CP-690,550 inhibited the activation of the Jak3/STAT5 pathway present in patients ATL cells. (A) CP-690,550 inhibited the phosphorylation status of STAT5 in isolated T cells from patients with ATL. The T cells from a patient with ATL and those from a normal control were immediately isolated from the freshly separated PBMCs and lysed with cell lysis buffer; alternatively, the T cells were isolated from PBMCs cultured for 2 days with and without 50nM CP-690,550. Cell lysates were immunoblotted with anti–phospho-STAT5 monoclonal antibody and anti–STAT5 antibody. β-actin was used as an input control. (B) CFSE staining of CD3^LowCD25⁺ lymphocytes was used to monitor cell division. ATL PBMCs were labeled with CFSE at day 0, and then cultured with (CP) or without (−) CP-690,550 for 6 days. FACS analysis of CD3^LowCD25⁺CFSE triple-positive ATL cells were performed on day 6. Normal donor PBMCs (Cont) were used as a control. Because normal PBMCs proliferated under stimulation of anti-CD3/CD28 and ATL PBMCs spontaneously proliferated regardless of such stimulation, the nonstimulated normal and ATL PBMCs were chosen for analysis.