作者：歐陽(yáng)為明 金伯泉 張赟 劉雪松 李琦,，夏海濱
單位：歐陽(yáng)為明 金伯泉 張赟 劉雪松 李琦,，夏海濱（第四軍醫(yī)大學(xué)免疫學(xué)教研室，陜西西安710032）
關(guān)鍵詞：NK細(xì)胞,；T細(xì)胞,；9.1C3分子
摘要：目的 探討9.1C3分子是否作為抑制型受體調(diào)節(jié)NK細(xì)胞和T細(xì)胞的殺傷功能。方法 用抗CD56抗體和羊抗鼠IgG免疫磁珠分離混合淋巴細(xì)胞培養(yǎng)中活化的淋巴細(xì)胞,，分選CD56＋細(xì)胞
和CD56－細(xì)胞分別作為效應(yīng)細(xì)胞,。采用重導(dǎo)向殺傷實(shí)驗(yàn)(redirected killing assay，RKA)觀察抗9.1C3抗體對(duì)效應(yīng)細(xì)胞殺傷小鼠肥大細(xì)胞瘤細(xì)胞P815作用的影響,。結(jié)果 發(fā)現(xiàn)人NK細(xì)胞和T細(xì)胞對(duì)P815細(xì)胞均有一定的殺傷作用,，在效靶比為4∶1，2∶1和1∶1時(shí),，NK細(xì)胞和T細(xì)胞的殺傷率分別為:6.4％,，3.4％，1.1％和21.2％，16.7％,，6.5％,。用抗CD16和抗CD3抗體分別刺激NK細(xì)胞和T細(xì)胞時(shí)，它們對(duì)P815細(xì)胞的細(xì)胞毒作用顯著增強(qiáng),；在相同的效靶比例,，它們對(duì)P815的殺傷率分別為:47.1％，32.2％,，19.1％和64.4％,，50.3％，39.5％,。但用抗9.1C3抗體刺激效應(yīng)細(xì)胞時(shí),，不僅NK細(xì)胞的殺傷作用完全被抑制，CD16介導(dǎo)的NK細(xì)胞的殺傷作用也被明顯下調(diào),，其殺傷率僅為18.5％,，9.7％和7.0％；但對(duì)CD3介導(dǎo)的T細(xì)胞的殺傷作用只輕度被抑制,。結(jié)論 9.1C3分子可能是一種新的抑制型殺傷細(xì)胞受體,，對(duì)NK細(xì)胞和T細(xì)胞細(xì)胞毒作用的負(fù)調(diào)節(jié)可能有所不同。
中圖號(hào)：R392.11 文獻(xiàn)標(biāo)識(shí)碼：A
文章編號(hào)：1007－8738(2000)02－0121－03
The inhibitory effect of 9.1C3 molecule on cytotoxicity mediated by human natural killer cells and T cells
OU- YANG Wei- ming JIN Bo- quan ZHANG Yun LIU Xue- song LI Qi XIA Hai- bin
（Department of Immunology, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China）
Abstract: Aim To investigate if 9.1C3 molecule acts as a killer cell inhibitory receptor regulating the cytotoxicities of human natural killer cells and T cells. Methods Redirected killing assay(RKA) was employed in which CD56＋ cells and T cells were isolated from mixed lymphocyte cultures by magnetic beads method and were used as effectors, and murine mastocytoma cell line P815 was used as target cells. Results We found that both human NK cells and T cells can lyse P815 cells, and the level of specific lysis were 6.4％ , 3.4％ and 1.1％ for NK cells, and 21.2％ , 16.7％ and 6.5％ for T cells at the effector to target ratios of 4, 2 and 1 respectively. The cytotoxicities were increased significantly when the effectors were pre- incubated with CD16 mAb and anti- CD3 mAb respectively. The specific lysis of CD16 triggered NK cells were 47.1％ , 32.2％ and 19.1％ , and 64.4％ , 50.3％ and 39.5％ for CD3-triggered T cells at the same ratios. When the anti- 9.1C3 mAb was added to coincubate effector cells in the presenceor absence anti- CD16 or anti- CD3 mAb, both native and induced cytotoxicities were inhibited obviously. Thespecific lysis of 9.1C3- triggered NK cells was too low to be detected, and the specific lysis of CD16- plus 9.1C3-triggered NK cells or CD3- plus 9.1C3- triggered T cells were 18.5％ , 9.7％ and 7.0％ versus 59.9％ , 41.9％ and27.9％ . Conclusion 9.1C3 molecule may be a kind of killing cell inhibitory receptor, and the inhibitory effect of 9.1C3on NK cell- mediated killing was much stronger than that on T cells which may be due to different signal pathwaysmediated by TCR and Fcγ RIII.
Keywords: natural killer cells; T cells; 9.1C3 molecule
9.1C3為主要表達(dá)在人NK細(xì)胞和T細(xì)胞表面的膜分子,，抗9.1C3單抗(mAb)能顯著抑制NK和LAK等殺傷細(xì)胞的細(xì)胞毒作用[1],。在重導(dǎo)向殺傷實(shí)驗(yàn)（redirect killing assay，RKA）中,，抗9.1C3mAb能抑制CD16和CD2等活化型分子介導(dǎo)的混合淋巴細(xì)胞培養(yǎng)(MLC)中淋巴細(xì)胞對(duì)P815細(xì)胞的殺傷作用,，提示該分子有可能同時(shí)參與NK細(xì)胞和T細(xì)胞殺傷功能的調(diào)節(jié)。本文應(yīng)用免疫磁珠的方法,，從MLC細(xì)胞中分離出CD56＋細(xì)胞作為NK效應(yīng)細(xì)胞,，CD56－的CD3細(xì)胞作為T(mén)效應(yīng)細(xì)胞，采用RKA方法,，研究9.1C3分子對(duì)NK細(xì)胞和T細(xì)胞細(xì)胞毒的抑制作用,，并比較了9.1C3對(duì)抗CD16和抗CD3mAb分別促 進(jìn)NK細(xì)胞和T細(xì)胞殺傷活性的影響。
1 材料和方法
1.1 材料 Na51CrO4購(gòu)自Amersham公司,?？?.1C3，抗CD16,，抗CD3和抗葡萄球菌腸毒素B(SEB)mAb為本室制備,。抗CD56mAb和羊抗鼠IgG交聯(lián)免疫磁珠購(gòu)自Coulter公司,。P815和Daudi細(xì)胞為本室凍存,，培養(yǎng)于 含10mL/LFCS的RPMI1640培養(yǎng)基中。
1.2 方法
1.2.1 混合淋巴細(xì)胞培養(yǎng)〔2〕 取健康人外周血，用Ficoll分離單個(gè)核細(xì)胞(PBMC)作為混合淋巴細(xì)胞培養(yǎng)（MLC）中的反應(yīng)細(xì)胞,。將1.5×106PBMC和0.5×106組照射的Daudi細(xì)胞接種于24孔板中,，置37℃ 50mL/LCO2孵箱培養(yǎng)5d后，加入1.0×105U/L的IL-2繼續(xù)培養(yǎng) 2d,。
1.2.2 NK細(xì)胞和T細(xì)胞的分離 收集培養(yǎng)7d的混合淋巴細(xì)胞,，用抗CD56mAb和免疫磁珠進(jìn)行分離，操作參照說(shuō)明書(shū),。將得到的CD56＋的NK細(xì)胞和CD56－細(xì)胞,，繼續(xù)培養(yǎng)3d后，去除磁珠的細(xì)胞作為RKA實(shí)驗(yàn)的效應(yīng)細(xì)胞,。另外,，取部分細(xì)胞進(jìn)行活細(xì)胞染色， 用FCM分析其表型,。
1.2.3 間接免疫熒光染色和FCM分析 收集細(xì)胞調(diào)整細(xì)胞密度至5×109/L,，100μL/管，加入5μL抗9.1C3等(見(jiàn)1.1)mAb腹水,，4℃作用30min,。洗去游離的一抗，加入FITC標(biāo)記的羊抗鼠IgG二抗,，4℃作用30min,，洗去游離的二抗,，加入200μL固定液進(jìn)行FCM分析,。
1.2.4 RKA實(shí)驗(yàn)[3] 收集對(duì)數(shù)生長(zhǎng)期的P815細(xì)胞，加入51Cr至3.7×106Bq/1.0×106細(xì)胞,，于37℃ 50mL/LCO2孵箱中標(biāo)記2h,，每隔15min搖勻1次。標(biāo)記完畢后洗滌3次,，調(diào)整細(xì)胞密度至1.0×108/L,，加入V型板中，100μL/孔,，作為RKA中的靶細(xì)胞,。按4∶1,2∶1和1∶1的效靶比加入相應(yīng)的效應(yīng)細(xì)胞(CD56＋或CD56－效應(yīng)細(xì)胞預(yù)先同相應(yīng)的mAb于37℃孵育30min)，于37℃ 50mL/LCO2孵箱中放置4h,。每孔取上清100μL,，測(cè)定γ射線的cpm值，并按以下公式計(jì)算殺傷率 ,。
2 結(jié)果
2.1 FCM檢測(cè)磁珠分離細(xì)胞的表型 用抗CD56mAb和免疫磁珠分離混合培養(yǎng)7d的淋巴細(xì)胞,，然后通過(guò)間接免疫熒光染色和FCM分析，分離前、后與免疫磁珠結(jié)合和與免疫磁珠不結(jié)合細(xì)胞的表型及陽(yáng)性百分率(圖1),。結(jié)果表明,，經(jīng)免疫磁珠分離后，可獲 得較純的NK細(xì)胞和T細(xì)胞,。
圖1 免疫磁珠分離前,、后混合培養(yǎng)細(xì)胞的表型
Fig1 Phenotypes of MLC cells before and after im-muno-bead isolation
2.2 抗9.1C3mAb對(duì)效應(yīng)細(xì)胞殺傷活性的影響 通過(guò)免疫磁珠分離得到NK細(xì)胞和T細(xì)胞后，應(yīng)用RKA觀察抗9.1C3mAb對(duì)NK細(xì)胞和T細(xì)胞殺傷活性的影響,。結(jié)果發(fā)現(xiàn),，抗9.1C3mAb能顯著抑制活化型受體CD16介導(dǎo)的NK細(xì)胞對(duì)P815細(xì)胞的殺傷作用。效靶比為4∶1,，2∶1和1∶1時(shí),，CD16介導(dǎo)的殺傷率分別為47.1％，32.2％和19.1％,；若同時(shí)加入抗9.1C3mAb時(shí)殺傷率則下降為：18.8％,，9.7％和7.0％(抑制率分別為：61.6％，69.9％和63.4％),；對(duì)照抗體(抗CD56mAb)對(duì)CD16介導(dǎo)的殺傷作用無(wú)明顯影響,。抗9.1C3mAb對(duì)T細(xì)胞殺傷作用也有一定的抑制作用,，但不如對(duì)NK細(xì)胞殺傷的抑制作用明顯,。在效靶比為4∶1，2∶1和1∶1時(shí),，CD3介導(dǎo)的T細(xì)胞對(duì)P815細(xì)胞的殺傷率分別為64.4％,，50.3％和39.5％；若同時(shí)加入抗9.1C3mAb時(shí),，則殺傷率分別為：59.5％,，41.9％和27.9％(圖2)。
圖2 抗9.1C3mAb對(duì)NK細(xì)胞和T細(xì)胞殺傷作用的影響
Fig2 Effects of anti-9.1C3 mAb on the cytotoxicities of NK and T cells
A:Effect on the cytotoxicity of NK cells;B:Effect on the cytotoxicity of T cells.
3 討論
90年代以來(lái),，有關(guān)NK細(xì)胞受體的研究有了突破,，并克隆和鑒定了一批NK細(xì)胞刺激型或抑制型受體?；罨褪荏w主要有:CD2,，CD16，CD44,，CD69,，NKR-P1和PTA1等。其中CD16是最常用的活化
型NK細(xì)胞受體的模型,；抑制型受體主要有KIRs(killer cell immunoglobulin-like receptors)家族(CD158a,，CD158b,，p70和p140)，p49,，LAIR-1和NKG2家族及Ly49家族(小鼠)等[4],。這些NK細(xì)胞受體的表達(dá)并不限于NK細(xì)胞，其中大部分也表達(dá)于T細(xì)胞上,，并參與調(diào)節(jié)T細(xì)胞的活化,，如KIR和NKG2A等[3－5]，這可能是由于NK細(xì)胞和T細(xì)胞來(lái)自相同的前體細(xì)胞所致,?？?.1C3mAb是用從外周血分離的人NK細(xì)胞免疫小鼠通過(guò)雜交瘤技術(shù)篩選到的1株mAb，其識(shí)別的分子9.1C3分布較廣,，除NK細(xì)胞外,，還表達(dá)于T細(xì)胞、中性粒細(xì)胞,、巨核細(xì)胞及單核細(xì)胞(資料未顯示),。從9.1C3分子的分布及相對(duì)分子質(zhì)量(Mr)(約70000）來(lái)看，9.1C3很可能是一種新的殺傷細(xì)胞抑制型 受體,。
本實(shí)驗(yàn)采用RKA觀察到抗9.1C3mAb能顯著抑制CD16介導(dǎo)的NK細(xì)胞對(duì)P815細(xì)胞的殺傷作用,；對(duì)CD3介導(dǎo)的T細(xì)胞對(duì)P815細(xì)胞的殺傷作用也有一定的抑制作用，但抑制程度相對(duì)較低,。這種現(xiàn)象可
能是9.1C3分子誘導(dǎo)的負(fù)調(diào)節(jié)信號(hào)不足以抑制CD3介導(dǎo)的活化信號(hào)所致,。有關(guān)9.1C3分子的信號(hào)轉(zhuǎn)導(dǎo)，以及與CD16和CD3介導(dǎo)的信號(hào) 轉(zhuǎn)導(dǎo)的關(guān)系值得進(jìn)一步研究,。
基金項(xiàng)目：國(guó)家自然科學(xué)基金資助,，No.39700133
作者簡(jiǎn)介：歐陽(yáng)為明，男,，29歲,，博士生西安市長(zhǎng)樂(lè)西路17號(hào)，Tel.(029)3374531Email:[email protected]
??2000byJCellmolImmunolPress ?www.immunology.net/jcmi;Email:[email protected]
參考文獻(xiàn)：
［1］Gordon FB, Tony T, Perry FB, et al. Human natural killer cells, activated lymphocyte killer cells and monocytes possess similar cytotoxic mechanisms［J]. Proc Natl Acad Sci USA, 1983; 80: 7606－ 7610.
［2］Aramburu J, Balboa MA, Ramirez A, et al. A novel functional cell surface dimer (Kp43) expressed by natural killer cells and T cell receptor- gamma/delta＋ T lymphocytes. I. Inhibition of the IL- 2- dependent proliferation by anti－ Kp43 monoclonal antibody［J]. J Immunol, 1990; 144: 3238－ 3247.
［3］Le Drean E, Vely F, Olcese L, et al. Inhibition of antigen- induced T cell response and antibody- induced NK cell cytotoxicity by NKG2A: association of NKG2A with SHP- 1 and SHP- 2 protein- tyrosine phosphatases[J].Eur J Immunol, 1998; 28: 264－ 276.
［4］Lanier LL. NK cell receptors[J]. Annu Rev Immunol, 1998; 16: 359－ 393.
［5］Lanier LL. Natural killer cells: from no receptor to too many[J]. Immunity, 1997; 6: 371－ 378.
