韓晨露，趙勇，李衛(wèi)國

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髓源性抑制細(xì)胞的體外誘導(dǎo)及其應(yīng)用

韓晨露，趙勇，李衛(wèi)國

作者單位：453007 新鄉(xiāng)，河南師范大學(xué)生命科學(xué)學(xué)院（韓晨露、李衛(wèi)國）；100101 北京，中國科學(xué)院動(dòng)物研究所膜生物學(xué)國家重點(diǎn)實(shí)驗(yàn)室（趙勇）

近來，髓源性抑制細(xì)胞（myeloid-derived suppressor cells，MDSCs）在固有免疫和適應(yīng)性免疫中發(fā)揮非常重要的作用，參與多種自身免疫疾病和移植排斥反應(yīng)[1-2]。MDSCs 是來源于骨髓的一群異質(zhì)性細(xì)胞，由一些髓系祖細(xì)胞及樹突狀細(xì)胞（dendritic cells，DCs）、巨噬細(xì)胞和粒細(xì)胞的前體細(xì)胞組成，具有顯著抑制免疫細(xì)胞應(yīng)答的能力[3]。小鼠 MDSCs 的表面標(biāo)志：粒樣 MDSCs（G-MDSCs）為 CD11b+Ly6G+Ly6Clow，單核樣 MDSCs（M-MDSCs）為CD11b+Ly6G?Ly6Chigh，在癌癥、自身免疫性疾病中這兩類細(xì)胞有不同的功能[4-5]。傳統(tǒng)上，人類 MDSCs 被定義為CD14?CD11b+CD33+CD15+細(xì)胞，細(xì)胞表達(dá) CD33 標(biāo)記，但不表達(dá)骨髓和淋巴細(xì)胞的一些成熟標(biāo)記和 HLA-DR 抗原[6]。

MDSCs 通過許多機(jī)制抑制 T 細(xì)胞激活，包括提高精氨酸酶-1（arginase-1，Arg-1）活性、增強(qiáng)一氧化氮（nitric oxide，NO）和活性氧（reactive oxygen species，ROS）的產(chǎn)生[3, 7-9]。不同亞型的 MDSCs通過不同機(jī)制發(fā)揮功能：G-MDSCs 主要通過 ROS，而 M-MDSCs 主要通過精氨酸酶和 NO 發(fā)揮其免疫抑制功能[4, 10]。MDSCs 還可以通過其他機(jī)制發(fā)揮免疫抑制功能，如分泌細(xì)胞因子 TGF-β 抑制免疫反應(yīng)[11-12]，消耗半胱氨酸[13]，高表達(dá) COX2 和前列腺素 E2（prostaglandin E2，PGE2）、血紅素氧合酶-1 （hemeoxygenase-1，HO-1）、酶吲哚胺 2,3 加雙氧酶（enzyme indoleamine 2,3 dioxygenase，IDO）、還原型煙酰胺腺嘌呤二核苷酸磷酸氧化酶（nicotinamide adenine dinucleotide phosphate oxidase-2，NOX-2）、下調(diào) T 細(xì)胞表面 TCR-ζ 鏈、質(zhì)膜分子選擇素（CD62L）的表達(dá)、誘導(dǎo)調(diào)節(jié)性 T 細(xì)胞（regulatory T cells，Treg）生成[14-21]，抑制自然殺傷細(xì)胞（natural killer cell，NK）的殺傷作用[22-24]，影響 DC 和巨噬細(xì)胞功能等發(fā)揮其抑制功能[25]。目前，調(diào)控MDSCs 的分子機(jī)制及 MDSCs 的細(xì)胞治療應(yīng)用為該領(lǐng)域的研究熱點(diǎn)。

1　MDSCs 體外誘導(dǎo)

研究表明，粒-巨噬細(xì)胞集落刺激因子（granulocytemacrophage colony-stimulating factor，GM-CSF）等因子可以在體外誘導(dǎo)骨髓細(xì)胞及外周單核細(xì)胞發(fā)育分化為CD11b+Gr-1+MDSCs[26]（表1）。下面分別進(jìn)行簡要介紹。

表1　不同細(xì)胞因子組合誘導(dǎo) MDSCs 的效率、表型

續(xù)表1

1.1骨髓細(xì)胞誘導(dǎo)分化 MDSCs

早期研究表明，體外用 GM-CSF、GM-CSF+ 白細(xì)胞介素-4（interleukin-4，IL-4）誘導(dǎo)小鼠骨髓細(xì)胞分化為 DCs。但根據(jù)細(xì)胞因子的濃度大小和刺激時(shí)間長短可以誘導(dǎo)免疫抑制性細(xì)胞產(chǎn)生[35-36]。體外高濃度GM-CSF 短期（3 ～ 4 d）/低濃度 GM-CSF 長期（8 ～ 10 d）刺激小鼠骨髓細(xì)胞產(chǎn)生 CD11b+Gr-1lowCD31+ER-MP58+F4/80+asialoGM1+CD11c?MDSCs，它們通過細(xì)胞接觸和 NO 機(jī)制抑制CD4+T 和CD8+T 細(xì)胞應(yīng)答反應(yīng)[37]。

粒細(xì)胞集落刺激因子（granulocyte colony-stimulating factor，G-CSF）（100 ng/ml）、GM-CSF（250 U/ml）短期（4 d）培養(yǎng)鼠骨髓細(xì)胞誘導(dǎo)生成 CD11b+Ly6GlowLy6C+MDSCs，其中絕大多數(shù)細(xì)胞還表達(dá) IL-4Rα(CD124)+和F4/80+，體外這些細(xì)胞通過 Arg-1 消耗精氨酸機(jī)制抑制同種異基因 T 細(xì)胞應(yīng)答。與 G-CSF 或 GM-CSF 相比，G-CSF + GM-CSF 或 G-CSF + GM-CSF + IL-13（80 ng/ml）培養(yǎng)骨髓細(xì)胞顯著增加 CD11b+Gr-1+細(xì)胞的產(chǎn)生。相對(duì)于其他誘導(dǎo) Arg-1 的細(xì)胞因子（IL-4 和 PGE2 等），外源性 IL-13體外誘導(dǎo) MDSC 亞型（MDSC-IL-13）產(chǎn)生，表達(dá) CD11c+（60%）、MHC-II+（65%）、Ly6C+（80%）、Ly6Glow（5%）、F4/80+（75%）、CD115+（55%）和 IL4Ra+（55%），顯著增強(qiáng) Arg-1 活力及抑制 T 細(xì)胞的同種異體反應(yīng)[27]。

G-CSF+GM-CSF、GM-CSF+IL-6 短期（4 d）培養(yǎng)鼠骨髓細(xì)胞生成 MDSCs，高表達(dá) CD11b、Gr-1 和 IL-4R，依賴于轉(zhuǎn)錄因子 CCAAT- 增強(qiáng)子結(jié)合蛋白 β（CCAAT enhancer binding proteinsβ，C/EBPβ）顯著增強(qiáng)其免疫抑制活性。同樣，GM-CSF+G-CSF、GM-CSF+IL-6 短期（4 d）處理人骨髓細(xì)胞誘導(dǎo)不成熟 CD11b+CD16?MDSCs 細(xì)胞的產(chǎn)生[29]，免疫抑制活性依賴 C/EBPβ 轉(zhuǎn)錄因子。進(jìn)一步研究表明，GM-CSF+G-CSF 誘導(dǎo)的 BM-MDSCs中 CD11blow/?/CD16?細(xì)胞亞群具有較強(qiáng)的免疫抑制能力，而 CD11b+/CD16?細(xì)胞和 CD11b+/CD16+細(xì)胞無免疫抑制能力[38]。G-CSF+GM-CSF+IL-6 培養(yǎng)小鼠骨髓細(xì)胞 3 d 誘導(dǎo)產(chǎn)生Ly6GhighLy6Cint/lowMDSCs，類似于滑膜液中的 MDSCs?；ひ?MDSCs 通過產(chǎn)生 NO，顯著抑制抗原特異性及多克隆 T 細(xì)胞增殖[39]。

研究表明，GM-CSF 可誘導(dǎo)小鼠骨髓細(xì)胞產(chǎn)生 DCs，但高濃度脂多糖（lipopolysaccharides，LPS）早期或長期處理 BM-DCs 培養(yǎng)體系能夠阻斷 DC 分化成熟，產(chǎn)生體外能夠誘導(dǎo)同種異體抗原特異性 T 細(xì)胞無應(yīng)答的不成熟細(xì)胞[30]。進(jìn)一步研究表明，LPS/IFN-γ 刺激 3 d BM-GM-CSF 培養(yǎng)體系，能顯著抑制骨髓細(xì)胞向 DCs 分化，提高 MDSCs 的免疫抑制功能[24]。體外 GM-CSF+LPS 誘導(dǎo) Lin-骨髓祖細(xì)胞生成 CD11b+Gr-1+細(xì)胞[40]。

GM-CSF+IL-4 處理視網(wǎng)膜色素上皮（retinal pigment epithelial，RPE）細(xì)胞與骨髓細(xì)胞共培養(yǎng) 6 d 可誘導(dǎo)產(chǎn)生CD11b+Gr-1+MDSCs，以劑量依賴的方式抑制 T 細(xì)胞增殖。深入研究表明視網(wǎng)膜色素上皮細(xì)胞表面蛋白和分泌的可溶性因子 IL-6 參與功能性 MDSCs 的誘導(dǎo)[31]。GM-CSF 聯(lián)合腫瘤外植體培養(yǎng)上清短期處理（3 d）EL-4 荷瘤鼠骨髓細(xì)胞中分離的 CD11b+Ly6ChighLy6G?M-MDSCs 可誘導(dǎo)產(chǎn)生免疫抑制性多型核白細(xì)胞（polymorphonuclear，PMN）樣MDSCs（CD11b+Ly6ClowLy6G+），抑制 T 細(xì)胞應(yīng)答，促進(jìn)腫瘤進(jìn)展。正常小鼠單核細(xì)胞不能分化為 PMNs，而荷瘤鼠中 M-MDSCs 可以轉(zhuǎn)化為 CD11b+Ly6ClowLy6G+PMN-MDSCs[41]。阿司匹林敏感性哮喘患者肺部CD11b+Gr-1highLy6G+Ly6CintPMN-MDSCs 具有免疫抑制性，可抑制氣道炎癥。體外實(shí)驗(yàn)表明，COX1-PGE2 通過PGE2 受體信號(hào)通路介導(dǎo) BM-MDSCs 擴(kuò)增，PGE2 促進(jìn)IL-4/GM-CSF 誘導(dǎo)的骨髓前體細(xì)胞產(chǎn)生大量 Ly6C+Ly6G+PMN-MDSCs，激活 PMN-MDSCs 成為治療支氣管哮喘的潛在治療策略[42]。肝星狀細(xì)胞（hepatic stellate cells，HSCs）加入到 GM-CSF+IL-4-DC 培養(yǎng)體系中可以促進(jìn) HSC-MDSCs產(chǎn)生，高表達(dá) iNOS 和 Arg-1，能顯著抑制混合淋巴細(xì)胞體系中 T 淋巴細(xì)胞的增殖反應(yīng)[43]。

1.2外周血單核細(xì)胞誘導(dǎo)分化 MDSCs

外周血單核細(xì)胞（peripheral blood mononuclear cells，PBMCs）由于在循環(huán)系統(tǒng)中具有相對(duì)豐富的細(xì)胞量，可為體外誘導(dǎo) MDSCs 提供一個(gè)方便的細(xì)胞來源。研究表明腫瘤模型中擴(kuò)增和激活 MDSCs 的相關(guān)因素，包括生長因子如（stem cell factor，SCF）、VEGF、GM-CSF、G-CSF 和M-CSF[32]；細(xì)胞因子如 IFN-γ、IL-1β、IL-6、IL-10、IL-12、IL-13、COX2 和 PGE2[3, 44]。體內(nèi)腫瘤微環(huán)境中誘導(dǎo)MDSCs 所需的細(xì)胞因子將為體外誘導(dǎo)具有免疫抑制功能MDSCs 的產(chǎn)生提供依據(jù)。進(jìn)一步研究表明，不同細(xì)胞因子培養(yǎng)健康人 PBMCs 可誘導(dǎo)不同功能、表型的 MDSCs 產(chǎn)生[45]。

外源性 PGE2 和不同 COX2 激活劑（如 LPS、IL-1β 和 IFN-γ）誘導(dǎo)單核細(xì)胞表達(dá) COX2，阻止其向 CD1a+DCs分化和誘導(dǎo) MDSCs 相關(guān)抑制因子 PGE2、IDO1、IL-4Rα、NOS2、IL-10 的產(chǎn)生，PGE2 和 COX2 在誘導(dǎo)人卵巢癌患者體內(nèi)分離的 CD1a+DCs 向 CD14+CD33+CD34+M-MDSCs 分化中發(fā)揮決定性作用[17]。更進(jìn)一步的研究發(fā)現(xiàn)，rhGM-CSF、IL-4、PGE2 處理健康人外周血分離的 CD14+單核細(xì)胞（6 d）阻斷 DCs 分化，誘導(dǎo)CD1a?DCSIGN?CD14+CD33+CD34+CD80?CD83?-MDSCs產(chǎn)生，高表達(dá) PGE2-COX2、IDO1、誘導(dǎo)型一氧化氮合酶（inducible nitric oxide synthase，iNOS）、IL-10、IL-4Rα 等MDSCs 相關(guān)細(xì)胞因子抑制細(xì)胞毒性 T 淋巴細(xì)胞的功能[46]。這些數(shù)據(jù)表明，COX2 和 PGE2 是參與調(diào)控 MDSCs 分化和影響 MDSCs 功能的關(guān)鍵信號(hào)分子。多聚肌苷酸多聚胞苷酸（polyiosinic-polycytidylic acid，polyI:C）或者 LPS 刺激 GM-CSF+IL-4 與人 PBMCs 培養(yǎng)體系 24 h，單核樣CD14+CD33+HLA-DR?MDSCs 顯著增多，通過 IDO 依賴的方式誘導(dǎo)異基因細(xì)胞毒性 T 淋巴細(xì)胞凋亡，顯著增強(qiáng)MDSCs 吞噬凋亡的能力[47]。LPS 或 poly（I:C）激活的MDSCs 成為移植潛在的治療策略。

人實(shí)體瘤細(xì)胞與 PBMCs 共培養(yǎng)，體外可產(chǎn)生兩種不同的 MDSCs 亞型：CD33+HLA-DRlowHIF1a+/STAT3+及CD11b+HLA-DRlowC/EBPβ+。CD33+MDSCs 的誘導(dǎo)主要依賴腫瘤來源細(xì)胞因子 GM-CSF、IL-1β、IL-6、VEGF、PGE2、TNF-α 高表達(dá)，CD11b+MDSCs 誘導(dǎo)與酪氨酸激酶受體 3配體（Fms-liket tyrosine kinase 3 ligand，F(xiàn)LT3L）、TGF-β 高表達(dá)相關(guān)[33]。肝星狀細(xì)胞通過 CD44 介導(dǎo)的細(xì)胞之間的彼此接觸來誘導(dǎo)人外周血單核細(xì)胞分化為 CD14+HLA?DR?/lowMDSCs[48]。而 HIV gp120 處理健康人外周血細(xì)胞產(chǎn)生CD11b+CD33+CD14+HLA-DR?/low細(xì)胞。gp120-CD33+細(xì)胞產(chǎn)生 IL-10 和誘導(dǎo) CD4+CD25+FoxP3+Treg 細(xì)胞擴(kuò)增，依賴 iNOS 和 ROS 介導(dǎo) T 細(xì)胞抑制。HIV 患者體內(nèi)MDSCs 細(xì)胞數(shù)明顯增多，因此探討 MDSCs 在 HIV 感染中的作用及其誘導(dǎo)因素可為治療 HIV 提供依據(jù)[49]。

1.3其他細(xì)胞誘導(dǎo)分化 MDSCs

此外，小鼠胚胎干細(xì)胞（embryonic stem cells，ESCs）體外也可誘導(dǎo)功能型 MDSCs 分化。ESCs 誘導(dǎo)的 MDSCs分泌 NO 和 IL-10，誘導(dǎo) CD4+CD25+Foxp3+Treg 細(xì)胞產(chǎn)生，表現(xiàn)較強(qiáng)的抑制活性[50]。IL-17（10 ng/ml）體外能夠促進(jìn) MMTV-PyMT FVB 荷瘤鼠脾臟中分離的CD11b+Gr-1+MDSCs 分泌 Arg-1、IDO、COX2，增強(qiáng)其抑制能力[51]。GM-CSF、G-CSF、IL-6 培養(yǎng)臍帶血來源的CD34+細(xì)胞產(chǎn)生不同功能 MDSCs。G-CSF 和（或）IL-6 能顯著增加 CD14+HLA-DRlow/?和 CD14+PD-L1+細(xì)胞產(chǎn)生，并顯著提高 Arg-1 和 C/EBPβ 的表達(dá)進(jìn)而調(diào)控 MDSCs 的功能。GM-CSF+IL-6 誘導(dǎo) Lin?CD34+CD38+CD123+CD45RA+粒-單核系祖細(xì)胞的產(chǎn)生，高表達(dá) CD11b、CD14、CD15。GM-CSF+G-CSF 或 GM-CSF+IL-6 誘導(dǎo)產(chǎn)生的CD11b+CD14+CB-MDSCs 能抑制 CD3+、CD3+CD4+、CD3+CD4?T 細(xì)胞的增殖，下調(diào) T 細(xì)胞表面 CD3+ξ 表達(dá)，并且誘導(dǎo) Foxp3+Treg 細(xì)胞的產(chǎn)生[52]。深入研究表明rh-GM-CSF 和 rh-G-CSF 處理人臍帶血細(xì)胞（4 d），可產(chǎn)生 MDSCs，表達(dá) IDO 選擇性誘導(dǎo)擴(kuò)增 Foxp3+Treg 細(xì)胞，具有較強(qiáng)的耐受活性和抑制活性[53]。

2　MDSCs 細(xì)胞免疫療法

過繼轉(zhuǎn)移體內(nèi)分離的功能性 MDSCs 可以有效降低器官移植排斥反應(yīng)，促進(jìn)同種異體皮膚、腎臟移植長期存活和預(yù)防自身免疫性疾病，如 I 型糖尿病、腦脊髓炎、敗血癥、腸炎、肝炎、關(guān)節(jié)炎和腎損傷的發(fā)生[34, 54-62]。

一些自身免疫性疾病小鼠模型中，過繼轉(zhuǎn)移體外誘導(dǎo)的 MDSCs 能夠抑制自身免疫性反應(yīng)，限制組織損傷。在實(shí)驗(yàn)性自身免疫性葡萄膜炎模型中，過繼轉(zhuǎn)移體外誘導(dǎo)的MDSCs 能抑制特異性 T 細(xì)胞應(yīng)答，減小葡萄膜炎的發(fā)病[31]。在蛋白聚糖誘導(dǎo)的關(guān)節(jié)炎小鼠模型中，過繼轉(zhuǎn)移體外誘導(dǎo)的 BM-MDSCs 可以減弱 PG 特異性 T 細(xì)胞應(yīng)答，減緩關(guān)節(jié)炎進(jìn)展[39]。將體外誘導(dǎo) BM-MDSC-IL-13 注入小鼠體內(nèi)能更有效地抑制 T 細(xì)胞增殖、激活以及分泌 IFN-γ的能力，并能依賴 Arg-1 機(jī)制有效抑制移植物抗宿主?。℅VHD）[27]。此外，給小鼠過繼體外誘導(dǎo)產(chǎn)生的 ES-MDSCs也可以有效防止同種反應(yīng)性 T 細(xì)胞介導(dǎo)的 GVHD[50]。同樣，過繼轉(zhuǎn)移體外骨髓細(xì)胞產(chǎn)生的 GM-CSF/G-CSF-MDSCs能有效抑制 GVHD 死亡率[62]。在 I 型糖尿病小鼠模型中，過繼轉(zhuǎn)移體外誘導(dǎo)的 MDSCs 聯(lián)合 UCB-T 淋巴細(xì)胞，能夠誘導(dǎo) Treg 細(xì)胞，維持血糖濃度正常，延緩糖尿病發(fā)病[53]。

MDSCs 參與移植免疫耐受的誘導(dǎo)，MDSCs 在器官（腎臟、心臟和皮膚）移植部位發(fā)揮局部免疫抑制效應(yīng)，誘導(dǎo)免疫耐受[2]。在同種異體胰島移植模型中，給糖尿病鼠過繼轉(zhuǎn)移體外誘導(dǎo)的 BM-MDSCs，能減少抗原特異性 CD8+T 細(xì)胞的功能，有效維持糖尿病小鼠正常血糖濃度，顯著地促進(jìn)胰島移植長期存活[29]。體內(nèi)共轉(zhuǎn)移 HSC-MDSCs，能通過Inos 介導(dǎo)的 T 細(xì)胞抑制顯著延長同種異體胰島移植存活[43]。過繼轉(zhuǎn)移LPS 或者 poly（I:C）激活的 MDSCs 同樣具有這種保護(hù)機(jī)制，成為潛在的移植治療策略[47]。過繼轉(zhuǎn)移實(shí)驗(yàn)表明，MDSCs 能夠在體內(nèi)誘導(dǎo)移植免疫耐受反應(yīng)，延緩移植排斥，使得 MDSCs 成為潛在的臨床治療移植排斥反應(yīng)的有效方法。

3　小結(jié)

體外誘導(dǎo)的 MDSCs 具有抑制自身免疫性疾病、移植排斥反應(yīng)的功效，利用該細(xì)胞過繼療法治療相關(guān)疾病具有潛在臨床應(yīng)用價(jià)值。建立穩(wěn)定的體外誘導(dǎo) MDSCs 體系將為MDSCs 的臨床應(yīng)用提供重要的技術(shù)支撐。然而，到目前為止，體外分化 MDSCs 體系效率低下，而且擴(kuò)增無法達(dá)到顯著水平[63]。此外，MDSCs 在實(shí)驗(yàn)和臨床前研究的治療效率，特異性及安全性仍待解決。因此，理解 MDSCs 的誘導(dǎo)通路和建立有效的擴(kuò)增 MDSCs 誘導(dǎo)體系及深入了解MDSCs 體外誘導(dǎo)的分子機(jī)制將大大推進(jìn) MDSCs 的臨床應(yīng)用。

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·協(xié)會(huì)之窗·

收稿日期：2015-10-08

通信作者：李衛(wèi)國，Email：liwg0618@htu.cn

基金項(xiàng)目：河南省重點(diǎn)科技攻關(guān)計(jì)劃（112102310320、122102310282）

DOI:10.3969/j.issn.1673-713X.2016.01.011