IGSF4^GT/GT mice show substantial blockade of intrathymic T cell development. (A) Thymocytes were stained with FITC-conjugated anti-CD4 and Cy5.5-conjugated anti-CD8 mAbs, and then the cells were analyzed by flow cytometry. On dot plots, the percentage of cells in each quadrant is indicated. Mean cell numbers of thymocyte subsets are shown in the bar graph (right). The results are the mean ± SD of triplicate experiments. *, P < 0.05, as compared with cells from IGSF4^+/+ mice. (B) Flow cytometric analysis of TCR-β, CD5, and CD69 in IGSF4^+/+ and IGSF4^GT/GT thymocytes. Numbers above bracketed lines indicate the percentage of TCR-β^hi, CD69^hi, and CD5^hi cells. (C) Surface expression of CD3ε, TCR ζ-chain, and TCR-β on thymocytes or lymphocytes from IGSF4^+/+ and IGSF4^GT/GT. (A–C) Data are representative of at least five to six independent experiments. (D) Thymocytes from IGSF4^+/+ and IGSF4^GT/GT mice were stimulated with anti-CD3/28 for the indicated time. The phosphorylated (p) and total (t) forms of Lck, Zap70, ERK, and p38 kinase were determined by Western blot analysis. Data are representative of two independent experiments. Molecular mass (M) is indicated in kilodaltons. (E) Thymocytes were stimulated with anti-CD3/28 for the indicated time, and the cytokine productions (IL-2) were measured by ELISA. The results are the mean ± SD of triplicate experiments. *, P < 0.05, as compared with cells from IGSF4^+/+ mice.