Distinct Tumor Necrosis Factor Alpha Receptors Dictate Stem Cell Fitness Versus Lineage Output in Murine Dnmt3a-Mutant Clonal Hematopoiesis. Distinct Tumor Necrosis Factor Alpha Receptors Dictate Stem Cell Fitness Versus Lineage Output in Murine Dnmt3a-Mutant Clonal Hematopoiesis

Clonal hematopoiesis resulting from enhanced fitness of mutant hematopoietic stem cells (HSCs) associates with both favorable and unfavorable health outcomes related to the types of mature mutant blood cells produced, but how this lineage output is regulated is unclear. Using a mouse model of DNMT3AR882/+ clonal hematopoiesis (Dnmt3aR878H/+), we found that aging-induced TNFα signaling promoted the selective advantage of mutant HSCs as well as stimulated mutant B lymphoid cell production. Genetic loss of TNFα receptor TNFR1 impaired mutant HSC fitness without altering lineage output, while loss of TNFR2 reduced lymphoid cell production and favored myeloid cell production from mutant HSCs without altering overall fitness. These results support a model where clone size and mature blood lineage production can be independently controlled to harness potential beneficial aspects of clonal hematopoiesis. Overall design: 1x10^6 CD45.2+ cells were competed against 1x10^6 CD45.1+ post-ficoll whole BM cells and transplanted into aged, lethally irradiated CD45.1+ recipient animals. One month post-transplant, the recipients received one IP injection of poly(I:C) and recombination was checked via PCR on PB. One month post poly(I:C), animals were bled monthly for 16 weeks. Bone marrow was harvested and 4x10^6 post-ficoll whole BM cells were used for secondary transplantation into aged, lethally irradiated recipients. Recipients were harvested 20 weeks post-transplant.

由突变造血干细胞（hematopoietic stem cells, HSCs）适应性增强所引发的克隆性造血（clonal hematopoiesis），与所产生的成熟突变血细胞类型相关的有益及不良健康结局均存在关联，但其谱系输出的调控机制仍不明确。我们利用DNMT3AR882/+型克隆性造血小鼠模型（Dnmt3aR878H/+）开展研究，发现衰老诱导的肿瘤坏死因子α（TNFα）信号通路不仅促进了突变HSCs的选择优势，还刺激了突变B淋巴细胞的生成。敲除TNFα受体TNFR1会损害突变HSCs的适应性，但不会改变其谱系输出；而敲除TNFR2则会减少突变HSCs的淋巴细胞生成，并偏向髓系细胞生成，但不会改变整体适应性。上述研究结果支持如下模型：克隆大小与成熟血液谱系生成可被独立调控，从而得以利用克隆性造血的潜在有益效应。
实验整体设计：将1×10^6个CD45.2+细胞与1×10^6个经聚蔗糖分离后的全骨髓（bone marrow, BM）CD45.1+细胞进行竞争性移植，植入经致死剂量照射的老年CD45.1+受体动物体内。移植1个月后，受体动物接受1次腹腔内（intraperitoneal, IP）注射聚肌胞苷酸（poly(I:C)），并通过外周血（peripheral blood, PB）的聚合酶链式反应（PCR）检测重组情况。聚肌胞苷酸注射1个月后，每月采集动物外周血，持续16周。收集骨髓，取4×10^6个经聚蔗糖分离后的全骨髓细胞用于二次移植，植入经致死剂量照射的老年受体动物体内。于移植后20周收获受体动物。