Hoxb5诱导重编程再生T细胞的效率优化及抗肿瘤活性研究

T cells play pivotal roles in immune responses against tumors and viral infections. Our preliminary data demonstrated that overexpression of the transcription factor Hoxb5 in mouse pro/pre-B cells reprogrammed these cells into functional early T cell progenitors (iETPs) in vivo. The iETPs subsequently differentiated into functional T cells in vivo in thymus. However, the reprogramming efficiency of this system remains very low (1/10000). In this study, we will test the potential range of other progenitor cells and B progenitor subpopulations among the hematopoietic hierarchy, including common lymphoid progenitor (CLP), pre-pro-B, pro-B, pre-B cells, by evaluating their reprogramming plasticities mediated by Hoxb5. Next, we will knockdown the B-cell identity transcription factor Pax5 in Hoxb5-overexpressing cells to investigate whether this combination strategy can promote B-to-T cell converting efficiency. To assess the anti-tumor activity of the regenerated T cells in our system, we will introduce the CD19-CAR or OT1 (MHC- I restricted antigen) coding sequence into B cell progenitors by viral infection, and reprogram these cells into T cells carrying the tumor-specific elements. B cell- and T cell-lymphoma models will be used for assessing the anti-tumor activity of these regenerative tumor-specific T cells. In addition, stromal cell-based co-culture strategy will be used to explore the feasibility of reprogramming B cells into T cells in vitro. Our study will optimize the protocol of Hoxb5-induced B-to-T cell reprogramming and confirm the anti-tumor activity of these de novo regenerated T cells, which may provide insights into obtaining alternative source of T cells for eradicating tumors.

T细胞在机体抗肿瘤、抗病毒感染方面发挥重要作用。前期研究揭示转录因子Hoxb5将pro/pre-B细胞体内重编程为早期T淋巴祖细胞，并在胸腺分化为成熟T细胞。然而，该体系重编程效率依然很低(1/10000)。拟进一步探索Hoxb5诱导重编程再生T细胞的起始靶细胞层级范围，包括共同淋系祖细胞、pre-pro-B、pro-B和pre-B等B祖细胞；利用RNAi技术沉默B细胞身份转录因子Pax5的表达，结合骨髓移植、流式免疫表型分析等研究下调Pax5后对Hoxb5诱导重编程再生T细胞效率的影响；通过病毒感染法将CD19-CAR或OT1（MHC-I限制性抗原）序列导入B祖细胞，利用B细胞和T细胞淋巴瘤模型研究重编程再生T细胞的抗肿瘤活性；利用基质细胞共培养策略探索体外诱导B-T转化。本研究将优化Hoxb5诱导再生T细胞技术体系，确认再生T细胞的抗肿瘤活性，为寻找抗肿瘤新来源T细胞提供理论指导。

T细胞疗法在临床的广泛应用对获得功能性再生T淋巴细胞提出新要求。前期研究揭示转录因子Hoxb5可以将pro/pre-B细胞体内重编程为T淋巴细胞。然而，该体系重编程效率低下，亟待改进，再生T细胞的抗肿瘤功能需要进一步验证。本项目利用构建的Hoxb5LSL/+Vav-cre模型鼠，分析了过表达Hoxb5对多能祖细胞（multipotent progenitor，MPP）、淋系潜能多能祖细胞（lymphoid-primed multipotent progenitor, LMPP）、共同淋系祖细胞（common lymphoid progenitor，CLP）等祖细胞的淋系谱系分化能力的影响。结果表明过表达Hoxb5不会影响LMPP的淋系分化能力，但可以促进CLP向T淋巴细胞分化；同时还发现Hoxb5可以诱导MPP重编程为具有多谱系分化潜能及长期移植重构造血能力的类似于造血干细胞（hematopietic stem cells, HSC）的细胞群，为获得可移植HSC提供了新的来源。本项目构建了包含Pax5-shRNA片段的质粒，通过病毒感染的方式导入到过表达Hoxb5的B细胞中，发现下调B细胞发育相关重要转录因子Pax5的表达不会提高Hoxb5诱导重编程再生T细胞的效率；而同时过表达Hoxb5和T细胞发育相关转录因子Gata3会抑制T细胞的再生，并诱导B细胞重编程为完整的髓系谱系细胞。此外，本项目还通过将Hoxb5诱导OT1转基因鼠的B细胞重编程再生的OT1-iT细胞与肿瘤细胞进行体外共培养和体内移植实验，证实重编程再生的T细胞具有抗肿瘤活性。本项目明确了Hoxb5诱导体内重编程再生T细胞的起始靶细胞范围，证实了再生T细胞的抗肿瘤活性，发现了Hoxb5可诱导MPP再生HSC，并构建了转录因子组合诱导B细胞重编程再生髓系细胞技术体系，可为获得再生的功能性血液细胞提供理论指导。