The Role Of IL-35in Acute Graft Versus Host Disease

Part one Construction of Mouse Single Chain Interleukin-35FusionGene by Overlap Extension PCRObjective：To construct mouse sigle chain interleukin-35(mscIL-35) fusion gene and itseukaryotic expression vector.Methods：The cDNA encoding mouse EBI3and IL-12p35were amplified by RT-PCRfrom the total RNA extracted from spleen cells of C57BL/6mice stimulated with LPS.EBI3gene and IL-12p35mature peptide gene were fused via a hydrophobic polypeptidelinker (Gly4Ser)3by overlap extension PCR to obtain mscIL-35fusion gene. ThemscIL-35fusion gene was cloned into eukaryotic expression vector pcDNA3.1(+) afteradded Igk signal sequence and restriction enzyme cutting site, then the positiverecombinant clone was analyzed by digestion of restriction endonuclease and DNAsequencing.Results：Sequence analysis showed that the splicing order, orientation and sequence ofmscIL-35fusion gene were completely correct.Conclusion：The mscIL-35fusion gene was constructed successfully, which will behelpful for the further research on its biological function. Part two The Role of IL-35in Mouse aGVHD of Allogeneic BoneMarrow TransplantationObjective: To explore the effects and mechanisms of hydrodynamic gene transfer of IL-35plasmids on mouse aGVHD of allo-BMT.Methods:①Fourteen SPF grade BALB/c mice were randomly assigned to two groups, The recipient BALB/c (H-2~d) mice were conditioned with hydrodynamic gene transfer ofpcDNA-3.1IL-35plasmids, the recipient mice conditioned with hydrodynamic genetransfer of pcDNA-3.1plasmids. All mice received an HGT injection24h before TBI.Irradiation was followed by the infusion of1×10~7C57BL/6bone marrow (TCD-BM)cells and whole spleen cells (5×10~6). Clinical manifestations of aGVHD and survivalwere observed after allogeneic bone marrow transplantation. Histopathology and the stateof chimera were detected in recipient mice after10days post allo-BMT. The survival ratewas compared by Log-rank test.②The percentage and counts of CD3、CD4、CD8、CD44、CD62L、Gr1、CD19、NK1.1、CD11b、CD11c of spleen、lung、liver、small intestine werealso measured by flow anlaysis after10days in allo-transplanted recipients. Lymphocytesisolated from spleen, liver, lung, and small intestine were stimulated for4hours with PMA(50ng/mL) and ionomycin (500ng/mL), with brefeldin A added for the final2hours.Intracellular cytokine IL-17、IL-10、IFN-γ、TNF-α on CD4~+T cell and CD8~+T cell wereanalyzed through flow analysis. The levels of IFN-γ、TNF-α、IL-10、IL-17and IL-6inserum were assessed by Cytometric Beads Array.③In vivo proliferation was assessedwith a BD Pharmingen BrdU allophycocyanin (APC) kit. In brief, mice were pulsed with1mg of BrdU i.p.4hours before euthanasia.4hours after BrdU injection, organs wereharvested and processed. Cells were stained with antibodies to cell surface CD4or CD8and nuclear BrdU and FoxP3, as per manufacturer’s instructions. The percentage andcounts of CD4~+FoxP3~+T cell、CD8~+FoxP3~+T cell of spleen、liver、lung、small intestinewere also measured by flow analysis. The percentage of CD4~+BrdU~+T cell、CD4~+FoxP3~+BrdU~+T cell、CD8~+BrdU~+T cell、CD8~+FoxP3~+BrdU~+T cell of spleen、liver、lung、smallintestine were also measured by flow analysis.④We used splenocytes from10days afterallo-transplanted BALB/C mice as responder cells, and the irradiated H2~dsplenocytesfrom BALB/c mice as stimulator cells to set up an in vitro MLR system. Tritiatedthymidine was pulsed to detect the proliferation of responder cells, cytotoxicity assay wasused to detect the killing capacity of responder cells against the H2~dtumor cells. Theallo-response of the host splenocytes was also determined by measuring IL-2secretion after the three day MLR reaction mentioned above by MTT.⑤For GVL experiments,Luc-B-cell leukemia/lymphoma1(A20) cells (1×10~6) were injected at the same timewhen donor bone marrow (BM) and spleen cells were injected intravenously. Mice weregiven an intraperitoneally injection of luciferin (150mg/kg) and imaged using the IVISImaging system (Xenogen) to assess bioluminescence10minutes after injection of thesubstrate once a week. The recipients were monitored daily for survival and every2daysfor body weight and clinical signs of GVHD.⑥Twenty SPF grade BALB/c mice wererandomly assigned to four groups, The recipient BALB/c (H-2~d) mice conditioned withHGT/IL-35, the recipient mice conditioned with HGT/control, The recipient BALB/c (H-2~d)mice conditioned with HGT/IL-35and was given400μg anti–IL-35antibody on days0(12hours after HGT), and200μg anti–IL-35antibody7days after BMT, The recipientBALB/c (H-2~d) mice conditioned with HGT/IL-35and was given400μg antibody controlon the same day(12hours after HGT), and200μg anti–IL-35antibody7thday after BMT.The recipients were monitored daily for survival and every2days for body weight andclinical signs of GVHD.Results:①Liver tissues from mice that received HGT/IL-35showed a higher level ofIL-35expression compared with those of control mice. HGT/control mice received5×10~6donor spleen cells induced severe clinical GVHD, the same dose of donor cells inducedonly moderate clinical GVHD in recipients administrated with IL-35. There was asignificant difference of mice survival between the two groups (P=0.0004). Ten days aftertransplantation the organs of recipient animals were harvested and pathology analysesconducted. Histological examination showed a significant reduction of inflammation inlung、liver and small intestine of HGT/IL-35recipients.②The percentage and cellnumbers of B、Mφ、DC、Neu cell decreased in spleen, lung, liver, small intestine ofHGT/IL-35recipients compared with HGT/control group(P<0.05); the proportion ofCD4~+CD44~+CD62L-T cell were decreased by administration with HGT/IL-35in the spleencompared to control group（P<0.05）; The percentage of CD3~+T、CD4~+T cell increased inHGT/IL-35group compared with HGT/control group（P<0.05）.③To address the protective role of HGT/IL-35in the pathogenesis of aGVHD, we tested T-cell subsets from the liver,small intestine, lung, and spleen in vivo10days after allogeneic HCT. We observed thatHGT/IL-35resulted in a significant decrease in the percentage of CD4~+T cells secretingIFN-γ in recipient spleen, liver, lung and small intestine（P<0.05）；HGT/IL-35resulted ina significant decrease in the percentage of CD4~+T cells secreting TNF-α in recipient spleenand lung(P<0.05)；HGT/IL-35resulted in a significant decrease in the counts of CD4~+Tcells secreting IL-17in recipient liver and small intestine(P<0.05)；but on the contrary, cellnumbers of CD4~+T cells secreting IL-17in recipient lung was increased in HGT/IL-35group(P<0.05). HGT/IL-35resulted in a significant increase in the counts of CD4~+T cellssecreting IL-10in recipient spleen, lung, liver and small intestine(P<0.05). The levels ofIL-10in serum increased dramatically in HGT/IL-35group compared with HGT/controlgroup (P<0.0001). The levels of TNF-α、IFN-γ、IL-6in serum decreased dramatically inHGT/IL-35group compared with HGT/control group (P<0.05), but the level of IL-17inserum did not reach significant difference in two groups(P>0.05).④The percentage ofCD4~+FoxP3~+T cells increased significantly in lung, liver and small intestine of recipientsadministrated with HGT/IL-35compared with HGT/control group(P<0.05)；we alsoobserved that HGT/IL-35resulted in a significant increase in the percentage ofCD8~+FoxP3~+T cells in recipient spleen, lung and liver(P<0.05)；The proliferation of CD4~+Tcell was decreased in liver and small intestine of recipients administrated withIL-35(P<0.05）；but the proliferation of CD8~+T cell did not reach significant differencebetween the two groups(P>0.05)；the proliferation of CD4~+FoxP3~+T cell was increasedsignificantly in liver and lung of recipients administrated with IL-35（P<0.05）；and theproliferation of CD8~+FoxP3~+T cell also increased significantly in spleen and lung ofrecipients administrated with IL-35(P<0.05)；⑤The proliferation of the splenocytes fromIL-35treated recipient mice was significantly lower than the splenocytes from the controlgroups in the MLR stimulated with irradiated H2~dsplenocytes from BALB/C mice(P<0.05). The ability for the mixed splenocyts to kill H2~dtumor targets and the level ofIL-2expression during the MLR had also been analyzed, the results of the recipient mice group administrated with IL-35were obviously lower than the control groups(P<0.05).⑥HGT/IL-35mitigate graft-versus-host disease without sacrificing graft-versus-leukemia.⑦Anti-IL-35antibody blockade diminished this suppressive effect of IL-35. Four groups ofmice survival were statistically difference (P<0.0001).Conclusion: Our study demonstrates that exogenous IL-35is capable of suppressing thedevelopment of aGVHD. The mechanisms were mainly through inhibition of B、Mφ、DC、Neu cell proportion and amounts, inhibition of CD4~+T cells secrete IFN-γ and TNF-α,promote the spleen、lung、liver and intestinal lymphocytes secretion of IL-10, inhibition ofCD4~+T cell proliferation, promotion of Treg augmentation and so on work together, and IL-35in GVHD inhibition did not weaken the GVL effect at the same time. Part three The Expression of Th17-Associated Cytokines in HumanGraft-versus-Host DiseaseObjective: To explore the role of Th17cells and Th17-associated cytokines in thedevelopment of graft-versus-host disease(GVHD) in clinical allogeneic hematopoietic stemcell transplantation (allo-HSCT) recipients.Methods: There were76patients including46males and30females received allo-HSCTin Department of Hematology, First Affiliated Hospital of Soochow University fromAugust2010to September2011. The median age was34years old (range from11to57).Matched sibling donor transplantation performed in44patients. Twenty-two patientsreceived matched unrelated donor peripheral blood stem cell transplantation, seven patientsreceived haploidentical donor stem cell transplantation and three patients receivedumbilical cord blood transplantation. Allo-HSCT patients was examined for thepercentages of Th17and FoxP3~+Treg cells and the expressions of RORγt and FoxP3inperipheral blood mononuclear cells (PBMCs). The expressions of Th17-associatedcytokines, including TGF-β, IL-6, IL-1β, IL-17, IL-21, IL-22, IL-23, IL-23R in the PBMCsof patients after allo-HSCT were detected using real-time PCR technique. And the concentration of IL-17and IL-23in the serum were examined by ELISA.Results: All patients achieved a sustained and stable donor engraftment. Among the76patients,45(59.2%) patients developed acute GVHD.7(9.2%) patients developed chronicGVHD. Among the45patients that developed aGVHD,12(26.7%) were grade I,16(35.5%)were grade II,5(11.1%) were grade III, and12(26.7%) were grade IV. The median day ofonset of aGVHD was32(15-94). The percentage of Th17and RORγt expression weresignificantly higher, while the percentage of Treg and FoxP3expression were significantlylower in acute GVHD (aGVHD)(grade II-IV) patients than in aGVHD (grade0-I) patientsand healthy donors. The expression of FoxP3was also decreased in chronic GVHD(cGVHD) patients as compared to healthy donors. The expressions of IL-6, IL-1β, IL-17,IL-21, IL-23and IL-23R were all increased, while IL-22expression was decreased inaGVHD patients. IL-1β and IL-23were also increased in cGVHD patients. In order toinvestigate the dynamic changes of Th17/Treg cells and Th17-associated cytokines,patients were examined for their expressions during the onset and resolution of aGVHD.The results demonstrated a reciprocal relationship between Treg and Th17cells.Th17-assocaited cytokine expression, namely IL-17and IL-23were closely related to theoccurrence and resolution of aGVHD.Conclusion: The dynamic balance between the Th17and Treg cells and the changes ofTh17-associated cytokines could be the indicators of the disease progression and promisingcandidates of prognostic biomarkers of aGVHD.