Introduction: Tissue injury releases alarmins like IL-33 which stimulate cells expressing ST2, the IL-33R. Mouse (mu) models have identified a beneficial role for IL-33 in tissue repair due to IL-33 stimulating the expansion and reparative function of ST2+ T regulatory cells (Tregs).  In mice, IL-33-stimulated ST2+ Treg repair mechanisms involve the secretion of the stem cell growth factor amphiregulin (Areg) and the type 2 cytokine IL-13. There are few investigations into the presence or functions of human (hu) ST2+ Tregs. These studies describe our recent efforts to molecularly characterize, and harness presumed reparative mu and hu IL-33-stimulated ST2+ Tregs as novel cell therapy after transplantation and tissue injury.

Methods: Isolated huTregs were expanded with L-cell-based artificial antigen-presenting cells (aAPCs) or CD3/CD28 beads with/without IL-33 for 14-21 days. Mu ST2+ or ST2- Tregs were isolated from splenocytes of IL-33-treated B6 FoxP3-RFP+ mice and expanded with CD3/CD28 beads with/without IL-33 for 14-21 days. Treg phenotype was assessed using flow cytometry and suppression with in vitro assays. Methylation profiles of Treg-specific genes were assessed. Adoptive transfer of IL-33-stimulated Treg was assessed in an acute lung injury (ALI) model.

Results:  ST2+ Tregs were rare in PBMCs, however, IL-33-stimulation increased the frequency of hu ST2+ Tregs in culture compared to those expanded IL-2 alone.  The addition of IL-33 to huTreg expansion cultures did not limit Treg expansion, and we could generate large numbers of polyclonal hu ST2+ Treg using aAPCs. IL-33-stimulated huTregs uniformly express CCR4 and FoxP3 and display increased IL-13 at day 21. Surprisingly, Areg expression was comparable between IL-2 and IL-2/IL-33 cultures. Assessing the methylation of Treg-specific genes suggested that huTregs expanded with IL-2/IL-33 are very similar to IL-2-expanded Treg, with the exception such as reduced methylation of the IL-13 and CTLA-4 genes being noted. IL-33-stimulated muTregs limited weight loss and local inflammation when delivered following ALI.

Conclusions: We show that IL-33 can be used in mu and hu Treg expansion cultures to expand suppressive and potentially reparative Tregs that retain expression of FoxP3 and CCR4, but have augmented ST2, CTLA4, PD-1, and produce IL-13. Methylation data of huTreg suggest that IL-33 stimulation does not result in profound Treg instability but increases the expression of IL-13 and other reparative genes. Importantly, the transfer of IL-33-stimulated muTreg provided protection after tissue injury.