Cellular and molecular requirements for the selection of in vitro-generated CD8 T cells reveal a role for Notch.

Differentiation of CD8 single-positive (SP) T cells is predicated by the ability of lymphocyte progenitors to integrate multiple signaling cues provided by the thymic microenvironment. In the thymus and the OP9-DL1 system for T cell development, Notch signals are required for progenitors to commit to the T cell lineage and necessary for their progression to the CD4(+)CD8(+) double-positive (DP) stage of T cell development. However, it remains unclear whether Notch is a prerequisite for the differentiation of DP cells to the CD8 SP stage of development. In this study, we demonstrate that Notch receptor-ligand interactions allow for efficient differentiation and selection of conventional CD8 T cells from bone marrow-derived hematopoietic stem cells. However, bone marrow-derived hematopoietic stem cells isolated from Itk(-/-)Rlk(-/-) mice gave rise to T cells with decreased IFN-γ production, but gained the ability to produce IL-17. We further reveal that positive and negative selection in vitro are constrained by peptide-MHC class I expressed on OP9 cells. Finally, using an MHC class I-restricted TCR-transgenic model, we show that the commitment of DP precursors to the CD8 T cell lineage is dependent on Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout T cell differentiation, including the final step of CD8 SP selection.

Hematopoiesis: from start to immune reconstitution potential.

The study of hematopoiesis has been a focus for developmental biologists for over 100 years. What started as a series of microscopic observations in different animal model systems has since evolved into studies of gene expression and regulation, and subsequent protein-protein interactions, cell surface protein expression profiling, and functional mapping of cell fates. In this review, we will discuss the milestone discoveries that have been achieved in the field of hematopoietic development, as well as the techniques that have been employed. Finally, we look toward the future and consider unresolved questions. We also reflect on one of the earliest realizations made in this area of study: that hematopoiesis is evolutionarily conserved, and as a consequence we reflect on the impacts of early and current discoveries and their clinical implications. The future direction of the study of hematopoietic stem cells will probably make use of pluripotent stem cells to yield specific immune cell lineages and eventual clinical applications.

Directed differentiation of embryonic stem cells to the T-lymphocyte lineage.

Hematopoiesis is the highly regulated and complex process by which blood cells are formed. Hematopoiesis can be achieved in vitro by the differentiation of embryonic stem cells (ESCs) into hematopoietic lineage cells. Differentiation of ESCs initially gives rise to mesoderm colonies that go on to form hemangioblast cells, which possess endothelial and hematopoietic lineage potential. While the differentiation of several hematopoietic lineages from ESCs, such as erythrocytes and macrophages, can be easily recapitulated in vitro, T-cell differentiation requires additional Notch-dependent signals for their generation. Keeping with this, ESCs induced to differentiate with OP-9 cells, a bone marrow-derived stromal cell line, give rise to erythro-myeloid cells and B lymphocytes, while the expression of an appropriate Notch ligand, such as Delta-like 1, on OP-9 cells (OP9-DL1) is required to support the generation of T-cells in vitro. Here, we describe an updated and streamlined protocol for the generation of T-lineage cells from mouse ESCs cultured on OP9-DL1 cells. This approach can facilitate studies aimed to assess the effects of environmental and genetic manipulations at various stages of T-cell development.