Project 1 - Dioxin-like Compounds Suppress IgM Responses by Targeting CD5+ (Innate-like) B cells, which can Serve as a Biomarker of Susceptibility to Environmental AHR Ligands

项目总结/摘要：环境污染物抑制体液免疫， 是原型芳烃受体（AHR）配体（例如，2，3，7，8-四氯二苯并二恶英 已经证明在实验室动物中抑制体液免疫， 流行病学证据。本研究的总体目标是确定分子机制（S） 负责AHR激动剂介导的人原代B细胞抗体产生的抑制。之前 比较活化的小鼠、大鼠和人原代B细胞的研究，RNAseq显示少量的 TCDD差异表达基因一个仅在人类B细胞中差异调节的基因 与小鼠和大鼠相比，淋巴细胞特异性蛋白酪氨酸激酶（LCK）。虽然LCK是 除了由T细胞表达的LCK外，B细胞亚群也表达LCK，特别是CD 5 + B细胞。我们已经表明 TCDD降低IgM分泌的部分原因是对CD 5 + B细胞的选择性作用，称为先天性样B (ILB)细胞尽管CD 5 + ILB构成循环B细胞的大约10-25%，并且是免疫缺陷的来源，但是， 在没有免疫反应的情况下，大部分循环IgM。此外，CD 5 + ILB是原发性B 在适应性免疫系统仍在发育的生命早期和晚期介导体液免疫的细胞类型 或者在生命的晚期，当适应性免疫力下降时。蛋白质分析还显示， CD 5 + ILB上LCK、抑制性受体PD-1及其配体PD-L2的TCDD。PD-1和PD-L2的诱导 尤其重要，因为它们提供了AHR抑制IgM应答的机制 在CD 5 + ILB中活化。LCK可以磷酸化PD-1受体的抑制区，促进免疫反应。 PD-1抑制。抑制LCK活性可保护CD 5 + B细胞免受TCDD对IgM的抑制， 证明LCK是至关重要的。因此，我们将测试假设：AHR介导的抑制 IgM是通过诱导抑制性受体PD-1和LCK（一种已知可启动PD-1的激酶）而产生的。 介导的免疫调节，优先作用于人原代CD 5+（先天性样）B细胞。具体目标（SA）1 将人CD 5+（先天性样）B细胞表征为对抑制敏感的高度敏感群体 通过AHR配体。SA 2旨在确定诱导检查点抑制剂PD-1和酪氨酸激酶LCK在 TCDD介导的对CD 5 + B细胞IgM产生的抑制实现上述目标具有很强的 可能：（a）确定对免疫关键的AHR配体损伤高度敏感的B细胞群 （B）鉴定一种新的免疫毒性机制，其涉及免疫原性的上调， 抑制性受体，PD-1;（c）使用IFNγ表征LCK在通过AHR活化的IgM抑制中的作用， （d）利用循环CD 5 + B细胞百分比作为敏感群体的生物标志物， AHR配体对IgM的抑制和（e）该实验计划的结果将用于开发一种 校准TCDD PBPK模型，与计算建模核心合作进行风险评估。

PROJECT SUMMARY/ABSTRACT: Suppression of humoral immunity by environmental contaminants that are prototypical aryl hydrocarbon receptor (AHR) ligands (e.g., 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)) have been demonstrated to suppress humoral immunity in laboratory animals and suggested by epidemiological evidence. The overall goal of this research is to define the molecular mechanism(s) responsible for AHR agonist-mediated suppression of antibody production by human primary B cells. Prior studies comparing activated mouse, rat and human primary B cells, RNAseq showed a small number of differentially expressed genes by TCDD. One gene that was only differentially regulated in human B cells compared to mouse and rat was the lymphocyte specific protein tyrosine kinase (LCK). While LCK is expressed by T cells, subpopulations of B cells also express LCK, specifically CD5+ B cells. We have shown that decreased IgM secretion by TCDD is due, in part, to selective effects on CD5+ B cells, termed innate-like B (ILB) cells. Although CD5+ ILBs constitute approximately 10-25% of circulating B cells and are the source for the majority of circulating IgM in the absence of an immune response. Further, CD5+ ILBs are the primary B cell type mediating humoral immunity early and late in life when the adaptive immune system is still developing or during late life stages when adaptive immunity is in decline. Protein analysis also showed induction by TCDD of LCK, the inhibitory receptor PD-1 and its ligand, PD-L2 on CD5+ ILBs. Induction of PD-1 and PD-L2 are especially important as they provide a mechanism for the suppression of the IgM response by AHR activation in CD5+ ILBs. LCK can phosphorylate the inhibitory region of the PD-1 receptor, facilitating immune suppression by PD-1. Inhibition of LCK activity protected CD5+ B cells from IgM suppression by TCDD, demonstrating LCK is critically involved. Therefore, we will test the hypothesis: AHR-mediated suppression of IgM occurs through induction of the inhibitory receptor PD-1 and LCK, a kinase known to initiate PD-1- mediated immune regulation, preferentially on human primary CD5+ (innate-like) B cells. Specific Aim (SA) 1 will characterize human CD5+ (innate-like) B cells as a highly sensitive population susceptible to suppression by AHR ligands. SA2 is to identify the role of induced checkpoint inhibitor, PD-1, and tyrosine kinase, LCK, in TCDD-mediated suppression of IgM production by CD5+ B cells. Completion of the above aims has a strong potential to: (a) define B cell population(s) highly sensitive to impairment by AHR ligands critical for immunity against common pathogens; (b) identify a novel mechanism of immunotoxicity involving upregulation of the inhibitory receptor, PD-1; (c) characterize the role of LCK in IgM suppression by AHR activation using IFNγ as a molecular probe; (d) utilize percent circulating CD5+ B cells as biomarker of sensitive populations to suppression of IgM by AHR ligands and (e) results for this experimental plan will be used to develop a calibrated TCDD PBPK model for risk assessment in collaboration with the Computational Modeling Core.