Cellular and molecular profiling of CD8 T cell differentiation with respect to positive and negative selection in vitro reveals a role for Notch signaling (111.45)

Abstract

Abstract Differentiation of functionally mature 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 thymus, signaling through Notch receptors directs T cell progenitors to commit to the T cell lineage, undergo early T cell differentiation, and progress to the CD4+CD8+ (DP) stage of T cell development. However, it is still unclear whether Notch signals are prerequisite for differentiation or commitment of DP cells to CD8 SP stage of T cell development. Here, we demonstrate that ectopic expression of the Notch-ligand by OP9 stromal cells allows for efficient differentiation of conventional effector CD8 T cells from SAP-/- bone marrow (BM)-derived hematopoietic stem cells (HSCs). We also prove that OP9-DL1 stromal cells restrict differentiation of innate CD8 T cells but allow for differentiation of IL17-producing CD8 T cells from BM-HSCs isolated from Itk-/-Rlk-/- (DKO) mice. We further reveal that the process of positive and negative selection in vitro is constrained by peptide-MHC (pMHC) class I expressed by the OP9 stromal cells. Finally, using a T cell receptor (TCR) transgenic model we disclose that the commitment of DP precursors to the CD8 T cell lineage is facilitated by Notch signaling. Our findings further establish the requirement for Notch receptor-ligand interactions throughout intrathymic T cell differentiation.