TCR基因修饰构建不同γδ T细胞功能亚群的抗AML免疫调节治疗研究

The γδ T cells play important roles in anti-tumor immune responses and immunomodulatory effects. Differing from major histocompatibility (MHC)-restricted αβ T cells, γδ T cells specifically recognize tumor cells independent of MHC class I expression. However, peripheral γδ T cells are still heterogeneous populations and they may contain many different subgroups with diverse functions. In our previous research, clonality of particular TCR γδ subfamilies were found and might be correlated with hematological malignancies, and the oligoclonal proliferation of TCR Vδ3、Vδ4 and Vγ2 subfamilies of T cells in acute myeloid leukemia (AML) were identified. To further study the antagonistic activity and regulatory functions of different clonal expansions of γδ T cells subsets in AML, we propose a new concept in the field of adoptive immunotherapy that different functional subset of γδ T cells will be constructed for adoptive immunomodulatory therapy of AML based on specific TCR γδ gene modification. The distribution and clonality of TCR repertoire of peripheral γδ T cells in AML will be identified by gene-scan analysis, and the specific TCR gene sequences of predominant expressed TCR γδ subfamilies will be determined by gene subcloning and sequencing. Using the specific TCR γδ gene sequences, the eukaryotic expression plasmids of specific TCR γδ subfamilies genes will be constructed. Different functional subsets of γδ T cells will be constructed after recombinant plasmids transfection. The anti-leukemia function, mechanism of immune regulation and proliferation capacity of specific γδ T cells subsets will be confirmed by analyzed the cell phenotypes, characteristics of cytokines secretion, specific cytotoxic activity of anti-AML in vitro and in vivo. This research project will be beneficial to confirm a new approach for targeting anti-AML based on non-MHC-restricted TCR gene modification and provide the evidence of the development of novel immunomodulatory therapy strategy of AML.

γδ T细胞具有特异性识别抗原而不受主要组织相容性复合物限制的特点，在抗肿瘤和免疫调节中起着重要作用。我们的前期研究发现部分T细胞受体（TCR）Vγ和Vδ亚家族T细胞克隆性增殖与血液肿瘤相关，急性髓性白血病（AML）的TCR Vδ 3、Vδ 4和Vγ2亚家族T细胞出现寡克隆增殖，为进一步了解克隆性增殖的不同的γδ T细胞亚群在AML中的特异拮抗或调节功能，本研究首先借助于鉴定AML中克隆性增殖的TCR γδ亚家族T细胞，获得优势表达的特异性TCR γδ亚家族基因序列，构建特异性TCR γδ亚家族基因的真核表达重组质粒；转染T细胞后构建具有不同功能的特异性γδ T细胞亚群，通过细胞表型、细胞因子分泌特点、体内外特异抗AML细胞毒活性等分析特异性γδ T细胞亚群的抗白血病作用及免疫调节机制，明确基于非限制性TCR基因修饰的靶向抗AML新途径，为建立新的抗AML免疫调节治疗策略提供依据。

本项目首先分析了初发AML患者外周血TCR γδ亚家族的谱系分布及克隆性增殖情况。初发AML患者TCR Vγ亚家族基因表达水平显著降低，且呈现出TCR Vγ I> III> II的表达模式，与正常组的TCR Vγ II> I> III表达模式不同。初发AML组外周血γδ T细胞中Vδ1、Vγ I和Vγ III亚家族表达率较低，而Vδ4亚家族表达率较高。AML患者外周血中存在克隆性增殖的Vγ和Vδ亚家族T细胞，可能与体内存在的白血病抗原有关；其中TCR Vγ I的重排方式为：TCR Vγ I- TRGJ1*01；TCR Vγ III的重排方式为：TCR Vγ III-TRGJ1*02；TCR Vδ1重排方式为：TRDV1*01-TRDD3*01或TRDD1*01-TRDJ1*01；TCR Vδ4重排方式为：TRDV4*01-TRDD2*01-TRDJ1*01。部分初发AML患者外周血中FoxP3+ TCR Vδ1+亚群比例较高，且临床转归差，易出现复发或治疗无效。为了进一步研究调节性γδ T细胞（γδ Treg）的功能，我们使用含有转化生长因子-β的培养体系，成功将外周血单个核细胞诱导生成γδ Treg，并且首次使用含有G-CSF的诱导培养体系成功诱导产生新型γδ Treg。本研究还首先建立了TCR基因修饰γδ T细胞的研究平台，将原有专利的特异性TCR基因重组质粒转导入γδ T细胞以及γδ Treg亚群，分析经TCR基因修饰后γδ T细胞的功能变化。证实经TCR基因修饰后的γδ T细胞以及γδ Treg能够发挥特异性杀伤作用，且TCR基因修饰的γδ Treg仍有免疫调节功能。为探讨经TCR基因修饰的γδ T细胞的抗白血病作用，我们首先利用所获得AML相关的Vδ4亚家族的TCR基因序列，通过TCR转基因专利技术平台，构建TCR Vδ4重组质粒并转染正常人的CD4 T细胞，成功构建了特异性TCR Vδ4 T细胞。这种特异性TCR Vδ4 T细胞能够杀伤HL-60细胞，且杀伤率高于CD8+ 杀伤性T细胞。我们使用HL-60细胞株构建了裸鼠成瘤实验模型，发现同时输注特异性TCR Vδ4 T细胞，可导致成瘤时间延迟。本项目为开展新的有效治疗AML的过继性免疫治疗策略奠定研究基础和提供实验依据，具有良好应用前景。