【作者】 李润美；

【导师】 郝希山； 任秀宝；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2009， 博士

【摘要】 目的：通过对Vav1与肿瘤浸润T淋巴细胞（tumor infiltrating T lymphocytes，TIL-T）活性的研究，提出肿瘤诱导的T细胞失能的分子机制；初步探讨TIL-T中Vav1基因的表达情况及其与肿瘤局部微环境中吲哚胺2，3双加氧酶（indoleamine-2，3-dioxygenase，IDO）的表达的相关性，提出肿瘤诱导的T细胞失能的一个可能的分子机制；分析IDO对T细胞活性、Vav1基因和蛋白表达水平，Vav1蛋白磷酸化及其下游信号分子[Ca2+]流、MAPK、NF-κB等的影响，同时测定IL-2基因的表达水平。提出TIL-T中Vav1信号异常的可能机制；提出调控TIL-T活性的方法，为纠正肿瘤诱导的免疫抑制提供新的解决方法。内容：1)检测TIL-T中Vav1 mRNA的表达水平，分析其与T细胞活性、肿瘤局部IDO表达水平的相关性。2)体外以稳定表达IDO的CHO细胞与T细胞共孵育，观察IDO对T细胞增殖、凋亡的影响，以及对Vav1基因、蛋白表达及Vav1蛋白活化的作用；观察IDO对T细胞中Vav1下游信号主要传导途径[Ca2+]流、MAPK及NF-κB的变化。探讨IDO在调控Vav1表达和活化中的作用，以及Vav1进一步调节T细胞活性的机制。3)将IDO抑制剂1-甲基色氨酸（1-MT）加入共孵育体系，观察T细胞增殖、凋亡以及Vav1信号途径的变化。方法：1)无菌条件下分离肺癌原位TIL-T及恶性胸腹水中的T淋巴细胞，在体外加入刺激活化因子，观察T淋巴细胞对刺激的反应性，同时检测T细胞中Vav1基因的表达水平；再用免疫组化的方法观察肺癌原位肿瘤组织中IDO蛋白的表达情况。2)体外以稳定表达IDO的CHO细胞株与外周血正常T细胞共同培养，MTT法检测T淋巴细胞的增殖率，流式细胞仪检测T细胞凋亡，Real-time PCR法检测Vav1和IL-2 mRNA水平，Western blot免疫印迹和免疫沉淀的方法检测Vav1蛋白表达及磷酸化水平，及其下游分子ERK1／2、p38和IκBα的活化；应用细胞钙离子成像系统检测单细胞内[Ca²⁺]i的动态变化。3)以IDO抑制剂1-MT加入共孵育体系，观察T细胞增殖、凋亡以及Vav1基因蛋白表达水平和下游信号途径的变化。结果：1)在11例肺癌患者肿瘤原位手术标本中，3例TIL-T对活化刺激信号的反应性低下，只表现为极微的增殖反应；其余8例TIL-T对刺激信号的反应正常，可正常增殖。同样用刺激活化信号在体外对10例癌症患者癌性胸腹水中T淋巴细胞进行激活，我们观察到，所有病例所采集的T细胞均能正常增殖。2)利用Real-time PCR的方法对肺癌原位TIL-T和癌性胸腹水中分离得到的T淋巴细胞中Vav1的mRNA含量进行了检测，结果显示可分为两组：对刺激信号反应正常的8例肺癌原位TIL-T和癌性胸腹水中的T细胞中，Vav1 mRNA含量与正常对照差异无统计学意义（P＞0.05）。而3例表现为“失能”状态的T细胞中Vav1的mRNA水平却非常低（P＜0.05）。3)免疫组化检测显示所观察的肺癌患者标本中IDO阳性表达率为27.3％，肺癌原位组织及转移性淋巴结中IDO表达呈一致性。3例IDO表达阳性的肺癌组织，其中浸润T淋巴细胞中Vav1基因表达水平较对照组下降；8例IDO表达阴性的肺癌组织Vav1基因表达水平与对照组的差异无统计学意义（P＞0.05）。IDO表达阳性的3例标本中TIL-T对活化刺激信号的反应性低下。4)稳定表达IDO的CHO细胞株经检测稳定表达IDO mRNA和蛋白，表达的IDO经氨基酸检测分析可分泌至细胞外并且发挥活性；将此细胞与T细胞共孵育后，T细胞增殖下降，凋亡增加；Vav1 mRNA及蛋白水平下降，磷酸化水平降低，而且Vav1下游分子的活化水平均降低；同时IL-2基因的表达下降。5) 1-MT加入共孵育体系后，T细胞增殖及凋亡均可部分恢复，Vav1 mRNA及蛋白水平也可部分恢复。结论：1) Vav1基因的表达与肿瘤局部浸润的T淋巴细胞的活性具有相关性，与肿瘤局部微环境中IDO的表达水平具有相关性。部分肺癌原位肿瘤局部浸润的T淋巴细胞呈现为功能抑制状态，在这些“失能”细胞中信号传导蛋白Vav1的表达下降。免疫组化结果提示IDO可能是肿瘤局部微环境中影响T细胞中Vav1蛋白表达和活化的重要因素之一。2) IDO抑制T细胞增殖，诱导T细胞凋亡。IDO对TCR信号传导通路具有调节作用。IDO及其代谢产物可能通过抑制T细胞中信号传导蛋白Vav1及其下游分子的表达和活化而抑制T细胞功能。3) Vav1的低表达和活化障碍，导致其下游分子的活化障碍，以致IL-2因子的合成和分泌减少，使T细胞增殖抑制。Vav1的下调与T细胞的凋亡是否有关仍需进一步探索。4) Vav1信号通路在IDO诱导的T细胞凋亡中的作用尚需进一步探索。本研究为进一步探讨IDO在病理或生理情况下对T细胞的抑制作用提供了另一个研究方向，初步探索了调控肿瘤局部T淋巴细胞功能的方法，同时为抗肿瘤免疫治疗方法的发展提供了新的治疗方法。更多还原

【Abstract】 Purpose:This study intendes to investigate the relationship between the signal transducerVavl and the activity of tumor infiltrating T lymphocytes (TIL-T).Indoleamine-2,3-dioxygenase (IDO), a tryptophan catabolic enzyme, plays animportant role in immune escape through suppressing T-cell function. Since Vavlsignaling pathway regulates T cell homeostasis, this study was designed to test thehypothesis that IDO induces T-cell immunosuppression through inhibiting Vavlsignaling.Materials and Methods:Firstly, we investigate the expression of Vavl gene in TIL-T. Then theexpression of IDO in the tumors is detected by immunohistochemistry (IHC).Chinese hamster ovary (CHO) cells were stably transfected with human IDO(CHO/IDO). The transcription and expression of IDO gene were detected by RT-PCRand Western blot analysis, respectively. The enzyme activity of IDO was measuredusing Hitachi amino acid automatic analyzer. CD3~+ T cells were isolated from humanperipheral blood monouclear cells and sorted by CD3~+ microbeads in MACS. Afterco-culture of CHO/IDO cells with T cells in the presence or absence of an anti-CD3antibody which can activate T cell receptor (TCR) and/or 1-methyl-L-tryptophan(1-MT) which can inhibit IDO activity, T cell proliferation and apoptosis weredetermined. T cell total RNA and cellular protein samples were isolated for detectingVavl gene and protein expression and activate state as well as the activation of thedownstream signal transducers of Vavl by realtime-PCR and immunoprecipitation orimmunoblotting.Results:T cells isolated from some patients have low response to the stimuli, and can notproliferate normally. Moreover, the level of Vavl expression in these TIL-T is lowerthan the T cells isolated from PBMC.The positive rate of IDO expression is 27.3% in our experiment. The IDOexpression in tumor tissues is consistent with that in metastatic lymph nodes. In the IDO positive tissues, the levels of Vavl in TIL-T are lower than normal(P＜0.05).The expression of IDO in transfected CHO cells was identified by RT-PCR andWestern blotting. And the IDO transgenic CHO cells yielded a high enzyme activityand resulted in complete depletion of tryptophan from the culture medium. We foundthat IDO produced by these IDO-expressing CHO cells significantly inhibitedinterleukin (IL)-2 expression and proliferative response in T cells and increased theapoptosis of T cells. IDO suppressed Vavl mRNA and protein production in T cells.Furthermore, IDO inhibited TCR activation-induced Vavl phosphorylation, whichrepresents Vavl’s activation state and the activation of the downstream signaltransducers of Vavl in T cells. These effects on T-cells induced by co-culture ofCHO/IDO can be were attenuated by 1-MT.Conclusions:The activities of TIL-T correlate with the expression of Vavl. Vavl expressionis defective in T cells in the microinviroment of some tumors.The low expression and activity of Vavl lead to the deficiency of IL-2 synthesis,and then T cells cannot proliferation normally in respond to stimuli.The inhibitory effects of IDO on T cell immune responses may be throughdown-regulating of Vavl protein expression and activation. These studies provideinsights into the mechanisms of immune escape induced by IDO and the therapeuticapplication of IDO inhibitors for cancer treatments.更多还原