PD-1信号通路参与aaDC诱导小鼠皮肤移植耐受的机制研究
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摘要
[研究背景]
树突状细胞(Dendritic cell，DC)是专职抗原提呈细胞之一。依据其成熟状态、表型或来源不同，在免疫或耐受的诱导中起着关键作用。DC捕捉，处理，传递抗原到淋巴器官，在淋巴器官与T细胞相互作用，引起抗原特异性的免疫反应或免疫耐受。T细胞受体(T cell receptor，TCR)识别抗原肽-MHC分子复合物、足够的共刺激分子及自分泌、旁分泌的细胞因子的参与，导致T细胞活化，诱导免疫应答。缺乏这些信号分子则使T细胞不能完全活化，诱导免疫耐受。成熟的树突状细胞表达高水平的MHC-Ⅱ类分子和共刺激分子，导致T细胞活化。与此相反，不成熟的树突状细胞缺乏共刺激信号，因此诱导同种异体抗原特异性低反应，延长同种异体移植物存活。阿司匹林，维生素D3或白细胞介素-10可体外诱导生成不成熟的树突状细胞。然而，这些体外培养的不成熟树突状细胞，当暴露于体内的炎性刺激时，可能转变成成熟的树突状细胞，导致免疫刺激而不是免疫抑制。最近研究表明，骨髓来源的树突状细胞与IL-10及细菌脂多糖(Lipopolysaccharides，LPS)培养刺激下表现为一种半成熟状态，即表达低水平的共刺激分子，或称为旁路活化的树突状细胞(Alternativelyactivated DC，aaDC)。这些半成熟的树突状细胞即使在炎性条件下也是不可逆转的，因此诱导抗原特异性T细胞无反应，延长同种异体移植存活。然而，这些半成熟树突状细胞免疫调节的分子机制仍不清楚。
PD-1(Programmed cell death protein 1，PD-1)受体是表达在活化T细胞上的抑制性共刺激分子，属于CD28家族成员，在免疫反应的调节及外周耐受方面起着重要作用。PD-L1表达在多种抗原提呈细胞。PD-L1可以通过与活化T细胞上的PD-1结合，抑制T细胞增殖，并促进活化T细胞的凋亡。最近的研究表明，PD-1／PD-L途径在动物同种异体移植模型中起着重要作用。虽然树突状细胞表达PD-1受体的两种配体，即PD-L1和PD-L2，但是PD-1／PD-L途径是否在半成熟树突状细胞引起的免疫耐受中起作用仍不清楚。本研究拟从PD-1／PD-L信号通路角度，探讨小鼠骨髓来源的半成熟树突状细胞(即aaDC)在小鼠皮肤移植模型中的耐受机制。
[研究方法]
1．不同类型小鼠骨髓来源的树突状细胞的制备培养与受者小鼠静脉输注
小鼠骨髓来源的树突状细胞(bone marrow-derived dendritic cell，BMDC)制备参照文献。分离小鼠后腿股骨和胫骨骨髓细胞，用NH4Cl低渗缓冲液裂解红细胞，细胞计数后以2×10~6个细胞接种在100mm的细胞培养皿中，培养基为含20 ng／ml rGM-CSF(Pepro Tech Inc，New Jersey，USA)的RPMI-1640完全培养基，37℃5％CO2培养箱中培养7天，获得所谓未处理的DC(untreated DC)，培养期间每间隔1天换1次培养基。为诱导产生不成熟DC(immature DC，imDC)，在培养第6天，加入20 ng／ml rmIL-10(Pepro Tech Asia，Rehovot，Israel)培养1天；而aaDC的制备是在第7天在imDC培养基中加入1μg／ml LPS(L4130，Sigma-Aldrich)，培养1天后即为aaDC；而成熟DC的获得采用1μg／ml LPS刺激未处理DC 1天。
为测试以上制备的4种不同类型DC对同种异体皮肤移植物存活时间的影响，在移植前7天，分别将BALB／c供者小鼠来源的上述4种DC经尾静脉输注到受者小鼠(C57BL／6)体内，细胞数为2×10~6／只小鼠。同时采用抗PD-1抗体阻断PD-1／PD-L信号通路以观察其对aaDC调节效应的影响，而以同型IgG为对照。抗体应用方法如下：在aaDC输注当天腹腔注射500μg／只(day 0)，在随后第2、4、6天各250μg／只腹腔注射，，共分7个实验组(每组6只小鼠)：group 1：untreated DC；group 2：imDC；group 3：aaDC；group 4：aaDC plus anti-PD-1 mAb；group 5：anti-PD-1 mAbalone：group 6：mDC and group 7：aaDC plus control IgG．
2．采用流式细胞术分析DC表型
DC首先与anti-CD16／CD32 mAb孵育以封闭FcγR，然后用荧光素标记的抗CD11c、CD80、CD86、MHCⅡ、PD-L1以及PD-L2分别孵育(4℃，30 min)，然后用PBS洗涤2次，采用流式细胞仪分析[FACSCalibur~(TM) flow cytometer(BectonDickinson)]。
3．小鼠皮肤移植术
参照Billingham and Medawar方法。受者小鼠(C57BL／6)麻醉后，剪除背部皮肤毛发，准备直径12mm的植床。颈椎断离法处死供者小鼠(BALB／c)，剪除躯干毛发，取直径12mm皮片，去除皮下脂肪，将皮片移植缝合到受者小鼠(用5—0#丝线采取8针不连续缝合法)，用创可贴包被伤口，每天观察1次移植皮肤的存活情况，如果50％皮片翻起或坏死即为排斥。同时选择性应用组织病理分析证实排斥的发生。
4．组织学分析
参照文献。皮肤移植物切下后，用4％福尔马林固定，石蜡包埋切片，HE染色后进行病理组织学分析，观察炎症细胞浸润程度。
5．ELISA检测细胞因子浓度
根据使用说明书介绍的方法，采用ELISA试剂盒(Biosource International，Camarillo，CA)检测混合淋巴细胞培养上清中细胞因子IFN-γ、IL-10含量。
6．混合淋巴细胞反应
刺激细胞为来源于BALB／c小鼠的各种类型DC。DC细胞经丝裂霉素C处理后接种于96孔V型底培养板。来源于C57BL／6受者小鼠脾脏细胞，经尼龙棉纯化获得T淋巴细胞，用CFSE标记后作为反应细胞。4×10~5个反应细胞与4×10~5刺激细胞混合后，在37℃、5％CO_2条件下培养5天，收获细胞，用流式细胞术分析CFSE荧光强度以评判T细胞增殖程度。
7．流式细胞术检测CD4~+CD25~+Foxp3~+Tregs
混合淋巴细胞培养5天后收集细胞，用anti-CD4-FITC、anti-CD25-PE进行细胞表面标记染色，细胞经透膜后用APC-labeled anti-Foxp3标记(Foxp3 StainingBuffer Set，eBioscience)，用流式细胞仪分析CD4~+CD25~+ T细胞表达Foxp3的水平(23)。
8．流式细胞术检测细胞内细胞因子
制备单个脾脏细胞，在体外37℃、5％CO_2培养条件下，用50 ng／ml PMA，1μg／ml ionomycin、2μM monensin(all Sigma-Aldrich)刺激处理4小时后收获细胞，经FITC标记抗CD4或CD8染色后，4％formaldehyde固定、透膜处理后用相应荧光素标记细胞因子抗体染色，流式细胞仪检测细胞因子表达水平。
9．统计分析
实验数据采用单向方差分析法，P＜0．05为统计学显著差异。不同处理组间移植物存活时间差异分析采用Kaplan and Meier法(24)。
[结果]
1．部分成熟为aaDC表型特点
对几种类型DC表型分析发现，与不成熟DC(imDC)和未处理DC(untreatedDC)相比较，aaDC表达CD80、CD86水平有一定程度增加，而MHCⅡ分子表达水平相当。然而，以上分子的表达水平比成熟DC(mDC)明显降低。值得注意的是，我们观察到aaDC表达PD-L1和PD-L2的水平与不成熟DC和未处理DC比较显著升高(图1)。
2．aaDC刺激同种反应T细胞增殖的能力降低，该特性与PD-1／PD-L信号通路有关
随后，我们通过混合淋巴细胞反应实验证实，如图2所示，aaDC(来源于BALB／c小鼠，H-2~d)刺激同种反应性T细胞(来源于C57BL／6，H-2~b)增殖的能力明显低于成熟DC，与未成熟DC相当。重要的是，当用抗PD-1抗体阻断PD-1／PD-L信号通路后，aaDC刺激同种T细胞增殖的活性增强，提示PD-1／PD-L共抑制信号通路参与aaDC对同种T细胞反应的调节。
3．在混合淋巴细胞培养体系中，aaDC诱导抗炎性细胞因子IL-10分泌增加而抑制IFN-γ的产生，该效应部分依赖于PD-1信号通路
我们采用ELISA检测上述混合淋巴细胞培养上清液中细胞因子IL-10和IFN-γ产生水平，发现aaDC作为刺激细胞时，能诱导出比成熟DC和未处理DC显著升高(p＜0．01)，几乎与未成熟DC相当浓度的IL-10；与此相反，与未处理DC相比较，aaDC、未成熟DC能显著抑制混合淋巴细胞反应炎症性细胞因子IFN-γ的产生(p＜0．01)。有趣的是当用抗PD-1抗体阻断后，可以逆转该调节效应(图3)，上述结果表明aaDC对以上细胞因子的调节也部分依赖于PD-1信号通路。
4．aaDC具有扩增CD4~+CD25~+Foxp3~+调节性T细胞的特性
因为已有文献报道调节性DC能诱导调节性T细胞(Treg)的产生。因此，我们进一步检测了混合淋巴培养体系中不同类型DC细胞诱导Treg产生的差异性。结果表明(图4)，与其它类型DC相比较，aaDC扩增CD4~+CD25~+Foxp3~+Treg的功能最强(p＜0．01)，该调节效应涉及PD-1信号通路，其次是不成熟DC，而成熟DC以及未处理DC对Treg的诱导产生能力弱。
5．静脉输注供者来源的aaDC能明显炎症皮肤移植物的存活时间，而且PD-1／PD-L信号通路参与aaDC的调节作用
依据上述体外实验结果，我们进一步探讨在同种异体皮肤移植前7天，输注供者来源的各种类型DC到受者体内(2×10~6个细胞／只小鼠)，观察其对皮肤移植物存活时间的影响。结果发现(图5)，与其它DC细胞{untreated DCs(MST 11．83±2．40days)，imDC(MST 16．00±3．10 days)or mDC(MST 7．50±1．64 days)}相比较，aaDC输注能显著延长移植物的存活时间(MST 22．67±4．08 days)(P＜0．01)。有趣的是，当用抗PD-1抗体阻断PD-1／PD-L信号通路后，aaDC对移植物的保护效应显著降低(MST22．67±4．08 vs 12．33±3．33 of aaDC+anti-PD．1，P＜0．01)。单独应用抗PD-1抗体对移植物的存活时间影响很小，与未处理DC的效应相当(MST 10．83±2．79 vs 11．83±2．40days，p＞0．05)。
6．组织病理学分析证实aaDC输注能显著减轻皮肤移植物中炎症细胞浸润
在皮肤移植后7天，对移植皮肤进行病理组织学分析，如图6所示，发现输注aaDC能显著减轻移植皮肤中炎症细胞浸润，imDC也能在一定程度上降低炎症细胞的浸润。而与此相对照，输注成熟DC(mDC)以及未处理DC后，皮肤移植物中仍有明显的炎症细胞浸润。aaDC输注加抗PD-1抗体能降低其对移植物的保护效应，而单独应用抗PD-1抗体或同型对照IgG对炎症细胞浸润的影响较小。以上病理组织学结果与移植物的存活时间相一致。
7．aaDC输注能改变受者小鼠脾脏T淋巴细胞产生细胞因子谱的特征
为了探讨供者来源的各种不同类型DC输注后，能否影响受者脾脏T淋巴细胞产生细胞因子的类型。进一步分离了输注不同类型DC后的各组受者小鼠的脾脏T淋巴细胞，体外在经PMA／ionomycin刺激后，采用流式细胞术分析T细胞内细胞因子(IL-2、IL-10和IFN-γ)表达水平。如图7A所示，与其它类型的DC相比，输注aaDC后受者小鼠脾脏CD4~+T细胞产生IL-2的能力降低(~(**)p＜0．01)，而IFN-γ有一定程度升高。与此相反，aaDC能增强受者脾脏CD4~+T细胞产生IL-10。有趣的是，我们观察到输注aaDC后，与其它组相比较，受者小鼠脾脏CD8~+T细胞IFN-γ的分泌潜能显著降低(_*~* p＜0．01)，而对CD8~+T细胞IL-2的产生无明显影响(图7B)。同样地，当应用抗PD-1抗体阻断PD-1／PD-L信号通路后，对aa DC的免疫负调节效应有明显的抑制作用。
[结论]
通过输注供者来源aaDC能显著延长异基因小鼠皮肤移植物存活时间，抑制受者同种反应性T细胞增殖，减轻移植物组织中单个核细胞浸润。aaDC的免疫调节机制可能涉及扩增CD4~+CD25~+Foxp3~+调节性T细胞，增强受者脾脏同种反应T细胞产生抗炎性细胞因子IL-10，而抑制与T细胞增殖密切相关的IL-2的产生，炎症性细胞因子IFN-γ也明显受到抑制，有趣的是，PD-1／PD-L信号通路对以上aaDC的免疫调节作用起着关键作用。本研究成果提示，采用aaDC过继输注可抑制同种移植物排斥反应，为防治同种异体器官移植排斥反应提供了新的干预策略，进一步深化了对aaDC免疫调节机制的认识。
[Background]
Alternatively activated DC (aaDC) can prolong allograft survival in the mousemodel. However, the molecular mechanism(s) by which these DCs function toregulate alloreactive-T cell responses remains to be clearly defined.
[Methods]
1. Preparation of various types of DCs and infusion into recipients mice
BMDCs were generated from bone marrow cells as described previously (19). Inbrief, bone marrow cells were obtained by aspirating femurs and tibial bones andthen seeded at 2×10~6 cells per 100 mm dish in 10 ml RPMI-1640 complete mediumsupplemented with 20 ng/ml rGM-CSF (Pepro Tech Inc, New Jersey, USA).Immature DCs (imDC) were generated by adding 20 ng/ml rmIL-10 (Pepro TechAsia, Rehovot, Israel) at day 6. To obtain alternatively activated DCs (aaDC), 1μg/ml LPS (L4130, Sigma-Aldrich) was added to imDC at day 7. Mature DCs (mDC)were generated by adding 1μg/ml LPS to untreated BMDCs 24 hr before harvesting.To test the in vivo regulatory effects of these DCs originated from BALB/c mice,each C57BL/6 recipient mouse was infused with 2x 10~6 untreated DC, imDC,aaDC, or mDC via tail vein at 7 days before the transplantation. As a result, 7experimental groups were established (6 mice per group) : group 1: untreated DC ;group 2: imDC ; group 3: aaDC; group 4: aaDC plus anti-PD-1 mAb; group 5:anti-PD-1 mAb alone ; group 6: mDC and group 7: aaDC plus control IgG. Anti-PD-1mAb or control IgG was given intraperitoneally after i.v. infusion of aaDC at 500μg on day 0, followed by 250μg on days 2, 4 and 6.
2. Analysis of DC phenotype by flow cytometry
For analysis of DC phenotype, cells were preincubated with anti-CD16/CD32 mAbto block FcγR. After staining with anti-CD11c, the cells were co-stained for CD80,CD86, MHCII, PD-L1 or PD-L2 antibody at 4℃for 30 min, washed twice withPBS, and analyzed on a FACSCalibur~(TM) flow cytometer (Becton Dickinson).
3. Procedures for skin transplantation
Full-thickness skin grafts were transplanted using a modified method of Billinghamand Medawar (20). In brief, the recipient mice were anesthetized and shaved aroundthe back. Skins were prepared from the trunk of BALB/c mice. After removing thesubcutaneous fat, 12-mm-diameter circles of full-thickness pieces weresimultaneously transplanted onto the alternate dorsal thorax of the C57BL/6 recipients.The graft bed was slightly larger than the skin allograft. The skin graft was sutured andcovered with a vaseline gauze and adhesive bandage for 7 days. Skin grafts wereinspected daily. Rejection was defined as the complete loss of viable epidermal grafttissue characterized by 50% of the graft surface became raised and necrotic by visualexamination. In selected animals, allograft rejection was further confirmed byhistological analysis.
4. Histology
Histological analysis was performed as previously described (21). The skinallografts were excised and stained with hematoxylin/eosin for the analysis ofpathological changes.
5. Determination of cytokine production by ELISA
IFN-γand IL-10 were measured in supematants harvested from co-cultures ofC57BL/6 splenic T cells with various types of DCs from BALB/c mice, using ELISAkits (Biosource International, Camarillo, CA) according to manufacturer'sinstructions.
6. Mixed lymphocyte reaction
Stimulator cells were various types of DCs originated from BALB/c mice. The DCswere treated with mitomycin C and seeded into 96-well plates with V-bottom. T cellsfrom recipient mice (C57BL/6) were purified by the nylon wool technique (22). ThoseT cells were stained with CFSE and used as responders. 4×10~5 responder and 4×10~5stimulator cells were mixed and incubated at 37℃under 5% CO_2. After culturing for5 days, the cells were harvested and subjected to flow cytometric analysis of CFSEfor T cell proliferation.
7. Detection of CD4~+CD25~+ Foxp3~+ Tregs by flow cytometry
C57BL/6 T cells after 5 days of coculture with BALB/c DCs were co-stained withanti-CD4-FITC and anti-CD25-PE, fixed and permeabilized, and then stained withAPC-labeled anti-Foxp3 by using commercial kits (Foxp3 Staining Buffer Set,eBioscience) according to the manufacturer's instructions. The cells were analyzed byflow cytometry as described (23). The collected data were analyzed with CellQuestsoftware (BD Biosciences, Mountain View, CA) as instructed.
8. Intracellular cytokine determination by flow cytometry
For determination of intracellular cytokine production, single-cell suspensions ofsplenocytes were prepared and stimulated for 4 hr in culture medium containing 50ng/ml PMA, 1μg/ml ionomycin, and 2μM monensin (all Sigma-Aldrich) at 37℃under 5% CO_2. Cells were then stained for surface marker, fixed in 4 % formaldehydein PBS, and permeabilized with 0.5 % saponin plus 1 % BSA PBS, followed bylabeling with specific cytokine antibodies or isotype controls. Cells were then analyzedon a FACSCalibur using CellQuest software.
9. Statistical analysis
Data were analyzed by one-way analysis of variance. Statistical significance wasdefined as P＜0.05. Allograft survival differences among groups were analyzed usingthe method of Kaplan and Meier (24). Comparisons of two means were carried out byone-way anova. Regression curves were fitted using SigmaPlot software (version 8.0;SPSS, Chicago, IL).
[Results]
1. Partially matured phenotype of aaDC
We first examined the phenotypic profiles of various types of DCs generated fromBALB/c BM progenitors. As shown in Fig. 1, we observed a slight increase of CD80and CD86 as compared with imDC and untreated DC, while a similar level ofMHCII expression was observed between aaDC and imDC or untreated DC. Incontrast, the levels of these molecules on aaDC were much lower than those on mDC.These analysis revealed that aaDC displayed a semi-mature phenotype. Of note, weobserved that aaDC had significantly higher levels of PD-L1 and PD-L2 as comparedto imDC and untreated DC (Fig. 1).
2. aaDC displayed a reduced alloreative T cell stimulating capacity that wasassociated with PD-1/PD-L pathway
Next, we performed an MLR to test the stimulatory capacity of aaDC preparedfrom BALB/c donor mice for the proliferation of allogeneic T cells from C57BL/6recipients. As expected, stimulation with mDC resulted in a strong proliferativeresponse (Fig.2). In contrast, stimulation with aaDC or imDC gave rise to a weakT-cell proliferation. Of important note, we observed that the decreased T-cellproliferative response to aaDC was largely restored when PD-1/PD-L pathway wasblocked by anti-PD-1 Ab.
3. aaDC enhanced the production of IL-10 while inhibited the secretion of IFN-γin an MLR
We also measured the production of IFN-γand IL-10 in the supernatant harvestedfrom the cocultures by ELISA (Fig.3). aaDC induced a reduced IFN-γand an enhancedIL-10 production as compared with mDC, which were reversed in the presence ofanti-PD-1 blocking Ab. Together, these results suggest that aaDC have an impairedallo-stimulating capacity and, PD-1/PD-L pathway plays an important role in theregulatory capacity of aaDC in this setting.
4. Augmented expansion of CD4~+CD25~+Foxp3~+ Tregs coculture with aaDC
Given the importance of regulatory DCs in the induction of regulatory T cells(Tregs), we next checked the differences in Tregs induced by various types of DC(imDC, aaDC, mDC). To this end, C57BL/6 T cells were cocultured with varioustypes of DC generated from BALB/c mice for 5 days. Expanded T cells originatedfrom each group were stained for intracellular Foxp3 after co-staining for surface CD4and CD25, and then subjected to flow cytometry analysis. As shown in Fig.4, aaDCinduced an increased expression of Foxp3 in CD4~+CD25~+ T-cell population ascompared mDC and untreated DC, and this effect was partially dependent onPD-1/PD-L pathway.
5. Prolonged skin allograft survival by aaDC infusion was reversed by blockadeof the PD-1/PD-L pathway
Based on the above results, we next sought to explore the feasibility of aaDC forinduction of antigen-specific allograft tolerance in transplantation. For this purpose,2 x 10~6 various types of DC (aaDC, imDC, mDC and untreated DC) originated fromBALB/c mice were injected into C57BL/6 recipient mice via tail vein respectively.At 7 days after injection, the mice were transplanted with BALB/c-derived skinallografts. As shown in Fig. 5, the mean graft survival time (MST 22.67±4.08 days)in the recipient mice pre-infused with donor-specific aaDC was significantly longerthan that in the recipients pre-treated with either untreated DCs (MST 11.83±2.40days) , imDC (MST 16.00±3.10 days) or mDC (MST 7.50±1.64 days) (P＜0.01).Notably, pre-treatment of the recipient mice with anti-PD-1 blocking Ab reversed theprolonged survival of skin allografts by aaDC (MST 22.67±4.08 vs 12.33±3.33 ofaaDC + anti-PD-1, P＜0.01), while administration of anti-PD-1 mAb alone showedonly a marginal effect on the allograft survival as compared with that untreated DCgroup ( MST 10.83±2.79 vs 11.83±2.40 days, p＞0.05 ).
6. Intravenous infusion of aaDCs attenuated the infiltration of leukocytes in thegrafts.
Consistently, histological analysis of allografts at 7 days after transplantation revealed much severe inflammatory infiltrates in the recipient mice pre-injected withmDC and untreated DC as compared to the recipients pre-infused with aaDC or imDC(Fig. 6). Allograft in the recipients treated with aaDC and PD-1 blocking Ab showed asevere infiltration and necrosis (Fig. 6). These results indicate that the PD-1/PD-Lpathway is essential for the aaDC-mediated allograft protection.
7. aaDC infusion changed the cytokine profiles produced by splenic T cells fromrecipient mice
Finally, we questioned whether infusion of donor-derived aaDCs affects thecytoldne profiles of recipient splenocytes. To address this question, splenocyteswere isolated from each group of recipient mice and then subjected to intracellularcytokine determination by flow cytometry. As shown in Figure 6A, we observed adecreased level for IL-2 (~(**)p＜0.01) (Fig. 7A) while an increase of IFN-γin thesplenic CD4~+ T cells from the recipient mice infused with donor-derived aaDCs ascompared to those in the other groups. On the contrary, an increase of IL-10production by CD4~+ T cells was detected from the recipient mice with aaDCs infusion.Interestingly, we observe a reduction of IFN-γproduction by splenic CD8~+ T cellsin the recipients treated with aaDCs as compared with other types of DCs (_*~*p＜0.01),whereas IL-2 secretion by CD8~+ T cells were not affected by infusion of aaDCs (Fig.7B). These regulatory effects of aaDC on the cytokine production by splenic T cellswere abrogated when PD-1/PD-L pathway was blocked by anti-PD-1(Fig. 6).
[Conclusions] Our data indicate that the PD-1/PD-L pathway plays an important rolein aaDC-mediated prolongation of skin allograft survival.
树突状细胞(Dendritic cell，DC)是专职抗原提呈细胞之一。依据其成熟状态、表型或来源不同，在免疫或耐受的诱导中起着关键作用。DC捕捉，处理，传递抗原到淋巴器官，在淋巴器官与T细胞相互作用，引起抗原特异性的免疫反应或免疫耐受。T细胞受体(T cell receptor，TCR)识别抗原肽-MHC分子复合物、足够的共刺激分子及自分泌、旁分泌的细胞因子的参与，导致T细胞活化，诱导免疫应答。缺乏这些信号分子则使T细胞不能完全活化，诱导免疫耐受。成熟的树突状细胞表达高水平的MHC-Ⅱ类分子和共刺激分子，导致T细胞活化。与此相反，不成熟的树突状细胞缺乏共刺激信号，因此诱导同种异体抗原特异性低反应，延长同种异体移植物存活。阿司匹林，维生素D3或白细胞介素-10可体外诱导生成不成熟的树突状细胞。然而，这些体外培养的不成熟树突状细胞，当暴露于体内的炎性刺激时，可能转变成成熟的树突状细胞，导致免疫刺激而不是免疫抑制。最近研究表明，骨髓来源的树突状细胞与IL-10及细菌脂多糖(Lipopolysaccharides，LPS)培养刺激下表现为一种半成熟状态，即表达低水平的共刺激分子，或称为旁路活化的树突状细胞(Alternativelyactivated DC，aaDC)。这些半成熟的树突状细胞即使在炎性条件下也是不可逆转的，因此诱导抗原特异性T细胞无反应，延长同种异体移植存活。然而，这些半成熟树突状细胞免疫调节的分子机制仍不清楚。
PD-1(Programmed cell death protein 1，PD-1)受体是表达在活化T细胞上的抑制性共刺激分子，属于CD28家族成员，在免疫反应的调节及外周耐受方面起着重要作用。PD-L1表达在多种抗原提呈细胞。PD-L1可以通过与活化T细胞上的PD-1结合，抑制T细胞增殖，并促进活化T细胞的凋亡。最近的研究表明，PD-1／PD-L途径在动物同种异体移植模型中起着重要作用。虽然树突状细胞表达PD-1受体的两种配体，即PD-L1和PD-L2，但是PD-1／PD-L途径是否在半成熟树突状细胞引起的免疫耐受中起作用仍不清楚。本研究拟从PD-1／PD-L信号通路角度，探讨小鼠骨髓来源的半成熟树突状细胞(即aaDC)在小鼠皮肤移植模型中的耐受机制。
[研究方法]
1．不同类型小鼠骨髓来源的树突状细胞的制备培养与受者小鼠静脉输注
小鼠骨髓来源的树突状细胞(bone marrow-derived dendritic cell，BMDC)制备参照文献。分离小鼠后腿股骨和胫骨骨髓细胞，用NH4Cl低渗缓冲液裂解红细胞，细胞计数后以2×10~6个细胞接种在100mm的细胞培养皿中，培养基为含20 ng／ml rGM-CSF(Pepro Tech Inc，New Jersey，USA)的RPMI-1640完全培养基，37℃5％CO2培养箱中培养7天，获得所谓未处理的DC(untreated DC)，培养期间每间隔1天换1次培养基。为诱导产生不成熟DC(immature DC，imDC)，在培养第6天，加入20 ng／ml rmIL-10(Pepro Tech Asia，Rehovot，Israel)培养1天；而aaDC的制备是在第7天在imDC培养基中加入1μg／ml LPS(L4130，Sigma-Aldrich)，培养1天后即为aaDC；而成熟DC的获得采用1μg／ml LPS刺激未处理DC 1天。
为测试以上制备的4种不同类型DC对同种异体皮肤移植物存活时间的影响，在移植前7天，分别将BALB／c供者小鼠来源的上述4种DC经尾静脉输注到受者小鼠(C57BL／6)体内，细胞数为2×10~6／只小鼠。同时采用抗PD-1抗体阻断PD-1／PD-L信号通路以观察其对aaDC调节效应的影响，而以同型IgG为对照。抗体应用方法如下：在aaDC输注当天腹腔注射500μg／只(day 0)，在随后第2、4、6天各250μg／只腹腔注射，，共分7个实验组(每组6只小鼠)：group 1：untreated DC；group 2：imDC；group 3：aaDC；group 4：aaDC plus anti-PD-1 mAb；group 5：anti-PD-1 mAbalone：group 6：mDC and group 7：aaDC plus control IgG．
2．采用流式细胞术分析DC表型
DC首先与anti-CD16／CD32 mAb孵育以封闭FcγR，然后用荧光素标记的抗CD11c、CD80、CD86、MHCⅡ、PD-L1以及PD-L2分别孵育(4℃，30 min)，然后用PBS洗涤2次，采用流式细胞仪分析[FACSCalibur~(TM) flow cytometer(BectonDickinson)]。
3．小鼠皮肤移植术
参照Billingham and Medawar方法。受者小鼠(C57BL／6)麻醉后，剪除背部皮肤毛发，准备直径12mm的植床。颈椎断离法处死供者小鼠(BALB／c)，剪除躯干毛发，取直径12mm皮片，去除皮下脂肪，将皮片移植缝合到受者小鼠(用5—0#丝线采取8针不连续缝合法)，用创可贴包被伤口，每天观察1次移植皮肤的存活情况，如果50％皮片翻起或坏死即为排斥。同时选择性应用组织病理分析证实排斥的发生。
4．组织学分析
参照文献。皮肤移植物切下后，用4％福尔马林固定，石蜡包埋切片，HE染色后进行病理组织学分析，观察炎症细胞浸润程度。
5．ELISA检测细胞因子浓度
根据使用说明书介绍的方法，采用ELISA试剂盒(Biosource International，Camarillo，CA)检测混合淋巴细胞培养上清中细胞因子IFN-γ、IL-10含量。
6．混合淋巴细胞反应
刺激细胞为来源于BALB／c小鼠的各种类型DC。DC细胞经丝裂霉素C处理后接种于96孔V型底培养板。来源于C57BL／6受者小鼠脾脏细胞，经尼龙棉纯化获得T淋巴细胞，用CFSE标记后作为反应细胞。4×10~5个反应细胞与4×10~5刺激细胞混合后，在37℃、5％CO_2条件下培养5天，收获细胞，用流式细胞术分析CFSE荧光强度以评判T细胞增殖程度。
7．流式细胞术检测CD4~+CD25~+Foxp3~+Tregs
混合淋巴细胞培养5天后收集细胞，用anti-CD4-FITC、anti-CD25-PE进行细胞表面标记染色，细胞经透膜后用APC-labeled anti-Foxp3标记(Foxp3 StainingBuffer Set，eBioscience)，用流式细胞仪分析CD4~+CD25~+ T细胞表达Foxp3的水平(23)。
8．流式细胞术检测细胞内细胞因子
制备单个脾脏细胞，在体外37℃、5％CO_2培养条件下，用50 ng／ml PMA，1μg／ml ionomycin、2μM monensin(all Sigma-Aldrich)刺激处理4小时后收获细胞，经FITC标记抗CD4或CD8染色后，4％formaldehyde固定、透膜处理后用相应荧光素标记细胞因子抗体染色，流式细胞仪检测细胞因子表达水平。
9．统计分析
实验数据采用单向方差分析法，P＜0．05为统计学显著差异。不同处理组间移植物存活时间差异分析采用Kaplan and Meier法(24)。
[结果]
1．部分成熟为aaDC表型特点
对几种类型DC表型分析发现，与不成熟DC(imDC)和未处理DC(untreatedDC)相比较，aaDC表达CD80、CD86水平有一定程度增加，而MHCⅡ分子表达水平相当。然而，以上分子的表达水平比成熟DC(mDC)明显降低。值得注意的是，我们观察到aaDC表达PD-L1和PD-L2的水平与不成熟DC和未处理DC比较显著升高(图1)。
2．aaDC刺激同种反应T细胞增殖的能力降低，该特性与PD-1／PD-L信号通路有关
随后，我们通过混合淋巴细胞反应实验证实，如图2所示，aaDC(来源于BALB／c小鼠，H-2~d)刺激同种反应性T细胞(来源于C57BL／6，H-2~b)增殖的能力明显低于成熟DC，与未成熟DC相当。重要的是，当用抗PD-1抗体阻断PD-1／PD-L信号通路后，aaDC刺激同种T细胞增殖的活性增强，提示PD-1／PD-L共抑制信号通路参与aaDC对同种T细胞反应的调节。
3．在混合淋巴细胞培养体系中，aaDC诱导抗炎性细胞因子IL-10分泌增加而抑制IFN-γ的产生，该效应部分依赖于PD-1信号通路
我们采用ELISA检测上述混合淋巴细胞培养上清液中细胞因子IL-10和IFN-γ产生水平，发现aaDC作为刺激细胞时，能诱导出比成熟DC和未处理DC显著升高(p＜0．01)，几乎与未成熟DC相当浓度的IL-10；与此相反，与未处理DC相比较，aaDC、未成熟DC能显著抑制混合淋巴细胞反应炎症性细胞因子IFN-γ的产生(p＜0．01)。有趣的是当用抗PD-1抗体阻断后，可以逆转该调节效应(图3)，上述结果表明aaDC对以上细胞因子的调节也部分依赖于PD-1信号通路。
4．aaDC具有扩增CD4~+CD25~+Foxp3~+调节性T细胞的特性
因为已有文献报道调节性DC能诱导调节性T细胞(Treg)的产生。因此，我们进一步检测了混合淋巴培养体系中不同类型DC细胞诱导Treg产生的差异性。结果表明(图4)，与其它类型DC相比较，aaDC扩增CD4~+CD25~+Foxp3~+Treg的功能最强(p＜0．01)，该调节效应涉及PD-1信号通路，其次是不成熟DC，而成熟DC以及未处理DC对Treg的诱导产生能力弱。
5．静脉输注供者来源的aaDC能明显炎症皮肤移植物的存活时间，而且PD-1／PD-L信号通路参与aaDC的调节作用
依据上述体外实验结果，我们进一步探讨在同种异体皮肤移植前7天，输注供者来源的各种类型DC到受者体内(2×10~6个细胞／只小鼠)，观察其对皮肤移植物存活时间的影响。结果发现(图5)，与其它DC细胞{untreated DCs(MST 11．83±2．40days)，imDC(MST 16．00±3．10 days)or mDC(MST 7．50±1．64 days)}相比较，aaDC输注能显著延长移植物的存活时间(MST 22．67±4．08 days)(P＜0．01)。有趣的是，当用抗PD-1抗体阻断PD-1／PD-L信号通路后，aaDC对移植物的保护效应显著降低(MST22．67±4．08 vs 12．33±3．33 of aaDC+anti-PD．1，P＜0．01)。单独应用抗PD-1抗体对移植物的存活时间影响很小，与未处理DC的效应相当(MST 10．83±2．79 vs 11．83±2．40days，p＞0．05)。
6．组织病理学分析证实aaDC输注能显著减轻皮肤移植物中炎症细胞浸润
在皮肤移植后7天，对移植皮肤进行病理组织学分析，如图6所示，发现输注aaDC能显著减轻移植皮肤中炎症细胞浸润，imDC也能在一定程度上降低炎症细胞的浸润。而与此相对照，输注成熟DC(mDC)以及未处理DC后，皮肤移植物中仍有明显的炎症细胞浸润。aaDC输注加抗PD-1抗体能降低其对移植物的保护效应，而单独应用抗PD-1抗体或同型对照IgG对炎症细胞浸润的影响较小。以上病理组织学结果与移植物的存活时间相一致。
7．aaDC输注能改变受者小鼠脾脏T淋巴细胞产生细胞因子谱的特征
为了探讨供者来源的各种不同类型DC输注后，能否影响受者脾脏T淋巴细胞产生细胞因子的类型。进一步分离了输注不同类型DC后的各组受者小鼠的脾脏T淋巴细胞，体外在经PMA／ionomycin刺激后，采用流式细胞术分析T细胞内细胞因子(IL-2、IL-10和IFN-γ)表达水平。如图7A所示，与其它类型的DC相比，输注aaDC后受者小鼠脾脏CD4~+T细胞产生IL-2的能力降低(~(**)p＜0．01)，而IFN-γ有一定程度升高。与此相反，aaDC能增强受者脾脏CD4~+T细胞产生IL-10。有趣的是，我们观察到输注aaDC后，与其它组相比较，受者小鼠脾脏CD8~+T细胞IFN-γ的分泌潜能显著降低(_*~* p＜0．01)，而对CD8~+T细胞IL-2的产生无明显影响(图7B)。同样地，当应用抗PD-1抗体阻断PD-1／PD-L信号通路后，对aa DC的免疫负调节效应有明显的抑制作用。
[结论]
通过输注供者来源aaDC能显著延长异基因小鼠皮肤移植物存活时间，抑制受者同种反应性T细胞增殖，减轻移植物组织中单个核细胞浸润。aaDC的免疫调节机制可能涉及扩增CD4~+CD25~+Foxp3~+调节性T细胞，增强受者脾脏同种反应T细胞产生抗炎性细胞因子IL-10，而抑制与T细胞增殖密切相关的IL-2的产生，炎症性细胞因子IFN-γ也明显受到抑制，有趣的是，PD-1／PD-L信号通路对以上aaDC的免疫调节作用起着关键作用。本研究成果提示，采用aaDC过继输注可抑制同种移植物排斥反应，为防治同种异体器官移植排斥反应提供了新的干预策略，进一步深化了对aaDC免疫调节机制的认识。
[Background]
Alternatively activated DC (aaDC) can prolong allograft survival in the mousemodel. However, the molecular mechanism(s) by which these DCs function toregulate alloreactive-T cell responses remains to be clearly defined.
[Methods]
1. Preparation of various types of DCs and infusion into recipients mice
BMDCs were generated from bone marrow cells as described previously (19). Inbrief, bone marrow cells were obtained by aspirating femurs and tibial bones andthen seeded at 2×10~6 cells per 100 mm dish in 10 ml RPMI-1640 complete mediumsupplemented with 20 ng/ml rGM-CSF (Pepro Tech Inc, New Jersey, USA).Immature DCs (imDC) were generated by adding 20 ng/ml rmIL-10 (Pepro TechAsia, Rehovot, Israel) at day 6. To obtain alternatively activated DCs (aaDC), 1μg/ml LPS (L4130, Sigma-Aldrich) was added to imDC at day 7. Mature DCs (mDC)were generated by adding 1μg/ml LPS to untreated BMDCs 24 hr before harvesting.To test the in vivo regulatory effects of these DCs originated from BALB/c mice,each C57BL/6 recipient mouse was infused with 2x 10~6 untreated DC, imDC,aaDC, or mDC via tail vein at 7 days before the transplantation. As a result, 7experimental groups were established (6 mice per group) : group 1: untreated DC ;group 2: imDC ; group 3: aaDC; group 4: aaDC plus anti-PD-1 mAb; group 5:anti-PD-1 mAb alone ; group 6: mDC and group 7: aaDC plus control IgG. Anti-PD-1mAb or control IgG was given intraperitoneally after i.v. infusion of aaDC at 500μg on day 0, followed by 250μg on days 2, 4 and 6.
2. Analysis of DC phenotype by flow cytometry
For analysis of DC phenotype, cells were preincubated with anti-CD16/CD32 mAbto block FcγR. After staining with anti-CD11c, the cells were co-stained for CD80,CD86, MHCII, PD-L1 or PD-L2 antibody at 4℃for 30 min, washed twice withPBS, and analyzed on a FACSCalibur~(TM) flow cytometer (Becton Dickinson).
3. Procedures for skin transplantation
Full-thickness skin grafts were transplanted using a modified method of Billinghamand Medawar (20). In brief, the recipient mice were anesthetized and shaved aroundthe back. Skins were prepared from the trunk of BALB/c mice. After removing thesubcutaneous fat, 12-mm-diameter circles of full-thickness pieces weresimultaneously transplanted onto the alternate dorsal thorax of the C57BL/6 recipients.The graft bed was slightly larger than the skin allograft. The skin graft was sutured andcovered with a vaseline gauze and adhesive bandage for 7 days. Skin grafts wereinspected daily. Rejection was defined as the complete loss of viable epidermal grafttissue characterized by 50% of the graft surface became raised and necrotic by visualexamination. In selected animals, allograft rejection was further confirmed byhistological analysis.
4. Histology
Histological analysis was performed as previously described (21). The skinallografts were excised and stained with hematoxylin/eosin for the analysis ofpathological changes.
5. Determination of cytokine production by ELISA
IFN-γand IL-10 were measured in supematants harvested from co-cultures ofC57BL/6 splenic T cells with various types of DCs from BALB/c mice, using ELISAkits (Biosource International, Camarillo, CA) according to manufacturer'sinstructions.
6. Mixed lymphocyte reaction
Stimulator cells were various types of DCs originated from BALB/c mice. The DCswere treated with mitomycin C and seeded into 96-well plates with V-bottom. T cellsfrom recipient mice (C57BL/6) were purified by the nylon wool technique (22). ThoseT cells were stained with CFSE and used as responders. 4×10~5 responder and 4×10~5stimulator cells were mixed and incubated at 37℃under 5% CO_2. After culturing for5 days, the cells were harvested and subjected to flow cytometric analysis of CFSEfor T cell proliferation.
7. Detection of CD4~+CD25~+ Foxp3~+ Tregs by flow cytometry
C57BL/6 T cells after 5 days of coculture with BALB/c DCs were co-stained withanti-CD4-FITC and anti-CD25-PE, fixed and permeabilized, and then stained withAPC-labeled anti-Foxp3 by using commercial kits (Foxp3 Staining Buffer Set,eBioscience) according to the manufacturer's instructions. The cells were analyzed byflow cytometry as described (23). The collected data were analyzed with CellQuestsoftware (BD Biosciences, Mountain View, CA) as instructed.
8. Intracellular cytokine determination by flow cytometry
For determination of intracellular cytokine production, single-cell suspensions ofsplenocytes were prepared and stimulated for 4 hr in culture medium containing 50ng/ml PMA, 1μg/ml ionomycin, and 2μM monensin (all Sigma-Aldrich) at 37℃under 5% CO_2. Cells were then stained for surface marker, fixed in 4 % formaldehydein PBS, and permeabilized with 0.5 % saponin plus 1 % BSA PBS, followed bylabeling with specific cytokine antibodies or isotype controls. Cells were then analyzedon a FACSCalibur using CellQuest software.
9. Statistical analysis
Data were analyzed by one-way analysis of variance. Statistical significance wasdefined as P＜0.05. Allograft survival differences among groups were analyzed usingthe method of Kaplan and Meier (24). Comparisons of two means were carried out byone-way anova. Regression curves were fitted using SigmaPlot software (version 8.0;SPSS, Chicago, IL).
[Results]
1. Partially matured phenotype of aaDC
We first examined the phenotypic profiles of various types of DCs generated fromBALB/c BM progenitors. As shown in Fig. 1, we observed a slight increase of CD80and CD86 as compared with imDC and untreated DC, while a similar level ofMHCII expression was observed between aaDC and imDC or untreated DC. Incontrast, the levels of these molecules on aaDC were much lower than those on mDC.These analysis revealed that aaDC displayed a semi-mature phenotype. Of note, weobserved that aaDC had significantly higher levels of PD-L1 and PD-L2 as comparedto imDC and untreated DC (Fig. 1).
2. aaDC displayed a reduced alloreative T cell stimulating capacity that wasassociated with PD-1/PD-L pathway
Next, we performed an MLR to test the stimulatory capacity of aaDC preparedfrom BALB/c donor mice for the proliferation of allogeneic T cells from C57BL/6recipients. As expected, stimulation with mDC resulted in a strong proliferativeresponse (Fig.2). In contrast, stimulation with aaDC or imDC gave rise to a weakT-cell proliferation. Of important note, we observed that the decreased T-cellproliferative response to aaDC was largely restored when PD-1/PD-L pathway wasblocked by anti-PD-1 Ab.
3. aaDC enhanced the production of IL-10 while inhibited the secretion of IFN-γin an MLR
We also measured the production of IFN-γand IL-10 in the supernatant harvestedfrom the cocultures by ELISA (Fig.3). aaDC induced a reduced IFN-γand an enhancedIL-10 production as compared with mDC, which were reversed in the presence ofanti-PD-1 blocking Ab. Together, these results suggest that aaDC have an impairedallo-stimulating capacity and, PD-1/PD-L pathway plays an important role in theregulatory capacity of aaDC in this setting.
4. Augmented expansion of CD4~+CD25~+Foxp3~+ Tregs coculture with aaDC
Given the importance of regulatory DCs in the induction of regulatory T cells(Tregs), we next checked the differences in Tregs induced by various types of DC(imDC, aaDC, mDC). To this end, C57BL/6 T cells were cocultured with varioustypes of DC generated from BALB/c mice for 5 days. Expanded T cells originatedfrom each group were stained for intracellular Foxp3 after co-staining for surface CD4and CD25, and then subjected to flow cytometry analysis. As shown in Fig.4, aaDCinduced an increased expression of Foxp3 in CD4~+CD25~+ T-cell population ascompared mDC and untreated DC, and this effect was partially dependent onPD-1/PD-L pathway.
5. Prolonged skin allograft survival by aaDC infusion was reversed by blockadeof the PD-1/PD-L pathway
Based on the above results, we next sought to explore the feasibility of aaDC forinduction of antigen-specific allograft tolerance in transplantation. For this purpose,2 x 10~6 various types of DC (aaDC, imDC, mDC and untreated DC) originated fromBALB/c mice were injected into C57BL/6 recipient mice via tail vein respectively.At 7 days after injection, the mice were transplanted with BALB/c-derived skinallografts. As shown in Fig. 5, the mean graft survival time (MST 22.67±4.08 days)in the recipient mice pre-infused with donor-specific aaDC was significantly longerthan that in the recipients pre-treated with either untreated DCs (MST 11.83±2.40days) , imDC (MST 16.00±3.10 days) or mDC (MST 7.50±1.64 days) (P＜0.01).Notably, pre-treatment of the recipient mice with anti-PD-1 blocking Ab reversed theprolonged survival of skin allografts by aaDC (MST 22.67±4.08 vs 12.33±3.33 ofaaDC + anti-PD-1, P＜0.01), while administration of anti-PD-1 mAb alone showedonly a marginal effect on the allograft survival as compared with that untreated DCgroup ( MST 10.83±2.79 vs 11.83±2.40 days, p＞0.05 ).
6. Intravenous infusion of aaDCs attenuated the infiltration of leukocytes in thegrafts.
Consistently, histological analysis of allografts at 7 days after transplantation revealed much severe inflammatory infiltrates in the recipient mice pre-injected withmDC and untreated DC as compared to the recipients pre-infused with aaDC or imDC(Fig. 6). Allograft in the recipients treated with aaDC and PD-1 blocking Ab showed asevere infiltration and necrosis (Fig. 6). These results indicate that the PD-1/PD-Lpathway is essential for the aaDC-mediated allograft protection.
7. aaDC infusion changed the cytokine profiles produced by splenic T cells fromrecipient mice
Finally, we questioned whether infusion of donor-derived aaDCs affects thecytoldne profiles of recipient splenocytes. To address this question, splenocyteswere isolated from each group of recipient mice and then subjected to intracellularcytokine determination by flow cytometry. As shown in Figure 6A, we observed adecreased level for IL-2 (~(**)p＜0.01) (Fig. 7A) while an increase of IFN-γin thesplenic CD4~+ T cells from the recipient mice infused with donor-derived aaDCs ascompared to those in the other groups. On the contrary, an increase of IL-10production by CD4~+ T cells was detected from the recipient mice with aaDCs infusion.Interestingly, we observe a reduction of IFN-γproduction by splenic CD8~+ T cellsin the recipients treated with aaDCs as compared with other types of DCs (_*~*p＜0.01),whereas IL-2 secretion by CD8~+ T cells were not affected by infusion of aaDCs (Fig.7B). These regulatory effects of aaDC on the cytokine production by splenic T cellswere abrogated when PD-1/PD-L pathway was blocked by anti-PD-1(Fig. 6).
[Conclusions] Our data indicate that the PD-1/PD-L pathway plays an important rolein aaDC-mediated prolongation of skin allograft survival.
引文
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