Figure 6.

TCR-induced activation of Rap1 and adhesion to ICAM-1 are ZAP-70 kinase-independent. (a) CD4⁺CD25⁻ Tconv and CD4⁺CD25⁺ T_REG cells were loaded with Fluo-3 and Fura Red for analysis of intracellular calcium levels by flow cytometry. Cells were stimulated in the presence of vehicle alone (red) or 3-MB-PP1 (blue). Arrow “1” indicates the addition of biotinylated anti-CD3ε and anti-CD4, arrow “2” indicates the addition of streptavidin, and arrow “3” indicates the addition of ionomycin. Tconv were >99% Foxp3⁻ and T_REG cells were >98% Foxp3⁺. (b) Splenocytes were stimulated ex vivo by crosslinking anti-CD3 mAbs and stained for phospho-ERK. Histograms are gated on Foxp3-negative or Foxp3-positive CD4⁺ cells. Cells were either unstimulated (filled gray histograms) or stimulated in the presence of vehicle alone (red), 5 µM 3-MB-PP1 (green), 10 µM 3-MB-PP1 (blue), or 10 µM 3-MB-PP1 plus PMA (gray line). (c) CrkII was immunoprecipitated (IP) from Zap70(AS) thymocyte lysates. Cells were stimulated by crosslinking anti-CD3 mAbs for 2 minutes. Immunoprecipitated proteins were immunoblotted (IB) for ZAP-70 and CrkII (top). Whole cell lysates were also immunoblotted for the presence of ZAP-70 and CrkII (bottom). (d) Pull-down assay for Rap1-GTP in Zap70^+/− and Zap70(AS) thymocytes. Cells were left unstimulated, or stimulated with crosslinked anti-CD3ε mAb for 2 minutes. The concentrations of 3-MB-PP1 are indicated. Total Rap1 levels in whole cell lysates are shown below. (e) CD4⁺ T cells from Zap70^+/− and Zap70(AS) were stimulated with anti-CD3ε for 10 minutes in wells coated with recombinant ICAM-1. Cells were then washed and counted by flow cytometry. Data in all panels are representative of 3 independent experiments.