| Objectives:To explore the efficacy of C5a/C5aR blockade in the treatment of cGVHD and provide a new experimental basis for immunotherapy of cGVHD.Methods:1.Collectting the sample of peripheral blood in cGVHD patients,no cGVHD patients after allogeneic hematopoietic stem cell transplantation(allo-HSCT)and donors and detecting the expression of C5aR in lymphocytes and CD 14+mononuclear leucocytes and the proportion of CD4+CD25+Foxp3+ regulatory T cells(Tregs)in peripheral blood lymphocytes by flow cytometry(FCM).Futhermore,the relationship between the expression of C5aR on peripheral blood lymphocytes and CD 14+mononuclear leucocytes and the frequency of CD4+CD25+Foxp3+ Tregs cells within CD4+ T cells in cGVHD patients was analysed.2.In vitro,first,we detected the expression of the regulatory T cells in the spleen cells in C5aR knockout(C5aR-/-)mice and wild-type BALB/c mice.Second,we cultured the spleen cells and invided into 4 groups:C5aR-/-spleen cells,wild-type BALB/c spleen cells,100ng/mL RmC5a + wild-type BALB/c spleen cells and 500ng/mL RmC5a + wild-type BALB/c spleen cells.The regulatory T cells of spleen cells were analysed by flow cytometer and the section of TGF-β1 were detected by ELISA.3.Sepatating the CD4+ T cells from the spleen cells from the C5aR KO mice and wild type BALB/c mice by magnetic activated cell sorting.The CD4+ T cells were divided into 2 groups.We polarized the Tregs cells with TGF-β1(5ng/mL),CD3(5ng/mL),CD28(2ng/mL)and IL-2(40ug/mL)and detected the expression of regulatory T cells.4.cGVHD mice model was established.Wild-type BALB/c mice were accepted major histocompatibility(MHC)antigen matched,minor histocompatibility antigens(miHA)mismatched of B10.D2 male mice spleen cells and bone marrow cells(8 x 106,1:1)by tail intravenous,can build a cGVHD model like human cGVHD.5.12 cGVHD mice model were equally divided into 2 groups at random:control group and PMX53 group.In the 29th day after transplantation,all mice were manfestied with cGVHD and PMX53 or PBS were administrated with intraperitoneal injection.PMX53 group mice were accepted intraperitoneal injection with PMX53 by lmg/kg at twice per week for 3 weeks and Control group mice were accepted intraperitoneal injection with PBS at the frequency of PMX53 group.The clinical manfestation and survival time after treatment with PMX53 were observed until the end point of observation(the 65th days)or the weight of mice less than 14 g.And the target organs like skin,liver for HE staining.The expression of C5aR and CD4+CD25+Foxp3+Tregs cells in spleen cells were measured by flow cytometry.The secretion of TGF-β1 in supernatant were detected by enzyme-linked immuno sorbent assay(ELISA).6.Statistical analysis was performed by SPSS 20.0 software.The One-Way ANOVA was used to compare multiple sets of data,LSD was acted to the comparison of single factor analysis between groups.Two sets of measurement data were compared by statistical analysis using two independent samples t test.Kapplan-Meier method was used to analysis the survival time.All statistics were 2-tailed,and siginicance was set at P<0.05.Results:1.The expression of C5aR on peripheral blood lymphocytes and CD 14+mononuclear leucocytes in cGVHD patients is significantly higher than no cGVHD patients after allogeneic hematopoietic stem cell transplantation(allo-HSCT)and healthy donors(lymphocytes:3.76±0.30%vs 2.36±0.55%,1.16±0.16%,P<0.001:CD 14+ mononuclear leucocytes:94.05±0.52%vs 89.51±0.40%,88.53±1.16%,<P<0.001),and the frequency of CD4+CD25+Foxp3+ regulatory T cells within CD4+ T cells in cGVHD patients is notely lower compared to no cGVHD patients and healthy donors(1.45±0.13%vs 2.97±0.18%,3.16±0.31%,P<0.001).Futhermore,the expression of C5aR on peripheral blood lymphocytes was significantly negatively correlation with the frequency of CD4+CD25+Foxp3+ regulatory T cells within CD4+T cells in cGVHD patients(R=-0.603,P<0.01),and the correlation betwen the C5aR on CD14+ mononuclear leucocytes and the tregs was not significant(R=-0.378,P>0.05).2.C5aR deficiency promoted the development of Tregs cells whearas Rm C5a abolished the section of TGF-β1 and the differentiation of Tregs cells.(1)The expression of CD4+CD25+Foxp3+ regulatory T cells in spleen cells is increased in C5aR KO mice compared with the wild type BALB/c mice(15.67±0.55 vs 11.3 ±0.60%,P<0.01).(2).The section of TGF-β1 was detected by ELISA,and the section level of TGF-β1 between the concentration of 100ng/mL and 500ng/mL was not different(322.67±25.33 vs 413±217.29,P>0.05),whereas there were decreased notably compared to the spleen cells group without Rm C5a(879.17±27.42,P<0.001)and the section of TGF-β1 in the C5aR KO spleen cells group(1668±109.79)was the highest than the other three groups(P<0.01).(3).The frequency of CD4+CD25+Foxp3+ Tregs cells was analysed by FCM.The Tregs cells decreased by recombined mouse C5a protein(Rm C5a)(P<0.001),and there were significantly between the concentration of 100g/mL and 500ng/mL compared to the WT group(5.59±0.08%,5.80±0.03%vs 6.91±0.30%,P>0.05).There were the higest frequency of Tregs at the C5aR-/-group(9.76±0.33%,P<0.001).(4).CD4+ T cells were separated from the spleen cells in C5aR-/-mice and wild type BALB/c mice and polarized Tregs cells with CD3,CD28,IL2 and TGF-β1 for 3 days in vitro.We found that the frequency of CD4+CD25+Foxp3+ regulatory T cells in C5aR-/-group was significantly increased by FCM(34.52±0.69%vs 5.60±0.26%,P<0.001).3.The cGVHD mice model was established and treated by PMX53.(1).The clinical score in the cGVHD mice after the treatment with PMX53 was gradual decline as the hours pass away and the clinical score in cGVHD control mice group continued to rise.And the survival time was longer in the group with PMX53 treated compared to the cGVHD control group(P<0.05).(2).The cGVHD pathological changes were observed at the target organ such as skin and liver at the end point of observation or the weight of mice less than 14 g.We found that the skin epidermis hyperplasia,dermal collagen deposition and skin extensive infiltration of inflammatory cells in the subcutaneous fat,severe atrophy,sparse hair,hair follicle number decreased significantly in the cGVHD control mice;but PMX53 treated mice showed relatively mild pathological changes.cGVHD control group liver HE staining showed that most infiltration of inflammatory cells in portal area with lymphocytic infiltration of bile duct,and PMX53 treatment group in portal area is less affected.(3).The expression of C5aR and CD4+CD25+Foxp3+ Treg cells on the spleen cells was analysed by FCM that shown that the expression of C5aR was decreased in the group mice with PMX53 treated compared to the cGVHD control group(1.36±0.96%vs 3.41±0.29%,P<0.05),and the frequency of the CD4+CD25+Foxp3+ Treg cells was significant increased(10.56 ± 0.40%vs 13.64±0.48%,P<0.001).(4).The secretion of TGF-β1 in peripheral blood supernatant was detected by ELISA.The results was shown that the secretion of TGF-β1 in cGVHD control group was significantly decreased compared with the PMX53 treated group(938.4±12.91 vs 745.6±24.48,P<0.001).Conclusions:1.The expression of C5aR is higher whereas the Tregs cells are lower on cGVHD patients,C5a/C5aR blockade may be a new target in treatment of cGVHD.2.The secretion of TGF-β1 and the frequency of Tregs were increased after downregulating the expression of C5aR in vitro;on the contrary,these were decreased after upregulating the expression of C5aR.3.The expression of C5aR cGVHD model mice increased.The C5aR antogonist PMX53 in vivo plays a important role in treatment of cGVHD by promoting the secrection of TGF-β1,polarzing the CD4+CD25+Foxp3+ Tregs cells and improving the clinical manifestion and the pathological lesion. |
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