Transcription Factor GATA2 Is Inevitable for the Survival and Proper Differentiation of Thymic Multipotent Progenitor Cells By Gene Expression Orchestration

Proper differentiation of T cells in the thymus is inevitable for the establishment of acquired immunity. Lymphoid-primed multipotent progenitors (LMPPs) emigrate from the bone marrow (BM) to the thymus for becoming earliest T cell progenitors, so-called early T cell progenitors (ETPs). Previous studies have shown that ETPs retain not only T cell but also innate immune cell (i.e., myeloid cells) differentiation potentials. Therefore, ETPs might be the bifurcation point of innate and adaptive immunity in thymus. However, still little is known about the mechanism by which the differentiation fates are determined in ETPs and the cellular heterogeneity in ETP subset. Since transcription factors that possess important roles in hematopoietic stem and progenitor cells (HSPCs) may play significant roles in the early differentiation of T cells as legacy stem cell genes, we sought to investigate the roles for GATA2, which is known to regulate the HSPC maintenance and myeloid differentiation, in ETPs.

First, we revealed the GATA2 expression is preserved in ETPs and sharply disappear with their differentiation (GATA2 expressions were almost undetectable from double-negative 2 cells even by qPCR). To reveal the roles for GATA2 in T cell hematopoiesis, we analyzed Gata2^fl/fl; Mx1-Cre mice in which GATA2 was deletable in hematopoietic cells by polyI:C administration (Gata2 KO). Gata2 KO mice showed a smaller thymus and a lower number of thymic T cells, including ETPs and double-positive (DP; CD4⁺CD8⁺) cells, than control. To rule out contributions of potential GATA2 deletions in non-hematopoietic cells, CD45.1⁺ wild-type unfractionated BM cells were transplanted into Gata2 KO or control mice. There were no particular differences in T cell hematopoiesis from CD45.1⁺ donor cells between them, supporting the notion that hematopoietic cell intrinsic GATA2 are inevitable for the proper T cell hematopoiesis. To further reveal the roles for GATA2 in ETPs, we performed global transcriptomic analysis of ETPs (RNA-seq; Gata2 KO vs control). Gene set enrichment analysis (GSEA) revealed that signatures associated with cell cycle regulation, hematopoietic progenitor cells and myeloid cells were down-regulated in Gata2 KO ETPs, suggesting that GATA2 is involved in the survival and myeloid differentiation potentials in ETPs. By taking the advantage of novel GATA2 reporter mice (GATA2-VENUS mice) in which GATA2 protein levels are detectable by flow cytometry, we revealed that the ETP fraction contained a mixture of cells with high or low GATA2 expression. To be noted, the protein expression intensity of GATA2 in GATA2-high ETPs was almost equivalent to that of LMPPs. These observations suggest that GATA2-high ETPs might be a thymic multipotent progenitors (T-MPPs), the earliest thymic progenitors in which T or myeloid cell differentiation commitment is defined. To elucidate the mechanism by which GATA2 regulates the survival and differentiation of T-MPPs, we integrated publicly available chromatin immunoprecipitation followed by sequencing data targeting GATA2 (GATA2 ChIP-seq) and our ETP RNA-seq data. We found several key genes in HSPCs maintenance (i.e. Lyl1) or myeloid differentiation (i.e. Itga2b) as potential targets of GATA2. Furthermore, we sought to reveal the effect of GATA2 deletion in progenitor cells to their progenies. To this end, we performed RNA-seq of DP cells (Gata2 KO vs control). GSEA revealed that cell cycle related gene signature was repressed in Gata2 KO DP cells compared to control. To be noted, in CD4-Cre dependent GATA2 deletion model (Gata2^fl/fl:CD4-Cre mice), there were almost no particular cell number and transcriptomic differences, which was also analyzed by RNA-seq, in DP cells compared to control. These results suggest that GATA2 in GATA2-high ETPs, but not in DP cells, may affect the feature of DP cells, potentially by epigenetic memory.

Collectively, this study revealed that GATA2 contributes to T cell hematopoiesis. GATA2 expression was maintained in a part of the ETP subset (GATA2-high ETPs) which might be equivalent to T-MPPs. GATA2 may affect the differentiation trajectory and function from GATA2-high ETPs to their progenies. Further study of this mechanism will help us to understand how the acquired and innate immune bifurcation is defined, which might pave the way to overcome T cell development related disorders, such as T-cell/myeloid mixed-phenotype acute leukemia.