阿婷曦，邵春益，傅 瑤

上海交通大學(xué)醫(yī)學(xué)院附屬第九人民醫(yī)院眼科，上海市眼眶病眼腫瘤重點(diǎn)實(shí)驗(yàn)室，上海 200011

調(diào)節(jié)性T 細(xì)胞（regulatory T cell，Treg 細(xì)胞）是一類具有免疫調(diào)節(jié)作用的T細(xì)胞，在抑制過度炎癥反應(yīng)、維持免疫平衡和誘導(dǎo)免疫耐受等方面發(fā)揮重要作用。GERSHON 和KONDO［1］最早在1970 年提出抑制性T 細(xì)胞這一概念，隨后SAKAGUCHI 等［2］首次發(fā)現(xiàn)CD4+CD25+的T細(xì)胞具有抑制自身免疫反應(yīng)的作用，將其命名為Treg 細(xì)胞，自此Treg 細(xì)胞成為免疫學(xué)領(lǐng)域的一個研究重點(diǎn)。

在解剖學(xué)上，眼表由角膜、結(jié)膜、眼瞼及其上面的瞼板腺、淚腺組成，長期暴露于各種環(huán)境刺激物、病原體和過敏原。為此，眼表具有成熟的免疫系統(tǒng)，包括先天性和適應(yīng)性免疫，以提供保護(hù)作用［3］。眼表的黏膜免疫反應(yīng)與其他部位的黏膜類似，可以簡述為以下步驟：①抗原與黏膜上皮的相互作用。②啟動先天性免疫系統(tǒng)。③眼表的抗原提呈細(xì)胞（antigen presenting cell，APC）捕獲并處理抗原，再將其提呈給T 細(xì)胞。④效應(yīng)T 細(xì)胞和Treg 細(xì)胞的分化、增殖、遷移和激活。⑤體液免疫反應(yīng)。⑥黏膜先天性免疫、適應(yīng)性免疫和神經(jīng)系統(tǒng)之間廣泛的相互作用［4］。然而為了使組織器官能發(fā)揮正常的功能，黏膜需維持無炎癥的生理狀態(tài)，這種無害抗原通過黏膜表面?zhèn)鬟f后引起的局部和全身免疫無應(yīng)答的狀態(tài)稱為黏膜免疫耐受，由Treg 細(xì)胞參與調(diào)控［3］。除黏膜免疫系統(tǒng)外，前房相關(guān)免疫偏倚（anterior chamber associated immune deviation，ACAID）也參與眼部免疫耐受的維持，Treg 細(xì)胞在其中發(fā)揮重要作用［5］。免疫調(diào)節(jié)功能障礙被認(rèn)為是許多眼表疾病的核心原因，因此本研究擬綜述Treg 細(xì)胞的生物學(xué)特性及其在多種眼表疾病中作用的相關(guān)研究進(jìn)展，并對靶向Treg 細(xì)胞的治療在眼表疾病中的應(yīng)用進(jìn)行展望。

1 Treg細(xì)胞的生物學(xué)特性

Treg 細(xì)胞占外周血中CD4+T 細(xì)胞的5%～10%，特征性表達(dá)細(xì)胞膜標(biāo)志物CD25 和細(xì)胞核轉(zhuǎn)錄因子叉頭狀蛋白P3（forkhead box P3，F(xiàn)OXP3）［6］。FOXP3的表達(dá)對Treg 細(xì)胞的發(fā)育和功能的維持起著至關(guān)重要的作用［7］。在某些條件如極端的炎癥情況下，Treg細(xì)胞會變得不穩(wěn)定，失去FOXP3 表達(dá)和免疫抑制功能，轉(zhuǎn)化為效應(yīng)T細(xì)胞，這種現(xiàn)象被稱為Treg細(xì)胞的可塑性（plasticity）［8］。另有研究［9］發(fā)現(xiàn)CD127 的表達(dá)與FOXP3的表達(dá)及Treg細(xì)胞的抑制功能呈負(fù)相關(guān)，因此也將CD4+CD25+CD127lowT 細(xì)胞定義為純化的Treg細(xì)胞。根據(jù)其來源不同Treg細(xì)胞被分為3類：胸腺衍生的Treg 細(xì)胞（tTreg 細(xì)胞，也稱天然Treg 細(xì)胞）、特定環(huán)境抗原刺激誘導(dǎo)外周的初始CD4+T 細(xì)胞分化成的Treg 細(xì)胞（pTreg 細(xì)胞）以及體外利用轉(zhuǎn)化生長因子-β（transforming growth factor β，TGF-β）等誘導(dǎo)初始CD4+T 細(xì)胞分化產(chǎn)生的Treg 細(xì)胞（iTreg細(xì)胞）［10］。目前尚無明確可區(qū)分人tTreg 細(xì)胞和pTreg細(xì)胞的表面標(biāo)志物，因此目前常用于實(shí)驗(yàn)和臨床研究的從人外周血中分離出來的Treg 細(xì)胞很可能同時(shí)含有tTreg細(xì)胞和pTreg細(xì)胞［11］。

Treg 細(xì)胞通過多種方式發(fā)揮免疫抑制作用，包括：①分泌產(chǎn)生抑制性細(xì)胞因子TGF-β、白細(xì)胞介素-35（interleukin-35，IL-35）和IL-10，參與調(diào)節(jié)多種譜系的T 細(xì)胞分化和功能，發(fā)揮免疫抑制作用［12-13］。②高表達(dá)CD25［又稱白介素-2 受體α（IL-2Rα）］，競爭性結(jié)合T 細(xì)胞增殖所必需的細(xì)胞因子IL-2，阻止T 細(xì)胞的繼續(xù)增殖，導(dǎo)致已有細(xì)胞的代謝中斷和細(xì)胞死亡［14］。③表達(dá)抑制性共刺激受體細(xì)胞毒性T 淋巴細(xì)胞相關(guān)抗原4（cytotoxic T-lymphocyteassociated antigen-4，CTLA-4），與效應(yīng)T細(xì)胞競爭結(jié)合APC 表面共刺激分子CD80 和CD86，控制效應(yīng)T細(xì)胞的數(shù)量和影響免疫應(yīng)答［15］；同時(shí)促進(jìn)APC 產(chǎn)生吲哚胺2，3-雙加氧酶（indoleamine 2，3-dioxygenase，IDO），其代謝物可以發(fā)揮免疫抑制作用［16］。④表達(dá)淋巴細(xì)胞活化基因3（LAG3，又稱CD223），通過與APC 表達(dá)的Ⅱ類主要組織相容性復(fù)合體（major histocompatibility complex class Ⅱ，MHC-Ⅱ）結(jié)合，誘導(dǎo)免疫耐受［17］。⑤高表達(dá)環(huán)磷酸腺苷（cyclic adenosine monophosphate，cAMP），調(diào)節(jié)效應(yīng)T細(xì)胞和APC 的功能活性［18］。⑥分泌顆粒酶B（granzyme B）和穿孔素-1（perforin-1）誘導(dǎo)自然殺傷細(xì)胞（natural killer cell，NK細(xì)胞）和細(xì)胞毒性T細(xì)胞的溶解［19］。

此外，最近的研究結(jié)果［20］表明，Treg 細(xì)胞也存在于健康組織，如骨骼肌、內(nèi)臟脂肪組織和皮膚的毛囊干細(xì)胞龕中，表達(dá)不同的歸巢和遷移標(biāo)志物，稱為組織調(diào)節(jié)性T 細(xì)胞（tissue regulatory T cell），且具有抑制炎癥以外的功能。

2 Treg細(xì)胞與眼表疾病

2.1 干眼癥

干眼癥是眼科常見疾病，淚膜和眼表協(xié)會（Tear Film and Ocularsurface Society，TFOS）干眼癥工作小組（Dry Eye Workshop，DEWS）發(fā)布的專家共識（TFOS DEWS Ⅱ）將其定義為以淚膜穩(wěn)態(tài)喪失并伴有淚膜不穩(wěn)定和高滲狀態(tài)、眼表炎癥和損傷以及神經(jīng)感覺異常等眼部癥狀的多因素眼表疾?。?1］。其核心驅(qū)動因素是干燥壓力誘發(fā)的炎癥惡性循環(huán)，與CD4+T細(xì)胞的活化和浸潤有關(guān)［22-23］。在膽堿能受體拮抗劑誘發(fā)的小鼠干眼模型和環(huán)境誘導(dǎo)的小鼠干眼模型中均可以觀察到引流區(qū)淋巴結(jié)中Treg細(xì)胞的抑制能力受損、輔助性T 細(xì)胞17（helper T cell 17，Th17）/Treg 穩(wěn)態(tài)失衡，體內(nèi)阻斷IL-17可以恢復(fù)Treg細(xì)胞的功能，并顯著降低干眼的嚴(yán)重程度、延緩疾病進(jìn)展［24-25］。

通過靶向Treg 細(xì)胞治療干眼癥已在動物模型上取得較好效果。SIEMASKO等［26］發(fā)現(xiàn)過繼體外擴(kuò)增產(chǎn)生的FOXP3+Treg 細(xì)胞至干眼模型小鼠可以有效減少淚液中炎癥因子的含量，抑制免疫介導(dǎo)的炎癥反應(yīng)。RATAY 等［27］通過增加淚腺中趨化因子CCL22的局部釋放，誘導(dǎo)內(nèi)源性Treg 細(xì)胞的募集，與未經(jīng)治療組相比，引流區(qū)效應(yīng)性CD4+T 細(xì)胞的數(shù)量和淚腺中CD4+IFN-γ+（γ 干擾素）Th1 細(xì)胞的浸潤減少，淚液分泌增加，杯狀細(xì)胞增多，上皮病變減少，說明局部增加功能正常的Treg 細(xì)胞數(shù)量也能改變免疫失衡，進(jìn)而有效減輕實(shí)驗(yàn)性干眼模型中的炎癥反應(yīng)，從而緩解干眼的相關(guān)癥狀。此外，靜脈注射色素上皮衍生 因 子（pigment epithelium-derived factor，PEDF）可以通過增加干眼小鼠的Treg 細(xì)胞數(shù)量和增強(qiáng)免疫抑制功能，減輕干眼的嚴(yán)重程度［28］。間充質(zhì)干細(xì)胞及其外泌體療法可以抑制Th17 細(xì)胞、誘導(dǎo)Treg 細(xì)胞的增殖，減輕干燥綜合征的嚴(yán)重程度［29-30］。由此可見，通過細(xì)胞療法或者藥物干預(yù)等手段增加Treg 細(xì)胞的循環(huán)或局部數(shù)量、增強(qiáng)其抑制功能，可能實(shí)現(xiàn)對干眼癥的治療和改善。

2.2 眼表過敏性疾病

眼表的過敏性疾病包括季節(jié)性過敏性結(jié)膜炎、常年性過敏性結(jié)膜炎、春季角膜結(jié)膜炎和特應(yīng)性角膜結(jié)膜炎等一系列疾病，與抗原特異性IgE 介導(dǎo)的Ⅰ型超敏反應(yīng)和抗原特異性T細(xì)胞介導(dǎo)的Ⅳ型超敏反應(yīng)密切相關(guān)。研究［31-32］發(fā)現(xiàn)過敏性結(jié)膜炎患者中存在免疫失調(diào)，與健康對照相比，常年性過敏性結(jié)膜炎人群外周血單核細(xì)胞中CD4+CD25+FOXP3+Treg 細(xì)胞的數(shù)量減少，CD4+CD25+FOXP3-T 細(xì)胞的數(shù)量增加，提示Treg 細(xì)胞受損可能參與過敏性結(jié)膜炎的發(fā)生發(fā)展。SUMI等［33］發(fā)現(xiàn)胸腺切除術(shù)和PC61（抗CD25 抗體）消耗小鼠體內(nèi)的CD25+T 細(xì)胞導(dǎo)致豚草（ragweed，RW）致敏的小鼠結(jié)膜嗜酸性粒細(xì)胞浸潤增多，增加實(shí)驗(yàn)性過敏性結(jié)膜炎 （experimental allergic conjunctivitis，EC）的嚴(yán)重程度，而過繼正常小鼠的CD4+CD25+T 細(xì)胞至致敏小鼠可以有效抑制EC 的發(fā)展。另一項(xiàng)研究［34］發(fā)現(xiàn)，具有免疫調(diào)節(jié)作用的合成糖脂α-半乳糖神經(jīng)酰胺（α-galactosylceramide，α-GalCer），可以通過增加CD4+CD25+FOXP3+Treg細(xì)胞的數(shù)量抑制EC 的發(fā)展，提示Treg 細(xì)胞有希望成為過敏性結(jié)膜炎的治療靶點(diǎn)。

2.3 眼表感染性疾病

發(fā)生感染時(shí)，Treg細(xì)胞的主要功能是控制過度的炎癥反應(yīng)以防止組織損害、減少對宿主的傷害，但在某些情況下Treg 細(xì)胞的免疫抑制能力會減弱機(jī)體的免疫監(jiān)測能力，促進(jìn)病毒的潛伏［35］。1型單純皰疹病毒（HSV-1）復(fù)發(fā)引起的角膜基質(zhì)炎為先天性免疫與適應(yīng)性免疫介導(dǎo)的慢性炎癥反應(yīng)，其中效應(yīng)性CD4+T細(xì)胞為主要驅(qū)動因素，而Treg細(xì)胞在其中也發(fā)揮著重要作用［36］。通過PC61 耗竭小鼠體內(nèi)的Treg 細(xì)胞后，HSV-1誘導(dǎo)的角膜基質(zhì)炎的嚴(yán)重程度增加，而疾病早期過繼Treg 細(xì)胞可以抑制角膜的免疫炎癥［37-38］。BHELA 等［39］運(yùn)用FOXP3 表達(dá)追蹤轉(zhuǎn)基因小鼠品系（FOXP3Cre-GFP：Rosa26lsl-Td-Tomato），觀察到病毒誘導(dǎo)的角膜炎癥狀態(tài)下角膜Treg 細(xì)胞可塑性的變化，發(fā)現(xiàn)HSV-1 感染眼部后，角膜中Treg 細(xì)胞是不穩(wěn)定的，可轉(zhuǎn)化為具有效應(yīng)Th1 細(xì)胞表型的ex-Treg細(xì)胞，分泌IFN-γ，參與角膜基質(zhì)炎的發(fā)生。此外，過繼的體外誘導(dǎo)的正常功能iTreg 細(xì)胞在角膜炎癥的環(huán)境下也高度不穩(wěn)定，部分轉(zhuǎn)化為促進(jìn)角膜基質(zhì)炎發(fā)生的Th1 表型的ex-Treg 細(xì)胞［39］。而在這種情況下，氮雜胞苷、視黃酸和維生素C 等藥物能夠維持FOXP3+Treg 細(xì)胞特異性去甲基化區(qū)（Treg-specific demethylated region，TSDR）的去甲基化，有助于促進(jìn)Treg 細(xì)胞的穩(wěn)定性并改善其功能，更有效抑制角膜基質(zhì)炎的進(jìn)展［39-40］。

2.4 角膜移植排斥

Treg細(xì)胞療法在誘導(dǎo)同種異體移植物的免疫耐受或者預(yù)防移植物抗宿主?。╣raftversushost disease，GVHD）方面已經(jīng)得到廣泛的研究［41］。Treg 細(xì)胞通過抑制宿主對移植物的免疫反應(yīng)，促進(jìn)機(jī)體對移植物的耐受，在降低角膜移植排斥過程中方面發(fā)揮著重要作用，CD25+CD4+Treg 細(xì)胞的耗竭可加速角膜移植排斥的發(fā)生［42-43］。植床存在炎癥或新生血管的宿主更易對移植的角膜產(chǎn)生排斥反應(yīng)，此類高危宿主的pTreg 細(xì)胞（而非tTreg 細(xì)胞）的數(shù)量和功能被抑制，表現(xiàn)為FOXP3 表達(dá)丟失，CTLA-4 表達(dá)降低，IL-10和TGF-β 的分泌減少，并且與pTreg 細(xì)胞向分泌IL-17 和IFN-γ 的ex-Treg 細(xì)胞的病理性轉(zhuǎn)換有關(guān)［44-46］。因此，通過不同途徑，靶向Treg 細(xì)胞來減少角膜移植的排斥反應(yīng)具有很好的發(fā)展前景。小劑量的IL-2治療可以顯著增加CD4+CD25+FOXP3+Treg 細(xì)胞的數(shù)量，增強(qiáng)其免疫抑制功能，進(jìn)而可提高角膜同種異體移植物的存活率［47］。在小鼠異體角膜移植模型中，結(jié)膜下注射Treg 細(xì)胞可以抑制角膜和淋巴組織中APC 的成熟，使角膜中IL-10、TGF-β 表達(dá)增加，CD45+炎癥細(xì)胞侵入減少，移植成功率增加［48］。

2.5 眼表組織修復(fù)

在機(jī)體受傷后，入侵的病原體、壞死的碎片、凝血反應(yīng)和組織內(nèi)的免疫細(xì)胞引發(fā)炎癥反應(yīng)，促進(jìn)組織修復(fù)和瘢痕形成，然而過度的炎癥反應(yīng)會導(dǎo)致病理性纖維化，損害組織功能。Treg細(xì)胞可以通過影響中性粒細(xì)胞、誘導(dǎo)巨噬細(xì)胞分化和抑制效應(yīng)T細(xì)胞參與的免疫反應(yīng)來間接調(diào)節(jié)再生［49］。近年來研究發(fā)現(xiàn)，Treg細(xì)胞除經(jīng)典的免疫抑制功能外，還能通過其他途徑在組織修復(fù)和再生方面發(fā)揮作用，包括促進(jìn)骨骼肌再生［50］、促進(jìn)皮膚傷口愈合［51］、促進(jìn)毛囊干細(xì)胞增殖和分化［52］、促進(jìn)心肌細(xì)胞增殖［53］等。然而目前對Treg 細(xì)胞在眼表組織損傷修復(fù)中的作用研究較少。YAN 等［54］發(fā)現(xiàn)在小鼠角膜堿燒傷急性期的結(jié)膜下注射Treg 細(xì)胞不僅能抑制過度炎癥反應(yīng)，改善眼表環(huán)境，還能促進(jìn)小鼠堿燒傷后角膜上皮修復(fù)，恢復(fù)角膜透明，推測這些作用與局部增高的雙調(diào)蛋白（amphiregulin，AREG）有關(guān)。AREG 是上皮生長因子受體（epidermal growth factor receptor，EGFR）的配體之一，通常由上皮細(xì)胞、間充質(zhì)細(xì)胞和淋巴細(xì)胞等分泌。AREG 與細(xì)胞上的EGFR 結(jié)合，可促進(jìn)這些細(xì)胞的增殖和遷移［55］。另一研究［56］發(fā)現(xiàn)，Treg 細(xì)胞通過分泌IL-10 而非細(xì)胞間直接接觸的方式抑制IFN-γ 和腫瘤壞死因子α （tumor necrosis factor α，TNF-α） 誘導(dǎo)的角膜內(nèi)皮細(xì)胞的死亡。此外，ALTSHULER 等［57］發(fā)現(xiàn)角膜緣外緣存在CD4+CD25+FOXP3+Treg 細(xì)胞，在結(jié)膜下注射PC61.5（也是抗CD25 抗體）消耗Treg 細(xì)胞后，靜止角膜緣干細(xì)胞（quiescent limbal stem cell，qLSC）的標(biāo)志物CD63 和糖蛋白激素α 亞基2（glycoprotein hormone subunit α 2，GPHA2）顯著下降，而細(xì)胞增殖水平上升，推測Treg 細(xì)胞的缺失或功能抑制導(dǎo)致了qLSC 靜止?fàn)顟B(tài)的喪失，傷口愈合延遲。

3 結(jié)語與展望

FOXP3+Treg 細(xì)胞是眼表微環(huán)境的重要組成部分，它們積極地參與抑制針對自身、微生物和環(huán)境抗原的異常或過度的免疫反應(yīng)，在眼表的免疫調(diào)節(jié)中發(fā)揮著重要作用。基于Treg 細(xì)胞誘導(dǎo)免疫耐受的能力，擴(kuò)增FOXP3+Treg 細(xì)胞或者增強(qiáng)其免疫抑制能力已成為治療自身免疫性疾病或者其他免疫相關(guān)疾病，以及防止器官移植排斥反應(yīng)的重要方法［58］。最簡單的方式為過繼細(xì)胞療法，即從患者體內(nèi)分離純化循環(huán)Treg細(xì)胞，在體外擴(kuò)增達(dá)到一定數(shù)量后回輸至患者體內(nèi)［58］。目前主流的Treg 細(xì)胞來源為患者的自體外周血或是臍帶血，通過流式細(xì)胞儀，或是帶標(biāo)記的磁珠分選CD4+CD25+T細(xì)胞或抑制能力更強(qiáng)的CD4+CD25+CD127lowT 細(xì) 胞［59］。Treg 細(xì) 胞 過 繼 療 法 在 治 療GVHD［60］、1 型糖尿病［61］、克羅恩?。?2］等疾病的臨床試驗(yàn)中已取得良好的結(jié)果。另有研究證明，小劑量的IL-2可安全有效增加丙型肝炎病毒相關(guān)性血管炎患者［63］和慢性GVHD患者［64］體內(nèi)Treg細(xì)胞的數(shù)量。

然而目前在眼表領(lǐng)域中Treg 細(xì)胞的研究還停留在基礎(chǔ)階段，缺乏基于眼表疾病的臨床試驗(yàn)，基礎(chǔ)研究和臨床研究之間存在脫節(jié)。要將Treg 細(xì)胞運(yùn)用于眼表的疾病還有許多科學(xué)問題需要解決。比如，SHAO 等［48］研究發(fā)現(xiàn)小鼠球結(jié)膜下注射Treg 細(xì)胞，6 h 后Treg 細(xì)胞即可遷移至角膜和同側(cè)淋巴結(jié)，48 h達(dá)高峰值，但7 d 后僅檢測到很少量的細(xì)胞。Treg 細(xì)胞是否需要多次注射使其能在眼表長期發(fā)揮生物學(xué)效應(yīng)仍待研究。此外，部分Treg 細(xì)胞的不穩(wěn)定性和可塑性也給其臨床應(yīng)用帶來挑戰(zhàn)。

總而言之，隨著對Treg 細(xì)胞領(lǐng)域的深入研究，使用新技術(shù)來改變細(xì)胞的基因組，以增強(qiáng)Treg 細(xì)胞功能、穩(wěn)定性、持久性和抗原特異性，提高Treg 細(xì)胞過繼療法的治療潛力是未來的發(fā)展方向。更進(jìn)一步地探究Treg 細(xì)胞在眼表疾病發(fā)生發(fā)展中的作用，針對性地開展靶向FOXP3+Treg 的治療方法在眼表疾病

領(lǐng)域具有廣闊的前景。

利益沖突聲明/Conflict of Interests

所有作者聲明不存在利益沖突。

All authors disclose no relevant conflict of interests.

作者貢獻(xiàn)/Authors'Contributions

阿婷曦負(fù)責(zé)論文初稿的撰寫，邵春益參與了論文的審閱和修訂，傅瑤提出構(gòu)思以及參與論文的審閱和修訂。所有作者均閱讀并同意了最終稿件的提交。

A Tingxi drafted the original manuscript；SHAO Chunyi participated in the reviewing and editing；FU Yao conceived the idea and participated in the reviewing and editing.All the authors have read the last version of paper and consented for submission.

·Received：2022-02-07

·Accepted：2022-05-23

·Published online：2022-08-12

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