IL-39/IL-39R signaling drives acute graft-versus-host disease

Abstract Background: Acute graft-versus-host disease (aGVHD) is a major cause of morbidity and non-relapse mortality (NRM) following allogeneic hematopoietic cell transplantation (allo-HCT), emphasizing the need for novel biomarkers and therapeutic interventions. Among cytokine families, IL-12 family cytokines play essential roles in the pathogenesis of aGVHD. IL-39, composed of IL-23p19 and EBI3, is a proposed IL-12 family member. We previously showed IL-23p19 has greater pathogenicity than IL-12/23p40 in aGVHD, and that IL-23Rα, but not IL-12Rβ1, is required for donor T cell–mediated aGVHD. These findings suggest IL-39 may drive aGVHD via IL-23Rα/gp130 signaling, but its role in graft-versus-host and graft-versus-leukemia (GVL) responses remains to be validated. Methods: We quantified IL-39 levels using ELISA at baseline and 7 timepoints post-HCT in 276 patients (138 with grade 2–4 aGVHD and 138 without aGVHD), from the prospectively BMT CTN 1202 study. Associations with aGVHD were assessed using linear mixed models, adjusting for clinical covariates. Landmark Cox regression models evaluated links of day 90 IL-39 levels with aGVHD, relapse, NRM and overall survival (OS). Human recombinant IL-39 (hrIL-39) was used to assess proliferation, cytokine production, T helper 1 (Th1)/T helper 17 (Th17)/regulatory T (Treg) cell differentiation, and STAT1/3 phosphorylation in murine and human Th17 cells. Murine and human IL-23Rα/gp130 heterodimerization was confirmed via protein ligation assays (PLA). MHC-mismatched murine BMT model, C57BL/6 into BALB/c, was used to establish aGVHD, and host-origin MLL-AF9 leukemia was inoculated for evaluating the GVL activity. A human xenogeneic GVHD model was used to evaluate IL-39 effect on disease progression and allogeneic T cells response. Results: Plasma IL-39 levels were significantly elevated in patients who developed aGVHD with the median onset at day 35 (p < 0.0001), particularly at days 14 (p = 0.022), 21 (p = 0.029), 28 (p = 0.026), 42 (p = 0.008), and 56 (p = 0.029). IL-39 levels positively correlated with grade 2-4 aGVHD onset (hazard ratio (HR) = 1.42, p = 0.01) and were independently associated with increased NRM (p = 0.008) and worse OS (p = 0.009). However, IL-39 levels were not associated with disease relapse (HR=1.01, p=0.97). HrIL-39 promoted proliferation and inflammatory cytokine production (IFN-γ, TNF-α, GM-CSF) in both murine and human T cells, enhanced Th1/Th17, and suppressed Treg cell differentiation. HrIL-39 stimulation induced robust STAT1 and STAT3 phosphorylation particularly in IL-23Rα high expressed murine and human Th17 cells. IL-23Rα/gp130 heterodimerization, the putative IL-39 receptors, was confirmed in murine and human cells via PLA assay. In murine aGVHD models, hrIL-39 significantly exacerbated GVHD severity, increased GVHD scores (p < 0.0001), decreased survival (p = 0.0097), and was associated with elevated IFN-γ⁺ (p = 0.003), TNF-α⁺ (p = 0.02), and IL-17⁺ (p = 0.030) CD4⁺ T cells while reducing Foxp3⁺ Tregs (p = 0.018). However, these effects mediated by hrIL-39 were absent when IL-23Rα was deficient in donor T cells, indicating that IL-23Rα is required for IL-39 signaling. The recipients of IL-23RαKO or IL-23Rα/IL-12Rβ1 DKO donor splenocytes showed similarly improved GVHD outcomes, both significantly better than IL-12Rβ1-/- or WT, which were comparable. The GVL activity was preserved in all groups, confirming that IL-23Rα drives aGVHD independently of IL-12Rβ1. In the human xenograft GVHD model, hrIL-39 similarly worsened disease, leading to reduced survival (p=0.0003), progressive weight loss (p<0.0001), and elevated GVHD scores (p<0.0001). Flow cytometry of splenic tissue revealed increased human T-cell infiltration (p=0.006) and elevated frequencies of IFN-γ⁺ (p=0.045), TNF-α⁺ (p=0.03), and IL-17⁺CD4⁺ (p=0.025) human T cells, along with a marked reduction in Foxp3⁺Tregs⁺ (p=0.007). These findings mirror murine data and confirm the conserved pathogenic role of IL-39 in human GVHD pathogenesis. Conclusions: Our findings establish that IL-39/IL-39R signaling drives aGVHD pathogenesis in both murine and human settings. This pathway represents a potential novel biomarker and therapeutic target for aGVHD prevention while preserving GVL responses.