Ct-CD45 has immuno-regulatory function. (A) Binding of ct-CD45 to resting T cells and to T cells activated for 24 hours with PMA/ionomycin was analyzed with Alexa Fluor 488-labeled recombinant ct-CD45 molecules and flow cytometry. Pretreatment of cells with unlabeled recombinant protein or mAb 8-301 but not with PTPase inhibitor-blocked binding of fluorescent ct-CD45 (filled histograms) to T cells. The figure shows overlay histogram profiles including Alexa Fluor 488-labeled human serum albumin used as a negative control (open histogram), which are representative of 3 independent experiments. (B) Binding of increasing amounts of Alexa Fluor 488 labeled recombinant ct-CD45 molecules analyzed by flow cytometry. The figure shows mean values plus or minus SEM of 4 experiments of mean fluorescence intensity. The values of nonspecific staining with Alexa-labeled human serum albumin were subtracted from the values obtained with Alexa-labeled ct-CD45. (C) Addition of recombinant ct-CD45 (▲) inhibits T-cell proliferation induced by allogeneic DC (○). The immunosuppressive cytokine IL-10 (■) and recombinant S100A4 proteins (•) were used as positive and negative control, respectively. Data represent mean cpm (± SEM) of 3 independent experiments. (D) T cells were cultured in the presence of immobilized anti-CD3 alone (mock, taken as 100%), anti-CD3 plus control-Ig (co-Ig), or as positive control anti-CD3 plus ICOS-L-Ig or anti-CD3 plus different CD45-Ig-fusion proteins (all Ig-fusion proteins coated at 0.5 or 2 μg/mL) for 72 hours, and proliferation was measured by [³H]thymidine incorporation. Mean values and SD of 3 different experiments are shown. A paired Student t test was performed (***P < .01; **P < .05). (E) Percentages of annexin- and PI-positive T cells cultured as in (D) with 2 μg/mL Ig-fusion proteins coated. Mean values and SD of 3 different experiments are shown.