PO.TB10.18 · 肿瘤生物学
利用体外模型中的CRISPR筛选定义树突状细胞的免疫调控分子驱动因素
Defining the immunoregulatory molecular drivers of dendritic cells using CRISPR screens in ex-vivo modelsvivo models
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
经典1型树突状细胞（cDC1）通过产生IL-12等细胞因子并表达包括PD-L1在内的关键调控分子，在协调抗肿瘤T细胞应答中发挥着核心且不可或缺的作用。尽管cDC1在塑造抗肿瘤免疫和影响现代免疫疗法应答方面十分重要，但控制cDC1激活和成熟的分子机制仍不明确。肿瘤微环境（TME）中cDC1的极低丰度使这一知识空白更加复杂，限制了在实验上研究其生物学特性以及大规模应用基因扰动工具的能力。为应对这些挑战，我们开发、优化并比较了稳健的体外分化系统，这些系统能够从小鼠和人类造血干细胞（HSC）中生成具有生理相关性的cDC1样细胞。通过精心设计的两阶段培养平台，结合Fms样酪氨酸激酶3配体（Flt3L）、DLL1介导的Notch信号和粒细胞-巨噬细胞集落刺激因子（GM-CSF），我们高效地从小鼠骨髓祖细胞中生成了CD103⁺ cDC1样细胞群，并从G-CSF动员的人类外周血CD34⁺ HSC中生成了Clec9a⁺CD141⁺ cDC1样细胞。这些体外来源的细胞与其体内对应物高度相似，表现出预期的表面免疫表型，经ELISA检测在刺激后有高水平的IL-12分泌，并对TLR激动剂和细胞相关抗原具有强大的功能反应性。激活后，细胞持续上调经典的成熟标志物，包括PD-L1、MHCII和CD40，证明其能够进行适当的免疫刺激驱动的成熟。为支持下游基因研究，我们优化了原代HSC的病毒转导策略，这是该领域的一大技术障碍。嗜亲性逆转录病毒系统结合retronectin显著提高了小鼠细胞的基因递送效率，而嗜双性假型逆转录病毒方法则增强了人类CD34⁺ HSC的转导。这些进展使得在分化前将基于CRISPR的扰动可靠地引入前体细胞成为可能。总之，这一可扩展、高产量的分化和基因递送平台为开展基于CRISPR的基因筛选提供了强大基础，旨在识别协调cDC1激活和成熟的分子调控因子。这些见解有望为利用或增强树突状细胞驱动的抗肿瘤免疫的下一代免疫疗法的开发提供依据。
查看英文原文 English abstract
Conventional dendritic cell type 1 (cDC1) plays a central and indispensable role in coordinating antitumor T-cell responses by producing cytokines such as IL-12 and expressing key regulatory molecules including PD-L1. Despite their importance in shaping antitumor immunity and influencing responses to modern immunotherapies, the molecular mechanisms controlling cDC1 activation and maturation remain poorly defined. This gap in knowledge is compounded by the extremely low abundance of cDC1 within the tumor microenvironment (TME), which limits the ability to experimentally interrogate their biology and apply genetic perturbation tools at scale. To address these challenges, we developed, optimized, and compared robust ex vivo differentiation systems capable of generating physiologically relevant cDC1-like cells from both murine and human hematopoietic stem cells (HSCs).Using a carefully designed two-stage culture platform incorporating Fms-like tyrosine kinase 3 ligand (Flt3L), DLL1-mediated Notch signaling, and granulocyte-macrophage colony-stimulating factor (GM-CSF), we efficiently produced CD103⁺ cDC1-like populations from mouse bone marrow progenitors and Clec9a⁺CD141⁺ cDC1-like cells from G-CSF-mobilized human peripheral blood CD34⁺ HSCs. These ex-vivo derived cells closely mirrored their in-vivo counterparts, exhibiting the expected surface immunophenotypes, high IL-12 secretion upon stimulation measured by ELISA, and strong functional responsiveness to TLR agonists and cell-associated antigens. Upon activation, cells consistently upregulated classical maturation markers including PD-L1, MHCII, and CD40, demonstrating their ability to undergo appropriate immune stimulation-driven maturation.To support downstream genetic studies, we optimized viral transduction strategies for primary HSCs, a major technical barrier in the field. An ecotropic retroviral system combined with retronectin substantially improved gene delivery efficiency in murine cells, while an amphotropic pseudotyped retroviral approach enhanced transduction of human CD34⁺ HSCs. These advances enable reliable introduction of CRISPR-based perturbations into precursor cells prior to differentiation.Together, this scalable, high-yield differentiation and gene-delivery platform provides a powerful foundation for conducting CRISPR-based genetic screens aimed at identifying the molecular regulators orchestrating cDC1 activation and maturation. These insights have the potential to inform the development of next-generation immunotherapies that harness or enhance dendritic cell-driven antitumor immunity.Defining the immunoregulatory molecular drivers of dendritic cells using CRISPR screens in ex-vivo models
利益披露 Disclosure
M. Asnani, None..
J. Yang, None.