Interaction of Ras with p110γ is required for thymic β-selection in the mouse

Abstract

Thymocytes are tested for productive rearrangement of the tcrb locus by expression of a pre-TCR in a process termed β-selection, which requires both Notch1 and CXCR4 signaling. It has been shown that activation of the GTPase Ras allows thymocytes to proliferate and differentiate in the absence of a Pre-TCR; the direct targets of Ras at this checkpoint have not been identified, however. Mice with a mutant allele of p110γ unable to bind active Ras revealed that CXCR4-mediated PI3K activation is Ras dependent. The Ras-p110γ interaction was necessary for efficient β-selection-promoted proliferation but was dispensable for the survival or differentiation of thymocytes. Uncoupling Ras from p110γ provides unambiguous identification of a Ras interaction required for thymic β-selection.

Figure 1. Reduced thymic cellularity in mice with a Ras-binding mutant of p110γ

(A) Flow cytometric analysis of CD4 and CD8 expression on T lymphocytes in the thymus from WT, p110γ^D/D and p110γ^D/D × p110δ^−/− mice. The percentages of CD4⁺CD8⁺ double positive (DP) and CD4⁻CD8⁻ double negative (DN) cells are shown. (B) Flow cytometric analysis of the DN thymocytes (identified as Thy1⁺ and [CD4/CD8/CD44/B220/CD11b/NK1.1/Gr1/Ter119/γδTCR]^neg) and (C) the DN3/4 subsets. The percentages of CD25^hiCD98^lo (DN3a), CD25^intCD98^hi (DN3b) and CD25^loCD98^hi (DN4) are shown. (D) Thymic cellularity and the numbers of DN3a, DN3b, DN4 and DP thymocytes for each genotype. Graphs represent the mean and SEM (n ≥ 5).

Figure 2. Competitve bone marrow chimeras reveal defective β-selection in p110γ^D/D thymocytes

(A) Example of FACS analysis used to distinguish B6.SJL cell origin based upon expression of the CD45.1 allotypic marker. The gating strategy for CD5^loCD69^lo DP and CD5^hiCD69^hi DP thymocytes is also shown. (B) The contribution of B6.SJL (CD45.1⁺) (■) and WT (B6), p110γ^D/D or p110γ^−/− CD45.1^neg cells (□) to thymic T cell subsets, splenic T cells and splenic B cells six weeks after bone marrow reconstitution. The graphs show the mean and SEM from analysis of 10 chimaeric mice.

Figure 3. The Ras-p110γ signalling pathway is required in DN3a and DN3b cells

(A) CD4 and CD8 expression following culture of DN3a (CD25^hiCD98^lo) and DN3b (CD25^intCD98^hi) cells on OP9-DL1. The percentages of cells that remain CD4⁻CD8⁻ are shown. (B) The number of live cells recovered from WT (■), p110γ^D/D and p110γ^−/− (□) cultures after three days. The seeding number for each cell type was 5 × 10⁴ DN3a and 2.5 × 10⁴ DN3b. Graphs show the mean and SD from 3 independent groups.

Figure 4. Analysis of differentiation, proliferation and survival responses at β-selection

(A) Division profiles of sorted WT, p110γ^D/D and p110γ^−/− DN3a cells (CD25^hiCD98^lo) after culture on OP9-DL1 for the indicated times. (B) Expression of CD8 (■) and CD4 (○) at each division cycle in WT, p110γ^D/D and p110γ^−/− cultures after 72 hours. (C) Detection of caspase-3⁺ cells after 14 and 60 hours of culture. FACS plots show the gates used to identify cells which have divided ≥ 2 times and those that remain undivided that are either caspase-3 positive or negative. Graphs show data from three independent experiments where WT = ■, p110γ^D/D = and p110γ^−/− = □. (D) DNA content profiles from cells cultured for 60hrs. The gating strategy used to identify divided and undivided populations is the same as in Figure 3D. Coloured background on FACS plots show the delineation of cells in G0/1 (blue), S (green) and G2/M (red). Numbers on FACS plots show percentage of cells in G0/1. Graphs show data from three independent experiments where G0/1 = ■, S = □ and G2/M = . For all statistical analysis a repeated measures ANOVA test was performed. NS = not significant, * = p<0.05, ** = p<0.01.

Figure 5. Defective induction of PI3K signalling in p110γ^D/D DN3 cells upon SDF1α stimulation

(A) FACS histograms showing the detection of phosphorylated-Akt (at S473) in DN3 cells from WT and p110γ^D/D thymi after stimulation with SDF1α. (B) Graph showing fold induction of p-Akt, (measured as median fluorescence of stimulated / median fluorescence of unstimulated), in WT (■) and p110γ^D/D () DN3 cells after SDF1α stimulation. Graph shows the mean and SD from analysis of five mice.

Figure 6. Intact PI3K but defective ERK activation in Rag2^−/− and Vav1/2/3^−/− DN3 cells following stimulation with SDF1α

Induction of (A) p-Akt (S473) and (B) p-Erk1/2 (T202/Y204) in WT (), Vav1/2/3^−/− (□) and Rag2^−/− (○) DN3 cells after stimulation with 10nM SDF1α. Graph shows the mean and SD from analysis of four-six mice.

Figure 7. CXCR4 inhibition affects the proliferative response of DN3 cells

(A) Division profiles of sorted WT DN3 cells (CD25^hiCD98^lo) cultured on OP9-DL1 in the presence of 0, 0.1, 1 and 10μM of the CXCR4 antagonist AMD3100. (B) Expression of CD8 (■) and CD4 (○) at each division cycle at 72 hours in untreated and cultured supplemented with 0.1μM, 1μM and 10μM of AMD3100. (C) FACS plots show the gates used to identify cells which have divided ≥ 2 times and those that remain undivided that are either caspase-3 positive or negative. Graphs show aggregate data from three independent experiments; no AMD3100 (■), 0.1μM , 1μM and 10μM (□). (D) DNA content profiles from cells cultured for 60hrs. The gating strategy used to identify divided and undivided populations is the same as in Figure 3D. Graphs show data from three independent experiments where G0/1 = ■, S = □ and G2/M = . For all statistical analysis a repeated measures ANOVA test was performed. NS = not significant, * = p<0.05.