Coordinate Expression of Beta 2 Integrins and the Interleukin 23 Receptor Define a Pathogenic CD4+ T Cell Population That Induces Colonic Inflammation during Graft Versus Host Disease
Authors: Agle Kimberle , Zhou Vivian , Chen Xiao , Beres Amy , Komorowski Richard , Taylor Carolyn , Haribhai Dipica , Williams Calvin , Verbsky James , Blumenschein Wendy , Sadekova Svetlana , Bowman Eddie , Weaver Casey , Cua Daniel , Drobyski William R. 
Published: October 2019

Abstract
Damage to the gastrointestinal (GI) tract is a major complication of graft versus host disease (GVHD). IL-23 has been shown to be a proximate mediator of GVHD-induced pathology within the GI tract; but the donor immune cell populations that mediate the downstream effects of this cytokine and how IL-23-induced inflammation is regulated are not well understood. To define the functional significance of IL-23R expression on donor cells, we first employed antibody-based and genetic approaches to examine the effect of interruption of IL-23R signaling on GVHD severity. Lethally irradiated Balb/c (H-2d) mice transplanted with C57BL/6 (H-2b) BM and spleen cells, and then treated with an anti-IL-23R antibody had significantly prolonged survival compared to mice administered control antibody (70% versus 0% survival at 60 days, p<0.001). Additionally, recipients reconstituted with IL-23R‾/‾ BM and spleen cells had increased survival and less pathological damage in the colon relative to mice transplanted with grafts from wild type B6 animals. Experiments performed to define the critical IL-23R-expressing population revealed that survival was significantly prolonged in animals transplanted with IL-23R‾/‾ T cells, whereas mortality was unaffected when IL-23R expression was present only on BM-derived cells indicating that IL-23R expression on T cells drove inflammation. Furthermore, transplantation with CD4+ T cells from IL-23R¯/¯ animals resulted in less mortality, demonstrating that the T cell-directed proinflammatory effects of IL-23 were mediated primarily by CD4+ IL-23R+ T cells. We observed that CD4+ IL-23R+ T cells could be identified in the colon of GVHD animals as early as five days post transplantation, and were present in significantly higher numbers when compared to syngeneic recipients. Unexpectedly, we discovered a population of donor-derived CD4+ T cells that expressed the β2 integrins, CD11b and CD11c, and determined that the highest percentage of IL-23R-expressing CD4+ T cells in the colon derived from cells that co-expressed these integrins. Experiments conducted to determine the functional significance of β2 integrin expression on CD4+ T cells revealed that animals transplanted with sorted CD4+ αb+ T cells that lacked expression of both CD11b and CD11c had a significantly reduced number of total donor-derived CD4+ and CD4+ IL-23R+ T cells in the colon, reduced pathological damage, and significantly prolonged survival from lethal GVHD. Furthermore, mice reconstituted with CD4+ IL-23R−/− T cells that expressed at least one β2 integrin had significantly greater survival than animals transplanted with CD4+ IL-23R+/+ T cells that had the same integrin profile, indicating that the pathogenicity of β2 integrin–expressing CD4+ T cells was dependent upon co-expression of the IL-23R. Since IL-10 has been shown to have a critical role in the maintenance of immune homeostasis within the colon and also signals through Stat3, we sought to define whether IL-10 was critical for the regulation of IL-23R-mediated inflammation. Using IL-10 reporter mice (10BiT.Foxp3EGFP) to delineate IL-10 expression in immune cell populations, we observed that thepreponderance of IL-10 in GVHD target tissues derived almost exclusively from donor non-Foxp3-expressing CD8+ T cells. The importance of IL-10 as a critical regulatory cytokine of IL-23R signaling was corroborated by the fact that transplantation with IL-10‾/‾ grafts augmented GVHD mortality, blockade of IL-23R signaling was ineffectual in the absence of donor-derived IL-10 production, and transplantation with CD4+ T cells from IL-23R‾/‾ IL-10‾/‾ animals abrogated the protection that was observed when animals were reconstituted with IL-23R‾/‾ T cells alone. Blockade of IL-23R signaling prolonged survival in the complete absence of functional CD4+ Tregs, indicating that, in contrast to IL-10, there was not an obligate requirement for CD4+ Tregs to observe a protective effect from IL-23R signaling inhibition. A role for IL-23/IL-23R signaling in humans was supported by studies demonstrating increased IL-23 and IL-23-dependent (IL-17A, IL-22, IL-17F, and Reg3γ) gene expression in the GI tract of patients with GVHD. In summary, these studies indicate that the coordinate expression of β2 integrins and the IL-23R in CD4+ T cells defines a novel, pathogenic T cell population that potently induces inflammation in the colon during GVHD.

Disclosures
No relevant conflicts of interest to declare.