Progression through the mammalian cell cycle is regulated by cyclins, cyclin- dependent kinases (CDKs), and cyclin-dependent kinase inhibitors (CKIs). The function of these proteins in the irreversible growth arrest associated with terminally differentiated cells is largely unknown. The function of Cip/Kip proteins p21Cip1and p27Kip1 during erythropoietin-induced terminal differentiation of primary erythroblasts isolated from the spleens of mice infected with the anemia-inducing strain of Friend virus was investigated. Both p21Cip1 and p27Kip1 proteins were induced during erythroid differentiation, but only p27Kip1 associated with the principal G1CDKs—cdk4, cdk6, and cdk2. The kinetics of binding of p27Kip1 to CDK complexes was distinct in that p27Kip1 associated primarily with cdk4 (and, to a lesser extent, cdk6) early in differentiation, followed by subsequent association with cdk2. Binding of p27Kip1 to cdk4 had no apparent inhibitory effect on cdk4 kinase activity, whereas inhibition of cdk2 kinase activity was associated with p27Kip1binding, accumulation of hypo-phosphorylated retinoblastoma protein, and G1 growth arrest. Inhibition of cdk4 kinase activity late in differentiation resulted from events other than p27Kip1 binding or loss of cyclin D from the complex. The data demonstrate that p27Kip1 differentially regulates the activity of cdk4 and cdk2 during terminal erythroid differentiation and suggests a switching mechanism whereby cdk4 functions to sequester p27Kip1 until a specified time in differentiation when cdk2 kinase activity is targeted by p27Kip1 to elicit G1 growth arrest. Further, the data imply that p21Cip1 may have a function independent of growth arrest during erythroid differentiation.

Mobilization failure rates were <5% across all thresholds. Mobilization costs were comparable. Conclusion: We demonstrate that a liberal strategy for plerixafor administration correlates with fewer apheresis days while having similar mobilization costs. There were 26 fewer days of collection per 100 mobilization attempts with a pCD34 threshold of 40/μL compared to 15/μL at our institution. More patients completed collection in one day, and more reached an optimal collection yield.

Background: Selective depletion of αβ T cells and CD 19 + B cells from a haploidentical stem cell graft is hypothesized to preserve graft versus tumor effect, while minimizing infection and graft versus host disease (GVHD). Limited data exists on the feasibility of this strategy in adult patients with lymphoma. We are conducting a phase II trial to assess the feasibility of this approach in adult patients with relapsed lymphoma. Here, we present preliminary data on graft processing, engraftment, and immune reconstitution in the first 8 patients enrolled on this study. Methods: Patients were eligible if they had relapsed Hodgkin or non-Hodgkin lymphoma after at least 1 prior line of therapy. Conditioning consisted of cyclophosphamide 60 mg/kg IV days -5 and -4; fludarabine 40 mg/m2 IV days -5 to -2, and total lymphoid irradiation 7 Gy on day -1. GCSF mobilized peripheral blood stem cell (PBSC) grafts were depleted of αβ T cells and CD 19 + B cells using the CliniMACS R system. Target αβ T cell content was ≤1.5 × 10 5 cells/kg ideal body weight (IBW) and target CD19+ cell content ≤1 × 10 5 cells/kg IBW. Target CD34+ content was ≥4 × 10 6 cells/kg IBW. GVHD prophylaxis consisted of mycophenolate mofetil from day -1 to day +30. The primary endpoint is successful engraftment (defined as absolute neutrophil count [ANC] > 500/μl and platelets > 20K/ μl without transfusion in the prior 7 days) before day +28. Results: Median age was 57 (range 29-69). Diagnoses included mantle cell lymphoma (n = 3), Hodgkin lymphoma (n = 2), diffuse large B cell lymphoma (n = 1), follicular lymphoma (n = 1), and chronic lymphocytic leukemia (n = 1). Minimum dose of required CD34 cells was achieved for all patients, with a median of 5.8 × 10 6 CD34+ cells/kg IBW infused. Graft processing resulted in αβ T cell and CD19+ cell content below target maximum for all patients with median log depletion of 3.7 (3.3-4.1) and 2.5 (2.2-3.3), respectively. All 8 patients successfully engrafted, with recovery of neutrophils at median 14.5 days (9-20) and platelets at median 16 days (13-22). Measurement from the peripheral blood at 1 month posttransplant showed a median αβ cell content of 11/μl (3-233) and γδ cells 39/μl (23-336). By 3 months, however, αβ cells were at 140/μl (87-236) and γδ cells 50/μl (29-82). There were no CD19+ cells detected in the peripheral blood lymphocytes of any patients at 1 month and median 66/μl (0-256) at 3 months. Conclusion: Selective depletion of αβ T/CD19+ B cells from haploidentical PBSC grafts leads to stable engraftment after conditioning with fludarabine, cyclophosphamide, and total lymphoid irradiation in adult lymphoma patients. At 1 month post-transplant, the majority of CD3+cells present in the pe-ripheral blood are γδ cells; however, by 3 months, the αβ cells start to show significant reconstitution. Longer term followup and further study are required to evaluate disease control, GVHD, and infection.