Rapid engraftment after allogeneic transplantation of density-enriched peripheral blood CD34+ cells in patients with advanced hematologic malignancies

BACKGROUND. Acute graft versus host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic cell transplantation. Pre-clinical studies have suggested that a T-cell subset with a CD4-/CD8- double-negative (DN) T-cell phenotype is capable of suppressing GVHD. Double-negative T cells can be mobilized into the peripheral blood with granulocyte colony-stimulating factor (G-CSF) and enriched by density centrifugation. The current study was performed to study the feasibility and safety of applying a density gradient separation technique for enrichment of CD34+ and DN T cells, while depleting CD4+ and CD8+ single-positive (SP) T cells from peripheral blood progenitor cells (PBPCs) for the purpose of allogeneic transplantation. METHODS. Twenty-five patients with advanced hematologic malignancies were treated with a myeloablative preparative regimen consisting of fractionated total body irradiation, etoposide, and cyclophosphamide. Human leukocyte antigen identical donors were mobilized with G-CSF PBPC collected by apheresis. The apheresis product was applied to a single-step density gradient, and the low-density cell population was collected. The low-density cell population was infused as the sole source of allogeneic cells after myeloablative therapy. Graft versus host disease prophylaxis consisted of cyclosporine with or without prednisone. RESULTS. CD34 cell recovery was efficient with a median 72% yield, providing for a median CD34+ cell dose of 6.5 × 10⁶/kg (range, 1.0-13.9 × 10⁶/kg). CD3+CD4+ or CD3+CD8+ SP T cells were depleted by a median of 94.4% (range, 58.8-99.2%), and the ratio of CD34+:SP T cells increased 10-fold. Double-negative T cells were depleted by 92% (range, 18.8-99.4%), thus the ratio of DN:SP T cells increased less than 2-fold in 71% of apheresis samples tested. Hematopoietic engraftment was rapid, and there was no occurrence of graft failure in examinable patients. Median time to absolute neutrophil count greater than 0.5 × 10⁹/L and platelet count greater than 20 × 10⁹/L was 10.5 and 12 days, respectively. The incidence of Grade 2-4 acute GVHD was 26% (95% confidence interval [CI], 6-45%), although not all patients were examinable due to an unexpectedly high nonrecurrence mortality that at Day 180 was 62% (95% CI, 40-83%). CONCLUSIONS. These data suggest that T-cell subset manipulation via density gradient separation is a safe procedure and allowed rapid hematopoietic recovery. Selective enrichment of a donor DN T-cell subset was observed in only a few and was not associated with a reduced incidence of GVHD. However, the low-density selected cells still resulted in GVHD, and there was a high treatment-related mortality.