Analysis of thymocyte development reveals that the GTPase RhoA is a positive regulator of T cell receptor responses in vivo

Abstract

Loss of function of the guanine nucleotide binding protein RhoA blocks pre-T cell differentiation and survival indicating that this GTPase is a critical signaling molecule during early thymocyte development. Previous work has shown that the Rho family GTPase Rac-1 can initiate changes in actin dynamics necessary and sufficient for pre-T cell development. The present data now show that Rac-1 actions in pre-T cells require Rho function but that RhoA cannot substitute for Rac-1 and induce the actin cytoskeletal changes necessary for pre-T cell development. Activation of Rho is thus not sufficient to induce pre-T cell differentiation or survival in the absence of the pre-T cell receptor (TCR). The failure of RhoA activation to impact on pre-TCR-mediated signaling was in marked contrast to its actions on T cell responses mediated by the mature TCR alpha/beta complex. Cells expressing active RhoA were thus hyperresponsive in the context of TCR-induced proliferation in vitro and in vivo showed augmented positive selection of thymocytes expressing defined TCR complexes. This reveals that RhoA function is not only important for pre-T cells but also plays a role in determining the fate of mature T cells.

Figure 1

Thymic phenotype of CD2-V14RhoA transgenic mice. (A) Diagram of the CD2-V14RhoA construct. A 0.7-kb fragment containing human V14RhoA myc-tagged cDNA was subcloned at Sma I site in the human CD2 vector. Position of hCD2 promoter and LCR are shown. Arrows indicate the position of transgene-specific primer pairs used in PCR-based screening for transgene-carrying mice. (B) Thymic expression of V14RhoA transgene. Proteins of thymocytes lysates from NLC mice and transgenic mice (V14RhoA) (10⁶ cells) were separated on a 15% SDS-PAGE. Detection of endogenous RhoA and transgene V14RhoA was performed by Western blot analysis using a specific monoclonal anti-RhoA antibody. Results are shown for the two different transgenic lines generated (2029.1 and 2036.F). Top arrowhead, myc-tagged transgenic V14RhoA; bottom arrowhead, endogenous RhoA. (C) Absolute thymocyte number in CD2-V14RhoA mice. Thymocyte cell numbers from 6–8-wk-old NLC (n = 8) and V14RhoA are shown (n = 10). (D) CD4/CD8 profiles on V14RhoA thymocytes. Thymocytes from NLC and V14RhoA mice were isolated, stained with anti-CD4-PE and anti-CD8-FITC, and analyzed by flow cytometry. Two dimensional dot plots representative of five independent experiments are shown.

Figure 1

Thymic phenotype of CD2-V14RhoA transgenic mice. (A) Diagram of the CD2-V14RhoA construct. A 0.7-kb fragment containing human V14RhoA myc-tagged cDNA was subcloned at Sma I site in the human CD2 vector. Position of hCD2 promoter and LCR are shown. Arrows indicate the position of transgene-specific primer pairs used in PCR-based screening for transgene-carrying mice. (B) Thymic expression of V14RhoA transgene. Proteins of thymocytes lysates from NLC mice and transgenic mice (V14RhoA) (10⁶ cells) were separated on a 15% SDS-PAGE. Detection of endogenous RhoA and transgene V14RhoA was performed by Western blot analysis using a specific monoclonal anti-RhoA antibody. Results are shown for the two different transgenic lines generated (2029.1 and 2036.F). Top arrowhead, myc-tagged transgenic V14RhoA; bottom arrowhead, endogenous RhoA. (C) Absolute thymocyte number in CD2-V14RhoA mice. Thymocyte cell numbers from 6–8-wk-old NLC (n = 8) and V14RhoA are shown (n = 10). (D) CD4/CD8 profiles on V14RhoA thymocytes. Thymocytes from NLC and V14RhoA mice were isolated, stained with anti-CD4-PE and anti-CD8-FITC, and analyzed by flow cytometry. Two dimensional dot plots representative of five independent experiments are shown.

Figure 1

Thymic phenotype of CD2-V14RhoA transgenic mice. (A) Diagram of the CD2-V14RhoA construct. A 0.7-kb fragment containing human V14RhoA myc-tagged cDNA was subcloned at Sma I site in the human CD2 vector. Position of hCD2 promoter and LCR are shown. Arrows indicate the position of transgene-specific primer pairs used in PCR-based screening for transgene-carrying mice. (B) Thymic expression of V14RhoA transgene. Proteins of thymocytes lysates from NLC mice and transgenic mice (V14RhoA) (10⁶ cells) were separated on a 15% SDS-PAGE. Detection of endogenous RhoA and transgene V14RhoA was performed by Western blot analysis using a specific monoclonal anti-RhoA antibody. Results are shown for the two different transgenic lines generated (2029.1 and 2036.F). Top arrowhead, myc-tagged transgenic V14RhoA; bottom arrowhead, endogenous RhoA. (C) Absolute thymocyte number in CD2-V14RhoA mice. Thymocyte cell numbers from 6–8-wk-old NLC (n = 8) and V14RhoA are shown (n = 10). (D) CD4/CD8 profiles on V14RhoA thymocytes. Thymocytes from NLC and V14RhoA mice were isolated, stained with anti-CD4-PE and anti-CD8-FITC, and analyzed by flow cytometry. Two dimensional dot plots representative of five independent experiments are shown.

Figure 1

Thymic phenotype of CD2-V14RhoA transgenic mice. (A) Diagram of the CD2-V14RhoA construct. A 0.7-kb fragment containing human V14RhoA myc-tagged cDNA was subcloned at Sma I site in the human CD2 vector. Position of hCD2 promoter and LCR are shown. Arrows indicate the position of transgene-specific primer pairs used in PCR-based screening for transgene-carrying mice. (B) Thymic expression of V14RhoA transgene. Proteins of thymocytes lysates from NLC mice and transgenic mice (V14RhoA) (10⁶ cells) were separated on a 15% SDS-PAGE. Detection of endogenous RhoA and transgene V14RhoA was performed by Western blot analysis using a specific monoclonal anti-RhoA antibody. Results are shown for the two different transgenic lines generated (2029.1 and 2036.F). Top arrowhead, myc-tagged transgenic V14RhoA; bottom arrowhead, endogenous RhoA. (C) Absolute thymocyte number in CD2-V14RhoA mice. Thymocyte cell numbers from 6–8-wk-old NLC (n = 8) and V14RhoA are shown (n = 10). (D) CD4/CD8 profiles on V14RhoA thymocytes. Thymocytes from NLC and V14RhoA mice were isolated, stained with anti-CD4-PE and anti-CD8-FITC, and analyzed by flow cytometry. Two dimensional dot plots representative of five independent experiments are shown.

Figure 4

Mature thymocytes expressing constitutively active RhoA are hyperresponsive to TCR/CD3 triggering. (A) Analysis of CD3/TCR expression on thymic populations. Thymocytes from NLC and V14RhoA mice were stained for CD4, CD8, and CD3ε. Histograms show expression of CD3 on CD4⁺CD8⁺ DP population, CD4⁺ single positive (CD4 SP), and CD8⁺ single positive (CD8 SP). Mean fluorescence intensity for CD3 expression is shown in the histograms. (B) TCR-stimulated in vitro proliferation of thymocytes. Total thymocytes from NLC and V14RhoA transgenic mice were prepared as described previously. Proliferation of thymic T cells in response to a range of concentrations of plate-bound anti-CD3 antibody, or a combination of PdBu (5 ng/ml) and ionomycin (0.5 ng/ml), was assessed by the incorporation of [³H]thymidine during the last 12 h of a 36-h assay. Graph shows mean of [³H]thymidine incorporation (± SEM) of triplicate samples. Circles represent proliferation of thymocytes to PdBu plus ionomycin. The experiment shown is representative of three different experiments. White circles, NLC; black circles, V14RhoA. (C) CFSE analysis of cell division in TCR-stimulated CD4 SP thymocytes. CFSE-labeled thymocytes from NLC and V14RhoA transgenic mice were stimulated with soluble anti-CD3 antibodies or PdBu (5 ng/ml) and ionomycin (0.5 ng/ml) as described previously. After 48 h in culture cells were stained for CD4 and CD8 and analyzed by flow cytometry for CD4 and CD8 expression and CFSE fluorescence. Histograms show CFSE fluorescence gated on CD4 SP cells of NLC (shaded) or V14RhoA (open) mice. (D) TCR-stimulated intracellular calcium flux in CD4 SP thymocytes. Thymocytes were preloaded with Indo-1 coated with anti-CD3 antibodies and stained for CD4 and CD8. Intracellular calcium flux upon CD3 triggering was analyzed by flow cytometry as described in Materials and Methods. Cells were stimulated with goat anti–hamster, to crosslink the anti-CD3, at the concentrations and times indicated. Panels show intracellular calcium concentration in CD4 SP thymocytes as a ratio of Indo-1 violet/blue fluorescence versus time. Each channel on the time axis represents 500 msec.

Figure 4

Mature thymocytes expressing constitutively active RhoA are hyperresponsive to TCR/CD3 triggering. (A) Analysis of CD3/TCR expression on thymic populations. Thymocytes from NLC and V14RhoA mice were stained for CD4, CD8, and CD3ε. Histograms show expression of CD3 on CD4⁺CD8⁺ DP population, CD4⁺ single positive (CD4 SP), and CD8⁺ single positive (CD8 SP). Mean fluorescence intensity for CD3 expression is shown in the histograms. (B) TCR-stimulated in vitro proliferation of thymocytes. Total thymocytes from NLC and V14RhoA transgenic mice were prepared as described previously. Proliferation of thymic T cells in response to a range of concentrations of plate-bound anti-CD3 antibody, or a combination of PdBu (5 ng/ml) and ionomycin (0.5 ng/ml), was assessed by the incorporation of [³H]thymidine during the last 12 h of a 36-h assay. Graph shows mean of [³H]thymidine incorporation (± SEM) of triplicate samples. Circles represent proliferation of thymocytes to PdBu plus ionomycin. The experiment shown is representative of three different experiments. White circles, NLC; black circles, V14RhoA. (C) CFSE analysis of cell division in TCR-stimulated CD4 SP thymocytes. CFSE-labeled thymocytes from NLC and V14RhoA transgenic mice were stimulated with soluble anti-CD3 antibodies or PdBu (5 ng/ml) and ionomycin (0.5 ng/ml) as described previously. After 48 h in culture cells were stained for CD4 and CD8 and analyzed by flow cytometry for CD4 and CD8 expression and CFSE fluorescence. Histograms show CFSE fluorescence gated on CD4 SP cells of NLC (shaded) or V14RhoA (open) mice. (D) TCR-stimulated intracellular calcium flux in CD4 SP thymocytes. Thymocytes were preloaded with Indo-1 coated with anti-CD3 antibodies and stained for CD4 and CD8. Intracellular calcium flux upon CD3 triggering was analyzed by flow cytometry as described in Materials and Methods. Cells were stimulated with goat anti–hamster, to crosslink the anti-CD3, at the concentrations and times indicated. Panels show intracellular calcium concentration in CD4 SP thymocytes as a ratio of Indo-1 violet/blue fluorescence versus time. Each channel on the time axis represents 500 msec.

Figure 4

Mature thymocytes expressing constitutively active RhoA are hyperresponsive to TCR/CD3 triggering. (A) Analysis of CD3/TCR expression on thymic populations. Thymocytes from NLC and V14RhoA mice were stained for CD4, CD8, and CD3ε. Histograms show expression of CD3 on CD4⁺CD8⁺ DP population, CD4⁺ single positive (CD4 SP), and CD8⁺ single positive (CD8 SP). Mean fluorescence intensity for CD3 expression is shown in the histograms. (B) TCR-stimulated in vitro proliferation of thymocytes. Total thymocytes from NLC and V14RhoA transgenic mice were prepared as described previously. Proliferation of thymic T cells in response to a range of concentrations of plate-bound anti-CD3 antibody, or a combination of PdBu (5 ng/ml) and ionomycin (0.5 ng/ml), was assessed by the incorporation of [³H]thymidine during the last 12 h of a 36-h assay. Graph shows mean of [³H]thymidine incorporation (± SEM) of triplicate samples. Circles represent proliferation of thymocytes to PdBu plus ionomycin. The experiment shown is representative of three different experiments. White circles, NLC; black circles, V14RhoA. (C) CFSE analysis of cell division in TCR-stimulated CD4 SP thymocytes. CFSE-labeled thymocytes from NLC and V14RhoA transgenic mice were stimulated with soluble anti-CD3 antibodies or PdBu (5 ng/ml) and ionomycin (0.5 ng/ml) as described previously. After 48 h in culture cells were stained for CD4 and CD8 and analyzed by flow cytometry for CD4 and CD8 expression and CFSE fluorescence. Histograms show CFSE fluorescence gated on CD4 SP cells of NLC (shaded) or V14RhoA (open) mice. (D) TCR-stimulated intracellular calcium flux in CD4 SP thymocytes. Thymocytes were preloaded with Indo-1 coated with anti-CD3 antibodies and stained for CD4 and CD8. Intracellular calcium flux upon CD3 triggering was analyzed by flow cytometry as described in Materials and Methods. Cells were stimulated with goat anti–hamster, to crosslink the anti-CD3, at the concentrations and times indicated. Panels show intracellular calcium concentration in CD4 SP thymocytes as a ratio of Indo-1 violet/blue fluorescence versus time. Each channel on the time axis represents 500 msec.

Figure 4

Mature thymocytes expressing constitutively active RhoA are hyperresponsive to TCR/CD3 triggering. (A) Analysis of CD3/TCR expression on thymic populations. Thymocytes from NLC and V14RhoA mice were stained for CD4, CD8, and CD3ε. Histograms show expression of CD3 on CD4⁺CD8⁺ DP population, CD4⁺ single positive (CD4 SP), and CD8⁺ single positive (CD8 SP). Mean fluorescence intensity for CD3 expression is shown in the histograms. (B) TCR-stimulated in vitro proliferation of thymocytes. Total thymocytes from NLC and V14RhoA transgenic mice were prepared as described previously. Proliferation of thymic T cells in response to a range of concentrations of plate-bound anti-CD3 antibody, or a combination of PdBu (5 ng/ml) and ionomycin (0.5 ng/ml), was assessed by the incorporation of [³H]thymidine during the last 12 h of a 36-h assay. Graph shows mean of [³H]thymidine incorporation (± SEM) of triplicate samples. Circles represent proliferation of thymocytes to PdBu plus ionomycin. The experiment shown is representative of three different experiments. White circles, NLC; black circles, V14RhoA. (C) CFSE analysis of cell division in TCR-stimulated CD4 SP thymocytes. CFSE-labeled thymocytes from NLC and V14RhoA transgenic mice were stimulated with soluble anti-CD3 antibodies or PdBu (5 ng/ml) and ionomycin (0.5 ng/ml) as described previously. After 48 h in culture cells were stained for CD4 and CD8 and analyzed by flow cytometry for CD4 and CD8 expression and CFSE fluorescence. Histograms show CFSE fluorescence gated on CD4 SP cells of NLC (shaded) or V14RhoA (open) mice. (D) TCR-stimulated intracellular calcium flux in CD4 SP thymocytes. Thymocytes were preloaded with Indo-1 coated with anti-CD3 antibodies and stained for CD4 and CD8. Intracellular calcium flux upon CD3 triggering was analyzed by flow cytometry as described in Materials and Methods. Cells were stimulated with goat anti–hamster, to crosslink the anti-CD3, at the concentrations and times indicated. Panels show intracellular calcium concentration in CD4 SP thymocytes as a ratio of Indo-1 violet/blue fluorescence versus time. Each channel on the time axis represents 500 msec.

Figure 2

Adhesion to fibronectin is enhanced in thymocytes expressing constitutively active RhoA. (A) Thymocyte adhesion to fibronectin. Cell adhesion of freshly isolated thymocytes to increasing concentrations of mouse fibronectin was assayed as described in Materials and Methods. Specific adhesion is expressed as the percentage of attached cells for NLC (black) and CD2 V14RhoA (white) thymocytes. Data represent means ± SD of triplicates. One experiment representative of three is shown. (B) Cell morphology of thymocytes attached to fibronectin. Freshly isolated thymocytes were attached to fibronectin or poly-L-lysin coated coverslips and analyzed by light microscopy as described previously. Panels show phase contrast images of thymocytes from nontransgenic mice (a and c) or V14RhoA transgenic mice (b and d), attached to fibronectin (a and b) or poly-L-lysin (c and d). Bar, 10 μm.

Figure 2

Adhesion to fibronectin is enhanced in thymocytes expressing constitutively active RhoA. (A) Thymocyte adhesion to fibronectin. Cell adhesion of freshly isolated thymocytes to increasing concentrations of mouse fibronectin was assayed as described in Materials and Methods. Specific adhesion is expressed as the percentage of attached cells for NLC (black) and CD2 V14RhoA (white) thymocytes. Data represent means ± SD of triplicates. One experiment representative of three is shown. (B) Cell morphology of thymocytes attached to fibronectin. Freshly isolated thymocytes were attached to fibronectin or poly-L-lysin coated coverslips and analyzed by light microscopy as described previously. Panels show phase contrast images of thymocytes from nontransgenic mice (a and c) or V14RhoA transgenic mice (b and d), attached to fibronectin (a and b) or poly-L-lysin (c and d). Bar, 10 μm.

Figure 3

Effect of activated RhoA on pre-T cell differentiation. (A) Analysis of CD4⁻8⁻ DN populations in V14RhoA transgenic mice. Thymocytes from V14RhoA and NLC mice were analyzed for expression of CD25 and CD44 by lineage exclusion of all CD4 and CD8 DP and SP as well as cells of non-T cell lineage using a panel of biotinylated antibodies to CD4, CD8, CD3, B220, Mac-1, NK, γδ, and Gr-1, revealed with streptavidin allophycocyanin, and costained with anti-CD44 CyChrome, anti-CD25 FITC, and Thy-1.2 PE. Dot plots show CD44/CD25 profiles in lineage-, Thy-1.2⁺ gated cells. Number indicates percentage of total DN thymocytes. (B) Activated RhoA does not restore CD4⁺CD8⁺ DP differentiation in Rag-1^−/− mice. Thymocytes from Rag-1^−/− and V14RhoA/Rag-1^−/− were stained for CD25 and CD44 (left panel) as described in A, for CD4 and CD8 (right panel) and analyzed by flow cytometry. Dot plots show CD44/CD25 profiles in Lineage-, Thy-1.2⁺ cells (left) and CD4/CD8 profiles in Thy-1.2⁺ cells (right). Results are representative of at least three independent experiments.

Figure 5

Activated RhoA enhances positive selection. (A) Analysis of thymic and splenic CD4/CD8 populations in female HY-TCR/V14RhoA double transgenic mice. Thymocytes and splenocytes of HY-TCR and HY-TCR/V14RhoA female mice were stained for CD4, CD8, and HY-TCR and analyzed by flow cytometry. Panels show CD4/CD8 profile gated on HY-TCR^high thymocytes (top) and splenocytes (bottom). Absolute numbers (10⁶ cells) of CD8 SP thymocytes and splenocytes expressing high levels of HY-TCR are indicated. (B) Analysis of thymic and splenic CD4/CD8 populations in F5-TCR/V14RhoA double transgenic mice. Thymocytes and splenocytes of F5-TCR and F5-TCR/V14RhoA mice were stained for CD4, CD8, and Vβ11 and analyzed by flow cytometry. Panels show CD4/CD8 profile gated on Vβ11^high thymocytes and splenocytes. Absolute numbers (10⁶ cells) of CD8 SP thymocytes and splenocytes expressing high levels of Vβ11 are indicated. Each two dimensional dot plot shown is representative of at least three independent experiments.

Figure 5

Activated RhoA enhances positive selection. (A) Analysis of thymic and splenic CD4/CD8 populations in female HY-TCR/V14RhoA double transgenic mice. Thymocytes and splenocytes of HY-TCR and HY-TCR/V14RhoA female mice were stained for CD4, CD8, and HY-TCR and analyzed by flow cytometry. Panels show CD4/CD8 profile gated on HY-TCR^high thymocytes (top) and splenocytes (bottom). Absolute numbers (10⁶ cells) of CD8 SP thymocytes and splenocytes expressing high levels of HY-TCR are indicated. (B) Analysis of thymic and splenic CD4/CD8 populations in F5-TCR/V14RhoA double transgenic mice. Thymocytes and splenocytes of F5-TCR and F5-TCR/V14RhoA mice were stained for CD4, CD8, and Vβ11 and analyzed by flow cytometry. Panels show CD4/CD8 profile gated on Vβ11^high thymocytes and splenocytes. Absolute numbers (10⁶ cells) of CD8 SP thymocytes and splenocytes expressing high levels of Vβ11 are indicated. Each two dimensional dot plot shown is representative of at least three independent experiments.

Figure 6

Differentiation of L61 Rac-1 pre-T cells is dependent of RhoA function. (A) Thymocytes from L61 Rac-1, CD2-C3, and L61 Rac-1/CD2-C3 transgenic mice were stained for CD4 and CD8, and analyzed by flow cytometry. Dot plots show CD4/CD8 profiles. Results are representative of three independent experiments. (B) Thymocytes from L61 Rac-1, CD2-C3, and L61 Rac-1/CD2-C3 transgenic mice were stained for CD25 and CD44, as described in Fig. 3 A, and analyzed by flow cytometry. Dot plots show CD44/CD25 profiles in lineage-null, Thy-1.2⁺ cells. Results are representative of three independent experiments.

Figure 6

Differentiation of L61 Rac-1 pre-T cells is dependent of RhoA function. (A) Thymocytes from L61 Rac-1, CD2-C3, and L61 Rac-1/CD2-C3 transgenic mice were stained for CD4 and CD8, and analyzed by flow cytometry. Dot plots show CD4/CD8 profiles. Results are representative of three independent experiments. (B) Thymocytes from L61 Rac-1, CD2-C3, and L61 Rac-1/CD2-C3 transgenic mice were stained for CD25 and CD44, as described in Fig. 3 A, and analyzed by flow cytometry. Dot plots show CD44/CD25 profiles in lineage-null, Thy-1.2⁺ cells. Results are representative of three independent experiments.

Figure 7

Activated RhoA cannot rescue thymocyte development in Vav-1^−/− mice. (A) Thymic cellularity in V14RhoA/Vav-1^−/− double transgenic mice. Thymocyte cell numbers from 6–8-wk-old Vav-1^+/− (n = 6), Vav-1^−/− (n = 5), and V14RhoA/Vav-1^−/− mice (n = 5) are shown. (B) CD25/CD44 profile in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD25 and CD44 as described previously. Dot plots for CD44/CD25 profiles in lineage⁻, Thy-1.2⁺ cell are shown. (C) CD4/CD8 profiles in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. Dot plots for CD4/CD8 profiles in Thy-1.2⁺ cells are shown. (D) CD5 expression on CD4⁺CD8⁺ DP thymocytes in V14RhoA/Vav-1^−/− double transgenic. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. CD5 expression was analyzed on CD4⁺CD8⁺ Thy1.2⁺ cells. Results are representative of at least three independent experiments.

Figure 7

Activated RhoA cannot rescue thymocyte development in Vav-1^−/− mice. (A) Thymic cellularity in V14RhoA/Vav-1^−/− double transgenic mice. Thymocyte cell numbers from 6–8-wk-old Vav-1^+/− (n = 6), Vav-1^−/− (n = 5), and V14RhoA/Vav-1^−/− mice (n = 5) are shown. (B) CD25/CD44 profile in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD25 and CD44 as described previously. Dot plots for CD44/CD25 profiles in lineage⁻, Thy-1.2⁺ cell are shown. (C) CD4/CD8 profiles in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. Dot plots for CD4/CD8 profiles in Thy-1.2⁺ cells are shown. (D) CD5 expression on CD4⁺CD8⁺ DP thymocytes in V14RhoA/Vav-1^−/− double transgenic. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. CD5 expression was analyzed on CD4⁺CD8⁺ Thy1.2⁺ cells. Results are representative of at least three independent experiments.

Figure 7

Activated RhoA cannot rescue thymocyte development in Vav-1^−/− mice. (A) Thymic cellularity in V14RhoA/Vav-1^−/− double transgenic mice. Thymocyte cell numbers from 6–8-wk-old Vav-1^+/− (n = 6), Vav-1^−/− (n = 5), and V14RhoA/Vav-1^−/− mice (n = 5) are shown. (B) CD25/CD44 profile in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD25 and CD44 as described previously. Dot plots for CD44/CD25 profiles in lineage⁻, Thy-1.2⁺ cell are shown. (C) CD4/CD8 profiles in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. Dot plots for CD4/CD8 profiles in Thy-1.2⁺ cells are shown. (D) CD5 expression on CD4⁺CD8⁺ DP thymocytes in V14RhoA/Vav-1^−/− double transgenic. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. CD5 expression was analyzed on CD4⁺CD8⁺ Thy1.2⁺ cells. Results are representative of at least three independent experiments.

Figure 7

Activated RhoA cannot rescue thymocyte development in Vav-1^−/− mice. (A) Thymic cellularity in V14RhoA/Vav-1^−/− double transgenic mice. Thymocyte cell numbers from 6–8-wk-old Vav-1^+/− (n = 6), Vav-1^−/− (n = 5), and V14RhoA/Vav-1^−/− mice (n = 5) are shown. (B) CD25/CD44 profile in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD25 and CD44 as described previously. Dot plots for CD44/CD25 profiles in lineage⁻, Thy-1.2⁺ cell are shown. (C) CD4/CD8 profiles in V14RhoA/Vav-1^−/− double transgenic mice. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. Dot plots for CD4/CD8 profiles in Thy-1.2⁺ cells are shown. (D) CD5 expression on CD4⁺CD8⁺ DP thymocytes in V14RhoA/Vav-1^−/− double transgenic. Thymocytes from Vav-1^+/−, Vav-1^−/−, and V14RhoA/Vav-1^−/− mice were analyzed for expression of CD4 and CD8 as described previously. CD5 expression was analyzed on CD4⁺CD8⁺ Thy1.2⁺ cells. Results are representative of at least three independent experiments.