Distinct Roles of Cdc42 in Thymopoiesis and Effector and Memory T Cell Differentiation
Figure 2
Defective migration, survival, and TCR signaling in Cdc42−/− thymocytes.
(A) Migration to SDF-1α of DP thymocytes or to MIP3β of SP thymocytes from wild type (WT) and Cdc42−/− mice. Data were expressed as numbers of cells migrated to the exterior of transwell chambers relative to numbers of cells initially seeded in the interior of transwell chambers (% of input)n = 5. *P <0.05; **P <0.01. Error bars represent SD. (B) Adhesion to fibronectin of thymocytes from WT and Cdc42−/− mice. Data were expressed as numbers of adhered cells relative to numbers of cells plated (% of input). n = 6. Error bars represent SD. (C) Apoptosis of Ex vivo thymocytes from WT and Cdc42−/− mice. Freshly isolated thymocytes were stained with anti-CD4 and -CD8 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. n = 4. *P <0.05. Error bars represent SD. (D) Apoptosis of cultured thymocytes from WT and Cdc42−/− mice. Isolated thymocytes were cultured for 24 hours with anti-CD3/-CD28 antibodies and stained with anti-CD4 and -CD8 antibodies followed by Annexin V staining. The cells were then analyzed by flow cytometry. Numbers outside the gates indicate percent cells in each gate and numbers inside the gates indicate absolute numbers of Annexin V+ cells analyzed in each gate; right, average frequency of Annexin V+ thymocytes. Data are representative of three independent experiments. n = 4. *P <0.05. Error bars represent SD. (E) Anti-CD3/-CD28-induced proliferation of thymocytes from wild type (WT) and Cdc42−/− mice. TCRβ+ thymocytes were plated on 96-well plates at 1×106/well in 200 µL culture media in the presence or absence of plate-coated anti-CD3 (10 µg/mL) plus soluble anti-CD28 (2 µg/mL). The cells were cultured for 3 days and assayed for growth rate. n = 6. **P <0.01. Error bars represent SD. (F) MAP kinase activities in thymocytes from WT and Cdc42−/− mice. TCRβ+ thymocytes were stimulated with or without anti-CD3 (10 µg/mL) and -CD28 (2 µg/mL) for indicated time. Western blotting was performed to assess the phosphorylation status of Erk, p38, and JNK. The result is a representative of three experiments.