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

 描述(由申请人提供)：小鼠体内的CD5+B1 B细胞构成了一个天然的自身反应性B细胞池，执行许多重要功能，包括清除凋亡细胞，对病原体做出快速反应，以及通过分泌IL-10调节炎症。然而，它们的自身反应性BCR使它们易于在老年动物中克隆性扩张和发展为淋巴瘤和白血病。因此，从正常免疫反应和病理免疫反应的角度阐明产生这些细胞的调控机制是很重要的。这些细胞的一个有趣的特征是，它们主要来自独特的胎儿/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，Lin28b/let-7轴在调节小鼠从胎儿发育到成人发育的过程中的作用已经变得明显，使得通过逆转录病毒转导Lin28b从小鼠骨髓前体细胞产生CD5+B1 B细胞成为可能。虽然自然自身反应性B细胞也存在于人类中，但识别它们并阐明其发育起源要具有更大的挑战性，因为人类B细胞中CD5的表达是暂时的，可能反映了最近的激活，因此不是胎儿B细胞谱系的有用标记物。因此，我们比较了Lin28b在人胎儿和成人造血祖细胞中的表达，发现它的表达与小鼠相同，存在于脐带血造血祖细胞中，而在成年造血祖细胞中缺失。这些结果表明，Lin28b/let-7轴在人类B细胞发育过程中也起着调节开关的作用。当我们改变人类B细胞前体细胞中Lin28b/let-7的表达时，我们将通过表征成年B细胞中诱导的细胞表面表型、基因表达和自身反应的变化来检验这一假设。具体地说，我们将探索：1)Lin28b和let-7 miR控制人类B细胞发育中命运选择的分子基础；以及2)胎儿命运的采用如何影响B细胞抗原受体的特异性。在目标1中，我们将通过慢病毒转导成年人祖细胞表达Lin28b并去除let-7miRNA，体外分化细胞，然后用流式细胞仪检测细胞表面蛋白的表达，用RNA-Seq检测祖细胞和新形成的B细胞中基因的变化。在目标2中，我们将类似转导的祖细胞转移到免疫缺陷的NOD/SCID/IL2R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，重新编程为类似胎儿的类型。我们还将测试这些小鼠在正常和Lin28b重编程发育过程中产生的B细胞上表达的B细胞抗原受体的自反应性差异。这项工作将为进一步研究人类B1B细胞奠定基础，揭示它们发育的独特机制，它们的选择，以及它们对免疫、自身免疫和白血病的贡献。此外，深入了解人类B1B细胞的表型将使它们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发生中的作用。

 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.