白细胞介素17调节弥漫大B细胞淋巴瘤小鼠主要组织相容性复合体Ⅱ的表达及其与肿瘤进展关系的研究

目的 通过体外培养的Th17细胞过继免疫治疗弥漫大B细胞淋巴瘤(DLBCL)小鼠,分析白细胞介素17(IL-17)水平与主要组织相容性复合体Ⅱ(MHCⅡ)表达的相关性及其与肿瘤发生发展的关系.方法 免疫磁珠法分离纯化BALB/c小鼠脾来源的CD4+CD62L+T细胞,加入抗CD3抗体、抗CD28抗体、转化生长因子β(TGF-β)、IL-6体外培养Th17细胞,人生发中心B细胞样(GCB)DLBCL细胞株SUDHL-4传代培养后接种到严重联合免疫缺陷(SCID)小鼠,建立DLBCL小鼠模型.将荷瘤小鼠分为Th17细胞实验组(30只)和对照组(20只),实验组荷瘤小鼠接种Th17细胞,对照组注射0.9％NaCl溶液.分别于预实验得到的小鼠肿瘤中位发病时间和小鼠中位生存时间处死半数小鼠.酶联免疫吸附法(ELISA)检测小鼠肿瘤组织IL-17表达,免疫组织化学法检测MHCⅡ的表达.结果 DLBCL小鼠肿瘤中位发病时间为8d,中位生存期为28d.Th17细胞接种后IL-17表达水平[(11.93±0.56)pg/ml]较对照组[(9.82±0.26)pg/ml]升高(P＜ 0.000 1),随着肿瘤病程进展,IL-17表达水平降低[(9.53±0.18)pg/ml](P＜ 0.000 1);Th17细胞接种后MHCⅡ阳性细胞比例[(69.13±0.36)％]较对照组[(42.59±0.12)％]升高(P＜ 0.000 1),随着肿瘤病程进展,MHCⅡ阳性细胞比例降低[(54.63±0.45)％](P＜ 0.000 1).IL-17与MHCⅡ之间存在正相关性(r=0.89,P=0.000).结论 IL-17可上调DLBCL小鼠肿瘤组织MHCⅡ表达,且随着肿瘤病程进展,MHCⅡ表达下降.MHCⅡ表达水平可作为DLBCL疾病状态的判断指标,而其正向调控因子IL-17表达水平的上调会影响DLBCL的疾病进程,联合检测IL-17和MHCⅡ表达水平对判断DLBCL疾病状态及进程可能更有参考价值.     

Th17细胞过继免疫治疗对荷弥漫大B细胞淋巴瘤小鼠肿瘤生长的影响

目的 探讨Th17细胞过继免疫治疗对荷弥漫大B细胞淋巴瘤(DLBCL)小鼠肿瘤生长的影响.方法 采用Mini MACS免疫磁珠分离纯化BALB/c小鼠脾来源的CD4+ CD62L+初始T细胞,采用"转化生长因子(TGF-β)、白细胞介素6(IL-6)、干扰素γ抗体(anti-IFN-γ)、白细胞介素4抗体(anti-IL-4)、白细胞介素23(IL-23)"因子组合体外诱导小鼠Th17细胞分化,采用ELISA法测定Th17细胞产生IL-17水平;采用DLBCL细胞株SUDHL-4接种SCID小鼠建立DLBCL荷瘤小鼠模型;选择0、8、18d(3组,5只小鼠/每组)对荷瘤小鼠进行Th17细胞过继免疫治疗,计算肿瘤体积,观察荷瘤小鼠生存期.结果 小鼠在接种SUDHL-4细胞8d左右均形成瘤结节,成瘤率为100％.与对照组小鼠相比,3个过继免疫治疗组小鼠肿瘤体积均明显缩小(P＜0.05),荷瘤小鼠生存期均显著延长(P＜0.05).结论 Th17细胞过继免疫治疗对DLBCL荷瘤小鼠具有抗肿瘤作用.     

初诊DLBCL患者外周血Th17细胞表达与国际预后指标的关系

目的:探讨初诊的弥漫大B细胞淋巴瘤(DLBCL)患者外周血Th17细胞的表达与国际预后指标(IPI)之间的关系.方法:初诊DLBCL组(45例)按照国际预后指标(IPI)积分分为4组,采用ELISA和流式细胞术检测各个DLBCL组与正常对照组(43例)的外周血中IL-17的浓度以及Th17阳性细胞比例,比较各组数值间的差异,并分析IPI的5个指标与IL-17的浓度以及Th17阳性细胞比例的相关性.结果:高危组的IL-17的浓度以及Th17阳性细胞比例较正常对照组及其他IPI组降低,有显著性差异;DLBCL四组的IL-17的浓度以及Th17阳性细胞比例均低于正常对照组;且可见随着IPI分值的增高,IL-17的浓度以及Th17阳性细胞比例呈降低的趋势;IPI指标中年龄、临床分期、全身状态与Th17细胞的表达有相关性.结论:初诊DLBCL患者外周血Th17细胞的表达与国际预后指标有关系;随着IPI积分的增加,DLBCL患者Th17细胞表达下降;临床上对于年龄60岁以上、临床分期Ⅲ期以上、长期卧床及需别人照顾的患者更要注意监测其外周血Th17细胞的表达情况.     

IL-27通过上调MIG和IP-10的表达抑制肿瘤血管形成

目的: 研究IL-27对肿瘤血管生成的抑制作用及其机制.方法:IL-27基因稳定转染的人食管癌细胞(Eca109/IL-27)接种于裸鼠,建立荷瘤裸鼠模型,观察肿瘤生长情况和裸鼠生存期.用ELISA法检测脾细胞IFN-γ的分泌水平;免疫组化法检测瘤组织中VEGF和CD34的表达,并通过CD34的水平计算微血管密度;用RT-PCR法检测肿瘤组织趋化因子IP-10、MIG mRNA的表达水平.结果:接种Eca109/IL-27细胞荷瘤小鼠的生存期较接种野生型Eca109细胞(未转染质粒)和Eca109/LXSN细胞(空载体质粒转染)小鼠的生存期明显延长(P<0.05).接种Eca109/IL-27细胞的裸鼠瘤组织中VEGF和CD34的表达水平显著性低于接种Eca109细胞和Eca109/LXSN细胞,微血管密度显著降低(均P<0.01).Eca109/IL-27组小鼠脾细胞产生较高水平的IFN-γ(P<0.05),趋化因子IP-10和MIG mRNA的表达水平也显著性高于接种Eca109细胞组和Eca109/LXSN细胞组(P<0.05).结论:IL-27在裸鼠体内通过上调IP-10和MIG表达抑制肿瘤血管生成,从而发挥抗肿瘤作用.     

急性白血病患者外周血Treg细胞与Th17细胞的相关性

目的:检测急性白血病患者外周血中Treg细胞与Th17细胞的比例以及相关细胞因子如IL-17、IL-6、TGF-β的变化,分析其相关性。方法:选取兰州大学第二医院血液科急性白血病初诊患者15例,另取15名健康志愿者为对照。流式细胞术检测外周血中CD3+CD4+TIL-17+辅助性T细胞(Th17细胞)、CD4+TCD25+Foxp3+调节性T细胞(Treg细胞)占CD4+T细胞的比例,ELISA法检测血清中细胞因子IL-17、TGF-β、IL-6的水平。结果:急性白血病患者外周血中Th17细胞占CD4+T细胞的(1.39±0.24)%,高于对照组的(0.26±0.11)%(P<0.05);急性白血病患者外周血Treg细胞占CD4+T细胞的(11.58±2.17)%,高于对照组的(2.47±0.72)%(P<0.05);且Treg细胞与Th17细胞呈正相关(γ=0.37)。急性白血病患者血清中TGF-β、IL-6、IL-17的水平分别为(26.06±2.43)、(14.66±2.47)、(18.63±2.38)pg/ml,高于对照组的(13.41±1.92)、(1.44±0.29)、(10.34±1.71)pg/ml(均P<0.05)。结论:急性白血病患者外周血中Treg、Th17细胞比例升高,且两者呈正相关;急性白血病患者血清中TGF-β、IL-6、IL-17水平升高,可能影响Treg与Th17细胞的平衡。     

肺癌患者肿瘤浸润淋巴细胞中IL-17的分布特征及临床意义

目的:探讨肺癌患者肿瘤浸润淋巴细胞中IL-17的分布特征及其临床意义。方法:流式细胞术检测15例肺癌组织和15例配对远癌正常组织中浸润淋巴细胞的IL-17表达水平。结果:肺癌患者肿瘤组织浸润淋巴细胞中IL-17A显著高于远癌正常组织（P＜0.000 1）,肿瘤组织中浸润产生IL-17的CD3+CD4+T细胞（Th17）（P＜0.001）、CD3+CD8+T细胞（Tc17）（P＜0.001）、γδT细胞（γδT17）(P＜0.000 1)均高于远癌正常组织;肺癌患者肿瘤组织浸润Th17和Tc17水平无明显差异（P＞0.05）,而γδT17水平显著高于Th17（P＜0.001）和Tc17（P＜0.000 1）;肿瘤组织中浸润的γδT17表达水平和淋巴结转移相关（P＜0.05）。结论:IL-17和肺癌发生相关,其早期主要来源是肿瘤浸润的γδT细胞;γδT17数量与淋巴结转移有关,提示γδT17细胞可能参与肺癌病程的进展。     

乳腺癌患者外周血Th1／Th2细胞因子的漂移及其与病理和临床病程的关系

 目的 检测乳腺癌患者外周血CD+3 T细胞分泌细胞因子的水平，分析Th1和Th2细胞的细胞因子免疫变化，为肿瘤的免疫治疗提供实验依据。方法 用刺激物刺激细胞，增加细胞内细胞因子的表达，加入荧光标记的特异性抗细胞因子单克隆抗体，特异性抗原抗体结合，应用流式细胞仪分析特异性细胞因子表达水平。同时用酶联免疫吸附法（ELISA）检测血清中相应的细胞因子。结果 CD+3 T细胞分泌的细胞因子水平及血清中的细胞因子皆表现Th1型细胞因子干扰素-γ（IFN-γ）、白细胞介素-2（IL-2）、白细胞介素-12（IL-12）表达水平较正常对照组显著降低，Th2型细胞因子白细胞介素-4（IL-4）和白细胞介素-10（IL-10）表达水平较正常对照组升高，差异有统计学意义。肿瘤坏死因子-α （TNF-α）表达水平较正常对照组升高，差异有统计学意义。结论 乳腺癌患者体内Th2型细胞因子模式占优势状态，这可能是肿瘤细胞发生免疫逃逸，从而导致肿瘤的发生或者转移的原因之一。     

利妥昔单抗对弥漫大B细胞淋巴瘤患者Th17细胞及相关细胞因子影响的体外实验

目的 探讨利妥昔单抗对弥漫大B细胞淋巴瘤(DLBCL)患者Th17细胞及相关细胞因子体外的影响及其意义.方法 DLBCL初治患者(DLBCL组)20例和健康对照者(健康对照组)20名,每个对象分别采集4份外周血标本,分离外周血单个核细胞(PBMC),按照培养条件的不同分成4个亚组:空白组(A亚组)、加入利妥昔单抗组(B亚组)、加入利妥昔单抗和血清组(C亚组)和极化组(D亚组)[加白细胞介素6(IL-6)和转化生长因子(TGF-β)].培养后分别采用流式细胞术检测各亚组外周血PBMC中Th17细胞比例,酶联免疫吸附法(ELISA)检测上述培养液中白细胞介素17(IL-17)的表达水平.结果 健康对照组中,D亚组的Th17细胞比例和IL-17水平分别为(17.12±4.90)％、(45.735±10.012)pg/ml,均高于A、B、C亚组(P＜0.05),A、B、C亚组之间比较差异均无统计学意义(P＞0.05).DLBCL组A亚组的Th17细胞比例及IL-17水平为(0.69±0.24)％、(6.012±1.312)pg/ml,均低于健康对照组A亚组的(2.43±0.61)％、(8.217± 1.681)pg/ml(P＜ 0.05);在与利妥昔单抗一起体外培养后,DLBCL组中B、C和D亚组的Th17细胞比例分别为(2.34±0.48)％、(2.31±0.53)％和(16.92±4.81)％,IL-17水平分别为(7.944±1.538) pg/ml、(7.957±1.533) pg/ml和(44.417±9.881) pg/ml,均高于A亚组,且D亚组明显高于B、C亚组(P＜0.05).B、C亚组之间比较差异无统计学意义(P＞0.05).结论 体外实验证实,DLBCL患者外周血PBMC中Th17细胞比例低于健康人,利妥昔单抗作用于DLBCL患者外周血PBMC后可升高Th17细胞比例.     

Th17细胞过继免疫对DLBCL小鼠CXCL6/CCL2表达的影响及其意义分析

目的 建立弥漫大B细胞淋巴瘤(diffuse large B lymphoma,DLBCL)小鼠模型,在肿瘤发生发展阶段中给予Th17细胞过继免疫治疗,检测肿瘤组织中趋化因子CXCL6/CCL2的表达,了解Th17细胞对DLBCL肿瘤微环境中CX-CL6/CCL2表达的影响,及其与DLBCL发生发展的相关性.方法 人生发中心B细胞(germinal center B cell,GCB)样DLBCL细胞株SUDHL-4进行传代培养后接种10只SCID小鼠建立DLBCL小鼠模型,观察小鼠肿瘤发生发展过程并得出DLBCL小鼠肿瘤中位发病时间T1及小鼠中位生存时间T2.体外培养Th17细胞,在接种肿瘤细胞株时予30只DLBCL小鼠注射Th17细胞行过继免疫,并分别于T0(接种Th17细胞后)、T1和T2这3个时间点处死小鼠,获得A0、A1和A2组,每组10只小鼠.实时定量PCR法检测DLBCL小鼠肿瘤组织CXCL6/CCL2表达,与同一时间点15只小鼠的生理盐水对照组(B0、B1和B2组,每组5只)进行对比,并对比T0、T1和T2这3个时间点各组CXCL6/CCL2的表达.结果 A0组、A1组和A2组CXCL6 mRNA表达量分别为0.115士0.021、0.657±0.142和0.935士0.322,差异有统计学意义,F=7.013,P=0.025.B0组、B1组和B2组CXCL6mRNA表达量分别为0.175±0.024、0.321±0.011和0.631±0.027,差异有统计学意义,F=6.007,P=0.008.A0组CXCL6mRNA表达量与B0组对比差异无统计学意义,t=2.03,P=1.25;A1组明显高于B1组,t=6.43,P＜0.01;而A2组也明显高于B2组,t=6.35,P＜0.01.A0组、A1组和A2组CCL2 mRNA表达量分别为0.133±0.015、0.654±0.534和0.928±0.322,差异有统计学意义,F=7.021,P=0.023.B0组、B1组和B2组CCL2 mRNA表达量分别为0.124±0.013、0.327±0.025和0.628±0.322,差异有统计学意义,F=6.005,P=0.007.A0组CCL2 mRNA表达量与B0组对比差异无统计学意义,t=1.98,P=1.75;A1组明显高于B1组,t=6.13,P＜0.01;A2组也明显高于B2组,t=7.59,P＜0.01.结论 随着DLBCL肿瘤进程,CXCL6和CCL2的表达上升,CXCL6和CCL2可能是影响DLBCL肿瘤发生发展的因子,而Th17过继免疫治疗可以上调DLBCL肿瘤组织CXCL6和CCL2的表达,可能会对DLBCL肿瘤的发生发展产生影响.     

Klotho 蛋白通过TGF-β1/Foxp3/RORγt 通路抑制Treg 和Th7 细胞介导的宫颈癌细胞的免疫逃逸

目的: 研究抗衰老蛋白Klotho 对荷宫颈癌小鼠体内调节性T细胞（Treg 细胞）和辅助性T细胞17（Th7）细胞介导宫颈癌细胞免疫逃逸的影响及其作用机制。方法: 建立宫颈癌U14 细胞移植瘤小鼠模型,并设Control 组（正常小鼠组）、Model 组（荷宫颈癌小鼠模型组）、Klotho 处理组（荷宫颈癌小鼠经Klotho 蛋白处理组,200 ng/d)。分别在处理7、14 d 时称取各组小鼠体内宫颈癌移植瘤质量,以流式细胞术检测各组小鼠脾淋巴细胞功能、Treg和Th7细胞比例变化,qPCR检测各组小鼠Treg和Th7细胞关键转录因子Foxp3、RORγt的表达情况,ELISA方法检测各组小鼠脾淋巴细胞培养液中IL-17、IL-6、IL-10、TGF-β、IL-23 等因子的含量变化,WB检测各组小鼠脾淋巴细胞中Klotho、TGF-β1、Foxp3、RORγt 蛋白表达的变化。结果: 14 d 时,Klotho 组宫颈癌荷瘤小鼠肿瘤抑瘤率显著高于Model 组[(52.16±8.25)% vs (23.33±6.29)%,P<0.05]。Model 组与Control 组相比,荷瘤小鼠脾淋巴细胞中Treg、Th7 细胞比例均显著升高（均P<0.05）,总T淋巴细胞（CD3+）、辅助/诱导T淋巴细胞（CD3+CD4+）比例及免疫指数（CD3+CD4+/CD3+CD8+）显著下降（均P<0.05）,Foxp3、RORγt 基因mRNA表达显著增加（均P<0.05）,IL-17、IL-6、IL-10、TGF-β、IL-23 等因子分泌显著增加（均P<0.05）,Klotho 蛋白水平明显下降（P<0.05）,TGF-β1、Foxp3、RORγt 蛋白表达均明显升高（均P<0.05）;而Klotho 处理组与Model 组相比,上述指标均出现了相反的变化（均P<0.05）,但与Control 组无显著差异（均P>0.05）。结论: Klotho 蛋白可能通过调控TGF-β1/Foxp3/RORγt信号通路抑制宫颈癌荷瘤小鼠体内Treg和Th7 细胞介导的免疫逃逸,从而发挥抗肿瘤作用。     

Inoculation of human interleukin-17 gene-transfected Meth-A fibrosarcoma cells induces T cell-dependent tumor-specific immunity in mice

OBJECTIVE: The biological activities of interleukin-17 (IL-17), a newly cloned cytokine, have not been fully elucidated. The present study was designed to assess the in vitro and in vivo effect of transfecting the IL-17 gene into tumor cells. METHODS: A complementary DNA (cDNA) encoding human IL-17 (hIL-17) was obtained by polymerase chain reaction amplification from the human CD4+ T cell cDNA library and inserted into the plasmid pRc/cytomegalovirus to construct an expression vector for the hIL-17 gene. Murine Meth-A fibrosarcoma cells were transfected with the hIL-17 gene using the lipofectin method. The hIL-17 gene-expressing clone (Meth-A/IL-17) was selected and analyzed for cytokine expression by Northern blot. RESULTS: There was no significant difference in the in vitro proliferation rate among parent Meth-A, cells transfected with vector alone and Meth-A/IL-17 cells. When the tumor cells were transplanted subcutaneously into BALB/c nude (nu+/nu+) mice, there was no difference in in vivo growth rates among the three cell lines. Challenge with tumor cells in conventional BALB/c mice, however, resulted in the rejection of Meth-A/IL-17 cells, but the other two lines did grow. After immunization with Meth-A/IL-17 cells, the mice were rechallenged by parent Meth-A or syngeneic MOPC-104E plasmacytoma cells; the immunized mice rejected the Meth-A cells, but not the MOPC-104E cells. Injecting the anti-thy 1,2 (CD90), anti-CD4 or anti-CD8 monoclonal antibody into conventional BALB/c mice resulted in the resumption of in vivo growth of Meth-A/IL-17 cells, but injecting the anti-asialo GM1 antibody did not. Furthermore, flow cytometric analysis demonstrated a significant increase in the expression of major histocompatibility complex (MHC) class I and class II antigens and lymphocyte function-associated antigen-1 on Meth-A/IL-17 cells. CONCLUSION: Meth-A cells transfected with the hIL-17 gene can induce tumor-specific antitumor immunity by augmenting the expression of MHC class I and II antigens, and both CD4+ and CD8+ T cells may play important roles in inducing antitumor immunity, suggesting the possibility of developing a tumor vaccine incorporating IL-17-transfected tumor cells.     

Anti-CD40-induced inflammatory E-cadherin+ dendritic cells enhance T cell responses and antitumour immunity in murine Lewis lung carcinoma

Background

Agonistic CD40 antibodies have been demonstrated to activate antigen-presenting cells (APCs) and enhance antitumour T cell responses, thereby providing a new therapeutic option in cancer immunotherapy. In agonistic CD40 antibody-mediated inflammatory responses, a novel subset of E-cadherin + dendritic cells (DCs) has been identified, and little is known about the role of these DCs in tumour immunity. This study investigated the effect of anti-CD40-mediated inflammatory E-cadherin + DCs in murine Lewis lung carcinoma (LLC).

Methods

The phenotype and characteristics of anti-CD40-mediated inflammatory E-cadherin + DCs isolated from the anti-CD40 model were assessed in vitro. The antitumour activity of E-cadherin + DCs were evaluated in vivo by promoting the differentiation of effector CD4+ T cells, CEA-specific CD8+ T cells and CD103+ CD8+ T cells and assessing their resistance to tumour challenge, including variations in tumour volume and survival curves.

Results

Here, we demonstrated that anti-CD40-mediated E-cadherin + inflammatory DCs accumulate in the lungs of Rag1 KO mice and were able to stimulate naïve CD4+ T cells to induce Th1 and Th17 cell differentiation and polarisation and to inhibit regulatory T cell and Th2 responses. Importantly, with the adoptive transfer of E-cadherin + DCs into the Lewis lung cancer model, the inflammatory DCs increased the Th1 and Th17 cell responses and reduced the Treg cell and Th2 responses. Interestingly, following the injection of inflammatory E-cadherin + DCs, the CD103+ CD8+ T cell and CEA-specific CD8+ T cell responses increased and exhibited potent antitumour immunity.

Conclusions

These findings indicate that anti-CD40-induced E-cadherin + DCs enhance T cell responses and antitumour activity in non-small cell lung cancer (NSCLC)-bearing mice and may be used to enhance the efficacy of DC-based peptide vaccines against NSCLC.

Electronic supplementary material

The online version of this article (doi:10.1186/s13046-015-0126-9) contains supplementary material, which is available to authorized users.     

Bispecific antibody-directed antitumor activity of human CD4+ helper/killer T cells induced by anti-CD3 monoclonal antibody plus interleukin 2.

Freshly isolated human CD4+ T cells can not respond to recombinant interleukin 2 (rIL-2) because of their lack of p75 IL-2 receptor expression. However, we succeeded in inducing a marked proliferation of purified CD4+ T cells by activation with rIL-2 plus anti-CD3 monoclonal antibody (mAb) cross-linked to a plastic plate. The proliferated CD4+ T cells produced a significant amount of IL-2 upon stimulation with phorbol ester plus A23187. Interestingly, CD4+ T cells activated with anti-CD3 mAb plus rIL-2 revealed a strong cytotoxic activity against Fc receptor (FcR)-positive tumor cells in the presence of anti-CD3 mAb. Moreover, the CD4+ T cells could lyse FcR-negative glioma cells by targeting with bispecific mAb containing anti-CD3 mAb and anti-glioma mAb. Thus, we demonstrated that rIL-2 and immobilized anti-CD3 mAb allowed the rapid generation of human CD4+ helper/killer T cells, which may be useful for the development of a new adoptive tumor immunotherapy.     

Combinations of tumor-specific CD8+ CTLs and anti-CD25 mAb provide improved immunotherapy

One new approach to cancer therapy is based on the adoptive transfer of tumor-specific cytotoxic T cells and anti-CD25 antibodies. In the present study, CD8+ and IFN-gamma secreting T lymphocytes (CTLs) were enriched as tumor-specific cytotoxic T cells from spleen lymphocytes of mice bearing the Renca tumor (a murine renal carcinoma line originating from a BALB/c mouse) after stimulation with tumor cells. An anti-CD25 IL-2Ralpha(anti-CD25) mAb from hybridoma PC61 was used for depletion for CD4(+)CD25(+) regulatory T (Treg) cells. Treatment-efficacy for tumor-bearing mice was compared using 4 systems: 1, whole spleen lymphocytes stimulated with tumor cells in vitro from tumor-bearing mice; 2, CTLs; 3, anti-CD25 mAbs; 4, CTLs and anti-CD25 mAbs. At the 50th day after tumor inoculation, in the group which received anti-CD25 mAb for depletion of T cells and inoculation of CTLs, tumors had disappeared and no re-growth was observed. In contrast, all mice of the non-treated and other three groups, treated with whole spleen cells alone, CTLs alone and anti-CD25 mAb alone, had died. These results showed that a combination of Treg cell-depletion using anti-CD25 mAbs and CTL administration is a feasible approach for treatment of cancers which warrants further exploration in the clinical setting.     

Effects of IL-17A on the occurrence of lung adenocarcinoma

The relationship between IL-17A and cancer, whether beneficial or antagonistic, continues to be a controversial issue. In this study, effects of IL-17A on lung adenocarcinoma were investigated using lung cancer cell lines, 95D and 95C. In the presence or absence of IL-17A, cell proliferation and VEGF secretion were detected. Effects of IL-17A on capillary networks and process of angiogenesis were also evaluated. In vivo, the level of IL-17A was assayed in the serum of lung adenocarcinoma patients. At the same time, slices of adenocarcinoma tissue were analyzed for expression of IL-17A, its receptor (IL-17RA), VEGF, CD4(+)-IL-17A+ cells and CD8(+)-IL-17A+ cells by immunohistochemistry and immunofluorescence assays. IL-17A did not have effect on the proliferation of 95D or 95C cells, however, the elevated expression of VEGF in supernatant of 95D or 95C cells was found to be IL-17A concentration-dependent. Supernatants from 95D or 95C cells treated with IL-17A could obviously facilitate angiogenesis, compared with IL-17A absence group (P < 0.01). Higher levels of IL-17A were detected in serum of patients with lung adenocarcinoma than healthy controls (P < 0.001). Higher positive expressions of IL-17A, IL-17RA and VEGF were confirmed in lung adenocarcinoma lesion tissues compared to pericancerous normal tissues (P < 0.001). Tnc17 cells, as well as Th17 cells were found in adenocarcinoma tissue, indicating a potential role of these cells in disease. In summary, IL-17A might affect lung adenocarcinoma by promoting angiogenesis, while the role of Tnc17 cells or Th17 cells remains to be elucidated.     

白细胞介素-17过表达胃癌细胞 在小鼠体内的成瘤效应研究

目的：建立小鼠荷瘤模型,研究白细胞介素-17（IL-17）过表达的胃癌细胞在小鼠体内的成瘤效应。方法：分别以MFC、MFC/pcDNA3.1和MFC/IL-17细胞接种615小鼠,细胞浓度为5×10⁶个/mL,按每只200 μL注射于小鼠背部皮下。每组10只,观察小鼠皮下肿瘤生长情况。3周后处死小鼠,分别采用RT-PCR和Western blot法检测肿瘤组织内IL-17、VEGF、Podoplanin mRNA和蛋白表达;免疫组化法检测微血管密度。结果：MFC/IL-17组小鼠于接种后5~7 d开始形成肿瘤结节,且生长迅速,肿瘤质量为（5.384±0.391）g,大于MFC组[（1.726±0.445）g]和MFC/pcDNA3.1组[（2.064±0.397）g]（P < 0.05）。RT-PCR和Western blot结果显示,与MFC和MFC/pcDNA3.1组相比,MFC/IL-17组小鼠肿瘤组织内IL-17、VEGF、Podoplanin mRNA和蛋白表达均明显增加（P < 0.05）。免疫组化结果显示,与MFC和MFC/pcDNA3.1组相比,MFC/IL-17组小鼠肿瘤组织内微血管密度明显增加（P < 0.05）。结论：IL-17可能通过上调VEGF、Podoplanin和CD31的表达促进血管和淋巴管形成,加速肿瘤生长。     

Further evidence for a role of tumor CD200 expression in breast cancer metastasis: decreased metastasis in CD200R1KO mice or using CD200-silenced EMT6

Previous studies reported that CD200 expression on cells of the transplantable EMT6 mouse breast cancer line was increased during growth in immunocompetent mice. Low levels of expression persisted in NOD-SCID.IL-2^??r?/? mice or mice with generalized over-expression of a CD200 transgene (CD200^tg mice), despite the faster tumor growth in both of these latter strains. We also showed that CD200 expression (by the host and/or tumor cells) led to increased seeding of tumor cells to DLN in immunocompromised (CD200^tg or NOD-SCID.IL-2^??r?/?) vs immunocompetent mice, using limiting dilution cloning of tumor cells from DLN (vs contralateral lymph nodes, CLN). Evidence for an important role for CD200 expression in this increased metastasis came from the observation that neutralization of CD200 by anti-CD200mAbs decreased tumor metastasis and increased levels of cytotoxic anti-tumor immune cells in DLN. In the current studies, we have extended these observations by exploring tumor growth/metastasis in CD200R1 KO mice in which we have previously shown, in a transplant model, that expression of CD200 fails to deliver an immunosuppressive signal. In addition, we have studied local and metastatic growth in healthy control mice of EMT6 tumor cells stably transduced with shRNA able to silence CD200 expression. In both scenarios, decreased metastasis was observed, with increased immunity to EMT6 detected by cytotoxicity assays. In addition, adoptive transfer of DLN to control mice attenuated EMT6 metastases implying a potential therapeutic benefit from neutralizing CD200 expression in breast cancer.     

Human c-erbB-2 proto-oncogene product as a target for bispecific-antibody-directed adoptive tumor immunotherapy.

To develop an efficient strategy for the targeting of anti-tumor effector cells, we prepared bispecific antibody (BsAb) containing anti-CD3 and an anti-c-erbB-2 proto-oncogene product. The prepared BsAb specifically reacts with both c-erbB-2-positive tumor cells and CD3+ CTL. Human CD4+ helper/killer T cells, induced from peripheral-blood mononuclear cells by activation with immobilized anti-CD3 monoclonal antibody (MAb) plus IL-2, showed no significant cytotoxicity against tumor cells. However, treatment of human CD4+ helper/killer cells with the BsAb caused the induction of specific cytotoxicity against c-erbB-2-positive tumor cells. CD4+ helper/killer cells also produced significant amounts of IL-2 during co-culture with c-erbB-2-positive tumor cells in the presence of the BsAb. Moreover, by combination with the BsAb, CD4+ helper/killer cells showed a strong in vivo anti-tumor effect against c-erbB-2 transfectant or human colon-cancer cells implanted in nude mice. Our results strongly suggest that the c-erbB-2 proto-oncogene product on human tumor cells may be a good target for BsAb-directed adoptive tumor immunotherapy.