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Abstract：
T helper-2 (Th2) cells and type 2 innate lymphoid cells (ILC2s) play crucial roles during type 2 immune responses; the transcription factor GATA3 is essential for the differentiation and functions of these cell types. It has been demonstrated that GATA3 is critical for maintaining Th2 and ILC2 phenotype in vitro; GATA3 not only positively regulates type 2 lymphocyte-associated genes, it also negatively regulates many genes associated with other lineages. However, such functions cannot be easily verified in vivo because the expression of the markers for identifying Th2 and ILC2s depends on GATA3. Thus, whether Th2 cells and ILC2s disappear after Gata3 deletion or these Gata3-deleted \"Th2 cells\" or \"ILC2s\" acquire an alternative lineage fate is unknown. In this study, we generated novel GATA3 reporter mouse strains carrying the Gata3 ZsG or Gata3 ZsG-fl allele. This was achieved by inserting a ZsGreen-T2A cassette at the translation initiation site of either the wild type Gata3 allele or the modified Gata3 allele which carries two loxP sites flanking the exon 4. ZsGreen faithfully reflected the endogenous GATA3 protein expression in Th2 cells and ILC2s both in vitro and in vivo. These reporter mice also allowed us to visualize Th2 cells and ILC2s in vivo. An inducible Gata3 deletion system was created by crossing Gata3 ZsG-fl/fl mice with a tamoxifen-inducible Cre. Continuous expression of ZsGreen even after the Gata3 exon 4 deletion was noted, which allows us to isolate and monitor GATA3-deficient \"Th2\" cells and \"ILC2s\" during in vivo immune responses. Our results not only indicated that functional GATA3 is dispensable for regulating its own expression in mature type 2 lymphocytes, but also revealed that GATA3-deficient \"ILC2s\" might be much more stable in vivo than in vitro. Overall, the generation of these novel GATA3 reporters will provide valuable research tools to the scientific community in investigating type 2 immune responses in vivo.
摘要：
辅助性T细胞2（Th2）和2型先天淋巴细胞（ILC2s）在2型免疫反应中起着至关重要的作用；转录因子GATA3对于这些细胞类型的分化和功能至关重要。已经证明，GATA3对维持Th2和ILC2表型至关重要;GATA3不仅积极调节2型淋巴细胞相关基因，它还负调节与其他谱系相关的许多基因。然而,这样的功能不能在体内容易地验证,因为用于鉴定Th2和ILC2s的标志物的表达依赖于GATA3。因此，Th2细胞和ILC2s是否在Gata3缺失后消失，或者这些Gata3缺失的“Th2细胞”或“ILC2s”是否获得替代谱系命运尚不清楚。在这项研究中,我们产生了携带Gata3ZsG或Gata3ZsG-fl等位基因的新型GATA3报告小鼠品系。这是通过在野生型Gata3等位基因或修饰的Gata3等位基因的翻译起始位点插入ZsGreen-T2A盒来实现的，该等位基因带有两个外显子4侧翼的loxP位点。ZsGreen忠实地反映了体内和体外Th2细胞和ILC2s中内源性GATA3蛋白的表达。这些报告小鼠还允许我们在体内观察Th2细胞和ILC2s。通过使Gata3ZsG-fl/fl小鼠与他莫昔芬诱导型Cre杂交来产生诱导型Gata3缺失系统。注意到即使在Gata3外显子4缺失后ZsGreen的连续表达，这使我们能够在体内免疫反应期间分离和监测GATA3缺陷的“Th2”细胞和“ILC2s”。我们的结果不仅表明功能性GATA3对于调节其在成熟2型淋巴细胞中的自身表达是不必要的。但也揭示了GATA3缺陷的“ILC2s”在体内可能比在体外稳定得多。总的来说,这些新型GATA3报告基因的产生将为科学界研究体内2型免疫反应提供有价值的研究工具.
