Thymic development beyond β-selection requires phosphatidylinositol 3-kinase activation by CXCR4

Abstract

T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110δ, and class IB, p110γ catalytic subunits. However, the receptors on immature T cells by which each of these PI3Ks are activated have not been identified, nor has the mechanism behind their functional redundancy in the thymus. Here, we show that PI3K signaling from the preTCR requires p110δ, but not p110γ. Mice deficient for the class IB regulatory subunit p101 demonstrated the requirement for p101 in T cell development, implicating G protein–coupled receptor signaling in β-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of β-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos. Finally, we demonstrate that CXCL12, the ligand for CXCR4, allows for Notch-dependent differentiation of DN3 thymocytes in the absence of supporting stromal cells. These findings establish a role for CXCR4-mediated PI3K signaling that, together with signals from Notch and the preTCR, contributes to continued T cell development beyond β-selection.

Retinoic Acid Receptor γ Regulates B and T Lymphopoiesis via Nestin-Expressing Cells in the Bone Marrow and Thymic Microenvironments

“…Homing properties and the numbers of early thymic progenitors were not determined but would be of interest in the future. However, our observation of a reduction of DN4 T cells upon Nes cell-specific deletion of Rarg fits with the importance of CXCL12 in DN3-to-DN4 transition (49,50). Because of significant germline deletion issues, we have not been able to obtain sufficient mice to determine whether loss of Rarg in Nesexpressing CDR1 + EpCAM 2 thymic stromal cells results in deregulated expression of Cxcl12 or Scf, but these studies would also be of interest.…”

“…5B). The reduction of DN1 and DN4 cells in the Nes:Rarg D/D R26eYFP mice was indicative of a CXCL12 defect, because CXCL12 is required for homing of immature T cell progenitors to the thymus (48), in addition to b selection and differentiation beyond the DN3 stage (49,50). To determine whether the CDR1 + stroma expressed Cxcl12, we separated CDR1 + stromal cells based on eYFP expression and performed gene expression analysis of Cxcl12 and other T cell growth factors.…”

The preTCR‐dependent DN3 to DP transition requires Notch signaling, is improved by CXCL12 signaling and is inhibited by IL‐7 signaling

“…Recovery of CD27 low cells was considerably reduced, again confirming that survival (Fig. 3) and consequent differentiation of DN3 cells are dependent upon a productive TCR-b rearrangement.While in vivo, CXCR4 was shown to be required for the progression of thymocyte differentiation beyond the DN1 stage [49], a recent report suggested that it also contributed to the b-selection-dependent transition from DN3 to DP [41,42]. We could show that the dependence on the CXCR4 ligand CXCL12 (also known as SDF-1a) was not absolute, but its addition did result in a four-fold increase in cell recovery.…”

“…It was recently suggested that PI3 kinase signaling mediated by CXCR4 is also involved in the DN3 to DP transition [40][41][42]. To evaluate the contribution of CXCL12 during this differentiation step in the present culture system, we sorted DN3 cells and cultured them in the presence of 10 nM CXCL12.…”

“…Thus, proliferation and differentiation of DN3 cells into DPs on plate-bound DL4 are preTCR dependent. The finding that sorted DN3 cells from Rag-2-deficient mice did not even survive for 3 days in the present culture system further supports this conclusion.It was recently suggested that PI3 kinase signaling mediated by CXCR4 is also involved in the DN3 to DP transition [40][41][42]. To evaluate the contribution of CXCL12 during this differentiation step in the present culture system, we sorted DN3 cells and cultured them in the presence of 10 nM CXCL12.…”