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

项目摘要/摘要：环境污染物对体液免疫的抑制 是典型的芳烃受体(AHR)配体(例如，2，3，7，8-四氯二苯并-对二恶英(TCDD)) 已经被证明在实验动物中抑制体液免疫，并建议 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激动剂介导的人类原代B细胞抗体产生的抑制。之前 比较激活的小鼠、大鼠和人类原代B细胞的研究表明，RNAseq显示了少量的 TCDD差异表达基因分析。一种仅在人类B细胞中差异调节的基因 与小鼠和大鼠比较的是淋巴细胞特异性蛋白酪氨酸激酶(LCK)。而LCK则是 由T细胞表达的B细胞亚群也表达LCK，尤其是CD5+B细胞。我们已经展示了 TCDD减少IgM分泌的部分原因是对CD5+B细胞的选择性作用，这种细胞被称为先天B细胞 (ILB)细胞。尽管CD5+ILB约占循环B细胞的10%-25%，是 大多数循环中的IgM在没有免疫反应的情况下发生。此外，CD5+ILB是主要的B细胞 在适应性免疫系统仍在发育的生命早期和晚期调节体液免疫的细胞类型 或者是在适应性免疫功能下降的生命后期。蛋白质分析还显示， LCK抑制受体PD-1及其配体PD-L2对CD5+ILB的TCDD作用。PD-1和PD-L2的诱导 尤其重要，因为它们为AHR抑制IgM反应提供了一种机制 CD5+ILB的激活。LCK可以磷酸化PD-1受体的抑制区域，促进免疫 PD-1的抑制作用。抑制LCK活性保护CD5+B细胞免受TCDD对IgM的抑制 证明LCK是至关重要的。因此，我们将检验假设：AHR介导的抑制 IgM通过诱导抑制受体PD-1和LCK发生，LCK是一种已知的启动PD-1- 介导的免疫调节，优先于人类原代CD5+(先天类)B细胞。特定目标(SA)1 将人类CD5+(先天类)B细胞定性为易受抑制的高度敏感群体 由AHR配体。SA2是为了确定诱导检查点抑制物PD-1和酪氨酸激酶LCK在 TCDD对CD5+B细胞产生IgM的抑制作用上述目标的完成具有很强的针对性 可能：(A)确定对对免疫至关重要的AHR配体的损伤高度敏感的B细胞群体(S) 抗常见病原体；(B)确定一种新的免疫毒性机制，涉及上调 抑制性受体PD-1；(C)表征LCK在干扰素γ激活AHR抑制免疫球蛋白M中的作用 分子探针；(D)利用循环CD5+B细胞百分比作为敏感人群的生物标志物 AHR配体对IgM的抑制和(E)本实验计划的结果将用于开发一种 经校准的TCDD PBPK模型，用于与计算建模核心协作进行风险评估。

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.