title: 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.
authors.affiliation: Department of Immunology, University of Toronto, Toronto, Ontario M4N 3M5, Canada.
authors.forename: Dzana D
authors.initials: DD
authors.lastname: Dervovic
authors.forename: Haydn C-Y
authors.initials: HC
authors.lastname: Liang
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Cannons
authors.forename: Alisha R
authors.initials: AR
authors.lastname: Elford
authors.forename: Mahmood
authors.initials: M
authors.lastname: Mohtashami
authors.forename: Pamela S
authors.initials: PS
authors.lastname: Ohashi
authors.forename: Pamela L
authors.initials: PL
authors.lastname: Schwartzberg
authors.forename: Juan Carlos
authors.initials: JC
authors.lastname: Zúñiga-Pflücker
mesh_terms: D000199:Actins / Q000276:immunology; D000818:Animals; D000956:Antigens, Viral / Q000276:immunology; D015704:CD4 Antigens / Q000032:analysis; D016827:CD8 Antigens / Q000032:analysis; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology*; D002135:Calcium-Binding Proteins; D019070:Cell Lineage; D002478:Cells, Cultured; D060833:Cellular Microenvironment; D060966:Clonal Selection, Antigen-Mediated; D018920:Coculture Techniques; D003433:Crosses, Genetic; D006183:H-2 Antigens / Q000276:immunology; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology; D064532:Histocompatibility Antigen H-2D / Q000276:immunology; D036341:Intercellular Signaling Peptides and Proteins / Q000276:immunology*; D038041:Lymphopoiesis / Q000276:immunology*; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008822:Mice, Transgenic; D010446:Peptide Fragments / Q000276:immunology; D011948:Receptors, Antigen, T-Cell / Q000096:biosynthesis / Q000276:immunology; D051880:Receptors, Notch / Q000502:physiology*; D015398:Signal Transduction / Q000276:immunology*; D013047:Specific Pathogen-Free Organisms; D017154:Stromal Cells / Q000166:cytology / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000276:immunology*
chemical_list: D000199:Actins; D000956:Antigens, Viral; D015704:CD4 Antigens; D016827:CD8 Antigens; D002135:Calcium-Binding Proteins; C512925:Dlk1 protein, mouse; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D064532:Histocompatibility Antigen H-2D; D036341:Intercellular Signaling Peptides and Proteins; D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D051880:Receptors, Notch