Inhibition of cytokine-mediated proliferation of 32Dβ cells by CP-690,550 showed specificity for the γc/Jak3/STAT5-signaling pathway. (A) After cytokine starvation for 24 hours, 32Dβ cells were stimulated with human IL-2 or murine IL-3 for 48 hours with and without CP-690,550. ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. (B) After cytokine starvation for 24 hours, 32Dβ cells were stimulated with 1 μg/mL of human IL-2 or 10 ng/mL of murine IL-3 for 2 hours with and without the addition of CP-690,550. The cell lysates were immunoblotted with an anti–phospho-STAT5 monoclonal antibody and an anti-STAT5 antibody. β-Actin was used as an input control. Data are presented as means ± SD (A) and are representative of 3 independent experiments.

CP-690,550 inhibited ATL and HAM/TSP PBMC 6-day culture supernatant–mediated NK92-cell proliferation. After cytokine starvation for 24 hours, NK92 cells were stimulated with a 6-day ex vivo culture supernatant of PBMCs from an ATL patient (A) or a HAM/TSP patient (B) for 48 hours. Antibodies directed against the cytokines IL-2, IL-9, or IL-15 or CP-690,550 were added to the plates 1 hour prior to cell seeding. ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. The percentage of inhibition of proliferation with CP-690,550 was determined for 11 ATL culture supernatants (C) or 8 HAM/TSP culture supernatants (D). Black bars indicate the mean percentage of inhibition.

CP-690,550 showed therapeutic efficacy in a mouse model of B1 IL-15–transgenic CD8 T-cell leukemia that manifested an autocrine IL-15/IL-15Rα loop that acts through Jak3/STAT5. (A) Serum levels of human IL-15 from the human IL-15–transgenic CD8 T-cell leukemia–bearing mice after inoculation of tumor cells. (B) Serum levels of mouse IL-15Rα from the IL-15–transgenic CD8 T-cell leukemia–bearing mice after inoculation of tumor cells. (C) Kaplan-Meier analysis demonstrating CP-690,550 prolongation of the survival of mice bearing the IL-15–transgenic CD8 T-cell leukemia. Mice in the control group received PEG300 by subcutaneous pump for 14 days. Mice in the CP-690,550-pre group (CP-pre) received a pump infusion with CP-690,550 at 30 mg/kg/d for 14 days that was placed into the mice 2 days before tumor cell inoculation. Mice in the CP-690,550-post group (CP-post) received a pump infusion with CP-690,550 at 30 mg/kg/d for 14 days that was placed into the mice 1 day after inoculation of the leukemic cells. Mice in the anti–human IL-15 monoclonal antibody M111 group received M111 at 100 μg intraperitoneally weekly for 2 weeks starting at 3 days after inoculation of the leukemic cells. (D) CP-690,550 inhibited the phosphorylation status of Jak3/STAT5 in splenocytes from IL-15–transgenic CD8 T-cell leukemia–bearing mice. Mice in the tumor-only group received PEG300 by a subcutaneous pump for 14 days. Mice in the CP-690,550 group (CP) received a pump infusion with CP-690,550 at 30 mg/kg/d for 14 days that was placed into the mice 1 day after inoculation of the leukemic cells. Mice in the M111 group received M111 at 100 μg intraperitoneally weekly for 2 weeks starting at 3 days after inoculation of the leukemic cells. Mice in the normal group (no tumor, no treatment) were used as a control. The splenic cells were separated 13 days after tumor cell inoculation and checked for the phosphorylation status of STAT5. *P < .00001.

CP-690,550 inhibited the proliferation of the cytokine-dependent NK92 cell line mediated by cytokines that signal through Jak3/STAT5 but not by cytokines that use other pathways. (A) After cytokine starvation for 24 hours, NK92 cells were stimulated by the addition of human IL-2 for 48 hours with and without the addition of serially increasing concentrations of CP-690,550. ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. (B) Cytokine-starved NK92 cells were stimulated with human IL-2, combined IL-6/IL-6R, or IL-12 for 48 hours with and without serially increasing concentrations of CP-690,550. (C) The NK92 cells were treated as those in panel B with and without a single 50nM dose of CP-690,550 (CP). Data are presented as means ± SD (A-C) and are representative of 3 independent experiments. (D) After cytokine starvation for 24 hours, NK92 cells were stimulated with 30 ng/mL of IL-2, 100 ng/mL of combined IL-6/IL-6R, or 100 ng/mL of IL-12 for 1 hour with and without the addition of CP-690,550 (CP). The cell lysates were immunoblotted with an anti–phospho-STAT5 monoclonal antibody and an anti-STAT5 antibody. β-Actin was used as an input control. Data are representative of 3 independent experiments.

CP-690,550 inhibited the 6-day ex vivo spontaneous proliferation of PBMCs from patients with ATL and with HAM/TSP. The PBMCs from a patient with ATL (A) or a patient with HAM/TSP (B) were cultured ex vivo for 6 days with and without CP-690,550 or with 10 μg/mL of antibody when a single antibody directed against the cytokine IL-2, IL-9, or IL-15 was used or with their combination (5 μg/mL each). ³H-thymidine was added during the last 6 hours of the cultures. Cells were then harvested and analyzed for ³H-thymidine incorporation. The percentage of inhibition of proliferation with CP-690,550 addition was determined for 12 patients with ATL (C) or 9 patients with HAM/TSP (D). The value was calculated as: % inhibition = (cpm of proliferation without CP − cpm of proliferation with CP)/(cpm of proliferation without CP) × 100. Black bars indicate the mean inhibition percentage.

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.