The Role Of IL-27 In Acute Graft-versus-host Disease After Allogeneic Hematopoietic Stem Cell Transplantation

Part?:The function and mechanism of IL-27 in acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantationObject:The current study aimed to detect the function of IL-27 in murine a GVHD after allogeneic hematopoietic stem cell transplantation.Methods:The BALB/C（H-2^d）mice,wild type（WT）C57BL/6（H-2^b）mice and IL-27（interleukin-27）knockout(IL-27^-/-)mice in B6 background were used as donor mice,and BALB/C（H-2^d）mice were used as recipients.BALB/C recipients were received lethally irradiation with 650c Gy by X-Ray and injected intravenously with 1x10⁷ bone marrow（BM）cells and 5 x10⁶ splenocytes（SP）from BALB/C mice or C57BL/6 mice respectively.Total body irradiation（TBI）was divided into 2 steps separated by 4 hours to reduce the gastrointestinal toxicity.Total RNA from GVHD target tissues including spleen,liver,lung were extracted and reverse transcribed into c DNA at day 3,7,14 after transplantation.Quantitative real-time PCR was performed to detected the relative IL-27expression.BALB/C recipients were received lethally irradiation with 650c Gy by X-Ray and respectively injected intravenously with 1x10⁷ bone marrow（BM）cells and 5 x10⁶splenocytes（SP）from C57BL/6 mice,1x10⁷bone marrow（BM）cells from C57BL/6 mice and 5 x10⁶splenocytes（SP）from IL-27^-/-mice.Mice survival and pathological features were monitored and recorded.Systemic GVHD score was assessed by a cumulative scoring system.For histology examination,2 weeks after transplantation,tissue was fixed and embedded for hematoxylin and eosin（H&E）staining.The histopathology score was assessed by a semi-quantitative scoring system.Lymphocytes in spleen,liver,lung and intestine were isolated and detected for allo-reactive T cell responses by flow cytometry 2weeks after transplantation.BALB/C recipients were received lethally irradiation with 650c Gy by X-Ray and respectively injected intravenously with 1x10⁷bone marrow（BM）cells and 5 x10⁶splenocytes（SP）from C57BL/6 mice,1x10⁷ bone marrow（BM）cells from C57BL/6 mice and 5 x10⁶ splenocytes（SP）from IL-27^-/-mice,Populations of Treg cells in WT and IL-27^-/-recipients in GVHD target tissues were detected by flow cytometry 2 weeks after transplantation.In vitro studies also revealed the function of IL-27 on Treg cell differentiation by CD3/CD28 stimulation.Results:（1）Compared to syngeneic controls,IL-27 expression was increased in spleen,liver,lung in allogeneic transplantation.（2）Compared to WT group,Recipients with C57BL/6 bone marrow and IL-27^-/-spleen grafts exhibited the highest mortality.（3）Donor IL-27^-/-significantly augmented GVHD pathogenesis and promotes GVHD related mortality.Histologic assessment revealed that significantly tissue damage increased in the liver,lung,small intestine,as well as skin in the IL-27^-/-recipients.（4）In vitro studies revealed that IL-27^-/-promoted the proliferation and early apoptosis of T cells.（5）Compared to recipients with WT group,populations of CD4⁺T cells were obviously increased.（6）Compared to recipients with WT group,activation of CD3⁺and CD8⁺T cells in recipients with IL-27^-/-grafts were significantly increased.（7）Populations and absolute numbers of IFN-γproducing CD4⁺and CD8⁺T cells were markedly elevated in recipients with IL-27^-/-group.（8）Compared to WT group,the expression of IFN-γ,TNF-α,IL-6 were decreased in IL-27 deficiency group.（9）The percentage of Treg cells were obviously decreased in IL-27 deficiency group.Conclusion:IL-27 expression was significantly increased in allogeneic transplantation when compared to syngeneic transplantation.IL-27 deficiency aggravated murine acute GVHD and shortened survival.Splenocytes derived IL-27 exhibited a protective effect in acute GVHD after hematopoietic stem cell transplantation.IL-27deficiency in donors promoted CD4⁺cells activation and inflammatory cytokine IFN-γproduction.Both in vivo and in vitro studies suggested that IL-27 promotes Treg cell differentiation.IL-27 regulated murine acute GVHD by a Treg cell dependent manner.Part Ⅱ: Interleukin-27 is a predictive biomarker for the development of acute graft-versus-host disease after allogeneic hematopoietic stem cell transplantation in humanObjective: To detect the predictive value of serum interleukin-27（IL-27）in acute Graft-Versus-Host Disease（a GVHD）after allogeneic hematopoietic stem cell transplantation.Methods: Serum were collected from 67 patients after allo-HSCT during January 2012 to December 2012.All of the patients received myeloablative conditioning.Cs A+MMF+MTX were adopted for GVHD prophylaxis.In this study,we detected serum interleukin-27（IL-27）)in patients with a GVHD by ELISA.Then,we retrospectively evaluated whether IL-27 had predictive value in allogeneic HSCT.Results: Serum IL-27 was significantly decreased in grade II–IV a GVHD patients（grade 0–I: 35.57 ± 14.34pg/ml vs grade II–IV: 27.66 ± 6.74pg/ml;P<0.05）.IL-27 had a predictive ability for grade II–IV a GVHD（AUC=0.70,95% CI 0.528-0.789,P=0.029）.Patients with lower serum IL-27（<26.85pg/ml）had higher cumulative incidence of grade II–IV a GVHD than patients with higher serum IL-27（P=0.01）.Multivariate analysis confirmed that low IL-27（HR=3.005,95%CI 1.14-6.86,P<0.01）was the most significant risk factor for predicting grade II-IV aGVHD.Conclusion: Lower IL-27 might predict the development of grade II-IV acute GVHD after allo-HSCT.