CN108004208A - 一种体外诱导抗原特异性t细胞的方法 - Google Patents
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- CN108004208A CN108004208A CN201711252131.9A CN201711252131A CN108004208A CN 108004208 A CN108004208 A CN 108004208A CN 201711252131 A CN201711252131 A CN 201711252131A CN 108004208 A CN108004208 A CN 108004208A
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Abstract
本发明涉及一种扩增抗原特异性调节性T细胞的方法，属于生物技术领域。本发明采用Rapamycin联合TGF‑β细胞DC细胞的作用下体外诱导人的T细胞成为具有免疫抑制功能的调节性T细胞，具有以下优点：1)数量充分；2)能抵抗Th17细胞分化；3)较其他方法诱导的调节性T细胞具有更强的抑制功能与生物学作用。克服了自然调节性T细胞的诸多缺陷，在炎症性疾病，自身免疫性疾病治疗和器官移植免疫排斥的预防方面有更大的治疗优势。
Description
技术领域
发明涉及一种体外诱导抗原特异性T细胞的方法，属于生物医学技术领域
背景技术
半个世纪以来，免疫抑制药物显著延长同种异体移植物存活时间的同时，也由于自身的毒副作用、昂贵的价格及药物诱导的非特异性免疫抑制所引发的临床并发症等一系列严重问题，极大限制了临床器官移植的进一步发展。通过诱导移植物特异性免疫耐受或达到类似应用免疫抑制剂的“临床几乎耐受”状态，从而减少甚至不用免疫抑制剂，成为移植免疫学研究领域的基本课题之一。虽然许多研究者已在不同动物中成功地诱导了同种异体移植的免疫耐受状态，但由于目前尚不清楚的原因，至今尚未找到一种扩增调节性T细胞的有效方法，能够在临床前期的大动物模型中重复这些实验成果，更谈不上应用于临床。因此发展有效诱导针对供体特异性免疫耐受的治疗策略已成为实验和临床研究的主要方向，其特点不仅仅是延长移植物的存活率，同时应最大限度消除免疫抑制剂非特异性广泛抑制的缺点[1]。
国内外学者一直在寻求可以替代免疫抑制药物的途径。如：(1)阻断共刺激通路：虽然在移植动物模型中，通过阻断协同刺激通路使同种移植物存活时间明显延长，但该方法并不能达到持续稳定的耐受。(2)非成熟的异体树突状细胞(DC):目前体外诱导产生的非成熟的DC在某些情况下诱导体内外抗原特异性T细胞无反应，体内输注能明显延长同种异体移植物的存活，但只能获得短期的耐受。(3)混合性嵌合：大量实验研究进展说明，混合性嵌合诱导免疫耐受已经非常接近于临床应用，但一个关键的问题就是供者细胞输注，会诱发移植物抗宿主病(graft versus host disease，GVHD)。上述措施并未达到令人满意的效果。同时又有研究表明上述途径诱导免疫耐受的机制与继发性诱导CD4+CD25+调节性T细胞(Treg)有关。
1995年Sakaguchi等研究发现小鼠被祛除CD4+CD25+调节性T细胞会引发多种自身免疫性疾病,而将这些调节性T细胞回输则可抑制疾病的发生[2]。在正常人和小鼠的外周血及淋巴组织中的CD4+T细胞中约有5％～10％的细胞持续高表达CD25分子(IL-2受体a链)，同时这一亚群细胞是CD45RB分子低表达的。调节性T细胞亚群由胸腺或由外周活化的细胞发展而来，其T细胞受体(TCR)的表达不同于常规T细胞，与常规CD4+T细胞一起在外周与特异性抗原发生反应。胸腺产生的CD4+CD25+调节性T细胞进入外周后需要外周自身抗原的刺激等多种因素的影响才能成为功能性调节性T细胞。胸腺上皮细胞对CD4+CD25+调节性T细胞的分化可能起着主要作用，抗原提呈细胞(APCs)特别是树突状细胞(DCs)的成熟或活化对CD4+CD25+调节性T细胞的功能发挥调节作用。其中，转录因子Foxp3是该调节性T细胞的特征性标记，是调节性T细胞发育和功能的特异性转录因子,CD28及其信号通路，IL-2和IL-2受体信号途径等均是其生存所必须的信号分子。转导Foxp3基因到CD4+和CD8+细胞均能使它们转变为调节性T细胞[3,4]。CD4+CD25+调节性T细胞已证实具有控制I型糖尿病和预防造血干细胞的急性GVHD[5]。在同种异体器官移植中已证明了它潜在的对移植物的保护作用[6,7]。但是，目前有关调节性T细胞的应用仍受到以下条件的限制[8]：1)自然产生的调节性T细胞(天然Treg)数量少。天然Treg在动物体内约占胸腺和外周血CD4+的5％～10％，而在正常人体中仅仅1-2％的CD25bright细胞具有免疫抑制功能。因此，缺乏足够数量的Treg制约着临床的应用的可行性；2)扩增后表型和抑制功能可能发生改变。虽然通过体外扩增有可能克服细胞数量的问题，但是，最近有报道显示，体外多轮扩增的Treg失去了免疫抑制能力’并且多克隆扩增的Treg并不能对人造血干细胞移植后的GVHD产生防治作用，同时也发现Treg在一定条件下可向效应T细胞转化；3)自然Treg—旦被抗CD3单抗或PHA等广泛激活，其介导的抑制功能没有抗原特异性，即它们既可抑制具有相同抗原特异性的T细胞，也可抑制对其他抗原特异性的T细胞；4)在致炎因子IL-6等的诱导下，天然Treg细胞能够转化成为Thl7细胞。Thl7细胞是一类产生IL-17A和IL-17F的Th细胞亚群。IL-17属于促炎因子，与许多炎症反应和自身免疫性疾病的发生和发展有关。此外，Thl7细胞也与移植的排斥反应有密切关系。
近年来，Treg主要由包被CD3/CD28的磁珠来扩增，虽然这种扩增方式可以获得大量的细胞，但是这种技术也有它的弊端：1)培养易导致细胞Foxp3降低，2)扩增时间越久，Treg功能越低，3)培养成本昂贵。成熟DC诱导的抗原特异性调节性T细胞提供单一的内抗原，并能够治疗异种移植物抗宿主病(xGVHD)[9]。最新研究表明这种DC诱导的抗原特异性T细胞具有一定的体内扩增能力，且有非凡的抑制细胞因子表达的能力[10,11]。因此，DC诱导的调节性T细胞对于临床治疗自身免疫性疾病以及器官移植术后抗排斥治疗都具有极高的临床运用价值。
参考文献
1.Wilkes DS1ET,Reynolds HY.(2005)Lung transplantation:opportunitiesfor research and clinical advancement.Am J Respir Crit Care Med 172:12.
2.Sakaguchi S,Sakaguchi N,Asano M,Itoh M,Toda M(1995)Immunologicself-tolerance maintained by activated T cells expressing IL-2 receptoralpha-chains(CD25).Breakdown of a single mechanism of self-tolerance causesvarious autoimmune diseases.Journal of immunology 155:1151-1164.
3.Chai JG,Xue SA,Coe D,Addey C,Bartok I,et al.(2005)Regulatory Tcells,derived from naive CD4+CD25-T cells by in vitro Foxp3 gene transfer,caninduce transplantation tolerance.Transplantation 79:1310-1316.
4.Hori S,Nomura T,Sakaguchi S(2003)Control of regulatory T celldevelopment by the transcription factor Foxp3.Science 299:1057-1061.
5.Tritt M,Sgouroudis E,d'Hennezel E,Albanese A,Piccirillo CA(2008)Functional waning of naturally occurring CD4+regulatory T-cells contributesto the onset of autoimmune diabetes.Diabetes 57:113-123.
6.Pu LY,Wang XH,Zhang F,Li XC,Yao AH,et al.(2007)Adoptive transfusionof ex vivo donor alloantigen-stimulated CD4(+)CD25(+)regulatory T cellsameliorates rejection of DA-to-Lewis rat liver transplantation.Surgery 142:67-73.
7.Zheng SG WJ,Wang P,Gray JD,Horwitz DA.(2007)IL-2is essential forTGF-beta to convert naive CD4+CD25-cells to CD25+Foxp3+regulatory T cells andfor expansion of these cells.J immunol 178:10.
8.Chai JG TJ,Lechler R,Simpson E,Dyson J,Scott D.(2002)CD4+CD25+Tcells as immunoregulatory T cells in vitro.Eur J Immunol 32:11.
9.M M(2003)Dendritic cells in immunity and tolerance-do they displayopposite functions？immunity 19:4.
10.Katarina Kulhankova TR,Mohamed E.Nasr,Elizabeth H.Field(2012)Dendritic Cells Control CD4+CD25+Treg Cell Suppressor Function In Vitrothrough Juxtacrine Delivery of IL-2PLos one 09.
11.Sela U1OP,Park A,Schlesinger SJ,Steinman RM(2011)Dendritic cellsinduce antigen-specific regulatory T cells that prevent graft versus hostdisease and persist in mice.J Exp Med 208:8.
发明内容
本发明的目的是针对以上现有技术存在的缺点，提出一种体外扩增抗原特异性调节性T细胞的方法，利用该方法产生的调节性T细胞不仅数量多且稳定不易转化,可作为良好的免疫抑制剂使用。
为了增加以上技术问题，本发明的方法包括以下步骤：
一种体外诱导抗原特异性T细胞的方法，其特征在于包括以下步骤：
步骤一，采血：采用肝素抗凝常规采血手段采集血液；
步骤二，分离：从采集到的血液中离心分离出外周淋巴细胞，再由外周血淋巴细胞分离得到原始CD4+CD45RA+T细胞；
步骤三，准备DC细胞：使用CD14标记流式分选DC细胞，并用粒细胞-巨噬细胞集落刺激因子(GM-CSF)与白细胞介素-4(il-4)预处理DC细胞；
步骤四，第一次扩增T细胞：使用辐照处理的所述步骤三中的DC细胞激活T细胞；
步骤五，第二次扩增T细胞：第11天根据细胞浓度，再次加入辐照处理的DC细胞、白细胞介素-2(IL-2)、白细胞介素-15(IL-15)和转化生长因子-β(TGF-β)对所述步骤四中的T细胞重新刺激，促进T细胞的再次激活扩增，培养至细胞数达到目的扩增量，收集细胞，得到CD4+CD25+调节性T细胞。
所述步骤三包括如下步骤：选用HLA表型且与所述步骤二中得到的原始CD4+CD45RA+T细胞相异的供者的血液对其经过淋巴细胞分离液分离后,分选得到CD14+细胞，并使用浓度为1000U/ml的粒细胞-巨噬细胞集落刺激因子(GM-CSF),以及浓度为1000U/ml白细胞介素-4(il-4)对所述CD14+细胞刺激6天，分选得到DC细胞，在扩增之前，分选后的DC细胞加入抗原肽刺激成熟，并于诱导与扩增T细胞的当天，进行辐照当量为30Gy的辐照处理。
所述步骤四包括如下步骤：对所述步骤二中分选所得的所述CD4+CD45RA+T细胞进行刺激，加入所述步骤三中经辐照处理的DC细胞、白细胞介素-2(IL-2)、白细胞介素-15(IL-15)和转化生长因子-β(TGF-β)培养11天，每三天计算细胞数，并根据细胞密度分盘，补充培养基。
所述步骤四中，所述培养基由完全RPMI-1640培养基中添加浓度100U/ml的青霉素、浓度100μg/ml的链霉素、摩尔浓度2mM的左旋谷氨酸、摩尔浓度10mM 4-的羟乙基哌嗪乙磺酸、摩尔浓度0.1mM的非必须氨基酸、摩尔浓度1mM的丙酮酸钠和摩尔浓度50mΜ的二羟基乙醇配制而成。
所述步骤四中第一次扩增T细胞的方式为T细胞表面受体刺激，通过DC细胞进行T细胞表面受体刺激，调节性T细胞与DC细胞比例为10:1，由于DC细胞经过辐照处理，因此将于5天后，死亡殆尽；所述的刺激剂包括浓度为100U/ml的白细胞介素-2(IL-2)、浓度为10ng/ml的白细胞介素-5(IL-15)、浓度为100ng/ml的转化生长因子-β(TGF-β)以及摩尔浓度为10nM的雷帕霉素(rapamycin)；每三天计算细胞数，并增加培养基，从而保持细胞浓度在0.5X 106/ml,同时需重新加入足量的白细胞介素-2(IL-2)，并适量加入雷帕霉素(rapamycin)以维持其浓度。
所述步骤五中第二次扩增T细胞的方式为T细胞表面受体刺激，通过DC细胞进行T细胞表面受体刺激，调节性T细胞与DC细胞比例为10:1，由于DC细胞经过辐照处理，因此将于5天后，死亡殆尽；所述的刺激剂包括浓度为100U/ml的白细胞介素-2(IL-2)、浓度为10ng/ml的白细胞介素-5(IL-15)、浓度为100ng/ml的转化生长因子-β(TGF-β)以及摩尔浓度为10nM的雷帕霉素(rapamycin)；每三天计算细胞数，并增加培养基，从而保持细胞浓度在0.5X 106/ml,同时需重新加入足量的白细胞介素-2(IL-2)，并适量加入雷帕霉素(rapamycin)以维持其浓度。
RPMI是RoswellParkMemorialInstitute的缩写，代指洛斯维。帕克纪念研究所。RPMI是该研究所研发的一类细胞培养基，1640是培养基代号。其中含有10％胎牛血清。
本发明采用雷帕霉素(Rapamycin)联合IL-2体外诱导抗原特异性调节性T细胞成为具有免疫抑制功能的调节性T细胞，具有以下优点：
1)数量充分，并可根据需要扩增至治疗数量；
2)所得的调节性T细胞可以抵抗向Th17细胞分化，克服了自然的调节性T细胞的诸多缺陷。在炎症疾病以及器官移植患者中，本发明所得到的调节性T细胞具有极大的临床应用价值。
本发明采用雷帕霉素(Rapamycin)联合TGF-β细胞在DC细胞的作用下体外诱导人的T细胞成为具有免疫抑制功能的抗原特异性调节性T细胞，使之成为足够数量用于临床研究与治疗的调节性T细胞，起作用是通过雷帕霉素(Rapamycin)联合TGF-β与体外处理的DC细胞激活人外周血淋巴细胞分选的幼稚T细胞的T细胞受体(TCR)信号通路，达到增加Foxp3的转录的目的。Foxp3是调节性T细胞现有的标记，所以雷帕霉素(Rapamycin)联合DC细胞能够有效促进T细胞的激活，促进抗原特异性调节性T细胞的体外扩增。
附图说明
图1为对照组T细胞与抗原特异性调节性T细胞诱导过程中Foxp3+细胞比例的变化曲线对比图；
图2为对照组T细胞与抗原特异性调节性T细胞体外扩增数量对比图；
图3为对照组T细胞与抗原特异性调节性T细胞扩增中CD25，Foxp3双阳性细胞数；
图4为对照组T细胞与抗原特异性调节性T细胞对T细胞的抑制效果统计图；
图5为对照组T细胞与抗原特异性调节性T细胞对T细胞的抑制效果流式图；
图6为对照组T细胞与抗原特异性调节性T细胞对LPS刺激的T细胞细胞因子表达的抑制效果统计图；
图7为对照组T细胞与抗原特异性调节性T细胞对LPS刺激的T细胞细胞因子表达的抑制效果流式图；
图8为异种移植物抗宿主病的生存情况图；
图9为异种移植物抗宿主病的体重变化图；
图10为异种移植物抗宿主病的病理学检测图
具体实施方式
下面结合附图对本发明作进一步的说明：
实施例一
材料：CD4+CD45RA+T细胞分离试剂盒(购自Miltenyi Biotec，USA)，anti-humanCD14细胞分离试剂盒(购自Miltenyi Biotec，USA)，系统免疫缺陷(SCID)小鼠(购自美国Jackson灾验室)。
仪器：磁珠分离器(德国美天旎公司的Auto MACS)，流式细胞仪(BD公司，型号：Vantage SE)。
试剂：，rhIL-2(加入后最终浓度为100IU/ml)，rhIL-15(10ng/ml),Rapamycin(加入后最终浓度为10nM)购自惠氏制药。培养基由完全RPMI-1640培养基中添加100U/ml青霉素，100u g/ml链霉素，2mM左旋谷氩酸，10mM4-羟乙基哌嗪乙磺酸，0.1mM非必需氨基酸，1mM丙酮酸钠(以上购于BioSourceInternational公司)和50uM二羟基乙醇(购于SigmaAldrich公司)配制而成。
实验步骤如下：
1.1体外扩增抗原特异性调节性T细胞
第一步，采集：采用肝素抗凝常规采血手段采集血液
第二步，分离：向30ml人淋巴细胞分离液(购于上海欧韦达仪器科技有限公司)上缓慢加入采集的血液20ml，在4度以1500-2000转/分离心25分钟。吸取离心后中间白色的细胞层即可得到外周血的淋巴细胞(PBMC)，再将PBMC放入CD4+CD45RA+T细胞分离试剂盒用阴性分选去除CD8，CD14，CD16，CD19，CD36，CD56，CD123，CD235和CD45RO,再在剩余的细胞中阳性分选CD45RA细胞。
第三步，DC细胞准备：选择与上述供者HLA表型不同的供者，使用相同方法分离获得外周血淋巴细胞(PBMC),再将PBMC放入CD14细胞分离试剂盒，阳性分选出DC细胞。DC细胞以0.5-1.0X 106/ml细胞浓度接种于培养盘中，加入GM-CSF(1000U/ml)，IL-4(1000U/ml)刺激6天
第四步，第一次刺激：第一天，DC细胞预先辐照处理(30Gy)以去除细胞的增殖能力，保留细胞免疫原性。在CD4+CD45RA+T细胞中加入辐照处理后的DC细胞，T细胞与DC的细胞数比例为10:1，同时加入IL-2，IL-15，Rapamycin刺激细胞增殖。之后第三天，第六天，第九天，计算细胞数,保持细胞浓度并分孔培养，同时重新加入IL-2，适量补充Rapamycin。
第五步，第二次刺激：细胞培养第11天收集细胞，重新接种在容积较大的细胞培养瓶中，加入与第一次刺激浓度一致的辐照处理的DC细胞，IL-2，IL-15以及Rapamycin。之后依旧每三天计算细胞数并分盘，补充刺激剂，直至获得目标数量的调节性T细胞。使用细胞前，将上述细胞在含有10％FBS和rh-IL-2(1000IU/ml)的RPMI 1640培养基中休息48小时，收集上述细胞，即为调节性T细胞
1.2 体外扩增抗原特异性调节性T细胞的分析鉴定
1.2.1将本发明所得的调节性T细胞，包被CD3/CD28磁珠诱导形成的调节性T细胞按照1:5，1:10，1:20的比例与CFSE荧光标记的T细胞共培养，结果如图四，图五所示，抗原特异性T细胞表现出超越其他方法诱导的调节性T细胞的抑制T细胞增殖的能力，在1:20时仅仅本抗原特异性调节性T细胞具有细胞抑制功能。结论：抗原特异性T细胞比磁珠诱导的诱导性调节性T细胞(iTreg)具有更强的免疫调节能力。
1.2.2将本发明所得的调节性T细胞，anti-CD3/CD28磁珠诱导形成的调节性T细胞与LPS刺激的外周血淋巴细胞共培养三天，并流式检测细胞因子，结果如图六，图七所示，PBMC在抗原特异性调节性T细胞作用下基本抑制了向Th1,Th17细胞的分化。结论：抗原特异性调节性T细胞具有比磁珠诱导的诱导性调节性T细胞(iTreg)更强的抑制炎症因子表达的功能。
1.2.3将上述方法扩增后的抗原特异性调节性T细胞单独，或者联合同种异体的荧光燃料CFSE标记的T细胞一起经尾静脉注入SCID小鼠体内，本方法所获得的的调节性T细胞自身并不致病，同时能够抑制效应T细胞的增殖，延缓或者预防GVHD的发生。如图八，九，十所示：对照组肝脏出现明显的肝硬化小叶，肾脏出现明显的肾小管坏死和炎症，肺部出现明显的纤维化，而以上病理变化均未出现在抗原特异性调节性T细胞治疗组。可见抗原特异性调节性T细胞相对于其它体外诱导的调节性T细胞，具有更强的体内抑制功能，且无毒副作用。
1.3临床应用举例
以健康志愿者为例，用抗原特异性调节性T细胞进行有无毒副作用检测的实验：5个健康的志愿者，采集外周静脉血按照1.1方法制备后，按照体表面积计算20X106/m2，每月注射一次。观察细胞体内存活周期和毒副作用。注射后的调节性T细胞在体内能存活30天-50天，本身未引起发热，过敏等不良反应。
由以上实验得出本发明的优点如下：
(1)抗原特异性调节性T细胞具有明显的抑制T细胞的增生；
(2)抗原特异性调节性T细胞本身没有明显的毒副作用，具有体内长期的保护作用能力。
(3)抗原特异性调节性T细胞可以代替免疫抑制剂或者减少免疫抑制剂的使用剂量，用于器官移植后期免疫维持治疗，同时可以用于自身免疫性疾病患者或I型糖尿病的治疗，这将是器官移植免疫治疗史上里程碑性的突破。
除上述实施例外，本发明还可以有其他实施方式。凡釆用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。
Claims (6)
1.一种体外诱导抗原特异性T细胞的方法，其特征在于包括以下步骤：
步骤一，采血：采用肝素抗凝常规采血手段采集血液；
步骤二，分离：从采集到的血液中离心分离出外周淋巴细胞，再由外周血淋巴细胞分离得到原始CD4+CD45RA+T细胞；
步骤三，准备DC细胞：使用CD14标记流式分选DC细胞，并用粒细胞-巨噬细胞集落刺激因子(GM-CSF)与白细胞介素-4(il-4)预处理DC细胞；
步骤四，第一次扩增T细胞：使用辐照处理的所述步骤三中的DC细胞激活T细胞；
步骤五，第二次扩增T细胞：第11天根据细胞浓度，再次加入辐照处理的DC细胞、白细胞介素-2(IL-2)、白细胞介素-15(IL-15)和转化生长因子-β(TGF-β)对所述步骤四中的T细胞重新刺激，促进T细胞的再次激活扩增，培养至细胞数达到目的扩增量，收集细胞，得到CD4+CD25+调节性T细胞。
2.根据权利要求1所述的体外诱导抗原特异性T细胞的方法，其特征在于所述步骤三包括如下步骤：选用HLA表型且与所述步骤二中得到的原始CD4+CD45RA+T细胞相异的供者的血液对其经过淋巴细胞分离液分离后,分选得到CD14+细胞，并使用浓度为1000U/ml的粒细胞-巨噬细胞集落刺激因子(GM-CSF),以及浓度为1000U/ml白细胞介素-4(il-4)对所述CD14+细胞刺激6天，分选得到DC细胞，在扩增之前，分选后的DC细胞加入抗原肽刺激成熟，并于诱导与扩增T细胞的当天，进行辐照当量为30Gy的辐照处理。
3.根据权利要求1所述的体外诱导抗原特异性T细胞的方法，其特征在于：所述步骤四包括如下步骤：对所述步骤二中分选所得的所述CD4+CD45RA+T细胞进行刺激，加入所述步骤三中经辐照处理的DC细胞、白细胞介素-2(IL-2)、白细胞介素-15(IL-15)和转化生长因子-β(TGF-β)培养11天，每三天计算细胞数，并根据细胞密度分盘，补充培养基。
4.根据权利要求3所述的体外诱导抗原特异性T细胞的方法，其特征在于：所述步骤四中，所述培养基由完全RPMI-1640培养基中添加浓度100U/ml的青霉素、浓度100μg/ml的链霉素、摩尔浓度2mM的左旋谷氨酸、摩尔浓度10mM 4-的羟乙基哌嗪乙磺酸、摩尔浓度0.1mM的非必须氨基酸、摩尔浓度1mM的丙酮酸钠和摩尔浓度50mΜ的二羟基乙醇配制而成。
5.根据权利要求1所述的体外诱导抗原特异性T细胞的方法，其特征在于：所述步骤四中第一次扩增T细胞的方式为T细胞表面受体刺激，通过DC细胞进行T细胞表面受体刺激，调节性T细胞与DC细胞比例为10:1，由于DC细胞经过辐照处理，因此将于5天后，死亡殆尽；所述的刺激剂包括浓度为100U/ml的白细胞介素-2(IL-2)、浓度为10ng/ml的白细胞介素-5(IL-15)、浓度为100ng/ml的转化生长因子-β(TGF-β)以及摩尔浓度为10nM的雷帕霉素(rapamycin)；每三天计算细胞数，并增加培养基，从而保持细胞浓度在0.5X106/ml,同时需重新加入足量的白细胞介素-2(IL-2)，并适量加入雷帕霉素(rapamycin)以维持其浓度。
6.根据权利要求1所述的体外诱导抗原特异性T细胞的方法，其特征在于：所述步骤五中第二次扩增T细胞的方式为T细胞表面受体刺激，通过DC细胞进行T细胞表面受体刺激，调节性T细胞与DC细胞比例为10:1，由于DC细胞经过辐照处理，因此将于5天后，死亡殆尽；所述的刺激剂包括浓度为100U/ml的白细胞介素-2(IL-2)、浓度为10ng/ml的白细胞介素-5(IL-15)、浓度为100ng/ml的转化生长因子-β(TGF-β)以及摩尔浓度为10nM的雷帕霉素(rapamycin)；每三天计算细胞数，并增加培养基，从而保持细胞浓度在0.5X106/ml,同时需重新加入足量的白细胞介素-2(IL-2)，并适量加入雷帕霉素(rapamycin)以维持其浓度。
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