Tr17: A Unique Subset of T Regulatory Cells with Distinct Molecular and Functional Properties

Abstract 1116

A major challenge of the immune system is to fight pathogens and abnormal cell growth while preserving immune tolerance to self-antigen. Active immune suppression by T regulatory (Treg) cells is one of the mechanisms that maintain peripheral T cell tolerance and homeostasis. Recent findings suggest that Treg cells are heterogeneous in functions and phenotypes. Moreover, Treg cells might not be at a terminal stage of differentiation but rather display a significant degree of plasticity particularly under inflammatory conditions. We have previously determined that co-culture of conventional CD4⁺ T cells and Treg cells in the presence of antigen presenting cells (APC) during T cell receptor mediated stimulation, induced Treg cells producing IL-17 (thereafter named Tr17 cells). IL-1β and IL-2, endogenous products of the co-culture, were essential for the conversion of Treg cells into Tr17 cells. In the present study we sought to identify the phenotypic and functional properties of Tr17 cells. First, we examined the role of IL-2 and IL-1β in the generation of Tr17 cells. Treg cells from Foxp3.GFP knock-in mice were stimulated with anti-CD3 and anti-CD28 in the presence of IL-1β-plus-IL-2. A fraction of purified Foxp3⁺ Treg cells produced significant amounts of IL-17 as determined by ELISA and by intracellular cytokine staining. Very few Tr17 cells were observed during T cell activation in the absence of either IL-1β or IL-2, suggesting that the combination of IL-1β and IL-2 was required for the development of Tr17 cells. Compared to IL-17-Treg, Tr17 cells expressed increased levels of RORγt, the key transcription factor for Th17 cell differentiation. Expression of CCR6 is regulated by RORγt and is one of the features of Th17 cells. To determine whether expression of CCR6⁺ correlates with the ability of Treg to produce IL-17, we depleted CCR6⁺ Treg from Foxp3.GFP⁺ populations and examined the ability of the resulting Treg to produce IL-17. Depletion of CCR6⁺ Treg resulted in significantly reduced IL-17 production compared with total Foxp3⁺ Treg cells, suggesting that CCR6⁺ Treg cells might represent Tr17 cells. Because inducible costimulatory molecule (ICOS) was recently shown to be critical for the differentiation, expansion and function of Th17 cells we examined its role in Tr17 cells. We determined that Tr17 cells expressed strikingly elevated levels of ICOS compared to IL-17⁻ Treg obtained from the same cultures. These results indicate that Tr17 cells acquire phenotypic properties of Th17 cells, which involve RORγt-dependent and independent mechanisms. To determine whether besides obtaining features of Th17 cells, Tr17 cells also retained properties of Treg we first assessed the expression of Foxp3. Compared to IL-17⁻ Treg, Tr17 cells displayed lower levels of Foxp3. However, expression of CTLA-4 and GITR, two other signature makers of Treg cells, was comparable in IL-17⁻ Treg and Tr17 cells. Similarly, both Tr17 and IL-17⁻ Treg cells displayed suppression capacity. Taken together, our results suggest that Tr17 is a unique subset of Treg cells that is differentiated under inflammatory conditions and displays combined phenotypic and functional properties of both Th17 and Treg cells. Because Treg use canonical CD4⁺ effector cell-associated transcription factors to regulate the immune responses of lineage-specific effectors, our findings suggest that Tr17 cells may have a unique role in regulating Th17-mediated inflammatory responses.