Recent developments of tools for targeted genome modification have led to new concepts in how multiple traits can be combined. Targeted genome modification is based on the use of nucleases with tailor-made specificities to introduce a DNA double-strand break (DSB) at specific target loci. A re-engineered meganuclease was designed for specific cleavage of an endogenous target sequence adjacent to a transgenic insect control locus in cotton. The combination of targeted DNA cleavage and homologous recombination–mediated repair made precise targeted insertion of additional trait genes (hppd, epsps) feasible in cotton. Targeted insertion events were recovered at a frequency of about 2% of the independently transformed embryogenic callus lines. We further demonstrated that all trait genes were inherited as a single genetic unit, which will simplify future multiple-trait introgression.

T cells are generated from hematopoietic stem cells through a highly organized developmental process, in which stage-specific molecular events drive maturation towards αβ and γδ T cells. Although many of the mechanisms that control αβ- and γδ-lineage differentiation are shared between human and mouse, important differences have also been observed. Here, we studied the regulatory dynamics of the E and ID protein encoding genes during pediatric human T cell development by evaluating changes in chromatin accessibility, histone modifications and bulk and single cell gene expression. We profiled patterns of ID/E protein activity and identified up- and downstream regulators and targets, respectively. In addition, we compared transcription of E and ID protein encoding genes in human versus mouse to predict both shared and unique activities in these species, and in prenatal versus pediatric human T cell differentiation to identify regulatory changes during development. This analysis showed a putative involvement of TCF3/E2A in the development of γδ T cells. In contrast, in αβ T cell precursors a pivotal pre-TCR-driven population with high ID gene expression and low predicted E protein activity was identified. Finally, in prenatal but not postnatal thymocytes, high HEB/TCF12 levels were found to counteract high ID levels to sustain thymic development. In summary, we uncovered novel insights in the regulation of E and ID proteins on a cross-species and cross-developmental level.

Lineage tracing studies or other genetic approaches have allowed investigators to obtain detailed insights into the molecular mechanisms that control T cell development in the mouse. However, our understanding of how this process is regulated in human has remained unclear. Using various genome-wide approaches, such as bulk RNAseq, ATACseq, ChIPseq and scRNAseq, we have recently obtained important novel insights into the molecular mechanisms through which human hematopoietic progenitor cells are converted into T-lineage committed precursors, and further into the TCRαβ and −γδ lineages. Specifically, we identified two distinct populations of thymus seeding precursor cells (TSPs) of which one population, TSP2, expressed IRF8, a transcription factor that is mainly associated with dendritic cell development. Consistently, IRF8-expressing TSPs have both T− and DC-linage potential and are characterised by the coexpression of CD127 and CD123. We discovered that Notch activation, the main initiator of T-lineage specification, induces IRF8 expression in human ETPs, in contrast to in mouse, and that IRF8 is transiently expressed during early human T cell development. As T cell precursors commit to the T-cell lineage, IRF8 is repressed by GATA3, while IRF8 levels rise for ETPs that differentiate into plasmacytoid dendritic cells. Importantly, IRF8 is required for the generation of CD34+ CD127+ CD123+ T/DC precursors and low level IRF8 activity enhances their generation from hematopoietic progenitors. Thus, using these approaches, we have identified an IRF8-dependent T cell precursor subset and revealed unique molecular events that control early T cell development in human. Supported by grants from the Fund for Scientific Research Flanders (FWO), the Foundation Against Cancer (STK), the Chan Zuckerberg Initiative (CZI) and Ghent University (BOF Ugent).

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