Prognostic Analysis Of Graft Versus Host Disease And The Study Of Immune Regulatory Effect Of Prostaglandin E2 On CD4+ T Cells

Background:Allogeneic stem cell transplantation(allo-SCT) is a widely accepted treatment choice for many hematologic malignancies and it offers better prognosis for some of them.However,acute and chronic graft versus host disease(GVHD) remains the main cause of non-relpase mortality and the leading contributing factor for impaired quality of life after transplantation.On the other hand,GVHD exerts a powerful graft versus leukemia(GVL) effect, and the latter constitutes the superiority of allo-SCT against autologous SCT. How to balancing the competing effects of GVHD and GVL was always the focus of study in the fields of allo-SCT.Objective and methods:In order to evaluate the impact of the GVHD on the outcome of allo-SCT and to study the ideal strategy to manipulate GVHD,we retrospectively reviewed a relatively uniform population of patients.MRD SCT recipients with acute leukemia in first complete remission or chronic myeloid leukemia(CML) in first chronic phase were enrolled in the present study.Five major endpoints of study were evaluated,that's ACM,TRM,cumulative incidence of relapses, disease free survival and overall survival.Results:1.From January 2000 to December 2007,171 eligible cases were identified. After a median follow-up of 730 days(23-2500 days),105 cases were alive and 97 of them were in continuous complete remission,with OS and DFS of 53.5%and 50.8%respectively.Sixty-six cases of mortality were recorded, which were due to treatment related causes in 47 cases and disease relapses in 19 cases,leading to incidence of 30.7%and 15.8%respectively.2.Patients with acute GVHD of gradeâ…¡-â…£demonstrated higher TRM and poor prognosis.In patients without acute GVHD(n=73),with gradeâ… acute GVHD (n=74) or with gradeâ…¡-â…£acute GVHD(n=24),3-year DFS were 59.7%, 62.6%and 25.0%respectively(p＜0.0001),TRM were 21.4%,28.3%and 63.5%respectively,while no difference of relapse rates were detected.3.Patients with extensive chronic GVHD showed higher TRM,lower relapse rate and poor prognosis.In patients without chronic GVHD(n=78),with limited chronic GVHD(n=47) or with extensive chronic GVHD(n=34),3-year DFS were 67.0%,70.7%and 36.9%respectively(p＜0.0001),the TRM were 12.9%,23.3%and 66.3%respectively(p＜0.0001),while relapse rate were 24.9%,17.7%and 2.9%respectively(p=0.06).4.For patients complicating acute GVHD of gradeâ…¡-â…£or chronic GVHD of extensive form,use of high dose steroid was related to poor prognosis,with 3-year DFS of 14.5%and 47.0%in patients with cumulative dose of steroid lower or higher than 14 mg/kg respectively.5.As to acute lymphoblastic leukemia(ALL),patients with gradeâ… acute GVHD (n=15) demonstrated better prognosis in comparison to patients free from acute GVHD(n=17),with 3-year DFS of 64.2%and 14.7%respectively (p=0.002).In patients with CML,acute GVHD of any grade led to poor prognosis,with 3-year OS of 86.0%,63.3%and 36.4%in patients without acute GVHD,with gradeâ… or gradeâ…¡-â…£acute GVHD respectively;limited chronic GVHD(n=18) offered better prognosis against patients without chronic GVHD(n=39) or with extensive form(n=18),with 3-year OS of 100%, 80.4%and 26.5%respectively(p＜0.0001).6.Multivariate analysis identified three independent risk factors of prognosis: underlying diseases(ALL vs AML vs CML),occurrence of severe GVHD (gradeâ…¡-â…£acute GVHD and extensive chronic GVHD vs control) and use of high dose steroid.Conclusion:1.TRM was the major cause of mortality in allo-SCT recipients.2.As to patients with ALL,mild acute GVHD was a protective factor for better prognosis and lower relapse risk.3.As to CML,chronic GVHD in the limited form was a protective factor;and acute GVHD of any grade led to poor prognosis.4.Regardless of underlying diseases,acute GVHD of gradeâ…¡-â…£or extensive chronic GVHD was a risk factor of poor prognosis,with dramatically increased TRM.High dose steroid can not change the poor prognosis of severe GVHD.5.Underlying diseases,occurrence of severe GVHD and use of high dose steroid were independent risk factors of prognosis. Background:Prostaglandin E₂(PGE₂) had long been established as an important mediator in the physiological processes of inflammation and anti-inflammatory reactions. Studies in the past decades had revealed its important role in the immune tolerance, autoimmune diseases,immune regulation during graft versus host disease(GVHD) and tumor immunotolerance.T lymphocytes are major effectors of acute GVHD and play key role in the development and progression of GVHD.Previous studies demonstrated that PGE₂ showed some regulatory effects on the immune activity of CD4+ T cell by inducing growth inhibition or by inducing differentiation of CD4+CD25+Foxp3+ regulatory T cells(Treg).Based on these results,PGE₂ was considered to be a promising candidate for the control of acute GVHD.Objective:To study the immune regulatory effects of PGE₂ on the human CD4+ T cells during in vitro culture.We intend to find new targets of PGE₂ by the method of gene expression profiling,and to address its immune regulatory mechanisms during the subsequent examination for the downstream products of the target gene.Methods:1.Buffy coats of healthy donors were used to provide mononuclear cells(MNCs), which was done by the method of density gradient centrifugation.CD4+ T cells were purified by the method of magnetic cell sorting(MACS),during which the MNCs were labeled by CD4+ antibody coated micro-beads.2.Purified CD4+ T cell were stimulated by anti-human CD3 monoclonal antibody (mAb) and anti-human CD28 mAb and incubated with complete medium (RPMI1640 with 10%FCS) at 37℃in 5%CO₂,with(treated group) or wihout (control group) the presence of the PGE₂.Twelve hours later,cells were collected and sent to microarray detection,whose results were subsequently confirmed by the method of real-time polymerase chain reactions(RT-PCR).3.When target genes were determined,ELISA and flow cytometry(FCM) were applied to exmine the downstream products. Results:1.FCM demonstrated that the CD4+ T cells after MACS had purity of≥99%.Gene expression profiling showed that,after 12 hours of in vitro culture in the presence of PGE2(13 umol/L),1716 genes were down regulated and 73 genes were up regulated with a fold change of 1.5.Several signal transduction pathways were involved,including TNF-αand NF-kB signaling pathway,T cell receptor signaling pathway,IL-2 signaling pathway,MAPK pathway and et al.2.The method of ELISA was used to detect the concentration of cytokines in the supernatant,and significant reduction of IFN-γand TNF-αwas found in different timepoints of culture after treatment of PGE2.At the timepoints of 24,48 and 72 hours after cell culture,IFN-γshowed concentration of 0,(2.3±4.7) pg/ml, (10.9±4.7) pg/ml in the treated group,and(399.6±332.1)pg/ml,(842.8±479.7) pg/ml, (2963.1±644.9) pg/ml in the control group respectively.Forty-eight hours and 72 hours after cell culture,TNF-αshowed concentration of(113.5±57.3) pg/ml, (189.7±32.0)pg/ml in the treated group,and(1093.7±353.3) pg/ml,(1627.2±498.6) pg/ml in the control group respectively.However,the concentration of IL-2 in all timepoints was below the detection threshold of ELISA kit in the treated as well as the control group.3.Results of FCM analysis showed that,in comparison to the control,the expression of CD25,CD69,HLA-DR and CD40 was reduced in the treated groups 48 and 72 hours after cell culture.Conclusion:1.In the in vitro culture of CD4+ T cells,the presence of PGE₂ led to down-regulation of gene expression,mainly involving pathways closely related to immune regulation.2.In the presence of PGE₂,the core inflammatory mediators involved in acute GVHD, as well as surface factors that played important role in the activation and co-stimulation of CD4+ T cells,were down regulated.3.In conclusion,the in vitro experiment demonstrated prominent negative regulatory activity of PGE₂ on CD4+ T cells,and this may lead to some clinical significance in the management of GVHD.