Generation and characterization of human B1 B cells induced by Lin28b reprogramming of adult hematopoietic progenitors

 描述（由申请人提供）：小鼠中的CD 5 + B1 B细胞构成天然自身反应性B细胞库，其执行许多重要功能，包括清除凋亡细胞、对病原体作出快速反应以及通过分泌IL-10调节炎症。然而，它们的自身反应性BCR使它们在老年动物中易于克隆扩增和进展为淋巴瘤和白血病。因此，从正常和病理免疫应答的角度阐明产生这些细胞的调节机制是重要的。这些细胞的一个有趣的特征是其主要来源于独特的胎儿/新生儿发育途径，与成年小鼠骨髓中的大多数B细胞发育不同。最近，Lin 28 B/Let-7轴在调节小鼠从胎儿到成年发育的转换中的作用已经变得明显，使得通过逆转录病毒转导Lin 28 B从小鼠骨髓前体产生CD 5 + B1 B细胞成为可能。虽然天然的自身反应性B细胞也存在于人类中，但鉴定它们并阐明它们的发育起源更具挑战性，因为人B细胞中的CD 5表达更短暂，可能反映最近的活化，因此不是胎儿B细胞谱系的有用标志物。因此，我们比较了Lin 28 b在人胎儿和成人造血祖细胞中的表达，发现其表达与小鼠相同，存在于脐带血祖细胞中，而不存在于成人祖细胞中。这些结果表明，Lin 28 B/Let-7轴在人B细胞发育中也起调节开关的作用。当我们改变人B细胞祖细胞中的Lin 28 B/Let-7表达时，我们将通过表征在成人B细胞中诱导的细胞表面表型、基因表达和自身反应性的变化来检验这一假设。具体而言，我们将探讨：1）Lin 28 B和Let-7 miR控制人B细胞发育中命运选择的分子基础;和2）胎儿命运的采用如何影响B细胞抗原受体的特异性。在目的1中，我们将通过慢病毒转导成年人祖细胞表达Lin 28 B b并耗尽Let-7 miRNA，体外分化细胞，然后进行流式细胞术以表征表面蛋白表达，并进行RNA-Seq以确定祖细胞和新形成的B细胞中改变的基因。在目标2中，我们将类似地转导的祖细胞转移到免疫缺陷的NOD/SCID/IL 2 R β c-小鼠中以促进成熟B细胞的分化。我们将再次通过RNA-Seq比较成人发育的成熟B细胞后代中基因表达的变化，这些细胞被重编程为类似于胎儿类型。我们还将测试在这些小鼠中通过正常和Lin 28 B重编程发育产生的B细胞上表达的B细胞抗原受体的自身反应性的差异。这项工作将为进一步研究人类B1 B细胞奠定基础，阐明其发育、选择及其对免疫、自身免疫和白血病的贡献的独特机制。此外，深入了解人类B1 B细胞的表型将使其能够在衰老过程中进行监测，特别是它们在慢性淋巴细胞白血病起源中的作用。

 DESCRIPTION (provided by applicant): CD5+ B1 B cells in mice constitute a pool of natural autoreactive B cells that carry out many important functions, including clearance of apoptotic cells, making rapid responses to pathogens, and regulating inflammation by secreting IL-10. However, their self-reactive BCRs predispose them to clonal expansion and progression to lymphoma and leukemia in aged animals. Hence, it is important to elucidate the regulatory mechanisms that give rise to these cells both from the point of view of normal and pathological immune responses. An interesting feature of these cells is their predominant origin from a distinctive fetal/neonatal developmental pathway, distinguishing it from most B cell development in bone marrow of adult mice. Recently the role of the Lin28b/Let-7 axis in regulating the switch from fetal to adult development in mice has become apparent, making it possible to generate CD5+ B1 B cells from mouse bone marrow precursors by retroviral transduction of Lin28b. While natural autoreactive B cells also exist in humans, identifying them and elucidating their developmental origins is much more challenging, as CD5 expression in human B cells is more transient and may reflect recent activation, and so is not a useful marker for the fetal B cell lineage. Hence, we have compared Lin28b expression in human fetal and adult hematopoietic progenitors and found that its expression is the same as in mice, present in cord blood progenitors and absent from adult progenitors. These results suggest that the Lin28b/Let-7 axis also functions in human B cell development as a regulatory switch. We will test this hypothesis by characterizing changes in cell surface phenotype, gene expression, and autoreactivity induced in adult B cells when we alter Lin28b/Let-7 expression in human B cell progenitors. Specifically, we will explore: 1) the molecular basis by which Lin28b and Let-7 miR control fate choices in human B cell development; and 2) how adoption of the fetal fate influences the specificity of the B cell antigen receptor. In Aim 1, we will express Lin28b and deplete Let-7 miRNA by Lentiviral transduction of adult human progenitors, differentiating the cells in vitro, then performing flow cytometry to characterize surface protein expression and RNA-Seq to determine genes altered in progenitors and newly-formed B cells. In Aim 2, we will transfer similarly transduced progenitors into immunodeficient NOD/SCID/IL2Rc- mice to facilitate the differentiation of mature B cells. We will compare changes in gene expression in the mature B cell progeny of adult development reprogrammed to resemble fetal type, again by RNA-Seq. We will also test for differences in autoreactivity of the B cell antigen receptors expressed on B cels generated in these mice by normal and Lin28b reprogrammed development. This work will form the basis for further investigation of human B1 B cells, shedding light on the distinctive mechanism of their development, their selection, and their contribution to immunity, autoimmunity, and leukemia. Moreover, gaining insights into the phenotype of human B1 B cells will enable their monitoring during aging, particularly their role in the origin of chronic lymphocytic leukemia.