【作者】 李鑫；

【导师】 任贺；

【作者基本信息】 天津医科大学 ， 肿瘤学， 2018， 硕士

【摘要】 背景胰腺导管腺癌即大家所知的胰腺癌是人类恶性肿瘤中最致命的疾病之一,尽管在手术、放化疗以及最近开展的靶向治疗方面取得阶段性进展,但胰腺癌的5年生存率仍低于10%[1]。近十年来,免疫治疗在几种类型的肿瘤中展现出良好的治疗效果,于是研究者开始探索胰腺癌中免疫细胞的作用以及免疫治疗方法应用于胰腺癌[2,3]。尽管由临床样本中可以观察到胰腺癌组织以突出的结缔组织纤维化为特征[4],但其微环境中仍有大量免疫细胞的浸润,并且胰腺癌的免疫微环境是免疫抑制性的,这就抑制了免疫效应细胞及具有抗肿瘤活性的细胞因子[5,6]。在胰腺癌起病的早期这种免疫抑制状态便开始形成[7]。虽然胰腺癌的发展需要遗传背景和环境因素的组合,但慢性炎症也被认为是一个主要的危险因素。当具有突变的新变异体持续累积并超过极限时,免疫消除反应变得不充分,并且肿瘤细胞变体获得抗性。此外,免疫炎症细胞表现出改变的功能,随后产生免疫抑制信号,以及炎症细胞因子促进肿瘤生长和侵袭[8,9],最终肿瘤免疫微环境具有高度免疫抑制成分,进一步促进肿瘤细胞的免疫逃逸。胰腺癌免疫微环境中具有众多T细胞,因此它被分类为富含T细胞的肿瘤。尽管先天免疫应答和适应性免疫应答都对肿瘤具有杀伤活性,但胰腺癌自身诱导局部和全身免疫功能障碍或免疫抑制,因此阻止了效应免疫细胞清除胰腺癌细胞[10,11]。胰腺癌细胞通过下调主要组织相容性复合体（MHC）I类分子或抗原插入MHC I类槽中的表达来干扰对效应T细胞的抗原交叉呈递[12]。同时胰腺癌细胞分泌可溶性免疫抑制因子,如IL-10,TGF-β,这些因子促进了抑制性免疫细胞的扩增以及抑制功能的发挥[13]。胰腺癌表达免疫检查点配体,比如PD-L1,这是效应T细胞受到抑制的另一个重要机制[14]。免疫检查点程序性死亡1（PD-1）受体及其配体PD-L1在癌症中常常被激活,并通过抑制细胞毒性T细胞的功能,介导免疫逃避方面发挥重要作用[15][16]。阻断PD-1/PD-L1轴在如黑素瘤,肾和非小细胞肺癌等多种恶性肿瘤中产生了良好的应答[15,17,18]。然而,在PDAC中同样存在PD-L1表达的[19,20],使用PD-L1抑制剂的单一疗法临床收效甚微[21]。深入分析其可能的原因:PDAC是以低突变负荷为特征的“非免疫原性”肿瘤[22],缺乏CD8⁺T细胞浸润[4]以及存在免疫抑制性骨髓细胞群体[23,24]。Anti-PD-1抑制Foxp3+Treg细胞,肿瘤特异性细胞毒性T细胞（CTL）可以通过分泌IFNγ以及TNF细胞因子发挥细胞毒作用杀伤肿瘤细胞。在黑色素瘤中,肿瘤特异性细胞毒性T细胞的数量与临床更好的预后相关,并且对免疫应答的响应更高[25],所以免疫治疗的主要目标是诱导出潜在的抗肿瘤免疫应答的T细胞。然而,肿瘤的发展伴随着调节性T细胞的聚集,并通过分泌抗炎性因子及共抑制分子抑制抗肿瘤免疫[26]。Treg细胞浸润的数量与病人的不良预后成正相关关系[27]。我们的研究发现PDAC中高表达转录因子FOXP3,FOXP3是抑制性免疫调节细胞Treg细胞的特征性表达蛋白,是其发挥抑制作用的阳性指标,我们前期的研究已经证明FOXP3表达在胰腺癌细胞的细胞核中,直接转录调控趋化因子CCL-5的表达和分泌,在动物实验可以观察到Pan02高表达FOXP3组的瘤体中Treg细胞数显著高于对照组,通过阻断实验证明,正是通过CCL-5将Treg细胞招募到肿瘤局部,并发挥免疫抑制作用,促进胰腺癌细胞的免疫逃逸过程[28]。方法1.细胞培养及稳系构建。Pan02、PANC-1、MIA Pa Ca-2以及相应过表达FOXP3、干扰FOXP3蛋白稳转细胞系为课题已有细胞系。细胞在37℃,5%CO₂环境中恒温培养,应用DMEM或者RPMI-1640培养基混合10%FBS。2.C57BL/6小鼠皮下成瘤模型及治疗方式。构建C57BL/6小鼠胰腺导管腺癌皮下移植瘤动物模型,小鼠胰腺癌细胞系Pan02-p LV-Control、Pan02-p LV-FOXP3均为1×10⁶个细胞/只,于小鼠腹股沟皮下移植,待皮下瘤体达到70mm3后,将两种不同细胞系的分别随机分2组,分别给予同型对照Ig G及200ug PD-L1m Ab（AF156）腹腔注射治疗,于皮下移植瘤细胞后第六天开始给于相应治疗,每3天给药,同时测量瘤体大小,绘制瘤体增长曲线,于成瘤后24天处死。3.瘤体组织及小鼠脾脏的单细胞悬液制备。我们应用美天旎（Miltenyi Biotec）的小鼠肿瘤组织解离试剂盒（No.130-096-730）分离并制备小鼠移植瘤的单细胞悬液以及脾脏单细胞悬液。4.流式细胞学分析。将细胞制成单细胞悬液,在100ul体系中加入3ul/样本anti-mouse CD8抗体,室温避光20分钟,加入1ml PBS,400g离心5分钟,弃上清,应用0.5ml IFNγ固定破膜液,室温避光30分钟,加入1ml PBS后400g离心5min,弃上清后加入100ul稀释后的IFNγ染色缓冲以及5ul/样本anti-mouse IFNγ抗体,室温避光1小时,加入1ml PBS 400g离心5分钟,重复加入1ml PBS400g离心5分钟,弃上清后用200ul PBS重悬后,避光置于4℃,待上机检测。数据应用Flow J软件分析作图。5.免疫组化。切片置入烤箱中,65℃,90min烤片,二甲苯1-2各30min脱蜡。无水乙醇1-2各10min,梯度酒精95%、85%、75%,各5min,迅速将水化过的片子浸入盛有抗原修复液（柠檬酸缓冲液）的抗原修复盒中,注意组织要完全浸入液面,将盒子一起置入盛有少量水的高压锅,盖紧锅盖后,打开电磁炉160℃加热至喷气,从喷气开始计时3min,关闭电磁炉。抗原经过高压热修复后,取出抗原修复盒,保证组织完全浸入液面,室温下自然冷却。滴加3%过氧化氢去离子水,室温下避光孵育20min,阻断内源性过氧化物酶活性。取出片子,PBS浸洗,5min,3次,Ki67抗体+抗体稀释液（抗体/稀释液=1:50/1:25）。甩去片子上多余的PBS,用吸水纸擦拭去片子背面及组织外围多余PBS,切片平行架于湿盒中,组织区域滴加一抗,抗体要覆盖过全部组织边缘1mm,4℃过夜。取出湿盒,恢复室温约1.5h,PBS浸洗15min,3次,加入二抗,室温孵育30min,PBS冲洗5min,3次。DAB显色,苏木素复染细胞,盐酸酒精分化,氨水返蓝。脱水,中性树胶封片,显微镜下观察。6.体外细胞功能学Ed U实验。取对数生长期细胞,以每孔4×10³细胞接种于96孔板中,培养至正常生长阶段,加入PD-L1中和抗体,37℃培养12小时,用细胞培养基按1000:1的比例稀释Ed U溶液（试剂A）,制备适量50μM Ed U培养基,每孔加入100μL 50μM Ed U培养基孵育2小时,弃培养基,PBS清洗细胞1～2次,每次5分钟。每孔加入50μL。细胞固定液（即含4%多聚甲醛的PBS）室温孵育30分钟,弃固定液,每孔加入50μL 2 mg/m L甘氨酸,脱色摇床孵育5分钟后,弃甘氨酸溶液,每孔加入100μL PBS,脱色摇床清洗5分钟,弃PBS。每孔加入100μL的1X Apollo（?）染色反应液,避光、室温、脱色摇床孵育30分钟后,弃染色反应液,加入100μL渗透剂（0.5%Triton X-100的PBS）脱色摇床清洗2～3次,每次10分钟,弃渗透剂,每孔加入100μL 1X Hoechst 33342反应液,避光、室温、脱色摇床孵育30分钟后,弃染色反应液;每孔每次加入100μL PBS清洗1～3次,染色完成后立即进行观测。7.从人外周血中分离获得单个核细胞（Peripheral Mononuclear Cells,PBMCs）。取新鲜抗凝血5ml,用等体积的PBS稀释血液,在离心管中加入5ml分离液,将稀释后的血液平铺到分离液液面上方,室温水平转子1000G,离心30min,小心的吸取中间白膜层细胞至新的离心管中,10ml PBS洗涤细胞,250g,离心10min,弃上清,细胞重悬备用。8.共培养体系。将在24孔板中铺1×10⁵个肿瘤细胞/孔,将单个核细胞中加入200U/ml的重组IL-2（Peprotech）及anti-CD3/CD28（Invitrogen）刺激,并于37℃中培养72小时,按照1:5的比例将肿瘤细胞与淋巴细胞混合培养,期间持续添加维持量的重组IL-2,48小时后收取上层淋巴细胞检测。9.磁珠分选人外周血CD8⁺T细胞。将500ul分离液含1×10⁷个的人外周血单个核细胞转移至小管中,加入100ul FBS后加入100ul抗体混合液（Catalog no.11348D）,混合后在4℃中孵育20分钟,加入4ml分离液,混合均匀后350g,4℃离心8分钟,用500ul分离液重悬,加入已经洗过的500ul分离磁珠,室温孵育15分钟,加4ml分离液重悬混合液,避免气泡,将小管置于分离磁柱中2分钟,将上清中的悬浮液转移至另一小管中。结果1.在小鼠皮下成瘤模型中应用PD-L1中和抗体治疗,经过实时测量瘤体大小发现,PD-L1中和抗体治疗均可使抑制瘤体生长,同等剂量的抗体治疗对Pan02-p LV-FOXP3组的瘤体抑瘤作用更加明显;进一步对瘤体进行免疫组化染色发现,应用PD-L1中和抗体治疗后,肿瘤细胞的Ki67增殖指数均有所下降,抑制了瘤体中肿瘤细胞的增殖,并且同样是Pan02-p LV-FOXP3组的肿瘤细胞增殖受到的抑制更强烈;将瘤体研磨制成单细胞悬液,检测其中的肿瘤浸润CD8⁺T细胞的数量,IFNγ含量以及其凋亡情况,结果表明,PD-L1中和抗体治疗后,增加了肿瘤组织中肿瘤浸润淋巴细胞CD8⁺T细胞的数量及抗肿瘤活性,CD8⁺T细胞的凋亡受到抑制,且这一现象在FOXP3高表达组更加显著。2.接下来我们利用体外实验验证PD-L1中和抗体的抑瘤效果,以及肿瘤细胞增殖指数的下降的原因,是PD-L1中和抗体对肿瘤细胞的直接作用还是通过阻断PD-1/PD-L1信号通路后,对肿瘤浸润T淋巴细胞的作用,间接影响了肿瘤细胞。体外实验中,于肿瘤细胞培养条件下单独加入PD-L1单克隆抗体进行阻断,通过Ed U染色细胞增殖实验发现,单独作用于胰腺癌细胞时并不影响肿瘤细胞增殖;通过流式细胞术分析PD-L1中和抗体单独应用于胰腺癌细胞后,肿瘤细胞标记Annexin V发现,单独PD-L1中和抗体作用于胰腺癌细胞,不能促进其凋亡发生。3.利用体外实验验证PD-L1中和抗体的抑瘤效果,进一步探索动物实验中anti-PD-L1中和抗体对FOXP3高表达组响应更佳的原因,将肿瘤细胞与PBMC共培养,加入PD-L1中和抗体后,可以增加共培养体系中CD8⁺T细胞的活性,且FOXP3高表达时,增加的比例更加明显,在共培养体系中anti-PD-L1中和抗体可以减少CD8⁺T细胞的凋亡率,且在FOXP3高表达肿瘤细胞共培养组,减少的比例更加显著;利用降表达FOXP3稳系,改变肿瘤细胞自身FOXP3蛋白表达后,观察共培养中CD8⁺T细胞的活性及凋亡情况变化,结果显示降表达FOXP3组的CD8⁺T细胞的活性比Control组显著增高,且凋亡也减少。结论1.anti-PD-L1治疗在胰腺癌小鼠移植瘤模型中可以发挥一定抑瘤效果;肿瘤细胞高表达FOXP3蛋白的情况下应用anti-PD-L1治疗能获得更加良好的抑制作用;2.在体外实验中应用PD-L1中和抗体对胰腺癌细胞本身的增殖没有影响;PD-L1中和抗体单独应用于胰腺癌细胞时并无显著诱导其凋亡发生;3.肿瘤细胞FOXP3蛋白表达不同引起肿瘤对PD-L1治疗敏感性的改变;在体外共培养条件下,利用PD-L1中和抗体阻断PD-1/PD-L1,均能起到部分恢复CD8⁺T细胞的活性,减少CD8⁺T的凋亡,同时在肿瘤细胞高表达FOXP3组效果更加显著;直接降低肿瘤细胞FOXP3蛋白表达时,肿瘤浸润CD8⁺T细胞凋亡减少抗肿瘤活性增加。更多还原

【Abstract】 BackgroundPancreatic ductal adenocarcinoma and pancreatic cancer are known as one of the most deadly diseases among human malignancies.Although recent advances have been made in surgery,chemotherapy,radiotherapy and recent targeted therapies,the5-year survival rate of pancreatic cancer is still less than 10%.In the past decade,immunotherapy has shown good therapeutic effects in several types of tumors,so researchers began to explore the role of immune cells in pancreatic cancer and immunotherapy methods applied to pancreatic cancer.Although clinically observed pancreatic cancer tissues are characterized by prominent connective tissue fibrosis,there are still a large number of infiltration of immune cells in the microenvironment,and the immune microenvironment of pancreatic cancer is immunosuppressive.This inhibits immune effector cells and cytokines with antitumor activity.In the early stages of the development of pancreatic cancer,this immunosuppressive state begins to form.Although the development of pancreatic cancer requires a combination of genetic background and environmental factors,chronic inflammation is also considered to be a major risk factor.When new variants with mutations continue to accumulate and exceed the limits,the immune elimination response becomes insufficient and the tumor cell variants gain resistance.In addition,immune inflammatory cells exhibit altered function,followed by immunosuppressive signals,and inflammatory cytokines that promote tumor growth and invasion.The final tumor immune microenvironment has a highly immunosuppressive component that further promotes immune escape of tumor cells.Pancreatic cancer has a large number of T cells in its immune microenvironment,so it is classified as a tumor rich in T cells.Although both innate and adaptive immune responses are active against tumors,pancreatic cancer itself induces local and systemic immune dysfunction or immunosuppression,thus preventing effector immune cells from clearing pancreatic cancer cells.Pancreatic cancer cells interfere with antigen cross-presentation of effector T cells by downregulating the expression of major histocompatibility complex（MHC）class I molecules or antigens inserted into MHC class I cells.At the same time,pancreatic cancer cells secrete soluble immunosuppressive factors,such as IL-10 and TGF-β,which promote the expansion of inhibitory immune cells and their inhibitory function.Pancreatic cancer expresses immune checkpoint ligands,such as PD-L1,which is another important mechanism by which effector T cells receive inhibition.Immune checkpoints the programmed death 1（PD-1）receptor and its ligand PD-L1 are often activated in cancer and play an important role in mediating immune evasion by inhibiting the function of cytotoxic T cells.Blocking the PD-1/PD-L1axis produces a very good response rate in various malignancies such as melanoma,renal and non-small cell lung cancer.However,PD-L1 was also expressed in human PDAC samples,but monotherapy with PD-L1 inhibitors was not effective in PDAC.Probable causes:PDACs are"non-immunogenic"tumors characterized by low mutational burden,lack of CD8⁺T cell infiltration and the presence of immunosuppressive bone marrow cell populations.Anti-PD-1 inhibits Foxp3+Treg cells,and tumor-specific cytotoxic T cells（CTL）can kill cytotoxic tumor cells by secreting IFNγand TNF cytokines.In melanoma,the number of tumor-specific cytotoxic T cells correlates with clinically better prognosis and a higher response to immune responses,so the main goal of immunotherapy is to induce a potential antitumor immune response.T cells.However,the development of tumors is accompanied by the accumulation of regulatory T cells and the suppression of anti-tumor immunity by the secretion of anti-inflammatory factors and co-suppressor molecules.The number of Treg cell infiltrates is positively correlated with the patient’s poor prognosis.Our study found that FOXP3,a highly expressed transcription factor in PDAC,is a characteristic expression protein of suppressor immunoregulatory Treg cells and is a positive indicator of its inhibition.FOXP3 is expressed in the nucleus of pancreatic cancer cells and direct transcriptional regulation and chemotaxis Factor CCL-5 expression and secretion,in the animal experiments can be observed Pan02high expression of FOXP3 group of tumor cells in Treg cells was significantly higher than the control group,through the blocking experiment proved that it is through CCL-5 Treg cells recruited to the tumor Locally and exert immunosuppressive effects to promote the immune escape process of pancreatic cancer cells.Method1.Cell culture and stable construction.Pan02,PANC-1,MIA Pa Ca-2,and correspondingly overexpressing FOXP3,interfere with the FOXP3 protein stabilizing cell line as a subject to have a cell line.The cells were incubated at 37°C in a 5%CO₂atmosphere and mixed with 10%FBS in DMEM or RPMI1640 medium.2.Subcutaneous tumorigenesis and treatment of C57BL/6 mice.The animal models of pancreatic duct adenocarcinoma subcutaneously transplanted in C57BL/6 mice were constructed.The pancreatic cancer cell lines Pan02-p LV-Control and Pan02-p LV-FOXP3 were all 1×10⁶ cells/body and transplanted into the mouse inguinal subcutaneously.After subcutaneous tumors reached a certain size,two different cell lines were randomly divided into 2 groups,given Ig G and 200ug anti-PD-L1（AF156）intraperitoneal injection treatment,and were given on the sixth day after subcutaneous transplantation of tumor cells.The corresponding treatment was given once every other day,and the tumor was measured and the volume curve was drawn and sacrificed at 24 days.3.Single cell suspension preparation of tumor tissue and mouse spleen.We used the Miltenyi Biotec mouse tumor tissue dissociation kit（No.130-096-730）to isolate and prepare a single cell suspension of mouse transplanted tumors and a single cell suspension of the spleen.4.Flow cytometry analysis.The cells were made into single cell suspension,3ul/sample of anti-mouse CD8 antibody was added to 100 ul of the system,protected from light at room temperature for 20 minutes,added 1 ml of PBS,centrifuged at 400g for 5 minutes,and the supernatant was discarded,and 0.5 ml of IFNγ-fixation membrane was used.Incubate for 30 minutes at room temperature in the dark,add 1ml of PBS,centrifugation at 400 g for 5 min,discard the supernatant,add 100μl diluted IFNγstaining buffer and 5 ul/sample of anti-mouse IFNγantibody,and protect from light at room temperature for 1 hour.Add 1 ml of PBS 400 g and centrifuge for5 minutes.Repeat adding 1ml PBS 400g centrifugation for 5 minutes,after discarding the supernatant and resuspended with 200ul PBS,protected from light at 4°C,to be tested on the machine.The data was analyzed using FLOEJO software.5.Immunohistochemistry.Slice into the oven,and bake at 65°C for 90 minutes,and dewax 1-2 each for 30 min.Anhydrous ethanol 1-2 each 10min,gradient alcohol 95%,85%,75%,each 5min,quickly hydrated film immersed in an antigen retrieval solution（citrate buffer）antigen repair kit,pay attention to the organization to fully immerse in the liquid surface,place the box in a pressure cooker with a small amount of water.After the lid is closed,open the induction cooker to 160°C and heat it to the jet.After 3 minutes from the jet,turn off the induction cooker.After the antigen was subjected to high pressure heat recovery,the antigen repair kit was removed to ensure that the tissue was fully immersed in the liquid surface and allowed to cool naturally at room temperature.3%H₂O₂ deionized water was added dropwise and incubated at room temperature in the dark for 20 minutes to block endogenous peroxidase activity.The slides were removed,immersed in PBS,5 min*3 times,Ki67 antibody+antibody dilution（antibody/dilution=1:50/1:25）.Remove excess PBS from the slides,wipe off excess PBS on the back of the slide with tissue paper,and place the slides parallel in the wet box.Add primary antibody to the tissue area.The antibody should cover the entire tissue edge（1mm）and overnight at 4°C.Take out the wet box,restore the room temperature for about 1.5 hours,immerse in PBS for 15 min*3 times,add the second antibody,incubate for 30 minutes at room temperature,wash with PBS for 5 min*3 times.DAB coloration,hematoxylin stained cells,hydrochloric acid,alcohol differentiation,ammonia back to blue.Dehydration,neutral gel seals,observation under a microscope.6.In vitro cell function Ed U experiments.Logarithmic growth phase cells were seeded and plated in 96-well plates at 4×10³ to 1×10⁵ cells per well,and cultured to a normal growth stage.Anti-PD-L1 neutralizing antibody was added and incubated at37°C.for 12 hours.The cell culture medium is diluted with Ed U solution（reagent A）in a ratio of 1000:1 to prepare an appropriate amount of 50μM Ed U medium.Each well is added with 100μL of 50μM Ed U medium for 2 hours.The medium is discarded and the cells are washed 1 or 2 times with PBS.5 minutes.Add 50μL per well.Incubate the cell fixative（ie,4%paraformaldehyde in PBS）for 30 minutes at room temperature,discard the fixative and add 50μL 2 mg/m L glycine to each well.After 5 minutes of incubation in a decolorizing shaker,discard the glycine solution and add 100μL to each well.Wash with PBS,bleach for 5 minutes and discard PBS.Add 100μL of 1X Apollo（?）Staining Reaction Solution to each well and incubate for30 minutes in a dark,shaker and shaker at room temperature.Discard the staining reaction solution and add 100μL of osmotic agent（0.5%Triton X-100 in PBS）to decolorize the shaker.2～3 times,10 minutes each time,discard the penetrant,add100μL of 1X Hoechst 33342 reaction solution to each well,incubate for 30 minutes in the light,room temperature,and decolorization shaker,then discard the staining reaction solution;add 100μL PBS per well.Wash 1～3 times,observe immediately after dyeing.7.Peripheral Mononuclear Cells（PBMCs）were isolated from human peripheral blood.Take 5ml of fresh anticoagulant and dilute the blood with equal volume of PBS.Add 5ml of the separation liquid to the centrifuge tube.Spread the diluted blood to the top of the liquid of the separation solution.Rotate the rotor 1000G at room temperature and centrifuge for 30min.Buffy coat cells to a new centrifuge tube,10ml PBS wash cells,250g,centrifuged 10min,discard the supernatant,resuspend cells for use.8.Co-cultivation system.1*105 tumor cells/well will be plated in 24-well plates,mononuclear cells will be stimulated with 200 U/ml Recombinant IL-2（Peprotech）and anti-CD3/CD28（Invitrogen）,and cultured at 37°C 72 Hours,the tumor cells and lymphocytes were mixed in a ratio of 1:5,and the maintenance amount of Recombinant IL-2 was continuously added.After 48 hours,the upper layer lymphocytes were detected.9.Magnetic beads sorting human peripheral blood CD8⁺T cells.Transfer 500μl of the separation solution containing 1*107 human peripheral blood mononuclear cells to a small tube,add 100μl of FBS and add 100μl of antibody mixture（Catalog no.11348D）,mix and incubate at 4°C.for 20 minutes,and add 4 ml of the separation solution.,350 g after mixing,centrifuge at 4°C for 8 minutes,resuspend with 500μl of the separation solution,add 500 ul of washed magnetic beads,incubate for 15minutes at room temperature,resuspend the mixture with 4 ml of the separation solution,avoid air bubbles,and place the tube The magnetic column was separated for 2 minutes and the suspension in the supernatant was transferred to another tube.Results1.In the mouse subcutaneous tumorigenesis model,PD-L1 neutralizing antibody treatment was used.After measuring the size of the tumor,PD-L1 neutralizing antibody treatment can reduce the volume of the tumor,and the Pan02-p LV-FOXP3group The anti-tumor effect of the tumor was even more pronounced.Histological staining of the tumor revealed that the Ki67 proliferation index of the tumor cells was decreased after treatment with the neutralizing antibody to PD-L1,which inhibited the proliferation of tumor cells in the tumor.In the Pan02-p LV-FOXP3 group,tumor cell proliferation was inhibited more strongly.The tumor was ground to a single cell suspension and the number of tumor infiltrating CD8⁺T cells,IFNγcontent,and its apoptosis were examined.After treatment with PD-L1 neutralizing antibody,the number and activity of tumor-infiltrating lymphocyte CD8⁺T cells in the tumor tissue increased,and apoptosis decreased,and this phenomenon was more pronounced in the FOXP3 high-expression group.2.Next,we used in vitro experiments to verify the anti-tumor effect of PD-L1neutralizing antibody,and the reasons for the decline of tumor cell proliferation index,whether it is the direct effect of PD-L1 neutralizing antibody on tumor cells or by blocking PD1/PD-L1 signal pathway,the role of tumor infiltrating T lymphocytes,indirectly affect the tumor cells,in vitro experiments,in the tumor cell culture conditions alone add PD-L1 monoclonal antibody to block,by Ed U staining cell proliferation experiments found The effect of pancreatic cancer cells alone on proliferation was not significant.After flow cytometric analysis of PD-L1neutralizing antibody alone applied to pancreatic cancer cells,Annexin V of tumor cells was found to act on pancreatic cancer cells alone.It has little effect on its apoptosis.3.Use in vitro experiments to verify the anti-tumor effect of PD-L1 neutralizing antibody,and further explore the reason why anti-PD-L1 neutralizing antibody responds better to FOXP3 high expression group in animal experiments.Co-culture tumor cells with PBMCs.After neutralizing the antibody with PD-L1,the activity of CD8⁺T cells in the co-culture system can be increased,and the increased proportion is more obvious when FOXP3 is highly expressed.In the co-culture system,anti-PD-L1 neutralizing antibody can reduce CD8⁺T.The number of apoptosis of T cells,and FOXP3 high expression,the proportion of reduction is more significant,the use of FOXP3 down-regulated,after changing the expression of FOXP3 protein in the tumor cells,observe the changes in activity and apoptosis of CD8⁺T cells in co-culture The results showed that the activity of CD8⁺T cells in the FOXP3 group was significantly higher than that in the Control group,and the apoptosis was also decreased.Conclusion1.Anti-PD-L1 therapy can exert a certain anti-tumor effect in a mouse model of pancreatic cancer xenografts;anti-PD-L1 treatment can obtain more favorable inhibition when tumor cells highly express FOXP3 protein;2.In vitro application of PD-L1 neutralizing antibody has no effect on the proliferation of pancreatic cancer cells;PD-L1 neutralizing antibody has little effect on pancreatic cancer cells when applied alone;3.Changes in the expression of FOXP3 protein in tumor cells caused changes in the sensitivity of PD-L1 treatment;In vitro co-culture conditions,using PD-L1neutralizing antibodies to block PD1/PD-L1,can partially restore CD8⁺T.The activity of T cells decreased the effect of apoptosis,but the effect of high expression of FOXP3 in tumor cells was more significant.When directly reducing the expression of FOXP3 protein in tumor cells,the activity of tumor infiltrating CD8⁺T cells increased,and apoptosis decreased.更多还原