Michaud Daniel, Mirlekar Bhalchandra, Bischoff Steven, Cowley Dale O, Vignali Dario A A, Pylayeva-Gupta Yuliya
Department of Cell Biology & Physiology, The University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, NC 27599, USA; Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC, USA.
Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, School of Medicine, Chapel Hill, NC, USA.
Cytokine. 2020 Jan;125:154817. doi: 10.1016/j.cyto.2019.154817. Epub 2019 Aug 28.
B cells are important modulators of immune responses both in autoimmunity and cancer. We have previously shown that B regulatory (Breg) cells promote pancreatic cancer via production of IL35, a heterodimeric cytokine comprised of the subunits p35 (Il12a) and Ebi3. However, it is not known how production of IL35 is regulated in vivo in the context of cancer-associated inflammation. To begin addressing this question, we have generated a knock-in mouse model, Il12a, where an IRES-emGFP gene was inserted within the 3' UTR of the Il12a locus. EmGFP signal in B cells from the Il12a mice correlated with expression of p35 mRNA and protein. Using this model, we observed that in addition to Bregs, expression of GFP (p35) is upregulated in several other B cell subtypes in response to cancer. We assessed the expression of the other IL35 subunit, Ebi3, using a published tdTomato reporter model. We determined that Ebi3 expression was more tightly regulated in vivo and in vitro, suggesting that stimuli affecting Ebi3 upregulation are more likely to result in production of full IL35 heterodimer. We were also able to detect GFP and Tomato signal in myeloid & T cell lineages suggesting that these reporter models could also be used for tracking IL12-, IL27- and IL35-producing cells. Furthermore, using primary B cells isolated from reporter mice, we identified BCR, CD40 and TLR pathways as potential drivers of IL35 expression. These findings highlight the importance of pancreatic cancer-associated inflammatory processes as drivers of cytokine expression and provide a tool to dissect both disease-associated regulation of IL12- and IL35-competent lineage cells as well as establish assays for pharmacological targeting of individual subunits of heterodimeric IL12 family cytokines.
B细胞在自身免疫和癌症中都是免疫反应的重要调节因子。我们之前已经表明，B调节（Breg）细胞通过产生IL35促进胰腺癌，IL35是一种由亚基p35（Il12a）和Ebi3组成的异二聚体细胞因子。然而，在癌症相关炎症的背景下，IL35的产生在体内是如何被调节的尚不清楚。为了开始解决这个问题，我们构建了一个敲入小鼠模型Il12a，其中一个IRES-emGFP基因被插入到Il12a基因座的3'UTR内。来自Il12a小鼠的B细胞中的EmGFP信号与p35 mRNA和蛋白质的表达相关。使用这个模型，我们观察到除了Bregs之外，响应癌症时，GFP（p35）在其他几种B细胞亚型中的表达也会上调。我们使用已发表的tdTomato报告模型评估了另一个IL35亚基Ebi3的表达。我们确定Ebi3的表达在体内和体外受到更严格的调节，这表明影响Ebi3上调的刺激更有可能导致完整的IL35异二聚体的产生。我们还能够在髓系和T细胞谱系中检测到GFP和Tomato信号，这表明这些报告模型也可用于追踪产生IL12、IL27和IL35的细胞。此外，使用从报告小鼠中分离的原代B细胞，我们确定BCR、CD40和TLR途径是IL35表达的潜在驱动因素。这些发现突出了胰腺癌相关炎症过程作为细胞因子表达驱动因素的重要性，并提供了一个工具来剖析与疾病相关的IL12和IL35功能谱系细胞的调节，以及建立针对异二聚体IL12家族细胞因子单个亚基的药理学靶向检测方法。
