The IL-27/Wsx-1 Axis Modulates T Cell Responses and Regulates Acute Graft-Versus-Host Disease after Allogeneic Bone Marrow Transplantation
Authors: Ma Shoubao , Gong Huanle , Liu Shuangzhu , Han Jingjing , Yin Zhinan , Liu Haiyan , Wu Depei 
Published: October 2019

Abstract
IL-27 is a member of the IL-12 cytokine family that consists of EBV-induced gene 3 (EBI3), and p28. IL-27 is produced by activated antigen-presenting cells such as dendritic cells (DCs) and macrophages, and it signals through a heterodimeric receptor (IL-27R) consisting of the WSX-1 and the gp130. Emerging evidence has shown that IL-27 has both pro- and anti-Inflammatory effects. Recent study demonstrated a protected role IL-27p28 in acute graft-versus-host disease (aGVHD) in parent-to-F1 murine aGVHD model (Marillier et al., Eur. J. Immunol. 2014). However, the precise role of IL-27 in the development of aGVHD remains largely unknown.
In this study, we carried out studies in a murine aGVHD model of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). Lethally irradiated BALB/c mice transplanted with WT B6 bone marrow (BM) plus IL-27-/-(IL-27p28flox/flox-CD11c-cre (Itgax-p28f/f)) splenocytes had significantly accelerated aGVHD mortality comparing with those received WT BM plus splenocytes (P<0.001). However, similar aGVHD mortality was observed between mice transplanted with WT BM plus WT splenocytes and IL-27-/-BM plus WT splenocytes, and IL-27-/-BM plus IL-27-/- splenocytes, suggesting that IL-27 from splenocytes, not BM-derived cells, had protective effect in aGVHD. Additionally, mice were transplanted with IL-27-/- BM and splenocytes, and then treated with rmIL-27 on days 0 and 7 post BMT. rmIL-27 treated mice had longer survival and alleviated the clinical signs of aGVHD compared to PBS-treated mice (P<0.05). We next explored the mechanisms by which IL-27 protects mice against aGVHD. We found the expression of the cell surface IL-27 receptor, WSX-1 and gp130, were increased on both CD4+ and CD8+T cells after allo-stimulation. Moreover, IL-27 inhibited cell proliferation of allo-reactive T cells in mix lymphocytes reactivation (MLR) in vitro. We also found that the percentages of CD69+CD4+, CD69+CD8+T cells, as well as IFN-γ expression by CD4+ and CD8+T, were significantly increased in the spleen, liver and intestinal intraepithelial lymphocytes (IEL) from mice transplanted with WT BM and IL-27-/- splenocytes. Furthermore, we observed increased frequencies and numbers of Treg cells and MDSCs in aGVHD target organs in mice reconstituted with IL-27-/-splenocytes. Together, these results indicated that IL-27 alleviated aGVHD may through suppressing Th1 cell responses and promoting immune suppressing cells, including MDSCs and Treg cells.
To further clarify the role of IL-27 in aGVHD, mice were transplanted with WT BM plus IL-27R-/-(B6N.129P2-Il27ratm1Mak/J) splenocytes, the results, however, showed that IL-27R deficiency in donor T cells significantly attenuated aGVHD, which was unpredicted, and was contrary to that of IL-27 deficiency results. Additional studies showed that IL-27R deficiency in T cells inhibited allo-reactive T cells proliferation and IL-2, IFN-γ production in MLR assay. The percentages of CD69+ T cells and IFN-γ+ CD4+ and CD8+T were significantly decreased, while the MDSCs and Treg cells wereincreased in aGVHD target organs from mice transplanted IL-27R-/-splenocytes. These results indicated that lack of IL-27R signaling resulted in downregulation of intrinsic immune responses which led to alleviation of aGVHD.
Previous study reported that soluble form of IL-27Ra is highly existed in human serum and may severs as a natural IL-27 antagonist (Dietrich et al., JI. 2014). We hypothesized that sIL-27Ra may function as a decoy receptor to regulate aGVHD through inhibiting IL-27 signaling. To test this hypothesis, WT recipients were given exogenous soluble mouse IL-27 Ra Fc chimera protein known to block the binding of IL-27 to the membrane bound form of IL-27R. WT recipients given IL-27 Ra-Fc to block IL-27R/IL-27 interaction had significantly exacerbated GVHD mortality. The percentages of CD69+ T cells and IFN-γ+ CD4+ and CD8+T were significantly increased in aGVHD target organs from mice received IL-27 Ra fusion protein.
In conclusion, this is the first demonstration in the same allogeneic BMT model that IL-27 deficiency augments but IL-27R deficiency alleviates acute GVHD. Our studies provide further evidence for the protective role of rmIL-27, and promoting effect of sIL-27R in the same aGVHD model. This study warrants further investigations of the possible therapeutic applications of IL-27 in acute GVHD.

Disclosures
No relevant conflicts of interest to declare.