Preliminary Study On The Role Of LIGHT-HVEM Co-stimulatory Pathway In Graft-versus-host Disease

Objective and BackgroundAllogeneic hematopoietic stem cell transplantation has been accepted as the most effective treatment for hematological malignancies,benign hematological disorders,solid tumors,inherited diseases and some other autoimmune diseases.Since nowadays the society is made up of more and more core families,the sibling donors are harder to find. Therefore,HLA haploidentical transplantation becomes one of important treatment options.Compared with HLA-identical transplantation,HLA haploidentical transplantation is complicated by a higher rate of graft-versus-host disease(GVHD), which is the major cause of morbility and mortality after transplantation.GVHD is a pathophysiological process caused by alloreactive donor T cells which recognize host allo-antigens and cause the damage of host tissues.Depleting T cells in allograft and the use of immunosuppressive drugs have shown to be effective to reduce the incidence of GVHD.However,these approaches may result in several severe consequences,including the increased incidence of relapse,opportunistic infection.How to modulate T cell polarization which would tolerate tissue alloantigens while remain the functions of anti-leukemia and anti-infection are still an open question.T cell activation requires two stimulatory signals.Signaling through T cell receptor (TCR),termed the first signal which is both antigen specific and major histocompatibility complex(MHC) restricted,is necessary but not sufficient to induce antigen-specific T cell activation and cytokine secretion.The other signaling pathway,termed co-stimulatory signal,providing by antigen presenting cells(APCs),is necessary to induce cytokine secretion,cell proliferation and effective function.Without co-stimulatory signal,T cells enter a state of anergy.Direct evidence shows the blockade of co-stimulatory signals inhibits T cell activation and decreases the incidence of GVHD.LIGHT-HVEM is a novel co-stimulatory pathway.They are members of tumor necrosis factor superfamily/tumor necrosis factor receptor superfamily (TNFSF/TNFRSF).LIGHT-HVEM pathway has been found to be involved in allograft rejection and some sorts of autoimmune diseases.But up to now,the role of LIGHT-HVEM co-stimulatory in GVHD is not clear.In the present project,the expression of LIGHT,HVEM and secretion of cytokines in patients with acute GVHD after allo-HSCT was studied.To further study their function in vivo,we established haploidentical acute GVHD(C57BL/6→CB6F1) model.We blocked LIGHT-HVEM pathway in mixed lymphocyte curture(MLC) by anti-LIGHT monoclony antibody(mAb) and inducing donor-specific T cell anergy.To investigate the role of LIGHT-HVEM in the pathogenesis of GVHD,we transplanted bone marrow cells and donor spleen T lymphocytes pre-cultured with anti-LIGHT mAb in the haploidentical GVHD model.Part one The expression of LIGHT/HVEM and the secretion of eytokines in allo-HSCT patients with acute GVHDMethods 1.Patients:From March 2007 to February 2008,23 cases of malignant hematological disease and 3 cases of aplastic anemia underwent allo-HSCT were analyzed,and followed-up for more than 3 months.The samples from 15 healthy donors were as normal controls.2.Conditioning regimes:protocol A,cyclophosphamide(CTX) + Etoposide(VP-16) + total body irradiation(TBI);protocol B,Busulfan(Bu) + fludarabine(Flu);protocol C,Busulfan(Bu) + fludarabine(Flu) + Cytarabine(Ara-C); protocol D,cyclophosphamide(CTX) for aplastic anemia patients.3.GVHD prophylaxis: A,related donor:cyclosporine(CsA) + short course of methotrexate(MTX) +mycophenolate mofetil(MMF);B,unrelated donors:CsA + short course of MTX + MMF + antithymocyteglobulin(ATG).4.GVHD diagnosis:the diagnosis and grading of GVHD were established according to clinical and pathologic criteria of Seattle.5.Flow cytometery strategy:The expression of LIGHT and HVEM were measured by FACS Calibur and the secretion of IL-1β,TNF-α,IL-2,IL-4,IL-6,IL-10 were detected at different time points after transplant.6.Values are mean + standard derivation(S.D.). The statistical difference were performed using the One-Way ANOVA.P value was 2-sided(α=0.05).All calculations were performed with SPSS 11.5 software.Results1.All patients achieved engraftment with 9 patients developing aGVHD and 7 patients developing cGVHD.2.LIGHT was not expressed on peripheral blood cells from healthy donors and patients without GVHD,while HVEM can be detected.Normal LIGHT of CD3+ CD4+ T cells and CD3+ CD8+ T cells:(0.94±0.49)%,(1.84±1.07)%.Normal HVEM:(13.88±3.68)%and(26.67±6.98)%.Pre-condition:LIGHT:(1.26±0.5)%and(1.74±0.86)%. HVEM:(14±3.73)%and(25.58±4.7)%.Post-transplantation(+15d):LIGHT:(1.34± 0.65)%and(2.00±0.93)%.HVEM:(12.87±2.16)%and(24.66±4.05)%.3.When aGVHD occurred,the expression level of LIGHT on T cells were significantly increased,while HVEM decreased.LIGHT:(18.12±3.36)%and(30.33±5.09)%.HVEM:(2.26±1.61)%and(3.44±2.00)%.The alteration of LIGHT/HVEM on CD3~+CD4~+T cells was consistent with the grading of aGVHD.4.With the GVHD remission,the expression of those co-stimulators was back to the normal.LIGHT:(1.81±0.92)%and(2.58±0.93)%.HVEM:(13.54±2.56)%and(23.10±3.17)%.5.The trend of LIGHT-HVEM on patients with cGVHD were same with patients with aGVHD.LIGHT:(16.42±4.31)%and(26.14±4.55)%.HVEM:(1.26±0.55)% and(2.70±0.99)%.6.The concentration of IL-1βin patients with aGVHD was higher than that in other groups,p＜0.05.The concentration of TNF-αin patients post-transplantion with aGVHD was higher than that in other groups,p＜0.05.There were no significance of other cytokines in different groups.Part two Donor-speeitie T cell anergy induced by blockade of LIGHT-HVEM pathway in vitroMethods 1.Spleen T cells of C57BL/6 origin were isolated as effector,and 25 Gy 60Coγ-ray irradiated spleen cells of CB6F1 origin as stimulator.Anti-LIGHT mAb at different dose were added to primary MLC(anti-LIGHT mAb group).Anti-IgG antibody was added in control group.Incubated for 3 days,the responsive rates were detected by MTT assays.2.ELISA assay was used to detect the level of cytokine production.3.T cells from the primary MLC were used as effector in secondary MLC,and 25 Gy 60Coγ-ray irradiated spleen cells of CB6F1 origin(recipient) or C3H origin(third part) as stimulator.Incubated for 3 days,the responsive rates were detected by MTT assays.4. Values are mean±S.D.or percentage(%).The statistical difference were performed with One-Way ANOVA.P value was 2-sided(α=0.05).All calculations were performed with SPSS11.5 software.Results1.Blockade of LIGHT-HVEM pathway inhibited MLR in a dose-independent pattern.1μg/ml anti-LIGHT mAb achieved the most pronounced inhibition rate of 29.9%.2.Blockade of LIGHT-HVEM pathway significantly reduced the production of IFN-γand TNF-α,while the level of IL-4 increased slightly.3.Blockade of LIGHT-HVEM pathway inhibited the donor T-cell proliferative reaction to the given antigens in secondary MLC,While the proliferative reaction to the third part were retained.Part Three Induction of immune tolerance ex vivo by blocking LIGHT-HVEM pathway to prevention of aGVHD in haploidentical transplantation mice modelMethods 1.Male C57BL/6 mice were used as donor,and female CB6F1 mice as recipients in haploidentical transplantation model.In primary MLC,cells co-cultured with anti-LIGHT mAb were harvested on day 3(anti-LIGHT mAb cultured cells).Cells treated with anti-IgG antibody were used as control.In haploidentical GVHD model, anti-LIGHT mAb cultured cells or control along with whole bone marrow cells were transplanted into the recipients.2.Groups:group A,PBS 0.5ml;group B,5×10~6 bone marrow cells;group C,5×10~6bone marrow cells + 6×10~7 control;group D,5×10~6bone marrow cells+ 6×10~7 anti-LIGHT mAb cultured cells.The severity of GVHD was evaluated with clinical GVHD scoring system and semi-quantitative scoring system with histologic examination.3.Within 56 days after transplantation,survival rate,weight, whole blood cell count,and GVHD scores were monitored.4.Values are mean±S.D.or percentage(%).The statistical difference were performed with One-Way ANOVA.P value was 2-sided(α=0.05).All calculations were performed with SPSS 11.5 software.Results1.In group A,the animals were dead in 15 days after TBI because of bone marrow failure.In group B,all animals were alive in 56 days.All animals in group C showed typical signs of aGVHD 19 days after transplantation.These signs included lethargy, hunched,rough fur and hair loss,diarrhea,weight loss.All animals were dead in 28 days. There were three animals dead on day 37,day 38,and day 42 in group D,owing to aGVHD.2.In 56 days,the survival rate of group A,B,C and D were 0,100%,0 and 50%, respectively.There were no difference in survival rate between group B and D.There were significant difference between groupD and group A,C.Conclusions1.The expression of LIGHT on both CD4+ and CD8+ T cells were increased in patients with aGVHD and/or cGVHD after allo-HSCT,more prominent on CD3+ CD4+ T cells.While the level of HVEM were decreased in the same condition.The intensities of LIGHT and HVEM went back to the normal level when GVHD was in remission.The concentration of IL- 1βin patients with aGVHD was higher than that in other groups.The concentration of TNF-αin patients post-transplantion with aGVHD was higher than that in other groups.There were no significance of other cytokines in different groups.These data indicated that LIGHT-HVEM pathway and cytokines such as IL-1β,TNF-αwere involved in the development of GVHD after allo-HSCT.2.With a MLC system in vitro,blockade of LIGHT-HVEM co-stimulatory pathway inhibited the given-antigen induced donor T cell proliferation and reduced the production of IFN-γ,TNF-α.While the responsiveness to third part were retained.3.Successfully established of the haploidentical transplantation mice model (C57BL/6→CB6F1).Co-transplantation of donor spleen T cells treated with anti-LIGHT mAb and bone marrow cells induced the immune tolerance and reduced the incidence of GVHD in vivo.