吉林大学学报(医学版)  2018, Vol. 44 Issue (03): 543-547

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王丹, 李怡飞, 历春, 董志恒, 董营, 盖晓东
WANG Dan, LI Yifei, LI Chun, DONG Zhiheng, DONG Ying, GAI Xiaodong
结直肠癌组织中B7-H1和B7-H4表达与Foxp3+调节性T细胞浸润的关联性
Relationships between expressions of B7-H1 and B7-H4 and Foxp3+ regulated T-cell infiltration in colorectal cancer tissue
吉林大学学报(医学版), 2018, 44(03): 543-547
Journal of Jilin University (Medicine Edition), 2018, 44(03): 543-547
10.13481/j.1671-587x.20180316

文章历史

收稿日期: 2017-09-21
结直肠癌组织中B7-H1和B7-H4表达与Foxp3+调节性T细胞浸润的关联性
王丹1 , 李怡飞2 , 历春1 , 董志恒1 , 董营1 , 盖晓东1     
1. 北华大学基础医学院病理教研室, 吉林 吉林 132013;
2. 吉林省吉林市中心医院骨关节科, 吉林 吉林 132011
[摘要]: 目的: 探讨B7-H1和B7-H4在结直肠癌(CRC)组织中的表达及其与肿瘤中Foxp3+调节性T细胞(Treg)浸润的关系,为判定CRC的生物学行为及预后提供依据。方法: 免疫组织化学法检测56例CRC患者肿瘤标本及癌旁正常组织样本中B7-H1、B7-H4蛋白的表达和Treg的浸润情况,并与CRC患者的临床病理特征进行关联性分析。结果: CRC患者CRC组织中B7-H1阳性表达率高于癌旁正常组织(χ2=72.34,P < 0.01)。不同浸润深度的CRC组织中B7-H1表达频数比较差异有统计学意义(P < 0.01);淋巴结转移阳性者CRC组织中B7-H1高表达频数高于淋巴结转移阴性者(P < 0.01);Duke's分期C+D者CRC组织中B7-H1高表达频数高于Duke's分期A+B者(P < 0.01)。CRC组织中B7-H4阳性表达率高于癌旁正常组织(χ2=78.92,P < 0.01)。不同浸润深度的CRC组织中B7-H4高表达频数比较差异有统计学意义(P < 0.05),淋巴结转移阳性者CRC组织中B7-H4高表达频数高于淋巴结转移阴性者(P < 0.05);肿瘤组织中Treg阳性细胞数明显高于癌旁正常组织(P < 0.01);分化程度不同CRC组织中Treg阳性细胞数比较差异有统计学意义(P < 0.01);淋巴结转移阳性者CRC组织中Treg阳性细胞数高于淋巴结转移阴性者(P < 0.01)。Treg浸润与B7-H1的表达有相关性(P < 0.01,Cramer's=0.463),Treg浸润与B7-H4的表达有相关性(P < 0.05,Cramer's=0.320)。结论: B7-H1和B7-H4在CRC组织中呈异常高表达,并与肿瘤中Treg浸润增加有关,可作为评估CRC预后的指标。
关键词: 结直肠肿瘤    B7-H1    B7-H4    调节性T细胞    
Relationships between expressions of B7-H1 and B7-H4 and Foxp3+ regulated T-cell infiltration in colorectal cancer tissue
WANG Dan1, LI Yifei2, LI Chun1, DONG Zhiheng1, DONG Ying1, GAI Xiaodong1     
1. Department of Pathology, School of Basic Medical Sciences, Beihua University, Jilin University, Jilin 132013, China;
2. Department of Orthopaedics, Central Hospital, Jilin City, Jilin Province, Jilin 132011, China
[Abstract]: Objective: To explore the expressions of B7-H1 and B7-H4 in colorectal cancer(CRC) tissue and their relationships with the Foxp3+ regulated T-cell (Treg) infiltrationin tumor tissue, and to provide the basis for judging the biological behaviours and prognosis of CRC. Methods: Immunohistochemistry was used to detect the expressions of B7-H1 and B7-H4 and the infiltration of Treg in 56 cases of CRC tissue samples and adjacent normal tissue samples, and their relationships with the clinicopathological features of CRC patients were analyzed. Results: The positive expression rate of B7-H1 in CRC tissue of the CRC patients was higher than that in adjacent normal tissue(χ2=72.34, P < 0.01).The differences of expression frequencies of B7-H1 in CRC tissues with different infiltration depths were significant (P < 0.01). The high expression frequency of B7-H1 in CRC tissue of the patients with positive lymphnode metastasis was higher than that in the patients with negative lymphnode metastasis(P < 0.01).The high expression frequency of B7-H1 in CRC tissue of the patients at Duke's stage (C+D) was higher than that of the patients at Duke's stage (A+B)(P < 0.01).The positive expression rate of B7-H4 in CRC tissue was higher than that in adjacent normal tissue(χ2=78.92, P < 0.01).The differences of expression frequencies of B7-H4 in CRC tissues with different infiltration depths were significant(P < 0.05).The high expression frequency of B7-H4 in CRC tissue of the patients with positive lymphnode metastasis was higher than that of the patients with negative lymphnode metastasis(P < 0.05).The number of Treg positive expression cells in CRC tissue was significantly higher than that in adjacent normal tissue (P < 0.01), the number of Treg postive expression cells in CRC tissues with different differentiation degrees were different(P < 0.01), and the number of Treg positive expression cells in CRC tissue of the patients with positive lymphnode metastasis was higher than that of the patients with negative lymph node metastasis(P < 0.01).The expression of B7-H1 was correlated with Treg infiltration(P < 0.01, Cramer's=0.463);the expression of B7-H4 was correlated with Treg infiltration(P < 0.05, Cramer's=0.320). Conclusion: B7-H1 and B7-H4 are highly expressed in human CRC tissue, and they are associated with the increasing of Treg infiltration; they can be used as the indicators to judge the prognosis of CRC.
Key words: colorectal neoplasms     B7-H1     B7-H4     regulatory T cells    

结直肠癌(colorectal cancer, CRC)是世界范围内最常见的恶性肿瘤之一。由于外科技术的进步、放疗和化疗药物的使用,临床上CRC患者的预后有明显的改善,但仍有复发和转移的危险。因此,有必要进一步探讨CRC免疫逃逸机制从而开发新的CRC治疗方法[1]。抗原提呈细胞调控T细胞免疫是通过B7家族中的一类共激活分子完成的。B7分子与T细胞表面相应受体结合后,在抗原特异性免疫应答过程中发挥正性或负性调控作用[2]。B7-H1是B7家族中的负性调控分子,其与受体PD-1的相互作用在肿瘤免疫应答中发挥重要作用[3]。B7-H4是B7家族中新发现的成员,其在卵巢癌、乳腺癌、子宫内膜癌和肺癌等多种肿瘤组织中表达,而在正常组织中几乎检测不到[4]。上述研究结果提示:B7-H1和B7-H4可能作为免疫反应负调节分子,通过不同途径在抗肿瘤免疫中发挥重要的作用[5]。调节性T细胞(Treg)是一个独特的CD4+辅助性T细胞亚群,其特点是CD4+及CD25+阳性表达。叉头转录因子(Foxp3+)是调节T细胞发育及其功能效应的关键因素,也是Treg的特异且可靠的标记[6]。Treg在许多人类肿瘤组织中均有表达,与肿瘤免疫逃避机制有密切关联[7]。研究者[8-9]发现:B7-H1与肿瘤微环境中Treg表达量增加相关,B7-H4对Treg功能具有调控作用。抑制性B7分子在肿瘤细胞中的表达与Treg浸润的相关性目前尚无研究报道。本研究选择56例CRC患者的癌组织作为研究对象,检测B7-H1和B7-H4在CRC组织中的表达及Treg在CRC组织中的浸润性,并且探讨二者之间的相关性,为CRC的诊断和预后判断提供参考。

1 资料与方法 1.1 研究对象

收集2007—2009年北华大学附属医院手术切除的经病理诊断为CRC患者的结肠癌标本56例,均取癌组织和癌旁正常组织(大于肿瘤边缘5cm处)。其中男性患者35例,女性患者21例,年龄34~79岁。24例患者有淋巴结转移,32例患者无淋巴结转移。所有患者均无术前放疗或化疗。根据国际癌症控制联盟(UICC 2002)标准进行病理分期。

1.2 免疫组织化学法检测CRC组织和癌旁正常组织中B7-H1和B7-H4的表达

将OCT胶包埋好的组织标本采用恒冷冰冻切片机(Leica CM1900,德国莱卡公司)切成5 μm厚度的连续切片,室温干燥后以多聚甲醛固定,PBS冲洗。以下步骤按说明书操作:0.3%过氧化氢室温下阻断内源性过氧化物酶活性,PBS冲洗,山羊血清封闭。切片分别滴加抗B7-H1抗体(1:30稀释)、抗B7-H4抗体(1:500稀释)和抗Foxp3+抗体(1:100稀释),4℃过夜。PBS洗涤切片, 以生物素标记的二抗处理,链霉亲和素-辣根过氧化物酶复合物孵育,DAB显色,切片脱水,清洗和封片。

1.3 结果判定标准

采用双盲法评估所有载玻片。高倍镜下(×400)随机取10个视野,每个视野计数100个细胞,以细胞质中有黄至棕褐色颗粒染色者为B7-H1和B7-H4染色阳性。按阳性细胞数计分:0分(无阳性肿瘤细胞),1分(阳性肿瘤细胞率 < 10%),2分(10%≤阳性肿瘤细胞率≤50%),3分(阳性肿瘤细胞率>50%)。按染色强度计分:未着色或与背景一致为0分,浅黄色为1分,黄褐色为2分,褐色为3分。2项指标的乘积分数小于4归类为低表达,其余为高表达。

根据随机选择的高倍视野(HPF)中阳性染色细胞的百分率来评估Treg细胞的表达情况,以细胞核中有黄至棕褐色颗粒者为判定Treg细胞阳性的标准。每张切片随机选择10个高倍视野(×400)计数阳性细胞数, 计算Treg阳性细胞数平均值。Treg阳性细胞数平均值≤20为低浸润,Treg阳性细胞数平均值>20为高浸润。

1.4 统计学分析

采用GraphPad Prism 5.0统计软件进行统计学分析。B7-H1和B7-H4表达频数与CRC患者临床病理参数的关系分析采用χ2检验;不同临床病理参数CRC患者Treg阳性细胞数以x±s表示,组间比较采用t检验;B7-H1表达与Treg浸润及B7-H4表达与Treg浸润的关联性分析采用χ2检验及Cramer’ s检验。以P < 0.05表示差异有统计学意义。

2 结果 2.1 CRC患者癌组织和癌旁正常组织中B7-H1和B7-H4的表达

B7-H1和B7-H4阳性表达呈棕黄色颗粒,主要定位于肿瘤细胞的细胞质和细胞膜(图 1AB,见插页四);癌旁正常组织中其为阴性表达(图 1CD,见插页四)或在肠腺上皮细胞质中呈弱阳性表达。56例癌旁正常组织中B7-H1呈弱阳性表达5例(8.93%),B7-H4呈弱阳性表达4例(7.14%)。CRC组织中B7-H1高表达27例,高表达率为48.21%;B7-H4高表达27例,高表达率为48.21%。CRC组织中B7-H1阳性表达率为89.29%,明显高于癌旁正常组织(8.93%)(χ2= 72.34,P < 0.01);CRC组织中B7-H4阳性表达率为91.07%,明显高于癌旁正常组织(7.14%)(χ2=78.92,P < 0.01)。

图 1 CRC和癌旁正常组织中B7-H1、B7-H4和Treg阳性细胞表达(×200) Figure 1 Expressions of B7-H1, B7-H4 and Treg positive cells in CRC and adjacent normal tissues(×200)
2.2 CRC患者癌组织和癌旁正常组织中Treg阳性细胞数

Treg细胞形态完整, 结构清晰,DAB显色核呈棕黄色,散在分布于CRC组织中,尤其在CRC组织和癌旁正常组织交界处分布密集。本研究56例CRC组织中均有Treg表达, 其阳性细胞数(23.5±7.0)明显高于癌旁正常组织(0.7±1.9)(P < 0.01)。见图 1EF(插页四)。

2.3 不同临床病理特征CRC患者癌组织中B7-H1、B7-H4和Treg的表达

B7-H1表达频数与肿瘤浸润深度有关联(P < 0.01),淋巴结转移阳性者CRC组织中B7-H1高表达频数高于淋巴结转移阴性者(P < 0.01),Duke’ s分期C+D者B7-H1高表达频数高于Duke’ s分期A+B者(P < 0.01)。B7-H1表达频数与患者年龄、性别、肿瘤位置和分化程度无关联(P> 0.05)。见表 1

表 1 不同临床病理特征CRC患者癌组织中B7-H1、B7-H4的表达频数和Treg阳性细胞数 Table 1 Expression frequencies of B7-H1 and B7-H4 and number of Treg positive expression cells in cancer tissue of CRC patients with different clinicopathological features
Clinicopathological feature n Expression frequency of B7-H1 Expression frequency of B7-H4 No.of Treg positive expression cells P
Low High P Low High P
Gender
  Men 35 19 16 0.628 8 21 14 0.112 2 21.8±8.0 0.312 7
  Women 21 10 11 8 13 18.7±6.2
Age(year)
  ≤60 26 13 13 0.803 4 15 11 0.410 2 27.1±8.1 0.522 9
   >60 30 16 14 14 16 19.9±5.8
Tumor position
  Colon 23 10 13 0.299 0 14 9 0.256 1 17.7±5.6 0.067 2
  Rectum 33 19 14 15 18 22.2±6.5
Differentiation degree
  High 27 16 11 0.209 7 15 12 0.197 3 18.2±4.3 0.002 9
  Moderate and low 24 10 14 9 15 25.8±6.5
Infiltration depth
  T1+T2 18 15 3 0.003 0 14 4 0.007 0 9.7±8.8 0.648 5
  T3+T4 38 14 24 15 23 21.0±7.5
Lymphnode metastasis
  Negative 32 22 10 0.003 4 21 11 0.016 7 18.9±7.5 0.006 5
  Positive 24 7 17 8 16 24.4±6.4
Duke’s stage
  A+B 30 21 9 0.003 4 19 11 0.063 2 19.3±7.6 0.162 8
  C+D 26 8 18 10 16 23.3±7.0

B7-H4的表达频数与肿瘤浸润深度有关联(P < 0.05),淋巴结转移阳性者CRC组织中B7-H4高表达频数高于转移阴性者(P < 0.05)。B7-H4的表达频数与患者性别、年龄、肿瘤位置、分化程度和Duke’ s分期无关联(P> 0.05)。Treg阳性细胞数与肿瘤分化程度有关联(P < 0.01),淋巴结转移阳性者CRC组织中Treg阳性细胞数高于转移阴性者(P < 0.01)。Treg阳性细胞数与患者性别、年龄、肿瘤位置、分化程度和Duke’ s分期无关联(P> 0.05)。见表 1

2.4 CRC患者癌组织中B7-H1、B7-H4表达与Treg浸润程度的关系

本研究56例CRC组织中Treg分布情况:Treg低浸润30例,Treg高浸润26例。Treg高浸润组B7-H1和B7-H4的高表达频数明显高于低浸润组,CRC组织中B7-H1表达与Treg有相关性(P < 0.01,Cramer’ s=0.463),CRC组织中B7-H4表达与Treg有相关性(P < 0.05,Cramer’ s=0.320)。见表 2

表 2 CRC患者癌组织中B7-H1和B7-H4表达与Treg浸润的关系 Table 2 Relationship between expressions of B7-H1 and B7-H4 and Treg infiltration in cancer tissue of CRC patients
Treg n B7-H1 χ2 P Cramer’s B7-H4 χ2 P Cramer’s
Low High Low High
Low infiltration High infiltration 30 22 8 12.020 0.000 5 0.463 20 10 5.731 0.016 7 0.320
High infiltration 26 7 19 20 9 10 17
3 讨论

肿瘤细胞可以被抗原特异性T细胞免疫应答识别和攻击。B7家族在抗原特异性T细胞介导的正性和负性免疫调控作用中起核心作用。负性共刺激分子B7表达上调能通过抑制T细胞功能负性调控T细胞的免疫应答,从而促进肿瘤免疫逃避[10]。本研究结果显示:负性共刺激分子B7-H1和B7-H4在CRC组织中均呈异常高表达,并且B7-H1在CRC组织中的高表达与浸润深度、淋巴结是否转移及Duke’ s分期有关联;B7-H4高表达频数随着肿瘤浸润深度、淋巴结转移也出现出升高趋势。但B7-H1和B7-H4与CRC患者其他病理特征(如性别、年龄、部位和分化程度等)无关联,提示B7-H1和B7-H4在CRC的转移和进展中起重要作用,但CRC进程的具体机制尚需进一步探讨。

Treg通过在肿瘤微环境中发挥肿瘤特异性免疫抑制功能而促进肿瘤生长,对肿瘤的发生发展有重要意义[11-12]。本研究探讨了CRC患者肿瘤组织中Treg细胞分布情况, 发现肿瘤组织中Treg表达水平明显高于相应的癌旁正常组织。对Treg浸润与临床病理特征关系分析结果显示:中-低分化患者CRC组织中Treg阳性细胞数明显高于高分化者, 肿瘤的异型性越大,Treg阳性细胞数越多, 恶性程度越高。这些结果提示Treg在CRC肿瘤免疫中有重要作用,Treg可能影响了肿瘤的分化,Treg的高表达可能是影响CRC预后不良的因素之一。

目前,在肿瘤微环境中如何趋化Treg细胞并维持Treg细胞的生存和功能机制尚不清楚。负性共刺激分子B7-H1和B7-H4能增加肿瘤特异性抗原激活的T细胞凋亡、抑制特异性T细胞免疫应答、减少白细胞介素2(IL-2)、白细胞介素10(IL-10)和干扰素γ(IFN-γ)的分泌,以及阻碍细胞周期的进程[13-15]。研究[16-17]显示:B7-H1在调节诱导性Treg中起关键作用,通过抑制活化T细胞中的细胞外信号调节蛋白激酶、C-Jun N端激酶、丝裂原激活蛋白激酶P38和AKT信号通路,以影响T细胞的增殖[18]。体内实验结果显示:抑制B7-H1信号通路可抑制肿瘤特异性抗原激活的Treg生成。Treg可诱导人卵巢癌细胞表达B7-H4,从而抑制抗原提呈细胞的活性[19],表明B7-H4对Treg功能具有调控作用[20]。本研究结果显示:Treg高浸润组B7-H1和B7-H4高表达频数明显高于低浸润组,B7-H1和B7-H4在CRC组织中的表达与Treg有关联性。随着CRC的发生发展、浸润及转移,肿瘤细胞增殖活性呈递增趋势。

综上所述,CRC患者癌组织中B7-H1、B7-H4和Treg高表达可能促进肿瘤的浸润和转移,并在CRC的发生发展中起抑制作用,B7-H1和B7-H4表达可能是诱导机体产生Foxp3+ Treg的机制之一。因此,联合检测B7-H1、B7-H4和Treg的表达对CRC预后评估有较高的临床参考价值。

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