Limits...
Thymic development beyond beta-selection requires phosphatidylinositol 3-kinase activation by CXCR4.

Janas ML, Varano G, Gudmundsson K, Noda M, Nagasawa T, Turner M - J. Exp. Med. (2009)

Bottom Line: Here, we show that PI3K signaling from the preTCR requires p110delta, but not p110gamma.We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos.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 beta-selection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Lymphocyte Signaling and Development, the Babraham Institute, Babraham, Cambridge, CB22 3AT England, UK. michelle.janas@bbsrc.ac.uk

ABSTRACT
T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110delta, and class IB, p110gamma 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 p110delta, but not p110gamma. 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 beta-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-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 beta-selection.

Show MeSH

Related in: MedlinePlus

Inhibition of CXCR4 signaling blocks DN3 cell differentiation. 4 × 104 DN3 cells from WT mice were cultured on OP9-DL1 stromal cells for 3 d in the presence of increasing concentrations of AMD3100 or GSK812397A. Cells were harvested, analyzed for the expression of CD4 and CD8 (A), and absolute numbers of CD4+CD8+ DP and CD4−CD8− DN cells recovered (B). Graphs show the mean and SD. n = 3. This experiment is representative of three independent replicates.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2812547&req=5

fig5: Inhibition of CXCR4 signaling blocks DN3 cell differentiation. 4 × 104 DN3 cells from WT mice were cultured on OP9-DL1 stromal cells for 3 d in the presence of increasing concentrations of AMD3100 or GSK812397A. Cells were harvested, analyzed for the expression of CD4 and CD8 (A), and absolute numbers of CD4+CD8+ DP and CD4−CD8− DN cells recovered (B). Graphs show the mean and SD. n = 3. This experiment is representative of three independent replicates.

Mentions: The in vivo and in vitro requirement for p101 at the β-selection checkpoint prompted us to seek evidence of a role for a GPCR in this process. We observed that CXCL12, the ligand for CXCR4, was secreted by the OP9-DL1 stromal line (Fig. S3 A). We examined whether CXCL12 was required for DN3 proliferation and differentiation by culturing sorted DN3 cells on OP9-DL1 in the presence of the CXCR4 antagonist AMD3100. This small molecule binds to CXCR4 with high specificity and inhibits chemotaxis of human PBMCs to CXCL12 with an IC50 of 0.16 µM (Hatse et al., 2002). Culturing DN3 cells in the presence of AMD3100 inhibited their ability to become CD4+CD8+ (Fig. 5). The numbers of DP cells recovered after 3 d of culture were decreased in a dose-dependent manner, indicating that in the absence of CXCR4 signaling DN3 cells had reduced proliferation and/or survival. This effect on DN3 cells was also seen with a chemically distinct small molecule inhibitor of CXCR4 termed GSK812397A, which inhibits the CXCL12-mediated chemotaxis of the U937 cell line with an IC50 of <1 nM (GlaxoSmithKline; personal communication). These results show that signaling through CXCR4 contributes to the development of DN3 cells to the DP developmental stage.


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

Janas ML, Varano G, Gudmundsson K, Noda M, Nagasawa T, Turner M - J. Exp. Med. (2009)

Inhibition of CXCR4 signaling blocks DN3 cell differentiation. 4 × 104 DN3 cells from WT mice were cultured on OP9-DL1 stromal cells for 3 d in the presence of increasing concentrations of AMD3100 or GSK812397A. Cells were harvested, analyzed for the expression of CD4 and CD8 (A), and absolute numbers of CD4+CD8+ DP and CD4−CD8− DN cells recovered (B). Graphs show the mean and SD. n = 3. This experiment is representative of three independent replicates.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2812547&req=5

fig5: Inhibition of CXCR4 signaling blocks DN3 cell differentiation. 4 × 104 DN3 cells from WT mice were cultured on OP9-DL1 stromal cells for 3 d in the presence of increasing concentrations of AMD3100 or GSK812397A. Cells were harvested, analyzed for the expression of CD4 and CD8 (A), and absolute numbers of CD4+CD8+ DP and CD4−CD8− DN cells recovered (B). Graphs show the mean and SD. n = 3. This experiment is representative of three independent replicates.
Mentions: The in vivo and in vitro requirement for p101 at the β-selection checkpoint prompted us to seek evidence of a role for a GPCR in this process. We observed that CXCL12, the ligand for CXCR4, was secreted by the OP9-DL1 stromal line (Fig. S3 A). We examined whether CXCL12 was required for DN3 proliferation and differentiation by culturing sorted DN3 cells on OP9-DL1 in the presence of the CXCR4 antagonist AMD3100. This small molecule binds to CXCR4 with high specificity and inhibits chemotaxis of human PBMCs to CXCL12 with an IC50 of 0.16 µM (Hatse et al., 2002). Culturing DN3 cells in the presence of AMD3100 inhibited their ability to become CD4+CD8+ (Fig. 5). The numbers of DP cells recovered after 3 d of culture were decreased in a dose-dependent manner, indicating that in the absence of CXCR4 signaling DN3 cells had reduced proliferation and/or survival. This effect on DN3 cells was also seen with a chemically distinct small molecule inhibitor of CXCR4 termed GSK812397A, which inhibits the CXCL12-mediated chemotaxis of the U937 cell line with an IC50 of <1 nM (GlaxoSmithKline; personal communication). These results show that signaling through CXCR4 contributes to the development of DN3 cells to the DP developmental stage.

Bottom Line: Here, we show that PI3K signaling from the preTCR requires p110delta, but not p110gamma.We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-selection and demonstrated a requirement for CXCR4 during thymic development in CXCR4-deficient embryos.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 beta-selection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Lymphocyte Signaling and Development, the Babraham Institute, Babraham, Cambridge, CB22 3AT England, UK. michelle.janas@bbsrc.ac.uk

ABSTRACT
T cell development requires phosphatidylinositol 3-kinase (PI3K) signaling with contributions from both the class IA, p110delta, and class IB, p110gamma 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 p110delta, but not p110gamma. 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 beta-selection. We found evidence of a role for CXCR4 using small molecule antagonists in an in vitro model of beta-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 beta-selection.

Show MeSH
Related in: MedlinePlus