【作者】 康世均；

【导师】 罗荣城；

【作者基本信息】 南方医科大学 ， 肿瘤学， 2009， 博士

【摘要】 研究背景趋化因子是能使细胞发生趋化运动的小分子细胞因子,分子量多在8-14kDa之间。趋化因子与趋化因子受体结合后传递多种细胞信息。趋化因子受体是7次跨膜G蛋白偶联受体中的一个家族。根据他们的两个保守的N-末端半胱氨酸残基,趋化因子及其受体分为CXC,CC,C,and CX3C家族。趋化因子基因分布在4q12-q13染色体（CXC主要趋化中性粒细胞）,4q21,和17q11.2（CC主要趋化单核细胞）。趋化因子受体的基因组存在于2和3染色体。许多趋化因子有着共同的受体和并能与多种受体结合。人们对趋化因子的最初认识是其能够趋化白细胞,现已被证明他们对多类型的细胞,包括T淋巴细胞均有趋化作用。趋化因子能诱导白细胞特别是T淋巴细胞,向炎症反应和肿瘤浸润局部移动。第一章致癌信号通路促进肿瘤的生长和入侵,也影响抗肿瘤免疫反应,包括免疫细胞动员（1-4）。Notch信号传递途径的自然活化见报于白血病,乳腺癌,肺癌,粘液瘤,卡波西氏肉瘤,神经母细胞瘤,胰腺癌,宫颈癌,以及黑色素瘤,此为探讨Notch信号传递途径作为一种治疗肿瘤的目标提供科学基础。然而,Notch信号传递途径的活化对趋化因子表达的影响尚未经研究调查。Notch信号途径传递从细胞表面的配体结合信号到细胞核转录系统。因此,人们试图用药物抑制gamma-secretase的活性以阻断Notch蛋白裂解,或siMAML1转染以阻断Notch信号转录活动。我们调查了人黑色素瘤细胞系M624经小siMAML1转染后多种基因的表达,发现Tweak受体（TweakR,Fn14）和几个趋化因子包括CCL2的表达显著上调。第二章CXCL16是一种跨膜趋化因子,在生理或病理状态的多种组织器官中都能检测到。在炎症和免疫反应中,CXCL16在巨噬细胞/树突状细胞（Dendriticcells,DC）与T细胞的反应和移动过程中起重要作用。细胞膜表面的CXCL16与T细胞表面的CXCR6结合还介导对T细胞的强力粘附作用。CXCL16不仅分布在细胞表面,这种跨膜受体蛋白经蛋白酶自然切割后也以可溶性蛋白小分子形式分泌,这个过程被称为脱落。LPA（Lysophosphatidic acid,LysoPtdOH）作为具有生物活性的磷脂酸,在创伤愈合及炎症反应中对DC细胞和上皮细胞功能有着重要影响。然而,CXCL16的表达与LPA的直接关系尚不清楚。用单核细胞来源的巨噬细胞/DC,我们研究了LPA刺激后CXCL16的基因和蛋白表达水平及其在细胞表面的分布变化。第三章树突状细胞（DC）分泌的趋化因子可调节DC自身的运动功能,也可以动员肿瘤免疫反应性T淋巴细胞。DC制造许多趋化因子,包括CCL2,CCL3,CCL4,CCL22。肿瘤细胞分泌的CCL2可能首先吸引肿瘤相关的单核细胞来源的DC（MoDC）/巨噬细胞到肿瘤微环境中,之后MoDC/巨噬细胞分泌的趋化因子又可以影响MoDC/巨噬细胞的进一步聚集和T淋巴细胞的浸润,从而调控肿瘤的生长行为。但是,最近在老鼠的实验中发现:未成熟的DC低表达MHC-II和不表达活化共刺激分子CD80/CD86等,他们被动员到肿瘤后,不能激活免疫反应,反而吸引更多的调节性T细胞（Treg）到肿瘤部位,结果导致免疫耐受。因此,在DC成熟过程中全面系统地监测DC的趋化因子的变化,将为发展用于肿瘤治疗的DC疫苗提供免疫学理论基础。血中单核细胞不断地产生巨噬细胞或者DC以维持体内环境稳定。利用纯化的单核细胞,我们在体外制备了MoDC,并研究它们在分化过程中趋化因子的表达特性,总体上,巨噬细胞和MoDC都继承了单核细胞的一系列趋化因子（CCL27,CXCL16,CXCL5,CCL24,CXCL3,CXCL8,CCL2,CCL4,CXCL7和CCL18）,MoDC显著上调CCL17,CCL22,CCL23的表达。研究目的调节肿瘤微环境中多种细胞来源的多种趋化因子的表达和功能。第一章研究用siMAML1阻断黑色素瘤细胞中Notch信号传递途径,诱导依赖于TweakR上调的CCL2的表达,引导更多免疫细胞动员入侵肿瘤微环境。第二章探索血清溶血磷脂酸（LPA）调节巨噬细胞/MoDC中CXCL16的生产和脱落的机理,了解LPA调节巨噬细胞对效应T细胞的趋化活性及其与G_i/o,Rho和NFκB通路的关系,探讨通过调节CXCL16以增加肿瘤局部免疫效应细胞的可行性。第三章观察来自单核细胞的不同分化阶段的MoDC产生的趋化因子表达水平,探讨调节CCL22和CCL17在细胞中转录表达的机制与信号传递途径,探索应用DC肿瘤疫苗治疗肿瘤时,特异性阻断CCL22和CCL17的表达与功能,减少Treg的趋化移动频率,同时吸引更多的T效应细胞到肿瘤部位去杀伤肿瘤细胞。研究方法第一章SiMAML1通过上调黑色素瘤细胞TweakR的表达诱导CCL2的产生1.用实时定量聚合酶链反应（RT-PCR）检测siMAML1,siHey1转染对靶基因（MAML1,Hey1）的抑制效果,及Tweak,TreakR和多种其他趋化因子基因的表达。2.用Western-blot检测以上分子相关蛋白的表达水平。3.用流式细胞术（FACS）分析TweakR在细胞表面的表达。4.用酶联免疫吸附试验（ELISA）测量CCL2在细胞培养上清中的表达水平。5.用单核细胞趋化实验检测黑色素瘤细胞分泌趋化因子（CCL2）对单核细胞的趋化功能。第二章LPA提高LPS刺激巨噬细胞产生CXCL16及调节细胞移动功能的研究1.用流式细胞术（FACS）分析LPA受体（LPA1,LPA2,LPA3）及趋化因子CXCL16在巨噬细胞,MoDC和单核细胞表面的表达。2.定性RT-PCR检测LPA受体（LPA1,LPA2,LPA3）基因在巨噬细胞,MoDC和单核细胞中的表达。3.用酶联免疫吸附试验（ELISA）测量CXCL16在细胞培养上清中的表达水平。4.用实时定量聚合酶链反应检测LPA/LPS对CXCL16基因表达的调节作用。5.用趋化实验检测巨噬细胞,MoDC和单核细胞分泌趋化因子（CXCL16）对T细胞的趋化功能。第三章在DC细胞分化和成熟过程中通过siRNA特异性抑制CCL22和CCL17的表达影响T淋巴细胞亚群的动员1.用流式细胞术分析CD11c,CD11b,CD14在MoDC,巨噬细胞和单核细胞表面的表达,及趋化因子受体CCR1,CCR2,CCR4,CCR8,CXCR3,CXCR6在CD4/CD8 T细胞表面的表达。2.用趋化因子蛋白芯片分析MoDC,巨噬细胞和单核细胞培养上清中趋化因子的广泛表达。3.用实时定量聚合酶链反应检测CCL22,CCL17,CCL2基因在巨噬细胞,MoDC和单核细胞中的表达及siCCL22,siCCL17对其调节作用。4.用酶联免疫吸附试验（ELISA）测量CCL22,CCL17,CCL2在细胞培养上清中的表达水平。5.用体外趋化实验检测siCCL22/siCCL17转染的MoDC对CD4/CD8 T细胞及Treg的趋化功能。6.建立人乳腺癌细胞裸鼠皮下移植瘤模型,体内检测siCCL22/siCCL17转染的MoDC对CD4/CD8 T细胞及Treg的趋化功能。注:统计分析:所有数据采用SPSS 11.5统计软件进行统计处理,实验数据用均数±标准差（（?）±s）表示,组间比较在有多种影响因素时采用析因分析,在只有一种影响因素时采用单因素方差分析;两组间比较采用独立样本t检验法进行分析,多组间两两比较在方差齐性时采用LSD法,方差不齐时采用近似F检验（Welch方法）及多重比较的Dunnett’s T3方法;P<0.05时有统计学意义。研究结果第一章SiMAML1通过上调黑色素瘤细胞TweakR的表达诱导CCL2的产生1.SiMAML1增加TweakR和CCL2的基因表达和蛋白生产SiMAML1成功转染黑色素瘤细胞系M624后,实时定量聚合酶链反应显示:与对照siRNA相比,siMAML1几乎完全抑制了MAML1 mRNA的转录。Western—blot检测,与对照肌动蛋白相比,MAML1蛋白翻译表达也被特异性抑制了。Notch下游Heyl的基因表达和蛋白生产也被特异性抑制了。实时定量聚合酶链反应显示SiMAML或siHeyl成功转染黑色素瘤细胞系M624后TweakR基因转录增强,流式细胞术分析发现TweakR蛋白受体在细胞表面的表达也显著增强。实时定量聚合酶链反应同时显示黑色素瘤细胞系M624被siMAML1或siHeyl转染72小时后,与对照siRNA相比,siMAML1和siHeyl显著上调CCL2 mRNA的转录,ELISA检测证实,CCL2蛋白因子在细胞培养上清的表达也显著增强。2.外源性Tweak刺激CCL2的生产外源性Tweak剂量和时间依赖性地刺激黑色素瘤细胞系M624和siMAML1转染后的黑色素瘤细胞系M624 CCL2的生产,应用anti-TweakR封闭抗体阻断Tweak-TweakR,却几乎完全抑制了外源性Tweak对CCL2的诱导产生,证明Tweak刺激CCL2生产需要Tweak—TweakR的交连。3.SiMAML1增加依赖于CCL2的单核细胞迁移趋化实验进一步研究M624生产的CCL2的功能发现,经siMAML1转染的M624细胞,其培养上清对单核细胞的趋化能力显著增强（P<0.01）。随外源性重组Tweak剂量增加,被刺激的siMAML1转染的M624细胞的培养上清对单核细胞的趋化能力显著增强。用抗体封闭CCL2的功能,严重阻断Tweak刺激上调的siMAML1转染的细胞诱导的单核细胞的迁移,证明siMAML1转染的M624对单核细胞迁移的趋化诱导能力依赖于CCL2.第二章LPA提高LPS刺激巨噬细胞产生CXCL16及调节细胞移动功能的研究1.巨噬细胞和MoDC细胞表面表达LPA受体流式细胞术分析显示巨噬细胞和MoDC细胞表面表达LPA受体1（LPA1）和LPA受体2（LPA2）,但没有LPA受体3（LPA3）。定性RT-PCR确证巨噬细胞/MoDC细胞,象他们的前体细胞CD14⁺单核细胞一样,表达LPA受体1（LPA1）和LPA受体2（LPA2）mRNA,只有MoDC细胞表达低水平的LPA受体3（LPA3） mRNA。2.巨噬细胞和MoDC细胞表面表达CXCL16流式细胞术分析显示巨噬细胞和MoDC细胞表面表达CXCL16,少数单核细胞表面表达CXCL16。几乎所有经单核细胞分化来的巨噬细胞获得CXCL16,而MoDC细胞表面表达最高水平的CXCL16。ELISA法测定巨噬细胞和MoDC分泌可溶性CXCL16,上述MoDC细胞表面表达最高水平的CXCL16相对照,而巨噬细胞释放更多的CXCL16到培养上清液。3.LPA增加巨噬细胞中CXCL16的生产和脱落用LPS刺激巨噬细胞以诱导趋化因子的释放,ELISA法检发现,LPA大大增加CXCL16在巨噬细胞的生产和释放,其水平超过对照LPS组2倍以上。然而流式细胞术分析显示LPA并不导致CXCL16在巨噬细胞表面的表达增强。定量实时RT-PCR显示LPA也上调CXCL16 mRNA在巨噬细胞的表达超过64倍。4.LPA刺激巨噬细胞CXCL16脱落的信号传递途径作为特定的G_i/o受体蛋白,Rho及NFkB通路抑制剂,ELISA分析显示PTx,exoC3,PDTC均能显著降低LPA刺激诱导的CXCL16蛋白的生产（P<0.01）,这表明LPA刺激诱导的CXCL16蛋白的生产需要G_i/o,Rho,NFkB通路的活化。RT-PCR检测证实LPA刺激诱导的CXCL16 mRNA的表达也依赖于G_i/o,Rho,和NFkB通路的调节。5.LPA增加巨噬细胞的趋化活性趋化实验结果显示,经LPA/LPS刺激的巨噬细胞培养上清对CD3⁺T细胞的趋化能力显著增强（P<0.01）,然而经CXCL16阻断抗体预处理过的培养上清液对CD3⁺T细胞的趋化能力显著降低（P<0.01）,与此同时,正常鼠对照抗体IgG没有影响LPA/LPS刺激的巨噬细胞培养上清对CD3⁺T细胞的趋化能力（>0.05）,这意味着LPA介导的巨噬细胞对CD3⁺T细胞的趋化能力增强是通过增加可溶性CXCL16的生产。与上述ELISA试验结果显示的LPA刺激CXCL16生产通过G_i/o,Rho和NFkB几个信号转导通路的活化相似,LPA增强的巨噬细胞对CD3⁺T细胞的趋化能力也需要G_i/o,Rho和NFkB通路的活化。第三章在MoDC细胞分化和成熟过程中通过siRNA特异性抑制CCL22和CCL17的表达影响T淋巴细胞亚群的动员1.MoDC表达的趋化因子与单核细胞不同由PBMC纯化的CD14⁺细胞在培养基中单独培养,或加GM-CSF和IL-4培养MoDC,或加LPS或其他的复合细胞因子诱导MoDC成熟,获得的细胞具有不同细胞的典型形态。MoDC表达共刺激分子CD86,CD40和HLA-DR,但失去了CD14抗原。趋化因子蛋白芯片定性检测发现:单核细胞表达大量的CCL27,CXCL16,CXCL5,CCL24,CXCL3,CXCL8,CCL2,CCL4,CXCL7和CCL18,少量的CXCL1,CCL3和CCL8;MoDC与单核细胞相比,不同之处在于不表达CCL8,高表达CCL17,CCL22和CCL23。ELISA法定量测定和实时定量聚合酶链反应显明与单核细胞相比,MoDC分泌高水平的CCL22,CCL17,CCL23。2.MoDC和单核细胞分泌的趋化因子对活化的T淋巴细胞的趋化能力不同MoDC的培养上清对CD4⁺和CD8⁺效应淋巴细胞的趋化能力显著高于单核细胞（P<0.01）,但对静止细胞的趋化能力相对较小（0.01<P<0.05）。单核细胞的条件培养基对CD4⁺和CD8⁺淋巴细胞的趋化功能相同,但MoDC对CD4⁺效应淋巴细胞的趋化功能较对CD8⁺效应淋巴细胞强。由于自单核细胞分化和成熟过程中MoDC增强表达趋化因子（CCL17,CCL22,CCL23）,分析其相应受体CCR1（CCL23受体）,CCR4（CCL17受体）,CCL22受体),CCR8（CCL17受体）与CCL2（CCL2受体）、CXCR6（CXCL16受体）在CD3⁺淋巴细胞的表达情况显示,CD4⁺淋巴细胞表达的CCR4较高,CD8⁺淋巴细胞主要表达CCR2和CXCR6。这些结果提示,激活的CD4⁺淋巴细胞可能根据他们表达的CCR4向CCL17和CCL22梯度方向运动,而CD8⁺淋巴细胞主要是根据在巨噬细胞和DC分化过程中形成的CCL2和CXCL16梯度移动。MoDC的培养上清对CD25⁺Foxp3⁺CD4⁺Treg的吸引作用明显强于单核细胞的培养上清。3.抗体阻断CCL22和CCL17能调节MoDC动员CD4⁺/CD8⁺比率和Treg的频率将MoDC与抗-CCL2,抗-CCL17,anti-CCL22,anti-CCL23,或相应非特异抗体对照作用后,趋化实验分析发现:阻断CCL2对CD4⁺/CD8⁺Treg的移动频率几乎没有影响（P>0.05）,但是阻断CCL17和CCL22显著地降低CD4⁺/CD8⁺比率和Treg的移动频率（P<0.05:P<O.01）,并意外地发现,阻断CCL23增加CD4⁺/CD8⁺比率和Treg的移动频率。4.用siRNA特异性的抑制CCL22和CCL17的表达可以调节CD4⁺/CD8⁺比率和MoDC动员Treg的频率应用CCL17和CCL22特异性siRNA转染后沉默了CCL17和CCL22基因和蛋白的表达,体外趋化实验分析发现其结果显著地降低CD4⁺/CD8⁺比率和Treg的移动频率（P<0.05）。5.体内瘤内注射siCCL22和siCCL17转染的MoDC招募更少Tregs但更多CD8⁺T细胞当人类乳腺癌细胞株于裸鼠长成实体瘤时,对照MoDC或经siCCL22和siCCL17转染的MoDC被注入肿瘤体内后,实时定量荧光RT-PCR和ELISA法证实,在这些siRNA转染的MoDC中,瘤体内5-7天内CCL22和CCL17几乎被完全抑制了,体外筛选的Tregs和激活CD8⁺T细胞混合液也被注入肿瘤体内。48小时后,手术摘取瘤体,经分离消化获得单个细胞悬液。流式细胞仪分析显示,与对照组MoDC相比,siCCL22和siCCL17转染的MoDC,显著招募更多的CD8+T细胞（10.5%比3.56%）和更少Tregs（0.24%比4.59%）。上述肿瘤样本经免疫组织化学染色证实,瘤体内存在相同数量的CD11c⁺MoDC,而经siCCL22和siCCL17转染的MoDC周围存在更多的CD8⁺T细胞和更少Tregs共存。结论:1.SiMAMLI通过上调黑色素瘤细胞TweakR的表达诱导CCL2的产生Notch信号传递途径在黑色素细胞中的高表达和天然活化,导致其下游转录抑制因子Heyl的转录和激活,后者抑制rweakR在细胞表面的表达而抑制CCL2的表达和分泌,因此降低免疫细胞在黑色素瘤肿瘤微环境的正常入侵和免疫监视,表明Notch信号传递途径代表肿瘤细胞摆脱自然免疫监视分子机制之一。siMAML1特异性阻断Notch信号传递途径下游的转录信号,增加TweakR表达,扩增了Tweak-TweakR之间的相互作用,诱导CCL2表达和自然免疫细胞的浸润。因此,针对Notch信号传递途径的siMAML1应作为一种探索治疗肿瘤的免疫治疗策略。2.LPA提高LPS刺激巨噬细胞产生CXCL16及调节细胞移动功能的研究LPA显著地增加巨噬细胞经LPS刺激产生的CXCL16。经LPA处理后,巨噬细胞/MoDe分泌产生的可溶性CXCL16对活化T细胞也具化学吸附能力。LPA不仅能增加其他炎性细胞因子的产生和脱落,也增加CXCL16的产生和脱落,进而引导更多的活化T细胞到炎症和肿瘤局部,发挥功能性免疫反应。因而LPA上调巨噬细胞和MoDC表达CXCL16,最有可能趋化诱导炎症和肿瘤部位的Thl和功能性细胞毒性效应。LPA和趋化因子之间的有机联系会促进我们对脂质介导的免疫反应的认识。3.在DC细胞分化和成熟过程中通过siRNA特异性抑制CCL22和CCL17的表达影响T淋巴细胞亚群的动员来自单核细胞的不同分化阶段的MoDC产生的趋化因子的高低不一,MoDC与巨噬细胞一样,产生一系列的T效应细胞趋化因子（高表达CCL2,CCL4,CXCL16）和Treg趋化因子（高表达CCL22,CCL17）。用siRNA特异性阻断CCL22和CCL17的转录表达,可有效地减少Treg的趋化移动频率,而吸引更多的T效应细胞到肿瘤部位去杀伤肿瘤细胞,为DC疫苗治疗肿瘤提供一种新思路。总之,siRNA或LPA调节肿瘤微环境中多种细胞（包括肿瘤细胞,巨噬细胞和DC等）来源的多种趋化因子（CCL2,CXCL16,CCL22/CCL17,et al.）的表达和功能,可有效地诱导自然免疫巨噬细胞本身和后天免疫T细胞移动到炎症及肿瘤反应部位,从而调控肿瘤免疫反应,为优化肿瘤免疫治疗提供了新思路。更多还原

【Abstract】 IntroductionChemokines are structurally related,small（8-14 kDa）polypeptide signaling molecules that bind to the chemokine receptors,a family of seven transmembrane G protein-coupled receptors.The chemokines and their respective receptors are divided into the CXC,CC,C,and CX₃C families,based upon the positions of their conserved two N-terminal cys residues.The chemokines genes are clustered on genomic loci, including chromosome 4q12-q13（CXC acting mainly on neutrophils）,4q21,and 17qll.2（CC chemokines acting mainly on monocytes）.The chemokine receptors gene clusters exist on chromosomes 2 and 3.Many chemokines share common receptors and bind to multiple receptors.Chemokines were originally characterized by their ability to induce chemotaxis of leukocytes.They have since been shown to act on multiple cell types,including T lymphocytes.Chemokines lead to the directed migration of leukocytes especially T lymphocytes to the inflammation and tumor sites.Chapter one Oncogenic signaling pathways promoting tumor growth and invasion also affect antitumor immune responses including immune cell recruitment. Constitutive activation of Notch signaling was found in leukaemia,breast cancer, lung cancer,mucoepidermoid tumors,Kaposi’s sarcoma,neuroblastoma,pancreas cancer,cervical cancers and melanoma,providing a basis to explore Notch signaling as a therapeutic target in cancers.However,the chemokine expression profile affected by Notch signaling activation has not been investigated.Since the Notch signaling pathway conveys the ligands-binding signal from the cell surface to the transcriptional machinery through multiple proteolytic cleavage and activation signaling,pharmacological inhibitors of gamma-secretase and small siRNA specifically taregtting Notch transcriptional signaling activities including the co-activitor mastermind-like proteins（MAMLs）.We investigated broad gene expressions affected by specific small siMAML1 transfection.We found a significant upregulation of Tweak receptor（TweakR,Fn14） and several chemokines including CCL2 after siMAML1 transfection in melanoma cells.Chapter two As a transmembrane chemokine,CXCL16 has been detected in various tissues and organs under normal and pathological conditions,also plays an important role in macrophages/DC and T cell interactions and trafficking during inflammation and immune responses.The membrane bound CXCL16 also mediates the firm adhesion of CXCR6-expressing T cells.CXCL16 is not exclusively expressed on the cell surface.It is also found as soluble molecule that is constitutively generated by proteolytic cleavage of its transmembrane variant,a process called shedding.LPA,a bioactive lipid mediator has been indicated to regulate DC and epithelial functions during wound healing and inflammation responses.However,the direct link of CXCL16 expression with LPA has not been established.Using monocyte-derived macrophages/DC（MoDC）,we investigated how LPA regulate CXCL16 production and shedding.Chapter three Chemokines secreted by DC are instrumental for DC to regulate their own migratory capacities and to recruit T lymphocytes during local tumor immune response.DC are the major producers of many chemokines including CCL2,CCL3, CCL4,CCL22.CCL2 may contribute to the initial migrating of monocyte derived DC（MoDC） into the inflammation and tumor sites,thereafter,chemokines secreted by MoDC themselves may influence DC further accumulation and T lymphocytes infiltrating.Recent experiments in mice showed that immature MoDC recruited into tumors weakly expressing MHC classⅡand not expressing activating costimulatory molecules B7,they were unable to activate the immune response,meanwhile these immature MoDC might recruit regulatory T cells（Tregs） into tumor sites and function as tolerogenic rather than immunogenic signals.Therefore,carefully and systemically monitoring MoDC chemokines expression profiles during their differentiation and maturation process will provide insight and overall guidance for DC vaccine development to improve the effectiveness of immunotherapy.Blood monocytes continuously repopulate DC populations to maintain homeostasis.We generated MoDC in vitro and monitored chemokines expression profiles during their differentiation and maturation.We found MoDC largely inherit a broad range of chemokines（CCL27,CXCL16,CXCL5,CCL24,CXCL3,CXCL8,CCL2,CCL4, CXCL7 and CCL18）from monocytes.Meanwhile,there was significant induction of CCL17,CCL22 and CCL23.Objective:To investigate the mechanisms which regulate the broad chemokines expression and functions.First,using siRNA targeting Notch signaling co-activator MAML1 to induce the functional CCL2 production regulated through the upregulation of TweakR in melanoma cells,this results in tumor cells to attract more innate immune cells into tumor microenvironment.Second,exploring the mechanisms of serum-borne LPA upregulating CXCL16 expression and shedding,this increased production of CXCL16 stimulated by LP A in macrophages/MoDC may involve multiple signaling transduction pathways,which would affect the chemotactic activity of macrophages/MoDC.Third,migration assays showed that spent culture supernatant from MoDC were chemotactic to activated T subtypes.Since the transcriptional regulation of CCL22 and CCL17 are similar to T helper chemokines like CCL3,CCL4,CCL5 and CXCL10 in several cell types,blocking antibodies or siRNA specifically targetting CCL17 and CCL22 would alter MoDC chemokines expression profiles as well as their abilities to recruit activated T subsets while reduce Tregs migration and infiltrating in vitro and in vivo,tipping the immune response toward the cytotoxic arm in tumor microenvironment and during DC vaccination.Methods and Results:Chapter one Induction of CCL2 by siMAMLl through upregulation of TweakR in melanoma cells1.SiMAML1 increased TweakR and CCL2 gene expression and protein production.Melanoma cell lines M624 were transfected with siMAMLl,along with control siRNA for 72 h,multiple genes expression analyzed by RT-PCR showed significant upregulation of TweakR and CCL2,with completely knocking down of siMAML1 and Hey1.TweakR ligand Tweak and CCL2 receptor CCR2 mRNA were both expressed at very low to no level and may not be significant in this cell system. MAML1 and Hey1 proteins were significantly decreased in siMAML1 transfected cells.The direct repressions of Hey1 on TweakR and CCL2 mRNA were verified using siHey1.Cell surface TweakR affected by siMAML1 and siHey1 were confirmed by FACS.CCL2 protein secretion regulated by siMAML1 and siHey1 were confirmed by ELISA.2.Exogenous Tweak upregualted CCL2 production.We thus examined the effect of recombinant Tweak on CCL2 production by M624 cells.Exogenous Tweak induced CCL2 production in a dose-and time-dependent manners,which was almost completely inhibited by blocking the Tweak/TweakR interaction with anti-TweakR antibody.3.SiMAML1 increased CCL2 dependent migration of monocytes.To study if CCL2 produced by M624 are functional we collected culture supernatant from siMAML1 transfected cells stimulated with exogenous Tweak,put it into the bottom wells of transwell system with monocytes in the top wells.We found that supernatant from siMAML1 transfected cells were much more chemoattractive to monocytes,and this chemoattraction increased with increasing doses of exogenous Tweak. Neutralizing CCL2 significantly blocked Tweak induced monocyte migration in siMAMLl transfected cells.Chapter two LPA enhances CXCL16 production stimulated by LPS from macrophages and regulates T cell migration1.Macrophages and MoDC express LPA receptors.Macrophages and MoDC were derived from CD14⁺monocytes from normal donor PBMC without age or gender discrimination.FACS confirmed the cell surface expression of LPA1 and LPA2,but not LPA3 on macrophages/MoDC,as well as their precursors CD14⁺monocytes. Similar results were observed when analyzing mRNA expression of LPA receptors by qualitative RT-PCR,where expression of both LPA1 and LPA2 mRNA were readily detectable.2.Macrophages and MoDC express and secrete CXCL16.FACS confirmed macrophages and MoDC expressed CXCL16 on the cell surface.A few monocytes expressed CXCL16 on their cell surface,almost all macrophages acquired CXCL16 after differentiation,MoDC expressed CXCL16 with the highest intensity on cell surface.ELISA showed both macrophages and MoDC secreted soluble CXCL16, while macrophages released more CXCL16 into their spent culture supernatants.3.LPA stimulates CXCL16 production from macrophages.To examine the effect of LPA on CXCL16 expression,we cultured macrophages,stimulated them with LPS to trigger chemokine release.We observed that LPA at 10μM significantly increased the production and release of CXCL16 from macrophages by more than 2 fold detected by ELISA.LPA did not affect CXCL16 cell surface presentation shown by FACS.Quantitative real time PCR showed LPA upregulated CXCL16 mRNA expressions in macrophages also.Control macrophages had basal CXCL16 mRNA （detectable at cycle 35）,LPA increased CXCL16 mRNA（detectable at cycle 32）,LPS induced CXCL16 mRNA（detectable at cycle 28）,however,LPA and LPS synergized in CXCL16 mRNA transcription（detectable at cycle 22）.By calculating the cycle threshold（CT）values of in each sample and ACT,we found that LPA enhanced CXCL16 mRNA transcription by 64 fold.4.Signaling pathways regulating CXCL16 production enhanced by LPA.By binding to specific LPA receptors,LPA activates complex cell signaling including PTx sensitive and-insensitive G proteins depending on cell types,triggering tyrosine phosphorylation and regulating rho-dependent actin reorganization.PTx,a specific inhibitor of G_i/oproteins,which has been shown to inhibit G_i/o-dependent LPA effects in different cell systems.Pretreatment of macrophages with 10 ng/ml of PTx for 16 hr before LPA suppressed CXCL16 protein production stimulated by LPS.In addition, pretreatment with 1μg/ml of exoC3,a specific inhibitor of Rho and 200μM of PDTC,an inhibitor of the NFκB-dependent pathway significantly decreased LPA enhanced CXCL16 protein production from macrophages,suggesting that the enhancement of LPA on CXCL16 production are G_i/o,Rho,and NFκB dependent. Similar results were also observed at the RNA level as detected by RT-PCR. y0,5.LPA enhances chemotactic activity of macrophages.Macrophages play an important role in immune cell trafficking into inflammation and tumor sites.To investigate CXCL16 production stimulated by LPA would functionally attract more T lymphocyte migration,chemotactic activity of spent culture supernatant collected from LPA/LPS treated macrophages was tested in trans-well system.Results showed that spent culture supernatant from LPA/LPS stimulated macrophages significantly increased CD3⁺T cells migration,and pre-incubation the spent culture supernatant with 10μg/ml blocking antibody against human CXCL16 significantly blocked CD3⁺cells migration.On the contrary,normal mouse IgG（10μg/ml）had no effects on CD3⁺cells migration,implying the enhanced chemotactic activity of macrophages stimulated by LPA was mediated through the increased production of soluble CXCL16.Consistent with the ELISA results of CXCL16 production regulated by LPA through several signaling transduction pathways above,the chemotactic activity of macrophages was also mediated through a G_i/o-,Rho-,and NF-κB-dependent pathways,since CD3⁺cells migration toward to spent culture supernatant collected from macrophages were blocked by pretreatment macrophages with PTx,exoC3,or PDTC.LPA and LPS alone had no significant enhancement on CD3⁺cells migration.Chapter three Specifically knocking down of CCL22 and CCL17 expression by siRNA during DC differentiation and maturation affect T subsets recruitment in vitro and in vivo1.Chemokine expression of MoDC.MoDC generated from monocytes exhibited typical DC morphologies acquired CD11c and lost CD14 antigen,these MoDC also expressed high intensity of CD83,CD86,CD40 and HLA-DR after maturation with LPS.MoDC or fresh purified monocytes were stimulated with LPS and spent culture supernatant were collected and a sensitive chemokine protein arrays were performed. The resulting spots that corresponded to each chemokine were analyzed by densitometry and compared to assay-specific positive controls and relative expression of each chemokine was analyzed.Monocytes constitutively express CCL27,CXCL16, CXCL5,CCL24,CXCL3,CXCL8,CCL2,CCL4,CXCL7,CCL18,and low levels of CXCL1 and CCL3.MoDC continue to express the above chemokines with significant upregulation of CCL17,CCL22 and CCL23 by quantified by ELISA.Real time PCR confirmed the upregulations of CCL17,CCL22 and CL23 were reflected at RNA levels compared with the constitutively expressed CCL2.2.Chemotaxis of MoDC to activated T cells.Phenotypes of naive and activated CD3⁺T cells were confirmed by FACS.CD4⁺to CD8⁺ratios were about 2 in these healthy donors tested.Spent culture supernatant from monocytes and MoDC were placed in the bottom wells of transwell and CD3⁺T cells were placed on the top wells. Spent culture supernatant from monocytes had basal chemotatic abilities to both naive and activated CD3⁺T cells compared to media control.Meanwhile,spent culture supernatant from MoDC exhibited significantly higher chemotatic abilities to activated CD4⁺and CD8⁺cells.There is no significant difference between the chemotactic recruitment of na（?）ve T cells between spent culture supernatant from MoDC and monocytes.Spent culture supernatant from monocytes had equal chemotatic abilities to both CD4⁺and CD8⁺T cells.However,spent culture supernatant from MoDC attracted significantly more activated CD4⁺than CD8⁺, meanwhile,fewer na（?）ve CD4⁺than CD8⁺T cells.There was no significant difference in the ratios of CD4⁺to CD8⁺in na（?）ve and activated CD3⁺T cells before they were loaded into the transwells.To investigate the expressions of chemokine receptors on CD3⁺T cells which would bind to those significantly upregulated chemokines（CCL17,CCL22,CCL23）during DC differentiation and maturations,CCR1（receptor for CCL23）,CCR4（receptor for CCL17 and CCL22）,and CCR8（receptor for CCL17）in comparison with CCR2 （receptor for CCL2）and CXCR6（receptor for CXCL16）expressions were analyzed on activated CD4⁺and CD8⁺cells.Result showed that higher levels of CCR4 and CCR8 expression on CD4⁺cells.CD8⁺cells mainly expressed CCR2 and CXCR6. These observations suggest that more activated CD4 T cells may migrate toward CCL17 and CCL22 gradients depend on their CCR4 and CCR8 expression,while CD8⁺T cells may travel according to CCL2 and CXCL16 gradients from MoDC during DC differentiation and maturation.3.MoDC are highly chemptactic to Tregs Activated CD3⁺T cells transwell assays were set up as above and activated CD4⁺cells migrated were stained for CD25 and Foxp3.Compared to spent culture supernatant from monocytes,spent culture supernatant from MoDC attracted significantly higher numbers of CD25⁺Foxp3⁺CD4⁺Tregs.This similar chemotactic ability was seen by MoDC to recruit natural Tregs.4.Blocking CCL22 and CCL17 can regulate the ratios of CD4⁺to CD8⁺as well as frequency of Tregs recruited by MoDC Spent culture supernatant from MoDC was pretreated with anti-CCL2,anti-CCL17,anti-CCL22,anti-CCL23,or isotype control,then were placed in the bottom of transwell and CD3⁺T cells were placed on the top wells.CD4⁺cells migrated to the bottom wells were analyzed.Blocking CCL2 had very little effect on the ratio of CD4⁺to CD8⁺and the frequency of Tregs migrated.While blocking CCL17 and CCL22 significantly decreased the ratio of CD4⁺to CD8⁺and the frequency of Tregs migrated.Surprisingly,blocking CCL23 increased the ratio of CD4⁺to CD8⁺and the frequency of Tregs migrated.5.Specifically knock-down CCL22 and CCL17 expression by siRNA can regulate the ratios of CD4⁺to CD8⁺as well as the frequency of Tregs recruited by MoDC We applied siRNA specific for CCL17 and CCL22 and evaluated the effect of silencing gene expression of CCL17 and CCL22 post-transcriptionally.Compare to the non-specific siRNA control with over 90%transfection efficiency,siRNA specifically knocked down either CCL22（＞80%）or CCL17（＞70%）at the mRNA levels during MoDC differentiation and maturation.Chemokine protein array showed that either CCL22 or CCL17 expression was significantly decreased after separate transfections with siRNA for CCL22 or CCL17,which were confirmed by ELISA. Spent culture supernatant from MoDC with specifically knocking down either CCL17 or CCL22 attracted fewer CD4⁺while more CD8⁺T cells and significantly lower numbers of Tregs.6.Intratumoral injections of MoDC transfected with siCCL22 and siCCL17 recruited fewer Tregs but more CD8⁺T cells When human breast cancers were established in athymic nude mice,control MoDC or MoDC transfected with siRNA for CCL22 and CCL17 during the last 2 days of differentiation and maturation were injected into the tumors followed by mixture of sorted Tregs and activated CD8⁺T cells.Tumors were dissected and digested into single cell suspensions,infiltrating lymphocytes were analyzed by FACS.Compared to the control MoDC,MoDC developed with siCCL22 and siCCL17 transfections significantly recruited more CD8⁺T cells（10.5%vs 3.56%）and fewer Tregs（0.24%vs 4.59%）.In these siRNA transfected MoDC,CCL22 and CCL17 were almost completely suppressed for 5-7 days analyzed by real time RT-PCR and ELISA of sorted human intratumor CD11⁺ cells.IHC performed on these tumor samples confirmed the localization CD11c⁺ MoDC in tumors and the reversed colocalizations of CD8⁺T cells and Tregs.Conclusion:Chapter one Our study found the transcription and activation of transcriptional repressor Hey1,resulted from the constitutive activation of Notch signaling might actively repress TweakR protein and cell surface expression which suppressed CCL2 expression and secretion,therefore might reduce immune cells normal recruitment and immune surveillance in melanoma tumor microenvironment,indicating Notch as one of the molecular mechanisms that mediate tumor escape from natural immune surveillance.Unlike gamma-secretase inhibitors,siMAML1 specifically block Notch transcriptional signaling,amplified Tweak-TweakR interaction to induce CCL2 production in melanoma cells,therefore increas the innate immune cells infiltration. Therefore,like small-molecule drugs,siMAML1 targeting Notch signaling should be explored as a therapeutic strategy for cancer immunotherapy.Chapter two LPA has been indicated to promote early T cell migration to tissue sites of immune responses and regulate T cell proliferation and secretion of numerous cytokines.LPA has also been reported to regulate the differentiation and activation of mononcytes/macrophages and their further interactions with endothelial cells.Here, we demonstrated that LPA increases CXCL16 production and shedding stimulated by LPS in addition to other inflammatory cytokines,therefore directing more activated T cells to the inflammation and tumor sites to develop functional immune responses.LPA enhanced CXCL16 production stimulated by LPS through activation of NF-κB,G_i/o-and Rho-signaling pathways.Since most of CXCL16 binding CXCR6 expressing cells in blood are IFN-γsecreting helper T cells,cytotoxic T cells,or CD56⁺T cells that are enriched for effector functions,thus upregulation of CXCL16 by LPA in macrophages and MoDC most likely leads to the（T helper 1）Th1 and cytotoxic effector function in inflammation and tumor sites.We hope this mechanic link between LPA and chemokines would advance our knowledge in lipid mediated immune responses.Chapter three As professional antigen presenting cells,DC stimulate T cell-mediated adaptive immune response,which largely rely on DCs maturation and activation status.Chemokines secreted by DC are instrumental to recruit T cells during local inflammation and tumor immune response.Tregs play a central role for the maintenance of peripheral tolerance by active suppression of T effector functions and for the immunosuppressive role in tumor microenvironment.Our results showed that MoDC at different differentiation stages from monocytes produce relative different levels of immune stimulating and suppressive chemokines.MoDC produce both T effector chemotatic chemokines（high CCL2,CCL4,CXCL16）and Tregs chemotatic chemokines（high CCL22,CCL17）,therefore they might recruit both Tregs and CD8⁺T effectors.By regulating the relative expression levels of CCL22 and CCL17 using siRNA might significantly reduce the frequency of Tregs while selectively increase CD8⁺T effectors migrated into tumor site to perform cytotolytic activities during DC priming and DC vaccination.In general,using siRNA or LPA to regulate multiple chemokines（CCL2,CXCL16, CCL22/CCL17,et al.）from different cell types in tumor microenvironment,may successfully increase both inate and adaptive T immune cells recruitment to local inflammation or tumor microenvironment,therefore enhance local immune responses. This study shall lead to a new strategy to improve the effectiveness of tumor immunotherapy.更多还原