【作者】 贺伟峰；

【导师】 吴军；

【作者基本信息】 第三军医大学 ， 外科学， 2006， 博士

【摘要】 γδT细胞在抗肿瘤保护性免疫中起十分重要的作用。缺乏γδT细胞小鼠(TCRδ^-/-小鼠)比野生型小鼠更容易被MCA诱导形成肿瘤。此外,通过对一些动物致癌模型的研究,发现γδT细胞在早期抗肿瘤免疫反应中起着比αβT细胞还重要的作用。Vγ1和Vγ4是两类主要的外周γδT细胞亚群,并且这两类γδT细胞亚群近来已经在许多感染模型中被阐明具有明确不同的功能。然而,Vγ1和Vγ4γδT细胞亚群各自在肿瘤免疫反应中所起的作用,以及相应的细胞和分子机制还尚未阐明。与αβT细胞不同,有相当比例的外周γδT细胞都表现出自发性激活型的细胞表型(CD44h^high。我们以前的研究表明小鼠体内自然存在大量的自发性激活型CD44^highγδT细胞,这些细胞自发性表达低水平的IFN-γ和T-bet,并且一旦经TCR触发后迅速大量表达IFN-γ。这进一步支持他们的确是处于活化状态的。此外,CD44分子能介导γδT细胞与内皮细胞、基质细胞和ECM的黏附,使CD44^highγδT细胞穿出血管壁到达肿瘤发生部位。因此,CD44^highγδT细胞在γδT细胞早期抗肿瘤免疫反应中起至关重要的作用。然而,自发性激活型CD44^highγδT细胞各亚群在肿瘤免疫反应中究竟起什么作用?与激活后产生的效应CD44^highγδT细胞究竟有什么不同?这些目前都尚不清楚。IFN-7和perforin是免疫监视中清除肿瘤细胞机制中十分关键的两个分子。IFN-γ在抗肿瘤免疫反应中发挥多重效应功能。内源性产生的IFN-γ能够抑制移植肿瘤的生长,此外还能终止化学诱导肿瘤和自发性肿瘤的形成。我们以前的研究也阐明了γδT细胞在抗肿瘤免疫反应中提供早期IFN-γ的来源,以调节αβT细胞抗肿瘤效应功能。perforin在抗肿瘤免疫反应中主要作为关键的肿瘤细胞杀伤分子。如果perforin产生缺陷,小鼠更容易被化学致癌物甲基胆葸（MCA）诱导成瘤。因此,十分有必要阐明IFN-γ和perforin在Vγ1或Vγ4γδT细胞抗肿瘤免疫反应中的作用。在分化为Th1型的CD4⁺T细胞中,能通过抗原特异性（获得性）和非抗原特异性（天然）方式诱导IFN-γ产生。现已明确.αβT细胞主要通过抗原特异性方式,即TCR-依赖性通路,激活产生IFN-γ,而NK细胞则主要通过非特异性方式,即细胞因子受体通路,激活而产生IFN-γ。作为链接天然免疫和获得性免疫反应桥梁,γδT细胞是否能够通过非特异性的细胞因子受体通路激活产生IFN-γ以及perfofin尚未见报道。另外,这两种方式中,哪一种方式在γδT细胞抗肿瘤作用中发挥更重要作用也是一个值得探讨的问题。阐明这些问题不仅能使我们更好的理解γδT细胞在肿瘤免疫监视的作用以及所涉及的分子机制,而且也有助于我们进一步发展新的抗肿瘤免疫治疗方案。我们将本项目研究获得的主要结果和结论归纳如下:一、Vγ4γδT细胞有强烈的抑制肿瘤形成和生长作用,但在野生型小鼠体内该细胞抗肿瘤作用可能被其他细胞调节,处于抑制状态;而VγlγδT细胞并不直接影响肿瘤的形成和生长,但能通过调节其他免疫细胞的抗肿瘤功能来发挥其负调功能。①抗体清除试验为了确定Vγ1和Vγ4γδT细胞各自在抗肿瘤免疫反应中的作用,性别和年龄匹配的B6小鼠分别用Vγ1-清除性抗体（2.11）或Vy4-清除性抗体（UC3）或PBS对照（n=15每组）在-5天和-1天进行静脉注射,然后在第0天,在小鼠皮下种植B16 FO肿瘤细胞（2×105/每只小鼠）。注射Vγ1或Vγ4抗体后能完全清除Vγ1或Vγ4γδT细胞并能保持这种清除状态至少3周。对比野生型小鼠,Vy1细胞清除小鼠肿瘤形成明显延后以及肿瘤生长明显减缓（p＜0.05）。然而Vy4细胞清除小鼠却与野生型小鼠肿瘤形成时间及肿瘤生长速度无明显差异。②重建试验为了进一步确定Vγ4γδT细胞抗肿瘤作用以及Vγ1γδT细胞无促进肿瘤生长作用,性别和年龄匹配的B6 TCRδ链缺陷小鼠分别通过静脉输入流式细胞仪分选的Vγ1或Vγ4γδT细胞（1×10⁵/每只小鼠）（n=6）,第二天在皮下种植B16 FO黑色素瘤细胞（2×10⁵/每只小鼠）,并观察肿瘤的形成时间。与上面的清除试验结果相契合,对比TCRδ链缺陷小鼠,Vγ4γδT细胞的重建小鼠肿瘤形成时间明显延后（p＜0.05）。然而Vγ1γδT细胞重建小鼠同样与TCRδ链缺陷小鼠肿瘤形成时间没有明显不同。二、自发性激活型Vγ1γδT细胞在体外培养激活后产生低水平的IFN-γ,并且在体内重建试验中表现出不直接影响肿瘤形成和生长;而初始型Vγ1γδT细胞在体外培养激活后产生高水平的IFN-γ,并且在体内重建试验中表现出一定的抗肿瘤保护作用;因而表明不同于效应Vγ1γδT细胞,自发性激活型VγlγδT细胞是一类具有独特功能的yδT细胞亚群。提示可能是自发性激活型Vγ1γδT细胞,而非所有Vγ1γδT细胞都参与抗肿瘤免疫反应的负调。①体外功能试验为证实自发性激活型Vγ1γδT细胞不同于激活后的效应型Vγ1γδT细胞（两者都高表达CD44分子）,是一类具有独特功能的细胞亚群,我们在体外通过固相培养法扩增通过流式细胞仪分选出的自发性激活型Vγ1、初始型Vγ1、白发性激活型Vγ4、初始型Vγ4γδT细胞,然后将这些扩增好的γδT细胞亚群用抗小鼠CD3抗体再刺激,通过流式细胞仪检测IFN-y表达情况。结果发现,仅有18.94%的自发性激活型Vγ1γδT细胞表达IFN-γ阳性,而有42.54%的初始型Vγ1、45.84%自发性激活型Vγ4和57.95%的初始型Vγ4γδT细胞表达IFN-γ阳性,几乎是表达IFN-γ阳性自发性激活型Vγ1γδT细胞的2.5-3倍。表明自发性激活型Vγ1γδT细胞是一类具有独特功能的γδT细胞亚群。②重建试验为了进一步确定自发性激活型(CD44^high)和初始型(CD44^lowVγ1及Vγ4γδT细胞各自在抗肿瘤免疫反应中的作用,性别和年龄匹配的B6 TCRδ链缺陷小鼠分别过继静脉输入通过流式细胞仪分选的自发性激活型(CD44^high)或初始型(CD44^lowVγl或Vγ4γδT细胞（1×10⁵/每只小鼠）后,皮下种植B16 FO黑色素瘤细胞系（2×10⁵/每只小鼠）。从第二天开始每天观察和记录肿瘤形成时间和生长速度。对比TCR 6链缺陷小鼠,重建自发激活型Vγ1、初始型Vγ4和Vγ1γδT细胞组小鼠肿瘤形成时间明显延后（p＜0.05）,并且在三组之间并无明显差异。然而重建自发性激活型(CD44^high)Vγ1γδT细胞小鼠与TCRδ链缺陷小鼠肿瘤形成时间和肿瘤生长速度无明显差异。表明除自发性激活型Vγ1γδT细胞亚群外,其他γδT细胞亚群都具有一定的抑制肿瘤生长作用。三、IFN-γ和perforin是自发性激活型CD44^high Vγ4γδT细胞在体内直接杀伤肿瘤作用所必须的;而自发性激活型CD44^high Vγ1γδT细胞在体内缺乏IFN-γ和perforin介导的抗肿瘤作用。①为证实自发性激活型Vγ1和Vγ4γδT细胞在直接杀伤肿瘤反应中的不同功能,我们在体外通过固相培养法扩增通过流式细胞仪分选出的自发性激活型Vγ1和Vγ4γδT细胞,然后将这些扩增好的CD44^high Vγ1和Vγ4γδT细胞分别与B16黑色素瘤细胞（2×10⁵/每侧）以1:4的比率混合后,分别接种到B6 TCRδ链缺陷小鼠背部皮下的左、右侧（n=5）。接种后每天观察并记录肿瘤的形成情况。结果发现,对比TCRδ链缺陷小鼠,CD44^high Vγ4γδT细胞共接种小鼠肿瘤形成时间明显延后（P＜0.05）并且肿瘤发生率明显降低（100%vs.40%）,然而CD44^high Vγ1γδT细胞共接种小鼠却都没有明显差异。表明CD44^high Vγ4γδT细胞在体内有强烈直接杀伤肿瘤作用,而CD44^high Vγ1γδT细胞在体内缺乏直接杀伤肿瘤作用。②为了评估IFN-γ和perforin在CD44^highVγ4γδT细胞抗肿瘤免疫反应中的作用。如上所述方法在体外扩增好野生型、IFN-γ缺陷型或perforin缺陷型的自发性激活型CD44^highVγ4γδT细胞,并分别与B16黑色素瘤细胞（2×10⁵/每侧）以1:4的比率混合后,分别接种到B6 TCRδ缺陷小鼠背部两侧皮下（每组n=5）。每天观察和记录肿瘤形成情况。结果发现,对比野生型CD44^high Vγ4细胞共接种小鼠,IFN-γ缺陷型（P＜0.05）和perforin缺陷型（P＜0.01）CD44^high Vγ4细胞共接种小鼠肿瘤形成时间明显提前,发生率明显增高（40%vs 100%）,表明IFN-γ和perforin是自发性激活型CD44^high Vγ4γδT细胞直接杀伤肿瘤作用所必须的;值得注意的是,共接种IFN-γ缺陷型Vγ4γδT细胞小鼠比共接种perforin缺陷型Vγ4γδT细胞小鼠肿瘤形成时间明显延后（P＜0.05）,表明perforin在Vγ4细胞直接杀伤肿瘤作用中发挥比IFN-γ更为重要的作用。③为了评估IFN-γ和perforin在CD44^high Vγ1γδT细胞抗肿瘤免疫反应中的作用。如上所述方法在体外扩增好野生型、IFN-γ缺陷型或perforin缺陷型的自发性激活型CD44^high Vγ1γδT细胞,并分别与B16黑色素瘤细胞（2×10⁵/每侧）以1:4的比率混合后,分别接种到B6 TCRδ缺陷小鼠背部两侧皮下（每组n=5）。每天观察和记录肿瘤发生情况。结果发现,野生型、IFN-γ缺陷型和perforin缺陷型CD44^high Vγ1细胞共接种小鼠肿瘤形成时间没有明显差异,提示自发性激活型CD44^high Vγ1γδT细胞可能在体内缺乏由IFN-γ和perforin介导的抗肿瘤作用。四、自发性激活型(CD44^high)Vγ4γδT细胞比自发性激活型(CD44^high)Vγ1γδT细胞在激活后产生更高水平的IFN-γ①CD3刺激ex vivo为了评估自发性激活型(CD44^high)Vγ1或Vγ4γδT细胞经TCR通路激活后IFN-γ产生情况,我们从初始B6小鼠脾脏细胞中通过流式细胞仪分选出自发性激活型(CD44^high)Vγ1和Vγ4γδT细胞,立即用抗小鼠CD3抗体体外刺激6小时。流式细胞仪分析细胞因子产生细胞,结果发现表达IFN-γ阳性Vγ4γδT细胞比例（4.27%）几乎是表达IFN-γ阳性Vγ1γδT细胞比例（2.78%）的2倍。②CD3刺激in vitro其次,我们在体外扩增CD44^high Vγ1和Vγ4γδT细胞后,再用抗小鼠CD3抗体体外重新刺激6小时。流式细胞仪分析细胞因子产生细胞,结果发现表达IFN-γ阳性Vγ4γδT细胞比例（45.84%）明显高于表达IFN-γ阳性Vγ1γδT细胞比例（18.94%）,约为2倍;但非常少比例的Vγ1γδT细胞（0.47%）和Vγ4γδT细胞（0.10%）表达IL-4,＜0.5%。③IL12+IL18刺激ex viro为了进一步评估了自发性激活型(CD44^high)Vγ1或Vγ4γδT细胞通过细胞因子受体通路激活后产生IFN-γ情况,通过流式细胞仪分别分选出CD44^high vγ1和Vγ4γδT细胞,体外在含有IL12和IL18的培养液中孵育6小时后进行细胞因子分析。结果发现,表达IFN-γ阳性的Vγ4γδT细胞比例（20.24%）明显高于表达IFN-γ阳性的Vγ1γδT细胞比例（12.5%）,几乎是其两倍。④IL12+IL18刺激in vitro体外扩增的CD44high Vγ1和Vy4γδT细胞进一步用IL12和IL18重新刺激后作细胞因子分析。结果发现,表达IFN-γ阳性的Vγ4γδT细胞比例（60.24%）明显高于表达IFN-γ阳性的Vγ1γδT细胞比例（29.16%）,几乎是其两倍。⑤自发性激活型CD44^high vγ1和Vγ4γδT细胞中T-bet和Eomes基因转录水平T-bet和Eomes都是调控CD8⁺T细胞产生IFN-γ的重要转录因子。为了评估CD44^highVγ1γδT细胞和CD44^high Vγ4γδT细胞中T-bet和Eomes的表达情况,通过流式细胞仪从B6小鼠脾脏细胞中分别分选出CD44^high VγlγδT细胞和CD44^high Vy4γδT细胞,直接用实施定量RT-PCR分析T-bet和Eomes基因的转录情况。结果显示出CD44^high Vγ1γδT细胞中T-bet的mRNA丰度大约是CD44^high Vγ4γδT细胞中的两倍（图15）;而CD44^high Vγ4γδT细胞中Eomes的mRNA丰度大约是CD44^high Vγ1γδT细胞中的20倍（图15）,表明Eomes对CD44^high Vy4γδT细胞的功能起更重要的作用。五、自发性激活型(CD44^high) Vγ4γδT细胞比自发性激活型(CD44^high)Vγ1γδT细胞在激活后产生更高水平的perforin①CTL试验为了评估Vγ4γδT细胞在体外对肿瘤细胞的细胞毒性,我们通过流式细胞仪分选出CD44^high Vγ4和Vy1γδT细胞在体外进行扩增,扩增后通过JAM试验来分析这些扩增细胞的CTL能力。使用YAC-1细胞作为靶细胞。结果显示,对比Vγ1γδT细胞,Vy4γδT细胞在高效靶比率（10:1,20:1 and 40:1）时有明显增加的CTL活性（P＜0.05）,表明CD44^high Vγ4γδT细胞比CD44^high Vγ1γδT细胞在体外对肿瘤细胞有更强的细胞毒性作用。②CD3刺激ex vivo由于CD44^high Vγ4γδT细胞被证实产生更高水平的IFN-γ,我们希望确定是否CD44^highh Vγ4γδT细胞和CD44^high vγ1γδT细胞也产生不同水平的perforin。因此我们首先通过流式细胞仪从B6小鼠脾脏细胞中分选出自发性激活型CD44^high Vγ1和Vγ4γδT细胞,直接用抗小鼠CD3抗体刺激6小时后进行细胞内细胞因子染色。流式细胞分析结果表明表达perforin阳性Vγ4γδT细胞比例（8.47%）明显少于表达perforin阳性Vγ1γδT细胞比例（2.67%）,几乎是其3倍。③IL12+IL18刺激ex vivo我们用IL12和IL18直接刺激通过流式细胞仪从B6小鼠脾脏细胞中分选出自发性激活型(CD44^high)Vγ1和Vγ4γδT细胞6小时,同样用流式细胞仪分析,结果表明表达perforin阳性Vγ4γδT细胞比例（20.27%）几乎是表达perforin阳性Vγ1γδT细胞比例（2.56%）的8倍。④原位检测ex vivo为了体内原位检测浸润到肿瘤区域的Vγ1和Vγ4γδT细胞表达perforin情况,B6小鼠皮下接种B16黑色素瘤细胞（2×10⁵/每只小鼠）,取下接种肿瘤部位的皮肤组织并消化为单细胞状态,直接标记anti-Vγ1或anti-Vγ4表面荧光抗体后固定并通过细胞内染色标记perforin荧光抗体。流式细胞分析结果阐明仅有0.07%的浸润Vγ1γδT细胞表达perforin,远远少于表达perforin的浸润Vγ4γδT细胞（1.09%）。更多还原

【Abstract】 γδT cells play a critical role in the protective immune response against tumor development.Mice lackingγδT cells(TCRδ^-/- mice) are more susceptible to MCA-induced tumor formation than wild-type mice.Moreover,it is shown thatγδT cells play a more important role in antitumor immune response thanαβT cells in carcinogenesis models.Vγ1 and Vγ4 are the two dominant subsets of peripheralγδT cells.More recently, it has been demonstrated that these two subsets have different roles in systemic listeriosis and asthma.However,the role of Vγ1 versus Vγ4γδT cells in tumor immune response is unclear.UnlikeαβT cells,most peripheralγδT cells exhibit a spontaneous activation phenotype,which is characterized by the surface expression of the activation marker (CD44^high) and by a fast turnover rate.Our previous studies also demonstrated that CD44^highγδT cells,but not CD44^low one,spontaneously express IFN-γand T-bet,and rapidly express IFN-γupon TCR triggering.This further supports the notion that they are indeed activated. However,the precise role of those CD44^highγδT cells in tumor immune response has not been clarified yet.Both the production of IFN-γand the ability to kill tumor cells were the critical physiologically relevant cellular effectors of immunosurveillance in eradicating developing tumors.IFN-γis a critical cytokine in antitumor immune responses.Endogenously produced IFN-γwas shown to inhibit the growth of transplanted tumors,in addition to cease the formation of primary chemically induced and spontaneous tumors.Our previous study also demonstrated thatγδT cells provided an early source of IFN-γwhich regulated theαβT cell effector function in the antitumor immune response.Early studies identified perforin as a critical cytolytic molecule in antitumor immune response.Compared with wild-type mice,the perforin^-/- mice developed tumors with greater frequency in response to the chemical carcinogen methylcholanthrene（MCA）.However,the precise role of IFN-γand perforin in Vγ1 versus Vγ4γδT cells in tumor immune response is not fully understood.In Th1 cells,at least two distinct receptor-mediated pathways can induce IFN-γproduction.T cell receptor（TCR）-induced IFN-γproduction is antigen-specific as a part of the adaptive immune response.The differentiated Th1 cells can also produce IFN-γdirectly in response to IL-12 and IL-18 as part of the innate immune response.Furthermore,inαβT cells,the primary way to produce IFN-γis TCR-dependent pathways,whereas the primary way to induce IFN-γproduction in NK cells is based on cytokine pathway.As the bridge between the innate（NK and macrophages） and the adaptive immune response,whether theγδT cells can produce IFN-γunder the condition of cytokine stimulation is unclear. Moreover,it is remain unclear whether the expression of perforin can be regulated through the TCR and cytokine pathways in Vγ1 and Vγ4γδT cells.To answer these questions will not only shed light on the molecular mechanisms involved in tumor immune surveillance,but may also lead to the development of new strategies for tumor immunotherapy.Our main findings and conclusions of this study are summarized as follows:1.Vγ4γδT cells possess powerful capacity against tumor,while Vγ1γδT cells may be involved in regulation of Vγ4γδT cells resistance to tumor development.①depletion experiment In order to define the roles of Vγ1 and Vγ4γδT cells in antitumor immune response,sex- and age-matched B6 mice were treated with Vγ1-depletion Ab（2.11） or Vγ4-depletion Ab（UC3） or the control antibody（n=15 per each group） at day -5 and -1,and on day 0,the mice were then inoculated subcutaneously with B16 F0 tumor cells（2×10⁵/mouse）.Tumor growth was monitored and recorded daily.In our preliminary studies,injection of Vγ1 or Vγ4 antibody completely depleted Vγ1 and Vγ4γδT cells and the depletion lasted for about 3 weeks.Compared to Wt depleted mice,Vγ1 depleted mice showed delayed tumor development（p＜0.05） as well as smaller tumor size （p＜0.05）;but it is not found significant difference between Wt depleted and Vγ4 depleted mice.②reconstitution experiment To further define the role of Vγ4γδT cells,sex- and age-matched B6 TCRδ^-/- mice were transferred with sorted Vγ1 or Vγ4γδT cells （1×10⁵/mouse）（n=6） and inoculated with the B16 F0 melanoma cell line（2×10⁵/mouse） on the following day,and the incidence of tumor growth was monitored.Similar to the depletion experiment above,mice reconstituted with Vγ4γδT cells,compared with Wt, were highly protective to tumor development（p＜0.05）;but it is not found significant difference between Wt mice and Vγ1γδT cells reconstituted mice.2.Spontaneously activated,but not effector,Vγ1γδT cells may play a role in regulating otherγδT cells’ anti-tumor activity①To access the production of IFN-γin Spontaneously Activated(CD44^high) Vγ1 or Vγ4γδT cells upon TCR stimulation,we sorted CD44^high Vγ1、CD44^high Vγ4、CD44^low Vγ1、CD44^low Vγ4γδT cells from naive B6 mice splenocytes and cultured them with anti-mouse CD3 antibody and IL-2 for 5 days,then the expanded cells were restimulated with anti-mouse CD3 antibody for 6 hours as described in material and method.Analysis of cytokine-producing cells revealed that just 18.94%Spontaneously Activated Vγ1γδT cells produced IFN-γ,which is much less than Spontaneously Activated Vγ4γδT cells（45.84%）, naive Vγ1γδT cells（42.54%） and na（i|¨）ve Vγ4γδT cells（57.93%）,indicating that Spontaneously Activated Vγ1γδT cells may have special function which is different from otherγδT cells.②Reconstitution experiment To define what different role of the spontaneously activated CD44^high and naive CD44^low Vγ1 or Vγ4γδT cells play in antitumor immune response,sex- and age-matched B6 TCRδ^-/- mice were reconstituted with the sorted activated(CD44^high) or na（i|¨）ve(CD44^low) Vγ1 and Vγ4γδT cells（1×10⁵/mouse） by i.v. injection and inoculated with B16 F0 melanoma cell line（2×10⁵/mouse）.Starting on the following day,tumor growth was monitored and recorded daily.The mice reconstituted with activated Vγ4,na（i|¨）ve Vγ4 and na（i|¨）ve Vγ1γδT cells showed more protective tumor growth than those reconstituted with activated Vγ1γδT cells（p＜0.05）,and no significant difference was observed in their tumor growth among mice reconstituted with activated Vγ4, na（i|¨）ve Vγ4 and na（i|¨）ve Vγ1γδT cells,indicating that,excepts the spontaneously activated Vγ1γδT cells population,otherγδT cells population have powerful effect in antitumor immune response. 3.Both IFN-γand perforin is required for spontaneously CD44^high Vγ4-mediated tumor protection;However,CD44^high Vγ1γδT cells do not show IFN-γ- or perforin-mediated antitumor effector function in vivo①tumor killing experiment in vivo To further confirm the role of the spontaneously activated Vγ1 and Vγ4γδT cells in tumor resistance,we expanded the spontaneously activated Vγ1 and Vγ4γδT cells in vitro,and then,the expanded CD44^high Vγ1 and Vγ4γδT cells populations were mixed with of B16 cells at ratio 1:4 respectively before coinjection into the skin of recipient B6 TCRδ^-/- mice.After injection,recipient mice were monitored for the presence of tumors daily.It can be seen that,compared to B6 TCRδ^-/- mice,CD44^high Vγ4γδT cells coinjection mice were more effective at controlling tumor formation and retarding tumor growth（P＜0.05）,but CD44^high Vγ1γδT cells coinjection mice did not,indicating that the CD44^high Vγ4γδT cells have directly effector function to tumor development,but CD44^high Vγ1γδT cells do not.②IFN-γand perforin are two key factors for tumor immune surveillance;we next assessed the requirement of IFN-γand perforin for the role of CD44^high Vγ4γδT cell in antitumor immune response.The expanded Wt,IFN-γ^-/- or perforin^-/- CD44^high Vγ4γδT cells populations were prepared,and mixed with of B16 cells at a ratio of 1:4 respectively before coinjection into the skin of recipient B6 TCRδ^-/- mice（n=5 per each group）.Tumor growth was monitored and recorded daily.In comparison to Wt CD44^high Vγ4 coinjection mice,both IFN-γ^-/-（P＜0.05） and perforin^-/-（P＜0.01） CD44^high Vγ4 coinjection mice were highly susceptible to tumor growth,therefore demonstrating that both IFN-γand perforin are required for the effector function of CD44^high Vγ4γδT cell in antitumor immune response.Notably,IFN-γ^-/- Vγ4γδT cells render B6 TCRδ^-/- mice more protective to tumor development than perforin^-/- Vγ4γδT cells（P＜0.05）,indicating that perforin plays a more important role in Vγ4-mediated local tumor protection than IFN-γ.③In order to assessed the requirement of IFN-γand perforin for the role of CD44^high Vγ1γδT cell in antitumor immune response.The expanded Wt,IFN-γ^-/- or perforin^-/- CD44^high Vγ1γδT cells populations were prepared as described above,and mixed with of B16 cells at a ratio of 1:4 respectively before coinjection into the skin of recipient B6 TCRδ^-/- mice（n=5 per each group）.Tumor growth was monitored and recorded daily as described above.No significant differences were detected between any groups of Vγ1γδT cells coinjection mice（Fig.3B）,it was suggested that CD44^high Vγ1γδT cells might lack both IFN-γand perforin-mediated antitumor effector function.4.Spontaneously activated(CD44^high) Vγ4γδT cells produce a much higher level of IFN-γthan spontaneously activated(CD44^high) Vγ1γδT cells does through either TCR pathway or cytokine pathway①CD3 stimulation ex vivo To access the production of IFN-γin spontaneously activated(CD44^high) Vγ1 or Vγ4γδT cells upon TCR stimulation,we firstly sorted the spontaneously activated(CD44^high) populations of Vγ1 and Vγ4γδT cells from naive B6 mice splenocytes and stimulated them immediately with anti-mouse CD3 antibody for 6 hours.Analysis of cytokine-producing cells revealed that the percentage of IFN-γ-producing Vγ1γδT cells（2.78%） were lower than that of IFN-γ-producing Vγ4γδT cells（4.27%）.②CD3 stimulation in vitro Secondly,we expanded CD44^high Vγ1 and Vγ4γδT cells in vitro.The expanded cells were restimulated with anti-mouse CD3 antibody for 6 hours.Analysis of cytokine-producing cells revealed that the percentage of IFN-γ-producing Vγ1γδT cells（18.94%） were much fewer than that of IFN-γ-producing Vγ4γδT cells（45.84%）,but the percentage of both IL-4-producing Vγ1γδT cells （0.47%）and IL-4-producing Vγ4γδT cells（0.10%） are less than 0.50%.③IL12+IL18 stimulation ex vivo We further assessed the production of IFN-γin spontaneously activated(CD44^high) Vγ1 or Vγ4γδT cells upon cytokine stimulation.The sorted activated(CD44^high) populations of Vγ1 and Vγ4γδT cells were cultured with IL12 and IL18 for 6 hours for cytokine analysis.The results showed that the percentage of IFN-γ-producing Vγ4γδT cells（20.24%） were almost two fold of that of IFN-γ-producing Vγ1γδT cells（12.5%）.④IL12+IL18 stimulation in vitro The expanded CD44^high Vγ1 and Vγ4γδT cells in vitro were restimulated with IL12 and IL18 for cytokine analysis.The results show that the percentage of IFN-γ-producing Vγ1γδT cells（29.16%） were much fewer than that of IFN-γ-producing Vγ4γδT cells（60.24%）.⑤Both T-bet and Eomes have been defined as the transcription factors that contribute to IFN-γproduction in CD8⁺ T cells.To assess the transcription of T-bet and Eomes in CD44^high Vγ1γδT Cells and CD44^high Vγ4γδT Cells,CD44^high Vγ1γδT Cells and CD44^high Vγ4γδT Cells sorted from B6 mice splenocytes were used for real-time PCR analysis.The transcription of T-bet in CD44^high Vγ4γδT Cells was approximately two fold below that in CD44^high Vγ1γδT Cells.Strikingly,The transcription of Eomes in CD44^high Vγ1γδT Cells was approximately twenty fold below that in CD44^high Vγ4γδT Cells, indicating that Eomes play more important role in function of CD44^high Vγ4γδT Cells.5.Spontaneously activated(CD44^high) Vγ4γδT cells have more CTL ability than spontaneously activated(CD44^high) Vγ1γδT cells does through producing more perforin①CTL assay To assess the cytotoxicity of Vγ4γδT cells to tumor cell in vitro, sorted CD44^high Vγ4 and Vγ1γδT cells were expanded,and the CTL ability of these expanded cells was analyzed using the JAM test.YAC-1 cells were used as the target cells. Vγ4γδT cells showed significantly increased CTL activity at higher E:T ratios（10:1,20:1 and 40:1）（P＜0.05）,suggesting that CD44^high Vγ4γδT cells have higher cytotoxicity to tumor cells than CD44^high Vγ1γδT cell in vitro.②CD3 stimulation ex vivo Given the information that CD44^high Vγ4γδT cells produce more IFN-γ,we wanted to determine whether there was any difference of the production of perforin between in CD44^high Vγ4 and Vγ1γδT cells.To do this, spontaneously activated(CD44^high) populations of Vγ1 and Vγ4γδT cells were sorted from B6 mice splenocytes,and stimulated with anti-mouse CD3 antibody for 6 hours,and used for intracellular cytokine staining.Analysis of cytokine-producing cells revealed that the percentage of perforin-producing Vγ1γδT cells（2.67%） were much fewer than that of perforin-producing Vγ4γδT cells（8.47%）.③IL12+IL18 stimulation ex vivo Sorted spontaneously activated(CD44^high) populations of Vγ1 and Vγ4γδT cells were then stimulated with IL 12 and IL 18 for 6 hours. The results shown that the percentage of perforin-producing Vγ4γδT cells（2.56%） were ahnost two fold of that of perforin-producing Vγ1γδT cells（20.27%）.The results demonstrate that CD44^high Vγ4γδT cells produce higher levels of perforin compared to Vγ1γδT cells upon TCR and cytokine stimulation.④Infiltrating Vγ1 and Vγ4γδT cells produce perforin in situ To test whether does the infiltrating Vγ1 and Vγ4γδT cells produce perforin in situ,B6 mice were injected with B16 melanoma cells,the tissue at the tumor injection site was then digested,and infiltrating cells were stained directly for anti-Vγ1 or anti-Vγ4 followed by intracellular perforin staining.The results also demonstrate that there are much more infiltrating perforin-producing Vγ4γδT cells（1.09%） than the infiltrating perforin-producing Vγ1γδT cells（0.07%）.更多还原