CN106795496B - 一种获得t细胞的方法及应用 - Google Patents
一种获得t细胞的方法及应用 Download PDFInfo
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Abstract
本发明涉及一种使用转录因子Hoxb5诱导B淋系细胞转分化为T淋系细胞的方法、其相关产品及应用。本发明的方法具体包括：将Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体引入至所述B淋系细胞，获得Hoxb5过表达的B淋系细胞；然后将所获得的B淋系细胞植入受试者体内，转分化方式获得再生T细胞祖细胞，T细胞祖细胞分化获得成熟具有功能的T淋系。使用本发明的方法所获得的再生T细胞不仅功能正常，而且没有致瘤风险或者致瘤风险极低。
Description
技术领域
本发明涉及医药生物工程技术领域，具体涉及一种使用Hoxb5诱导B细胞转分化为T细胞的方法、其相关产品及应用。
背景技术
T细胞对于免疫系统不可或缺。目前，国内外已有的基础研究表明，利用Pax5缺失(Pax5-/-)可以将小鼠成熟B细胞转分化为T细胞。Ebf1和Pax5复合型杂合子小鼠(Ebf1+/-Pax5+/-)的B细胞也能转分化为T细胞。此外，通过逆分化途径将B祖细胞返祖至多能祖细胞阶段，可以再次分化得到T细胞。然而，以上途径得到的T细胞功能均不健全，甚至会克隆化产生淋巴瘤。归根结底，以上研究采用的关键基因大多为造血谱系命决定关键因子(lineage master regulators，缺失或过表达这些因子均会导致再生T细胞的功能异常或癌变。因此，在造血干祖细胞中寻找新的谱系潜力决定性因子，从细胞表观遗传组学上彻底改变靶向细胞以实现转分化T细胞获得全部的T细胞潜力。只有完全转分化的再生T祖细胞才能再次循着类似生理T细胞的发育轨迹，在体内分化成熟为功能性T细胞以及尽量避免致瘤风险。
发明内容
发明目的
本发明的目的在于提供一种使用转录因子Hoxb5诱导B淋系细胞转分化为T淋系细胞的方法、其相关产品及应用。
本发明的发明人通过RNA-Seq及生物信息学技术，深度解析小鼠造血干祖细胞及成熟血细胞的转录表达谱，筛选出候选转录因子——Hoxb5；然后，通过一系列生物实验发现，该转录因子不仅可使B淋系细胞成功转分化为功能性T细胞，还可避免再生T细胞的致瘤风险。
技术方案
为实现上述目的，一方面，本发明提供了Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体在制备(i)用于将B淋系细胞转分化为功能性T细胞的制剂，(ii)用于增强免疫响应、优选地增强T细胞相关的免疫响应的药物，和/或(iii)用于预防或治疗免疫缺陷、优选地用于预防或治疗T细胞免疫缺陷的药物中的用途。
上述用途中，作为优选，所述B淋系细胞为祖B细胞或前B细胞。
第二方面，本发明提供了一种转化的B淋系细胞，所述B淋系细胞中引入了Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体以过表达Hoxb5，并且所述B淋系细胞具有转分化为T细胞潜能。
作为优选，所述B淋系细胞为祖B细胞(Pro-B)或前B细胞(Pre-B)。
第三方面，本发明提供了如第二方面所述的转化的B淋系细胞在制备(i)用于再生T细胞的药物，(ii)用于增强免疫响应、优选地增强T细胞相关的免疫响应，和/或(iii)用于预防或治疗免疫缺陷、优选地用于预防或治疗T细胞免疫缺陷的药物中的用途。
第四方面，本发明提供了一种药物组合物，其包括作为活性成分的、如第二方面所述的转化的B淋系细胞，以及药学上可接受的载体、赋形剂或稀释剂。
第五方面，本发明提供了一种用于将B淋系细胞转分化为功能性T细胞的方法，其包括：
(1)将Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体引入至所述B淋系细胞，获得Hoxb5过表达的B淋系细胞；
(2)将步骤(1)获得的B淋系细胞植入受试者体内，以诱导转分化获得T细胞祖细胞，再经分化获得功能性T细胞。
上述方法中，作为优选，所述B淋系细胞为祖B细胞或前B细胞。
作为优选，在步骤(1)中，所述Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体携带追踪剂，优选荧光蛋白追踪剂，更优选EGFP荧光蛋白追踪剂。
作为优选，在步骤(1)中，通过转染或病毒感染、优选地通过逆转录病毒感染，将编码Hoxb5的核酸分子或包含所述核酸分子的构建体引入B淋系细胞。
在一个具体的实施方案中，发明人首先构建了Hoxb5逆转录病毒表达载体，具体地，通过设计酶切位点，把Hoxb5基因重组到逆转录表达载体(例如，pMYs-IRES-EGFP)；利用逆转录病毒包装体系，包被出含有Hoxb5基因的高滴度逆转录病毒；接着，借助逆转录病毒整合功能将Hoxb5基因在小鼠pro-/pre-B细胞内实现过表达；然后，通过眼静脉移植，将转导Hoxb5的Pro-/Pre-B细胞移植到清髓后的受体鼠；移植4周后，借助流式细胞术，Southernblot，RNA-Seq测序及生物信息学分析，鉴定再生T细胞的来源和身份，结果表明：再生T细胞确实来自Pro-/Pre-B细胞的转分化；进一步结合分析再生T细胞的免疫表型、在淋巴组织的分布、TCR受体重排及体外抗体刺激增殖实验，证明再生T细胞的功能正常；此外，连续追踪移植受体鼠健康状态，特别是T淋系造血，评估再生T细胞的致瘤风险，结果表明：使用本发明的方法所获得的再生T细胞没有致瘤风险或者致瘤风险极低。
附图说明
图1显示：(A)再生T细胞的实验设计流程图；(B)荧光EGFP检测显示Hoxb5重组质粒转染至逆转录病毒包装plat-E细胞的效率超过85％(转染后48小时)；(C)流式检测分析显示Hoxb5逆转录病毒感染Pro-/Pre-B细胞转导效率超过50％(感染后48小时)。
图2显示：(A)流式细胞术分析移植后第2至第4周受体鼠胸腺内再生T细胞的比例及构成。移植第4周时，流式细胞术分析受体鼠外周血(B)、脾脏(C)、淋巴结(D)中供体来源细胞(GFP+)的构成、再生T细胞的分类。其中，右侧为3次独立重复实验的统计图。(E)Northern blot检测单个再生T细胞中的B细胞特征VDJ重排。
图3显示：(A)细胞转录组学的聚类分析受体鼠胸腺内各类再生T细胞与野生型T细胞(B)B细胞Ig重链VDJ重排PCR检测单个再生T细胞中不同位点的TCR-β链重排(C)抗CD3和抗CD28抗体体外刺激再生T细胞增殖的代表性形态图。其中，WT为野生型对照。(D)ELISA法检测体外刺激增殖后的再生T细胞分泌到培养基上清中的代表性细胞因子。
图4显示：(A)Hoxb5敲进模型小鼠(LSL-Hoxb5)在ROSA26位点打靶构建示意图。(B)动态分析4周龄、8周龄、12周龄的CD19-Cre LSL-Hoxb5复合模型小鼠胸腺中再生T细胞(GFP+)的比例及构成。(C)B细胞Ig重链VDJ重排PCR分析单个和10个再生T细胞的不同重链VDJ重排位点(VHJ558、VHQ52、VHGAM3、DHQ52)，证实再生T细胞来源于B细胞。
具体实施方式
为便于理解本发明，本发明列举实施例如下。本领域技术人员应该明了，所述实施例仅仅是帮助理解本发明，不应视为对本发明的具体限制。
实施例1
首先，设计体内Hoxb5转分化B细胞产生再生T整个实验流程图(图1A)。接着，通过Lasergene软件分析Hoxb5基因的酶切位点信息，选取XhoI/SnaBI作为上下游重组酶切位点，将Hoxb5基因重组构建到逆转录病毒载体(pMYs-IRES-EGFP)。DH5α感受态转化连接产物后，利用氨苄平板筛选阳性重组克隆。进一步结合菌液PCR及提取重组质粒进行DNA测序，确定重组成功的pMY-Hoxb5-IRES-EGFP逆转录病毒质粒进行去内毒素大提后备用。随后，采用磷酸钙转染法把Hoxb5重组载体转入逆转录病毒包装细胞系(Plat-E细胞)。24小时后，借助荧光显微镜查看转染率，确保转染率≥85％(图1B)。48h后，收集含有Hoxb5的逆转录病毒上清备用。同时，牺牲4-6周小鼠，取出小鼠骨髓并制备成单细胞悬液。接着，借助磁珠富集法，将骨髓单细胞悬液中的B220+细胞富集出来。通过Pro-/Pre-B抗体组合(CD19/B220/CD93/IgM)染色后，用超高速流式分选仪(Moflo Astrios)将Pro-/Pre-B细胞(CD 19+B220+CD93+IgM-)分选出来。500g低温离心后收集分选的Pro-/Pre-B细胞。随后，把富集的Pro-/Pre-B细胞放入B淋系细胞完全培养基中预刺激培养12-14小时。将逆转录病毒按1∶1体积加入opti-MEM基础培养基，同时加入8μg/ml polybrene混匀好后备用。通过350g离心5分钟收集预刺激后的Pro-/Pre-B细胞。随后，用重新配置好的逆转录病毒液重悬Pro-/Pre-B细胞后置于低粘附6孔板，在35℃恒温水平离心机中805g离心感染90分钟。离心结束后，将6孔培养板放回细胞培养箱(37℃；5％CO2)中静置培养2小时。随后，350g离心收集Pro-/Pre-B细胞。用预热后的完全培养基重悬培养Pro-/Pre-B细胞，保持密度在每毫升2-4百万细胞的密度。24小时后，重复一次离心感染。第二次感染24小时后，取少量细胞用台盼蓝计数及流式仪分析Pro-/Pre-B细胞感染率。结果表明：无论对照组，还是Hoxb5组的感染率都超过50％(图1C)。随后，350g离心收集悬浮细胞，按每只受体鼠移植6-8百万的活细胞量进行移植，受体鼠提前4小时进行亚致死剂量(6.5Gy)的辐照处理。同时，在小鼠的饲养水中添加1.14g/L的新霉素硫酸盐预防辐照后小鼠肠道感染。
移植2-4周后，牺牲移植受体鼠，分析胸腺，脾脏，淋巴结、外周血的T细胞生成。借助T淋巴细胞表面抗原CD3和内源EGFP荧光蛋白追踪再生T淋巴细胞。结果发现：在移植2周后，即可在受体鼠胸腺发现高达10％的EGFP和CD3双阳性的T细胞：进一步分析证实，这群T细胞中包含：CD4单阳性、CD8单阳性、CD4CD8双阳性(Double positive，DP)和CD4CD8双阴性(Double negative，DN)T细胞(图2A)。
此外，通过对DN细胞的进一步分析，其可分类为似生理状态比例下的四群细胞：DN1细胞(CD44+CD25-)、DN2细胞(CD44+CD25+)、DN3细胞(CD44-CD25+)、DN4细胞(CD44-CD25-)(图2A)。
接着，对受体鼠胸腺细胞的连续分析表明：随着时间的推移，受体鼠胸腺中CD3+EGFP+的再生T细胞比例在逐步增高。在移植4周，超过80％的胸腺细胞都为再生T细胞(图2A)。
此外，对受体鼠外周血、脾脏、淋巴结的再生T细胞进行分析，结果表明：在外周血中再生T细胞群体中能够检测到CD4单阳性辅助T(Th)细胞、CD8单阳性细胞毒性T细胞及表达T细胞受体(TCR)β链(图2B)。在受体鼠脾脏内能够检测到Foxp3+CD4+的调节性T细胞(图2C)。而在淋巴结中能够检测到TCR-γδ阳性T细胞(图2D)。
为了进一步确定再生T细胞(CD3+EGFP+)是否从Pro-/Pre-B细胞来源，借助分析B细胞Ig重链VDJ及轻链(κ、λ)重排加以鉴定。通过流式分选单个CD3+EGFP+细胞进行PCR检测。随后，将PCR片段回收连接到T载体进行测序分析。结果显示：不同的单个CD3+EGFP+的T细胞具有B细胞Ig重链VDJ及轻链(κ、λ)重排，表明所述T细胞是由B细胞转分化而来的(图2E)。
此外，发明人分选了移植4周后受体鼠胸腺内再生T细胞(EGFP+)发育的7个细胞群体：DN1、DN2、DN3、DN4、DP、CD4+单阳性、CD8+单阳性细胞进行RNA-Seq测序分析。接着，生物信息学解析7群细胞的转录表达谱，并进行了树枝聚类分析。结果表明，再生的7群T细胞与野生型具有接近的转录表达谱的相对应T细胞群聚类到一起(图3A)。进一步借助流式细胞分选受体鼠脾脏单个成熟T细胞(CD4+/CD8+)进行TCR-β重排进行分析。结合PCR技术和T载体测序，实验结果表明：不同的再生T细胞具有不同的TCR-β重排(图3B)。并且，发明人将脾脏成熟的再生T细胞分选出来进行了体外刺激增殖实验，联合CD3和CD28抗体刺激培养6天，再生T细胞能够对刺激做出反应并大量增殖(图3C)。采用ELISA技术分析培养上清，结果表明：刺激增殖后的再生T细胞能够分泌大量的白介素2(IL-2)、白介素10(IL10)、γ干扰素(IFN-γ)、肿瘤坏死因子α(TNF-α)(图3D)；最后，连续追踪移植受体鼠的健康状态，尤其是T淋系造血，结果表明：小鼠体内再生T细胞没有致瘤风险，或致瘤风险极低。
实施例2
为了排除逆转录病毒整合位点不确定及表达水平不均一等潜在问题，发明人还构建了Hoxb5敲进动物模型(LSL-Hoxb5)(图4A)。通过Hoxb5敲进小鼠与B淋系特异性表达Cre模式小鼠CD19-Cre进行杂合，得到LSL-Hoxb5CD19-Cre(以下简称Hoxb5小鼠)。通过对Hoxb5小鼠的胸腺进行分析，发现其中有少量CD3+EGFP+的再生T细胞(图4B)。同样将再生T细胞分选出来进行B细胞Ig重链VDJ及轻链(κ、λ)重排加以鉴定(图4C)。结合T载体测序，结果表明：再生T细胞具有B细胞特征的重排，表明其源自B细胞的转分化。
申请人声明，本发明通过上述实施例来说明本发明的产品、方法及用途，但本发明并不局限于此，所属技术领域的技术人员应该明了，对本发明的任何改进，对本发明产品的等效替换及辅助成分的添加、具体方式的选择等，均落在本发明的保护范围和公开范围之内。
Claims (14)
1.Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体在制备(i)用于将B淋系细胞转分化为功能性T细胞的制剂，(ii)用于增强免疫响应的药物，和/或(iii)用于预防或治疗免疫缺陷的药物中的用途；
所述B淋系细胞为祖B细胞或前B细胞。
2.如权利要求1所述的用途，其特征在于，所述增强免疫响应为增强T细胞相关的免疫响应。
3.如权利要求1所述的用途，其特长在于，所述免疫缺陷为T细胞免疫缺陷。
4.一种转化的B淋系细胞，其特征在于，所述B淋系细胞中引入了Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体以过表达Hoxb5，并且所述B淋系细胞具有转分化为T细胞潜能；
所述B淋系细胞为祖B细胞或前B细胞。
5.如权利要求4所述的转化的B淋系细胞在制备(i)用于再生T细胞的药物，(ii)用于增强免疫响应，和/或(iii)用于预防或治疗免疫缺陷的药物中的用途。
6.如权利要求5所述的用途，其特征在于，所述增强免疫响应为增强T细胞相关的免疫响应。
7.如权利要求5所述的用途，其特征在于，所述免疫缺陷为T细胞免疫缺陷。
8.一种药物组合物，其包括作为活性成分的、如权利要求4所述的转化的B淋系细胞，以及药学上可接受的载体、赋形剂或稀释剂。
9.一种B淋系细胞转分化得到的功能性T细胞，其转分化方法包括：
(1)将Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体引入至所述B淋系细胞，获得Hoxb5过表达的B淋系细胞；
(2)将步骤(1)获得的B淋系细胞植入受试者体内，以诱导转分化获得T细胞祖细胞，再经分化获得功能性T细胞；
所述B淋系细胞为祖B细胞或前B细胞。
10.如权利要求9所述的功能性T细胞，其特征在于，在步骤(1)中，所述Hoxb5、编码Hoxb5的核酸分子或包含所述核酸分子的构建体携带追踪剂。
11.如权利要求10所述的功能性T细胞，其特征在于，所述追踪剂包括荧光蛋白追踪剂。
12.如权利要求11所述的功能性T细胞，其特征在于，所述荧光蛋白追踪剂包括EGFP荧光蛋白追踪剂。
13.如权利要求9-12任一项所述的功能性T细胞，其特征在于，在步骤(1)中，通过转染或病毒感染将编码Hoxb5的核酸分子或包含所述核酸分子的构建体引入B淋系细胞。
14.如权利要求13所述的功能性T细胞，其特征在于，所述病毒感染为逆转录病毒感染。
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