Notch signalling inhibits CD4 expression during initiation and differentiation of human T cell lineage.
The Delta/Notch signal transduction pathway is central to T cell differentiation from haemopoietic stem cells (HSCs). Although T cell development is well characterized using expression of cell surface markers, the detailed mechanisms driving differentiation have not been established. This issue becomes central with observations that adult HSCs exhibit poor differentiation towards the T cell lineage relative to neonatal or embryonic precursors. This study investigates the contribution of Notch signalling and stromal support cells to differentiation of adult and Cord Blood (CB) human HSCs, using the Notch signalling OP9Delta co-culture system. Co-cultured cells were assayed at weekly intervals during development for phenotype markers using flow cytometry. Cells were also assayed for mRNA expression at critical developmental stages. Expression of the central thymocyte marker CD4 was initiated independently of Notch signalling, while cells grown with Notch signalling had reduced expression of CD4 mRNA and protein. Interruption of Notch signalling in partially differentiated cells increased CD4 mRNA and protein expression, and promoted differentiation to CD4(+) CD8(+) T cells. We identified a set of genes related to T cell development that were initiated by Notch signalling, and also a set of genes subsequently altered by Notch signal interruption. These results demonstrate that while Notch signalling is essential for establishment of the T cell lineage, at later stages of differentiation, its removal late in differentiation promotes more efficient DP cell generation. Notch signalling adds to signals provided by stromal cells to allow HSCs to differentiate to T cells via initiation of transcription factors such as HES1, GATA3 and TCF7. We also identify gene expression profile differences that may account for low generation of T cells from adult HSCs.
Blood Stem Cells and Cancer Research, St Vincent's Centre for Applied Medical Research, Sydney, New South Wales, Australia.
Stephen M
SM
Carlin
Melissa L M
ML
Khoo
David D
DD
Ma
John J
JJ
Moore
D000328:Adult; D015704:CD4 Antigens / Q000096:biosynthesis*; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology* / Q000502:physiology; D002454:Cell Differentiation / Q000502:physiology*; D019070:Cell Lineage; D018920:Coculture Techniques; D004151:Dipeptides / Q000494:pharmacology; D005312:Fetal Blood / Q000166:cytology; D006801:Humans; D012333:RNA, Messenger / Q000378:metabolism; D051880:Receptors, Notch / Q000502:physiology*; D015398:Signal Transduction
D015704:CD4 Antigens; D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D012333:RNA, Messenger; D051880:Receptors, Notch