Galpha13 mediates a signal that is essential for proliferation and survival of thymocyte progenitors

V McNeil Coffield, Whitney S Helms, Qi Jiang, Lishan Su, V McNeil Coffield, Whitney S Helms, Qi Jiang, Lishan Su

Abstract

G protein signaling via the Galpha12 family (Galpha12 and Galpha13) has not been well studied in T cells. To investigate whether Galpha12 and Galpha13 are involved in thymopoiesis, we expressed the regulator of G protein signaling domain of p115RhoGEF to inhibit Galpha12 and Galpha13 during thymopoiesis. Fetal thymus organ cultures seeded with p115DeltaDH-expressing progenitor cells showed impaired thymopoiesis with a block at the CD4-CD8-CD44-CD25+ (DN3) stage. Using Galpha13 or Galpha12 minigenes, we demonstrated that Galpha13, but not Galpha12, is required for thymopoiesis. T progenitor cells expressing p115DeltaDH showed reduced proliferation and increased cell death. T cell receptor stimulation of the fetal thymus organ cultures did not rescue the block. Overexpression of the antiapoptotic gene Bcl2 rescued the defect in DN3 cells and partially rescued T cell development. Therefore, Galpha13-mediated signaling is necessary in early thymocyte proliferation and survival.

Figures

Figure 1.
Figure 1.
p115ΔDH is stably expressed in both human T cell lines and mouse FL progenitor cells. (a) The DH domain of the p115RhoGEF was deleted to produce p115ΔDH, which contains only the RGS and PH domains. The p115ΔDH was cloned into a retroviral vector, which also encodes enhanced GFP under the control of a phosphoglycerate kinase (PGK) promoter. (b) Flow cytometric analysis of GFP expression in transduced Jurkat T cells at 2, 11, and 21 d posttransduction. (c) Flow cytometric analysis of GFP expression in transduced E14/15 FL progenitor cells at 2 and 7 d posttransduction in culture. (d) 105 transduced Jurkat T cells and 105 transduced fetal liver cells were analyzed by Western blot for p115ΔDH expression at 21 and 8 d posttransduction, respectively.
Figure 2.
Figure 2.
The expression of p115ΔDH inhibits T cell development. 2-dG–depleted E15 thymic lobes were seeded with transduced E14/15 FL progenitor cells and cultured for 3 wk. 24 lobes from four different experiments were analyzed for the percentage (a and b) and the number (b) of GFP+ and GFP− cells. The percentage (c) and number of GFP+ (d) and GFP− (e) CD4−CD8−, CD4+CD8+, CD4+CD8−, and CD4−CD8+ cells generated per lobe were also analyzed. Bars indicate SE; **P < 0.005 compared with vector.
Figure 3.
Figure 3.
The expression of p115ΔDH inhibits early thymopoiesis. 2-dG–depleted E15 thymic lobes were seeded with transduced E14/15 FL progenitor cells and cultured as above. (a) Thymocytes that were negative for expression of CD3, CD4, and CD8 were analyzed for percentages of CD44+CD25− (DN1), CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4). (b) The total number of DN1, DN2, DN3, and DN4 cells generated per lobe from four lobes for vector or p115ΔDH is summarized. The data is representative of four independent experiments. Bars indicate SE; *P < 0.05; **P < 0.005 compared with vector.
Figure 4.
Figure 4.
The expression of the Gα13 minigene, but not the Gα12 minigene, inhibits early thymopoiesis. Six FTOC lobes for vector or p115ΔDH were individually analyzed to determine the total number of GFP+ (a), total CD3−CD4−CD8− (b), and DN1, DN2, DN3, and DN4 (c) cells generated per lobe. The data is representative of two independent experiments. Bars indicate SE; *P < 0.05 compared with vector.
Figure 5.
Figure 5.
The inhibition of Gα13 signaling results in decreased DN proliferation and increased cell death. 2-dG–depleted E15 thymic lobes were seeded with transduced E14/15 FL progenitor cells. (a) Duplicate pooled samples consisting of six lobes for both vector and p115ΔDH were stained with Hoechst 33342 and CD3/CD4/CD8/CD25/CD44. At least 5 × 105 GFP+ cells were analyzed to determine the percentage of cells with greater than 2N DNA (S/G2 phases). (b and c) Three lobes for vector or p115ΔDH were harvested after 2 wk in FTOC culture when most thymocytes are between DN1-DN3. Thymocytes were gated on CD25+ (DN2, DN3) or CD25− (DN1) and individually analyzed for annexin V mean fluorescence intensity (MFI) and percentage of annexin V+ cells generated per lobe. Bars indicate SE, and p-values are indicated.
Figure 6.
Figure 6.
TCR stimulation does not rescue the block in thymopoiesis. 2-dG–depleted E15 thymic lobes were seeded with transduced E14/15 FL progenitor cells and cultured for 2 wk. Lobes were stimulated with 10 μg/ml of anti-CD3 mAb from day 14 to 21 in culture. Fresh media containing anti-CD3 was added every 2 d. Six lobes per sample were individually analyzed to determine the total number of GFP+ (a) and DN1, DN2, DN3, and DN4 (b) cells generated per lobe. Bars indicate SE; *P < 0.05; **P < 0.005 as vector or p115DDH samples compared with unstimulated controls.
Figure 7.
Figure 7.
Overexpression of Bcl2 rescues the block in DN3 development. 2-dG–depleted E15 thymic lobes were seeded with wild-type or littermate Bcl2-tg E15 FL progenitor cells transduced with vector or p115ΔDH and cultured for 3 wk. Six lobes per sample were individually analyzed to determine the number of total GFP+ (a), total CD3−CD4−CD8− (b), and DN1, DN2, DN3, and DN4 (c) cells generated per lobe. Bars indicate SE; *P < 0.05; **P < 0.005 compared with vector.

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