B7-H3在人肝癌细胞株HepG2对外周血CD8+T细胞活化、周期及分泌IL-17调节中的作用

目的 研究B7-H3在人肝癌细胞株HepG2对人外周血CD8+T细胞活化、周期及IL-17分泌等调节中的作用.方法 RT-PCR及FCM检测B7-H3在HepG2细胞上的表达;应用脂质体法将PGPU6/GFP/neo-B7-H3shRNA质粒转入肝癌细胞株HepG2,阻断B7-H3的表达;免疫磁珠分选健康人外周血CD8+T细胞;FCM分析B7-H3分子在HepG2细胞对PHA刺激下CD8+T细胞活化、周期及PMA刺激下CD8+T细胞分泌IL-17调节中的作用.结果 肝癌细胞株HepG2高表达B7-H3分子,PGPU6/GFP/neo-B7-H3 shRNA质粒能有效阻断B7-H3在HepG2细胞上的表达;FCM分析结果显示,肝癌细胞株HepG2对CD8+T细胞活化及周期均有抑制作用;阻断B7-H3的表达后,明显减弱HepG2细胞对CD8+T细胞早期活化表型CD69表达的抑制作用,且能够通过下调CD8+T细胞Go/G1期细胞数量,上调S期细胞数量逆转HepG2细胞对CD8+T细胞周期的阻滞作用;在HepG2存在条件下,CD8+T细胞对IL-17的分泌明显增加,阻断B7-H3的表达后,IL-17的分泌被进一步上调.结论 HepG2细胞高表达B7-H3分子;B7-H3能够协同HepG2细胞对CD8+T细胞活化表型CD69的表达及细胞周期的抑制作用;HepG2细胞上调CD8+T细胞对IL-17的分泌作用,但B7-H3可抑制该上调作用.     

乳腺癌异常表达B7-H4对T细胞分泌细胞因子和凋亡的影响

目的:探讨B7-H4在乳腺癌中的表达及其对外周血T细胞分泌细胞因子及增殖、凋亡的影响。方法:应用SP免疫组化法检测B7-H4在乳腺浸润性癌、癌旁组织和纤维腺瘤的表达。流式细胞术分析B7-H4对乳腺癌患者外周血活化T细胞增殖和凋亡的作用。ELISA芯片检测T细胞培养上清液细胞因子的含量。结果:B7-H4在乳腺浸润性癌的阳性表达率为84.62%(44/52),显著高于癌旁组织和纤维腺瘤组织(P0.01,P0.01)。体外淋巴细胞混合培养结果显示,与空白组比较,B7-H4对乳腺癌患者外周血活化T细胞的增殖指数Ki67无明显影响;但B7-H4诱导CD8~+T细胞凋亡的作用强于CD4~+T细胞。B7-H4组FOXP3~+T/CD4~+T也高于空白组(P0.05)。B7-H4组较空白组细胞培养上清液中TGF-β1、IL-17含量均显著增高(P0.05,P0.05)。结论:乳腺浸润性癌异常表达B7-H4。B7-H4在体外能促进CD8~+T细胞凋亡、促进T细胞分泌TGF-β1和IL-17。B7-H4在削弱乳腺癌微环境的抗肿瘤细胞免疫中发挥一定作用。     

协同刺激分子B7-H4在胃癌组织中的表达及定位

探讨负性协同刺激分子B7-H4在胃癌组织及浸润免疫细胞亚群中的表达及定位。应用免疫组织化学、免疫荧光染色及激光共聚焦技术检测B7-H4蛋白在胃癌组织中的表达及定位;流式细胞术检测分析胃癌患者PBMC亚群(CD3+T细胞、CD19+B细胞、CD14+单核细胞及粒细胞)中B7-H4的表达。免疫组织化学检测结果表明,B7-H4在胃癌组织中呈阳性表达。免疫荧光染色及激光共聚焦检测结果显示,B7-H4在胃癌组织中呈阳性表达,并主要定位于胃癌细胞的细胞膜,特别是在Ki67+高度增殖的胃癌细胞中B7-H4呈高表达,且B7-H4在CD34+血管内皮细胞中呈高表达;B7-H4在浸润CD19+B细胞和CD68+巨噬细胞中呈高表达;而在胃癌浸润CD4+T细胞、CD8+T细胞及CD57+NK细胞中呈弱表达。流式细胞术检测结果表明,B7-H4在胃癌患者外周血浸润CD19+B细胞及粒细胞中呈阳性表达,而在浸润CD3+T细胞及CD14+单核细胞中呈弱表达。本研究结果提示B7-H4在胃癌组织中可表达于肿瘤细胞和部分浸润免疫细胞,并可通过其介导的信号途径参与胃癌的发生与发展。检测B7-H4的表达可作为潜在的胃癌诊断、治疗及预后判断的新靶点。     

乳腺癌异常表达B7-H4对T细胞分泌细胞因子和凋亡的影响 乳腺癌异常表达B7-H4对T细胞分泌细胞因子和凋亡的影响

目的：探讨B7-H4在乳腺癌中的表达及其对外周血T细胞分泌细胞因子及增殖、凋亡的影响。方法：应用S-P免疫组化法检测B7-H4在乳腺浸润性癌、癌旁组织和纤维腺瘤的表达。流式细胞术分析B7-H4对乳腺癌患者外周血活化T细胞增殖和凋亡的作用。ELISA芯片检测T细胞培养上清液细胞因子的含量。结果：B7-H4在乳腺浸润性癌的阳性表达率为84.62%(44/52),显著高于癌旁组织和纤维腺瘤组织（P<0.01,P<0.01 ）。体外淋巴细胞混合培养结果显示,与空白组比较,B7-H4对乳腺癌患者外周血活化T细胞的增殖指数Ki67无明显影响;但B7-H4诱导CD8+T细胞凋亡的作用强于CD4+T 细胞。B7-H4组FOXP3+T/CD4+T也高于空白组（P<0.05 ）。B7-H4组较空白组细胞培养上清液中TGF-β1、IL-17含量均显著增高（P<0.05,P<0.05）。结论：乳腺浸润性癌异常表达B7-H4。B7-H4在体外能促进CD8+T细胞凋亡、促进T细胞分泌TGF-β1 和IL-17。B7-H4在削弱乳腺癌微环境的抗肿瘤细胞免疫中发挥一定作用。     

链球菌CpG DNA对寻常型银屑病患者T细胞活化的影响

研究链球菌核酸成分对寻常型银屑病患者T细胞活化的影响。利用层析法去除A型β溶血型链球菌超声粉碎产物中的核酸成分,分别用链球菌全菌抗原(streptococcal antigen,SA)和去除核酸的链球菌抗原(nucleic acid depleted-streptococ-cal antigen,non-NASA)刺激寻常型银屑病患者(20例)和正常人(12例)外周血单个核细胞(PBMC);同时non-NASA联合人工合成的CpG ODN(CpG-A和CpG-B)以及单用CpG ODN刺激患者PBMC(12例),24 h后采用流式细胞术检测并比较总T细胞及皮肤淋巴细胞抗原阳性(CLA+)T细胞活化表达CD69+及患者B细胞活化表达CD86+的百分率的差异。结果显示,在银屑病患者中,SA刺激后总T细胞和CLA+T细胞活化表达CD69+的百分率均高于non-NASA(P=0.012和0.042),而在正常人中两者无统计学差异,且均不激活T细胞表达CD69+(P>0.05);同时non-NASA联合CpG-A刺激患者PBMCs后总T细胞及CLA+T细胞表达CD69+的百分率亦高于non-NASA单独刺激(P=0.031和0.022),但联合CpG-B未发现差异(P>0.05),且CpG-A和B单独刺激对T细胞活化表达CD69+的百分率无影响(P>0.05)。另一方面,SA、non-NASA以及后者联合CpG-A刺激患者B细胞活化表达CD86+的百分率无统计学差异(P>0.05),但non-NASA联合CpG-B刺激可显著增加B细胞CD86+的表达率(P<0.01),同时CpG-B可激活B细胞表达CD86+,CpG-A无此作用。研究表明,去除核酸的链球菌抗原降低银屑病患者总T细胞以及CLA+T细胞的活化,但对B细胞活化无影响,提示链球菌CpG DNA可协同菌体蛋白诱导病理性T细胞的活化,参与银屑病的发生。     

IL-27诱导原发性胆汁性肝硬化患者CD4+T细胞增殖分化作用机制研究

目的 探讨IL-27对原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)患者外周血CD4+T细胞的增殖分化作用及其相关免疫学机制.方法 收集PBC患者、慢性乙肝患者(choronic hepatitis B,CHB)、健康体检者(health controls,HCs)外周血,磁珠分离CD4+T细胞.IL-27体外作用后,CCK-8测定细胞增殖情况,ELISA法检测细胞因子,定量PCR分析T-bet和GATA3基因表达情况,免疫印迹测定p-STAT-1和p-STAT-3的表达.结果 IL-27作用后,PBC组、CHB组和HCs组CD4+T细胞增殖能力均显著增强,PBC组CD4+T细胞增殖能力强于CHB和HCs组,差异有统计学意义(P＜0.001),同时PBC组细胞培养液中IL-2和IFN-γ在IL-27作用后较CHB和正常对照组均显著增高(P＜0.001),IL-10表达无明显变化.未经IL-27诱导情况下,PBC组T-bet表达高于CHB组(P=0.007),IL-27诱导后PBC组CD4+T淋巴细胞T-bet基因表达显著增加,同时对GATA3具有抑制作用,作用前后差异有统计学意义(P＜0.001),CHB组作用前后无显著变化(P=0.3).免疫印迹测定p-STAT-1、p-STAT-3发现,正常情况下各研究组均不表达p-STAT-1、p-STAT-3,IL-27作用后,表达明显升高,其中PBC组升高尤为明显.结论 IL-27可以诱导PBC患者CD4+T细胞增殖,并通过活化p-STAT-1、p-STAT-3信号通路,诱导CD4+T细胞向Th1分化,同时分泌相关细胞因子,可能在PBC早期免疫炎症反应过程中具有重要作用.     

再生障碍性贫血患者外周血CD4~＋CD25~＋调节性T细胞及Foxp3的变化及临床意义

目的:探讨再生障碍性贫血(AA)患者外周血中CD4+CD25+调节性T细胞(Tress)及Foxp3的表达及临床意义.方法:25例AA病人,其中含非重型再障(nSAA)18例、重型再障(SAA)7例,采用四色流式细胞检测技术分析从患者外周血单个核细胞(PBMC)中CD4+T细胞、CD4+CD25+/CD4+、CD4+CD25high/CD4+T细胞百分比及绝对计数,并进一步分析AA患者PBMC的CD4++CD25+、CD4+CD25low及CD4+CD25highT细胞中Foxp3+T细胞的百分比,同时利用RT-PCR方法检测PBMC中Foxp3mRNA表达水平,并与29例正常对照组的上述指标进行比较.结果:与正常对照组相比,SAA患者及nSAA患者PBMC中CDM+T细胞、CD4+CD25+/CD4+、CD4+CD25high/CD4+百分比及绝对计数减低(P<0.05),并且SAA组明显低于nSAA组(P<0.001);从患者CD+CD25+、CD4+CD25low及CD4+CD25highT细胞中Foxp3的表达较正常对照组减低(P<0.05);AA患者PBMC中Foxp3 mRNA表达水平与正常对照组没有明显差别(P>0.05).结论:CD4+CD25+调节性T细胞减低可能与从的发病有关,SAA的Treg表达低于nSAA,为其作为从病情变化的判断指标提供进一步的依据.     

不同类型HBV感染者外周血Treg/Th17细胞的变化及意义

目的:探讨不同类型HBV感染者外周血中CD4+ Foxp3+ Treg/Th17细胞的变化及意义.方法:选取15例急性乙型肝炎(Acute Hepatitis B,AHB)患者、40例慢性乙型肝炎(Chronic hepatitis B,CHB)患者、40例无症状携带者(Asymptomatic HBV carriers,AsC)及30例健康对照者,分别采用流式细胞术、RT-PCR和ELISA检测外周血CD4+ Foxp3+ Treg/Th17细胞百分率、核转录因子foxhead winged-helix box protein 3 (Foxp3)/retinoid-related orphan receptor gamma-t (RORγt) mRNA的表达以及血浆转化生长因子-β1(Transforming growth factor-31,TGF-β1)/IL-17的水平.结果:AHB组患者CD4+ Foxp3+ Treg/CD4+T细胞百分率、Foxp3 mRNA及TGF-β1水平与正常对照组相比无明显差异(P＞0.05);而CD4+ IL-17 +/CD4+T细胞百分率、RORγtmRNA及IL-17水平与正常对照组相比明显升高,差异有统计学意义(P＜0.05).CHB组患者CD4+ Foxp3+ Treg/CD4+T细胞百分率、Foxp3 mRNA、TGF-31水平及CD4+ IL-17 +/CD4+T细胞百分率、RORγt mRNA、IL-17水平与正常对照组相比均明显升高,差异有统计学意义(P＜0.05);与正常对照组相比,AsC组患者CD4+ Foxp3+ Treg/CD4+T细胞百分率、Foxp3 mR-NA、TGF-β1水平及CD4+ IL-17 +/CD4+T细胞百分率、RORγt mRNA、IL-17水平无明显差异(P＞0.05).结论:在不同类型HBV感染者外周血中Treg/Th17细胞失衡,Treg/Th17细胞可能与HBV感染的状态有关.     

IL-27介导的炎症反应在原发性胆汁性肝硬化中的作用

目的 探讨IL-27介导的免疫炎症性反应在原发性胆汁性肝硬化(primary biliary cirrhosis,PBC)发病中的作用.方法 荧光定量PCR、流式细胞术、酶联免疫吸附试验(ELISA)、免疫组化等方法检测PBC患者IL-27的表达;生化常规测定PBC患者和健康对照者的谷草转氨酶(AST)、碱性磷酸酶(ALP)、谷丙转氨酶(ALT)、总胆红素(TBIL)、谷氨酰基转移酶(GGT)水平,分析IL-27与它们之间的相关性.结果 PBC患者外周血IL-27/P28亚基mRNA的表达显著高于慢性乙型肝炎组和健康对照组(P＜0.05),IL-27/EBI3 mRNA未有明显改变;免疫组化结果显示IL-27在PBC患者肝脏呈阳性表达;流式细胞仪检测结果表明IL-27在PBC患者的CD4+T细胞表达率(72.40%±6.22%)较慢性乙型肝炎组(59.40%±7.03%,P＜0.01)和健康对照组(1.70%±0.55%,P＜0.01)明显升高,而IL-27在各研究组的CD8+T细胞、单核细胞、B细胞中的表达无明显变化;PBC患者外周血血清IL-27的表达[(126.25±36.00)pg/ml]显著高于慢性乙型肝炎组[(51.81±23.30)pg/ml,P＜0.01)和健康对照组[(34.19±9.70)pg/ml,P＜0.01],PBC患者IL-27的血清蛋白表达水平与GGT(r=0.554,P＜0.01)和TBIL(r=0.559,P＜0.01)水平呈显著正相关,与ALT、AST和ALP无相关性.结论 IL-27在PBC中表达升高,与PBC的发生发展存在一定的相关性,可能参与了PBC的发病机制.     

吸入糖皮质激素对哮喘儿童外周血Foxp3~+调节性T细胞及IL-2、IL-6的影响及其机制研究

研究吸入丙酸氟替卡松(FP)对哮喘患儿外周血单个核细胞(PBMC)中CD4+Foxp3+调节T细胞、细胞因子IL-2、IL-6以及转录因子STAT5的影响。以30例确诊为支气管哮喘的患儿为研究对象,随机分为未治疗哮喘组(15例)、吸入FP缓解组(15例),10例同期正常儿童为对照组。流式细胞仪检测外周血PBMC中的CD4+Foxp3+调节T细胞比率,ELISA检测血浆或培养上清中IL-2、IL-6细胞因子水平,Western blot检测PBMC中磷酸化及非磷酸化STAT5的水平。结果1.未治疗哮喘组PBMC中CD4+Foxp3+T细胞百分率在PHA刺激培养前后均明显低于正常对照组,吸入FP缓解组明显升高,与正常对照组没有差异;各组刺激后CD4+Foxp3+T细胞百分率均有升高,吸入FP缓解组、正常对照组分别升高约1.89、2.01倍,而未治疗哮喘组升高仅1.56倍;2.未治疗哮喘组血浆中IL-6水平明显高于正常组及吸入FP缓解组,而IL-2水平没有明显差异;3.PHA刺激24 h后未治疗哮喘组磷酸化STAT5(p-STAT)表达水平明显低于吸入FP组及正常对照组,而各组STAT5表达水平没有明显差异,结论吸入FP能增加哮喘患儿外周血PBMC中CD4+Foxp3+调节T细胞数量,其机制可能与降低血浆IL-6,上调STAT5磷酸化水平有关。     

艾滋病患者T淋巴细胞表面归巢分子的表达在抗病毒治疗后升高

目的 探讨艾滋病(AIDS)患者高效抗反转录病毒治疗(HAART)前后T淋巴细胞表面归巢分子CD49d、CCR9、CD62L表达的变化情况.方法 采用流式细胞术检测42例艾滋病患者和18例HIV阴性健康对照的外周血T淋巴细胞表面CD49d、CCR9和CD62L表达,用BD FACSDiva软件分析计算各组细胞表达的百分率.结果 治疗后组外周血的平均CD4~+T淋巴细胞明显高于治疗前组(P<0.01);治疗前组CD3~+CD49d~+、CD3~+ CCR9~+、CD3~+CD62L~+、CD3~+CD4~+、CD4~+CD49d~+、CD4~+CCR9~+、CIM~+CD62L~+、CD8~+CD49d~+、CD8~+CD62L~+T淋巴细胞的百分率显著低于治疗后组和阴性对照组(P<0.05);CD3~+CD8~+T淋巴细胞的百分率高于治疗后组(P<0.05).治疗后组CD3~+CCR9~+、CD8~+CCR9~+、CD8~+CD62L~+T淋巴细胞的百分率均低于阴性对照组(P均<0.001).结论 AIDS患者外周血T淋巴细胞亚群不仅比例失调,而且其表面表达肠道归巢分子CD49d、CCR9,淋巴结归巢分子CD62L的数量发生异常改变.抗病毒治疗可以逆转以上部分免疫病理变化.建议肠道归巢分子CD49d、CCR9和淋巴结归巢分子CD62L可作为艾滋病疾病进展和评价机体HAART后免疫重建的指标.     

B7-H4在人子宫颈癌的表达及其与肿瘤内浸润T细胞亚群的相关性研究

目的:研究共刺激分子B7-H4在人子宫颈癌的表达及其与肿瘤内浸润的FOXP3+、CD4+T、CD8+T细胞数量和分泌细胞因子的关系。方法:采用SP免疫组织化学染色检测B7-H4在30例正常人宫颈组织、30例高级别宫颈上皮内瘤变(Cervical intraepithelial neoplasiaⅡ-Ⅲ,CINⅡ-Ⅲ)和67例子宫颈癌的表达;间接免疫荧光双标(Indirect immunofluorescentdouble-staining)观察肿瘤内浸润的FOXP3+、CD4+T、CD8+T细胞数量及其TGF-β1和IFN-γ分泌情况。结果:B7-H4不表达于正常人宫颈上皮,仅在部分瘤变宫颈上皮微弱表达;子宫颈癌B7-H4的阳性表达率为46%(31/67),显著高于正常人宫颈上皮和瘤变宫颈上皮(P<0.01,P<0.05);子宫颈癌B7-H4阳性组病灶内浸润的CD8+T细胞以及分泌IFN-γ的CD8+T数量显著低于B7-H4阴性组(P<0.001,P<0.035);B7-H4的表达与肿瘤内浸润的FOXP3+细胞、CD4+T细胞以及分泌TGF-β1的CD4+T细胞数量无关(P>0.05,P>0.05)。结论:B7-H4在人子宫颈癌细胞异常高表达,并与肿瘤内浸润的CD8+T细胞数量减少以及分泌IFN-γ减少有关,提示B7-H4在抑制肿瘤微环境的细胞免疫中发挥作用。     

Blocking of monocyte-associated B7-H1 (CD274) enhances HCV-specific T cell immunity in chronic hepatitis C infection

The establishment of a chronic hepatitis C (CHC) infection is associated with defective HCV-specific T cell responses. Recent studies suggest that negative T cell regulators such as programmed death 1 (PD-1) contribute to the impairment of virus-specific T cell functions in chronic viral infections. However, the implication of peripheral monocytes from CHC patients in the inhibition of HCV-specific T cell responses is only partially defined. In this study, we found that B7-H1, a ligand of PD-1, was significantly up-regulated on monocytes of CHC patients. Proliferation of T cells in response to anti-CD3 antibody was directly suppressed by B7-H1+CD14+ monocytes, and this suppression was reversed by addition of antagonistic B7-H1 mAb. Furthermore, blocking of monocyte-associated B7-H1 (moB7-H1) significantly enhanced the frequency of IFN-gamma-producing, HCV-specific CD4+ and CD8+ effector T cells and the production of Th1 cytokines, such as IL-2 but not Th2 cytokines, including IL-4 and IL-10. Upon B7-H1 blockade, production of perforin was also increased in CD8+ T cells stimulated with HCV peptides. Our findings suggest that moB7-H1 inhibits HCV-specific CD4+ and CD8+ T lymphocyte proliferation and suppresses Th1 cytokine production and perforin secretion. Blockade of the B7-H1 pathway thus represents an attractive approach in the treatment of chronic HCV infection.     

Clinical observation of immunity in patients with secondary infection from severe acute pancreatitis

Objectives

To observe immune system changes in patients with secondary infection from severe acute pancreatitis (SAP).

Methods

Seventy-nine patients were recruited. The percentages of CD4+, CD8+, natural killer (NK), HLA-DR+ cells and B lymphocytes, and the CD4+/CD8+ ratio, were determined. In addition, tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-10 (IL-10), and interleukin-4 (IL-4) serum levels were determined on days 1, 7, 14, and 28.

Results

Fifteen patients had a secondary infection. The immune response of the infected group was quite different from the non-infected group, with a higher percentage of CD4+ and HLA-DR+ cells on days 1, 7, 14 and 28, a higher percentage of CD8+ and NK cells on days 14 and 28, a reduced CD4+/CD8+ ratio, and a reduction in B lymphocytes. The cytokine levels in the infected group were different from the non-infected group, with a rise in TNF-α and IL-6 through the first 2?weeks, but dropping at 1?month. IL-10 and IL-4 increased initially, but then dropped over the next 3?weeks.

Conclusions

An early excessive immune response followed by a subsequent immune deficiency is closely related to secondary SAP infection.     

晚期肺癌患者调节性T细胞和IL-10表达的变化

探讨晚期肺癌患者的CD4＋CD25＋调节性T细胞（Treg）、IL-10及其他T细胞亚群表达的临床意义。检测晚期肺癌患者92例和正常对照者64名的IL-10（ELISA法）、CD4＋CD25＋调节性T细胞及其他T细胞亚群（流式细胞仪法）。结果显示：肺癌组血清IL-10明显大于对照组（278±34ng/L vs 122±65ng/L,P〈0.01）、CD4＋CD25＋Treg细胞明显大于对照组[（17.8±5.2）%vs（7.1±0.4）%,P〈0.01]、CD3＋、CD4＋、CD8＋CD28＋和NK细胞明显小于对照组[（58.4±7.8）%vs（78.2±6.4）%,P≤0.05;（34.4±7.6）%vs（44.9±8.4）%,P〈0.01;（9.4±3.6）%vs（16.5±2.7）%,P〈0.01;（9.4±3.6）%vs（18.5±7.2）%,P〈0.01]。CD8＋T细胞明显高于对照组[（37.8±6.5）%vs（31.8±5.1）%,P〈0.01]。CD4＋CD25＋Treg细胞、IL-10与CD8＋CD28＋细胞、NK细胞呈明显负相关。这些结果表明,CD4＋CD25＋Treg细胞与IL-10增多为晚期肺癌患者免疫功能受损的表现。     

Distribution of beta 7 integrins in human intestinal mucosa and organized gut-associated lymphoid tissue.

Two alternative integrins involved in mucosal homing (alpha 4 beta 7) or epithelial retention (alpha E beta 7) of lymphocytes were examined in the human gut. The distribution of the beta 7 subunit [monoclonal antibody (mAb) M301] was bimodal in that it was strongly expressed by alpha E beta 7 + cells but weakly by alpha 4 beta 7 + cells. More than 90% of intraepithelial lymphocytes (IEL), including the minor subsets of CD4+, T-cell receptor (TCR) gamma/delta +, and CD3- cells, expressed alpha E beta 7 as did most lamina propria CD8+ (88%) and a fraction (36%) of CD4+ lymphocytes. Conversely, B-lineage cells (CD19+) and macrophages (CD68+) were negative. In gut-associated lymphoid tissue (GALT: Peyer's patches and appendix) only a few (< 5%) cells were positive for alpha E beta 7 (confined to CD8+ lymphocytes and CD11c+ putative dendritic cells). A relatively small fraction of IEL (30-50%) expressed alpha 4 beta 7 (mAb Act-1), while most (70%) lamina propria T and B lymphocytes, blasts, plasma cells and macrophages were positive. In GALT, T lymphocytes expressed similar levels of alpha 4 beta 7 as in the lamina propria whereas relatively few B lymphocytes (< 50%) were positive. Isolated lamina propria CD8+, CD4+, CD19+, and CD38+ cells contained mRNA for alpha 4 and the former three subsets as well as appendix CD8+ cells also for beta 7 while only lamina propria CD8+ cells had mRNA for alpha E. Together, the results suggested that alpha E beta 7 and alpha 4 beta 7 are differentially regulated in inductive sites and effector sites of the human gut. Because lymphoid cells at both sites expressed mainly alpha 4 beta 7, this integrin may be a homing receptor on memory and effector cells bound for lamina propria as well as on naive lymphocytes extravasating in GALT. Conversely, because alpha E beta 7 was mainly expressed by CD8+ cells in epithelium and lamina propria, it was probably induced after extravasation, in agreement with the observation that IEL and a fraction of lamina propria T lymphocytes (mainly CD8+ cells) generally expressed higher levels of beta 7 than most CD4+ and B cells. Also a subset of putative dendritic cells located near the follicle-associated epithelium of GALT expressed alpha E beta 7, perhaps reflecting epithelial interaction during primary immune responses.     

膜型B7-H1分子诱导产生的T抑制细胞及其免疫学特性的初步研究

为了探讨B7-H1分子诱导产生的T抑制细胞免疫生物学特性,我们首先构建了人B7-H1基因,筛选出细胞膜表面稳定表达B7-H1蛋白的人ECV304细胞系,并在体外建立了由这种非专职抗原提呈细胞系刺激诱导纯化的CD4+T细胞,并使其具有抑制功能的新方法。通过检测其增殖效应和上清细胞因子水平的变化,观察到这群细胞对同种异体混合淋巴细胞反应(MLC),具有明显抑制作用,该作用与大量产生IL-10、TGF-β细胞因子有关。实验表明,ECV304/B7-H1可有效诱导具有免疫抑制的功能T细胞,为其进一步应用于临床治疗打下基础。     

B7-H4 overexpression impairs the immune response of T cells in human cervical carcinomas

Objective

To investigate B7-H4 expression and its correlation with the number of infiltrating T lymphocytes and cytokine production by those lymphocytes in human cervical cancer and to determine the effect of recombinant B7-H4 on the active peripheral blood T cells of the patients in vitro.

Methods

B7-H4 expression was detected in 67 cases of cervical cancer using immunohistochemical staining. Tumor-infiltrating CD8⁺T, CD4⁺T, and FOXP3⁺ (Forkhead Box P3) T lymphocytes and their levels of IFN-γ and TGF-β₁ production were determined by immunofluorescent double-staining. After the peripheral blood T lymphocytes of patients were co-cultured with B7-H4, proliferation, apoptosis, and cell subtypes were analyzed using flow cytometry. Cytokines in the supernatant were detected by ELISA.

Results

B7-H4 was expressed in 46% (31/67) of the cases of cervical cancer. The number of infiltrating CD8⁺T lymphocytes and their IFN-γ production in positive B7-H4 expression cervical cancers was significantly lower than in negative B7-H4 cases (P < 0.01, P < 0.05), but there was no significant difference between cases positive and negative for B7-H4 with respect to infiltrating FOXP3⁺T and CD4⁺T cells or TGF-β1 production. After co-culture with B7-H4 for 48 h, the patients’ activated T lymphocytes were arrested at G1/G2 phase. The Ki67 positive rates of CD4⁺T and CD8⁺T cells were 2.13 ± 0.13% and 1.03 ± 1.33%, and they were lower than in the blank group. The proportion of CD4⁺T and CD8⁺T cells decreased, but CD4⁺T/CD8⁺T and the proportion of CD4⁺CD25⁺Foxp3⁺T cells increased. In addition, concentrations of IL-10 and TGF-β1 in the supernatant of co-cultured T cells increased significantly (P < 0.05, P < 0.05), but that of IFN-γ decreased. B7-H4 had no significant effect on apoptosis of the T cells.

Conclusion

B7-H4 is overexpressed in human cervical cancers, and it is associated with lower numbers of tumor-infiltrating CD8⁺T lymphocytes and therefore less IFN-γ production. In vitro, B7-H4 inhibits the proliferation of CD4⁺T and CD8⁺T but promotes the proliferation of Tregs and the secretion of IL-10 and TGF-β1. B7-H4 plays an important role in depressing the anti-tumor immunity of CD8⁺T cell in microenvironments of cervical cancer.