Blockade of Interleukin 27 Signaling Attenuates Graft Versus Host Disease By Augmenting CD4+ and CD8+ Regulatory T Cell Reconstitution

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-2^d) mice were transplanted with C57BL/6J (H-2^b) 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 Foxp3^EGFP 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^−/−Foxp3^EGFP 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.BitFoxp3^EGFP 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.