An Interleukin 27 Dependent Lysosomal Thiol Reductase Regulates Regulatory T Cell Survival and Mitochondrial Fitness during Graft Versus Host Disease

Graft versus host disease (GVHD) is a proinflammatory syndrome driven by alloreactive donor T cells and inflammatory cytokine production which results in tissue destruction and immunological impairment. A critical element of the pathophysiology of GVHD is the failure to reconstitute the regulatory T cell (Treg) compartment. Re-establishment of an effective regulatory network to counterbalance the pro inflammatory milieu by enhancing Treg survival and augmenting suppressive capability therefore remains a major goal in the field. To unveil therapeutically targetable inflammatory cytokine-driven pathways that adversely impact Treg reconstitution during GVHD, our studies focused on the role of interleukin 27 (IL-27) which is produced by activated antigen presenting cells. To examine this question, we employed major (C57BL/6 [H-2b]→Balb/c [H-2d]) and minor (B6 [H-2b]→Balb.B [H-2b]) histocompatibility mismatched murine bone marrow transplantation models and observed that blockade of IL-27 signaling using an IL-27p28-specific antibody significantly improved survival and reduced pathological damage in the liver, lung, and colon when compared to isotype control antibody-treated mice. Similarly, transplantation with IL-27R^-/- marrow grafts significantly reduced GVHD lethality and decreased the number of proinflammatory T cells in GVHD target tissues, supporting findings observed after p28 antibody administration. Notably, we observed that the genetic deletion of the IL-27R as well as p28-specfic antibody blockade augmented the reconstitution of peripherally induced and thymically derived CD4⁺ Tregs in recipient animals, and stabilized Foxp3 expression, preventing reversion to a more pathogenic cellular phenotype. Furthermore, adoptive transfer of IL-27R^-/- Tregs was more potent at suppressing GVHD lethality than wild type (WT) Tregs. To identify downstream IL-27-dependent pathways that regulated CD4⁺ Treg function, donor CD4⁺ Foxp3⁺ EGFP⁺ T cells were flow sorted from pooled livers and colons of mice transplanted with either WT (B6) or IL-27R^-/- Foxp3⁺ EGFP⁺ marrow grafts and subjected to bulk RNA sequencing. Bioinformatic analysis of RNA sequencing results revealed that the gene IFI30, which encodes the protein known as GILT, a gamma interferon inducible lysosomal thiol reductase, was significantly over expressed in WT when compared to IL-27R^-/- Tregs in both tissue sites. To confirm a direct linkage between IL-27 and GILT expression, in vitro-induced CD4⁺ Foxp3⁺ Tregs cultured in IL-27 were subjected to western blot analysis which demonstrated a linear correlation between increasing IL-27 concentrations and GILT expression. In vivo studies also confirmed that GILT expression was constitutively increased in naïve splenic WT when compared to IL-27R^-/- CD4⁺ Tregs. Competitive repopulation studies revealed that GILT^-/- CD4⁺ Tregs were present in significantly higher frequencies (9:1 ratio) than WT Tregs in all GVHD target organs, indicating that these cells had superior in vivo survival. To determine if GILT expression was functionally significant, WT or GILT^-/- CD4⁺ Foxp3⁺ EGFP⁺ Tregs were adoptively transferred into transplant recipients to prevent GVHD. These studies demonstrated increased Treg persistence in GVHD target organs and improved survival in mice reconstituted with GILT^-/- CD4⁺ Tregs, confirming that these cells were more potent at suppressing GVHD lethality and that the functional inhibitory effects of IL-27/IL-27R signaling were mediated by downstream expression of GILT. Since Treg function and survival is critically dependent upon mitochondrial metabolism, we comparatively assessed metabolic function in WT versus GILT^-/- Tregs and observed that polyclonally activated GILT^-/- CD4⁺ Tregs had superior glycolytic activity, oxidative phosphorylation, and fatty acid oxidation when compared to WT CD4⁺ Tregs. Mechanistically, augmented mitochondrial fitness was attributable to increased mTor signaling leading to inhibition of autophagy. Additionally, GILT^-/- CD4⁺ Tregs had a significant reduction in mitochondrial reactive oxygen species, indicating that GILT exacerbated oxidative stress in these cells. Thus, these results demonstrate that IL-27/IL-27R signaling inhibits CD4⁺ Treg survival, suppressive capability, and mitochondrial fitness by regulating intracellular lysosomal thiol reductase expression.