Abstract
The development of functional T cells requires receptor-mediated transition through multiple checkpoints in the thymus. Double negative 3 (DN3) thymocytes are selected for the presence of a rearranged TCR beta chain in a process termed β-selection which requires signalling via the pre-TCR, Notch1 and CXCL12. Signal integration by these receptors converges on core pathways including the Phosphatidylinositol–3-kinase (PI3K) pathway. Glycogen Synthase Kinase 3 (GSK3) is generally thought to be negatively regulated by the PI3K pathway but its role in β-selection has not been characterised. Here we show that developmental progression of DN3 thymocytes is promoted following inhibition of GSK3 by the synthetic compound CHIR99021. CHIR99021 allows differentiation in the absence of pre-TCR-, Notch1- or CXCL12-mediated signalling. It antagonizes IL-7-mediated inhibition of DP thymocyte differentiation and increases IL-7-promoted cell recovery. These data indicate a potentially important role for inactivation of GSK3 during β-selection. They might help to establish an in vitro stromal cell-free culture system of thymocyte development and offer a new platform for screening regulators of proliferation, differentiation and apoptosis.
Highlights
T cells expressing the ab T cell receptor (TCR) are formed in the thymus
We have previously reported that inhibition of Glycogen Synthase Kinase 3 (GSK3) with CHIR99021 promoted proliferation and differentiation of DN3a thymocytes cultured on plate-bound Delta-like 4 (DL4) in the presence of CXCL12 [11]
To further investigate the effect of CHIR99021, we have used an established model where Double negative 3 (DN3) cells are cultured on OP9-Delta-like 1 (OP9-DL1) stromal cells that are deficient in macrophage-colony stimulating factor (M-CSF) and transduced by a retrovirus expressing DL1 [25]
Summary
T cells expressing the ab T cell receptor (TCR) are formed in the thymus. Stage thymocytes are termed doublenegative (DN) as they do not express the cell surface glycoproteins CD4 and CD8. DN thymocytes develop into immature CD8+ single positive (iSP) cells prior to expression of both CD4 and CD8 that defines the double positive (DP) stage. Prior to b-selection, thymocytes can be defined by cell surface staining as CD442CD25highCD98lowCD27low DN3 cells (referred to as DN3a) [3,4]. Cells in which TCRb has been rearranged successfully to form a complex with precursor TCR a-chain and CD3 increase in size as well as cell surface expression of CD5, CD98 and CD27 and are referred to as DN3b [3,4]. Failure to rearrange TCRb and by consequence to undergo b-selection results in apoptosis [5]
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