CN116262779A - 一种单细胞筛选鉴定人乳头瘤病毒特异性tcr的方法 - Google Patents

一种单细胞筛选鉴定人乳头瘤病毒特异性tcr的方法 Download PDF

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CN116262779A
CN116262779A CN202111524432.9A CN202111524432A CN116262779A CN 116262779 A CN116262779 A CN 116262779A CN 202111524432 A CN202111524432 A CN 202111524432A CN 116262779 A CN116262779 A CN 116262779A
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hpv
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王蒲
汪亚兰
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Shenzhen Institute of Advanced Technology of CAS
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Abstract

本发明涉及TCR‑T细胞免疫治疗，具体公开一种单细胞筛选鉴定人乳头瘤病毒特异性TCR的方法。该方法包括如下步骤：制备标记的MHC H‑2‑Db限制性HPV多肽四聚体；将待筛选鉴定的人乳头瘤病毒TCR细胞用抗CD3、抗CD8和标记的MHC H‑2‑Db限制性HPV多肽四聚体标记，通过流式细胞术筛选四聚体+/CD8+的TCR阳性单细胞；将筛选得到的四聚体+/CD8+的TCR阳性单细胞用细胞裂解液裂解后，以TCRα链和TCRβ链的引物进行PCR扩增，扩增产物进行基因测序。该方法可以实现特异性TCR的快速筛选鉴定，为HPV TCR‑T免疫治疗提供了快速鉴定TCR的方法。

Description

一种单细胞筛选鉴定人乳头瘤病毒特异性TCR的方法
技术领域
本发明属于肿瘤免疫治疗领域，涉及TCR-T细胞免疫治疗，具体而言，本发明涉及一种单细胞筛选鉴定人乳头瘤病毒特异性TCR的方法。
背景技术
人乳头瘤病毒(human papillomavirus,HPV)属乳头瘤病毒科乳头瘤病毒属，是一组嗜上皮组织双链小分子DNA病毒的总称。HPV感染人类上皮组织，能引起人类皮肤和黏膜的增生性病变，根据危险程度的不同，除导致多种乳头状瘤或疣以外，还与多种肿瘤的发生发展密切相关，如宫颈癌(cervical cancer)、膀胱癌、甲状腺癌、大肠癌、乳腺癌和皮肤癌等。在诸多HPV相关肿瘤中，宫颈癌是女性生殖系统中最常见的恶性肿瘤，发病率居全球女性肿瘤第二位，被称为“危害健康女性的杀手”，占女性癌症患者总数的15％，对女性健康威胁极大。99％以上的宫颈癌患者的癌组织中可以检测到HPV存在，高危型HPV的持续感染是宫颈癌最主要的发病因素。据WHO最新数据显示，全球每年因宫颈癌而死亡的人数达27.3万，新发病例数约53万；我国每年约有13.2万宫颈癌新发病例，且患者年轻化趋势越来越明显，在25-45岁的中国年轻女性中，宫颈癌死亡率高居所有疾病死亡率第二位。
宫颈癌发生与高危型人乳头状瘤病毒(HR-HPV)感染密切相关。HPV已成为宫颈癌免疫治疗的主要靶点。HPV的某些蛋白片段和肿瘤蛋白多肽会被展示在肿瘤细胞表面的MHC上，被T细胞受体(T cell receptor,TCR)识别，触发免疫系统对肿瘤的杀伤。但在肿瘤患者体内，这类能有效识别和杀伤HPV肿瘤细胞的T细胞数目往往很低，不足以发挥抗肿瘤的效果。因此，增强TCR对MHC呈递的病毒或肿瘤多肽的亲和力和增加这类T淋巴的数量，如TCR-T细胞，是抗肿瘤治疗的重要途径。TCR-T细胞治疗的原理是提取患者外周血中的T细胞，经基因工程改造和扩大培养，使T细胞表达高亲和力的特异性TCR，回输到患者体内，识别肿瘤细胞MHC多肽，从而杀伤癌细胞。基因改造的T细胞具有更为优良的靶向性、杀伤性和持久性，已很快成为细胞免疫治疗的最前沿技术。HPV-16E6/E7致瘤基因能在HPV阳性肿瘤细胞中持续表达并维持肿瘤细胞的生长，是T细胞最好的靶点。已有研究报道以HPVE6和E7病毒抗原为靶点，采用TCR-T治疗技术清除被HPV转化的宫颈癌细胞，显示出良好的治疗效果，但鉴定筛选TCR耗时。
发明内容
为了解决现有技术中的不足，本发明的目的在于提供一种单细胞筛选鉴定人乳头瘤病毒特异性TCR的方法。具体方案如下：
本发明提供一种单细胞筛选鉴定人乳头瘤病毒特异性TCR的方法，包括如下步骤：
制备标记的MHC H-2-Db限制性HPV多肽四聚体；
将待筛选鉴定的人乳头瘤病毒TCR细胞用抗CD3、抗CD8和标记的MHC H-2-Db限制性HPV多肽四聚体标记，通过流式细胞术筛选四聚体+/CD8+的TCR阳性单细胞；
将筛选得到的四聚体+/CD8+的TCR阳性单细胞用细胞裂解液裂解后，以TCRα链和TCRβ链的引物进行PCR扩增，扩增产物进行基因测序。
进一步地，所述HPV多肽为HPV-16 E6或HPV-16 E7。
进一步地，所述标记的MHC H-2-Db限制性HPV多肽四聚体为用荧光基团标记。
进一步地，所述待筛选鉴定的人乳头瘤病毒TCR细胞为：HPV表位肽免疫小鼠后采集脾脏制备的单细胞悬液。
进一步地，所述HPV表位肽为HPV-16 E6表位肽或HPV-16 E7表位肽。
本发明还提供所述方法获得的HPV表位特异性的TCR阳性单细胞。
本发明还提供一种人乳头瘤病毒特异性TCR-T细胞的制备方法，包括如下步骤，根据测序确定的人乳头瘤病毒多肽特异性TCR基因序列，合成TCR基因，将TCR基因克隆到逆转录病毒中，用逆转录病毒转导T细胞，即得到人乳头瘤病毒特异性TCR-T细胞。
本发明进一步提供一种所述制备方法制备得到的人乳头瘤病毒特异性TCR-T细胞。
本发明的有益效果为：
本发明提供的单细胞筛选鉴定人乳头瘤病毒特异性T细胞受体的方法，将待筛选鉴定的人乳头瘤病毒TCR细胞用抗CD3、抗CD8和标记的MHC H-2-Db限制性HPV多肽四聚体标记，通过流式细胞术筛选四聚体+/CD8+的TCR阳性单细胞，进一步以TCRα链和TCRβ链的引物进行PCR扩增，扩增产物进行基因测序，可以实现特异性TCR的快速筛选鉴定。该方法为HPVTCR-T免疫治疗提供了快速鉴定TCR的方法。
附图说明
图1为HPV-16E7/H-2-Db四聚体+/CD8+T细胞分选。
具体实施方式
为了更清楚地理解本发明，现参照下列实施例及附图进一步描述本发明。实施例仅用于解释而不以任何方式限制本发明。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
easYmer H2-Db MHC Tetramers Kit，Eagle Biosciences，AmberstNH，USA。
TC-1细胞系从ATCC购买。
实施例1
1.小鼠MHC H-2-Db限制性HPV-16E7多肽四聚体(HPV-16E7/H-2-Db四聚体)制备。使用easYmer H2-Db MHC Tetramers Kit(Eagle Biosciences，Amberst NH，USA)，easYmer简单地说，H-2-Db的细胞外结构域通过添加一段序列，用生物素标记。HPV-16E7/H-2-Db四聚体制备方法按照使用说明操作，具体步骤如下：
(1)HPV-16 E7多肽稀释为100μM
(2)制作90μL的H-2Db多肽单体复合物
Figure BDA0003409554910000031
(3)上述反应液装入PCR反应管，彻底混合，18℃孵育48h。
(4)将60μL H-2-Db多肽单体复合物转移到一个新反应管中，加入4.8μl链霉亲和素荧光基团(Streptavidin-fluorophore)，彻底混合。在4℃的黑暗中孵育至少1小时，即得到小鼠MHC H-2-Db限制性HPV-16E7多肽四聚体。
2.HPV-16 E7表位肽免疫小鼠，以便进一步进行HPV-16E7特异性TCRs的筛选。
HPV-16 E7转化TC-1和小鼠脾细胞，在RPMI-1640或DMEM培养基中培养，辅以10％FBS、100U/ml青霉素和100μg/ml链霉素，备用。雌性C57BL/6小鼠皮下接种5×105TC-1细胞。体外扩增的肿瘤细胞表达HPV-16E7，表现出与其他宫颈癌细胞系相似的形态，支原体检测呈阴性。HPV-16 E7表达稳定。
3.单个HPV-16 E7阳性特异性TCRs细胞的筛选分离
雌性C57BL/6小鼠接种TC-1细胞第28天，采集脾脏制备单细胞悬液，分离的细胞培养在RPMI 1640培养基(1×106细胞/mL)中。小鼠脾细胞培养一天后，脾细胞用抗CD3、抗CD8和HPV-16E7/H-2-Db四聚体标记。通过流式细胞术对T细胞克隆进行四聚体结合筛选。通过流式细胞技术鉴定出具有高亲和性的四聚体结合单细胞，将四聚体+/CD8+T细胞(如图1所示，方框中为四聚体+/CD8+阳性的细胞及比例)用分选流式仪分选到96孔PCR反应板。
4、单细胞TCR基因扩增及测序
将单个细胞裂解液加入到上述96孔PCR反应板的单个细胞孔中，Oligo(dT)与扩增引物、dNTP混合物在72℃下孵育。然后将这些混合物添加到含有M-MLVRTase、RT缓冲液、RNAse抑制剂、DTT和氯化镁的溶液中，用TCRα链和TCRβ链的引物扩增。扩增产物进行琼脂糖凝胶电泳，将目的条带进行凝胶纯化(Zymo研究试剂盒)，并进行基因测序。通过测序确定HPV E7多肽特异性TCR基因序列。
实施例2
本实施例提供一种HPV TCR-T细胞的制备，其制备方法包括：
根据测序确定的HPV-16 E7多肽特异性TCR基因序列，合成TCR基因，将TCR基因克隆到逆转录病毒中，用逆转录病毒转导T细胞，即得到HPV TCR-T细胞。
本发明制备得到的HPV TCR-T细胞对HPV-16E7多肽的亲和力增强。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。

Claims (8)

1.一种单细胞筛选鉴定人乳头瘤病毒特异性TCR的方法，其特征在于，包括如下步骤：
制备标记的MHC H-2-Db限制性HPV多肽四聚体；
将待筛选鉴定的人乳头瘤病毒TCR细胞用抗CD3、抗CD8和标记的MHC H-2-Db限制性HPV多肽四聚体标记，通过流式细胞术筛选四聚体+/CD8+的TCR阳性单细胞；
将筛选得到的四聚体+/CD8+的TCR阳性单细胞用细胞裂解液裂解后，以TCRα链和TCRβ链的引物进行PCR扩增，扩增产物进行基因测序，确定HPV多肽特异性TCR基因序列。
2.根据权利要求1所述的方法，其特征在于，所述HPV多肽为HPV-16E6或HPV-16E7。
3.根据权利要求1所述的方法，其特征在于，所述标记的MHC H-2-Db限制性HPV多肽四聚体为用荧光基团标记。
4.根据权利要求1所述的方法，其特征在于，所述待筛选鉴定的人乳头瘤病毒TCR细胞为：HPV表位肽免疫小鼠后采集脾脏制备的单细胞悬液。
5.根据权利要求4所述的方法，其特征在于，所述HPV表位肽为HPV-16E6表位肽或HPV-16E7表位肽。
6.权利要求1-4任一项所述方法获得的HPV表位特异性的TCR阳性单细胞。
7.一种人乳头瘤病毒特异性TCR-T细胞的制备方法，其特征在于，包括如下步骤，根据权利要求1测序确定的人乳头瘤病毒多肽特异性TCR基因序列，合成TCR基因，将TCR基因克隆到逆转录病毒中，用逆转录病毒转导T细胞，即得到人乳头瘤病毒特异性TCR-T细胞。
8.一种权利要求7所述制备方法制备得到的人乳头瘤病毒特异性TCR-T细胞。
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