TH17 Cells Mediate Extensive Skin Pathology in Acute GVHD.

INTRODUCTION: Cytokines play an integral role in the development of acute graft-versus-host disease (GVHD). In particular, IFN-gamma has been ascribed both pathologic as well as protective roles in murine models of GVHD. IFN-gamma is the trademark cytokine produced by differentiated T helper (TH) type 1 CD4+ T cells, whereas IL-4 is a characteristic cytokine produced by TH2 cells. Although a number of studies have investigated the role each of these cytokines have in development or inhibition of GVHD, little evidence has been gathered regarding the role of IL-17 producing T cells (TH17). TH17 cells have been attributed to mediating pathology in several autoimmune conditions that once were thought to be uncontrolled TH1 responses. IL-17 is a proinflammatory cytokine secreted exclusively by T cells that induces production of other proinflammatory cytokines by epithelial cells, macrophages, endothelial cells and fibroblasts. Elevated levels of IL-17 have been observed in rheumatoid arthritis, multiple sclerosis, and inflammatory bowl disease.

METHODS: Given the highly proinflammatory properties of IL-17 we sought to examine the role of TH17s in acute GVHD. Sort purified naive CD4+ effectors (CD4+/CD25-/CD62Lhi) from C57BL/6 mice were cultured under TH17 polarizing conditions for 11 days. Two rounds of anti-CD3 + anti-CD28 stimulation in the presence of TH17 inducing cytokines yielded a culture in which >90% of the cells were producing IL-17 with minimal production of IFN-gamma. These cells were transferred into lethally irradiated B6D2 recipients at various doses with whole splenic T cells. The results demonstrated that a sub-lethal dose of T cells could be made lethal by substituting 1×10⁶ naive whole T cell effectors for TH17s. Interestingly, substitution of 2×10⁶ TH17s did not cause significant mortality, however typical signs of GVHD (hunching, weight loss, fur ruffling) were observed. In addition, transfer of TH17s alone caused significant morbidity with skin ulceration and extensive loss of fur. In a minority of mice TH17s alone were sufficient to induce lethality. To investigate the trafficking of TH17s in GVHD, we culture CD4+ naive effectors from GFP+ mice under optimal TH17 conditions and transferred these cells into irradiated B6D2 recipients. Fluorescent microscopy revealed extensive infiltration by GFP+ TH17 cells in GVHD target tissues, specifically the entire GI tract, lung, as well as liver. Additionally these cells were found extensively in secondary lymphoid organs seven days after transfer. By day 14 post-transfer the emigrated cells had undergone significant expansion. We confirmed these findings by characterizing GFP+ cells at these sites by flow cytometric analysis.

CONCLUSIONS: These data demonstrate that in vitro differentiated TH17s alone can cause lethal acute GVHD and can synergize to cause lethal GVHD when given with a suboptimal dose of naive T cells. The trafficking data show that TH17s infiltrate and expand robustly in lymphoid and GVHD target organs in the first two weeks post transplanataion. Collectively these results describe a pathogenic role of TH17s in acute GVHD.

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