B細胞活化因子的雙向性：促進調節性T細胞分泌白細胞介素-35

張亞敏 王瑞云 李 軍 陶 娟 涂亞庭

(華中科技大學同濟醫學院附屬協和醫院皮膚科，武漢 430022)

·皮膚病性病診療與研究·

B細胞活化因子的雙向性：促進調節性T細胞分泌白細胞介素-35

張亞敏 王瑞云 李 軍 陶 娟 涂亞庭*

(華中科技大學同濟醫學院附屬協和醫院皮膚科，武漢 430022)

目的研究B細胞活化因子(B cell activation factor of the tumor necrosis factor family, BAFF)對調節性T細胞(Tregs)分泌抑制性細胞因子白細胞介素-35(interleukin-35, IL-35)的影響，驗證系統性紅斑狼瘡(systemic lupus erythematosus， SLE)中BAFF對免疫反應的雙向調節機制。方法磁珠分選狼瘡模型鼠Tregs，經BAFF刺激后采用qRT-PCR及流式細胞術檢測其分泌IL-35濃度。比較正常對照鼠、狼瘡模型鼠及經尾靜脈注射抗BAFF蛋白后狼瘡模型鼠脾臟Tregs頻率及分泌IL-35情況。結果BAFF能夠有效促進Tregs分泌IL-35。相對于正常對照鼠，狼瘡模型鼠(血清中BAFF升高)脾臟Tregs分泌較高濃度的IL-35，而中和其循環中BAFF后，Tregs分泌IL-35減少。結論在SLE發病機制中，BAFF不僅是重要的致病因素，還能夠通過促進Tregs細胞分泌IL-35發揮負向免疫調節效應。

系統性紅斑狼瘡；B細胞活化因子；調節性T細胞；白細胞介素-35

系統性紅斑狼瘡(system lupus erythematosus， SLE)是一種復雜的、累及全身多臟器的系統性自身免疫性疾病[1-2]。B細胞活化因子(B cell activation factor of the tumor necrosis factor family, BAFF)是對自身反應性 B 細胞增生分化至關重要的關鍵細胞因子。在 SLE 病人的血漿中 BAFF 水平明顯升高，且與疾病的嚴重程度正相關[3-4]。然而，越來越多的研究[5-6]提示BAFF可能具有免疫抑制功能。如BAFF能夠誘導正常C57BL/6小鼠脾臟B細胞分泌白細胞介素-10 (interleukin-10， IL-10)，促進慢性淋巴細胞白血病病人外周血及動物模型脾臟中B細胞分泌IL-10進而介導免疫抑制。此外，外源性BAFF能夠共刺激人類及鼠的T細胞活化及存活[7-8]。在狼瘡模型鼠中，BAFF可促進輔助性濾泡T細胞 (T follicular helper, TFH)增生并分泌干擾素-γ(interferon-γ，IFN-γ)[9]，提示BAFF可能參與調節T細胞免疫反應。另一方面，BAFF通過上調Tregs介導移植耐受，而BAFF能否直接促進Tregs產生及其相關機制目前仍存在爭議[10]。

IL-35是IL-12家族的最新成員，在自身免疫性疾病中發揮重要調節作用[11]。它是由p35和EBI3 (Epstein-Barr virus-induced gene 3)組成的異源性二聚體，是一類重要的免疫抑制性細胞因子，主要由Tregs分泌并介導Tregs的抑制功能[12-13]。研究[14]表明，重組IL-35在狼瘡模型鼠中能夠有效上調Tregs與調節性B細胞(Bregs)比例，減輕狼瘡腎損害，有效控制疾病進展。然而，在SLE中，BAFF是否影響Tregs產生IL-35尚不清楚。因此，該實驗通過磁珠分選法提取狼瘡模型鼠Tregs，并經外源性BAFF刺激后檢測IL-35亞基p35和EBI3的表達。同時經尾靜脈注射抗BAFF蛋白體內觀察并分析BAFF對Tregs分泌IL-35的影響。

1 材料與方法

1.1動物與試劑

成熟健康的雌性MRL/MpJ對照鼠(WT, wild-type control) 及MRL/MpJ-Faslpr/J 品系狼瘡模型鼠由湖南省湘雅二醫院醫學表觀基因組學重點實驗室饋贈。體質量22～24 g，均正常飲食、光照，飼養于華中科技大學同濟醫學院動物中心清潔級動物房。體內動物實驗共分3組：MRL/MpJ正常對照組 [n=5，尾靜脈注射磷酸鹽緩沖液(phosphate buffer saline, PBS)]，MRL/MpJ-Faslpr/J狼瘡模型鼠組 (n=5，尾靜脈注射PBS)以及抗BAFF治療MRL/MpJ-Faslpr/J狼瘡模型鼠組 (n=5，尾靜脈注射抗BAFF蛋白，100 μg/只)。抗BAFF蛋白購自美國Adipogen公司。

1.2細胞提取與培養

脾臟Tregs及CD4+T細胞均經CD4+CD25+調節性T細胞磁珠分選試劑盒(德國美天旎公司)提取后，加入BAFF 50 ng/mL進行細胞培養。每組均加入IL-2 (400U/mL)。細胞培養于含有16%(體積分數)胎牛血清的1640完全培養基中。

1.3流式細胞術

純化的脾臟Tregs培養60 h后行流式細胞術檢測，檢測前5 h加入佛波酯(Phorbol 12-myristate 13-acetate，PMA) (50 ng/mL)、離子霉素 (50 ng/mL)及高爾基阻斷劑。流式細胞術檢測過程中所用到的流式抗體如下：抗鼠 EBI3 (355022，美國R&D公司); 抗鼠p35 (27537，美國R&D公司)；BAFFR (eBio7H22-E16，美國eBioscience公司); TACI (ebio8F10-3，美國eBioscience公司)；CD25 (PC61，美國BD公司) 和 CD4(GK1.5，美國BD公司)。

1.4熒光定量-聚合酶鏈反應(qRT-PCR)

純化的脾臟Tregs培養48 h后，用Trizol提取細胞內總的RNA，采用PrimeScript RT試劑盒(日本Takara公司)反轉錄合成cDNA。作為模板，p35、EBI3及IL-10的RNA水平采用SYBR Green RT-PCR 試劑盒 (日本Takara公司)進行PCR擴增及定量。所有目的RNA的表達均以GAPDH作為內參。實驗過程中所采用的引物序列如下：GAPDH FP: 5′ -GGTGAAGGTCGGAGTCAACGG-3′, RP: 5′ -CCTGGAAGATGGTGATGGGATT-3′; EBI3 FP: 5′-CGGTGCCCTACATGCTAAAT-3′, EBI3 RP: 5′-GCGGAG TCGGTACTTGAGAG-3′; p35 FP: 5′- CATCGATGAGCTGATGCAGT-3′, p35 RP: 5′-CAGATAGCCCATCACCCTGT-3′; IL-10 FP: 5′-AGCCGGGAAGACAATAACTG-3′, IL-10 RP: 5′-CATTTCCGATAA GGCTTGG-3′。

1.5統計學方法

2 結果

2.1Tregs表達BAFF受體BAFF-R及TACI

為研究BAFF是否直接參與調節Tregs的生物學行為，筆者首先檢測了Tregs表面BAFF受體的表達情況。磁珠分選CD4+T細胞后采用流式細胞術檢測CD4+CD25+Tregs表面BAFF-R及TACI表達情況。流式結果顯示Tregs表面兩種受體均有一定程度的表達(圖1)，為BAFF對Tregs的直接作用提供了一定的理論依據。

2.2外源性BAFF能夠直接促進Tregs分泌IL-35

采用磁珠分選法提取CD4+CD25+Tregs的純度均能達到80%以上(圖2A)。于純化的Tregs中加入BAFF 50 ng/mL進行共培養，每組均加入IL-2(400U/mL)。48 h后實時定量PCR法檢測p35及EBI3的RNA表達。結果顯示，相對于未處理組，BAFF 50 ng/mL刺激組的p35及EBI3 RNA的表達水平明顯升高，差異有統計學意義(P<0.001)(圖2B)。

圖1 調節性T細胞表達BAFF受體BAFF-R及TACIFig.1 The expression of BAFF-R and TACI onregulatory T cells

Splenic CD4+T cells from MRL-Faslpr/lprmice were sorted and and the expression of BAFF-R and TACI in CD4+CD25+T cells were analyzed by flow cytometry. The black line indicates isotype control.BAFF: B cell activation factor of the tumor necrosis factor family;BAFF-R: B cell activation factor receptor;TACI: transmembrane activator and calcium-modulating cyclophilin ligand interactor.

同樣，將純化的Tregs經BAFF 50 ng/mL刺激60 h后，流式細胞術檢測胞內p35及EBI3蛋白水平的變化。結果顯示，在BAFF作用下，p35+EBI3+T細胞的頻率明顯升高且差異具有統計學意義(P<0.01)，其比例由(3.163±0.479)%上升到(7.903±0.874)%(圖2C)。說明BAFF能夠直接作用于Tregs并促進其分泌IL-35。

2.3體內實驗驗證BAFF對Tregs分泌IL-35的影響

動物實驗如圖3A所示，共分3組：尾靜脈注射PBS/抗BAFF蛋白3 d后，取小鼠脾臟，流式細胞術檢測Tregs頻率以及Tregs中產IL-35 的T細胞頻率。結果顯示，尾靜脈注射PBS的正常對照鼠與狼瘡模型鼠脾臟Tregs比例分別為(4.005±0.647)%和(8.117 ±1.015)%。相對于正常鼠，狼瘡模型鼠脾臟Tregs比例明顯升高且差異具有統計學意義(P<0.05)。而經抗BAFF蛋白降低狼瘡鼠體內BAFF濃度后，Tregs比例下降為(4.765±0.117)%，且差異亦具有統計學意義(P<0.05)(圖3B)，表明狼瘡鼠中BAFF 與Tregs的產生呈正相關。

同時，筆者也經流式細胞術對不同實驗組小鼠脾臟Tregs中p35+EBI3+T細胞的比例進行檢測及分析。

圖2 BAFF促進調節性T細胞分泌IL-35Fig.2 BAFF promoted regulatory T cells to produce IL-35

Splenic CD4+CD25+T cells from MRL-Faslpr/lprmice were sorted, and the purity was identified by flow cytometry (A).Sorted CD4+CD25+T cells were cultured with BAFF 50 ng/mL for 48 h, and the expression of p35 and EBI3 mRNA were detected by real-time PCR (B).Flow cytometric analysis of frequencies of splenic p35+EBI3+Tergs treated with or without BAFF 50 ng/mL for 60 h (C).Results are representative of at least three independent experiments.**P<0.01;***P<0.001；NS： non-significant；BAFF: B cell activation factor of the tumor necrosis factor family;EBI3: Epstein-Barr virus-induced gene 3；IL-35:interleukin-35.

圖3 體內驗證BAFF對Tregs分泌IL-35的促進效應Fig.3 BAFF promoted regulatory T cells to produce IL-35 in vivo

Schematic illustration of the experimentinvivo(A).Splenic CD4+CD25+T cells were gated and assessed for the frequency of Tregs by flow cytometry (B).CD4+CD25+regulatory T cells were gated and evaluated the frequency of p35+EBI3+T cells (C). Data are representative of at least three independent experiments.*P<0.05,NS： non-significant.BAFF: B cell activation factor of the tumor necrosis factor family;WT: wild-type control;EBI3: Epstein-Barr virus-induced gene 3;IL-35:interleukin-35;PBS:phosphate buffer saline.

結果表明，狼瘡模型鼠脾臟Tregs中p35+EBI3+T細胞的頻率明顯高于正常對照鼠，分別為(10.85±0.708)%和(16.00±1.155)%。當中和了狼瘡鼠血液循環中BAFF后，脾臟Tregs中p35+EBI3+T細胞頻率降低為(10.36±2.037)%(圖3C)，盡管在統計學分析中未顯示差異有統計學意義。綜上所述，本體內研究結果表明BAFF對Tregs細胞分泌抑制性細胞因子IL-35具有顯著的促進作用。

3 討論

文獻[3-4]表明BAFF有3個受體，即B細胞活化因子受體(B cell activation factor receptor, BAFF-R), 轉膜激活劑、鈣調節劑和親環素配體相互作用物(transmembrane activator and calcium-modulating cyclophilin ligand interactor, TACI)和B細胞成熟抗原(B cell maturation antigen, BCMA)。 BAFF主要與BAFF-R和TACI高親和力結合。BAFF-R信號通路對B細胞的成熟及存活意義重大，TACI主要介導IgG及IgA抗體類別轉換，B細胞對Ⅰ及Ⅱ類抗原的T細胞非依賴免疫反應，促進漿細胞生存及分化并抑制B細胞增生。BCMA主要分布于漿細胞表面，促進漿細胞存活[3,15]。近年來，大量研究[16-18]數據表明BAFF參與調節輔助性T細胞及調節性T細胞的多種免疫反應。如BAFF通過BAFF-R受體促進輔助性T細胞及記憶性T細胞的生存與活化，促進Th1介導的遲發型過敏反應，在實驗性自身免疫性腦脊髓炎模型中通過促進Th17細胞的產生促進疾病的進展[19]。然而BAFF是否能夠直接調節Tregs的生物學效應及相關機制尚不清楚。

該研究中，筆者證實Tregs表面表達BAFF受體BAFF-R及TACI，與以前的報道[16,18]相符。同時，筆者發現外源性BAFF能夠直接作用于Tregs，促進其分泌抑制性細胞因子IL-35。體內實驗亦得到相似的結論。研究[20-21]表明NZBxNZWF1、BXSB以及MRL-lpr/lpr三種狼瘡模型鼠血清中BAFF水平均明顯升高，而相對于正常對照鼠而言，其Tregs比例以及Tregs中分泌IL-35的T細胞比例均上升。當中和了狼瘡模型鼠循環中的高BAFF水平后，二者均有明顯的下降。本研究首次表明BAFF能夠直接作用于Tregs并進一步促進其分泌IL-35，從而發揮免疫抑制效應，證明了在SLE中，BAFF具有獨特的雙向免疫調節機制。2011 年 3 月，針對 BAFF 的單克隆抗體Belimumab被美國食品藥品監督管理局(Food and Drug Administration, FDA)認證并批準用于臨床治療 SLE。然而，盡管Belimumab在Ⅲ期臨床試驗中取得良好治療效應與安全性，仍有40%的病人病情未能得到有效緩解[22]。本研究結果可能為這一臨床試驗結果提供一個潛在的理論依據。

該研究中，BAFF促進Tregs分泌IL-35的相關機制還有待進一步探討，如BAFF是通過促進Tregs生存還是直接促進IL-35的產生，何種受體及信號通路介導該效應等。同時在其他自身反應性疾病模型或狼瘡病人中是否存在相似的機制仍有待進一步研究。綜上，本研究證實了BAFF對Tregs分泌IL-35的促進效應，明確了BAFF對T細胞的雙向調節效應，豐富了BAFF系統在SLE中的發病機制。

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DichotomouseffectofBAFFinducingIL-35productionbyregulatoryTcells

Zhang Yamin, Wang Ruiyun, Li Jun, Tao Juan,Tu Yating*

(DepartmentofDermatology,UnionHospital,TongjiMedicalCollege,HuazhongUniversityofScienceandTechnology(HUST),Wuhan430022，China)

ObjectiveThe purpose of this study was to determine whether B cell activation factor of the tumor necrosis factor (TNF) family (BAFF) has an effect on the production of interleukin-35 (IL-35) by regulatory T cells (Tregs), further identifying the dichotomous regulator of BAFF on immune responses in systemic lupus erythematosus (SLE).MethodsSplenic CD4+CD25+T cells were sorted by magnetic isolation and stimulated with BAFF. The production of IL-35 was determined by flow cytometry and quantitative RT-PCR. Furthermore, the frequency of IL-35 producing Tregs in spleen from wild-type (WT) controls, MRL-Faslpr/lprmice and anti-BAFF protein treated MRL-Faslpr/lprmice was also analyzed by flow cytometry.ResultsWe found that BAFF could promote Tregs to produce IL-35invitro. Flow cytometric analysis showed that the frequency of IL-35 producing Tregs was much higher in spleen of MRL-Faslpr/lprmice which have increased serum level of BAFF compared with those in WT controls. Whereas anti-BAFF treatment weakened this effect.ConclusionIn the pathogenesis of SLE, BAFF is not only a key pathogenic factor but also has immunosuppressive effect by promoting Tregs to produce IL-35.

systemic lupus erythematosus (SLE)；B cell activation factor of the tumor necrosis factor family (BAFF)；regulatory T cells；interleukin-35 (IL-35)

國家自然科學基金(81573047，81602760)。This study was supported by National Natural Science Foundation of China (81573047， 81602760).

*Corresponding author， E-mail：yatingtu@yahoo.com.cn

時間：2017-10-14 16∶19

http://kns.cnki.net/kcms/detail/11.3662.R.20171014.1619.032.html

10.3969/j.issn.1006-7795.2017.05.002]

R751

2017-05-09)

編輯 陳瑞芳