同种移植急性排斥反应中Pim2的作用及机制研究
|
摘要
研究目的
器官移植目前已成为治疗多种终末期疾病的的有效手段。然而发生于移植术后的数周或数月的急性排斥反应仍是导致治疗失败的主要因素。因此器官移植术的成败在很大程度上取决于对急性移植排斥反应的防治。既往研究表明T细胞,尤其是CD4+T细胞介导的细胞免疫应答在移植排斥反应的效应机制中发挥关键作用。T细胞存活的信号通路主要包括AKT(also known as protein kinase B, PKB)-mTOR(mammalian Target of Rapamycin)信号通路和Pim (Proviral integration site of murine)激酶信号通路。既往研究表明,在细胞因子或抗原激活的T细胞信号通路中,T细胞的生长及存活不完全依赖AKT-mTOR通路,阻断AKT-mTOR信号通路虽可部分抑制T细胞激活,但不能完全阻断；Pim激酶信号通路可作为选择性通路。但是有关Pim激酶信号通路在同种反应性T细胞的作用尚未见报道。推测同时阻滞这两条信号通路,有可能更好地抑制移植受者同种反应性T细胞介导的急性排斥,有利于诱导长期移植耐受。己知CD4+T细胞可分为CD4+CD25-效应T细胞和CD4+CD25+Foxp3+调节性T细胞(Treg)两种亚群。其中同种抗原特异性Treg可通过抑制效应T细胞的激活、增殖和促进其凋亡,诱导移植物长期耐受。近来有研究报道Treg细胞可在雷帕霉素(Rapamycin, RAPA)的作用下优势扩增并能很好地保持其抑制功能；而自然调节性Treg中Foxp3基因能够诱导pim2基因的表达。以上研究提示pim2与Treg细胞功能密切相关。本实验室前期研究中,应用SAGE (sequencing-based serial analysis of gene expression,序列基因表达分析)技术建立了同种移植及同系移植组CD4+T细胞mRNA标签库,分析发现pim2基因在同种移植急性排斥组明显高表达,是同系移植组表达量的5倍,进一步提示pim2在同种移植急性排斥中具有重要作用。
Pim家族是原癌基因编码三种丝／苏氨酸激酶,包括Piml, Pim2and Pim3,属于钙调蛋白依赖性蛋白激酶且在序列上具有很高的同源性。pim2基因位于X染色体上(Xpll),编码三种分子量的蛋白质(34kD,37kD和40kD)。Pim激酶缺乏调节结构域,不需磷酸化激活。可能是翻译后组成性激活。Pim激酶由转录翻译和蛋白体降解调控。STAT (Signal transducers and activators of transcription,信号传导及转录激活因子)通过与其同源配体衔接而成为pim基因的转录因子。Pim蛋白激酶活性依赖于mRNA稳定性,Pim通过与SOCS (Suppressor of cytokine signaling,细胞因子信号抑制物)结合从而抑制JAK (Janus Kinase)/STAT信号。多项研究表明Pim激酶通过调控信号转导在许多生理病理过程中发挥重要作用,包括细胞周期、转录因子、细胞凋亡以及细胞代谢和蛋白质翻译。在许多造血系统恶性疾病及实体肿瘤中均发现pim原癌基因的高表达(尤其是Piml和Pim3),并与肿瘤细胞生长存活及化疗药物抵抗密切相关。据报道目前有几种Pim特异小分子抑制剂正用于肿瘤治疗临床前期实验。尽管已有pim2通路在造血系统肿瘤和实体肿瘤中作用的研究报道,但是有关pim2在同种移植急性排斥中的作用及分子机制尚未见报道。Pim2与CD4+T细胞,尤其是Treg密切相关,有可能作为诱导移植免疫耐受的靶向分子；此外Pim2靶向小分子化合物正在研制中,因此Pim2有可能作为同种移植急性排斥诊断和治疗的分子靶点。
综上所述,本论文的科学假设是：pim2可能通过靶向CD4+T细胞的凋亡和调节CD4+Treg增殖及抑制,在同种移植急性排斥反应过程中发挥重要作用。本论文研究目的是试图阐明Pim2激酶在同种移植急性排斥反应中的作用及分子机制,从而为靶向Pim2诱导诱导移植免疫耐受提供理论和实验依据,为临床器官移植排斥的治疗提供新的靶点,因此具有重要的理论和实际意义。
第一部分Pim2在小鼠同种皮肤移植急性排斥反应中的动态表达
研究方法
1、建立小鼠同种皮肤移植模型
无菌条件下将C57BL/6小鼠背部全厚度皮肤移植到BALB/c小鼠背部即为同种移植；将BALB/c小鼠的背部全厚度皮肤移植到BALB/c小鼠背部即为同系移植,以此作为对照组。
2、分析pim2基因在排斥高峰时的表达状况
移植术后14天左右,同种移植皮片完全排斥而同系移植存活完好。利用RT-PCR技术分别检测两组小鼠脾细胞、脾CD4+T细胞及移植皮片中的pim2基因表达。
3、同种排斥组与同系对照组的脾脏和移植皮片的病理学表现以及Pim2蛋白的
原位表达
通过病理组织切片HE染色及抗Pim2抗体对组织切片的免疫组化染色进一步比较同种排斥组与同系对照组的脾脏和移植皮片的病理学表现差异以及Pim2蛋白原位表达差异情况。
4、Pim2蛋白在同种移植排斥反应过程中的动态表达情况
移植后不同时间,取受体小鼠的脾及移植皮片,Western Blot检测Pim2蛋白表达状况。
研究结果
1、pim2mRNA在同种移植组的CD4+T细胞、脾脏和移植皮片中的表达水平均明显高于同系对照组；而且在同种移植组中,pim2mRNA在脾脏中的的表达水平高于移植皮片,但是同系对照组中二者之间无明显差异。结果提示：同种移植急性排斥反应较强时,pim2基因高表达,pim2基因是同种移植急性排斥反应正相关基因。
2、与同系对照组相比,同种排斥组的脾和移植皮片均发生明显组织病理改变,细胞浸润明显；Pim2蛋白高表达于同种排斥组的脾和移植皮片的细胞质中,但同系对照组脾和移植皮片中表达微弱或不表达。结果提示Pim2激酶参与同种急性排斥反应所导致的移植物损害和组织损伤过程。
3、 Western Blot结果显示同种移植术后Pim2蛋白的动态表达趋势与同种移植急性排斥反应的发生过程几乎完全一致,即脾或移植皮片中的Pim2蛋白表达量随着同种急性排斥反应的增强而升高。该结果进一步提示Pim2蛋白激酶参与同种移植急性排斥反应并参与对移植物的排斥。
第二部分抑制pim2基因表达对延长同种移植中移植物存活的影响
第一部分研究证实pim2在同种移植急性排斥期高表达,因此本部分进一步研究抑制Pim2后对同种移植排斥的效应及作用机制。
研究方法
1、确定pim抑制剂4a对pim2抑制的最适浓度和时间
首先制备野生型BALB/c小鼠脾细胞悬液,然后将该脾细胞分别与不同浓度的Pim抑制剂4a混合,培养不同时间,最后利用RT-PCR检测4a处理后脾细胞中pim2基因的表达水平并以pim2mRNA相对灰度值最低来确定Pim抑制剂4a对pim2抑制的最适浓度和时间。
2、T细胞中的pim2被4a抑制后对T细胞介导的同种移植急性排斥的影响将B6小鼠的全厚度皮肤移植到SCID (Severe Combined Immunodeficiency,严重联合免疫缺陷)小鼠背部而建立SCID小鼠移植模型,3周左右移植皮片愈合,生长完好。此时将预先经过4a或DMSO(对照)体外处理的野生型BALB/c小鼠脾脏T细胞通过腹腔注射过继到上述已行移植术的SCID鼠体内,构建同种移植急性排斥反应模型,T细胞过继后,每天观察两组移植皮片的生存状况直至对照组移植皮片排斥。
3、pim2对同种移植急性排斥反应中的同种反应性T细胞的作用及通路
利用T细胞分离柱分离纯化出同种移植排斥高峰期小鼠脾脏T细胞,然后利用Pim特异抑制剂4a体外处理24h,利用流式细胞术分析T细胞的凋亡率。采用WesternBlot方法检测4a对同种反应性T细胞中磷酸化BAD (Ser112)的影响。
4、同种移植急性排斥高峰期脾CD4+CD25-T细胞和CD4+CD25+T细胞中pim2mRNA的表达状况
利用免疫磁珠分选法分离出同种移植排斥高峰期BALB/c小鼠脾CD4+CD25-T细胞和CD4+CD25+T细胞两个亚群,然后利用RT-PCR方法检测两群细胞中pim2mRNA的表达状况。
研究结果
1、pim2mRNA的表达量与4a呈剂量依赖关系,但非时间依赖关系。最终选定5M4a和24h作为4a对pim2抑制的最适浓度和最适作用时间。
2、4a处理组小鼠移植皮片的平均生存时间约为31.2±2.3天,而DMSO对照组小鼠移植皮片的平均生存时间约为19.5±1.7天。与对照组比较,4a处理组同种移植物生存期明显延长约12天(P<0.05),而且其脾脏重量和大小明显减小。结果表明4a通过阻滞T细胞中的pim2,明显延长了同种移植物存活,降低了受体对同种移植物的排斥,提示pim2通过T细胞介导了对同种移植物的急性排斥。
3、4a处理组的T细胞凋亡率明显升高,与对照组有显著差异,P<0.05,提示阻滞T细胞中的pim2,增加了同种反应性T细胞的凋亡；4a处理组的BAD(Ser112)磷酸化水平明显降低,提示4a通过降低Pim2激酶的下游通路BAD (Ser112)磷酸化水平诱导同种反应性T细胞的凋亡,从而部分阻断由同种反应性T细胞介导的针对移植物的特异性免疫应答。结果提示：Pim2通过磷酸化BAD Ser112位点发挥抗同种反应性T细胞凋亡作用,进而介导对同种移植物的急性排斥。
4、在同种移植排斥高峰期,pim2基因在CD4+CD25-T细胞中高表达,约为CD4+CD25+T细胞中pim2基因含量的两倍,提示pim2优势介导同种反应性CD4+CD25-T细胞的存活,从而发挥增强同种移植急性排斥的作用。第三部分Pim2对同种抗原诱导的CD4+CD25+Treg细胞活性的影响前两部分研究证明pim2增强同种移植急性排斥反应,但是结果提示同种移植急性排斥中pim2在CD4+CD25+Treg中也有表达,因此不能排除pim2对CD4+CD25+Treg细胞的影响。为此,本部分研究了pim2对同种抗原诱导的CD4+CD25+Treg细胞活性的影响：
研究方法
1、pim2抑制对CD4+CD25+Treg细胞关键转录因子Foxp3的影响
利用T细胞分离柱纯化分离同种移植急性排斥反应高峰期小鼠脾T细胞,与5M4a混合培养24h后,采用RT-PCR方法检测细胞Foxp3mRNA的变化。
2、pim2抑制对CD4+CD25+T细胞抑制功能的影响
利用免疫磁珠分选方法分离出同种移植排斥高峰期BALB/c小鼠脾脏中的CD4+CD25-T细胞和CD4+CD25+T细胞。先将CD4+CD25+T细胞与4a体外共同培养24h,然后再与CD4+CD25-T细胞按照1：6的比例混合培养12h,最后利用流式细胞术分析CD4+CD25-T细胞的凋亡率。
3、Pim2激酶对雷帕霉素选择性扩增Foxp3+Treg细胞的影响
建立BALB/c小鼠同种移植模型,移植术后第1天开始每天腹腔注射雷帕霉素,放线菌酮,或雷帕霉素+放线菌酮,同时设生理盐水注射对照组。直至对照组小鼠移植皮片发生排斥时停止注射。此时收集不同处理组小鼠脾细胞,利用流式细胞术分析细胞表面的CD4、CD25及细胞质中的Foxp3,以此确定CD4+CD25+Foxp3+Treg所占总细胞比例的变化。随后,将纯化出的雷帕霉素组和联合药物组CD4+CD25+Foxp3+T细胞与Pim2单克隆抗体或4a在体外共培养12h,流式细胞术分析抑制Pim2对CD4+CD25+Foxp3+Treg细胞比例的影响。
研究结果
1、4a在抑制同种反应性T细胞中pim2表达的同时,也降低了Foxp3的基因表达水平,表明阻滞pim2能够降低同种抗原诱导的CD4+CD25+Foxp3+T细胞的增殖活化。结果提示Pim2激酶对同种抗原诱导的CD4+CD25+Foxp3+T细胞的增殖活化是必要的。
2、4a显著降低同种抗原诱导的CD4+CD25+Foxp3+T细胞诱导效应性CD4+CD25-T细胞凋亡的能力,表明阻滞pim2降低了CD4+CD25+Foxp3+T细胞的抑制功能,提示Pim2激酶对同种抗原诱导的CD4+CD25+Foxp3+T细胞的抑制功能也是必要的。
3、与对照组相比,雷帕霉素组和联合药物组小鼠脾CD4+CD25+Foxp3+T细胞比例明显升高,而同种反应性CD4+T细胞比例却显著下调,提示在同种移植模型中,雷帕霉素选择性扩增CD4+CD25+Foxp3+T细胞。抗Pim2单克隆抗体或4a都明显下调了雷帕霉素组和联合药物组CD4+CD25+Foxp3+T细胞的比例。结果提示在同种移植模型中,阻滞Pim2激酶可明显降低CD4+CD25+Foxp3+T细胞在雷帕霉素的作用下的扩增水平,提示Pim2激酶对雷帕霉素选择性扩增同种抗原诱导的CD4+CD25+Foxp3+T细胞同样是必要的。
第四部分放线菌酮抑制同种移植急性排斥及与Pim2的相关性研究鉴于文献报道放线菌酮可以抑制Pim激酶活性,因此推测放线菌酮可能通过抑制Pim激酶抑制急性同种移植排斥。
研究方法
1、放线菌酮对pim2表达的影响
为探索放线菌酮抑制pim2的最适浓度,利用小鼠T细胞分离柱从野生型BALB/c小鼠脾细胞中分离纯化T细胞,分别与不同浓度的放线菌酮混合培养24h,最后利用RT-PCR分析药物处理后T细胞中pim2基因的表达并以pim2mRNA相对灰度值来确定放线菌酮抑制pim2的最适浓度。
2、放线菌酮体外阻滞T细胞pim2后对同种移植物存活的影响
将B6小鼠的背部全厚度皮片移植到SCID小鼠背部建立SCID小鼠移植模型,大约3周后移植皮片愈合生长完好。此时将预先经过放线菌酮或DMSO(对照)体外处理的野生型BALB/c小鼠脾T细胞通过腹腔注射过继到上述己行移植术的SCID鼠体内。构建同种移植急性排斥反应模型,细胞过继后每天观察移植皮片的生存状况。
3、药物体内处理对同种移植物存活的影响
建立BALB/c小鼠同种移植模型,移植术后第1天开始每天腹腔注射放线菌酮、雷帕霉素或雷帕霉素十放线菌酮,直至对照组小鼠移植皮片发生排斥时结束药物注射。每天观察每组移植皮片的生存状况并绘制同种移植皮片生存曲线。
4、放线菌酮延长同种移植物存活的机制
建立BALB/c小鼠同种移植模型,移植术后第1天开始每天腹腔注射放线菌酮、雷帕霉素或雷帕霉素+放线菌酮,直到对照组(腹腔注射生理盐水)小鼠皮片完全被排斥。此时制备脾细胞悬液并采用流式细胞术分析放线菌酮、雷帕霉素或雷帕霉素+放线菌酮体内给药分别对同种反应性CD4+T细胞和CD4+CD25+Foxp3+Treg细胞比例的影响。
研究结果
1、与对照组相比,放线菌酮明显抑制pim2基因的表达,呈剂量依赖性,即50μM的放线菌酮能够更有效地抑制pim2,且无明显的细胞毒性。
2、与对照组相比,放线菌酮处理组的移植皮片延长存活约7天(p<0.05),提示放线菌酮可通过抑制T细胞中的pim2延长同种移植物的存活时间。
3、结果显示,对照组小鼠移植皮片的平均生存时间约为12.4±1.10天,注射放线菌酮组小鼠皮片的平均生存时间约为17.6±0.89天,注射雷帕霉素组小鼠皮片的平均生存时间约为19.6±1.52天,放线菌酮+雷帕霉素联合组小鼠皮片的平均生存时间约为16.8±1.64天,比对照组分别延长约5天,7天和4天,具有显著差异,P<0.05。但是,联合注射组与单独注射放线菌酮组之间无显著差异,P>0.05。提示放线菌酮体内应用明显延长同种移植物的存活时间,但与雷帕霉素联合没有增强效应。
4、与对照组比较,放线菌酮对CD4+T细胞扩增无明显影响(分别为8.0%和8.7%),而雷帕霉素显著降低CD4+T细胞比例(2.8%),雷帕霉素+放线菌酮联合亦可显著降低CD4+T细胞比例(3.2%)；与对照组(4.3%)比较,放线菌酮可降低CD4+CD25+Foxp3+T细胞比例(3.2%),但是无统计学差异,P>0.05；雷帕霉素明显诱导CD4+CD25+Foxp3+T细胞扩增(5.6%)；雷帕霉素+放线菌酮联合显著上调CD4+CD25+Foxp3+T细胞比例(7.6%),且联合应用组CD4+CD25+Foxp3+T细胞的比例明显高于雷帕霉素单独注射组,P<0.05。结果提示在同种移植模型中放线菌酮增强了雷帕霉素优势扩增CD4+CD25+Foxp3+T细胞的能力,而放线菌酮延长同种移植物存活除CD4+T细胞外,可能存在其它分子机制。
全文结论
1.pim2在同种移植急性排斥高峰期高表达,Pim2蛋白的动态表达趋势与同种移植急性排斥反应的发生过程一致,且在脾脏及移植物部位高表达。提示Pim2激酶参与同种移植急性排斥反应并介导移植物损害和组织损伤过程。
2.Pim2通过磷酸化BAD Ser112位点对同种反应性T细胞发挥抗凋亡作用,并优势介导效应性CD4+CD25-T细胞的存活,从而发挥其增强同种移植排斥反应的作用；抑制pim2可明显延长同种移植物的存活时间。
3.Pim2对同种抗原诱导的CD4+CD25+Foxp3+T细胞的增殖活化和发挥抑制功能是必要的；Pim2激酶对雷帕霉素选择性扩增同种抗原诱导的CD4+CD25+Foxp3+T细胞也是必要的。
4.放线菌酮通过抑制pim2可有效延长同种移植物存活,放线菌酮体内应用显著增强雷帕霉素优势扩增CD4+CD25+Foxp3+T细胞的能力。
本文创新点
1.首次利用小鼠同种皮肤移植模型,证实pim2在同种移植模型中高表达并且通过细胞凋亡信号通路增强同种移植急性排斥反应。研究结果为进一步阐明同种移植急性排斥反应的分子机制提供了理论依据。
2.首次证实pim2蛋白对同种抗原诱导的CD4+CD25+Foxp3+Treg细胞的增殖、抑制效应以及在雷帕霉素作用下的优势扩增是必要的；放线菌酮可增强雷帕霉素体内选择性扩增CD4+CD25+Foxp3+Treg细胞的能力。研究结果为CD4+CD25+Foxp3+Treg细胞的体外扩增和细胞治疗提供了新的途径。
3.首次证实pim激酶抑制剂4a和放线菌酮可通过抑制pim2明显延长同种移植物的存活,提示pim2有可能成为同种移植急性排斥诊断和治疗的分子靶点,研究结果为临床靶向Pim2抗移植排斥治疗提供了实验基础和理论依据。
Background and Objectives
Organ transplantation is the most effective therapy for patients with end-stage organ failure. However, acute rejection, which occurs within the first few weeks or months, remains a major cause of treatment failure. T cells, especially CD4+T cells play critical roles in the activation of the immune responses to alloantigens that cause graft rejection. Two survival pathways for T cells have been predominantly targeted: the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway and the PIM pathway. T-cell growth and survival following ligation of cytokine or antigen receptors is mediated, in part, through activation of the effector enzymes AKT and mTOR in the phosphatidylinositol3-kinase pathway and PIM kinases may provide the alternative pathway for T-cell survival. Even through the immunosuppressive therapies that block the PI3K/AK.T/mTOR pathway have been proven to be uniquely effective for anti-allotransplant in clinic, there has been no reports on the effects of Pim pathway blockade in either mice allotransplant models or human transplant recipients. This has led to the speculation that combined blockade of these two pathways might have synergistic effects in inducing immune tolerance, and thus, there has been a long-standing interest in translating about these results to the clinic. The CD4+T cells can be differentiated into various subsets. CD4+CD25+Foxp3+T cells, an important subset of CD4+T cells, can potently suppress the activation or function of conventional CD4+CD25-T effector cells. It has been reported that donor antigen-specific CD4+CD25+Foxp3+regulatory T (Treg) cells can regulate alloresponses and promote donor-specific tolerance in a skin transplantation model. Of the standard immunosuppressive agents that have been combined with Tregs, perhaps the most detailed work has been accomplished with rapamycin. Studies have shown that Tregs are preferentially able to retain their suppressive function in the presence of rapamycin. Interestingly, Foxp3induced pim2expression in natural Tregs, which caused Foxp3-expressing Treg cells to be selected in the presence of rapamycin. These studies suggested that pim2may be associated with the function of Treg cells in allograft rejection. Furthermore, in our previous study, an approximately five-fold overexpression of the pim2gene, as determined by sequencing-based serial analysis of gene expression, was detected in the allograft-activated CD4+T cells, which suggests a potential role of the Pim2kinase in allograft rejection.
The pim genes represent a family of proto-oncogenes, which encode three different serine/threonine protein kinases (piml, pim2and pim3) that are related to the calcium/calmodulin kinase superfamily by weak sequence homology. pim2is an X chromosome gene (Xp11) and transcribe for three isoforms (34kD,37kD, and40kD). Pim kinases lack a regulatory domain and as a result they do not require phosphorylation for activation, and are likely to be constitutively activated following translation. The Pim kinases are regulated by rates of transcription, translation and proteosomal degradation and the STAT proteins serve as transcription factors for the pim genes following engagement of their cognate ligands. The kinase activity of these proteins is proportional to and dependent on mRNA stability, which also serves to inhibit JAK/STAT signaling through binding to the SOCS proteins, which inhibit JAK-dependent signaling. The Pim kinases play essential roles in the regulation of signal transduction cascades, included cell cycle progression, transcription factors, apoptosis and cellular metabolism and protein translation in a large variety of tissues. The pim oncogenes are over-expressed in several hematopoietic malignancies and solid cancers, and promote the cell growth, survival and drug resistance. Pim kinases showed a unique crystal structure and several specific small-molecule inhibitors currently in development are showing interesting preclinical activity in multiple cancer histology. Although pim2pathway can contribute to hematological and solid tumors developing, the physiologic role of this kinase and the precise mechanism by which inhibition of Pim2signals attenuates donor-reactive T-cell response remains uncertain. Considering of the relationship between Pim2and Treg, targeting Pim2may serve as an alternative way to induce allograft tolerance in preclinical models. Moreover, with the continuing development of Pim2-specific small-molecule inhibitors for clinical translation, immunosuppressive therapies that block the Pim2pathway would have proven to be uniquely effective in preclinical allotransplant models.
Considering all of the aforementioned results, we hypothesize that Pim2kinase may participate in allograft rejection through targeting the apoptosis of CD4+T cells and modulating Treg-suppressing activities. In this study, we aimed to investigate the role and the underlying mechanism of Pim2during allograft rejection in murine skin allograft models so as to provide a new target for the diagnosis and therapy of allorejection for clinic.
Part One The expression of Pim2during allograft acute rejection in murine skin allograft models
Methods
1. Establishment of mice skin graft models
In this model, the dorsal skin of C57BL/6mice was allotransplanted onto the BALB/c mice under sterile conditions (allogenic transplantation) and the dorsal skin of BALB/c mice was isotransplanted onto the BALB/c mice (isogenic transplantation) as the control.
2. The pim2gene expression at the peak of allograft rejection analyzed with RT-PCR
At day14post-transplantation (allografts rejected completely, while isografts remained normal), pim2mRNA of splenic CD4+T cells, spleens and skin grafts of different recipient mice was examined by RT-PCR.
3. The expression of Pim2protein in situ analyzed with Immunohistology staining
The difference in the pathology of spleens and skin grafts between allorejected and isografted mice was conducted by the histology analysis. The in situ expression of Pim2protein of spleens and skin grafts from allorejected and isografted mice were conducted by immunohistochemical staining.
4. The dynamic expression of Pim2protein post allotransplantation analyzed with Western Blotting
The dynamic expression of Pim2protein in spleens and grafted skins at various times post-allotransplantation was also confirmed using Western Blotting.
Results
1. It was found that the expression of pim2mRNA in CD4+T cells was significantly higher both in the allografted mice and rapamycin-treated allograft mice with the RT-PCR analysis, but rapamycin treatment in vivo did not significantly change the expression of pim2in allograft mice. Our results also showed that pim2mRNA of spleens and skin grafts was significantly elevated in allograft mice compared with that from isografted mice. Moreover, in allograft mice, pim2mRNA was markedly higher in the spleens than in the skin grafts, while in isografted mice, there was no significant difference between these two tissues. These data indicated that pim2gene was over-expressed when the allorejection reached the peak, suggesting that pim2gene serves as a positively related to allorejection.
2. The histology observation of spleens and skin grafts from allorejected and isografted mice showed an obvious difference in the pathology between the two groups. By immunohistochemical staining, we found that Pim2protein was markedly expressed in the cytoplasm of allografted skins and spleens, but only weakly or negatively expressed in isografted skins and spleens, suggesting that Pim2was responsible for the immune-mediated tissue destruction processes.
3. The immunoblotting results showed that Pim2protein was upregulated at day7in the spleens of recipient mice and reached the highest levels at day10. The accumulation of Pim2in allograft skins showed the same tendency as in the spleens, except that upregulation occurred at day10and that the highest level observed was at day14. This pattern of Pim2expression overlapped with the progression of graft rejection in response to the allogeneic antigen, indicating that Pim2kinase involved in the alloresponse and allograft rejection.
Part Two The role and mechanism of Pim2in allograft acute rejection
As shown in part one, pim2gene and protein were overexpressed during allograft rejection. Then, we further investigate how pim2work during the allorejection.
Methods
1. The determination of a suitable concentration and time-points for4a The spleen cells of naive B ALB/c mice were treated with different concentration of4a for different time, followed by pim2mRNA analyzed with RT-PCR
2. The effect of inhibiting Pim2kinase on allograft survival
We established the SCID mouse skin acute allograft rejection model by allografting the skin of B6mice to the SCID mice. After the wounds healed3weeks later, the alloimmune response was then reconstituted with the adoptive transfer of naive BABL/c splenic T cells treated with4a for24h or with such cells treated with DMSO as a control. The grafts were observed daily after the removal of the bandages until that the graft in control group was completely rejected.
3. The potential effect of Pim2inhibiting on the apoptosis of alloreactive T cells
T cells were purified with Mouse T cell Enrichment Columns from recipient spleens at the height of allorejection, co-cultured with4a for24h and then were subjected to apoptosis analysis by flow cytometry. In addition, the phosphorylation of Bad (Ser112), a Pim2kinase substrate and apoptotic regulator, was detected by western blot in T cells treated by4a.
4. The gene expression of Pim2in different CD4+T-cell subsets
Based on the finding that pim2was overexpressed in unfractionated CD4+T cells during acute allograft rejection, we analyzed the gene expression of Pim2in different CD4+T-cell subsets. After that CD4+CD25-T cells and CD4+CD25+T cells were purified and collected with MagCellect Mouse CD4+CD25+Regulatory T Cell Isolation Kit, from recipient spleens at the height of allorejection, pim2expression was detected by RT-PCR separately.
Results
1. Firstly, we performed RT-PCR and found that pim2mRNA was decreased in a dose-dependent manner, with a significant effect shown in the group treated with5μM4a but that little change was observed among the different treatment time points. So5μM4a and24h were chosen as the suitable concentration and time for4a.
2. The T cells adoptive transfer assay showed that all of the mice in the control group (T cells treated by5μM DMSO for24h) had severe rejection at day19.5±1.7post cell transfer, while the allograft rejection in the experimental group (T cells treated by5μM4a for24h) was observed at day31±2.3. At same time, we found that there was a two-fold decrease in spleen weight compared with the control group. Therefore, blockade of Pim2significantly postponed acute rejection mediated by T cells.
3. The result demonstrated a notable increase in apoptotic cells in the4a-treated group. In addition, the phosphoryla-tion of Bad (Ser112), a Pim2kinase substrate and apoptotic regulator, was significantly reduced in the4a-treated group. These results suggested that the absence of Pim2activity increased the level of T-cell apoptosis by reducing the phosphorylation of apoptotic regulator Bad (Ser112) during allograft rejection, which indicates that pim2may promote the allograft rejection by inhibiting T-cell apoptosis.
4. By RT-PCR analysis of Pim2expression in different CD4+T-cell subsets during acute allograft rejection, we found that expression of pim2mRNA in CD4+CD25-T cells was2.66-fold higher than that in CD4+CD25+T cells, indicating that Pim2kinase more strongly promoted CD4+CD25-T-cell survival.
Part Three The effect of Pim2on the alloantigen-induced CD4+CD25+T cells
Considering the negative regulation of CD4+CD25+T cells observed during allorejection and the expression of pim2gene in alloantigen-induced CD4+CD25+T cells as shown in part two, we further investigated the effect of Pim2on this subset.
Methods
1. The effect of Pim2blocking on Foxp3gene expression analyzed with RT-PCR Gene expression of Foxp3in splenic T cells isolated from the allograft mice and treated with different concentration of4a for24h was analyzed by RT-PCR.
2. The effect of Pim2blocking on the function of alloantigen-induced CD4+CD25+T cells analyzed by Flow cytometry
Next, we explored whether Pim2was required for alloantigen-induced CD4+CD25+T-cell suppression activity. CD4+CD25-T cells and CD4+CD25+T cells were purified from the freshly isolated spleens of allograft mice with rejection. CD4+CD25+T cells treated with or without4a for24h were cocultured with CD4+CD25-T cells for12h at a ratio of1:6separately, and then examined for cell apoptosis by flow cytometric analysis.
3. The effect of Pim2blocking on the preferential expansion of alloantigen-induced Tregs in the presence of rapamycin analyzed by Flow cytometry
Since rapamycin selectively allowed for proliferation and fostering the suppressive function of Foxp3+Treg cells, next we explored whether Pim2kinase contributed to the observed an enrichment of circulating CD4+CD25+Foxp3+Treg in the presence of rapamycin. Flow cytometry of the surface expression of CD4, CD25and Foxp3on spleen cells from rapamycin-treated allografted mice was conducted after stimulated for12h with anti-pim2or with4a.
Results
1. By analyzing the expression of Foxp3and pim2in splenic T cells isolated from the allograft mice and treated with5or10μM4a for24h by RT-PCR, we found that4a treatment resulted in the significant decrease of Foxp3and pim2expression. Since Foxp3is the key transcriptional factor of CD4+CD25+T cells, the result showed that blockage of pim2by4a reduced the proliferation of alloantigen induced CD4+CD25+T cells, indicating that Pim2is indispensable for the ex vivo activation of alloantigen-induced CD4+CD25+T cells.
2. The results of flow cytometric analysis showed that the alloantigen induced CD4+CD25+T cells treated with5μM4a led to an obvious decrease in the percentage of alloantigen induced CD4+CD25-T-cell apoptosis compared with those untreated with4a, suggesting that the blocking of Pim2impaired the function of CD4+CD25+T cells to induce CD4+CD25-T-cell apoptosis. The data indicated that Pim2is indispensable for the ex vivo function of alloantigen-induced CD4+CD25+T cells.
3. The results of flow cytometric analysis showed that treatment with either anti-pim2or4a led to a substantially reduced expansion of the observed enrichment of CD4+CD25+Foxp3+Treg in the presence of rapamycin alone or combined with cycloheximide, suggesting that pim2allowed alloantigen-induced CD4+CD25+T cells to evade many rapamycin-imposed signaling blocks and to expand preferentially.
Part Four Cycloheximide served as an immunosuppressive drug to prevent allorejection through the pim2pathway
Considering that cycloheximide could inhibit pim2to function in anti-apoptosis, and the blockade of Pim2prolonged skin graft survival through the apoptosis regulation pathway in our previous study, we hypothesize that cycloheximide may prolong skin graft survival.'
Methods
1. The effect of cycloheximide in inhibiting pim2in vitro analyzed by RT-PCR For determination of a suitable concentration of cycloheximide, spleen T cells from naive BALB/c mice were treated with different concentrations of cycloheximide for24h, followed by RT-PCR analysis of pim2mRNA.
2. The effect of blocking pim2by cycloheximide on the allograft survival We established the SCID mouse skin acute allograft rejection model by allografting the skin of B6mice to the SCID mice. After the wounds healed3weeks later, the alloimmune response was then reconstituted with the adoptive transfer of naive BABL/c splenic T cells treated with cycloheximide for24h or with such cells treated with DMSO as a control. The grafts were observed daily after the removal of the bandages until that the graft in control group was completely rejected.
3. The effect of cycloheximide administrated in vivo on the allograft survival
We also confirmed the above result by in vivo administration of cycloheximide daily post allotransplantation. After the establishment of BALB/c mice skin allograft models, we performed the administration of cycloheximide, rapamycin and the combination of cycloheximide and rapamycin via intraperitoneal injection and then observed the grafts daily.
4. Flow cytometry analysis of the effect of cycloheximide combined with rapamycin
By the use of flow cytometry, we further investigated the immunological effect on alloantigen-reactive CD4+T-cells expansion of the joint administration of cycloheximide and rapamycin. To understand whether cycloheximide resulted in the expansion of Treg, we used the flow cytometry to analyze the frequency of Foxp3Treg cells in the spleens of cycloheximide-treated mice when the control mice developed a donor reactive T-cell response peaked.
Results
1. We found that Pim2mRNA was decreased by cycloheximide in a dose-dependent manner, with a significant effect shown in the group treated with50μM cycloheximide for24h.
2. The result of T cells adoptive transfer assay showed that all of the allograft SCID mice in the control group (T cells treated by50μM DMSO for24h) had severe rejection at day19.0±1.00post cell transfer, while the allograft rejection in the experimental group (T cells treated by50μM cycloheximide for24h) was observed at day26.3±1.53. The result suggested that cycloheximide significantly postponed acute rejection mediated by T cells through blockade of Pim2.
The in vivo administration of cycloheximide also showed that in the allogeneic BABL/c models, control mice rapidly rejected allograft with a median survival time (MST) of12.4±1.10d, whereas treatment with either cycloheximide or rapamycin significantly prolonged survival to more than17.6±0.89d and19.6±1.52d respectively. However, although the combination of cycloheximide and rapamycin has been shown to significantly delay rejection (16.8±1.64), it was no more effective in inducing allograft tolerance than cycloheximide alone. The survival advantage conferred by treatment with cycloheximide provides further evidence for the importance of cycloheximide in preventing alloimmune responses.
3.The results of flow cytometry analysis of the spleen of transplanted and treated mice revealed that the number and the frequency of circulating alloantigen-reactive CD4+T cells was reduced both in mice treated with the combined protocol (from8%to3.2%)and in those treated with rapamycin alone (from8%to2.8%). Nevertheless, treatment with cycloheximide alone in vivo did not led to a substantially reduced accumulation of donor-reactive CD4+T-cells compared with untreated controls(p>0.05), suggesting there may exist another mechanism for cycloheximide to postpone allorejection. Notably, no differences in the total number of CD4+T cells were observed among untreated and treated mice in vitro by either anti-Pim2or4a. Finally, the flow cytometry analyses showed an obvious increase of Foxp3+Treg cells in the spleen of mice treated both with the combined protocol (from4.3%to7.6%) and with rapamycin alone (from4.3%to5.6%). In contrast, we found slight decrease in the frequency of CD4+CD25+Foxp3+Treg cells in mice treated with cycloheximide alone (from4.3%to3.2%) compared with untreated controls. Interestingly, the combination of cycloheximide/rapamycin treatment in vivo led to a dramatic increase in the frequency of CD4+CD25+Foxp3+Treg compared with treatment by rapamycin alone (p<0.05) which has opened a new method for the ex vivo expansion of CD4+CD25+Foxp3+Treg.
Conclusions
1. Pim2was highly expressed in allografted mice at the peak of rejection and the pattern of Pim2expression overlapped with the progression of graft rejection in response to the allogeneic antigen, suggesting that Pim2played important roles in the alloresponse and graft survival and mediated tissue destruction processes during allograft rejection.
2. Blockade of Pim2kinase by4a significantly prolonged the allograft survival through reducing the phosphorylation of apoptotic regulator Bad (Ser112) so as to induce the apoptosis of the alloreactive T cells, indicating that pim2may promote the allograft rejection by inhibiting T-cell apoptosis.
3. Pim2enforced an alloantigen-induced CD4+CD25+Foxp3+T-cell phenotype and promoted the ex vivo expansion of CD4+CD25+Foxp3+T cells. Alloantigen-induced CD4+CD25+T cells appear to display high suppressive activity in the presence of Pim2. Pim2blockade inhibits alloantigen-reactive CD4+CD25+Foxp3+Treg expansion in the presence of rapamycin, indicating that the pim2pathway was partly responsible for the expansion of CD4+CD25+Foxp3+Treg observed in the presence of rapamycin. Collectively, Pim2is indispensable for the ex vivo activation and function of alloantigen-induced CD4+CD25+T cells, which has opened a new method for the ex vivo expansion of CD4+CD25+T cells.
4. Cycloheximide was able to prolong skin allograft survival through inhibition of pim2, with little effect on the expansion of CD4+T cells or CD4+CD25+Foxp3+Treg. However cycloheximide could significantly enhance the ability of rapamycin to expanse CD4+CD25+Foxp3+Treg. Although treatment with the combined protocol led to increased frequency of splenic CD4+CD25+Foxp3+Treg, it was no more efficient in controlling graft rejection than the alone protocol.
Innovations
1. It is the first time to investigate the role of pim2in murine skin allograft models. We confirmed that Pim2gene and protein were highly expressed in allografted mice and pim2may promote the allograft rejection by inhibiting T-cell apoptosis in murine skin allograft models. These results provide a mean to further understand the mechanisms and developing therapeutic approaches to prevent allograft acute rejection.
2. It is the first time to characterize the relationship between pim2kinase and CD4+CD25+Foxp3+Treg expansion. We confirmed that Pim2enforced an alloantigen-induced CD4+CD25+Foxp3+T-cell phenotype and Treg-mediated suppression may be partly related to pim2pathway. Pim2pathway was partly responsible for the expansion of CD4+CD25+Foxp3+Treg observed in the presence of rapamycin. Moreover, cycloheximide could significantly enhance the ability of rapamycin to expanse CD4+CD25+Foxp3+Treg. These results provide a mean to optimize the methods for ex vivo expansion of CD4+CD25+Foxp3+Treg cells and promote Foxp3+Treg-based immunosuppression strategies.
3. The allograft survival was successfully prolonged by the use of4a or Cycloheximide and we found that both of the two drugs prevented the allograft rejection partly through blockade of Pim2kinase. The survival advantage performed by treatment with4a or cycloheximide provides further evidence for the importance of blockade of the pim2pathway in preventing alloimmune responses.
器官移植目前已成为治疗多种终末期疾病的的有效手段。然而发生于移植术后的数周或数月的急性排斥反应仍是导致治疗失败的主要因素。因此器官移植术的成败在很大程度上取决于对急性移植排斥反应的防治。既往研究表明T细胞,尤其是CD4+T细胞介导的细胞免疫应答在移植排斥反应的效应机制中发挥关键作用。T细胞存活的信号通路主要包括AKT(also known as protein kinase B, PKB)-mTOR(mammalian Target of Rapamycin)信号通路和Pim (Proviral integration site of murine)激酶信号通路。既往研究表明,在细胞因子或抗原激活的T细胞信号通路中,T细胞的生长及存活不完全依赖AKT-mTOR通路,阻断AKT-mTOR信号通路虽可部分抑制T细胞激活,但不能完全阻断；Pim激酶信号通路可作为选择性通路。但是有关Pim激酶信号通路在同种反应性T细胞的作用尚未见报道。推测同时阻滞这两条信号通路,有可能更好地抑制移植受者同种反应性T细胞介导的急性排斥,有利于诱导长期移植耐受。己知CD4+T细胞可分为CD4+CD25-效应T细胞和CD4+CD25+Foxp3+调节性T细胞(Treg)两种亚群。其中同种抗原特异性Treg可通过抑制效应T细胞的激活、增殖和促进其凋亡,诱导移植物长期耐受。近来有研究报道Treg细胞可在雷帕霉素(Rapamycin, RAPA)的作用下优势扩增并能很好地保持其抑制功能；而自然调节性Treg中Foxp3基因能够诱导pim2基因的表达。以上研究提示pim2与Treg细胞功能密切相关。本实验室前期研究中,应用SAGE (sequencing-based serial analysis of gene expression,序列基因表达分析)技术建立了同种移植及同系移植组CD4+T细胞mRNA标签库,分析发现pim2基因在同种移植急性排斥组明显高表达,是同系移植组表达量的5倍,进一步提示pim2在同种移植急性排斥中具有重要作用。
Pim家族是原癌基因编码三种丝／苏氨酸激酶,包括Piml, Pim2and Pim3,属于钙调蛋白依赖性蛋白激酶且在序列上具有很高的同源性。pim2基因位于X染色体上(Xpll),编码三种分子量的蛋白质(34kD,37kD和40kD)。Pim激酶缺乏调节结构域,不需磷酸化激活。可能是翻译后组成性激活。Pim激酶由转录翻译和蛋白体降解调控。STAT (Signal transducers and activators of transcription,信号传导及转录激活因子)通过与其同源配体衔接而成为pim基因的转录因子。Pim蛋白激酶活性依赖于mRNA稳定性,Pim通过与SOCS (Suppressor of cytokine signaling,细胞因子信号抑制物)结合从而抑制JAK (Janus Kinase)/STAT信号。多项研究表明Pim激酶通过调控信号转导在许多生理病理过程中发挥重要作用,包括细胞周期、转录因子、细胞凋亡以及细胞代谢和蛋白质翻译。在许多造血系统恶性疾病及实体肿瘤中均发现pim原癌基因的高表达(尤其是Piml和Pim3),并与肿瘤细胞生长存活及化疗药物抵抗密切相关。据报道目前有几种Pim特异小分子抑制剂正用于肿瘤治疗临床前期实验。尽管已有pim2通路在造血系统肿瘤和实体肿瘤中作用的研究报道,但是有关pim2在同种移植急性排斥中的作用及分子机制尚未见报道。Pim2与CD4+T细胞,尤其是Treg密切相关,有可能作为诱导移植免疫耐受的靶向分子；此外Pim2靶向小分子化合物正在研制中,因此Pim2有可能作为同种移植急性排斥诊断和治疗的分子靶点。
综上所述,本论文的科学假设是：pim2可能通过靶向CD4+T细胞的凋亡和调节CD4+Treg增殖及抑制,在同种移植急性排斥反应过程中发挥重要作用。本论文研究目的是试图阐明Pim2激酶在同种移植急性排斥反应中的作用及分子机制,从而为靶向Pim2诱导诱导移植免疫耐受提供理论和实验依据,为临床器官移植排斥的治疗提供新的靶点,因此具有重要的理论和实际意义。
第一部分Pim2在小鼠同种皮肤移植急性排斥反应中的动态表达
研究方法
1、建立小鼠同种皮肤移植模型
无菌条件下将C57BL/6小鼠背部全厚度皮肤移植到BALB/c小鼠背部即为同种移植；将BALB/c小鼠的背部全厚度皮肤移植到BALB/c小鼠背部即为同系移植,以此作为对照组。
2、分析pim2基因在排斥高峰时的表达状况
移植术后14天左右,同种移植皮片完全排斥而同系移植存活完好。利用RT-PCR技术分别检测两组小鼠脾细胞、脾CD4+T细胞及移植皮片中的pim2基因表达。
3、同种排斥组与同系对照组的脾脏和移植皮片的病理学表现以及Pim2蛋白的
原位表达
通过病理组织切片HE染色及抗Pim2抗体对组织切片的免疫组化染色进一步比较同种排斥组与同系对照组的脾脏和移植皮片的病理学表现差异以及Pim2蛋白原位表达差异情况。
4、Pim2蛋白在同种移植排斥反应过程中的动态表达情况
移植后不同时间,取受体小鼠的脾及移植皮片,Western Blot检测Pim2蛋白表达状况。
研究结果
1、pim2mRNA在同种移植组的CD4+T细胞、脾脏和移植皮片中的表达水平均明显高于同系对照组；而且在同种移植组中,pim2mRNA在脾脏中的的表达水平高于移植皮片,但是同系对照组中二者之间无明显差异。结果提示：同种移植急性排斥反应较强时,pim2基因高表达,pim2基因是同种移植急性排斥反应正相关基因。
2、与同系对照组相比,同种排斥组的脾和移植皮片均发生明显组织病理改变,细胞浸润明显；Pim2蛋白高表达于同种排斥组的脾和移植皮片的细胞质中,但同系对照组脾和移植皮片中表达微弱或不表达。结果提示Pim2激酶参与同种急性排斥反应所导致的移植物损害和组织损伤过程。
3、 Western Blot结果显示同种移植术后Pim2蛋白的动态表达趋势与同种移植急性排斥反应的发生过程几乎完全一致,即脾或移植皮片中的Pim2蛋白表达量随着同种急性排斥反应的增强而升高。该结果进一步提示Pim2蛋白激酶参与同种移植急性排斥反应并参与对移植物的排斥。
第二部分抑制pim2基因表达对延长同种移植中移植物存活的影响
第一部分研究证实pim2在同种移植急性排斥期高表达,因此本部分进一步研究抑制Pim2后对同种移植排斥的效应及作用机制。
研究方法
1、确定pim抑制剂4a对pim2抑制的最适浓度和时间
首先制备野生型BALB/c小鼠脾细胞悬液,然后将该脾细胞分别与不同浓度的Pim抑制剂4a混合,培养不同时间,最后利用RT-PCR检测4a处理后脾细胞中pim2基因的表达水平并以pim2mRNA相对灰度值最低来确定Pim抑制剂4a对pim2抑制的最适浓度和时间。
2、T细胞中的pim2被4a抑制后对T细胞介导的同种移植急性排斥的影响将B6小鼠的全厚度皮肤移植到SCID (Severe Combined Immunodeficiency,严重联合免疫缺陷)小鼠背部而建立SCID小鼠移植模型,3周左右移植皮片愈合,生长完好。此时将预先经过4a或DMSO(对照)体外处理的野生型BALB/c小鼠脾脏T细胞通过腹腔注射过继到上述已行移植术的SCID鼠体内,构建同种移植急性排斥反应模型,T细胞过继后,每天观察两组移植皮片的生存状况直至对照组移植皮片排斥。
3、pim2对同种移植急性排斥反应中的同种反应性T细胞的作用及通路
利用T细胞分离柱分离纯化出同种移植排斥高峰期小鼠脾脏T细胞,然后利用Pim特异抑制剂4a体外处理24h,利用流式细胞术分析T细胞的凋亡率。采用WesternBlot方法检测4a对同种反应性T细胞中磷酸化BAD (Ser112)的影响。
4、同种移植急性排斥高峰期脾CD4+CD25-T细胞和CD4+CD25+T细胞中pim2mRNA的表达状况
利用免疫磁珠分选法分离出同种移植排斥高峰期BALB/c小鼠脾CD4+CD25-T细胞和CD4+CD25+T细胞两个亚群,然后利用RT-PCR方法检测两群细胞中pim2mRNA的表达状况。
研究结果
1、pim2mRNA的表达量与4a呈剂量依赖关系,但非时间依赖关系。最终选定5M4a和24h作为4a对pim2抑制的最适浓度和最适作用时间。
2、4a处理组小鼠移植皮片的平均生存时间约为31.2±2.3天,而DMSO对照组小鼠移植皮片的平均生存时间约为19.5±1.7天。与对照组比较,4a处理组同种移植物生存期明显延长约12天(P<0.05),而且其脾脏重量和大小明显减小。结果表明4a通过阻滞T细胞中的pim2,明显延长了同种移植物存活,降低了受体对同种移植物的排斥,提示pim2通过T细胞介导了对同种移植物的急性排斥。
3、4a处理组的T细胞凋亡率明显升高,与对照组有显著差异,P<0.05,提示阻滞T细胞中的pim2,增加了同种反应性T细胞的凋亡；4a处理组的BAD(Ser112)磷酸化水平明显降低,提示4a通过降低Pim2激酶的下游通路BAD (Ser112)磷酸化水平诱导同种反应性T细胞的凋亡,从而部分阻断由同种反应性T细胞介导的针对移植物的特异性免疫应答。结果提示：Pim2通过磷酸化BAD Ser112位点发挥抗同种反应性T细胞凋亡作用,进而介导对同种移植物的急性排斥。
4、在同种移植排斥高峰期,pim2基因在CD4+CD25-T细胞中高表达,约为CD4+CD25+T细胞中pim2基因含量的两倍,提示pim2优势介导同种反应性CD4+CD25-T细胞的存活,从而发挥增强同种移植急性排斥的作用。第三部分Pim2对同种抗原诱导的CD4+CD25+Treg细胞活性的影响前两部分研究证明pim2增强同种移植急性排斥反应,但是结果提示同种移植急性排斥中pim2在CD4+CD25+Treg中也有表达,因此不能排除pim2对CD4+CD25+Treg细胞的影响。为此,本部分研究了pim2对同种抗原诱导的CD4+CD25+Treg细胞活性的影响：
研究方法
1、pim2抑制对CD4+CD25+Treg细胞关键转录因子Foxp3的影响
利用T细胞分离柱纯化分离同种移植急性排斥反应高峰期小鼠脾T细胞,与5M4a混合培养24h后,采用RT-PCR方法检测细胞Foxp3mRNA的变化。
2、pim2抑制对CD4+CD25+T细胞抑制功能的影响
利用免疫磁珠分选方法分离出同种移植排斥高峰期BALB/c小鼠脾脏中的CD4+CD25-T细胞和CD4+CD25+T细胞。先将CD4+CD25+T细胞与4a体外共同培养24h,然后再与CD4+CD25-T细胞按照1：6的比例混合培养12h,最后利用流式细胞术分析CD4+CD25-T细胞的凋亡率。
3、Pim2激酶对雷帕霉素选择性扩增Foxp3+Treg细胞的影响
建立BALB/c小鼠同种移植模型,移植术后第1天开始每天腹腔注射雷帕霉素,放线菌酮,或雷帕霉素+放线菌酮,同时设生理盐水注射对照组。直至对照组小鼠移植皮片发生排斥时停止注射。此时收集不同处理组小鼠脾细胞,利用流式细胞术分析细胞表面的CD4、CD25及细胞质中的Foxp3,以此确定CD4+CD25+Foxp3+Treg所占总细胞比例的变化。随后,将纯化出的雷帕霉素组和联合药物组CD4+CD25+Foxp3+T细胞与Pim2单克隆抗体或4a在体外共培养12h,流式细胞术分析抑制Pim2对CD4+CD25+Foxp3+Treg细胞比例的影响。
研究结果
1、4a在抑制同种反应性T细胞中pim2表达的同时,也降低了Foxp3的基因表达水平,表明阻滞pim2能够降低同种抗原诱导的CD4+CD25+Foxp3+T细胞的增殖活化。结果提示Pim2激酶对同种抗原诱导的CD4+CD25+Foxp3+T细胞的增殖活化是必要的。
2、4a显著降低同种抗原诱导的CD4+CD25+Foxp3+T细胞诱导效应性CD4+CD25-T细胞凋亡的能力,表明阻滞pim2降低了CD4+CD25+Foxp3+T细胞的抑制功能,提示Pim2激酶对同种抗原诱导的CD4+CD25+Foxp3+T细胞的抑制功能也是必要的。
3、与对照组相比,雷帕霉素组和联合药物组小鼠脾CD4+CD25+Foxp3+T细胞比例明显升高,而同种反应性CD4+T细胞比例却显著下调,提示在同种移植模型中,雷帕霉素选择性扩增CD4+CD25+Foxp3+T细胞。抗Pim2单克隆抗体或4a都明显下调了雷帕霉素组和联合药物组CD4+CD25+Foxp3+T细胞的比例。结果提示在同种移植模型中,阻滞Pim2激酶可明显降低CD4+CD25+Foxp3+T细胞在雷帕霉素的作用下的扩增水平,提示Pim2激酶对雷帕霉素选择性扩增同种抗原诱导的CD4+CD25+Foxp3+T细胞同样是必要的。
第四部分放线菌酮抑制同种移植急性排斥及与Pim2的相关性研究鉴于文献报道放线菌酮可以抑制Pim激酶活性,因此推测放线菌酮可能通过抑制Pim激酶抑制急性同种移植排斥。
研究方法
1、放线菌酮对pim2表达的影响
为探索放线菌酮抑制pim2的最适浓度,利用小鼠T细胞分离柱从野生型BALB/c小鼠脾细胞中分离纯化T细胞,分别与不同浓度的放线菌酮混合培养24h,最后利用RT-PCR分析药物处理后T细胞中pim2基因的表达并以pim2mRNA相对灰度值来确定放线菌酮抑制pim2的最适浓度。
2、放线菌酮体外阻滞T细胞pim2后对同种移植物存活的影响
将B6小鼠的背部全厚度皮片移植到SCID小鼠背部建立SCID小鼠移植模型,大约3周后移植皮片愈合生长完好。此时将预先经过放线菌酮或DMSO(对照)体外处理的野生型BALB/c小鼠脾T细胞通过腹腔注射过继到上述己行移植术的SCID鼠体内。构建同种移植急性排斥反应模型,细胞过继后每天观察移植皮片的生存状况。
3、药物体内处理对同种移植物存活的影响
建立BALB/c小鼠同种移植模型,移植术后第1天开始每天腹腔注射放线菌酮、雷帕霉素或雷帕霉素十放线菌酮,直至对照组小鼠移植皮片发生排斥时结束药物注射。每天观察每组移植皮片的生存状况并绘制同种移植皮片生存曲线。
4、放线菌酮延长同种移植物存活的机制
建立BALB/c小鼠同种移植模型,移植术后第1天开始每天腹腔注射放线菌酮、雷帕霉素或雷帕霉素+放线菌酮,直到对照组(腹腔注射生理盐水)小鼠皮片完全被排斥。此时制备脾细胞悬液并采用流式细胞术分析放线菌酮、雷帕霉素或雷帕霉素+放线菌酮体内给药分别对同种反应性CD4+T细胞和CD4+CD25+Foxp3+Treg细胞比例的影响。
研究结果
1、与对照组相比,放线菌酮明显抑制pim2基因的表达,呈剂量依赖性,即50μM的放线菌酮能够更有效地抑制pim2,且无明显的细胞毒性。
2、与对照组相比,放线菌酮处理组的移植皮片延长存活约7天(p<0.05),提示放线菌酮可通过抑制T细胞中的pim2延长同种移植物的存活时间。
3、结果显示,对照组小鼠移植皮片的平均生存时间约为12.4±1.10天,注射放线菌酮组小鼠皮片的平均生存时间约为17.6±0.89天,注射雷帕霉素组小鼠皮片的平均生存时间约为19.6±1.52天,放线菌酮+雷帕霉素联合组小鼠皮片的平均生存时间约为16.8±1.64天,比对照组分别延长约5天,7天和4天,具有显著差异,P<0.05。但是,联合注射组与单独注射放线菌酮组之间无显著差异,P>0.05。提示放线菌酮体内应用明显延长同种移植物的存活时间,但与雷帕霉素联合没有增强效应。
4、与对照组比较,放线菌酮对CD4+T细胞扩增无明显影响(分别为8.0%和8.7%),而雷帕霉素显著降低CD4+T细胞比例(2.8%),雷帕霉素+放线菌酮联合亦可显著降低CD4+T细胞比例(3.2%)；与对照组(4.3%)比较,放线菌酮可降低CD4+CD25+Foxp3+T细胞比例(3.2%),但是无统计学差异,P>0.05；雷帕霉素明显诱导CD4+CD25+Foxp3+T细胞扩增(5.6%)；雷帕霉素+放线菌酮联合显著上调CD4+CD25+Foxp3+T细胞比例(7.6%),且联合应用组CD4+CD25+Foxp3+T细胞的比例明显高于雷帕霉素单独注射组,P<0.05。结果提示在同种移植模型中放线菌酮增强了雷帕霉素优势扩增CD4+CD25+Foxp3+T细胞的能力,而放线菌酮延长同种移植物存活除CD4+T细胞外,可能存在其它分子机制。
全文结论
1.pim2在同种移植急性排斥高峰期高表达,Pim2蛋白的动态表达趋势与同种移植急性排斥反应的发生过程一致,且在脾脏及移植物部位高表达。提示Pim2激酶参与同种移植急性排斥反应并介导移植物损害和组织损伤过程。
2.Pim2通过磷酸化BAD Ser112位点对同种反应性T细胞发挥抗凋亡作用,并优势介导效应性CD4+CD25-T细胞的存活,从而发挥其增强同种移植排斥反应的作用；抑制pim2可明显延长同种移植物的存活时间。
3.Pim2对同种抗原诱导的CD4+CD25+Foxp3+T细胞的增殖活化和发挥抑制功能是必要的；Pim2激酶对雷帕霉素选择性扩增同种抗原诱导的CD4+CD25+Foxp3+T细胞也是必要的。
4.放线菌酮通过抑制pim2可有效延长同种移植物存活,放线菌酮体内应用显著增强雷帕霉素优势扩增CD4+CD25+Foxp3+T细胞的能力。
本文创新点
1.首次利用小鼠同种皮肤移植模型,证实pim2在同种移植模型中高表达并且通过细胞凋亡信号通路增强同种移植急性排斥反应。研究结果为进一步阐明同种移植急性排斥反应的分子机制提供了理论依据。
2.首次证实pim2蛋白对同种抗原诱导的CD4+CD25+Foxp3+Treg细胞的增殖、抑制效应以及在雷帕霉素作用下的优势扩增是必要的；放线菌酮可增强雷帕霉素体内选择性扩增CD4+CD25+Foxp3+Treg细胞的能力。研究结果为CD4+CD25+Foxp3+Treg细胞的体外扩增和细胞治疗提供了新的途径。
3.首次证实pim激酶抑制剂4a和放线菌酮可通过抑制pim2明显延长同种移植物的存活,提示pim2有可能成为同种移植急性排斥诊断和治疗的分子靶点,研究结果为临床靶向Pim2抗移植排斥治疗提供了实验基础和理论依据。
Background and Objectives
Organ transplantation is the most effective therapy for patients with end-stage organ failure. However, acute rejection, which occurs within the first few weeks or months, remains a major cause of treatment failure. T cells, especially CD4+T cells play critical roles in the activation of the immune responses to alloantigens that cause graft rejection. Two survival pathways for T cells have been predominantly targeted: the PI3K/AKT/mTOR (mammalian target of rapamycin) pathway and the PIM pathway. T-cell growth and survival following ligation of cytokine or antigen receptors is mediated, in part, through activation of the effector enzymes AKT and mTOR in the phosphatidylinositol3-kinase pathway and PIM kinases may provide the alternative pathway for T-cell survival. Even through the immunosuppressive therapies that block the PI3K/AK.T/mTOR pathway have been proven to be uniquely effective for anti-allotransplant in clinic, there has been no reports on the effects of Pim pathway blockade in either mice allotransplant models or human transplant recipients. This has led to the speculation that combined blockade of these two pathways might have synergistic effects in inducing immune tolerance, and thus, there has been a long-standing interest in translating about these results to the clinic. The CD4+T cells can be differentiated into various subsets. CD4+CD25+Foxp3+T cells, an important subset of CD4+T cells, can potently suppress the activation or function of conventional CD4+CD25-T effector cells. It has been reported that donor antigen-specific CD4+CD25+Foxp3+regulatory T (Treg) cells can regulate alloresponses and promote donor-specific tolerance in a skin transplantation model. Of the standard immunosuppressive agents that have been combined with Tregs, perhaps the most detailed work has been accomplished with rapamycin. Studies have shown that Tregs are preferentially able to retain their suppressive function in the presence of rapamycin. Interestingly, Foxp3induced pim2expression in natural Tregs, which caused Foxp3-expressing Treg cells to be selected in the presence of rapamycin. These studies suggested that pim2may be associated with the function of Treg cells in allograft rejection. Furthermore, in our previous study, an approximately five-fold overexpression of the pim2gene, as determined by sequencing-based serial analysis of gene expression, was detected in the allograft-activated CD4+T cells, which suggests a potential role of the Pim2kinase in allograft rejection.
The pim genes represent a family of proto-oncogenes, which encode three different serine/threonine protein kinases (piml, pim2and pim3) that are related to the calcium/calmodulin kinase superfamily by weak sequence homology. pim2is an X chromosome gene (Xp11) and transcribe for three isoforms (34kD,37kD, and40kD). Pim kinases lack a regulatory domain and as a result they do not require phosphorylation for activation, and are likely to be constitutively activated following translation. The Pim kinases are regulated by rates of transcription, translation and proteosomal degradation and the STAT proteins serve as transcription factors for the pim genes following engagement of their cognate ligands. The kinase activity of these proteins is proportional to and dependent on mRNA stability, which also serves to inhibit JAK/STAT signaling through binding to the SOCS proteins, which inhibit JAK-dependent signaling. The Pim kinases play essential roles in the regulation of signal transduction cascades, included cell cycle progression, transcription factors, apoptosis and cellular metabolism and protein translation in a large variety of tissues. The pim oncogenes are over-expressed in several hematopoietic malignancies and solid cancers, and promote the cell growth, survival and drug resistance. Pim kinases showed a unique crystal structure and several specific small-molecule inhibitors currently in development are showing interesting preclinical activity in multiple cancer histology. Although pim2pathway can contribute to hematological and solid tumors developing, the physiologic role of this kinase and the precise mechanism by which inhibition of Pim2signals attenuates donor-reactive T-cell response remains uncertain. Considering of the relationship between Pim2and Treg, targeting Pim2may serve as an alternative way to induce allograft tolerance in preclinical models. Moreover, with the continuing development of Pim2-specific small-molecule inhibitors for clinical translation, immunosuppressive therapies that block the Pim2pathway would have proven to be uniquely effective in preclinical allotransplant models.
Considering all of the aforementioned results, we hypothesize that Pim2kinase may participate in allograft rejection through targeting the apoptosis of CD4+T cells and modulating Treg-suppressing activities. In this study, we aimed to investigate the role and the underlying mechanism of Pim2during allograft rejection in murine skin allograft models so as to provide a new target for the diagnosis and therapy of allorejection for clinic.
Part One The expression of Pim2during allograft acute rejection in murine skin allograft models
Methods
1. Establishment of mice skin graft models
In this model, the dorsal skin of C57BL/6mice was allotransplanted onto the BALB/c mice under sterile conditions (allogenic transplantation) and the dorsal skin of BALB/c mice was isotransplanted onto the BALB/c mice (isogenic transplantation) as the control.
2. The pim2gene expression at the peak of allograft rejection analyzed with RT-PCR
At day14post-transplantation (allografts rejected completely, while isografts remained normal), pim2mRNA of splenic CD4+T cells, spleens and skin grafts of different recipient mice was examined by RT-PCR.
3. The expression of Pim2protein in situ analyzed with Immunohistology staining
The difference in the pathology of spleens and skin grafts between allorejected and isografted mice was conducted by the histology analysis. The in situ expression of Pim2protein of spleens and skin grafts from allorejected and isografted mice were conducted by immunohistochemical staining.
4. The dynamic expression of Pim2protein post allotransplantation analyzed with Western Blotting
The dynamic expression of Pim2protein in spleens and grafted skins at various times post-allotransplantation was also confirmed using Western Blotting.
Results
1. It was found that the expression of pim2mRNA in CD4+T cells was significantly higher both in the allografted mice and rapamycin-treated allograft mice with the RT-PCR analysis, but rapamycin treatment in vivo did not significantly change the expression of pim2in allograft mice. Our results also showed that pim2mRNA of spleens and skin grafts was significantly elevated in allograft mice compared with that from isografted mice. Moreover, in allograft mice, pim2mRNA was markedly higher in the spleens than in the skin grafts, while in isografted mice, there was no significant difference between these two tissues. These data indicated that pim2gene was over-expressed when the allorejection reached the peak, suggesting that pim2gene serves as a positively related to allorejection.
2. The histology observation of spleens and skin grafts from allorejected and isografted mice showed an obvious difference in the pathology between the two groups. By immunohistochemical staining, we found that Pim2protein was markedly expressed in the cytoplasm of allografted skins and spleens, but only weakly or negatively expressed in isografted skins and spleens, suggesting that Pim2was responsible for the immune-mediated tissue destruction processes.
3. The immunoblotting results showed that Pim2protein was upregulated at day7in the spleens of recipient mice and reached the highest levels at day10. The accumulation of Pim2in allograft skins showed the same tendency as in the spleens, except that upregulation occurred at day10and that the highest level observed was at day14. This pattern of Pim2expression overlapped with the progression of graft rejection in response to the allogeneic antigen, indicating that Pim2kinase involved in the alloresponse and allograft rejection.
Part Two The role and mechanism of Pim2in allograft acute rejection
As shown in part one, pim2gene and protein were overexpressed during allograft rejection. Then, we further investigate how pim2work during the allorejection.
Methods
1. The determination of a suitable concentration and time-points for4a The spleen cells of naive B ALB/c mice were treated with different concentration of4a for different time, followed by pim2mRNA analyzed with RT-PCR
2. The effect of inhibiting Pim2kinase on allograft survival
We established the SCID mouse skin acute allograft rejection model by allografting the skin of B6mice to the SCID mice. After the wounds healed3weeks later, the alloimmune response was then reconstituted with the adoptive transfer of naive BABL/c splenic T cells treated with4a for24h or with such cells treated with DMSO as a control. The grafts were observed daily after the removal of the bandages until that the graft in control group was completely rejected.
3. The potential effect of Pim2inhibiting on the apoptosis of alloreactive T cells
T cells were purified with Mouse T cell Enrichment Columns from recipient spleens at the height of allorejection, co-cultured with4a for24h and then were subjected to apoptosis analysis by flow cytometry. In addition, the phosphorylation of Bad (Ser112), a Pim2kinase substrate and apoptotic regulator, was detected by western blot in T cells treated by4a.
4. The gene expression of Pim2in different CD4+T-cell subsets
Based on the finding that pim2was overexpressed in unfractionated CD4+T cells during acute allograft rejection, we analyzed the gene expression of Pim2in different CD4+T-cell subsets. After that CD4+CD25-T cells and CD4+CD25+T cells were purified and collected with MagCellect Mouse CD4+CD25+Regulatory T Cell Isolation Kit, from recipient spleens at the height of allorejection, pim2expression was detected by RT-PCR separately.
Results
1. Firstly, we performed RT-PCR and found that pim2mRNA was decreased in a dose-dependent manner, with a significant effect shown in the group treated with5μM4a but that little change was observed among the different treatment time points. So5μM4a and24h were chosen as the suitable concentration and time for4a.
2. The T cells adoptive transfer assay showed that all of the mice in the control group (T cells treated by5μM DMSO for24h) had severe rejection at day19.5±1.7post cell transfer, while the allograft rejection in the experimental group (T cells treated by5μM4a for24h) was observed at day31±2.3. At same time, we found that there was a two-fold decrease in spleen weight compared with the control group. Therefore, blockade of Pim2significantly postponed acute rejection mediated by T cells.
3. The result demonstrated a notable increase in apoptotic cells in the4a-treated group. In addition, the phosphoryla-tion of Bad (Ser112), a Pim2kinase substrate and apoptotic regulator, was significantly reduced in the4a-treated group. These results suggested that the absence of Pim2activity increased the level of T-cell apoptosis by reducing the phosphorylation of apoptotic regulator Bad (Ser112) during allograft rejection, which indicates that pim2may promote the allograft rejection by inhibiting T-cell apoptosis.
4. By RT-PCR analysis of Pim2expression in different CD4+T-cell subsets during acute allograft rejection, we found that expression of pim2mRNA in CD4+CD25-T cells was2.66-fold higher than that in CD4+CD25+T cells, indicating that Pim2kinase more strongly promoted CD4+CD25-T-cell survival.
Part Three The effect of Pim2on the alloantigen-induced CD4+CD25+T cells
Considering the negative regulation of CD4+CD25+T cells observed during allorejection and the expression of pim2gene in alloantigen-induced CD4+CD25+T cells as shown in part two, we further investigated the effect of Pim2on this subset.
Methods
1. The effect of Pim2blocking on Foxp3gene expression analyzed with RT-PCR Gene expression of Foxp3in splenic T cells isolated from the allograft mice and treated with different concentration of4a for24h was analyzed by RT-PCR.
2. The effect of Pim2blocking on the function of alloantigen-induced CD4+CD25+T cells analyzed by Flow cytometry
Next, we explored whether Pim2was required for alloantigen-induced CD4+CD25+T-cell suppression activity. CD4+CD25-T cells and CD4+CD25+T cells were purified from the freshly isolated spleens of allograft mice with rejection. CD4+CD25+T cells treated with or without4a for24h were cocultured with CD4+CD25-T cells for12h at a ratio of1:6separately, and then examined for cell apoptosis by flow cytometric analysis.
3. The effect of Pim2blocking on the preferential expansion of alloantigen-induced Tregs in the presence of rapamycin analyzed by Flow cytometry
Since rapamycin selectively allowed for proliferation and fostering the suppressive function of Foxp3+Treg cells, next we explored whether Pim2kinase contributed to the observed an enrichment of circulating CD4+CD25+Foxp3+Treg in the presence of rapamycin. Flow cytometry of the surface expression of CD4, CD25and Foxp3on spleen cells from rapamycin-treated allografted mice was conducted after stimulated for12h with anti-pim2or with4a.
Results
1. By analyzing the expression of Foxp3and pim2in splenic T cells isolated from the allograft mice and treated with5or10μM4a for24h by RT-PCR, we found that4a treatment resulted in the significant decrease of Foxp3and pim2expression. Since Foxp3is the key transcriptional factor of CD4+CD25+T cells, the result showed that blockage of pim2by4a reduced the proliferation of alloantigen induced CD4+CD25+T cells, indicating that Pim2is indispensable for the ex vivo activation of alloantigen-induced CD4+CD25+T cells.
2. The results of flow cytometric analysis showed that the alloantigen induced CD4+CD25+T cells treated with5μM4a led to an obvious decrease in the percentage of alloantigen induced CD4+CD25-T-cell apoptosis compared with those untreated with4a, suggesting that the blocking of Pim2impaired the function of CD4+CD25+T cells to induce CD4+CD25-T-cell apoptosis. The data indicated that Pim2is indispensable for the ex vivo function of alloantigen-induced CD4+CD25+T cells.
3. The results of flow cytometric analysis showed that treatment with either anti-pim2or4a led to a substantially reduced expansion of the observed enrichment of CD4+CD25+Foxp3+Treg in the presence of rapamycin alone or combined with cycloheximide, suggesting that pim2allowed alloantigen-induced CD4+CD25+T cells to evade many rapamycin-imposed signaling blocks and to expand preferentially.
Part Four Cycloheximide served as an immunosuppressive drug to prevent allorejection through the pim2pathway
Considering that cycloheximide could inhibit pim2to function in anti-apoptosis, and the blockade of Pim2prolonged skin graft survival through the apoptosis regulation pathway in our previous study, we hypothesize that cycloheximide may prolong skin graft survival.'
Methods
1. The effect of cycloheximide in inhibiting pim2in vitro analyzed by RT-PCR For determination of a suitable concentration of cycloheximide, spleen T cells from naive BALB/c mice were treated with different concentrations of cycloheximide for24h, followed by RT-PCR analysis of pim2mRNA.
2. The effect of blocking pim2by cycloheximide on the allograft survival We established the SCID mouse skin acute allograft rejection model by allografting the skin of B6mice to the SCID mice. After the wounds healed3weeks later, the alloimmune response was then reconstituted with the adoptive transfer of naive BABL/c splenic T cells treated with cycloheximide for24h or with such cells treated with DMSO as a control. The grafts were observed daily after the removal of the bandages until that the graft in control group was completely rejected.
3. The effect of cycloheximide administrated in vivo on the allograft survival
We also confirmed the above result by in vivo administration of cycloheximide daily post allotransplantation. After the establishment of BALB/c mice skin allograft models, we performed the administration of cycloheximide, rapamycin and the combination of cycloheximide and rapamycin via intraperitoneal injection and then observed the grafts daily.
4. Flow cytometry analysis of the effect of cycloheximide combined with rapamycin
By the use of flow cytometry, we further investigated the immunological effect on alloantigen-reactive CD4+T-cells expansion of the joint administration of cycloheximide and rapamycin. To understand whether cycloheximide resulted in the expansion of Treg, we used the flow cytometry to analyze the frequency of Foxp3Treg cells in the spleens of cycloheximide-treated mice when the control mice developed a donor reactive T-cell response peaked.
Results
1. We found that Pim2mRNA was decreased by cycloheximide in a dose-dependent manner, with a significant effect shown in the group treated with50μM cycloheximide for24h.
2. The result of T cells adoptive transfer assay showed that all of the allograft SCID mice in the control group (T cells treated by50μM DMSO for24h) had severe rejection at day19.0±1.00post cell transfer, while the allograft rejection in the experimental group (T cells treated by50μM cycloheximide for24h) was observed at day26.3±1.53. The result suggested that cycloheximide significantly postponed acute rejection mediated by T cells through blockade of Pim2.
The in vivo administration of cycloheximide also showed that in the allogeneic BABL/c models, control mice rapidly rejected allograft with a median survival time (MST) of12.4±1.10d, whereas treatment with either cycloheximide or rapamycin significantly prolonged survival to more than17.6±0.89d and19.6±1.52d respectively. However, although the combination of cycloheximide and rapamycin has been shown to significantly delay rejection (16.8±1.64), it was no more effective in inducing allograft tolerance than cycloheximide alone. The survival advantage conferred by treatment with cycloheximide provides further evidence for the importance of cycloheximide in preventing alloimmune responses.
3.The results of flow cytometry analysis of the spleen of transplanted and treated mice revealed that the number and the frequency of circulating alloantigen-reactive CD4+T cells was reduced both in mice treated with the combined protocol (from8%to3.2%)and in those treated with rapamycin alone (from8%to2.8%). Nevertheless, treatment with cycloheximide alone in vivo did not led to a substantially reduced accumulation of donor-reactive CD4+T-cells compared with untreated controls(p>0.05), suggesting there may exist another mechanism for cycloheximide to postpone allorejection. Notably, no differences in the total number of CD4+T cells were observed among untreated and treated mice in vitro by either anti-Pim2or4a. Finally, the flow cytometry analyses showed an obvious increase of Foxp3+Treg cells in the spleen of mice treated both with the combined protocol (from4.3%to7.6%) and with rapamycin alone (from4.3%to5.6%). In contrast, we found slight decrease in the frequency of CD4+CD25+Foxp3+Treg cells in mice treated with cycloheximide alone (from4.3%to3.2%) compared with untreated controls. Interestingly, the combination of cycloheximide/rapamycin treatment in vivo led to a dramatic increase in the frequency of CD4+CD25+Foxp3+Treg compared with treatment by rapamycin alone (p<0.05) which has opened a new method for the ex vivo expansion of CD4+CD25+Foxp3+Treg.
Conclusions
1. Pim2was highly expressed in allografted mice at the peak of rejection and the pattern of Pim2expression overlapped with the progression of graft rejection in response to the allogeneic antigen, suggesting that Pim2played important roles in the alloresponse and graft survival and mediated tissue destruction processes during allograft rejection.
2. Blockade of Pim2kinase by4a significantly prolonged the allograft survival through reducing the phosphorylation of apoptotic regulator Bad (Ser112) so as to induce the apoptosis of the alloreactive T cells, indicating that pim2may promote the allograft rejection by inhibiting T-cell apoptosis.
3. Pim2enforced an alloantigen-induced CD4+CD25+Foxp3+T-cell phenotype and promoted the ex vivo expansion of CD4+CD25+Foxp3+T cells. Alloantigen-induced CD4+CD25+T cells appear to display high suppressive activity in the presence of Pim2. Pim2blockade inhibits alloantigen-reactive CD4+CD25+Foxp3+Treg expansion in the presence of rapamycin, indicating that the pim2pathway was partly responsible for the expansion of CD4+CD25+Foxp3+Treg observed in the presence of rapamycin. Collectively, Pim2is indispensable for the ex vivo activation and function of alloantigen-induced CD4+CD25+T cells, which has opened a new method for the ex vivo expansion of CD4+CD25+T cells.
4. Cycloheximide was able to prolong skin allograft survival through inhibition of pim2, with little effect on the expansion of CD4+T cells or CD4+CD25+Foxp3+Treg. However cycloheximide could significantly enhance the ability of rapamycin to expanse CD4+CD25+Foxp3+Treg. Although treatment with the combined protocol led to increased frequency of splenic CD4+CD25+Foxp3+Treg, it was no more efficient in controlling graft rejection than the alone protocol.
Innovations
1. It is the first time to investigate the role of pim2in murine skin allograft models. We confirmed that Pim2gene and protein were highly expressed in allografted mice and pim2may promote the allograft rejection by inhibiting T-cell apoptosis in murine skin allograft models. These results provide a mean to further understand the mechanisms and developing therapeutic approaches to prevent allograft acute rejection.
2. It is the first time to characterize the relationship between pim2kinase and CD4+CD25+Foxp3+Treg expansion. We confirmed that Pim2enforced an alloantigen-induced CD4+CD25+Foxp3+T-cell phenotype and Treg-mediated suppression may be partly related to pim2pathway. Pim2pathway was partly responsible for the expansion of CD4+CD25+Foxp3+Treg observed in the presence of rapamycin. Moreover, cycloheximide could significantly enhance the ability of rapamycin to expanse CD4+CD25+Foxp3+Treg. These results provide a mean to optimize the methods for ex vivo expansion of CD4+CD25+Foxp3+Treg cells and promote Foxp3+Treg-based immunosuppression strategies.
3. The allograft survival was successfully prolonged by the use of4a or Cycloheximide and we found that both of the two drugs prevented the allograft rejection partly through blockade of Pim2kinase. The survival advantage performed by treatment with4a or cycloheximide provides further evidence for the importance of blockade of the pim2pathway in preventing alloimmune responses.
引文
1. Issa F, Schiopu A, Wood KJ. Role of T cells in graft rejection and transplantation tolerance. Expert Rev Clin Immunol.2010; 6:155-169.
2. Rocha PN, Plumb TJ, Crowley SD, Coffman TM. Effector mechanisms in transplant rejection. Immunol Rev.2003; 196:51-64.
3. Thompson P, Badell IR, Lowe M, et al. Alternative immunomodulatory strategies for xenotransplantation:CD40/154 pathway-sparing regimens promote xenograft survival. Am J Transplant.2012 Jul; 12(7):1765-75.
4. Lowe M, Badell IR, Thompson P, et al. A novel monoclonal antibody to CD40 prolongs islet allograft survival. Am J Transplant.2012 Aug; 12(8):2079-87.
5. Ferrer IR, Wagener ME, Song M, et al. Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade. Proc Natl Acad Sci U S A.2011 Dec 20; 108(51):20701-6.
6. Fox CJ, Hammerman PS, Thompson CB. The Pim kinases control rapamycin-resistant T cell survival and activation. J Exp Med.2005 Jan 17; 201(2):259-66.
7. Huttmann A, Klein-Hitpass L, Thomale J, et al. Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status. Leukemia.2006; 20(10):1774-82.
8. Hammerman PS, Fox CJ, Birnbaum MJ, Thompson CB. Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. Blood.2005 Jun 1; 105(11):4477-83.
9. Ayala GE, Dai H, Ittmann M, Li R, et al. Growth and survival mechanisms associated with perineural invasion in prostate cancer. Cancer Res.2004 Sep 1; 64(17):6082-90.
10. Dai H, Li R, Wheeler T, Diaz de Vivar A, et al. Pim-2 upregulation:biological implications associated with disease progression and perinueral invasion in prostate cancer. Prostate.2005 Nov 1; 65(3):276-86.
11. Gong J, Wang J, Ren K, et al. Serine/threonine kinase Pim-2 promotes liver tumorigenesis induction through mediating survival and preventing apoptosis of liver cell. J Surg Res.2009 May 1; 153(1):17-22.
12. Aho TL, Lund RJ, Ylikoski EK, et al. Expression of human pim family genes is selectively up-regulated by cytokines promoting T helper type 1, but not T helper type 2, cell differentiation. Immunology.2005 Sep; 116(1):82-8.
13. Xu J, Wang D, Zhang C, et al. Alternatively expressed genes identified in the CD4+ T cells of allograft rejection mice. Cell Transplant.2011; 20(2):333-50.
14. Woodland RT, Fox CJ, Schmidt MR, et al. Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival. Blood.2008 Jan 15; 111(2):750-60.
15. Spivey TL, Uccellini L, Ascierto ML, et al. Gene expression profiling in acute allograft rejection:challenging the immunologic constant of rejection hypothesis. J Transl Med.2011 Oct 12; 9:174.
16. llet N, Dieude'M,CailhierJ,He'bert M. The molecular legacy of apoptosis in transplantation. Am J Transplant.2012; 12:1378-1384.
17. Allen JD, Verhoeven E, Domen J, et al. Pim-2 transgene induces lymphoid tumors, exhibiting potent synergy with c-myc. Oncogene.1997 Sep 4;15(10):1133-41.
18. Breuer ML, Cuypers HT, Berns A. Evidence for the involvement of pim-2, a new common proviral insertion site, in progression of lymphomas. EMBO J.1989 Mar; 8(3):743-8.
19. Mizuki M, Schwable J, Steur C, et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific F1t3 mutations. Blood. 2003; 101(8):3164-73.
20. Tamburini J, Green AS, Bardet V, et al. Protein synthesis is resistant to rapamycin and constitutes a promising therapeutic target in acute myeloid leukemia. Blood. 2009 Aug 20; 114(8):1618-27.
21. Cohen AM, Grinblat B, Bessler H, et al. Increased expression of the hPim-2 gene in human chronic lymphocytic leukemia and non-Hodgkin lymphoma. Leuk Lymphoma.2004 May; 45(5):951-5.
22. Huttmann A, Klein-Hitpass L, Thomale J, et al. Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status. Leukemia.2006 Oct; 20(10):1774-82.
23. Li J, Peet GW, Balzarano D, et al. Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition. J Biol Chem.2001 May 25;276(21):18579-90.
24. Ayala GE, Dai H, Ittmann M, et al. Growth and survival mechanisms associated with perineural invasion in prostate cancer. Cancer Res.2004 Sep 1; 64(17):6082-90.
25. Wang Z, Bhattacharya N, Weaver M, et al. Pim-1:a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis. J Vet Sci. 2001 Dec; 2(3):167-79.
26. Lilly M, Sandholm J, Cooper JJ, et al. The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway. Oncogene.1999 Jul 8; 18(27):4022-31.
27. Yan B, Zemskova M, Holder S, et al. The PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD-induced cell death. J Biol Chem.2003 Nov 14; 278(46):45358-67.
28. Aho TL, Sandholm J, Peltola KJ, et al. Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site. FEBS Lett.2004 Jul 30; 571(1-3):43-9.
29. Fox CJ, Hammerman PS, Cinalli RM, et al. The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes Dev.2003 Aug 1; 17(15):1841-54.
30. Macdonald A, Campbell DG, Toth R, et al. Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL. BMC Cell Biol.2006 Jan 10; 7:1.
31. Zhang F, Beharry ZM, Harris TE, et al. PIM1 protein kinase regulates PRAS40 phosphorylation and mTOR activity in FDCP1 cells. Cancer Biol Ther.2009 May; 8(9):846-53.
32. Bullock AN, Russo S, Amos A, et al. Crystal structure of the PIM2 kinase in complex with an organoruthenium inhibitor. PLoS One.2009 Oct 20; 4(10):e7112.
33. Bullock AN, Debreczeni J, Amos AL, et al. Structure and substrate specificity of the Pim-1 kinase. J Biol Chem.2005 Dec 16; 280(50):41675-82.
34. Kumar A, Mandiyan V, Suzuki Y, et al. Crystal structures of proto-oncogene kinase Piml:a target of aberrant somatic hypermutations in diffuse large cell lymphoma. J Mol Biol.2005 Apr 22; 348(1):183-93.
35. Jacobs MD, Black J, Futer O, et al. Pim-1 ligand-bcund structures reveal the mechanism of serine/threonine kinase inhibition by LY294002. J Biol Chem.2005; 280(14):13728-34.
36. Qian KC, Wang L, Hickey ER, et al. Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase. J Biol Chem.2005 Feb 18;280(7):6130-7.
37. Gourley E, Liu XH, Lamb J et al. A potent small molecule PIM kinase inhibitor with activity in cell lines from hematological and solid malignancies. AACR Meeting Abstracts 2008,4974.
38. Chen LS, Redkar S, Bearss D, Wierda WG, Gandhi V. PIM kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells. Blood.2009; 114(19),4150-4157.
39. Mumenthaler SM, Ng PY, Hodge A et al. Pharmacologic inhibition of PIM kinases alters prostate cancer cell growth and resensitizes chemoresistant cells to taxanes. Mol. Cancer Ther.2009; 8(10),2882-2893.
40. Kelly KR, Espitia CM, Taverna P et al. Targeting PIM kinase activity significantly augments the efficacy of cytarabine. Br. J. Haematol.2012; 156(1),129-132.
41. Natarajan K, Burcu M, Baer MR. Inhibition of the serine/threonine kinase PIM-1 has anti-proliferative effects in acute myeloid leukemia (AML) and sensitizes multidrug resistant cells to chemotherapy. ASH Annual Meeting Abstracts 2010; 116(21),1832.
42. Herreros B, Bischoff JR, Rodriguez ME et al. Pharmacological inhibition of PIM kinases in chronic lymphocytic leukemia cases with unfavorable prognosis markers. ASH Annual Meeting Abstracts.2010; 116(21),2468.
43. Lin YW, Beharry ZM, Hill EG et al. A small molecule inhibitor of PIM protein Kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. Blood.2010; 115(4),824-833.
44. Beharry Z, Mahajan S, Zemskova M et al. The PIM protein kinases regulate energy metabolism and cell growth. Proc. Natl Acad. Sci. USA 2011; 108(2), 528-533.
45. Lin Y, Graham AM, Costa LJ et al. Dual inhibition of mTOR and PIM kinase pathways using small molecule inhibitors synergistically kills myeloid leukemic cells in vitro and in vivo. ASH Annual Meeting Abstracts.2010; 116(21),293.
46. Ebens AJ, Berry L, Chen YH et al. A selective PIM kinase inhibitor is highly active in multiple myeloma:the biology of single agent and PI3K/AKT/mTOR combination activity. ASH Annual Meeting Abstracts.2011; 116(21),3001.
47. O'Brien S, Haddach M, Borsan C et al. Discovery of selective small molecule pan-PIM kinase inhibitors with potent oral efficacy in murine xenograft models. ASH Annual Meeting Abstracts.2009; 114(22),1726.
48. Bamett AL, Ding S, Murray C et al. Anti-tumor activity of CXR1002, a novel anti-cancer compound that induces ER stress:human tumor xenograft efficacy and in vitro mode of action. Program and Abstracts of the AACR Meeting.2010.
49. hang M, Kan war N, Feng E, et al. PIM kinase inhibitors downregulate STAT3(Tyr705) phosphorylation. Mol. Cancer Ther.2010; 9(9),2478-2487.
50. Tao ZF, Hasvold LA, Leverson JD, et al. Discovery of 3H-benzo[4,5] thieno[3,2-d]pyrimidin-4-ones as potent, highly selective, and orally bioavailable inhibitors of the human protooncogene proviral insertion site in Moloney murine leukemia virus (PIM) kinases. J. Med. Chem.2009; 52(21),6621-6636.
51.Beharry Z, Zemskova M, Mahajan S, et al. Novel benzylidene-thiazolidine-2,4-diones inhibit Pirn protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells. Mol Cancer Ther.2009 Jun;8(6):1473-83.
52. Basu S, Golovina T, Mikheeva T, June CH, Riley JL. Foxp3-mediated induction of pim2 allows human T regulatory cells to preferentially expand in rapamycin. J Immunol.2008; 180:5794-5798.
53. Learn CA, Fecci PE, Schmittling RJ, Xie W, Karikaril, Mitchell DA, et al. Profiling of CD4+, CD8+, and CD4+CD25+CD45RO+FoxP3+T cells in patients with malignant glioma reveals differential expression of the immunologic transcriptome compared with T cells from healthy volunteers. Clin Cancer Res 2006; 12:7306-7315.
54. Sugimoto N, Oida T, Hirota K, Nakamura K, Nomura T, Uchiyama T, et al. Foxp3-dependent and-independent molecules specific for CD25+CD4+natural regulatory T cells revealed by DNAmicroarray analysis. Int Immunol 2006; 18:1197-1209.
55. Feldman JD, Vician L, Crispino M, et al. KID-1, a protein kinase induced by depolarization in brain. J Biol Chem.1998 Jun 26;273(26):16535-43.
56. Bullock AN, Debreczeni JE, Fedorov OY, et al. Structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM-1) kinase. J Med Chem.2005 Dec 1;48(24):7604-14.
57. Koyasu, S. The role of PI3K in immune cells. Nat. Immunol.2003;4:313-319.
58. Cho, H., J. Mu, J.K. Kim, et al. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science.2001; 292:1728-1731.
59. Cho, H., J.L. Thorvaldsen, Q. Chu, et al. Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J. Biol. Chem.2001; 276:38349-38352.
60. Sehgal, S.N. Rapamune (RAPA, rapamycin, sirolimus):mecha-nism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin. Biochem.1998; 31:335-340.
61. Mikkers H, Nawijn M, Allen J, et al. Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol Cell Biol.2004 Jul;24(13):6104-15.
62. Alvarado Y, Giles FJ, Swords RT. The PIM kinases in hematological cancers. Expert Rev Hematol.2012 Feb; 5(1):81-96.
63.Macintyre AN, Finlay D, Preston G, Sinclair LV, Waugh CM, Tamas P et al Protein kinase B controls transcriptional programs that directcytotoxic T cell fate but is dispensable for T cell metabolism. Immunity 2011; 34:224-236.
64. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science.2003 Feb 14;299(5609):1057-61.
65. Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995 Aug 1; 155(3):1151-64.
66. Ono M, Yaguchi H, Ohkura N, et al. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1. Nature.2007 Apr 5;446(7136):685-9.
67. Kaser T, Gerner W, Hammer SE, et al. Phenotypic and functional characterisation of porcine CD4(+)CD25(high) regulatory T cells. Vet Immunol Immunopathol. 2008 Mar 15;122(1-2):153-8.
68. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol.2003 Apr;4(4):330-6.
69. D'Cruz LM, Klein L. Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nat Immunol.2005 Nov;6(11):1152-9.
70. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol.2005 Nov;6(11):1142-51.
71.Maloy KJ, Powrie F. Fueling regulation:IL-2 keeps CD4+Treg cells fit. Nat Immunol.2005 Nov;6(11):1071-2.
72. Zippo A, De Robertis A, Bardelli M, et al. Identification of Flk-1 target genes in vasculogenesis:Pim-1 is required for endothelial and mural cell differentiation in vitro. Blood.2004 Jun 15;103(12):4536-44.
73. Losman J, Chen XP, Jiang H, et al. IL-4 signaling is regulated through the recruitment of phosphatases, kinases, and SOCS proteins to the receptor complex. Cold Spring Harb Symp Quant Biol.1999;64:405-16.
74. Chen XP, Losman JA, Cowan S, et al. Pim serine/threonine kinases regulate the stability of Socs-1 protein. Proc Natl Acad Sci U S A.2002 Feb 19;99(4):2175-80.
75. Peltola KJ, Paukku K, Aho TL, et al. Pim-1 kinase inhibits STAT5-dependent transcription via its interactions with SOCS1 and SOCS3. Blood.2004 May 15;103(10):3744-50.
76. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol.2003 Apr;4(4):330-6.
77. Hori, S., Nomura, T. and Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science.2003; 299:1057-1061.
78. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol.2003 Apr;4(4):337-42.
79. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol.2005 Nov;6(11):1142-51.
80. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol.2001 Mar 15;166(6):3789-96.
81. Graca L, Cobbold SP, Waldmann H. Identification of regulatory T cells in tolerated allografts. J Exp Med.2002 Jun 17;195(12):1641-6.
82. Tang Q, Adams JY, Tooley AJ, et al. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol.2006 Jan;7(1):83-92.
83. Mempel TR, Pittet MJ, Khazaie K, et al. Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity.2006 Jul;25(1):129-41.
84. Carvalho-Gaspar M, Jones ND, Luo S, et al. Location and time-dependent control of rejection by regulatory T cells culminates in a failure to generate memory T cells. J Immunol.2008 May 15;180(10):6640-8.
85. Pandiyan P, Zheng L, Ishihara S, et al. CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nat Immunol.2007 Dec;8(12):1353-62.
86. Bluestone JA. Regulatory T-cell therapy:is it ready for the clinic? Nat Rev Immunol.2005 Apr;5(4):343-9.
87. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood.2005 Jun 15;105(12):4743-8.
88. Battaglia M, Stabilini A, Migliavacca B, et al. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol.2006 Dec 15;177(12):8338-47.
89. Coenen JJ, Koenen HJ, van Rijssen E, et al. Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27+subset of human CD4+CD25+regulatory T cells. Blood.2006 Feb 1; 107(3):1018-23.
90. Zeiser R, Leveson-Gower DB, Zambricki EA, et al. Differential impact of mammalian target of rapamycin inhibition on CD4+CD25+Foxp3+regulatory T cells compared with conventional CD4+ Tcells. Blood.2008 Jan 1;111(1):453-62.
91. Strauss L, Whiteside TL, Knights A, et al. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin. J Immunol.2007 Jan 1;178(1):320-9.
92. Obrig TG, Culp WJ, McKeehan WL, Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Biol Chem.1971 Jan 10;246(1):174-81.
93. Hershko DD, Robb BW, Wray CJ, et al. Superinduction of IL-6 by cycloheximide is associated with mRNA stabilization and sustained activation of p38 map kinase and NF-kappaB in cultured caco-2 cells. J Cell Biochem.2004 Apr 1:91(5):951-61.
94. Ogura H, Tsukumo Y, Sugimoto H, et al. ERK and p38 MAP kinase are involved in downregulation of cell surface TNF receptor 1 induced by acetoxycycloheximide. Int Immunopharmacol.2008 Jun;8(6):922-6.
95. Kim do K, Hwang CK, Wagley Y, et al. p38 mitogen-activated protein kinase and PI3-kinase are involved in up-regulation of mu opioid receptor transcription induced by cycloheximide. J Neurochem.2011 Mar; 116(6):1077-87.
96. Shih SR, Weng KF, Stollar V, Li ML. Viral protein synthesis is required for Enterovirus 71 to induce apoptosis in human glioblastoma cells. J Neurovirol. 2008 Jan;14(1):53-61.
97. Eriksson D, Lofroth PO, Johansson L, et al. Apoptotic signalling in HeLa Hep2 cells following 5 Gy of cobalt-60 gamma radiation. Anticancer Res.2009 Nov;29(11):4361-6.
98. Liu DX, Greene LA. Regulation of neuronal survival and death by E2F-dependent gene repression and derepression. Neuron.2001 Nov 8;32(3):425-38.
99. Alessenko AV, Boikov PYa, Filippova GN, et al. Mechanisms of cycloheximide-induced apoptosis in liver cells. FEBS Lett.1997 Oct 13；416(1):113-6.
100. Ito K, Kiyosawa N, Kumagai K, et al. Molecular mechanism investigation of cycloheximide-induced hepatocyte apoptosis in rat livers by morphological and microarray analysis. Toxicology.2006 Feb 15;219(1-3):175-86.
101. Blom WM, de Bont HJ, Meijerman I, et al. Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors. Biochem Pharmacol.1999 Dec 15;58(12):1891-8.
102. Zhao X, Cao M, Liu JJ, et al. Reactive oxygen species is essential for cycloheximide to sensitize lexatumumab-induced apoptosis in hepatocellular carcinoma cells. PLoS One.2011 Feb 10;6(2):e16966.
103. Liu X, Yang JM, Zhang SS, et al. Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide. BMC Cancer.2010 Dec 15; 10:684.
104. Edinger AL, Thompson CB. Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol Biol Cell.2002 Jul;13(7):2276-88.
2. Rocha PN, Plumb TJ, Crowley SD, Coffman TM. Effector mechanisms in transplant rejection. Immunol Rev.2003; 196:51-64.
3. Thompson P, Badell IR, Lowe M, et al. Alternative immunomodulatory strategies for xenotransplantation:CD40/154 pathway-sparing regimens promote xenograft survival. Am J Transplant.2012 Jul; 12(7):1765-75.
4. Lowe M, Badell IR, Thompson P, et al. A novel monoclonal antibody to CD40 prolongs islet allograft survival. Am J Transplant.2012 Aug; 12(8):2079-87.
5. Ferrer IR, Wagener ME, Song M, et al. Antigen-specific induced Foxp3+ regulatory T cells are generated following CD40/CD154 blockade. Proc Natl Acad Sci U S A.2011 Dec 20; 108(51):20701-6.
6. Fox CJ, Hammerman PS, Thompson CB. The Pim kinases control rapamycin-resistant T cell survival and activation. J Exp Med.2005 Jan 17; 201(2):259-66.
7. Huttmann A, Klein-Hitpass L, Thomale J, et al. Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status. Leukemia.2006; 20(10):1774-82.
8. Hammerman PS, Fox CJ, Birnbaum MJ, Thompson CB. Pim and Akt oncogenes are independent regulators of hematopoietic cell growth and survival. Blood.2005 Jun 1; 105(11):4477-83.
9. Ayala GE, Dai H, Ittmann M, Li R, et al. Growth and survival mechanisms associated with perineural invasion in prostate cancer. Cancer Res.2004 Sep 1; 64(17):6082-90.
10. Dai H, Li R, Wheeler T, Diaz de Vivar A, et al. Pim-2 upregulation:biological implications associated with disease progression and perinueral invasion in prostate cancer. Prostate.2005 Nov 1; 65(3):276-86.
11. Gong J, Wang J, Ren K, et al. Serine/threonine kinase Pim-2 promotes liver tumorigenesis induction through mediating survival and preventing apoptosis of liver cell. J Surg Res.2009 May 1; 153(1):17-22.
12. Aho TL, Lund RJ, Ylikoski EK, et al. Expression of human pim family genes is selectively up-regulated by cytokines promoting T helper type 1, but not T helper type 2, cell differentiation. Immunology.2005 Sep; 116(1):82-8.
13. Xu J, Wang D, Zhang C, et al. Alternatively expressed genes identified in the CD4+ T cells of allograft rejection mice. Cell Transplant.2011; 20(2):333-50.
14. Woodland RT, Fox CJ, Schmidt MR, et al. Multiple signaling pathways promote B lymphocyte stimulator dependent B-cell growth and survival. Blood.2008 Jan 15; 111(2):750-60.
15. Spivey TL, Uccellini L, Ascierto ML, et al. Gene expression profiling in acute allograft rejection:challenging the immunologic constant of rejection hypothesis. J Transl Med.2011 Oct 12; 9:174.
16. llet N, Dieude'M,CailhierJ,He'bert M. The molecular legacy of apoptosis in transplantation. Am J Transplant.2012; 12:1378-1384.
17. Allen JD, Verhoeven E, Domen J, et al. Pim-2 transgene induces lymphoid tumors, exhibiting potent synergy with c-myc. Oncogene.1997 Sep 4;15(10):1133-41.
18. Breuer ML, Cuypers HT, Berns A. Evidence for the involvement of pim-2, a new common proviral insertion site, in progression of lymphomas. EMBO J.1989 Mar; 8(3):743-8.
19. Mizuki M, Schwable J, Steur C, et al. Suppression of myeloid transcription factors and induction of STAT response genes by AML-specific F1t3 mutations. Blood. 2003; 101(8):3164-73.
20. Tamburini J, Green AS, Bardet V, et al. Protein synthesis is resistant to rapamycin and constitutes a promising therapeutic target in acute myeloid leukemia. Blood. 2009 Aug 20; 114(8):1618-27.
21. Cohen AM, Grinblat B, Bessler H, et al. Increased expression of the hPim-2 gene in human chronic lymphocytic leukemia and non-Hodgkin lymphoma. Leuk Lymphoma.2004 May; 45(5):951-5.
22. Huttmann A, Klein-Hitpass L, Thomale J, et al. Gene expression signatures separate B-cell chronic lymphocytic leukaemia prognostic subgroups defined by ZAP-70 and CD38 expression status. Leukemia.2006 Oct; 20(10):1774-82.
23. Li J, Peet GW, Balzarano D, et al. Novel NEMO/IkappaB kinase and NF-kappa B target genes at the pre-B to immature B cell transition. J Biol Chem.2001 May 25;276(21):18579-90.
24. Ayala GE, Dai H, Ittmann M, et al. Growth and survival mechanisms associated with perineural invasion in prostate cancer. Cancer Res.2004 Sep 1; 64(17):6082-90.
25. Wang Z, Bhattacharya N, Weaver M, et al. Pim-1:a serine/threonine kinase with a role in cell survival, proliferation, differentiation and tumorigenesis. J Vet Sci. 2001 Dec; 2(3):167-79.
26. Lilly M, Sandholm J, Cooper JJ, et al. The PIM-1 serine kinase prolongs survival and inhibits apoptosis-related mitochondrial dysfunction in part through a bcl-2-dependent pathway. Oncogene.1999 Jul 8; 18(27):4022-31.
27. Yan B, Zemskova M, Holder S, et al. The PIM-2 kinase phosphorylates BAD on serine 112 and reverses BAD-induced cell death. J Biol Chem.2003 Nov 14; 278(46):45358-67.
28. Aho TL, Sandholm J, Peltola KJ, et al. Pim-1 kinase promotes inactivation of the pro-apoptotic Bad protein by phosphorylating it on the Ser112 gatekeeper site. FEBS Lett.2004 Jul 30; 571(1-3):43-9.
29. Fox CJ, Hammerman PS, Cinalli RM, et al. The serine/threonine kinase Pim-2 is a transcriptionally regulated apoptotic inhibitor. Genes Dev.2003 Aug 1; 17(15):1841-54.
30. Macdonald A, Campbell DG, Toth R, et al. Pim kinases phosphorylate multiple sites on Bad and promote 14-3-3 binding and dissociation from Bcl-XL. BMC Cell Biol.2006 Jan 10; 7:1.
31. Zhang F, Beharry ZM, Harris TE, et al. PIM1 protein kinase regulates PRAS40 phosphorylation and mTOR activity in FDCP1 cells. Cancer Biol Ther.2009 May; 8(9):846-53.
32. Bullock AN, Russo S, Amos A, et al. Crystal structure of the PIM2 kinase in complex with an organoruthenium inhibitor. PLoS One.2009 Oct 20; 4(10):e7112.
33. Bullock AN, Debreczeni J, Amos AL, et al. Structure and substrate specificity of the Pim-1 kinase. J Biol Chem.2005 Dec 16; 280(50):41675-82.
34. Kumar A, Mandiyan V, Suzuki Y, et al. Crystal structures of proto-oncogene kinase Piml:a target of aberrant somatic hypermutations in diffuse large cell lymphoma. J Mol Biol.2005 Apr 22; 348(1):183-93.
35. Jacobs MD, Black J, Futer O, et al. Pim-1 ligand-bcund structures reveal the mechanism of serine/threonine kinase inhibition by LY294002. J Biol Chem.2005; 280(14):13728-34.
36. Qian KC, Wang L, Hickey ER, et al. Structural basis of constitutive activity and a unique nucleotide binding mode of human Pim-1 kinase. J Biol Chem.2005 Feb 18;280(7):6130-7.
37. Gourley E, Liu XH, Lamb J et al. A potent small molecule PIM kinase inhibitor with activity in cell lines from hematological and solid malignancies. AACR Meeting Abstracts 2008,4974.
38. Chen LS, Redkar S, Bearss D, Wierda WG, Gandhi V. PIM kinase inhibitor, SGI-1776, induces apoptosis in chronic lymphocytic leukemia cells. Blood.2009; 114(19),4150-4157.
39. Mumenthaler SM, Ng PY, Hodge A et al. Pharmacologic inhibition of PIM kinases alters prostate cancer cell growth and resensitizes chemoresistant cells to taxanes. Mol. Cancer Ther.2009; 8(10),2882-2893.
40. Kelly KR, Espitia CM, Taverna P et al. Targeting PIM kinase activity significantly augments the efficacy of cytarabine. Br. J. Haematol.2012; 156(1),129-132.
41. Natarajan K, Burcu M, Baer MR. Inhibition of the serine/threonine kinase PIM-1 has anti-proliferative effects in acute myeloid leukemia (AML) and sensitizes multidrug resistant cells to chemotherapy. ASH Annual Meeting Abstracts 2010; 116(21),1832.
42. Herreros B, Bischoff JR, Rodriguez ME et al. Pharmacological inhibition of PIM kinases in chronic lymphocytic leukemia cases with unfavorable prognosis markers. ASH Annual Meeting Abstracts.2010; 116(21),2468.
43. Lin YW, Beharry ZM, Hill EG et al. A small molecule inhibitor of PIM protein Kinases blocks the growth of precursor T-cell lymphoblastic leukemia/lymphoma. Blood.2010; 115(4),824-833.
44. Beharry Z, Mahajan S, Zemskova M et al. The PIM protein kinases regulate energy metabolism and cell growth. Proc. Natl Acad. Sci. USA 2011; 108(2), 528-533.
45. Lin Y, Graham AM, Costa LJ et al. Dual inhibition of mTOR and PIM kinase pathways using small molecule inhibitors synergistically kills myeloid leukemic cells in vitro and in vivo. ASH Annual Meeting Abstracts.2010; 116(21),293.
46. Ebens AJ, Berry L, Chen YH et al. A selective PIM kinase inhibitor is highly active in multiple myeloma:the biology of single agent and PI3K/AKT/mTOR combination activity. ASH Annual Meeting Abstracts.2011; 116(21),3001.
47. O'Brien S, Haddach M, Borsan C et al. Discovery of selective small molecule pan-PIM kinase inhibitors with potent oral efficacy in murine xenograft models. ASH Annual Meeting Abstracts.2009; 114(22),1726.
48. Bamett AL, Ding S, Murray C et al. Anti-tumor activity of CXR1002, a novel anti-cancer compound that induces ER stress:human tumor xenograft efficacy and in vitro mode of action. Program and Abstracts of the AACR Meeting.2010.
49. hang M, Kan war N, Feng E, et al. PIM kinase inhibitors downregulate STAT3(Tyr705) phosphorylation. Mol. Cancer Ther.2010; 9(9),2478-2487.
50. Tao ZF, Hasvold LA, Leverson JD, et al. Discovery of 3H-benzo[4,5] thieno[3,2-d]pyrimidin-4-ones as potent, highly selective, and orally bioavailable inhibitors of the human protooncogene proviral insertion site in Moloney murine leukemia virus (PIM) kinases. J. Med. Chem.2009; 52(21),6621-6636.
51.Beharry Z, Zemskova M, Mahajan S, et al. Novel benzylidene-thiazolidine-2,4-diones inhibit Pirn protein kinase activity and induce cell cycle arrest in leukemia and prostate cancer cells. Mol Cancer Ther.2009 Jun;8(6):1473-83.
52. Basu S, Golovina T, Mikheeva T, June CH, Riley JL. Foxp3-mediated induction of pim2 allows human T regulatory cells to preferentially expand in rapamycin. J Immunol.2008; 180:5794-5798.
53. Learn CA, Fecci PE, Schmittling RJ, Xie W, Karikaril, Mitchell DA, et al. Profiling of CD4+, CD8+, and CD4+CD25+CD45RO+FoxP3+T cells in patients with malignant glioma reveals differential expression of the immunologic transcriptome compared with T cells from healthy volunteers. Clin Cancer Res 2006; 12:7306-7315.
54. Sugimoto N, Oida T, Hirota K, Nakamura K, Nomura T, Uchiyama T, et al. Foxp3-dependent and-independent molecules specific for CD25+CD4+natural regulatory T cells revealed by DNAmicroarray analysis. Int Immunol 2006; 18:1197-1209.
55. Feldman JD, Vician L, Crispino M, et al. KID-1, a protein kinase induced by depolarization in brain. J Biol Chem.1998 Jun 26;273(26):16535-43.
56. Bullock AN, Debreczeni JE, Fedorov OY, et al. Structural basis of inhibitor specificity of the human protooncogene proviral insertion site in moloney murine leukemia virus (PIM-1) kinase. J Med Chem.2005 Dec 1;48(24):7604-14.
57. Koyasu, S. The role of PI3K in immune cells. Nat. Immunol.2003;4:313-319.
58. Cho, H., J. Mu, J.K. Kim, et al. Insulin resistance and a diabetes mellitus-like syndrome in mice lacking the protein kinase Akt2 (PKB beta). Science.2001; 292:1728-1731.
59. Cho, H., J.L. Thorvaldsen, Q. Chu, et al. Akt1/PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J. Biol. Chem.2001; 276:38349-38352.
60. Sehgal, S.N. Rapamune (RAPA, rapamycin, sirolimus):mecha-nism of action immunosuppressive effect results from blockade of signal transduction and inhibition of cell cycle progression. Clin. Biochem.1998; 31:335-340.
61. Mikkers H, Nawijn M, Allen J, et al. Mice deficient for all PIM kinases display reduced body size and impaired responses to hematopoietic growth factors. Mol Cell Biol.2004 Jul;24(13):6104-15.
62. Alvarado Y, Giles FJ, Swords RT. The PIM kinases in hematological cancers. Expert Rev Hematol.2012 Feb; 5(1):81-96.
63.Macintyre AN, Finlay D, Preston G, Sinclair LV, Waugh CM, Tamas P et al Protein kinase B controls transcriptional programs that directcytotoxic T cell fate but is dispensable for T cell metabolism. Immunity 2011; 34:224-236.
64. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science.2003 Feb 14;299(5609):1057-61.
65. Sakaguchi S, Sakaguchi N, Asano M, et al. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol.1995 Aug 1; 155(3):1151-64.
66. Ono M, Yaguchi H, Ohkura N, et al. Foxp3 controls regulatory T-cell function by interacting with AML1/Runx1. Nature.2007 Apr 5;446(7136):685-9.
67. Kaser T, Gerner W, Hammer SE, et al. Phenotypic and functional characterisation of porcine CD4(+)CD25(high) regulatory T cells. Vet Immunol Immunopathol. 2008 Mar 15;122(1-2):153-8.
68. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol.2003 Apr;4(4):330-6.
69. D'Cruz LM, Klein L. Development and function of agonist-induced CD25+Foxp3+ regulatory T cells in the absence of interleukin 2 signaling. Nat Immunol.2005 Nov;6(11):1152-9.
70. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol.2005 Nov;6(11):1142-51.
71.Maloy KJ, Powrie F. Fueling regulation:IL-2 keeps CD4+Treg cells fit. Nat Immunol.2005 Nov;6(11):1071-2.
72. Zippo A, De Robertis A, Bardelli M, et al. Identification of Flk-1 target genes in vasculogenesis:Pim-1 is required for endothelial and mural cell differentiation in vitro. Blood.2004 Jun 15;103(12):4536-44.
73. Losman J, Chen XP, Jiang H, et al. IL-4 signaling is regulated through the recruitment of phosphatases, kinases, and SOCS proteins to the receptor complex. Cold Spring Harb Symp Quant Biol.1999;64:405-16.
74. Chen XP, Losman JA, Cowan S, et al. Pim serine/threonine kinases regulate the stability of Socs-1 protein. Proc Natl Acad Sci U S A.2002 Feb 19;99(4):2175-80.
75. Peltola KJ, Paukku K, Aho TL, et al. Pim-1 kinase inhibits STAT5-dependent transcription via its interactions with SOCS1 and SOCS3. Blood.2004 May 15;103(10):3744-50.
76. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol.2003 Apr;4(4):330-6.
77. Hori, S., Nomura, T. and Sakaguchi, S. Control of regulatory T cell development by the transcription factor Foxp3. Science.2003; 299:1057-1061.
78. Khattri R, Cox T, Yasayko SA, Ramsdell F. An essential role for Scurfin in CD4+CD25+ T regulatory cells. Nat Immunol.2003 Apr;4(4):337-42.
79. Fontenot JD, Rasmussen JP, Gavin MA, Rudensky AY. A function for interleukin 2 in Foxp3-expressing regulatory T cells. Nat Immunol.2005 Nov;6(11):1142-51.
80. Hara M, Kingsley CI, Niimi M, et al. IL-10 is required for regulatory T cells to mediate tolerance to alloantigens in vivo. J Immunol.2001 Mar 15;166(6):3789-96.
81. Graca L, Cobbold SP, Waldmann H. Identification of regulatory T cells in tolerated allografts. J Exp Med.2002 Jun 17;195(12):1641-6.
82. Tang Q, Adams JY, Tooley AJ, et al. Visualizing regulatory T cell control of autoimmune responses in nonobese diabetic mice. Nat Immunol.2006 Jan;7(1):83-92.
83. Mempel TR, Pittet MJ, Khazaie K, et al. Regulatory T cells reversibly suppress cytotoxic T cell function independent of effector differentiation. Immunity.2006 Jul;25(1):129-41.
84. Carvalho-Gaspar M, Jones ND, Luo S, et al. Location and time-dependent control of rejection by regulatory T cells culminates in a failure to generate memory T cells. J Immunol.2008 May 15;180(10):6640-8.
85. Pandiyan P, Zheng L, Ishihara S, et al. CD4+CD25+Foxp3+ regulatory T cells induce cytokine deprivation-mediated apoptosis of effector CD4+ T cells. Nat Immunol.2007 Dec;8(12):1353-62.
86. Bluestone JA. Regulatory T-cell therapy:is it ready for the clinic? Nat Rev Immunol.2005 Apr;5(4):343-9.
87. Battaglia M, Stabilini A, Roncarolo MG. Rapamycin selectively expands CD4+CD25+FoxP3+ regulatory T cells. Blood.2005 Jun 15;105(12):4743-8.
88. Battaglia M, Stabilini A, Migliavacca B, et al. Rapamycin promotes expansion of functional CD4+CD25+FOXP3+ regulatory T cells of both healthy subjects and type 1 diabetic patients. J Immunol.2006 Dec 15;177(12):8338-47.
89. Coenen JJ, Koenen HJ, van Rijssen E, et al. Rapamycin, and not cyclosporin A, preserves the highly suppressive CD27+subset of human CD4+CD25+regulatory T cells. Blood.2006 Feb 1; 107(3):1018-23.
90. Zeiser R, Leveson-Gower DB, Zambricki EA, et al. Differential impact of mammalian target of rapamycin inhibition on CD4+CD25+Foxp3+regulatory T cells compared with conventional CD4+ Tcells. Blood.2008 Jan 1;111(1):453-62.
91. Strauss L, Whiteside TL, Knights A, et al. Selective survival of naturally occurring human CD4+CD25+Foxp3+ regulatory T cells cultured with rapamycin. J Immunol.2007 Jan 1;178(1):320-9.
92. Obrig TG, Culp WJ, McKeehan WL, Hardesty B. The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J Biol Chem.1971 Jan 10;246(1):174-81.
93. Hershko DD, Robb BW, Wray CJ, et al. Superinduction of IL-6 by cycloheximide is associated with mRNA stabilization and sustained activation of p38 map kinase and NF-kappaB in cultured caco-2 cells. J Cell Biochem.2004 Apr 1:91(5):951-61.
94. Ogura H, Tsukumo Y, Sugimoto H, et al. ERK and p38 MAP kinase are involved in downregulation of cell surface TNF receptor 1 induced by acetoxycycloheximide. Int Immunopharmacol.2008 Jun;8(6):922-6.
95. Kim do K, Hwang CK, Wagley Y, et al. p38 mitogen-activated protein kinase and PI3-kinase are involved in up-regulation of mu opioid receptor transcription induced by cycloheximide. J Neurochem.2011 Mar; 116(6):1077-87.
96. Shih SR, Weng KF, Stollar V, Li ML. Viral protein synthesis is required for Enterovirus 71 to induce apoptosis in human glioblastoma cells. J Neurovirol. 2008 Jan;14(1):53-61.
97. Eriksson D, Lofroth PO, Johansson L, et al. Apoptotic signalling in HeLa Hep2 cells following 5 Gy of cobalt-60 gamma radiation. Anticancer Res.2009 Nov;29(11):4361-6.
98. Liu DX, Greene LA. Regulation of neuronal survival and death by E2F-dependent gene repression and derepression. Neuron.2001 Nov 8;32(3):425-38.
99. Alessenko AV, Boikov PYa, Filippova GN, et al. Mechanisms of cycloheximide-induced apoptosis in liver cells. FEBS Lett.1997 Oct 13；416(1):113-6.
100. Ito K, Kiyosawa N, Kumagai K, et al. Molecular mechanism investigation of cycloheximide-induced hepatocyte apoptosis in rat livers by morphological and microarray analysis. Toxicology.2006 Feb 15;219(1-3):175-86.
101. Blom WM, de Bont HJ, Meijerman I, et al. Prevention of cycloheximide-induced apoptosis in hepatocytes by adenosine and by caspase inhibitors. Biochem Pharmacol.1999 Dec 15;58(12):1891-8.
102. Zhao X, Cao M, Liu JJ, et al. Reactive oxygen species is essential for cycloheximide to sensitize lexatumumab-induced apoptosis in hepatocellular carcinoma cells. PLoS One.2011 Feb 10;6(2):e16966.
103. Liu X, Yang JM, Zhang SS, et al. Induction of cell cycle arrest at G1 and S phases and cAMP-dependent differentiation in C6 glioma by low concentration of cycloheximide. BMC Cancer.2010 Dec 15; 10:684.
104. Edinger AL, Thompson CB. Akt maintains cell size and survival by increasing mTOR-dependent nutrient uptake. Mol Biol Cell.2002 Jul;13(7):2276-88.