MTS/ΔSLP CD4⁺ T cells have impaired TCR signaling and are not poised to produce pro-inflammatory cytokines. (A) 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. (B) Peripheral cells from +/ΔSLP (black line) and MTS/ΔSLP (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 YFP⁺CD4⁺ T cells. Data are representative of 3 independent experiments using 1 or 2 mice per group. (C) Splenocytes isolated from either +/ΔSLP (black line) or MTS/ΔSLP (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 YFP⁺CD4⁺ T-cell population. Data shown are representative of 3 independent experiments. P + I indicates PMA plus ionomycin. (D) Peripheral cells isolated from +/ΔSLP and MTS/ΔSLP mice after tamoxifen treatment were stimulated with PMA plus ionomycin and stained for the presence of intracellular inflammatory cytokines. Populations are gated on YFP⁺CD4⁺ T cells. Data are representative of 5 independent experiments using 2 or 3 mice per group.