Reduced IL-35 Levels Are Associated with Increased Platelet Aggregation and Activation in Patients with Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation

Introduction: Acute graft-versus-host disease (aGVHD) is a major limitation of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The mechanism of aGVHD has not been completely elucidated. aGVHD is primarily caused by immune responses to allogeneic disparities between the donor and recipient organs (Nakamura K, et al. Bone marrow transplantation 2005). aGVHD can also lead to thrombotic complications through endothelial damage. Following endothelial injury, inflammation triggers platelet activation and leads to enhanced expression of CD62P, which can then interact with the P-selectin glycoprotein ligand 1 receptor expressed on monocytes and mediate the rolling of monocytes on activated endothelium, facilitating platelet-leukocyte aggregation and inducing platelet thrombus formation. IL-35 is a novel anti-inflammatory cytokine that suppresses the immune response. Previous work has demonstrated that IL-35 is an anti-inflammatory cytokine that suppresses the immune response through the expansion of regulatory T cells and suppression of Th17 cell development that has a protective function in various autoimmune disorders. In this study, we hypothesized that patients with aGVHD may have increased platelet aggregation, which is associated with an increased risk of thrombus formation in aGVHD. Because the increased platelet aggregation is caused by inflammation during aGVHD, and IL-35 is a novel anti-inflammatory cytokine, IL-35 could also inhibit platelet activation and aggregation in aGVHD patients.

Methods: In this study, we prospectively studied a total of 65 patients who received allo-HSCT. We measured plasma levels of IL-35 inpatients just at the onset of aGVHD. We collected time-matched samples from patients without aGVHD to serve as controls after HSCT. We also detected the levels of IL-35 in granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells (PBPC) and G-CSF-primed bone marrow (GBM) to explore the relationship between IL-35 levels and the occurrence of aGVHD. Then, we analyzed platelet aggregation in patients with and without aGVHD. We also added IL-35 to the plasma of these patients to test its function on platelet aggregation and platelet activation.

Results: The plasma levels of IL-35 were reduced in the patients with grade III-IV aGVHD (23.46 ng/ml) compared with the patients with grade I-II aGVHD (40.26 ng/ml, p<0.01) and no aGVHD (41.40 ng/ml, p<0.05). Allografts from 38 patients were analyzed for IL-35 levels with respect to aGVHD. The patients were divided into either a high IL-35 group or a low IL-35 group according to the median levels of IL-35 in the patient’s GBM (28.0 pg/ml) and PBPC (53.1 pg/ml). We found that patients in the low IL-35 group demonstrated a higher cumulative incidence of aGVHD compared with patients in the high IL-35 group. In addition, patients with aGVHD have increased platelet aggregation compared with patients without aGVHD. In vitro, platelet aggregation was inhibited by IL-35 in a dose-dependent manner. The markers of platelet activation (CD62P/PAC-1) were also inhibited by IL-35.

Conclusions: The results imply that IL-35 may predict the occurrence of aGVHD, as well as inhibit platelet activation and aggregation. These data suggest that IL-35 might represent a potential effective therapeutic agent against aGVHD and prevent thrombotic complications after allo-HSCT.