郭軼先 張藍(lán)方 孫雪靜 萬(wàn)歲桂 夏長(zhǎng)青,2*

(1.首都醫(yī)科大學(xué)宣武醫(yī)院血液科, 北京 100053; 2.佛羅里達(dá)大學(xué)醫(yī)學(xué)院病理學(xué)、免疫學(xué)和實(shí)驗(yàn)醫(yī)學(xué)系，佛羅里達(dá) 32610)

· 基礎(chǔ)研究 ·

紫外線照射未成熟樹(shù)突狀細(xì)胞誘導(dǎo)小鼠同種抗原免疫耐受及其機(jī)制研究

郭軼先1張藍(lán)方1孫雪靜1萬(wàn)歲桂1夏長(zhǎng)青1,2*

(1.首都醫(yī)科大學(xué)宣武醫(yī)院血液科, 北京 100053; 2.佛羅里達(dá)大學(xué)醫(yī)學(xué)院病理學(xué)、免疫學(xué)和實(shí)驗(yàn)醫(yī)學(xué)系，佛羅里達(dá) 32610)

目的 探討應(yīng)用紫外線照射未成熟樹(shù)突狀細(xì)胞誘導(dǎo)C3H小鼠對(duì)Balb/c小鼠同種抗原免疫耐受及其免疫學(xué)機(jī)制。方法 ①用紫外線B(ultraviolet B, UVB)照射Balb/c(H-2d)小鼠骨髓來(lái)源的未成熟樹(shù)突狀細(xì)胞(immature dendritic cells，imDC)，誘導(dǎo)其凋亡，隨后將其輸注到C3H(H-2k)小鼠體內(nèi)，誘導(dǎo)C3H小鼠對(duì)Balb/c抗原免疫耐受。②檢測(cè)誘免疫耐受小鼠體內(nèi)調(diào)節(jié)T細(xì)胞以及細(xì)胞因子白細(xì)胞介素-10(interleukin-10, IL-10)和干擾素-γ(interferon-γ，IFN-γ)分泌以研究其耐受機(jī)制。結(jié)果 ①UVB-Balb/c imDC免疫的C3H小鼠對(duì)Balb/c抗原產(chǎn)生了免疫耐受，不能產(chǎn)生抗Balb/c抗體。②免疫耐受C3H小鼠不能排異注射到體內(nèi)的Balb/c脾細(xì)胞。③UVB-Balb/c imDC免疫的C3H小鼠T細(xì)胞分泌IL-10增加，并且產(chǎn)生更多的FOX-P3+調(diào)節(jié)T細(xì)胞。結(jié)論 ①應(yīng)用UVB-Balb/c imDC免疫C3H小鼠可以使C3H小鼠對(duì)Balb/c抗原產(chǎn)生完全徹底的免疫耐受。②免疫耐受C3H小鼠對(duì)Balb/c抗原產(chǎn)生免疫耐受的可能原因是由于其T細(xì)胞分泌IL-10增加，并且產(chǎn)生更多的FOX-P3+調(diào)節(jié)T細(xì)胞。

樹(shù)突細(xì)胞；凋亡；調(diào)節(jié)T細(xì)胞；免疫耐受

免疫耐受重建和免疫耐受誘導(dǎo)是自身免疫病和異基因移植研究領(lǐng)域的重要研究課題和最終目標(biāo)。此目標(biāo)的實(shí)現(xiàn)將克服目前無(wú)選擇性免疫抑制治療帶來(lái)的嚴(yán)重的健康問(wèn)題。在這些維持自身免疫耐受的機(jī)制中，自身組織細(xì)胞在自我更新中不斷發(fā)生的穩(wěn)態(tài)凋亡發(fā)揮著非常重要的作用[1]?，F(xiàn)有研究[2-3]表明，凋亡是機(jī)體維持免疫系統(tǒng)穩(wěn)定的重要作用機(jī)制，凋亡細(xì)胞誘導(dǎo)耐受的獨(dú)特之處是溫和而高效。因此應(yīng)用凋亡細(xì)胞誘導(dǎo)免疫耐受的治療策略具有極好的臨床前景。樹(shù)突狀細(xì)胞(dendritic cells，DC)是一類(lèi)專(zhuān)職的抗原呈遞細(xì)胞, 它本身并不起到直接殺傷病原的作用，但是在免疫應(yīng)答及免疫耐受中發(fā)揮雙向調(diào)節(jié)作用，穩(wěn)態(tài)DC遞呈抗原將會(huì)導(dǎo)致T細(xì)胞無(wú)應(yīng)答，產(chǎn)生免疫耐受[4-6]。在本研究中，筆者擬通過(guò)凋亡樹(shù)突狀細(xì)胞誘導(dǎo)不同品系小鼠間同種免疫耐受，探求其免疫耐受機(jī)制。

1 材料與方法

1.1 實(shí)驗(yàn)動(dòng)物

SPF級(jí)健康雌性Balb/c (H-2d)小鼠和雄性C3H(H-2k)小鼠購(gòu)自北京維通利華實(shí)驗(yàn)動(dòng)物技術(shù)有限公司。實(shí)驗(yàn)動(dòng)物許可證號(hào)為SCXK-京-2014-0001。在首都醫(yī)科大學(xué)實(shí)驗(yàn)動(dòng)物科學(xué)部飼養(yǎng)繁殖。所有的小鼠均在SPF環(huán)境下IVC飼養(yǎng)系統(tǒng)中飼養(yǎng)。本研究通過(guò)首都醫(yī)科大學(xué)宣武醫(yī)院動(dòng)物實(shí)驗(yàn)及實(shí)驗(yàn)動(dòng)物倫理委員會(huì)審查批準(zhǔn)。

1.2 樹(shù)突狀細(xì)胞培養(yǎng)及誘導(dǎo)

將冷凍在-80 ℃冰箱的Balb/c小鼠骨髓細(xì)胞復(fù)蘇，將細(xì)胞接種到6孔板中，按10 ng/mL 加入重組小鼠粒細(xì)胞巨噬細(xì)胞集落刺激因子(recombinant murine granulocyte-macrophage colony stimulating factor，rmGM-CSF)，按5 ng/mL加入重組小鼠白細(xì)胞介素(recombinant murine interleukin-4，rmIL-4),放37 ℃、飽和濕度、5%(體積分?jǐn)?shù))CO2培養(yǎng)箱中培養(yǎng)，倒置顯微鏡觀察細(xì)胞形態(tài)及增生情況。細(xì)胞在體外培養(yǎng)4～5 d時(shí)增生顯著，可見(jiàn)到大量具有典型樹(shù)枝狀突起的懸浮細(xì)胞，觸突呈放射狀，并有懸浮細(xì)胞呈集落生長(zhǎng)，這些細(xì)胞為未成熟DC細(xì)胞(immature DC,imDC)，將其中3孔加入脂多糖(lipopolysachrid，LPS)0.5 μg/mL誘導(dǎo)成熟DC細(xì)胞(mature DC,mDC)生成。加入 LPS繼續(xù)培養(yǎng)24 h后，可見(jiàn)到LPS刺激的細(xì)胞體積有所增大，突起變長(zhǎng)，這部分細(xì)胞為mDC細(xì)胞，此時(shí)將imDC及mDC收獲。

1.3 樹(shù)突細(xì)胞鑒定

1.3.1 倒置相差顯微鏡光鏡及光鏡分析

將上述培養(yǎng)收集的imDC及mDC 懸液甩片后鏡下觀察形態(tài)。

1.3.2 免疫表型分析

將上述培養(yǎng)收集的imDC及mDC 懸液離心沉淀，用 PBS 緩沖液調(diào)整細(xì)胞密度為 5×105/L，取100 μL標(biāo)記CD11c、主要組織相容性復(fù)合體Ⅱ(major histocompatibility complex-Ⅱ, MHC-Ⅱ)、CD80和CD86熒光抗體，后行流式細(xì)胞術(shù)檢測(cè)。

1.4 誘導(dǎo)C3H(H-2k)和Balb/c(H-2d)小鼠間免疫耐受

1)誘導(dǎo)Balb/c小鼠imDC凋亡：采用的紫外線B(ultraviolet, UVB)照射 (1 200 mJ/cm2)的方法照射使其由于DNA的交聯(lián)而啟動(dòng)細(xì)胞凋亡的過(guò)程。然后立即將UVB-Balb/c imDC及Balb/c mDC經(jīng)尾靜脈輸注到C3H小鼠體內(nèi)。

2)C3H小鼠分組詳見(jiàn)表1。

1.5 流式細(xì)胞術(shù)檢測(cè)C3H小鼠免疫耐受情況

1.5.1 外周血抗Balb/c細(xì)胞抗體檢測(cè)

取免疫結(jié)束后C3H小鼠內(nèi)眥抗凝血，取血漿20 μL加入Balb/c脾細(xì)胞20 μL，混勻，室溫下孵育30 min后用PBS徹底洗細(xì)胞3遍。標(biāo)記抗小鼠Ig-FITC、CD4-Per-CP、CD8-PE，待流式細(xì)胞儀檢測(cè)。

1.5.2 體內(nèi)檢測(cè)供者免疫耐受情況

將羧基熒光素琥珀酰亞胺酯(carboxyfluorescein succinimidyl ester,CFSE)標(biāo)記的Balb/c小鼠脾細(xì)胞尾靜脈輸注到C3H小鼠體內(nèi)。24 h后，將C3H小鼠處死，取脾細(xì)胞和腹股溝、腘窩淋巴結(jié)細(xì)胞，標(biāo)記抗小鼠CD4-Per-CP、CD8-PE和B220-APC抗體，待流式細(xì)胞儀檢測(cè)。

1.5.3 淋巴細(xì)胞增生實(shí)驗(yàn)

1)取之前經(jīng)UVB-Balb/c imDC和Balb/c mDC免疫的免疫結(jié)束后的C3H小鼠脾細(xì)胞，CFSE染色后種植在96孔細(xì)胞培養(yǎng)板，分別用Balb/c mDC及C57 mDC在細(xì)胞比例為20∶1條件下刺激，對(duì)照組為空白組cell only和植物血凝素(phytohemagglutinin, PHA)刺激組。置37 ℃，5%(體積分?jǐn)?shù))CO2溫箱培養(yǎng)4～5 d。培養(yǎng)4～5 d后，收集培養(yǎng)的細(xì)胞懸液，標(biāo)記抗CD4單抗流式細(xì)胞術(shù)分析。2)實(shí)驗(yàn)分組詳見(jiàn)表2。

1.5.4 細(xì)胞內(nèi)因子檢測(cè)

淋巴細(xì)胞增生實(shí)驗(yàn)步驟及分組同前，只是培養(yǎng)的細(xì)胞不經(jīng)過(guò)CFSE染色。細(xì)胞收獲前5 h加入含BolgiPlug白細(xì)胞活化混合物。收獲細(xì)胞標(biāo)記抗CD4抗體、干擾素-γ(interferon-γ，IFN-γ)和IL-10抗體，流式細(xì)胞儀檢測(cè)。

表1 C3H小鼠分組Tab.1 Grouping of C3H mice

imDC: immature dendritic cells; mDC: mature dendritic cells.

表2 C3H小鼠脾臟細(xì)胞分組Tab.2 Grouping of spleen cells of C3H mice

mDC: mature dendritic cells; imDC: immature dendritic cells.

1.5.5 調(diào)節(jié)T細(xì)胞檢測(cè)

淋巴細(xì)胞增生實(shí)驗(yàn)步驟及分組同前，只是培養(yǎng)的細(xì)胞不經(jīng)過(guò)CFSE染色。細(xì)胞收獲標(biāo)記CD4-Per-CP、FOX-P3抗體和CD25抗體，流式細(xì)胞儀檢測(cè)。

1.6 統(tǒng)計(jì)學(xué)方法

2 結(jié)果

2.1 小鼠DC體外誘導(dǎo)

2.1.1 細(xì)胞形態(tài)

小鼠骨髓細(xì)胞在體外經(jīng)rmGM-CSF 和rmIL-4誘導(dǎo)24 h，倒置相差顯微鏡下可見(jiàn)呈簇狀生長(zhǎng)的細(xì)胞團(tuán)。培養(yǎng)3 d，細(xì)胞簇較前增多，大多數(shù)細(xì)胞仍然貼壁，少數(shù)細(xì)胞呈半懸浮狀態(tài)。培養(yǎng)5 d，大量細(xì)胞呈半懸浮生長(zhǎng)，細(xì)胞體積較以前增大，呈圓形或梭型，細(xì)胞表面可見(jiàn)樹(shù)突狀突起(圖1A、B)。將第5天的DC經(jīng)LPS刺激24 h，細(xì)胞體積較前顯著增大，細(xì)胞呈圓形、星形或梭形，細(xì)胞核明顯，細(xì)胞表面突起較前增多、分支明顯(圖1C、1D)。

2.1.2 細(xì)胞免疫表型

流式細(xì)胞術(shù)分析表明，小鼠骨髓來(lái)源DC高表達(dá)CD11c,其中imDC低表達(dá)MHCⅡ、CD80和CD86，而mDC高表達(dá)MHCⅡ、CD80和CD86(圖2)。

2.2 Balb/c(H-2d)和C3H(H-2K)小鼠間免疫耐受

2.2.1 輸注了UVB-Balb/c imDC的C3H小鼠不能產(chǎn)生抗Balb/c抗體

輸注Balb/c UVB-imDC的C3H小鼠不能產(chǎn)生抗Balb/c抗體，即徹底完全的免疫耐受。而輸注未照射的Balb/c mDC的C3H小鼠體內(nèi)可以檢測(cè)出較高的抗Balb/c抗體。詳見(jiàn)圖3。

2.2.2 輸注了UVB-Balb/c imDCs的C3H小鼠未能排斥CFSE染色的Balb/c脾細(xì)胞

輸注UVB-Balb/c imDC的C3H小鼠脾臟和淋巴結(jié)可以檢測(cè)到CFSE染色的Balb/c脾細(xì)胞，即對(duì)Balb/c脾細(xì)胞產(chǎn)生免疫耐受。而輸注未照射的Balb/c imDC及mDC的C3H小鼠脾臟和淋巴結(jié)沒(méi)有檢測(cè)到CFSE染色的Balb/c脾細(xì)胞，即將Balb/c脾細(xì)胞排異掉了(圖4)。

圖1 小鼠BMDC倒置顯微鏡下及甩片后吉姆薩染色涂片光學(xué)顯微鏡下圖像Fig.1 Images of BMDC under inverted microscope and Giemsa stained smears under optical microscope

A： immature DC under inverted microcope (20×); B: immature DC by Giemsa staining (60×); C: mature DC under inverted microcope (20×);D: mature DC by Giemsa staining (60×). Dendritic bulge was seen on the cell surface (A, B). Cell volume increased significantly compared with the former, cell surface protrusions increased more than before (C, D). DC: dendritic cells; BMDC:bone marrow derived dendritic cells.

圖2 流式細(xì)胞術(shù)分析小鼠骨髓來(lái)源DC免疫表型Fig.2 Flow cytometry analysis of DC immunophenotype of bone marrow derived from mice

CD11c was highly expressed in bone marrow-derived DCs, MHC Ⅱ, CD80 and CD86 were lowly expressed in imDC, while MHC Ⅱ, CD80 and CD86 were highly expressed in mDCs; BM-imDC: bone marrow immature dendritic cells; BM-mDC: bone marrow mature dendritic cells; MHC-Ⅱ:major histocompatibility complex-Ⅱ.

圖3 Balb/C(H-2d) 和C3H(H-2k)小鼠間抗原特異性的免疫耐受誘導(dǎo)Fig.3 Antigen-specific immunotolerance induction in Balb/C (H-2d) and C3H (H-2k) mice

A：C3H； B：VVB-imDC; C:mDC; Antibody levels in C3H mice immunized by UVB- Balb/c imDCs(B), Balb/c mDCs(C), in non-immunized C3H mice(A) detected by flow cytometry. imDC: immature dendritic cells; mDC: mature dendritic cells.

圖4 流式細(xì)胞術(shù)檢測(cè)靜脈注射UVB-Balb/c imDC或mDC的C3H小鼠脾細(xì)胞及淋巴結(jié)中 CFSE染色的Balb/c小鼠脾細(xì)胞分布情況Fig.4 Distribution of spleen cells in CFSE-stained splenocytes of C3H mice injected with UVB-Balb/c imDCs or mDCs by intravenous injection of Balb/c mice by flow cytometry

The red cells were CFSE-stained Balb/c mouse spleen cells; imDC: immature dendritic cells; mDC: mature dendritic cells;CFSE:carboxyfluoresce in succinimidyl ester; LN:lymph node.

2.3 免疫耐受機(jī)制分析

流式細(xì)胞術(shù)分析結(jié)果提示UVB-Balb/c imDCs免疫的C3H小鼠脾臟中FOX-P3陽(yáng)性的調(diào)節(jié)T細(xì)胞比例增高(圖5)。

筆者將Balb/c UVB-imDC及mDC免疫的C3H小鼠脾細(xì)胞經(jīng)CFSE染色后與Balb/c小鼠mDC細(xì)胞培養(yǎng)5 d后，標(biāo)記抗體后用流式細(xì)胞儀檢測(cè)發(fā)現(xiàn)各組淋巴細(xì)胞均有明顯的增生(圖6)，隨后對(duì)增生的細(xì)胞標(biāo)記IFN-γ、IL-10、CD4，發(fā)現(xiàn)Balb/c UVB-imDC免疫的C3H小鼠T細(xì)胞較其他組T細(xì)胞分泌更多的IL-10(圖7)。

3 討論

自身免疫耐受的維持是機(jī)體預(yù)防自身免疫病的自我防御機(jī)制。當(dāng)這一防御機(jī)制因某些原因被打破，便可誘發(fā)自身免疫病[7]。研究者[8]在對(duì)自身免疫的研究中發(fā)現(xiàn)，DC吞噬攜帶某種抗原的凋亡細(xì)胞后可以誘導(dǎo)機(jī)體對(duì)該抗原產(chǎn)生特異性免疫耐受。本實(shí)驗(yàn)結(jié)果顯示，通過(guò)給C3H(H-2k)小鼠輸注UVB-Balb/c(H-2d) imDC可以誘導(dǎo)C3H(H-2k)小鼠對(duì)Balb/c(H-2d)小鼠MHC抗原免疫耐受。C3H小鼠外周血中抗Balb/c抗體水平反映了C3H抗Balb/c MHC抗原的反應(yīng)能力，較高的抗體水平提示較強(qiáng)的同種異體反應(yīng)。接受UVB-Balb/c imDC輸注的C3H小鼠不產(chǎn)生任何針對(duì)Balb/c的抗體。筆者還通過(guò)C3H小鼠靜脈注射CFSE染色的Balb/c脾細(xì)胞，觀察其是否對(duì)Balb/c抗原產(chǎn)生免疫耐受，這一結(jié)果和抗體檢測(cè)結(jié)果一致，均證明了UVB-Balb/c(H-2d)imDC免疫的C3H(H-2k)小鼠對(duì)Balb/c(H-2d)小鼠MHC抗原產(chǎn)生了免疫耐受。并且，通過(guò)分析C3H小鼠脾臟和淋巴結(jié)中CFSE標(biāo)記的Balb/c脾細(xì)胞，發(fā)現(xiàn)CD4+T細(xì)胞和CD8+T細(xì)胞較B細(xì)胞更易產(chǎn)生免疫耐受，因?yàn)樵诋a(chǎn)生免疫耐受的C3H小鼠脾臟和淋巴結(jié)中B細(xì)胞數(shù)量較CD4+T和CD8+T細(xì)胞數(shù)量明顯要少。這一結(jié)果提示UVB-Balb/c(H-2d)小鼠imDCs更傾向誘導(dǎo)MHC-Ⅰ抗原的免疫耐受，因?yàn)門(mén)細(xì)胞僅表達(dá)MHC-Ⅰ抗原，而B(niǎo)細(xì)胞不僅表達(dá)MHC-Ⅰ還表達(dá)MHC-Ⅱ。由于T細(xì)胞和B細(xì)胞是同時(shí)注射、歸巢到脾臟和淋巴結(jié)的，所以這一結(jié)果并不能用B細(xì)胞較T細(xì)胞更多存在外周循環(huán)中來(lái)解釋。

圖5 流式細(xì)胞術(shù)檢測(cè)靜脈注射UVB-Balb/c imDC或 Balb/c mDC的C3H小鼠脾細(xì)胞中POX-P3陽(yáng)性細(xì)胞比例Fig. 5 The ratio of FOX-P3 positive regulatory T cells in spleen cells of C3H mice treated by UVB-Balb/c imDC or Balb/c mDC

This observation was verified from three independent experiments;n=2;imDC: immature dendritic cells; mDC: mature dendritic cells.

圖6 Balb/c抗原誘導(dǎo)免疫后的C3H小鼠脾細(xì)胞Fig. 6 The immune responses of treated C3H mouse spleen cells induced by Balb/c antigen

C3H mouse spleen cells (1×106) treated by UVB-Balb/c imDC or Balb/c mDC intravenously were stained with

CFSE and culture with Balb/c mDCs. The proliferation was analyzed by flow cytometry after four days; imDC: immature dendritic cells; mDC: mature dendritic cells；CFSE:carboxyfluoresce in succinimidyl ester.

圖7 流式細(xì)胞術(shù)分析免疫后C3H小鼠增生的CD4+T細(xì)胞分泌細(xì)胞因子的情況Fig.7 Flow cytometry analysis of cytokines secreted by CD4+ T cells in C3H mice after immunization

Cytokines secreted by proliferating CD4+T cells were analyzed by flow cytometry. This observation was verified from three independent experiments.n=2; CFSE: carboxyfluoresce in succinimidyl ester; IL-10:interleukin-10; IFN-γ:interferon-γ.

DC誘導(dǎo)免疫耐受的機(jī)制尚不完全清楚，已知的機(jī)制包括誘導(dǎo)T細(xì)胞無(wú)能、釋放免疫抑制性細(xì)胞因子、介導(dǎo)T細(xì)胞的克隆清除、誘導(dǎo)和募集調(diào)節(jié)性T細(xì)胞等[9]。凋亡細(xì)胞所攜帶的抗原被DC吞噬后形成抗原肽-MHC-Ⅰ復(fù)合物，該復(fù)合物與 CD8+T細(xì)胞相互作用誘導(dǎo)其成為細(xì)胞毒T淋巴細(xì)胞(cytotoxic T lymphocyte,CTL)[10]。但是如果DC細(xì)胞沒(méi)有同時(shí)激活CD4+T細(xì)胞，則CD8+T細(xì)胞表達(dá)腫瘤壞死因子相關(guān)凋亡誘導(dǎo)配體(tumor necrosis factor related apoptosis inducing ligand，TRAIL)死亡，從而誘導(dǎo)了免疫耐受[11- 12]。凋亡細(xì)胞免疫途徑不同可以產(chǎn)生不同的免疫效果[13]。靜脈注射凋亡細(xì)胞可以誘導(dǎo)機(jī)體對(duì)該凋亡細(xì)胞所攜帶的抗原產(chǎn)生免疫耐受[14]，而皮下注射凋亡細(xì)胞則會(huì)產(chǎn)生抗原特異性的免疫應(yīng)答[15]。脾臟CD8+DC細(xì)胞誘導(dǎo)免疫耐受而CD8-DC誘導(dǎo)免疫應(yīng)答[13]。mDC由于表達(dá)MHC-Ⅱ和共刺激分子(CD80/CD86)增加而誘導(dǎo)免疫應(yīng)答，而imDC則誘導(dǎo)免疫耐受[16]。筆者將UVB-Balb/c imDC、 Babl/c mDC免疫的C3H小鼠脾細(xì)胞CFSE染色后與Balb/c小鼠mDC細(xì)胞培養(yǎng)5 d后，發(fā)現(xiàn)UVB-Balb/c imDC免疫的C3H小鼠T細(xì)胞較其他組小鼠T細(xì)胞分泌更多的IL-10，并且FOX-P3+的調(diào)節(jié)T細(xì)胞比例也增高。研究結(jié)果提示FOX-P3+的調(diào)節(jié)T細(xì)胞在UVB-imDC誘導(dǎo)的免疫耐受中起到一定作用。

總之，凋亡樹(shù)突狀細(xì)胞具有很強(qiáng)的免疫耐受誘導(dǎo)作用，在免疫病治療及移植免疫耐受中值得進(jìn)一步研究。

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[12]Griffith T S, Kazama H, Vanoosten R L, et al. Apoptotic cells induce tolerance by generating helpless CD8+T cells that produce TRAIL[J]. J Immunol,2007,178(5):2679-2687.

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編輯 孫超淵

Alloantigen immune tolerance and its mechanism in mice immunized with the ultraviolet radiation immature dendritic cells

Guo Yixian1, Zhang Lanfang1, Sun Xuejing1, Wan Suigui1, Xia Changqing1,2*

(1.DepartmentofHematology,XuanwuHospital,CapitalMedicalUniversity,Beijing100053,China;2.DepartmentofPathology,ImmunologyandLaboratoryMedicine,UniversityofFloridaCollegeofMedicine,Florida32610,USA)

Objective To induce alloantigen immune tolerance between C3H mice and Balb/c mice by infusing the ultraviolet B(UVB) radiation immature dendritic cells (imDC) and and study the immunological mechanisms of this process.Methods ① The authors induced alloantigen tolerance in C3H mice (H-2k) by intravenous injecting ultraviolet B (UVB) irradiated Balb/c immature dendritic cells (UVB-Balb/c imDC) derived from the cultures of Balb/c bone marrow cells. ②Detection of immune tolerance induced regulatory T cells, interleukin-10 (IL-10) and interferon-γ (IFN-γ) in mice in order to study the tolerance mechanism. Results ①C3H mice immunized with UVB-Balb/c imDC can produce immune tolerance to Balb/c antigens and can not produce anti-Balb/c antibody. ②C3H mice immunized with UVB-Balb/c imDC can not clear the Balb/c spleen cells in vivo. ③The T cells of C3H mice immunized with UVB-Balb/c imDC can secrete more IL-10 and produce more FOX-P3+regulatory T cells than the control group in vitro. Conclusion ①I(mǎi)mmunization of C3H mice with UVB-Balb/c imDC allows C3H mice to fully tolerate Balb/c antigen. ②The possible cause of immune tolerance to Balb/c antigens in C3H mice is due to the increased secretion of IL-10 from T cells and the production of more FOX-P3+regulatory T cells.

dendritic cells; apoptosis; regulatory T cells; immune tolerance

國(guó)家自然科學(xué)基金(81172854，81240015)。This study was supported by National Natural Science Foundation of China (81172854， 81240015).

時(shí)間：2017-06-09 18∶30 網(wǎng)絡(luò)出版地址：http://kns.cnki.net/kcms/detail/11.3662.r.20170609.1830.060.html

10.3969/j.issn.1006-7795.2017.03.020]

R3

2016-01-15)

*Corresponding author， E-mail：bjxwgyx@163.com