Peripheral CD4⁺ T cells in MTS mice are greatly decreased in number, possess an activated-like phenotype, and have impaired TCR signaling. (A) Total number of cells obtained from the spleen and peripheral LNs of WT (□) and MTS (▩) mice and percentages of lymphocyte subsets as determined by flow cytometry. Data are mean ± SEM and are representative of more than 5 independent experiments using 2 or 3 mice per group. Significance was determined using the 2-tailed Student t test. **P < .01. ***P < .001. (B) Surface expression of activation markers of WT (solid line) and MTS (shaded) mice. Histograms are gated on CD4⁺ T cells. Data are representative of more than 5 independent experiments using 2 or 3 mice per group. (C) Intracellular staining for the incorporation of BrdU in WT and MTS mice. Histograms are gated on CD4⁺ T cells. Data are representative of 2 independent experiments using 2 or 3 mice per group. (D) Purified peripheral T cells were stimulated for indicated lengths of time with a TCR cross-linking antibody, lysed, and blotted for phospho-PLC-γ1, total PLC-γ1, phospho-SLP-76, total SLP-76, and phospho-ERK. Data are representative of 3 independent experiments. (E) Peripheral cells from WT (black line) and MTS (gray line) mice were loaded with the calcium indicator Indo-1 and analyzed for their ability to flux calcium in response to CD3/CD4 cross-linking and ionomycin at the indicated time points. Histograms are gated on CD4⁺ T cells. Data are representative of 4 independent experiments using 1 or 2 mice per group. (F) Splenocytes isolated from either WT (black line) or MTS (gray line) mice were cultured in the presence of the indicated stimulus and stained for the early activation markers CD25 and CD69. Percentages are taken from positive gates set on the total CD4⁺ T-cell population. Data are mean ± SEM and are representative of 2 independent experiments. P + I indicates PMA plus ionomycin.