Toward that end, we observed that there was a higher percentage of CD8+, as opposed to CD4+, T cells that secreted IFN- after polyclonal stimulation or TH1 polarization in vitro, and an increase in the absolute number of CD8+IFN-+ T cells in GVHD tissue sites. murine transplant models. Moreover, protection from GVHD was attributable to augmented global reconstitution of CD4+ natural regulatory T cells (nTregs), CD4+ induced Tregs (iTregs), and CD8+ iTregs, and was more potent than temporally concordant blockade of IL-6 signaling. Inhibition of IL-27p28 also enhanced the suppressive capacity of adoptively transferred CD4+ nTregs by increasing the stability of Foxp3 expression. Notably, blockade of IL-27p28 signaling reduced T-cellCderived-IL-10 production in conventional T cells; however, there was no corresponding effect in CD4+ or CD8+ Tregs, indicating that IL-27 HI TOPK 032 inhibition had differential effects on IL-10 production and preserved a mechanistic pathway by which Tregs are known to suppress GVHD. Targeting of IL-27 therefore represents a novel strategy for the in vivo expansion of Tregs and subsequent prevention of GVHD without the requirement for ex vivo cellular manipulation, and provides additional support for the critical proinflammatory role that members of the IL-6 and IL-12 cytokine families play in GVHD biology. Introduction Graft-versus-host disease (GVHD) is usually characterized by the increased production of inflammatory cytokines, activation and expansion of alloreactive donor T cells, and the failure of existing regulatory mechanisms to counterbalance this proinflammatory milieu.1-3 The latter, in particular, has been a major focus of inquiry given that GVHD HI TOPK 032 is characterized by impaired reconstitution of regulatory T cells (Tregs) which contributes substantially to the pathophysiology of this disease.4-6 This observation has been the impetus for strategies directed at the reestablishment of an effective Treg network by the adoptive transfer of ex vivoCexpanded Tregs.7-9 Although these studies have demonstrated feasibility, there have been no controlled studies demonstrating efficacy, and the technology necessary for this approach is not widely available to all transplant centers.10 Thus, alternative strategies designed to facilitate the in HI TOPK 032 vivo expansion of existing Treg populations by modulating the inflammatory cytokine milieu via antibody blockade11,12 or exogenous cytokine administration13 have intrinsic merit given the potential broader clinical availability of these approaches. Interleukin-6 HI TOPK 032 (IL-6), along with other IL-6 cytokine superfamily members such as IL-23, has been shown to have an important proinflammatory role in GVHD in both preclinical murine models11,14-16 and recent clinical studies.17,18 IL-27, another member of the IL-6 cytokine family, is a heterodimeric cytokine that is composed of p28 and Epstein-BarrCinduced gene 3 (EBI3) subunits and signals through a heterodimeric receptor composed of WSX-1 and gp13019 which is part of the IL-6 signaling complex.20 Like IL-23, IL-27 is secreted by activated antigen-presenting cells (APCs) such as macrophages, monocytes, and dendritic cells and signals through Stat3.21 The IL-27R is highly expressed on effector memory CD4+ and CD8+ T cells, 22 and ligation of the receptor leads to Stat1 and Stat3 activation. 23 Although initially thought to have proinflammatory effects, more recent studies have uncovered an immunoregulatory role for IL-27 which has been derived from data showing that IL-27 suppresses retinoid-related orphan receptor t (RORt) T helper 17 (TH17) development24 and increases GPR44 T-cell production of IL-10.25 Notably, IL-27 has also been shown to affect Treg biology, although whether IL-27 inhibits or enhances Treg expansion remains controversial and appears to be dependent, in part, upon the experimental conditions.19,22,26-29 The goal of the current report therefore was to determine whether IL-27 exerted proinflammatory or immune-suppressive effects during GVHD, and to examine specifically the effect of IL-27 around the reconstitution of the Treg compartment under these inflammatory conditions. Methods Mice C57BL/6 (B6) (H-2b), Balb/c (H-2d), Balb.B (H-2b), and B6 Foxp3EGFP mice were bred in the Animal Resource Center (ARC) at the Medical College of Wisconsin (MCW) or purchased from The Jackson Laboratory (Bar Harbor, ME). IL-27p28?/?, IL-27R?/?, and Foxp3EGFP mice in which there is mutation in the Foxp3 coding region which renders the Foxp3 gene nonfunctional have been described.24,30,31 IL-10BiT-Foxp3EGFP.