Abstract
The interleukin-6 (IL-6) cytokine superfamily (i.e. IL-6, IL-12, and IL-23) plays a major role in the modulation of inflammatory and regulatory pathways during graft versus host disease (GVHD). IL-27, a recently discovered member of this family, is a heterodimeric cytokine that is composed of the p28 and EBI3 subunits and signals through a heterodimeric receptor composed of WSX-1 and gp130. Notably, IL-6 also uses gp130 as a signaling component which biologically links IL-27 and IL-6. IL-27 has been shown to have opposing proinflammatory and immunoregulatory effects, but its role in GVHD is not well understood. To define the functional significance of IL-27, lethally irradiated Balb/c (H-2d) mice were transplanted with C57BL/6J (H-2b) BM and spleen cells, and then treated with an anti-IL-27p28-specific antibody on days 0 and +6. p28 antibody-treated animals had significantly improved weight recovery and overall survival (47% versus 0% survival at day 60, p=0.002), as well as reduced numbers of proinflammatory CD4+ and CD8+ IFN-γ+ T cells in GVHD target organs, when compared to isotype control antibody-treated mice. A similar outcome was observed in an MHC-matched, minor antigen disparate model (B6→Balb.B), indicating that this was not a strain-specific phenomenon. Given the similarities between IL-6 and IL-27, we examined whether blockade of IL-27 promoted regulatory T cell (Treg) reconstitution as has been observed with inhibition of IL-6 signaling. Recipients transplanted with BM grafts from B6 Foxp3EGFP reporter animals and treated with p28 antibody had a significant increase in the number of CD4+ nTregs, CD4+ iTregs and CD8+ iTregs in GVHD target organs, indicating that blockade of IL-27 augmented global Treg reconstitution. In fact, inhibition of IL-27 was more effective at augmenting Treg reconstitution than comparable antibody blockade of IL-6. To further elucidate the role of IL-27, we employed transgenic IL-27−/− and IL-27R−/− animals to dissect the relevant contributions of donor and recipient populations. Paradoxically, we observed that transplantation with IL-27−/− donor grafts exacerbated GVHD mortality and augmented accumulation of proinflammatory T cells, whereas transplantation of recipient IL-27−/− mice with wild type grafts had no effect on transplant outcomes. This discordance between antibody-based and genetic studies was unexpected and led us to consider whether there were steady state alterations in T cells from IL-27−/− animals that biased these cells towards a proinflammatory phenotype. To that end, we observed that naive CD8+ T cells from IL-27−/− mice had greater IFN-γ production than wild type cells after in vitro polyclonal stimulation and CD4+ nTregs from these animals had diminished expression of CXCR3 which is critical for Treg trafficking into inflamed tissue sites. Thus, the lack of endogenous IL-27 resulted in intrinsic immune dysregulation which led to an exacerbation of GVHD after transfer of these T cells into recipients. To resolve this paradox, we employed IL-27R−/− (WSX-1−/−) mice and demonstrated that mice transplanted with IL-27R−/− grafts had enhanced weight recovery and survival providing confirmation that blockade of IL-27 signaling reduced GVHD. In addition, using IL-27R−/− Foxp3EGFP reporter mice, we observed increased frequencies and numbers of CD4+ and CD8+ Foxp3+ T cells in mice reconstituted with IL-27R−/− grafts, confirming results observed with p28 antibody blockade. Since IL-10 is a mechanism by which CD4+ Tregs suppress GVHD and IL-27 has been shown to enhance T cell-derived IL-10 secretion in nontransplant models, we examined whether IL-27 blockade adversely affected IL-10 production by Tregs. Recipients transplanted with marrow grafts from IL-10.BitFoxp3EGFP dual reporter animals and treated with p28 antibody had a significant reduction in the frequency of IL-10-producing conventional CD4+ and CD8+ T cells in GVHD target organs. Notably, however, there was no difference in the frequency of CD4+ Foxp3+ IL-10+ T cells, indicating that blockade of IL-27 signaling preferentially affected conventional T cells and had no adverse effect on CD4+ Foxp3+ T cell-derived IL-10 production. In summary, these studies demonstrate that blockade of IL-27 signaling potently augments Treg reconstitution leading to a reduction in the severity of GVHD and may therefore represent a novel strategy to reduce mortality from this disease in man.
Disclosures
No relevant conflicts of interest to declare.
Blockade of Interleukin 27 Signaling Attenuates Graft Versus Host Disease By Augmenting CD4+ and CD8+ Regulatory T Cell Reconstitution
