Figure 2

sAbs, but not iAbs, induce inhibitory feedback loops. Purified human T cells were treated with either soluble (sAbs) or immobilized (iAbs) CD3xCD28 mAbs for the indicated time points. A) Measurement of TCR internalization. The expression of the TCR was assessed by PE-conjugated anti-TCRαβ mAb staining analysis by flow cytometry. Data represent the % of the mean fluorescence intensity (MFI) of the TCR expression relative to time 0 of 4 independent experiments. B) The phosphorylation of c-Cbl on Y⁷³¹ and Dok2 on Y³⁵¹ was determined by Western blotting. The phosphorylated c-Cbl and Dok2 bands were quantified using the 1D ImageQuant software and the values were normalized to the corresponding β-actin signal. Data represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of at least 4 independent experiments. C) The expression of ZAP70 was determined by Western blotting. Equal loading is shown by reprobing immunoblots with antibodies specific for β-actin. ZAP70 bands were quantified as above. Data represent the mean of the expression levels shown as arbitrary units ± SEM of 6 independent experiments. D) Tyrosine phosphorylation of Fyn and Lck in the activation loop was determined by Western blotting using the pSrc (Y⁴¹⁶) antibody. Bands were quantified using the 1D ImageQuant software and values were normalized to the corresponding total Fyn and Lck signals. Data on the graph represent the mean of the phosphorylation levels shown as arbitrary units ± SEM of 4 independent experiments. Asterisks (*) indicate the Ig heavy chain. Statistical analysis ** P<0.01; ns, not statistically significant.