Lung Parenchyma–Derived IL-6 Induces Alloantigen Specific Th17 Differentiation Within The Lung and Idiopathic Pneumonia Syndrome After Allogeneic Stem Cell Transplantation

Idiopathic Pneumonia Syndrome (IPS) is a fulminant and largely untreatable form of GVHD of the lung following allogeneic stem cell transplantation (SCT). It characteristically develops in the third week after SCT in patients receiving prior total body irradiation (TBI). We have previously shown that IFNγ signaling within lung parenchyma is critical for preventing disease (Burman AC et al, Blood 2007, 110:1064). Using IL-17A-fate reporter grafts (where YFP is expressed if a T cell has ever made IL-17) we now show that Th17 cells preferentially accumulate in the lung (but not other sites) after experimental allo-SCT in recipients in which IFNγ is neutralized (lung Th17 cells: 6.6x10⁴ ± 0.2 vs. 0.8x10⁴ ± 0.01, anti-IFNγ vs. control treated, P = 0.002). To understand whether this accumulation of Th17 cells in the lung and IPS in general was an alloreactive (i.e. GVHD) dependent phenomena, we transplanted luciferase expressing B6 TEa TCR transgenic CD4⁺ T cells specific for alloantigen (I-E^d) expressed in B6D2F1 recipients in the presence or absence of IFNγ neutralization. Remarkably, allospecific donor TEa T cells again accumulated in the lung in the absence of IFNγ (BLI: 2.2x10⁵ ± 0.6 vs. 0.54x10⁵ ± 0.08 ph/sec/cm²/sr, P = 0.008), but not other sites, excluding responses to other antigens (e.g. pathogen-derived) as a cause for IPS. Critically, we show that donor T cell-derived IFNγ was vital for inhibiting IL-6, TGFβ and IL-21 mRNA expression in lung parenchymal cells after SCT. Protein analysis confirmed that IL-6 was markedly elevated in lung tissue lysate 5 days after allo-SCT in IFNγR^–/– recipients compared to WT (128 ± 25 pg/ml vs. 20 ± 5 pg/ml, P = 0.002). In contrast, IL-6 was elevated to equivalent levels in sera of both WT and IFNγR^–/– SCT recipients. Studies using IL-6^–/– recipients demonstrated that IL-6 was critical for Th17 differentiation within the lung and subsequent IPS. In the absence of IFNγ, IL-6 promoted Th17 cells to preferentially expand in the lungs (14.6x10⁴ ± 0.03 vs. 3.8x10⁴ ± 0.01, WT vs. IL-6^–/–, P = 0.04), produce IL-17A (3734 ± 736 pg/ml vs. 521 ± 142 pg/ml, WT vs. IL-6^–/–, P = 0.003) and induce IPS (lung pathology scores: 7.3 ± 0.9 vs. 5.0 ± 0.3, WT vs. IL-6^–/–; P = 0.02). Consistent with Th17 differentiation as the final pathological pathway, systemic blockade of IL-17A or the transplantation of grafts lacking the IL-17 receptor also profoundly attenuated disease (lung pathology scores: 3.9 ± 0.5 vs. 6.7 ± 0.6, anti-IL-17A vs. control treated, P = 0.002; and 4.5 ± 0.4 vs. 8.1 ± 1.0, IL-17RA^–/–- vs. WT, P= 0.003, respectively). Thus IFNγ is the critical cytokine for suppressing an environment conducive to Th17 differentiation within the lung and IPS after allogeneic SCT.

To study the clinical relevance of these findings we examined IL-6 dysregulation in a cohort of patients receiving allogeneic peripheral blood stem cell transplantation at our institution. We confirm that plasma levels of IL-6 are also significantly elevated in SCT recipients within the first 2 weeks of transplantation (30.6 ± 8.8 pg/ml vs. 8.3 ± 3.5 pg/ml, day +7 vs. day -7, P = 0.008), particularly in patients receiving Cy/TBI (12 Gy) compared to reduced intensity Flu/Mel (120mg/m²) conditioning (74.4 ± 38.4 pg/ml vs. 23.4 ± 7.1 pg/ml, P = 0.04). Furthermore, standard calcineurin and methotrexate–based immune suppression completely prevented systemic IFNγ generation after SCT, thus inducing an IL-6–high, IFNγ–deplete environment highly conducive to the development of IPS. These data confirm IL-6 and IL-17 as logical therapeutic targets to prevent IPS after clinical SCT utilizing TBI-based conditioning.