The size of CD45 ectodomain modulates its inhibitory effect on T-cell activation. (A) Schematic depiction of the CD45 constructs used in this study. They comprised the mouse CD45 transmembrane and cytosolic domains and the ectodomains of rat Thy1 (1 Ig-like domain), rat CD2 (2 Ig-like domains), or rat CD43. A catalytically inactive form of Thy1-CD45 (Thy1-CD45 PTPneg) had a cysteine-to-serine substitution (C840S) in the membrane-proximal phosphatase domain. All constructs contained an N-terminal FLAG tag to stain and match surface expression levels by FACS and a C-terminal GFP tag for imaging. 2B4 T-cells expressing comparable levels of the indicated CD45 construct (supplemental Figure 5) were stimulated with either (B) plate-immobilized anti-mouse CD3ε or (C) CHO cells expressing I-E^k presenting the cognate MCC peptide (left) and IL-2 secretion analyzed after 14 to 18 hours. Error bars represent the SD of the mean from at least 3 replicates. Data normalized to vector-transduced controls as in Figure 2.