OPTIMIZING EX-VIVO EXPANDED AUTOLOGOUS TREG INFUSIONS AND DISCOVERING THEIR FATE USING AN IN VIVO PRIMATE MODEL

Abstract

Introduction: Cell Therapy with regulatory Tcells (Tregs) is a promising tool for tolerance induction in solid organ transplantation. Despite encouraging results in humans and monkeys, questions about Treg phenotype, function, survival and migration after in vivo infusion remain. To better understand Treg fate, we have used a CFSE-labeled tracking method for autologous, polyclonal expanded Tregs in cynomolgus macaques. Methods: Polyclonal Tregs were grown from sorted (CD4+CD25hi) PBMCs and ex vivo expanded on allogeneic CD40-expressing Bcells and cryopreserved. 10-30x106 Tregs/kg were thawed and labeled with CFSE dye, then Tregs were administered intravenously. Phenotype and function of Tregs were specified via flow cytometry and suppression assays prior to in vivo infusion into recipient cynomolgus macaques. Labeled Tregs were then tracked in vivo with flow cytometry on peripheral blood and iliac crest bone marrow aspirates over the first month. At the time of sacrifice (Day 30), lymphoid organs (thymus, mesenteric and inguinal lymph nodes, spleen) and bone marrow were harvested and analyzed. Results: The first cynomolgus macaque received 12x106 CFSE-labeled Tregs/kg that retained their phenotype (CD4+CD25hi, FoxP3+ expression ≥95%) and suppressive capacities (1:16 suppression titer) throughout the expansion and restimulation process. CFSE-labeled Tregs were detected in circulation at Day 0, 2, 3, 6, 9 and 14 and were undetectable by day 15 post infusion. Dividing CFSE-labeled Tregs were seen at day 2,6 and 9 in the bone marrow. At day 30, no CFSE-labeled cells were found in any tissues. Treg infusions are upcoming in 4 more cynomolgus macaques. Conclusion: CFSE labeling of autologous, ex vivo expanded polyclonal Tregs is a valid model that allows in vivo tracking of migration and survival of Treg infusions in cynomolgus macaques.