唐春蓮,申志琴,嚴(yán)玲霞,劉曉宏

細(xì)胞毒性T淋巴細(xì)胞相關(guān)蛋白4在日本血吸蟲免疫逃避中的作用及其機(jī)制研究

唐春蓮,申志琴,嚴(yán)玲霞,劉曉宏

目的 探討細(xì)胞毒性T淋巴細(xì)胞相關(guān)蛋白4(CTLA-4)在日本血吸蟲免疫逃避中的作用及其機(jī)制。方法 雌性BALB/c小鼠隨機(jī)分成3組，即正常對照組、感染對照組和抗CTLA-4單克隆抗體(Anti-CTLA-4 mAb)組。各感染組每只小鼠經(jīng)腹部皮膚感染日本血吸蟲尾蚴40條，感染2周后，Anti-CTLA-4 mAb組每只小鼠連續(xù)3 d腹腔注射300 μg anti-CTLA-4 mAb，對照組注射等體積的PBS。感染后6周殺鼠沖蟲，計(jì)數(shù)每只小鼠蟲荷及每克肝臟蟲卵數(shù)。流式細(xì)胞術(shù)檢測各組小鼠脾淋巴細(xì)胞中調(diào)節(jié)性T細(xì)胞(CD4+CD25+Tregs)百分比。ELISA法檢測各組小鼠脾淋巴細(xì)胞培養(yǎng)上清中細(xì)胞因子IFN-γ、IL-4、IL-5和IL-10的含量。肝組織石蠟切片HE染色觀察感染組小鼠肝臟蟲卵肉芽腫病理變化。結(jié)果 Anti-CTLA-4 mAb組減蟲率為18.99%，脾淋巴細(xì)胞中CD4+CD25+Tregs百分比顯著升高(P<0.05)。Anti-CTLA-4 mAb組脾細(xì)胞培養(yǎng)上清中細(xì)胞因子IFN-γ、IL-4、IL-5和IL-10的含量均高于感染對照組，肝臟蟲卵肉芽腫直徑較感染對照組明顯增大。結(jié)論 Anti-CTLA-4 mAb使用同時增強(qiáng)Th1型和Th2型免疫反應(yīng)，有利于機(jī)體清除日本血吸蟲但以損傷機(jī)體為代價。研究結(jié)果提示宿主CTLA-4利于日本血吸蟲免疫逃避。

日本血吸蟲; 細(xì)胞毒性T淋巴細(xì)胞相關(guān)蛋白4; 免疫逃避; 細(xì)胞因子

Supported by the Schistosomiasis Control Project from Hubei Provincial Health and Family Planning Commission(No. WJ2015XB016) Corresponding author: Liu Xiao-hong, Email: 494527907@qq.com

血吸蟲病是一種常見的人獸共患寄生蟲病，全球76個國家至少23億人感染血吸蟲[1]，我國主要是日本血吸蟲。寄生蟲具有免疫逃避功能能在宿主體內(nèi)長期存在，其逃避機(jī)制可能與抗原偽裝、序列變異等相關(guān)[2]。新近研究證實(shí)，細(xì)胞毒性T淋巴細(xì)胞相關(guān)蛋白-4(cytotoxic T-lympnocyte-associated protein 4，CTLA-4)與病原體逃避宿主免疫攻擊有關(guān)，如HIV感染患者，感染利什曼原蟲后導(dǎo)致CD4+CTLA-4+調(diào)節(jié)性T細(xì)胞比例明顯增高，導(dǎo)致原蟲復(fù)發(fā)[3]。實(shí)驗(yàn)性腦瘧原蟲模型中，F(xiàn)OXP3+細(xì)胞損傷了抗原特異性CD4+T細(xì)胞及CD8+T細(xì)胞反應(yīng)，因此利于瘧原蟲逃避宿主免疫攻擊。而FOXP3+細(xì)胞調(diào)節(jié)的保護(hù)作用依賴于CTLA-4[4]。CTLA-4在日本血吸蟲免疫逃避中的作用尚無報(bào)道。本文采用CTLA-4 mAb，觀察其在日本血吸蟲免疫逃避中的作用及其機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)動物分組及處理 18只6～8周齡雌性BALB/c小鼠購自湖北省疾病預(yù)防控制中心，隨機(jī)分成3組，每組6只。即正常對照組、感染對照組、Anti-CTLA-4 mAb組。日本血吸蟲陽性釘螺(購自江西省寄生所)光照下逸出尾蚴，各感染組每只小鼠經(jīng)腹部皮膚感染尾蚴40條。感染后2周，抗體組每只小鼠連續(xù)3 d腹腔注射300 μg anti-CTLA-4 mAb，對照組注射等體積的PBS。感染后6周剖殺所有小鼠。

1.2 試劑器材 調(diào)節(jié)性T細(xì)胞染色試劑盒試劑盒購自eBioscience公司；細(xì)胞因子IFN-γ、IL-4、IL-5、IL-10檢測試劑盒購自eBioscience公司；流式細(xì)胞儀(FACSCalibur型)購自美國Becton Dickinson公司。

1.3 蟲荷及每克肝臟蟲卵計(jì)數(shù) 日本血吸蟲感染后6周，剖殺各感染組小鼠。生理鹽水灌注沖蟲[5]，計(jì)算各感染組每只小鼠蟲荷及減蟲率。稱取各感染組每只小鼠肝臟右葉0.5 g，研磨，5% KOH 20 mL 37 ℃消化2 h，取250 μL計(jì)數(shù)，重復(fù)4次取平均值，計(jì)算每克肝臟蟲卵數(shù)。剩余肝組織用于病理學(xué)檢測。

1.4 脾細(xì)胞流式細(xì)胞術(shù)檢測無菌取脾，根據(jù)文獻(xiàn)報(bào)道的方法[6]制備單個脾淋巴細(xì)胞懸液。以含10%胎牛血清的RPMI-1640培養(yǎng)液重懸，顯微鏡下計(jì)數(shù)淋巴細(xì)胞數(shù)量，調(diào)整其濃度為5×106/mL。流式細(xì)胞術(shù)染色使用小鼠調(diào)節(jié)性T細(xì)胞染色，其中CD4抗體、CD25抗體和Foxp3抗體分別以異硫氰酸熒光素、別藻青蛋白和藻紅蛋白標(biāo)記。流式細(xì)胞儀檢測脾淋巴細(xì)胞中CD4+CD25+Tregs的百分比。

1.5 脾細(xì)胞培養(yǎng)上清中細(xì)胞因子檢測 無菌取脾，研磨，制備脾淋巴細(xì)胞懸液并調(diào)整細(xì)胞濃度至5×106/mL，置37 ℃、5% CO2培養(yǎng)箱培養(yǎng)72 h，收集上清液。雙抗體夾心ELISA法檢測脾細(xì)胞培養(yǎng)上清中細(xì)胞因子IFN-γ、IL-4、IL-5和IL-10的水平，根據(jù)標(biāo)準(zhǔn)曲線計(jì)算待測樣品中特定細(xì)胞因子含量(pg/mL)。

1.6 病理學(xué)檢測 用于病理學(xué)檢測的肝組織經(jīng)10%福爾馬林固定后，石蠟包埋。切片，厚度約5 μm，常規(guī)HE染色，觀察各感染組小鼠肝臟蟲卵肉芽腫的變化。

2 結(jié) 果

2.1 Anti-CTLA-4 mAb對小鼠體內(nèi)蟲荷及每克肝臟蟲卵數(shù)的影響 Anti-CTLA-4 mAb使用組蟲荷及每克肝臟蟲卵數(shù)明顯低于感染對照組(P<0.05)(表1)。

表1 Anti-CTLA-4 mAb對小鼠體內(nèi)蟲荷及每g肝臟蟲卵數(shù)的影響Tab.1 The effect of anti-CTLA-4 mAb on the worm burden and eggs per gram liver in BALB/c mice

注：數(shù)值均為均數(shù)±標(biāo)準(zhǔn)差。N代表每組6只小鼠。*與感染對照組比較，P<0.05。

Note:All results were presented as mean ± S.D; n represents 6 mice in each group. *P<0.05, versus infected control group.

2.2 Anti-CTLA-4 mAb對脾淋巴細(xì)胞中CD4+CD25+Tregs百分比的影響 CTLA-4表達(dá)于CD4+CD25+Tregs，為了研究日本血吸蟲感染模型中CTLA-4在CD4+CD25+Tregs中的作用，流式細(xì)胞術(shù)檢測各組小鼠脾淋巴細(xì)胞中的CD4+CD25+Tregs百分比。既然Foxp3是CD4+CD25+Tregs的特異性標(biāo)志，因此，用CD4+CD25+Foxp3+T 細(xì)胞表示CD4+CD25+Tregs。無菌取抗體處理組和對照組小鼠脾臟，常規(guī)制備脾淋巴細(xì)胞懸液，小鼠調(diào)節(jié)性T細(xì)胞染色試劑盒抗體標(biāo)記。流式檢測結(jié)果見圖1，anti-CTLA-4 mAb組脾淋巴細(xì)胞中CD4+CD25+Tregs比例明顯高于對照組(P<0.05)。

2.3 脾細(xì)胞培養(yǎng)上清中細(xì)胞因子的含量 Anti-CTLA-4 mAb組脾淋巴細(xì)胞中細(xì)胞因子IFN-γ、IL-4、IL-5和IL-10的含量均明顯高于感染對照組(圖2)。

正常對照組(A), 感染對照組(B)和Anti-CTLA-4 mAb組 (C).P2門為CD4+CD25+T細(xì)胞在脾淋巴細(xì)胞中的百分比，Q2-2為P2門中的Foxp3+T細(xì)胞的百分比(即CD4+CD25+Foxp3+%)。

Normal control (A), infected control (B) and anti-CTLA-4 mAb (C). Upper panels, the cells in P2 gate denote the percentage of CD4+CD25+T cells in splenic lymphocytes. The cells in Q2-2 gate in the lower panels denote the percentage of Foxp3+lymphocytes in P2 gate.

圖1 流式細(xì)胞染色顯示CD4+CD25+Tregs在脾淋巴細(xì)胞中的的百分比。

Fig.1 FACS analysis demonstrates the percentage of CD4+CD25+Tregs in splenocytes

* 與感染對照組比較，P<0.05。* P<0.05, versus infected control group.圖2 BALB/c小鼠脾細(xì)胞培養(yǎng)上清中IFN-γ(A)、IL-4(B)、IL-5(C)和IL-10(D)的含量Fig.2 The expression of IFN-γ, IL-4, interleukin-5 and IL-10 in splenic lymphocytes culture supernatant

2.4 肝組織病理切片結(jié)果 肝組織HE染色結(jié)果見圖3，anti-CTLA-4 mAb組肝組織蟲卵肉芽腫直徑(503.2±53.6)較感染對照組(356.8±43.6)增大，組間差異有統(tǒng)計(jì)學(xué)意義。

3 討 論

CTLA-4為共刺激分子，與CD28分子結(jié)構(gòu)具有76%的同源性。CTLA-4能競爭性抑制CD28分子與B7復(fù)合物結(jié)合，防止CD28分子促進(jìn)T細(xì)胞激活[7]。CTLA-4最重要的功能是抑制T細(xì)胞激活，CTLA-4基因缺陷小鼠出現(xiàn)淋巴增殖及多器官淋巴細(xì)胞浸潤，出生后3～4周死亡[8]。Blank等[6]報(bào)道，采用抗CTLA-4單克隆抗體(anti-CTLA-4 mAb)能增強(qiáng)機(jī)體的免疫反應(yīng)，有利于機(jī)體抗黑色素瘤免疫反應(yīng)。CTLA-4在轉(zhuǎn)移性的黑色素瘤患者中，有利于腫瘤細(xì)胞的免疫逃避，使用相應(yīng)抗體阻斷則有利于病情改善[9]。Martins等[10]報(bào)道，錐蟲感染模型中采用anti-CTLA-4 mAb有利于機(jī)體對病原體的清除，使寄生蟲血癥下降及利于宿主存活。本實(shí)驗(yàn)證實(shí)，anti-CTLA-4 mAb使用時機(jī)體對日本血吸蟲的清除率為18.99%。CTLA-4可能有利于日本血吸蟲在宿主體內(nèi)的免疫逃避。

感染對照組(A)、anti-CTLA-4 mAb組(B)Infected control group (A), anti-CTLA-4 mAb group (B).圖3 各感染組蟲卵肉芽腫Fig.3 Schistosoma egg granuloma in infected groups

既然CTLA-4表達(dá)于CD4+CD25+Tregs，則很容易認(rèn)為其免疫逃避作用是通過CD4+CD25+Tregs來實(shí)現(xiàn)的。本實(shí)驗(yàn)使用流式方法檢測anti-CTLA-4 mAb對CD4+CD25+Tregs的影響，結(jié)果顯示：CD4+CD25+Tregs不降反升，表明CTLA-4在日本血吸蟲免疫逃避中的作用不是通過CD4+CD25+Tregs來實(shí)現(xiàn)的，Suarez N等[11]也報(bào)道，封閉CD4+CD25+Tregs與CTLA-4阻斷為不同的作用機(jī)制，兩者能起協(xié)同作用利于機(jī)體清除腫瘤?？笴TLA-4抗體作用的靶點(diǎn)在于增強(qiáng)效應(yīng)T細(xì)胞的反應(yīng)或者增強(qiáng)其對CD4+CD25+Tregs的抵抗。

CTLA-4為抑制性受體，能提高效應(yīng)T細(xì)胞激活的閾值[12]，用相應(yīng)抗體阻斷后，則能使各種類型細(xì)胞增殖及各種類型細(xì)胞因子分泌增加。本實(shí)驗(yàn)證實(shí)，anti-CTLA-4 mAb引起脾細(xì)胞培養(yǎng)上清中細(xì)胞因子IFN-γ明顯上升。Hoffman等[13]報(bào)道，Th1型免疫反應(yīng)對機(jī)體清除日本血吸蟲很重要。因此，抗體使用后的殺蟲效應(yīng)可能是通過增強(qiáng)Th1型免疫反應(yīng)來實(shí)現(xiàn)的。

病理組織學(xué)檢測結(jié)果顯示，與感染對照組相比較，anti-CTLA-4 mAb及聯(lián)合組單個蟲卵肉芽腫直徑明顯增大。蟲卵肉芽腫主要是由日本血吸蟲卵引起的Th2型免疫反應(yīng)[14]，anti-CTLA-4 mAb導(dǎo)致Th2型細(xì)胞因子上升，因而引起蟲卵肉芽腫反應(yīng)增強(qiáng)。Torino F等[15]報(bào)道，anti-CTLA-4 mAb具有明顯地抗腫瘤反應(yīng)，同時加重對機(jī)體的病理損傷。Anti-CTLA-4 mAb增強(qiáng)機(jī)體免疫反應(yīng)有利于機(jī)體清除寄生蟲的同時，以對機(jī)體的損傷增強(qiáng)為代價。

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Effect and mechanism of CTLA-4 on the immune evasion ofSchistosomajaponicumin mice

TANG Chun-lian,SHEN Zhi-qin,YAN Lin-xia,LIU Xiao-hong

(DepartmentofClinicalLaboratory,WuchangHospital,Wuhan430063,China)

In order to explore the effect and mechanism of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on the immune evasion ofSchistosomajaponicum(S.japonicum) in mice, the 18 female BALB/c mice were divided randomly into 3 groups: normal control, infected control and anti-CTLA-4 mAb-treated group. Each mouse in infected control and anti-CTLA-4 groups was challenged with 40 cercarie ofS.japonicum. Mice in anti-CTLA-4 mAb group were treated with 300 μg of anti-CTLA-4 mAb for 3 days consecutively, equal volume PBS as control. At 6 weeks post-infection, all mice were succumbed to measure worm burden and eggs per gram liver. The levels of IFN-γ, IL-4, IL-5 and IL-10 were detected by sandwich-ELISA. Flow cytometry was performed to measure the percentages of CD4+CD25+tregs in splenic cells. Liver sections were stained with hematoxylin and eosin to detect egg granuloma. The results showed that reduction rate of worm burden in anti-CTLA-4 mAb group were 18.99%. Compared with that of control group, the percentage of CD4+CD25+Tregs was extremely higher in anti-CTLA-4 mAb group. The egg granuloma size in anti-CTLA-4 mAb group were extremely larger than those of infected control group, while the levels of IFN-γ, IL-4, IL-5, IL-10 in anti-CTLA-4 mAb group were extremely higher than those of control group. It’s indicated that anti-CTLA-4 mAb favors the host to clearS.japonicumby enhancing Th1 type immune response, but at the cost of elevated pathological damage by enhancing Th2 type immune response. It hints that CTLA-4 contribute to the escape ofS.japonicumfrom the host immune responses.

Schistosomajaponicum; CTLA-4; immune evasion; cytokines

劉曉宏，Email: 494527907@qq.com

武漢市武昌醫(yī)院檢驗(yàn)科，武漢 430063

10.3969/j.issn.1002-2694.2015.11.016

R383

A

1002-2694(2015)11-1061-04

2015-05-08；

2015-08-26

湖北省衛(wèi)生和計(jì)劃生育委員會血防專項(xiàng)(No. WJ2015XB016)資助