phenotypic integrity of the infused Tregs. Tregs persisted in the peripheral blood to >35 days, and better maintained expression of CD25 and FoxP3 (with 75% of infused CD4+CFSE+ cells being CD25+FoxP3+ at Day 10).

Together, these data provide novel information about the availability of the accessible Treg pool in primates, enhance our understanding of the in-vivo dynamics of infused Tregs and suggest that sirolimus may extend their survival and phenotypic integrity. This information will be critical to making informed decisions about how best to incorporate Treg adoptive immunotherapy into new immunomodulatory regimens for transplantation.

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