IL-35在急性移植物抗宿主病中的作用研究
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摘要
一、小鼠IL-35的克隆及真核表达载体的构建
目的：构建小鼠IL-35单链融合基因及其真核表达载体。
方法：取小鼠脾细胞，体外LPS刺激6小时后提取总RNA，通过RT-PCR克隆小鼠EBI3和IL-12p35cDNA；采用重叠延伸PCR，通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3基因和IL-12p35成熟肽基因，构建小鼠IL-35单链融合基因，添加Igk信号肽及酶切位点，并将其克隆至pcDNA3.1（+）载体。通过酶切和测序鉴定阳性重组载体。
结果：DNA序列分析结果表明：小鼠IL-35单链融合基因中EBI3、IL-12p35和linker的连接顺序、方向及序列完全正确。
结论：成功构建了小鼠IL-35单链融合基因及其真核表达载体，为进一步探讨IL-35的生物学功能奠定了基础。
二、IL-35抑制急性移植物抗宿主病的作用及作用机制
目的：研究IL-35抑制急性移植物抗宿主病的作用及作用机制。
方法：①建立C57BL/6→BALB/c小鼠清髓性异基因骨髓移植aGVHD模型。14只BALB/c小鼠经预处理后随机分为2组：实验组水流动力学注射IL-35质粒（HGT/IL-35）；对照组水流动力学注射空质粒（HGT/control）；各组小鼠于HGT后24小时分别接受Co~608.5Gy全身照射，照射4小时后尾静脉输注1×10~7个骨髓细胞+5×10~6个脾细胞建立急性移植物抗宿主病模型。移植后观察aGVHD表现，Log-rank检验各组生存率；移植后10d取肺、肝脏、回肠进行病理组织学检查，移植后10d嵌合度检测。②应用流式细胞仪检测移植后10d脾、肝、肺、小肠淋巴细胞CD3、CD4、CD8、CD44、CD62L、Gr1、CD19、NK1.1、CD11b、CD11c阳性细胞表达率；胞内染色检测移植后10d脾、肝、肺、小肠CD4+T淋巴细胞分泌IL-10、IL-17、TNF-α、IFN-γ的比例及数量以及CD8+T淋巴细胞分泌IL-10、IL-17、TNF-α、IFN-γ的比例及数量；流式CBA检测移植后第10天小鼠血清中的IL-10、IFNγ、TNFα、IL-6、IL-17水平。③流式细胞仪检测移植后10d脾、肝、肺、小肠CD4~+FoxP3~+T细胞、CD8~+FoxP3~+T细胞的比例及数量；BrdU掺入法检测移植后10d脾、肝、肺、小肠CD4~+、CD8+T细胞的增殖以及CD4~+FoxP3|~+T细胞、CD8~+FoxP3~+T细胞的增殖。④建立体外混合淋巴细胞反应（MLR）体系，以BALB/C鼠来源的脾细胞Co~6030Gy辐照后作为刺激细胞，以移植后10d2组小鼠脾细胞为反应细胞，培养3d，3~H掺入法检测H2~b脾细胞（反应细胞）的增殖效应；细胞毒性试验检测脾细胞对H-2~d抗原包被的肿瘤细胞CT-26的杀伤作用；CTLL-2细胞增殖实验检测脾细胞在H-2~d抗原刺激下所表达的IL-2水平。⑤建立小鼠移植物抗白血病模型，实验分3组：第一组：输注异基因骨髓细胞及带luciferaceA20（H-2~d）（1×10~6）；第二组：HGT/IL-35+输注异基因骨髓细胞+脾细胞及带luciferaceA20（H-2~d）（1×10~6）；第三组：HGT/control+输注异基因骨髓细胞+脾细胞及带luciferaceA20（H-2~d）（1×10~6）；每周在小动物活体成像仪上观察3组小鼠的体内肿瘤荧光，每2d给小鼠称重，观察小鼠生存，绘制生存曲线。⑥在小鼠急性移植物抗宿主病模型基础上，实验分4组：第一组：水流动力学注射IL-35质粒（HGT/IL-35）；第二组：水流动力学注射空质粒（HGT/control）；第三组：水流动力学注射IL-35质粒（HGT/IL-35），12小时后腹腔注射anti-IL-35抗体400μg，7d后腹腔注射anti-IL-35抗体200μg；第三组：水流动力学注射IL-35质粒（HGT/IL-35），12小时后腹腔注射IgG control抗体400μg，7d后腹腔注射IgG control抗体200μg；移植后观察小鼠aGVHD表现，每2d给小鼠称重，观察小鼠生存，绘制生存曲线。
结果：①HGT/IL-35小鼠免疫组化在肝脏检测到IL-35的高效表达，HGT/control组小鼠出现中到重度的aGVHD表现，HGT/IL-35组小鼠GVHD程度轻，HGT/control组小鼠出现明显的肺部、肝脏和肠道病理改变。两组小鼠生存有明显统计学差异（P<0.0001）。②HGT/IL-35组小鼠B、Mφ、DC、Neu细胞比例及数量与HGT/control组比较有统计学差异（P<0.05）；HGT/IL-35组小鼠脾CD4+效应T细胞比例下降，与HGT/control组相比有统计学差异（P<0.05）；HGT/IL-35组小鼠CD3、CD4细胞比例高于HGT/control组（P<0.05）。③HGT/IL-35组小鼠脾、肝、肺、小肠CD4+T淋巴细胞分泌IFN-γ较HGT/control组明显下降（P<0.05）；脾、肺CD4+T淋巴细胞分泌TNF-α较HGT/control组明显下降（P<0.05）；肝、小肠CD4+T淋巴细胞分泌IL-17比例较HGT/control组明显下降（P<0.05）；肺CD4+T淋巴细胞分泌IL-17比例较HGT/control组明显升高（P<0.05）；HGT/IL-35组小鼠脾、肝、肺、小肠CD4+T淋巴细胞分泌IL-10较HGT/control组明显上升（P<0.05）。HGT/IL-35组小鼠血清中TNF-α、IFN-γ、IL-6含量较HGT/control组下降（P<0.05），而IL-10含量有明显上升（P<0.05）。IL-17血清浓度2组无统计学差异。④小鼠肺、肝、小肠CD4+FoxP3+T细胞比例HGT/IL-35组与HGT/control组比较有统计学差异（P<0.05）；脾、肺、肝CD8~+FoxP3~+T细胞比例HGT/IL-35组与HGT/control组比较有统计学差异（P<0.05）；肝、小肠CD4+BrdU+细胞比例HGT/IL-35组明显低于HGT/control组（P<0.05）；而CD8~-BrdU~-细胞比例二组间无统计学差异（P＞0.05）；肝、肺CD4~+FoxP3~+BrdU~+T细胞比例HGT/IL-35组明显高于HGT/control组（P<0.05）；脾、肺CD8~+FoxP3~+BrdU~+T细胞比例HGT/IL-35组明显高于HGT/control组（P<0.05）；⑤3H嵌入、细胞毒性实验、及IL-2水平的检测结果二组间有显著性差异（P<0.05）。⑥IL-35组小鼠在降低GVHD的同时并没有削弱GVL效应。⑦应用anti-IL-35抗体能阻断IL-35的免疫抑制作用，四组小鼠生存有统计学差异（P<0.05）。
结论：IL-35能明显抑制GVHD的病理进程、明显改善生存率。其机制主要是通过抑制B、Mφ、DC、Neu细胞比例及数量，抑制CD4+T细胞分泌IFN-γ、TNF-α，抑制肝、小肠CD4+T细胞分泌IL-17，促进肺CD4+T细胞分泌IL-17，促进脾、肺、肝、小肠分泌IL-10，抑制CD4+T细胞增殖，促进Treg扩增等共同发挥作用，并且IL-35在抑制GVHD的同时并没有削弱GVL效应。
三、Treg和Th17相关细胞因子在移植物抗宿主病中的作用研究
目的：研究Treg细胞、Th17细胞及相关细胞因子在临床移植物抗宿主病患者中的作用。
方法：2010年8月至2011年9月在苏州大学附属第一医院血液科接受异基因造血干细胞移植（allo-HSCT）的76例患者中，男性46例、女性30例，中位年龄34（11～57）岁。实施同胞全相合异基因移植（allo-HSCT）患者44例，无关供者全相合外周血干细胞移植（MUD-PBSCT）22例，单倍型骨髓移植（HID-BMT）7例，脐血移植3例。流式细胞术检测GVHD发生时GVHD改善后外周血Treg细胞、Th17细胞比例，应用real-timePCR技术检测Th17相关细胞因子，应用ELISA检测血浆中IL-17、IL-23含量，分析与GVHD相关性。
结果：所有患者均获造血重建，其中45(59.2%)例患者发生aGVHD，7(9.2%)例为cGVHD患者，45例aGVHD患者中，I°aGVHD12(26.7%)例；II°aGVHD16(35.5%)例；III°aGVHD5(11.1%)例；IV°aGVHD12(26.7%)例；aGVHD发生的中位时间为32(15-94)d。aGVHD（II°-IV°）患者Th17细胞比例及RORγt基因表达量明显高于aGVHD（O°-I°）患者及正常对照组。而Treg细胞比例及FoxP3基因表达量aGVHD（II°-IV°）患者明显低于aGVHD（O°-I°）患者及正常对照组。cGVHD患者FoxP3基因表达量低于正常对照组。aGVHD（II°-IV°）患者IL-6, IL-1β, IL-17,IL-21, IL-23和IL-23R基因表达量明显高于aGVHD（O°-I°）患者，而IL-22基因的检测结果则相反，aGVHD（II°-IV°）患者低于aGVHD（O°-I°）患者，cGVHD患者中IL-1β和IL-23明显升高。患者GVHD发生时Th17上升，Treg下降，血浆中IL-17，IL-23含量上升，GVHD改善后Th17下降，Treg上升，血浆中IL-17，IL-23含量下降。
结论：异基因造血干细胞移植患者动态检测Th17细胞、Treg细胞及相关细胞因子，能早期预警GVHD发生，有望成为GVHD的临床监测指标。
Part one Construction of Mouse Single Chain Interleukin-35FusionGene by Overlap Extension PCR
Objective：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 Transplantation
Objective: 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 Disease
Objective: 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.
目的：构建小鼠IL-35单链融合基因及其真核表达载体。
方法：取小鼠脾细胞，体外LPS刺激6小时后提取总RNA，通过RT-PCR克隆小鼠EBI3和IL-12p35cDNA；采用重叠延伸PCR，通过编码疏水性多肽接头(Gly4Ser)3的DNA序列连接小鼠EBI3基因和IL-12p35成熟肽基因，构建小鼠IL-35单链融合基因，添加Igk信号肽及酶切位点，并将其克隆至pcDNA3.1（+）载体。通过酶切和测序鉴定阳性重组载体。
结果：DNA序列分析结果表明：小鼠IL-35单链融合基因中EBI3、IL-12p35和linker的连接顺序、方向及序列完全正确。
结论：成功构建了小鼠IL-35单链融合基因及其真核表达载体，为进一步探讨IL-35的生物学功能奠定了基础。
二、IL-35抑制急性移植物抗宿主病的作用及作用机制
目的：研究IL-35抑制急性移植物抗宿主病的作用及作用机制。
方法：①建立C57BL/6→BALB/c小鼠清髓性异基因骨髓移植aGVHD模型。14只BALB/c小鼠经预处理后随机分为2组：实验组水流动力学注射IL-35质粒（HGT/IL-35）；对照组水流动力学注射空质粒（HGT/control）；各组小鼠于HGT后24小时分别接受Co~608.5Gy全身照射，照射4小时后尾静脉输注1×10~7个骨髓细胞+5×10~6个脾细胞建立急性移植物抗宿主病模型。移植后观察aGVHD表现，Log-rank检验各组生存率；移植后10d取肺、肝脏、回肠进行病理组织学检查，移植后10d嵌合度检测。②应用流式细胞仪检测移植后10d脾、肝、肺、小肠淋巴细胞CD3、CD4、CD8、CD44、CD62L、Gr1、CD19、NK1.1、CD11b、CD11c阳性细胞表达率；胞内染色检测移植后10d脾、肝、肺、小肠CD4+T淋巴细胞分泌IL-10、IL-17、TNF-α、IFN-γ的比例及数量以及CD8+T淋巴细胞分泌IL-10、IL-17、TNF-α、IFN-γ的比例及数量；流式CBA检测移植后第10天小鼠血清中的IL-10、IFNγ、TNFα、IL-6、IL-17水平。③流式细胞仪检测移植后10d脾、肝、肺、小肠CD4~+FoxP3~+T细胞、CD8~+FoxP3~+T细胞的比例及数量；BrdU掺入法检测移植后10d脾、肝、肺、小肠CD4~+、CD8+T细胞的增殖以及CD4~+FoxP3|~+T细胞、CD8~+FoxP3~+T细胞的增殖。④建立体外混合淋巴细胞反应（MLR）体系，以BALB/C鼠来源的脾细胞Co~6030Gy辐照后作为刺激细胞，以移植后10d2组小鼠脾细胞为反应细胞，培养3d，3~H掺入法检测H2~b脾细胞（反应细胞）的增殖效应；细胞毒性试验检测脾细胞对H-2~d抗原包被的肿瘤细胞CT-26的杀伤作用；CTLL-2细胞增殖实验检测脾细胞在H-2~d抗原刺激下所表达的IL-2水平。⑤建立小鼠移植物抗白血病模型，实验分3组：第一组：输注异基因骨髓细胞及带luciferaceA20（H-2~d）（1×10~6）；第二组：HGT/IL-35+输注异基因骨髓细胞+脾细胞及带luciferaceA20（H-2~d）（1×10~6）；第三组：HGT/control+输注异基因骨髓细胞+脾细胞及带luciferaceA20（H-2~d）（1×10~6）；每周在小动物活体成像仪上观察3组小鼠的体内肿瘤荧光，每2d给小鼠称重，观察小鼠生存，绘制生存曲线。⑥在小鼠急性移植物抗宿主病模型基础上，实验分4组：第一组：水流动力学注射IL-35质粒（HGT/IL-35）；第二组：水流动力学注射空质粒（HGT/control）；第三组：水流动力学注射IL-35质粒（HGT/IL-35），12小时后腹腔注射anti-IL-35抗体400μg，7d后腹腔注射anti-IL-35抗体200μg；第三组：水流动力学注射IL-35质粒（HGT/IL-35），12小时后腹腔注射IgG control抗体400μg，7d后腹腔注射IgG control抗体200μg；移植后观察小鼠aGVHD表现，每2d给小鼠称重，观察小鼠生存，绘制生存曲线。
结果：①HGT/IL-35小鼠免疫组化在肝脏检测到IL-35的高效表达，HGT/control组小鼠出现中到重度的aGVHD表现，HGT/IL-35组小鼠GVHD程度轻，HGT/control组小鼠出现明显的肺部、肝脏和肠道病理改变。两组小鼠生存有明显统计学差异（P<0.0001）。②HGT/IL-35组小鼠B、Mφ、DC、Neu细胞比例及数量与HGT/control组比较有统计学差异（P<0.05）；HGT/IL-35组小鼠脾CD4+效应T细胞比例下降，与HGT/control组相比有统计学差异（P<0.05）；HGT/IL-35组小鼠CD3、CD4细胞比例高于HGT/control组（P<0.05）。③HGT/IL-35组小鼠脾、肝、肺、小肠CD4+T淋巴细胞分泌IFN-γ较HGT/control组明显下降（P<0.05）；脾、肺CD4+T淋巴细胞分泌TNF-α较HGT/control组明显下降（P<0.05）；肝、小肠CD4+T淋巴细胞分泌IL-17比例较HGT/control组明显下降（P<0.05）；肺CD4+T淋巴细胞分泌IL-17比例较HGT/control组明显升高（P<0.05）；HGT/IL-35组小鼠脾、肝、肺、小肠CD4+T淋巴细胞分泌IL-10较HGT/control组明显上升（P<0.05）。HGT/IL-35组小鼠血清中TNF-α、IFN-γ、IL-6含量较HGT/control组下降（P<0.05），而IL-10含量有明显上升（P<0.05）。IL-17血清浓度2组无统计学差异。④小鼠肺、肝、小肠CD4+FoxP3+T细胞比例HGT/IL-35组与HGT/control组比较有统计学差异（P<0.05）；脾、肺、肝CD8~+FoxP3~+T细胞比例HGT/IL-35组与HGT/control组比较有统计学差异（P<0.05）；肝、小肠CD4+BrdU+细胞比例HGT/IL-35组明显低于HGT/control组（P<0.05）；而CD8~-BrdU~-细胞比例二组间无统计学差异（P＞0.05）；肝、肺CD4~+FoxP3~+BrdU~+T细胞比例HGT/IL-35组明显高于HGT/control组（P<0.05）；脾、肺CD8~+FoxP3~+BrdU~+T细胞比例HGT/IL-35组明显高于HGT/control组（P<0.05）；⑤3H嵌入、细胞毒性实验、及IL-2水平的检测结果二组间有显著性差异（P<0.05）。⑥IL-35组小鼠在降低GVHD的同时并没有削弱GVL效应。⑦应用anti-IL-35抗体能阻断IL-35的免疫抑制作用，四组小鼠生存有统计学差异（P<0.05）。
结论：IL-35能明显抑制GVHD的病理进程、明显改善生存率。其机制主要是通过抑制B、Mφ、DC、Neu细胞比例及数量，抑制CD4+T细胞分泌IFN-γ、TNF-α，抑制肝、小肠CD4+T细胞分泌IL-17，促进肺CD4+T细胞分泌IL-17，促进脾、肺、肝、小肠分泌IL-10，抑制CD4+T细胞增殖，促进Treg扩增等共同发挥作用，并且IL-35在抑制GVHD的同时并没有削弱GVL效应。
三、Treg和Th17相关细胞因子在移植物抗宿主病中的作用研究
目的：研究Treg细胞、Th17细胞及相关细胞因子在临床移植物抗宿主病患者中的作用。
方法：2010年8月至2011年9月在苏州大学附属第一医院血液科接受异基因造血干细胞移植（allo-HSCT）的76例患者中，男性46例、女性30例，中位年龄34（11～57）岁。实施同胞全相合异基因移植（allo-HSCT）患者44例，无关供者全相合外周血干细胞移植（MUD-PBSCT）22例，单倍型骨髓移植（HID-BMT）7例，脐血移植3例。流式细胞术检测GVHD发生时GVHD改善后外周血Treg细胞、Th17细胞比例，应用real-timePCR技术检测Th17相关细胞因子，应用ELISA检测血浆中IL-17、IL-23含量，分析与GVHD相关性。
结果：所有患者均获造血重建，其中45(59.2%)例患者发生aGVHD，7(9.2%)例为cGVHD患者，45例aGVHD患者中，I°aGVHD12(26.7%)例；II°aGVHD16(35.5%)例；III°aGVHD5(11.1%)例；IV°aGVHD12(26.7%)例；aGVHD发生的中位时间为32(15-94)d。aGVHD（II°-IV°）患者Th17细胞比例及RORγt基因表达量明显高于aGVHD（O°-I°）患者及正常对照组。而Treg细胞比例及FoxP3基因表达量aGVHD（II°-IV°）患者明显低于aGVHD（O°-I°）患者及正常对照组。cGVHD患者FoxP3基因表达量低于正常对照组。aGVHD（II°-IV°）患者IL-6, IL-1β, IL-17,IL-21, IL-23和IL-23R基因表达量明显高于aGVHD（O°-I°）患者，而IL-22基因的检测结果则相反，aGVHD（II°-IV°）患者低于aGVHD（O°-I°）患者，cGVHD患者中IL-1β和IL-23明显升高。患者GVHD发生时Th17上升，Treg下降，血浆中IL-17，IL-23含量上升，GVHD改善后Th17下降，Treg上升，血浆中IL-17，IL-23含量下降。
结论：异基因造血干细胞移植患者动态检测Th17细胞、Treg细胞及相关细胞因子，能早期预警GVHD发生，有望成为GVHD的临床监测指标。
Part one Construction of Mouse Single Chain Interleukin-35FusionGene by Overlap Extension PCR
Objective：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 Transplantation
Objective: 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 Disease
Objective: 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.
引文
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19. Rezvani K, Mielke S, Ahmadzadeh M, et al. High donor FOXP3-positive regulatoryT-cell (Treg) content is associated with a low risk of GVHD following HLA-matchedallogeneic SCT. Blood,2006,108:1291-1297.
20. Semple K, Yu Y, Wang D, Anasetti C, Yu XZ. Efficient and selective prevention ofGVHD by antigen-specific induced Tregs via linked-suppression in mice. Biol BloodMarrow Transplant,2011,17:309-318.
21. Rieger K, Loddenkemper C, Maul J, et al. Mucosal FOXP3+regulatory T cells arenumerically deficient in acute and chronic GVHD. Blood,2006;107:1717-1723.
22. Gaidot A, Landau DA, Martin GH, et al. Immune reconstitution is preserved inhematopoietic stem cell transplantation coadministered with regulatory T cells forGVHD prevention. Blood,2011,117:2975-2983.
23. Di Ianni M, Falzetti F, Carotti A, et al. Tregs prevent GVHD and promote immunereconstitution in HLA-haploidentical transplantation. Blood,2011,117:3921-3928.
24. Zorn E, Kim HT, Lee SJ, et al. Reduced frequency of FOXP3+CD4+CD25+regulatory T cells in patients with chronic graft-versus-host disease. Blood,2005,106:2903-2911.
25. Laurence A, Amarnath S, Mariotti J, et al. STAT3transcription factor promotesinstability of nTreg cells and limits generation of iTreg cells during acute murinegraft-versus-host disease.Immunity,2012,37(2):209-222.
26. Collison L W, Workman C J, Kuo T T, et al. The inhibitory cytokine IL-35contributesto regulatory T-cell function. Nature,2007,450:566-569.
27. Pflanz S, Timans J C, Cheung J, et al. IL-27, a heterodimeric cytokine composed ofEBI3and p28protein, induces proliferation of naive CD4(+) T cells. Immunity,2002,16:779-790.
28. Hibbert L, Pflanz S, Waal Malefyt R De, et al. IL-27and IFN-alpha signal via Stat1and Stat3and induce T-Bet and IL-12Rbeta2in naive T cells. J Interferon CytokineRes,2003,23:513-522.
29. Bardel E, Larousserie F, Charlot-Rabiega P, et al. Human CD4+CD25+Foxp3+regulatory T cells do not constitutively express IL-35. J Immunol,2008,181(10):6898-6905.
30. Chaturvedi V, Collison LW, Guy CS, et al. Human regulatory T cells require IL-35tomediate suppression and infectious tolerance. J Immunol,2011,186(12):6661-6666.
31. Collison LW, Chaturvedi V, Henderson AL, et al. IL-35-mediated induction of a potentregulatory T cell population. Nat Immunol,2010,11(12):1093-1101.
32. Chua A O, Chizzonite R, Desai B B, et al. Expression cloning of a human IL-12receptor component. A new member of the cytokine receptor superfamily with stronghomology to gp130. J Immunol,1994,153:128-136.
33. Chua A O, Wilkinson V L, Presky D H, et al. Cloning and characterization of a mouseIL-12receptor-beta component. J Immunol,1995,155:4286-4294.
34. Parham C, Chirica M, Timans J, et al. A receptor for the heterodimeric cytokine IL-23is composed of IL-12Rbeta1and a novel cytokine receptor subunit, IL-23R. JImmunol,2002,168:5699-5708.
35. Collison LW, Delgoffe GM, Guy CS, et al. The composition and signaling of the IL-35receptor are unconventional. Nat Immunol,2012,13(3):290-299.
36. Thierfelder WE, van Deursen JM, Yamamoto K, et al. Requirement for Stat4ininterleukin-12-mediated responses of natural killer and T cells. Nature,1996,382(6587):171-174.
37. O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses.Immunity,2008,28(4):477-487.
38. Niedbala W, Wei X Q, Cai B, et al. IL-35is a novel cytokine with therapeutic effectsagainst collagen-induced arthritis through the expansion of regulatory T cells andsuppression of Th17cells. Eur J Immunol,2007,37:3021-3029.
39. Huang CH, Loo EX, Kuo IC, et al.Airway inflammation and IgE production inducedby dust mite allergen-specific memory/effector Th2cell line can be effectivelyattenuated by IL-35. J Immunol,2011,187(1):462-471.
40. Kochetkova I, Golden S, Holderness K, et al. IL-35stimulation of CD39+regulatory Tcells confers protection against collagen II-induced arthritis via the production ofIL-10. J Immunol,2010,184(12):7144-7153.
41. Bettini M, Castellaw A H, Lennon G P, et al. Prevention of Autoimmune Diabetes byEctopic Pancreatic b-Cell Expression of Interleukin-35. Diabetes,2012,61(6):1519-1526.
1. Collison LW, Pillai MR, Chaturvedi V, et al. Regulatory T cell suppression ispotentiated by target T cells in a cell contact, IL-35-and IL-10-dependent manner. JImmunol,2009,182(10):6121-6128.
2. Collison L W, Workman C J, Kuo T T, et al. The inhibitory cytokine IL-35contributesto regulatory T-cell function. Nature,2007,450:566-569.
3. Niedbala W, Wei X Q, Cai B, et al. IL-35is a novel cytokine with therapeutic effectsagainst collagen-induced arthritis through the expansion of regulatory T cells andsuppression of Th17cells. Eur J Immunol,2007,37:3021-3029.
4. Mattner F, Fischer S, Guckes S, et al. The Interleukin-12subunit p40specificallyinhibits effects of the Interleukin-12heterodimer. Eur J Immunol,1993,23(9):2202-2208.
5.张梅，司履生，王一理等.重组人单链白细胞介素12融合基因(rhscIL-12)的构建和真核表达。西安医科大学学报，2000，21(4):298-302.
1. Collison L W, Workman C J, Kuo T T, et al. The inhibitory cytokine IL-35contributesto regulatory T-cell function. Nature2007,450:566-569.
2. Niedbala W, Wei X Q, Cai B, et al. IL-35is a novel cytokine with therapeutic effectsagainst collagen-induced arthritis through the expansion of regulatory T cells andsuppression of Th17cells. Eur J Immunol2007,37:3021-3029.
3. Huang CH, Loo EX, Kuo IC, et al.Airway inflammation and IgE production inducedby dust mite allergen-specific memory/effector Th2cell line can be effectivelyattenuated by IL-35. J Immunol2011Jul1;187(1):462-471.
4. Kochetkova I, Golden S, Holderness K, et al. IL-35stimulation of CD39+regulatory Tcells confers protection against collagen II-induced arthritis via the production ofIL-10. J Immunol2010Jun15;184(12):7144-7153.
5. Stanzani M, Martins S, Saliba R, et al. CD25expression on donor CD4+or CD8+Tcells is associated with an increased risk for graft-versus-host disease afterHLA-identical stem cell transplantation in humans. Blood2004;103:1140-1146.
6. Brunstein CG, Miller JS, Cao Q, et al. Infusion of ex vivo expanded T regulatory cellsin adults transplanted with umbilical cord blood: safety profile and detection kinetics.Blood2011;117:1061-1070.
7. Dieckmann D, Plottner H, Berchtold S, et al. Ex vivo isolation and characterization ofCD4(+)CD25(+) T cells with regulatory properties from human blood. J Exp Med2001;193(11):1303-1310.
8. Collison LW, Chaturvedi V, Henderson AL, et al. IL-35-mediated induction of apotent regulatory T cell population. Nat Immunol2010Dec;11(12):1093-1101.
9. Carlson MJ, West ML, Coghill JM, Panoskaltsis-Mortari A, Blazar BR, Serody JS. Invitro-differentiated TH17cells mediate lethal acute graft-versus-host disease withsevere cutaneous and pulmonary pathologic manifestations. Blood2009;113:1365-1374.
10. Kappel LW, Goldberg GL, King CG, et al. IL-17contributes to CD4-mediatedgraft-versus-host disease. Blood2009;113:945-952.
11. Yi T, Zhao D, Lin CL, et al. Absence of donor Th17leads to augmented Th1differentiation and exacerbated acute graft-versus-host disease. Blood2008;112:2101-2110.
12. Iclozan C,Yu Y, Liu C, et al. T helper17cells are sufficient but not necessary toinduce acute graft-versus-host disease. Biol Blood Marrow Transplant;16:170-178.
13. Yi T, Chen Y, Wang L, et al. Reciprocal differentiation and tissue-specificpathogenesis of Th1, Th2, and Th17cells in graft-versus-host disease. Blood2009;114:3101-3112.
14. Bettini M, Castellaw A H, Lennon G P, et al. Prevention of Autoimmune Diabetes byEctopic Pancreatic b-Cell Expression of Interleukin-35. Diabetes.2012Jun;61(6):1519-1526.
15. Yu Y, Wang D, Liu C, et al. Prevention of GVHD while sparing GVL effect bytargeting Th1and Th17transcription factor T-bet and RORγt in mice. Blood.2011Nov3;118(18):5011-20.
1. Welniak LA, Blazar BR, Murphy WJ. Immunobiology of allogeneic hematopoieticstem cell transplantation. Annu Rev Immunol2007;25:139-170.
2. Shlomchik WD. Graft-versus-host disease. Nat Rev Immunol2007;7:340-352.
3. Ferrara JL, Reddy P. Pathophysiology of graft-versus-host disease. Semin Hematol2006;43:3-10.
4. Weaver CT, Hatton RD, Mangan PR, Harrington LE. IL-17family cytokines and theexpanding diversity of effector T cell lineages. Annu Rev Immunol2007;25:821-852.
5. Park H, Li Z, Yang XO, et al. A distinct lineage of CD4T cells regulates tissueinflammation by producing interleukin17. Nat Immunol2005;6:1133-1141.
6. Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17and Th17Cells. Annu Rev Immunol2009;27:485-517.
7. Weaver CT, Harrington LE, Mangan PR, Gavrieli M, Murphy KM. Th17: an effectorCD4T cell lineage with regulatory T cell ties. Immunity2006;24:677-688.
8. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for thegeneration of pathogenic effector TH17and regulatory T cells. NATURE2006;441:235-238.
9. Aggarwal S, Ghilardi N, Xie MH, de Sauvage FJ, Gurney AL. Interleukin-23promotes a distinct CD4T cell activation state characterized by the production ofinterleukin-17. J Biol Chem2003;278:1910-1914.
10. Nurieva R, Yang XO, Martinez G, et al. Essential autocrine regulation by IL-21in thegeneration of inflammatory T cells. NATURE2007;448:480-483.
11. Yi T, Zhao D, Lin CL, et al. Absence of donor Th17leads to augmented Th1differentiation and exacerbated acute graft-versus-host disease. Blood2008;112:2101-2110.
12. Kappel LW, Goldberg GL, King CG, et al. IL-17contributes to CD4-mediatedgraft-versus-host disease. Blood2009;113:945-952.
13. Iclozan C, Yu Y, Liu C, et al. T helper17cells are sufficient but not necessary toinduce acute graft-versus-host disease. Biol Blood Marrow Transplant;2010,16:170-178.
14. Dander E, Balduzzi A, Zappa G, et al. Interleukin-17-producing T-helper cells as newpotential player mediating graft-versus-host disease in patients undergoing allogeneicstem-cell transplantation. Transplantation2009;88:1261-1272.
15. Zhao XY, Xu LL, Lu SY, Huang XJ. IL-17-producing T cells contribute to acutegraft-versus-host disease in patients undergoing unmanipulated blood and marrowtransplantation. Eur J Immunol;2011,41:514-526.
16. Ratajczak P, Janin A, Peffault de Latour R, et al. Th17/Treg ratio in humangraft-versus-host disease. Blood;2010,116:1165-1171.
17. Chen X, Vodanovic-Jankovic S, Johnson B, Keller M, Komorowski R, Drobyski WR.Absence of regulatory T-cell control of TH1and TH17cells is responsible for theautoimmune-mediated pathology in chronic graft-versus-host disease. Blood2007;110:3804-3813.
18. Chen X, Das R, Komorowski R, et al. Blockade of interleukin-6signaling augmentsregulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease.Blood2009;114:891-900.
19. Tawara I, Koyama M, Liu C, et al. Interleukin-6modulates graft-versus-host responsesafter experimental allogeneic bone marrow transplantation. Clin Cancer Res;2011,17:77-88.
20. Das R, Chen X, Komorowski R, Hessner MJ, Drobyski WR. Interleukin-23secretionby donor antigen-presenting cells is critical for organ-specific pathology ingraft-versus-host disease. Blood,2009;113:2352-2362.
21. Thompson JS, Chu Y, Glass JF, Brown SA. Absence of IL-23p19in donor allogeneiccells reduces mortality from acute GVHD. Bone Marrow Transplant;2010,45:712-722.
22. Das R, Komorowski R, Hessner MJ, et al. Blockade of interleukin-23signaling resultsin targeted protection of the colon and allows for separation of graft-versus-host andgraft-versus-leukemia responses. Blood;2010,115:5249-5258.
23. Bucher C, Koch L, Vogtenhuber C, et al. IL-21blockade reduces graft-versus-hostdisease mortality by supporting inducible T regulatory cell generation. Blood2009;114:5375-5384.
24. Hanash AM, Kappel LW, Yim NL, et al. Abrogation of donor T-cell IL-21signalingleads to tissue-specific modulation of immunity and separation of GVHD from GVL.Blood;2011,118:446-455.
25. Hanash AM, Dudakov JA, Hua G, et al. Interleukin-22protects intestinal stem cellsfrom immune-mediated tissue damage and regulates sensitivity to graft versus hostdisease. Immunity;2012,37:339-350.
26. Rezvani K, Mielke S, Ahmadzadeh M, et al. High donor FOXP3-positive regulatoryT-cell (Treg) content is associated with a low risk of GVHD following HLA-matchedallogeneic SCT. Blood2006;108:1291-1297.
27. Semple K, Yu Y, Wang D, Anasetti C, Yu XZ. Efficient and selective prevention ofGVHD by antigen-specific induced Tregs via linked-suppression in mice. Biol BloodMarrow Transplant;2011,17:309-318.
28. Rieger K, Loddenkemper C, Maul J, et al. Mucosal FOXP3+regulatory T cells arenumerically deficient in acute and chronic GvHD. Blood2006;107:1717-1723.
29. Gaidot A, Landau DA, Martin GH, et al. Immune reconstitution is preserved inhematopoietic stem cell transplantation coadministered with regulatory T cells forGVHD prevention. Blood;2011,117:2975-2983.
30. Di Ianni M, Falzetti F, Carotti A, et al. Tregs prevent GVHD and promote immunereconstitution in HLA-haploidentical transplantation. Blood;2011,117:3921-3928.
31. Zorn E, Kim HT, Lee SJ, et al. Reduced frequency of FOXP3+CD4+CD25+regulatory T cells in patients with chronic graft-versus-host disease. Blood2005;106:2903-2911.
32. Miura Y, Thoburn CJ, Bright EC, et al. Association of Foxp3regulatory geneexpression with graft-versus-host disease. Blood2004;104:2187-2193.
33. Kawano Y, Kim HT, Matsuoka K, et al. Low telomerase activity in CD4+regulatoryT cells in patients with severe chronic GVHD after hematopoietic stem celltransplantation. Blood;2011,118:5021-5030.
34. Carlson MJ, West ML, Coghill JM, Panoskaltsis-Mortari A, Blazar BR, Serody JS. Invitro-differentiated TH17cells mediate lethal acute graft-versus-host disease withsevere cutaneous and pulmonary pathologic manifestations. Blood2009;113:1365-1374.
35. Yi T, Chen Y, Wang L, et al. Reciprocal differentiation and tissue-specificpathogenesis of Th1, Th2, and Th17cells in graft-versus-host disease. Blood2009;114:3101-3112.
36. Bettelli E, Oukka M, Kuchroo VK. T(H)-17cells in the circle of immunity andautoimmunity. Nat Immunol2007;8:345-350.
37. McCarthy PL, Jr., Abhyankar S, Neben S, et al. Inhibition of interleukin-1by aninterleukin-1receptor antagonist prevents graft-versus-host disease. Blood1991;78:1915-1918.
38. Antin JH, Weisdorf D, Neuberg D, et al. Interleukin-1blockade does not prevent acutegraft-versus-host disease: results of a randomized, double-blind, placebo-controlledtrial of interleukin-1receptor antagonist in allogeneic bone marrow transplantation.Blood2002;100:3479-3482.
39. Hippen KL, Bucher C, Schirm DK, et al. Blocking IL-21signaling amelioratesxenogeneic GVHD induced by human lymphocytes. Blood;2012,119:619-628.
1. Collison L W, Workman C J, Kuo T T, et al. The inhibitory cytokine IL-35contributesto regulatory T-cell function. Nature2007,450:566-569.
2. Pflanz S, Timans J C, Cheung J, et al. IL-27, a heterodimeric cytokine composed ofEBI3and p28protein, induces proliferation of naive CD4(+) T cells. Immunity2002,16:779-790.
3. Hibbert L, Pflanz S, Waal Malefyt R De, et al. IL-27and IFN-alpha signal via Stat1and Stat3and induce T-Bet and IL-12Rbeta2in naive T cells. J Interferon CytokineRes2003,23:513-522.
4. Bardel E, Larousserie F, Charlot-Rabiega P, et al. Human CD4+CD25+Foxp3+regulatory T cells do not constitutively express IL-35. J Immunol2008Nov15;181(10):6898-6905.
5. Chaturvedi V, Collison LW, Guy CS, et al. Human regulatory T cells require IL-35tomediate suppression and infectious tolerance. J Immunol2011Jun15;186(12):6661-6666.
6. Collison LW, Chaturvedi V, Henderson AL, et al. IL-35-mediated induction of a potentregulatory T cell population. Nat Immunol2010Dec;11(12):1093-1101.
7. Chua A O, Chizzonite R, Desai B B, et al. Expression cloning of a human IL-12receptor component. A new member of the cytokine receptor superfamily with stronghomology to gp130. J Immunol1994,153:128-136.
8. Chua A O, Wilkinson V L, Presky D H, et al. Cloning and characterization of a mouseIL-12receptor-beta component. J Immunol1995,155:4286-4294.
9. Parham C, Chirica M, Timans J, et al. A receptor for the heterodimeric cytokine IL-23is composed of IL-12Rbeta1and a novel cytokine receptor subunit, IL-23R. JImmunol2002,168:5699-5708.
10. Collison LW, Delgoffe GM, Guy CS, et al. The composition and signaling of the IL-35receptor are unconventional. Nat Immunol2012Feb5;13(3):290-299.
11. Thierfelder WE, van Deursen JM, Yamamoto K, et al. Requirement for Stat4ininterleukin-12-mediated responses of natural killer and T cells. Nature1996Jul11;382(6587):171-174.
12. O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses.Immunity2008Apr;28(4):477-487.
13. Niedbala W, Wei X Q, Cai B, et al. IL-35is a novel cytokine with therapeutic effectsagainst collagen-induced arthritis through the expansion of regulatory T cells andsuppression of Th17cells. Eur J Immunol2007,37:3021-3029.
14. Huang CH, Loo EX, Kuo IC, et al.Airway inflammation and IgE production inducedby dust mite allergen-specific memory/effector Th2cell line can be effectivelyattenuated by IL-35. J Immunol2011Jul1;187(1):462-471.
15. Kochetkova I, Golden S, Holderness K, et al. IL-35stimulation of CD39+regulatory Tcells confers protection against collagen II-induced arthritis via the production ofIL-10. J Immunol2010Jun15;184(12):7144-7153.
16. Bettini M, Castellaw A H, Lennon G P, et al. Prevention of Autoimmune Diabetes byEctopic Pancreatic b-Cell Expression of Interleukin-35. Diabetes.2012Jun;61(6):1519-1526.
2. Stanzani M, Martins S, Saliba R, et al. CD25expression on donor CD4+or CD8+Tcells is associated with an increased risk for graft-versus-host disease afterHLA-identical stem cell transplantation in humans. Blood,2004,103:1140-1146.
3. Bettelli E, Carrier Y, Gao W, et al. Reciprocal developmental pathways for thegeneration of pathogenic effector TH17and regulatory T cells. Nature,2006,441:235-238.
4. Yi T, Zhao D, Lin CL, et al. Absence of donor Th17leads to augmented Th1differentiation and exacerbated acute graft-versus-host disease. Blood,2008;112:2101-2110.
5. Kappel LW, Goldberg GL, King CG, et al. IL-17contributes to CD4-mediatedgraft-versus-host disease. Blood,2009,113:945-952.
6. Iclozan C, Yu Y, Liu C, et al. T helper17cells are sufficient but not necessary to induceacute graft-versus-host disease. Biol Blood Marrow Transplant,2010,16:170-178.
7. Dander E, Balduzzi A, Zappa G, et al. Interleukin-17-producing T-helper cells as newpotential player mediating graft-versus-host disease in patients undergoing allogeneicstem-cell transplantation. Transplantation,2009,88:1261-1272.
8. Zhao XY, Xu LL, Lu SY, Huang XJ. IL-17-producing T cells contribute to acutegraft-versus-host disease in patients undergoing unmanipulated blood and marrowtransplantation. Eur J Immunol,2011,41:514-526.
9. Ratajczak P, Janin A, Peffault de Latour R, et al. Th17/Treg ratio in humangraft-versus-host disease. Blood,2010,116:1165-1171.
10. Chen X, Vodanovic JS, Johnson B, Keller M, Komorowski R, Drobyski WR. Absenceof regulatory T-cell control of TH1and TH17cells is responsible for theautoimmune-mediated pathology in chronic graft-versus-host disease. Blood,2007,110:3804-3813.
11. Chen X, Das R, Komorowski R, et al. Blockade of interleukin-6signaling augmentsregulatory T-cell reconstitution and attenuates the severity of graft-versus-host disease.Blood,2009,114:891-900.
12. Tawara I, Koyama M, Liu C, et al. Interleukin-6modulates graft-versus-host responsesafter experimental allogeneic bone marrow transplantation. Clin Cancer Res,2011,17:77-88.
13. Das R, Chen X, Komorowski R, Hessner MJ, Drobyski WR. Interleukin-23secretionby donor antigen-presenting cells is critical for organ-specific pathology ingraft-versus-host disease. Blood,2009,113:2352-2362.
14. Thompson JS, Chu Y, Glass JF, Brown SA. Absence of IL-23p19in donor allogeneiccells reduces mortality from acute GVHD. Bone Marrow Transplant,2010,45:712-722.
15. Das R, Komorowski R, Hessner MJ, et al. Blockade of interleukin-23signaling resultsin targeted protection of the colon and allows for separation of graft-versus-host andgraft-versus-leukemia responses. Blood,2010,115:5249-5258.
16. Bucher C, Koch L, Vogtenhuber C, et al. IL-21blockade reduces graft-versus-hostdisease mortality by supporting inducible T regulatory cell generation. Blood,2009,114:5375-5384.
17. Hanash AM, Kappel LW, Yim NL, et al. Abrogation of donor T-cell IL-21signalingleads to tissue-specific modulation of immunity and separation of GVHD from GVL.Blood,2011,118:446-455.
18. Hanash AM, Dudakov JA, Hua G, et al. Interleukin-22protects intestinal stem cellsfrom immune-mediated tissue damage and regulates sensitivity to graft versus hostdisease. Immunity,2012,37:339-350.
19. Rezvani K, Mielke S, Ahmadzadeh M, et al. High donor FOXP3-positive regulatoryT-cell (Treg) content is associated with a low risk of GVHD following HLA-matchedallogeneic SCT. Blood,2006,108:1291-1297.
20. Semple K, Yu Y, Wang D, Anasetti C, Yu XZ. Efficient and selective prevention ofGVHD by antigen-specific induced Tregs via linked-suppression in mice. Biol BloodMarrow Transplant,2011,17:309-318.
21. Rieger K, Loddenkemper C, Maul J, et al. Mucosal FOXP3+regulatory T cells arenumerically deficient in acute and chronic GVHD. Blood,2006;107:1717-1723.
22. Gaidot A, Landau DA, Martin GH, et al. Immune reconstitution is preserved inhematopoietic stem cell transplantation coadministered with regulatory T cells forGVHD prevention. Blood,2011,117:2975-2983.
23. Di Ianni M, Falzetti F, Carotti A, et al. Tregs prevent GVHD and promote immunereconstitution in HLA-haploidentical transplantation. Blood,2011,117:3921-3928.
24. Zorn E, Kim HT, Lee SJ, et al. Reduced frequency of FOXP3+CD4+CD25+regulatory T cells in patients with chronic graft-versus-host disease. Blood,2005,106:2903-2911.
25. Laurence A, Amarnath S, Mariotti J, et al. STAT3transcription factor promotesinstability of nTreg cells and limits generation of iTreg cells during acute murinegraft-versus-host disease.Immunity,2012,37(2):209-222.
26. Collison L W, Workman C J, Kuo T T, et al. The inhibitory cytokine IL-35contributesto regulatory T-cell function. Nature,2007,450:566-569.
27. Pflanz S, Timans J C, Cheung J, et al. IL-27, a heterodimeric cytokine composed ofEBI3and p28protein, induces proliferation of naive CD4(+) T cells. Immunity,2002,16:779-790.
28. Hibbert L, Pflanz S, Waal Malefyt R De, et al. IL-27and IFN-alpha signal via Stat1and Stat3and induce T-Bet and IL-12Rbeta2in naive T cells. J Interferon CytokineRes,2003,23:513-522.
29. Bardel E, Larousserie F, Charlot-Rabiega P, et al. Human CD4+CD25+Foxp3+regulatory T cells do not constitutively express IL-35. J Immunol,2008,181(10):6898-6905.
30. Chaturvedi V, Collison LW, Guy CS, et al. Human regulatory T cells require IL-35tomediate suppression and infectious tolerance. J Immunol,2011,186(12):6661-6666.
31. Collison LW, Chaturvedi V, Henderson AL, et al. IL-35-mediated induction of a potentregulatory T cell population. Nat Immunol,2010,11(12):1093-1101.
32. Chua A O, Chizzonite R, Desai B B, et al. Expression cloning of a human IL-12receptor component. A new member of the cytokine receptor superfamily with stronghomology to gp130. J Immunol,1994,153:128-136.
33. Chua A O, Wilkinson V L, Presky D H, et al. Cloning and characterization of a mouseIL-12receptor-beta component. J Immunol,1995,155:4286-4294.
34. Parham C, Chirica M, Timans J, et al. A receptor for the heterodimeric cytokine IL-23is composed of IL-12Rbeta1and a novel cytokine receptor subunit, IL-23R. JImmunol,2002,168:5699-5708.
35. Collison LW, Delgoffe GM, Guy CS, et al. The composition and signaling of the IL-35receptor are unconventional. Nat Immunol,2012,13(3):290-299.
36. Thierfelder WE, van Deursen JM, Yamamoto K, et al. Requirement for Stat4ininterleukin-12-mediated responses of natural killer and T cells. Nature,1996,382(6587):171-174.
37. O'Shea JJ, Murray PJ. Cytokine signaling modules in inflammatory responses.Immunity,2008,28(4):477-487.
38. Niedbala W, Wei X Q, Cai B, et al. IL-35is a novel cytokine with therapeutic effectsagainst collagen-induced arthritis through the expansion of regulatory T cells andsuppression of Th17cells. Eur J Immunol,2007,37:3021-3029.
39. Huang CH, Loo EX, Kuo IC, et al.Airway inflammation and IgE production inducedby dust mite allergen-specific memory/effector Th2cell line can be effectivelyattenuated by IL-35. J Immunol,2011,187(1):462-471.
40. Kochetkova I, Golden S, Holderness K, et al. IL-35stimulation of CD39+regulatory Tcells confers protection against collagen II-induced arthritis via the production ofIL-10. J Immunol,2010,184(12):7144-7153.
41. Bettini M, Castellaw A H, Lennon G P, et al. Prevention of Autoimmune Diabetes byEctopic Pancreatic b-Cell Expression of Interleukin-35. Diabetes,2012,61(6):1519-1526.
1. Collison LW, Pillai MR, Chaturvedi V, et al. Regulatory T cell suppression ispotentiated by target T cells in a cell contact, IL-35-and IL-10-dependent manner. JImmunol,2009,182(10):6121-6128.
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