IGSF4^GT/GT mice show defective T cell function. (A) IGSF4 expression in purified CD3⁺ T cells from the spleens and lymph nodes of WT and IGSF4^GT/GT mice. Molecular mass (M) is indicated in kilodaltons. (B) The number of each cell type in the lymph node and spleen. The results are the mean ± SD of triplicate experiments. *, P < 0.05, as compared with IGSF4^+/+ mice. (C) T cells purified from the spleen 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 three independent experiments. (D) Allogeneic DCs and CD3⁺ T cells from WT or IGSF4^GT/GT mice were incubated in SEB-containing medium for 3 h to allow conjugation. The cells were fixed and stained with anti–p-tyrosine (4G10; arrows). Polarization of p-tyrosine in T cells was quantified by cell counting (n > 50). Data are representative of at least three independent experiments. DIC, differential interference contrast. (E) CD3⁺ T cells were mixed with SEB-pulsed CD11C⁺ DCs (red) or CD19⁺ B cells from WT mice, and then conjugate formation was determined by confocal microscopy (left) and flow cytometry (bar graphs). (F) Purified splenic CD3⁺ T cells from WT and IGSF4^GT/GT mice were incubated with anti-CD3/28 for 72 h. Cell proliferation was assessed by [³H]thymidine incorporation. (G) CD4⁺ or CD8⁺ T cells were stimulated with anti-CD3/28 or PMA/A23187 for 24 h, and the cytokine productions (IL-2, IL-4, and IFN-γ) were measured by ELISA. (E–G) The results are the mean ± SD of triplicate experiments. *, P < 0.05, as compared with cells from IGSF4^+/+ mice. Bars: (D) 5 µm; (E) 10 µm.