Introduction: To achieve successful translation of T regulatory cell (Treg) therapy in human organ transplantation, it will be essential to generate large quantities of Treg ex vivo. This is necessary as rodent studies suggest an increased number of Treg relative to T effectors (Teff) is required for experimental allograft tolerance. IL-33 administration controls alloimmunity and promotes graft survival by increasing endogenous Treg in rodent models. IL-33 also selectively expands mouse Treg expressing the IL-33 receptor ST2 when added to Treg/allogeneic DC cultures. We attempted to translate these rodent findings to expansion of human Treg and to determine whether IL-33 could enhance the ability of allogeneic DCs (allo-DCs) to stimulate expansion of flow-sorted Treg.
Methods: Peripheral blood immunobead-isolated CD14+ cells were cultured with GM-CSF and IL-4 for 7 days to generate mono-DC. Irradiated DCs or anti-CD3/28 beads were co-cultured with flow-sorted peripheral blood CD4+CD25+CD127− Treg with or without 50 ng/ml IL-33 for 12 days in the presence of IL-2. Anti-CD3 Ab-loaded L cells were used to stimulate 2nd round Treg expansion, with or without IL-33 in the presence of IL-2 for an additional 7 days. DCs were incubated with or without IL-33 or IL-33 plus IL-2 for 18 hours for phenotypic analysis. Proliferation of autologous Teff in the presence or absence of the expanded Treg was used to test Treg suppressive function. Expanded alloreactive Treg were stimulated with anti-CD3/28 beads for 3 days to assess cytokine production.
Results: Treg stimulated by DCs in the presence of IL-33 were expanded approximately 40% more in the 1st round and 50% more in the 2nd round than in the absence of IL-33. However, such differences were not observed when Treg were stimulated by anti-CD3/28 beads. Allo-DC increased expression of CD86 when co-cultured with IL-2 and IL-33, suggesting stronger stimulatory potency. Interestingly, while IL-33 cultured Treg expressed lower Helios, modestly downregulated Foxp3, and higher levels of IFNγ expression relative to control allo-DC Treg (Fig. 1), they retained a potent ability to suppress T effector proliferation (Fig. 2).
Conclusion: IL-33 enhances DC-induced expansion of human Treg exhibiting strong suppressive capacity, but enhanced IFNγ production. Future studies will determine the functional relevance of increased IFNγ in Treg expanded during IL-33/Allo-DC culture.
This work was supported by funding from the NIH (R01HL122489: HRT).
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