Gata3ZsG and Gata3ZsG-fl: Novel murine Gata3 reporter alleles for identifying and studying Th2 cells and ILC2s in vivo

Gurram, Rama Krishna; Wei, Danping; Yu, Qiao; Kamenyeva, Olena; Chung, Hee Soon; Zheng, Mingzhu; Butcher, Matthew J.; Kabát, Juraj; Liu, Chengyu; Khillan, Jaspal S.; Zhu, Jinfang

doi:10.3389/fimmu.2022.975958

Cited by 4 publications

(2 citation statements)

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“…Tbx21 Cre mice [Jax line 024507 ( 39 )] were crossed with Gata3 fl/fl mice to generate Tbx21 Cre Gata3 fl/fl mice. Rorc E2-Crimson mice ( 35 ) were crossed with Gata3 ZsGreen ( 40 ) and Foxp3 RFP [Jax line 008374 ( 41 )] reporter mice to generate Rorc E2Crimson Gata3 ZsGreen Foxp3 RFP triple reporter mice. 2D2 mice were purchased from the Jackson Laboratory (JAX line 006912).…”

Section: Methodsmentioning

confidence: 99%

GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression

et al. 2023

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T-bet-expressing Th17 (T-bet+RORγt+) cells are associated with the induction of pathology during experimental autoimmune encephalomyelitis (EAE) and the encephalitic nature of these Th17 cells can be explained by their ability to produce GM-CSF. However, the upstream regulatory mechanisms that control Csf2 (gene encoding GM-CSF) expression are still unclear. In this study, we found that Th17 cells dynamically expressed GATA3, the master transcription factor for Th2 cell differentiation, during their differentiation both in vitro and in vivo. Early deletion of Gata3 in three complimentary conditional knockout models by Cre-ERT2, hCd2Cre and Tbx21Cre, respectively, limited the pathogenicity of Th17 cells during EAE, which was correlated with a defect in generating pathogenic T-bet-expressing Th17 cells. These results indicate that early GATA3-dependent gene regulation is critically required to generate a de novo encephalitogenic Th17 response. Furthermore, a late deletion of Gata3 via Cre-ERT2 in the adoptive transfer EAE model resulted in a cell intrinsic failure to induce EAE symptoms which was correlated with a substantial reduction in GM-CSF production without affecting the generation and/or maintenance of T-bet-expressing Th17 cells. RNA-Seq analysis of Gata3-sufficient and Gata3-deficient CNS-infiltrating CD4+ effector T cells from mixed congenic co-transfer recipient mice revealed an important, cell-intrinsic, function of GATA3 in regulating the expression of Egr2, Bhlhe40, and Csf2. Thus, our data highlights a novel role for GATA3 in promoting and maintaining the pathogenicity of T-bet-expressing Th17 cells in EAE, via putative regulation of Egr2, Bhlhe40, and GM-CSF expression.

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Section: Methodsmentioning

confidence: 99%

GATA3 induces the pathogenicity of Th17 cells via regulating GM-CSF expression

et al. 2023

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“…While the transcription factor staining can confirm the identity of individual ILC populations, it requires fixing and permeabilization of cells and cannot be applied to isolate live ILCs. There are fluorescent reporter mice that can be used to identify ILCs without fixing/permeabilization of cells, including reporters for GATA3 ( 11 ), IL-5 and IL-13 ( 12 ), RORγt ( 13 ), and NKp46 ( 14 ). Because those genes are not specific to ILCs but are also expressed in some other lymphocytes, combinations of other cell surface markers are required to identify individual ILC populations.…”

Section: Introductionmentioning

confidence: 99%

Flow cytometric analysis of innate lymphoid cells: challenges and solutions

Sadeghalvad,

Khijakadze,

Orangi

et al. 2023

Front. Immunol.

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IntroductionThe three groups of helper innate lymphoid cells (ILCs), namely ILC1, ILC2 and ILC3, have been identified by flow cytometry by combinations of cell surface markers. Here, we review various ways ILCs are currently identified, focusing on potential problems and their solutions. The first step to identify all ILCs is to exclude other lymphocytes and myeloid cells by their lineage-specific markers (Lin). However, the Lin cocktail varies in various studies, and the definition of Lin- population containing ILCs is often ambiguous, resulting in contamination of Lin+ cells, particularly T cells.MethodWe have designed combinations of cell surface markers to identify ILC populations in various tissues of B6 mice by flow cytometry. To minimize T cell contamination, TCR/CD3ϵ antibodies were used separately from the Lin cocktail. ILCs identified by surface markers are confirmed by the expression of the transcription factors GATA3, RORγt, T-bet and Eomes.ResultILC1s in the B6 mouse liver are identified by Lin-NKp46+NK1.1+TCR/CD3ϵ−CD49a+CD49b−. However, defining ILC1s in other tissues remains a challenge. ILC2s in the lung are identified by Lin−TCR/CD3ϵ− Thy1+CD127+ST2+ whereas ILC2s in the small intestine and liver are identified by Lin−TCR/CD3ϵ−Thy1+GATA3+RORγt−. ILC3s in B6 mouse spleen, liver, lung and small intestine are identified by Lin−TCR/CD3ϵ− Thy1+CD127+RORγt+.DiscussionThe ILC population is heterogeneous and the strategies to identify ILCs have to be designed for each ILC population and tissue. Excluding T cells in all cases is crucial, and a combination of transcription factors GATA3, RORγt, T-bet, and Eomes should be used to identify ILCs. Using CD3ϵ/TCRs in a different fluorochrome not in Lin cocktail minimizes contamination of T cells specifically identify individual ILC populations in various tissues.

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