CTL诱导及杀伤实验

一、抗原肽诱导CTL制备
1.PBMCs were separated from the whole blood of HLA-A2+ healthy controls. PBMCs (2 x106/ml) were cultured with each of the HLAA* 0201 refolding peptides at a concentration of 10 uM in RPMI 1640 medium containing 10% FCS and 20 U/ml recombinant human IL-2 (rhIL-2) in 24-well culture plate. Half of the medium was changed at day 4 with supplementation of rhIL-2 at 20 U/ml. At day 7, cells were harvested and tested for the presence of peptide-specific CD8+ T cells by an IFN-γrelease ELISPOT assay.
---------------Zhou M, Xu D, Li X, Li H, Shan M, Tang J, Wang M, Wang FS, Zhu X, Tao H et al: Screening and identification of severe acute respiratory
syndrome-associated coronavirus-specific CTL epitopes. J Immunol2006,
177(4):2138-2145.
2. PBMCs were separated from heparinized venous blood by Ficoll-Hypaque density gradient centrifugation from HLAA*0201 normal donors and HCC patients. DCs were generated from peripheral blood monocytes as described by Romani. Briefly, PBMCs were seeded into six-well culture plates containing 3 ml RPMI-1640 and 10% FCS at5–10x106/well. Plates were incubated in a 37o C incubator for 2 h, then the non-adherent cells were removed and the adherent cells were cultured at 37o C in RPMI-1640 supplemented with 10% FCS, 1000 U/ml human recombinant GM-CSF and 500 U/ml human recombinant IL-4. All T cell stimulation was with day 7 DCs. As described below, DCs were matured with lipopolysaccharide (LPS) on day 6 and HCA587 antigen was added for effective processing at this time.
Day 6 DCs were resuspended in RPMI-1640 with 1000 U/ml GM-CSF, 500 U/ml IL-4 and 10 ng/ml LPS and incubated at 37 o C. After 24 h cultivation the DCs were collected, washed and pulsed with 10 m g/ml HCA587 peptide for 3 h at 37 o C. The DCs were washed twice for the following stimulation.
CD8+T cells were isolated by positive selection with CD8-Dynal immunomagnetic beads according to the manufacturer’s instructions.After washing, CD8+T cells were co-cultured with HCA587peptide-loaded DCs in 2 ml RPMI-1640 medium
supplemented with 10% AB serum, recombinant human IL-2(10 ng/ml) and IL-6 (500 U/ml). Seven days later, the cultured T cells were restimulated with freshly prepared peptide-pulsed DCs and cultured for another 7 days. After four consecutive rounds of stimulation, cultures were tested for the presence of HCA587-specific CTLs. -----------Li B, Wang Y, Chen J, Wu H, Chen W: Identification of a new HLA-A*0201-restricted CD8+ T cell epitope from hepatocellular carcinoma-associated antigen HCA587. Clin Exp Immunol2005, 140(2):310-319.
3.Generation of CTLs in PBMCs from healthy donors：
Dendritic cells (DCs) have the unique capacity of activating naive T cells and initiating primary T-cell response. Antigen-specific T-cell responses from peripheral blood mononuclear cells (PBMCs) can be elicited with antigenic peptide-pulsed autologous DCs. We obtained PBMCs from the buffy coat of heparinized whole blood samples of healthy donors by density gradient centrifugation on the Histopaque 1077. These cells were resuspended in serum-free RPMI 1640 and allowed to adhere to six well plates at a final concentration of 1 x 107cells/3 ml/ well. After 2 h of incubation at 37°C, non-adherent cells were gently removed with warm medium by gently pipetting. The non-adherent cells (effector lymphocytes) were cryopreserved in FCS supplemented by 10% DMSO. The resultant adherent cells containing DCs were cultured in medium supplemented with 800 U/ml GMCSF and 1,000 U/ml IL-4 in 37°C/5% CO2. Every 2 days, one-half of the medium was replaced by fresh medium containing a double concentration of GM-CSF and IL-4 as indicated above. On day 5, 10 ng/ml of recombinant human tumor necrosis factor a (TNF-a) was added to the medium to induce phenotypic and functional maturation of DCs. After 48 h, DCs were pulsed with 20 ug/ml peptide in the presence of 3 ug/ml b2- microglobulin at 37°C for 3 h and irradiated at 30 Gy before use. The thawed 2 x106 of non-adherent effector lymphocytes were cocultured with 2 x105 peptidepulsed irradiated autologous DCs in a 24-well plate in the presence of 10 ng/ml recombinant human interleukin-7 (IL-7). After 7 days, lymphocytes were restimulated with peptide-pulsed autologous PBMCs in medium containing 10 ng/ml IL-7 and 20 U/ml IL-2. About 20 U/ml of IL-
2 was added 24 h later at regular intervals, 2 days after each restimulation. Lymphocytes were restimulated each week in the same manner. On the seventh day, after the three rounds of restimulation, cells were harvested and tested by ELISPOT assay.
--------------An altered peptide ligand for nave cytotoxic T lymphocyte epitope of TRP-2(180–188) enhanced immunogenicity.
Cancer Immunol Immunother (2007) 56:319–329 第三军医大吴玉章
4.Generation of CTLs in healthy donors
Dendritic cells are characterized by the unique capacity to activate naive T cells and initiate primary T-cell response. Antigen-specific T-cell responses from peripheral blood mononuclear cells (PBMCs) can be elicited with antigenic peptide- or protein-pulsed autologous DCs. Here, PBMCs were isolated from whole blood of 11 healthy HLA-A2.1_ volunteer donors by Ficoll/Hypaque density gradient centrifugation. Human peripheral blood monocyte-derived DCs were generated as previously described by us. On day 5 of culture, 10 ng/mL recombinant human tumor necrosis factor was added to the medium to induce phenotypic and functional maturation. Then, 48 hours later, DCs were pulsed with 20 ug/mL peptide in the presence of 3 ug/mL B2-microglobulin at 37°C for 3 hours and irradiated at 30 Gy before use. Peripheral blood lymphocytes (PBLs, 2 x106) were cocultured with 2 x 105peptide-pulsed irradiated autologous DCs in a 24-well plate in the presence of 10 ng/mL recombinant human interleukin-7. The next day, recombinant human IL-10 was added to the culture medium, to give a final concentration of 10 ng/mL. After 7 days, lymphocytes were restimulated with peptide-pulsed irradiated autologous DCs in medium containing 10 ng/mL IL-7 and 10 ng/mL IL-10, and then supplemented with 20 IU/mL IL-2 24 hours later. Lymphocytes were restimulated each week in the same manner. At 7 days after the fourth round of restimulation, cells were harvested and tested by ELISPOT assay, cytotoxicity assay, and tetramer staining. CD8T lymphocytes were purified by CD4_ cell–negative depletion using human CD4 microbeads.
---------Wang B, Chen H, Jiang X, Zhang M, Wan T, Li N, Zhou X, Wu Y, Yang F, Yu Y et al: Identification of an HLA-A*0201-restricted CD8+ T-cell epitope SSp-1 of SARS-CoV spike protein.Blood2004, 104(1):200-206.第二军医大曹雪涛
5. 抗原肽诱导CTL制备：
密度梯度离心法分离经EDAT抗凝的静脉血中的外周血单个核细胞（peripheral blood mononuclear cells，PBMC)，分离得到的PBMC种于6孔细胞培养板，在含10%FCS的RPMI1640培养基中，37℃5%CO2孵育1.5h。

收集悬浮细胞(主要为淋巴细胞)，以10%二甲亚矾90%小牛血清保存于液氮备用。

贴壁细胞为抗原提呈细胞-树突状细胞(DC)前体细胞，补充含GM-CSF(l000U/ml，R&D)、IL-4(l000U/ml，R&D)及含10%FCS的RPMI1640培养液，37，C5%COZ培养7d，需要时补充新鲜培养液。

5d后，加入LPS(lug/ml，Sigma)培养2d后形成成熟DC，r照射使其失去增殖活性。

将经过照射的DC与抗原肽(10u mol/l)在培养液中，37℃5%CO2共孵过夜。

将l*105负载有抗原肽的DC与1x106自体PBMC 种于含10%FCS的RPMI1640完全培养液的24孔细胞培养板，37℃5%coZ混合培养3d后，加入rIL-2(20U/ml，Sigma)继续培养10d，进行细胞增殖实验和细胞活性检测。

---------------结核杆菌抗原CD8+T细胞多表位“串珠式”肽疫苗研究第二军医大学长征医院实验诊断科仲人前
6.（一）人外周血单核细胞来源的DCs的培养
1）．外周血单核细胞的分离
HAL-A2.1+小PEBP+4乳腺癌病人抗凝外周全血来自长海医院和长征医院外科。

通过淋巴细胞分离液(Ficoll-Histopaque 1.077)密度梯度离心(室温，400×g，30分钟)，取界面细胞，置入50ml离心管中，用无钙镁PBS(pH7.2)-EDAT(2mM) 悬浮细胞，之后离心(300×g，10分钟)洗细胞一次，弃上清，无钙镁PBS重悬细胞，再此离心(200×g，5分钟)洗细胞2次，以便洗尽血小板。

之后所获得的细胞即为外周血单个核细胞(PBMC)。

2）. 人外周血单核细胞来源的DCs的培养
参照文献[60]的方法，人外周血单个核细胞用完全培养基(含10%胎牛血清的RPMI1640)悬浮PBMC，按1×107细胞/孔铺于6孔板，37℃、5%CO2孵育2小时后，轻晃板子，吸出悬浮细胞，用预温的培养基小心洗6孔板三遍(此部分细胞连同吸出的悬浮细胞冻存备用)，剩下的细胞即为获得的贴壁的单核细胞，贴壁细胞在含人重组rhGM-CSF(500U/ml)和人重组rhIL-4(10ng/ml)的完全培养基中37℃、5%CO2进行培养。

第三天，补充完全培养基，继续培养。

3）.相应候选表位肽体外致敏乳腺癌患者(hPEBP4+，HLA.A*0201+)外周血单核细胞来源的DCs
收集上述培养至第六天的hPEBP4+/HLA-A*0201+乳腺癌患者外周血单核细胞
来源的DCs，用人DCs完全培养基(RPMI1640完全培养基，500U/m1rhGM-CSF、
10ng/ml rhIL-4)调整细胞浓度为2x105细胞/ml浓度，lml/孔分入24孔板，按照实验分组加入20uM P40-48，4小时后收集细胞，弃培养基上清，用RPMI1640培养基离心洗涤细胞两次以除去原培养基中存在的刺激物，最后将2x105细胞悬浮在0.5ml人DCs完全培养基中，随即用于刺激同体的T淋巴细胞。

4）．乳腺癌病人外周血淋巴细胞抗原特异性CTL的体外诱导[61.68]
复苏本部分2中所收集冻存的非贴壁细胞(富含淋巴细胞)，悬浮于10%胎牛血清的RPMl6l40培养基中，调整细胞浓度为4x106细胞/m1，分别取0.5ml加
入至上述收集的各组经肽致敏的自体DCs中，于37℃、5%二氧化碳条件下共培养(淋巴细胞:DCs=10:1)，此为第一轮的刺激。

之后于共培养的第五天加入20U/ml
rhIL-2，培养7天后收集上述淋巴细胞，同样以10:1的比例再与新鲜制备的2xl0 5/ml肽致敏的同体DCs用10%胎牛血清RPMIl640培养液共培养进行第二轮的刺激。

同样的刺激每周一次共刺激三次。

培养过程中每隔3天加入一次20U/ml rhIL-2，2-3天半量更换新鲜培养基，并依据需要进行细胞的分孔扩增，末次刺激后的7天收集细胞，使用免疫磁珠阳性选择分选CD8+T细胞，方法严格依照厂商提供的说明书进行。

分选出的CD8+T细胞进行51Cr释放法和INF- r ELISPOT检测。

-----------NY-BR-1在中国乳腺癌患者中的表达及其HLA-A2限制性CTL表位免疫鉴定第三军医大吴玉章
7. DC刺激的T细胞对K562细胞的杀伤作用
按上述方法培养DC (在培养第4 天的DC中加入100μl K562细胞冻融抗原) 。

用RPM I 1640培养液调整DC、同种异体T淋巴细胞浓度分别为1 ×105 /ml、1 ×106 /ml。

各取DC和T淋巴细胞100μl,加入96孔培养板共培养,对照组以100μl RPM I1640培养液代替DC, 37℃、5% CO2 培养72 小时后,加入K562细胞作为靶细胞,效∶靶= 10∶1,设3个复孔。

37℃、5 % CO2孵育48小时后弃悬液,每孔加入20μlMTT溶液(5 mg/ml) , 37℃、5% CO2孵育4小时,终止培养, 40 ×g离心5分钟,弃上清,每孔加入二甲亚砜溶液150μl,振荡10分钟,在酶标仪上测定各组在570 nm 处A值(结果用3 孔均值表示) ,并计算其杀伤率(% ) 。

公式为:
细胞杀伤率(% ) = [ (效应细胞孔的A值+单纯靶细胞孔的A值- 细胞毒实验孔的A值) /
单纯靶细胞孔的A值] ×100%
二、Ficoll密度梯度离心法分离外周血单个核细胞
1）．原理
外周血单个核细胞（Peripheral blood mononuclear cell,PBMC)即外周血中具有单个核的细胞，包括淋巴细胞和单核细胞。

体外检测淋巴细胞首先要分离外周血单个核细胞，目前主要的分离方法是Ficoll-hypaque（葡聚糖－泛影葡胺）密度梯度离心法，因为血液中各有形成分的比重存在差异，因此得以分离。

红细胞和粒细胞密度大于分层液，同时因红细胞遇到Ficoll而凝集成串钱状而沉积于管底。

血小板则因密度小而悬浮于血浆中，唯有与分层液密度相当的单个核细胞密集在血浆层和分层液的界面中，呈白膜状，吸取该层细胞递经洗涤高心重悬。

本法分离单个核细胞纯度可达95％，淋巴细胞约占90％～95％，细胞获得率可达80％以上，其高低与室温有关，超过25℃时会影响细胞获得率。

2）.方法
1.在短中管中加入适量淋巴细胞分离液。

2.取肝素抗凝静脉血与等量Hank's液或RPMI1640充分混匀，用滴管沿管壁缓慢叠加于分层液面上，注意保持清楚的界面。

水平离心2000rpm×20分钟。

3.离心后管内分为三层，上层为血浆和Hank's液，下层主要为红细胞和粒细胞。

中层为淋巴细胞分离液，在上、中层界面处有一以单个核细胞为主的白色云雾层狭窄带(白膜层)，单个核细胞包括淋巴细胞和单核细胞。

此外，还含有血小板。

4.用毛细血管插到云雾层，吸取单个核细胞。

置入另一短中管中，加入５倍以上体积的Hank's液或RPMI1640，1500rpm×10分钟，洗涤细胞两次。

5.末次离心后，弃上清，加入含有10％小牛血清的RPMI1640，重悬细胞。

取一滴细胞悬液与一滴0.2％台盼兰染液混合，于血球计数板上，计数四个大方格内的细胞总数。

6.细胞活力检测：死的细胞可被染成兰色，活细胞不着色。

计数200个淋巴细胞。

计算出活细胞百分率。

3）人淋巴细胞分离液的使用方法：
使用方法例：取抗凝血1ml，与Hank’s液1：1混匀后，小心加于2ml的细胞分离液之液面上，以2000转／分离心（半径15cm水平转子）15分钟，收集界面上的细胞，放入含Hank’s液4-5毫升的试管中，充分混匀后，以1500-2000转／分离心10分钟。

吸去上清液，沉淀经反复洗2次即得所需细胞。

（此方法效果较好，推荐使用）
总结：
抗原肽特异性CTL的诱导和制备：
一、诱导特异性CTL的基本方法: T细胞-刺激细胞共培养。

二、一般步骤：
1.外周血单个核细胞(PBMCs)的分离
密度梯度离心法分离PBMCs
2.树突状细胞（DC）的制备
3.抗原提呈细胞（APCs）的准备
抗原肽与DC、TAP缺陷细胞或自体PBMCs共孵育，r照射或丝裂霉C处理后，用作APCs
4.效应细胞的制备
（1）将分离获得的PBMCs铺于细胞培养板
（2）常用的诱导CTL的方法
①多肽刺激：加入多肽后共孵育
②抗原肽孵育后TAP缺陷细胞刺激：PBMCs的第一次用多肽刺
激，一周后加入处理过的TAP缺陷细胞刺激
③抗原肽孵育后DC刺激：加入处理过的DCs
（3）共刺激2-4次
三、流程：
四、树突状细胞（DC）是专职的APC，其主要功能是摄取、加工处理和提呈抗原，它能诱导初始T细胞的活化，因此是机体适应性T细胞免疫应答的始动者。

未成熟的DC可表达MHC I/II 类分子，其摄取、加工和处理抗原的能力强，而提呈抗原激发免疫应答能力弱，成熟DC（表面特异性标志是CD1a，CD11c和CD83）可高表达MHC I/II 类分子，其摄取、加工处理抗原能力弱，而提呈抗原、启动免疫应答能力强。

五、 DC在摄取抗原或受到某些刺激（主要是炎症信号如LPS，TNF-α等）后，可以分化成熟。

细胞因子刺激能加速其成熟。

研究表明，GM-CSF主要是刺激
髓样单核细胞特别是树突状细胞的增殖和分化；IL-4的功能是B细胞的激活、增殖和分化，并抑制Th1细胞（Th1细胞主要介导巨噬细胞免疫应答，Th2细胞主要介导体液免疫应答）；IL-10抑制巨噬细胞；IL-7的主要功能是T、B细胞前体细胞的增殖和分化；IL-2的主要功能是促进T细胞的增殖；TNF-α主要是参与局部炎症，杀伤或抑制肿瘤。