Cellular and Molecular Requirements for the Selection of In Vitro–Generated CD8 T Cells Reveal a Role for Notch

Abstract

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.