CN102216446A - 制备t细胞的体系和方法 - Google Patents
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Abstract
本发明涉及由干细胞合成T细胞的体系和方法。更具体的说,本发明涉及使用造血干细胞,由CD4谱系合成CD4细胞和/或T细胞子类型的培养体系和方法。成人造血干细胞(HPC)是所有免疫细胞的前体。然而,采用上述便捷的培养体系成功合成功能性CD4T细胞的先例屈指可数。此外,本发明还涉及可表达DL1、白细胞介素-7(IL-7)和FMS型酪氨酸激酶3配体(Flt3-L)的新型基质细胞系。上述改进培养体系可显著促进T细胞的发展研究以及免疫治疗应用。
Description
政府支持项目说明
本发明为政府支持项目,项目号NIH,资金号HL59412。政府对本发明具有一定的专利所有权。
相关应用交叉引用
本发明与2008年9月11日提交的美国临时申请61/096,240有关,且根据35USC 119的规定,本发明享有该临时申请的优先权。
简介
T细胞对哺乳动物免疫系统的建立具有举足轻重的作用。对于身患慢病毒感染或癌症的病人而言,其体内免疫系统常无法正常工作(1)。大规模生产用于治疗感染和癌症的T细胞一直广受人们的关注。体内扩张的抗原特异性淋巴细胞的自体移植受健康的功能性T细胞来源的限制(2)。异性抗原特异性效应T细胞的继承性转移受上述活性T细胞适用性的限制,且受移植物抗宿主病(GVHD)的影响(3)。因此,由成人骨髓(BM)衍生的体内CD34造血干细胞(HPC)合成大量抗原特异性T细胞有助于克服上述部分局限。
较前发表的合成人体T淋巴细胞,如上皮细胞的胸腺器官培养和三维培养体系的体内培养系统较为复杂,且难以操作(4-6)。这些体内培养体系表明了鼠和人体器官胚胎干细胞的早期T细胞分化(7,8)。近期报道了一种简单的T细胞发育培养体系,该体系使用可表达Notch配体的δ-1型1(OP9-DL1)鼠胚胎基质细胞,且上述细胞为分化胸腺细胞提供了一种均一的二维环境(9)。OP9-DL1培养体系可以支持从鼠的胚胎肝脏(10)、成人骨髓(BM)(11,12)和人体脐带血和儿童骨髓(13,14)中分离的原体分化。
使用OP9-DL1培养体系,由成人造血干细胞合成成熟T细胞的先例为数不多(13,15)。近期我们的研究发现使用慢病毒载体(LV)工程OP9-DL1(LmDL1)培养体系,成人骨髓提取的CD34造血干细胞,其T细胞生长速率较胚胎和血液慢(16)。已有原理验证研究对人体CD8T细胞受体(TCR)与OP9-DL1培养体系一起进入人体脐带血或胸腺造血干细胞的逆转录病毒药物转移进行了验证(17,18)。如没有成人T细胞发育体系从患者自身造血干细胞合成与成人白细胞抗原(HLA)匹配的T细胞,则上述应用需进行同种异基因移植(19)。
发明内容
本发明阐述了较前利用体内成人T细胞发育体系的至少三个局限性:preT细胞的有限扩展,双阳(DP)阶段的无效分化和缺乏正选择和谱系提交。本发明开发出一种可表达DL1,Flt3-L和/或IL-7的工程基质细胞的改进型体系,上述体系可提高CD34造血干细胞的preT细胞扩展。更为重要的是,本发明发现连续的IL-7信号可破坏未成熟的单阳(ISP)胸腺细胞进一步分化为双阳胸腺细胞,进而导致淋巴细胞功能发育不完全。正选择过程受IL-7受体(IL-7R)和TCR信号的影响较大。有趣的是,通过对IL-7R信号和进一步TCR结合的灼烧,CD4T细胞的正选择和谱系提交可在体内发生。此外,本发明发现这些CD4T细胞功能成熟。由成人造血干细胞合成功能性CD4T细胞的简单体内培养体系使得一系列变换免疫治疗方案成为可能。
附图说明
图1.慢病毒载体改性的鼠胚胎基质细胞系。(A)慢病毒载体结构。(B)ELISA分析LmDL1和LmDLFL7细胞引发的IL-7分泌。(C)鼠δ型-1(DL1)表面表达的流式细胞仪分析。(D)流式细胞仪分析慢病毒载体改性基质细胞系LmDL1-FL和LmDL1-FL7的Flt3L表达。
图2.慢病毒载体改性LmDL1-FL7基质细胞支持早期T淋巴细胞的扩张生长(A)培养在LmDL1上,且含有IL-7和Flt3L,或培养在LmDL1-FL7上的成人骨髓CD34+造血干细胞的T细胞生长动力学。如图所示,不同的培养天数下,从共培养体上取样以观察造血干细胞的生长,选用抗-CD4和抗-CD8抗体进行染色,并采用流式细胞仪进行分析。(B)培养在LmDL1上,含有IL-7和Flt3L,或培养在LmDL1-FL7上成人骨髓CD34+造血干细胞的CD3和TCRαβ表达动力学。(C)培养在LmDL1上,含有IL-7和Flt3L,或培养在LmDL1-FL7上的分化T细胞的繁殖曲线。(D)自42天共培养开始,经过两周抗-CD3/CD28刺激后的T细胞发育标记和核Ki67的流式细胞仪分析。采用PBMCs(未刺激)进行控制。
图3.改进型体内培养体系内的成熟CD4,无CD8T细胞生长(A)实验设计。在LmDL1-FL7中培养24天,且转移到LmDL1-FL培养中的成人骨髓CD34+造血干细胞生长曲线。(B)CD8、CD4、CD3和TCRαβ表达动力学的流式细胞仪分析。(C)在LmDL1-FL7培养24天,且转移到LmDL1-FL培养中的成人骨髓CD34+造血干细胞。在第42天对细胞进行刺激并培养14天,随后进行进一步分析。成熟标记和核Ki67的流式细胞仪分析。采用上述条件下刺激过的PBMCs进行控制。
图4.体内衍生的CD4T细胞具有功能性,且具有一定的Vβ谱系(A)刺激2周的T细胞再次经PMA和伊屋诺霉素刺激5-6小时,选用可检测免疫效应细胞因子和蛋白质的抗体进行染色。移除IL-7后,从LmDL1-FL7/L-mDL1-FL共培养的两个独立供体骨髓CD34+造血干细胞中提取的T淋巴细胞可以合成IFN-γ、IL-4和IL-17,表达FoxP3且不调节CD25。采用正常PBMC和主要单细胞衍生CD4T细胞克隆进行控制。(B)与控制成人PBMC相比,从三组不同成人骨髓CD34+造血干细胞供体衍生的体内T淋巴细胞的V β谱系窄而扭曲。
图5.改进型体内T细胞发育体系可从成人造血干细胞合成成熟的CD4T细胞。上图表明DL1、Flt3L和IL-7T细胞生长共培养体系中缺乏功能性T细胞。下图表明伴随着慢病毒载体-工程共表达DL1、Flt3L和IL-7,以及IL-7的间歇移除,成熟的功能性CD4T细胞数量增加。
图S1-3(S3)流式细胞仪分析表明,与刺激PBMC控制相比,T细胞前体(在OP9FL7上培养42天)表现出较高水平的I级HLA和较低水平的HLADR DQ DP。(S1)CD3ε分析表明,与控制物相似,CD8细胞确实表达T细胞受体的CD3ε链,显示较低水平的GATA3a CD4谱系标记,且其表达的PU.1意味着非成熟阶段的分化受到抑制。
具体描述
成人骨髓提取的造血干细胞(HSCs)是所有功能性免疫细胞的前体。然而,指导不同造血干细胞发育成成熟T细胞所需的分子信号却一直难以攻克。可表达δ型1(OP9-DL1)的鼠类胚胎基质细胞系支持早期T细胞分化,但却无法支持由成人骨髓提取的CD34+造血干细胞发育成成熟的T淋巴细胞。不受胸腺影响,成功合成成熟CD4T淋巴细胞的先例屈指可数。本发明的一个具体实施方式包括一种病毒载体改性的培养体系,上述体系支持成人CD34+造血干细胞分化为成熟的体内CD4T淋巴细胞。表达DL1、白细胞-7(IL-7)和FMS型络氨酸激酶3配体(FL)的工程基质细胞支持早期分化T细胞的生长。然而,持续的IL-7信号导致非成熟单阳(ISP)CD8状态下的分化受阻。本发明通过暂时性终止IL-7受体信号和活化CD3/CD28信号转导通路相结合的方法,解决了上述问题。上述改性导致成熟CD4T细胞的合成,而上述成熟CD4T细胞通过刺激可合成包括IFN-γ和TNF-α在内的效应细胞因子。
本发明的一个实施方案包含一种培养体系,上述培养体系支持成人CD34+造血干细胞(HSCs)分化成完全成熟的体内CD4T淋巴细胞。
在一个更为具体的实施方案中包括在IL-7中培养造血干细胞,并在一定培养时间范围内对其进行终止。在一个更为具体的实施方案中造血干细胞与OP-9基质细胞等可表达IL-7、mDL1和Flt3L(通常通过与病毒载体进行转染,如慢病毒)的细胞进行共培养,培养时间为14-24天。14-30天后造血干细胞不再经IL-7培养。随后造血干细胞经TCR刺激。造血干细胞进而发育成完全成熟的功能性CD4T细胞。
本发明还涉及诱导抗癌免疫反应的方法。在某些实施方案中,上述方法包括向受体给药包括多种T细胞及一种或多种药物可接受载体或赋形剂的组合物。在某些具体实施方案中,抗肿瘤免疫反应具有以下性能(a)避免肿瘤生成;(b)延缓肿瘤生成;(c)减缓肿瘤生长速度;(d)避免肿瘤复发;(e)抑制肿瘤生长;或者(f)上述几种功效的组合。在某些具体实施方案中,抗肿瘤免疫反应包括对抗存在于肿瘤细胞内或肿瘤细胞上的抗原的细胞毒T细胞反应。在某些具体实施方案中,细胞毒T细胞反应受CD8+T细胞协调。
本发明公开的组合物和方法可用作多组分抗肿瘤和/或抗癌治疗中的一部分。在某些具体实施方案中,本发明公开的方法进一步包括提供选自辐射、化疗、手术切除、免疫治疗中的一种或几种的额外抗癌治疗。在某些具体实施方案中,额外抗癌治疗可在给药步骤前、同时、之后或它们的组合进行。在某些具体实施方案中,额外抗癌治疗在给药步骤前进行,且给药组合物可用作辅助性治疗。
本发明公开的组合物和方法用于预防和/或治疗任何肿瘤和/或任何癌症。在某些具体实施方案中,上述癌症选自膀胱癌、乳腺癌、宫颈癌、胆管癌、直肠癌、胃肉瘤、神经胶质瘤、肺癌、淋巴瘤、黑色素瘤、多发性骨髓瘤、骨肉瘤、卵巢癌、胰腺癌、前列腺癌、胃癌、脑瘤、颈部肿瘤和实体肿瘤。在某些具体实施方案中,上述癌症包括肺癌。
本发明公开的组合物和方法可用于预防和/或治疗肿瘤和/或癌症。在某些具体实施方案中,预防/治疗的对象为哺乳动物。在某些具体实施方案中,上述哺乳动物为人。
下列术语为本领域技术人员所熟知,下列定义用于进一步解释本发明。
除非另行说明,本发明所用的所有科技术语为本领域技术人员通常理解的含义。本发明引用参考文献旨在说明这些技术是本领域所熟知技术,包括那些技术的变体或同等技术的替代,对于本领域技术人员来说都是显而易见的。下列术语为本领域技术人员所熟知,下列定义用于进一步解释本发明。
除非另行说明,所有表示成分数量、反应条件以及说明书和权利要求中使用的数字应被理解为通过词语“约”可以在所有情况下进行调整。相应地,除非有相反的指示,说明书及权利要求中所列的数字参数均为约数,可依本发明要求进行调整。
根据专利法长久以来的惯例,本文中,包括权利要求中所用“a”,“an”和“the”意思是指一个或多个。如,词语“细胞”意为一个或多个细胞。同样,本文使用的词语“另一个”是指至少第二个或更多。
当本文中使用如用于质量、时间、剂量(如细胞数)等测量值时,本发明所用的“约”一词表示包括在一些实施方案中有一定变化。在某些具体实施方案中比指定的数量浮动+-.20%、在某些具体实施方案中浮动+-.10%,、在某些具体实施方案中浮动+-.5%、在某些具体实施方案中浮动+-.1%及在某些具体实施方案中浮动+-.0.1%,这些变化均符合本发明的方法。
本发明中“包括”(及其同义词,如“包含”和“含有”),“具有”(及其同义词,如“拥有”和“有”),“含”(及其同义词,如“包含”和“包括”)或“含有”(及其同义词,如“包括”和“包含”)均为广义词,且不排除还包括其他、未引用方法步骤等。
本发明中“有效治疗剂量”“药物有效剂量”“治疗剂量”和“有效剂量”均可互换,且意为足以产生可测定反应(如被治疗者的相关生物或临床反应)的组合物用量(如药物可接受载体或辅料的ES细胞和/或其他功能性细胞数量)。如本发明所用成分中CD4T细胞的实际用量随不同待测物所需达到的理想免疫反应不同而变化。所选剂量受几种因素的影响,其中包括但不限于给药途径、与其他药物共同服用或进行治疗、被治疗状况的严重程度及患者健康状况和病史。
本发明中“IL-7”指一种与IL-7至少具有95、96、97或98%相似性的已知IL-7分子或多肽。已知几种不同物种的IL-7序列。基因库登录号实例包括AAI 10554、BC110553、AAH47698和BC047698。根据传统技术和计算机程序确定其相似度。如当使用默认空位权重,采用GAP或BESTFIT(肽)程序进行最优排列,或使用计算机算法BLASTX或BLASTP进行测定时,两个序列具有特定的相似性。优选与保守氨基酸取代具有一定相似性的残留位置。如具有电荷或极性等相似化学性质的氨基酸取代基本不会影响蛋白质特性。非限制性实施例包括天冬酰胺所需谷氨酰胺或天冬氨酸所需谷氨酸。
本发明中“癌症”和“肿瘤”可互换使用,可指原发肿瘤、转移肿瘤及患者的任何组织癌症,其中上述组织包括但不限制于乳腺、结肠、直肠、肺、口腔、下咽部、食管、胃、胰脏、肝、胆囊、胆管、小肠、泌尿系统包括肾、膀胱和尿道;女性生殖系统包括宫颈、子宫、卵巢(如绒膜癌和妊娠滋养细胞疾病);男性生殖系统包括前列腺、精囊、睾丸和生殖细胞肿瘤;内分泌腺包括甲状腺、肾上腺和脑垂体;皮肤(如血管瘤和黑色素瘤),骨或软组织;血管(如卡波西肉瘤);脑、神经、眼睛和脑膜(如星型细胞瘤、神经胶质瘤、成胶质母细胞瘤、成视网膜细胞瘤、神经瘤、成神经细胞瘤、神经鞘瘤和脑膜瘤)。本发明中“癌症”和“肿瘤”也包括白血病等造血细胞恶性肿瘤所引起的实体瘤,其中包括绿色瘤、浆细胞瘤、蕈样肉芽肿病肿瘤和皮肤T细胞淋巴瘤/白血病,和包括霍奇金淋巴瘤和非霍奇金淋巴瘤在内的淋巴瘤。本发明中“癌症”和“肿瘤”也指多细胞肿瘤及癌细胞或癌前期细胞。在某些具体实施方案中,肿瘤为腺瘤和/或腺癌,在某些具体实施方案中,肿瘤为肺腺瘤和/或肺腺癌。
在某些具体实施方案中,本发明所公开的组合物包括药物可接受载体。任意适合的配方均可用于制备本发明所公开的组合物,用以向患者给药。在某些具体实施方案中,药物可接受载体在人体内具有药物可接受性。
例如,上述适宜的配方可以包括水溶或非水溶消毒注射液,该注射液可以包括抗氧化剂、缓冲剂、抑菌剂、杀菌性抗菌剂和溶质,且上述成分使得上述配方与受体体液等张;上述配方还可以包括水溶或非水溶消毒悬浮物,上述悬浮物可以含有悬浮剂和增稠剂。上述配方可为单剂量或多剂量包装,如密封安瓶和小瓶,可储存于冷冻或干冻(冻干)状态,且仅需加入消毒用液态载体,如使用前所需的注射用水。一些典型的成分为十二烷基硫酸钠(SDS),在某些具体实施方案中其浓度为0.1-10mg/ml,而在某些具体实施方案中,其浓度约为2.0mg/ml;典型成分还/或包括甘露醇或其他糖类,在某些具体实施方案中,其浓度为10-100mg/ml,而在某些具体实施方案中,其浓度约为30mg/ml,此外,上述典型成分还/或包括磷酸盐缓冲液(PBS)。
应注意的是,除上面特别提到的成分外,根据配方类型,本发明所公开的配方还可包括其他本领域常用制剂。如配方中可含有灭菌无热注射用水和无水溶液。
可以向需要的患者以任何方式给药本发明公开的组合物,有望产生并提高患者的免疫反应。给药本发明公开的组合物合适的方法包括但不限于静脉注射(i.v.)、腹腔注射(i.p.)、皮下注射(s.c.)、皮下注射(s.d.)、肌内注射(i.m.)和/或瘤体内注射及口服。
本发明公开的方法包括向需要的患者给药一定药物有效剂量的本发明公开的组合物。如上所定义的,“有效剂量”为足以产生可测定反应的组合物的量(如促进治疗患者细胞溶解反应和/或细胞霉素反应)。
实施例
实施例1:在简化的慢病毒载体改性的基质培养体系中,成人CD34+祖细胞中的早期T淋巴细胞得到快速生长
我们已经报道过慢病毒载体改性的可表达鼠δ型1配体(DL1)的鼠胎基质细胞(LmDL1)支持来源于人体脐带血、胚胎胸腺、胚胎肝脏和成人骨髓中的CD34+HPC的早期T细胞分化(16)。为开发具有稳定细胞因子环境且不受外部添加的生长因子影响的培养体系,我们采用表达人体Flt3L或Flt3L和IL-7的慢病毒载体,对LmDL1进行进一步转导,分别合成LmDL1-FL和LmDL1-FL7细胞系(图1A)。LmDL1-FL7所引发的IL-7分泌采用ELISA进行测量,培养48小时后,其分泌值应在10-14ng/mL范围内(图1B)。三个慢病毒载体转导的细胞系(LmDL1、LmDL1-FL和LmDL1-FL7)的表面DL1表达显著高于OP9上内源水平的表达,如流式细胞仪所示(图1C)。使用抗-Flt3-L抗体,高表面表达Flt3L也可显示于LmDL1-FL和LmDL1-FL7细胞系(图1D)。
使用培养于含有人体重组IL-7和Flt3-L细胞,或不含生长因子补充物的LmDL1细胞上的高纯(>97%)成人CD34+BM细胞对T细胞生长进行图示说明(图2)。LmDL1-FL7培养中的T细胞生长与具有稍高含量CD8表达的LmDL1培养中的T细胞生长相同(图2A)。CD3和TCRαβ表达在上述两种培养体系中略有不同(图2B)。两种体系均支持成熟BM CD34+细胞在50~60天内生长为CD3-TCRαβ-SP CD8+T细胞(图2)。然而,我们注意到与LmDL1体系相比,LmDL1-FL7系统中的pre-T细胞保持5倍增长(图2C)。因此,LmDL1-FL7细胞系在不改变T细胞分化能力的前提下,支持T细胞前体的生长。
对于本领域技术人员,可采用其他方法转录细胞,对IL-7进行表达,上述方法包括但不限于其他病毒载体,上述病毒载体包括但并不局限于腺病毒、逆转录病毒或AAV病毒或裸DNA。此外,除胚胎基质细胞外,其他类型细胞可用于表达共培养用IL-7。可替代的,IL-7还可以手动方式加入到培养介质中作用于目标细胞。
实施例2:LmDL1-FL7细胞系不支持BM CD34HPC分化为成熟T细胞
从双阴性(DN)到DP阶段和CD4及CD8细胞系的不同T细胞转换需要Notch信号和pre-TCR信号(22,23)。DP T细胞的存活完全依靠TCR下游信号;此阶段中,上述细胞对细胞因子引发的生存信号无响应(24,25)。我们观察到T细胞前体表达CD3,但是上述细胞前体在IL-7、Flt3L和Notch信号共培养中仅存活约40天(图2C)。为了弄清这些生长中的T细胞是否可以成为成熟的SP T细胞,我们在第42天采用抗-CD3/抗-CD28微珠对这些T细胞施加TCR信号(图2D)。CD3/CD28刺激后,细胞在表面表达较低水平的CD8。由于成熟T细胞表达CD3、TCR αβ和共刺激分子CD28,而缺少CD1a(26),我们在生长CD8SP细胞上对这些标记物进行了测试。抗体染色结果显示低水平CD3、CD28、低于检测量的TCR αβ和大量的CD1a(图2D),上述结果意味着这些CD8SP细胞并未完全成熟。培养细胞并未表现出成熟迹象,且增殖相关抗原Ki67的核染色实验表明上述细胞对TCR信号无响应(图2D)。共培养50天和60天的刺激细胞液表现出相同的结果(数据未给出)。简而言之,上述结果表明采用LmDL1-FL7细胞培养的人体BM HPC无法发育成功能性CD8或CD4单阳T细胞。
实施例3:移除IL-7后,pre-T到DP T细胞的分化加速
上述结果表明LmDL1-FL7培养体系不支持ISP到DP T细胞和全成熟T细胞的分化。共培养体系中,只有少量CD3+T细胞共表达为少量的TCR αβ,这意味着TCR重排或处理不当(图2B)。由于干扰发育成成熟CD4CD8DP阶段所需的转录因子,鼠的pre-T淋巴细胞的进一步分化需要IL-7受体信号的减量调节(27-30)。尽管IL-7信号在DP T细胞中被堵塞,上述细胞与极低限度的IL-7生成细胞共存于胸腺内(31)。我们假设出现ISP细胞后,通过移除IL-7可促进人体内大部分T细胞分化到DP阶段。为验证上述假设,我们将成人BM CD34+细胞在LmDL1-FL7中培养24天,并将上述细胞转移至不含IL-7的LmDL1-FL中(图3A)。移除IL-7后,我们在第30天观察到快速转化到DP阶段的现象(图2A对比3B)。上述转化随供体不同而变化,在某些供体存在下,细胞在第35天转化为DP。随着DP细胞的出现,观察到CD3共表达和大量TCR αβ,这说明这些细胞在移除IL-7后发生正选择。有趣的是,此方式的进一步分化导致增殖抑制和细胞死亡(图3A)。
实施例4:CD4T细胞谱系提交可通过对IL-7抑制分化T细胞进行TCR刺激而获得
T细胞谱系提交需要细胞因子和共受体信号(24)。我们假设IL-7抑制DP T细胞发出TCR信号时进行谱系提交。当在30-42天(供体差异)内检测到CD3和TCR αβ共表达时,我们采用抗-CD3/抗-CD28微珠对IL-7抑制T细胞前体进行刺激。TCR信号后,Ki67核染色显示T细胞激增(图3C)。此外,T细胞分化和成熟标记物,包括CD3、CD28和TCR αβ,但不包括CD1a,表明T细胞分化已超出ISP阶段(图3C,与相同刺激的PBMC进行比较)。因此,IL-7的持续出现抑制了T细胞进一步分化到ISP阶段以外,并破坏了发育成人T细胞的功能性成熟。此外,上述体内衍生成熟T细胞绝大部分均为CD4T细胞。由于发育成CD8+T细胞需要IL-7信号,因此IL-7的移除可能抑制细胞分化为CD4+T细胞中间体。由于延长的TCR信号(或更好强度和持续时间)可阻止共受体反转为CD8+SP,因此随后的TCR信号可能促进CD4+CD8-胸腺细胞中间体转为CD4+T细胞(20,32)。
实施例5:CD4T细胞在改性体内培养体系中的功能性发育
为了考察体内衍生CD4+T细胞是否具有效应T细胞功能,我们采用PMA和伊屋诺霉素对CD3/CD28活化42天。6-8h后,通过细胞内和表面染色,对效应细胞因子IFN-γ、IL-17和IL-4的分泌进行分析;除此之外,我们还对T调节细胞相关的CD25和FoxP3表达进行评估。如两个不同供体所示,体内衍生CD4+T淋巴细胞可分泌IFN-γ、IL-17和IL-4,并与控制PBMC衍生CD4T细胞或纯化CD4T细胞原体克隆相比,可表达表面CD25和低量细胞内FoxP3(图4A)。上述结果表明即使在缺少极化培养条件下,这些细胞也可受内在程序作用分化成不同CD4效应T细胞子类型(33)。
实施例6:体内产生的CD4SP T细胞的V β谱系变窄且扭曲
为了评估体内衍生T淋巴细胞的TCR多样性,使用Beta MarkTCR V β谱系设备对23个Vβ谱系进行了Vβ谱系分析。采用Abs的板对第42天发育成CD4+SP T细胞的T细胞进行染色。体内衍生CD4+T细胞表现出较窄的V β使用量,并向V β谱系方向扭曲(图4B)。如供体1表现为Vb5.1、Vb7.1、Vb13.1和Vb18的中等扭曲使用(>10%);供体2表现出Vb2(15%)和Vb5.2(29%)的扭曲使用;供体3表现出Vb7.2(29%)和Vb4(44%)的高扭曲使用。上述数据表明体内衍生T淋巴细胞的Vβ谱系较正常成人PBMC更为受到约束。
有关实施例1-6的讨论
不局限于任何原理、机理或定理,本发明的发明人对以上实施例1-6所涉及结果提出下列讨论:
OP9-DL1培养体系支持脐带血和胚胎肝HPC发育为早期T细胞,但并不支持由成人HPC发育成成熟T细胞(8-10,13,34)。大量研究表明OP9-DL1体系只支持早期T细胞分化成双阳(DP)阶段,但尚无超出DP阶段后这些T细胞的具体表征和功能性分析数据(10,13)。尽管OP9-DL1培养体系极大的促进了人体T细胞发育研究,但从成人HPC体内合成大量成熟T细胞仍然是个难题(35)。本发明的发明人公开了一种改性基质培养体系LmDL1-FL7,该体系无需外源细胞因子,支持成人CD34+HPC中的早期T细胞生长。然而LmDL1-FL7细胞系自身并不支持CD34+HPC中T细胞的完整生长;分化T细胞在免疫单阳(ISP)CD8T细胞阶段被抑制。如图5所述,通过进一步对DN到DP和SP T细胞生长阶段进行共培养条件改进,使得上述问题得以解决。
已知文献均无法从成人CD34+HPC中得出全成熟MHC II级限制CD4SP T细胞发育体系(10,15,35-38)。本发明的培养体系支持成人体内CD34+HPC中CD4T细胞的分化和成熟。IL-7-抑制DP T细胞的CD3/CD28刺激促进CD34+HPC完全分化为CD4T细胞。活化时,这些体内发育的CD4T细胞分泌IFN-γ、IL-7、IL-4,表达CD25和FoxP3,并表现为成熟且具有功能性的T细胞。更为重要的是,体内发育的T细胞的功能性反应与鼠和人体内携带次形态RAG突变的非常规未调节CD4T细胞不同,上述细胞在DN3阶段受到抑制,经非常规活化,且对CD3无响应(39-41)。
较前针对鼠类的研究表明DN3阶段后发育T淋巴细胞中的IL-7受体信号的减量调节需要pro-T充分分化为DP T淋巴细胞(27,28,30,42,43)。由于IL-7的连续性信号,成人HPC中CD8+ISP T淋巴细胞在LmDL1-FL7共培养中的聚集最可能影响DP阶段前的分化区域,这主要是因为上述细胞在T细胞分化早期阶段保留转录因子PU.1表达(图S1A)。IL-7有助于鼠体内T细胞生存及体内扩展,但在T细胞发育过程中也会阻碍ISP进一步发育为DP T淋巴细胞(27-29,42,44)。IL-7R信号可以抑制鼠体内对ISP到DP转录至关重要的转录因子的表达,上述转录因子包括转录因子-1(TCF-1)、淋巴增强结合因子1(LEF1)和孤激素受体RORγt(28)。本发明的研究结果表明由于影响ISP到DP间的转录,IL-7R信号在人体T细胞发育过程中的作用与鼠类相似(27-29,42,44,45)。表现为IL-7并非完全阻碍发育T细胞向DP阶段的转录,但却使非正常分化的DP T细胞无法响应TCR刺激,进而无法具备功能性。因此还需要对IL-7对DP T细胞的功能性和成熟性的作用做进一步的研究。
本发明所述具体实施方案中,本发明的发明人通过CD8T细胞获得成熟CD4T细胞。OP9基质细胞不对I级或II级人体白细胞抗原(HLA)进行表达,人体胸腺细胞可能为成熟DP T细胞提供足够的I级或II级HLA接触,并诱发正选择(图S 1B)(46,47)。事实上,II级MHC分子在人体DP T细胞上的表达对其自身正选择至关重要(48)。向CD4T细胞的谱系提交可用动态信号模型进行解释,上述模型认为在接受到正选择TCR信号和持续的TCR刺激后,DP T细胞采用CD4T细胞路径,而当TCR信号停止后,DP细胞采用CD8T细胞路径(20,24)。在某些体系的具体实施方案中,本发明的发明人通过抗-CD3/CD28抗体,向IL-7抑制分化T细胞前体提供长期TCR信号,上述可用于解释CD4谱系选择。
与实施例1-6相关的材料和方法
人体CD34+细胞和细胞系。成人骨髓或从健康供体获得的动员外周血CD34+造血干细胞(HPC)和脐带血CD34+细胞购自AllCell公司(美国加州圣马特奥)或Cambrex(马里兰州,沃克斯维尔)。鼠胚胎基质细胞(OP9)购自American Type Culture Collection(ATCC,弗吉尼亚州马纳萨斯)。工程LmDL1和LmDL1-FL7细胞系由带有慢病毒载体的转染细胞制得,上述慢细胞病毒可分别解码鼠类δ型1(DL1)、DL1、人体Fl t3L和人体IL-7。基质细胞保留在α-MEM内(Invitrogen/Gibco BRL,纽约格兰德埃兰),并带有20%的胎牛血清(FBS,Invitrogen/Gibco BRL)和1%的青霉素-链霉素(Mediatech公司,弗吉尼亚州马纳萨斯)。采用人体IL-7ELISA包对IL-7细胞因子分泌进行测定。在含有1ml介质(Ray Biotech公司)的12孔板上培养48h后的LmDL1和LmDLFL7细胞中(80-90%合流)得到不含细胞的上清液。测试结果由680型测微读数器(Bio-Rad)上读取。DL1和Flt3L的表面表达采用流式细胞仪进行分析,根据使用手册说明(Invitrogen),上述分析中施加Alexa Fluor 647-共轭抗-DL1Ab(Biolegend)和与zenon-alexa 488共轭的纯化抗-Flt3L Ab(Abcam公司,马萨诸塞州剑桥)。
LmDL1基质细胞-CD34+HPC共培养。CD34+HPC放入含有1x105细胞/孔的24孔板中,上述孔板中含有LmDL1或LmDL1-FL7细胞的合流单层。共培养自第一天起保存于完整介质中,上述介质为含有20%FBS和1%青霉素-链霉素的α-MEM,此外还含有5ng/ml IL-7(PeproTech公司,新泽西洛基山)和5ng/ml Flt3L(PeproTech公司)。每隔2-3天对共培养进行补给。当单层开始分化或当发育细胞达到80-90%合流时,将悬浮液中的细胞转移到新的合流基质单层上。采用移液的方式转移细胞,随后采用70μm滤纸(BD/Falcon,BD Biosciences,马里兰州斯帕克斯)进行过滤,并对所得的250g悬浮液在室温下离心10分钟。细胞束转移至新鲜合流单层上。T细胞发育指定时间点上收集上述细胞,以备分析。
单株抗体和流式细胞仪。表面染色用抗体包括CD4(克隆RPA-T4、PE、FITC、PE-Cy7和太平洋蓝)、CD8(克隆RPA-T8PE、FITC、PE-Cy7和太平洋蓝)、CD3(克隆SK7、PE-Cy7)、TCR αβ(克隆T10B9.1A-31、FITC),上述抗体购自BD biosciences,加州圣荷西。细胞先用PBS和2%FBS清洗,并在4℃下用鼠和人体血清堵塞30min。对抗体染色而言,根据使用说明采用相应的抗体进行孵化。每种荧光染料标记的Ab均含有一定量的同型对照。抗体染色后,清洗细胞两次,并注入2%多聚甲醛。使用BDFACSAria中的BD FACS Diva软件(5.0.1版本)进行处理获取数据,并采用Flowjo软件(7.1.3.0版本,Tree Star公司,德克萨斯州帕萨迪纳)进行数据分析。
抗-CD3/CD28微珠引发的T细胞刺激。为了刺激未成熟的T细胞,根据使用指南,采用抗-CD3/CD28微珠(Dynal/Invitrogen,加州圣地亚哥)进行长期刺激。细胞与微珠混合,并于37℃下,在含有X-vivo 20(BioWhittaker,Cambrex,马里兰州沃克斯维尔)介质的96孔板中放置2-3天,第3天,加入12.5UIL-2,5ng/mlIL-7和20ng/ml IL-15,细胞在上述环境下继续培养11-12天。表面染色采用如上所述方法,其中抗体CD4(克隆RPA-T4、PE、FITC、PE-Cy7和太平洋蓝)、CD8(克隆RPA-T8PE、FITC、PE-Cy7和太平洋蓝)、CD3(克隆SK7、PE-Cy7)、TCRαβ(克隆T10B9.1A-31、FITC)、CD1a(克隆HI149、APC)购自BD biosciences。CD28(克隆CD28.2、APC)购自eBioscience公司(加州圣地亚哥)。采用BD Biosciences公司的抗-Ki67(克隆B56、FITC)和同型IgG1κ进行细胞内染色。采用抗-Ki67FITC和同型IgG1κ(BD Biosciences)进行细胞内染色。根据使用说明,采用BD cytofix/cytoperm设备进行细胞内染色。
体内产生的CD4+T细胞的效应功能分析。采用PMA和伊屋诺霉素(Sigma-Aldrich,密苏里州圣路易斯)对含有CD3/CD28的CD4T细胞进行刺激,并分析IFN-γ、IL-4和IL-17的释放量。细胞在25ng/ml PMA和1μg/ml伊屋诺霉素中孵化1h,随后加入6μg/ml莫能菌素(Sigma-Aldrich)以抑制药用Golgi的细胞因子的分泌。孵化4-5h后,制得所需细胞,表面染色所用CD4(克隆RPA-T4、太平洋蓝)、CD8(克隆SK1、APC-Cy7)、CD3(克隆SK7PE-Cy7)、CD25(克隆M-A251、PE)和细胞内染色所需的IFN-γ-(克隆25723.11、FITC)、IL-4-(克隆MP425D2、APC)、FOXP3(克隆PCH101、Alexa 647)购自BD Biosciences,IL-17(克隆64CAP17、PE)购自e-Biosciences。使用BD FACSAria软件对流式细胞仪所得数据进行收集,并采用Flowjo进行数据分析。
体内衍生CD4+T细胞的V β谱系分析。采用Beta Mark TCRVβ谱系设备(Beckman Coulter,加州富勒顿)对体内发育T淋巴细胞的Vβ谱系进行分析。并根据使用指南进行24Vβ族染色。
与补充图表相关的材料和方法。
抗体
本发明所使用的抗体包括Clatag公司I级HLA(克隆TU149,PE),BDbiosciences HLA DR DQ DP(克隆TU39,FITC)。
RT-PCR
RNA来自于CD8、CD4单细胞克隆、体内发育DN+CD8、使用TRI试剂的体内发育CD4T细胞(Sigma-Aldrich)。使用两步AMV RT-PCR设备(Gene choice,马里兰州)将lug RNA反向转录在cDNA内。PCR反应中使用下列引物:GAPDH-F-5’CCG ATG GCA AAT TCG ATG GC 3’和R-5’GAT GACCCT TTT GGC TCC CC 3’、PU.1F-5’TGG AAG GGT TTC CCC TCG TC 3’和R-5’TGC TGT CCT TCA TGT CGC CG 3’、CD3e F-5’TGA AGC ATCATC AGT AGT CAC AC 3’和R-5’GGC CTC TGT CAA CAT TTA CC 3’、GATA-3F-5’GAC GAG AAA GAG TGC CTC AAG 3’和R-5’TCC AGA GTGTGG TTG TGG TG 3’。30次循环放大后(95℃进行30s,55℃进行30s,72℃进行60s),PCR产品在2%的琼脂糖凝胶上进行分离。
参考文献
这里所引用的所有参考文献包括相关的申请全部并入本发明,且与本发明的教导无冲突。
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上述具体实施方式和构成并非是完全和无一遗漏的。也可使用其他具体实施方案,单独使用或与以上所述或以下所述的一个或多个特征结合使用。除此之外,本发明所涉及的各种具体实施方案中包括如这里所述的组分、方法、工艺、体系和/或设备等,含有各种具体实施例、次级组合和子集等。本领域技术人员在了解本发明所公开的内容后可掌握怎样使用和制备本发明。本发明,在各种实施方案中,包括提供装置和工艺,尽管有一些项在这里没有提到和/或没有被描述;或者在各种实施方案中包括这些缺少的这些项,因为它们在以前的装置或过程中已经被使用,例如改善性能、提高容易度和/或降低实施成本等等。
此外,本发明包括了一个或多个具体实施方式及某些变化和改进的描述,但其他变化和改进也在本发明的范围内,如在了解本发明公开的内容后,本领域技术人员对本发明所做出的改进和变化。本发明旨在允许的程度内对包括可替代的实施方案在内享有专利权,包括改变、互换和/或等同结构、功能、范围或步骤,且不论上述改变、互换和/或等同结构、功能、范围或步骤是否在这里公开,本发明均对其享有专利权,且本发明无意涉及其他专利保护内容。
Claims (14)
1.一种从造血干细胞(HSCs)合成全成熟的功能性CD4T细胞的方法,其中所述方法包括在培养条件下培养造血干细胞,将所述造血干细胞直接培养成功能性CD4T细胞,所述培养条件包括:
将造血干细胞在IL-7中至少培养2周,以及
在约2-4周内的某个时间点终止所述干细胞在IL-7中的培养。
2.根据权利要求1所述的方法,其中所述方法包括在约3-4周内的某个时间点终止所述干细胞在IL-7中的培养。
3.根据权利要求1所述的方法,其中所述方法包括在20-28天内的某个时间点终止所述干细胞在IL-7中的培养。
4.根据权利要求1所述的方法,其中所述培养包括共同培养所述造血干细胞和改性胚胎基质细胞,所述胚胎基质细胞用于表达δ型1配体和IL-7和/或Flt3l。
5.根据权利要求4所述的方法,其中所述改性胚胎基质细胞为哺乳动物细胞。
6.根据权利要求5所述的方法,其中所述改性胚胎基质细胞来自于鼠、大鼠、兔或豚鼠。
7.根据权利要求4所述的方法,其中所述改性胚胎基质细胞经含可解码IL-7的多核苷酸载体或与所述IL-7至少有95%相似性的多肽分子所转染。
8.根据权利要求7所述的方法,其中所述载体为病毒载体。
9.根据权利要求8所述的方法,其中所述载体为慢病毒载体。
10.一种药物组合物,含有培养并产生于成人骨髓的功能性CD4T细胞和药学可接受载体,赋形剂或稀释剂。
11.一种治疗癌症的方法,通过向需要的患者给药治疗有效量的如权利要求10所述的药物组合物来治疗癌症。
12.根据权利要求10所述的方法,其中所述癌症为黑色素瘤或白血病。
13.用于表达δ型1配体和IL-7和/或Flt3l的改性胚胎基质细胞的隔离细胞样品。
14.根据权利要求13所述隔离细胞样品,其中所述细胞为鼠、大鼠、兔或豚鼠细胞。
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CN104789529A (zh) * | 2015-04-28 | 2015-07-22 | 济南劲牛生物科技有限公司 | 促进小鼠骨髓造血干细胞体外克隆形成及分化能力的方法 |
CN112795539A (zh) * | 2020-12-31 | 2021-05-14 | 中山大学 | 一种细胞流式分析干细胞细胞因子的方法 |
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EP3783098A1 (en) | 2013-05-14 | 2021-02-24 | Board Of Regents, The University Of Texas System | Human application of engineered chimeric antigen receptor (car) t-cells |
WO2014190273A1 (en) | 2013-05-24 | 2014-11-27 | Board Of Regents, The University Of Texas System | Chimeric antigen receptor-targeting monoclonal antibodies |
KR20160068960A (ko) | 2013-10-25 | 2016-06-15 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | 면역요법을 위한 다클론성 감마 델타 t 세포 |
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JP6983771B2 (ja) * | 2015-10-30 | 2021-12-17 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニアThe Regents Of The University Of California | 幹細胞からt細胞を作製する方法および該t細胞を用いた免疫療法的方法 |
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CN104789529A (zh) * | 2015-04-28 | 2015-07-22 | 济南劲牛生物科技有限公司 | 促进小鼠骨髓造血干细胞体外克隆形成及分化能力的方法 |
CN112795539A (zh) * | 2020-12-31 | 2021-05-14 | 中山大学 | 一种细胞流式分析干细胞细胞因子的方法 |
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