Abstract
Background : Ex vivo selective depletion (SD) is a strategy to prevent GvHD, in which host-reactive donor lymphocytes are selectively eliminated from a PBSC allograft while conserving useful donor immune function. Prior to testing this strategy in patients, our goal was to develop a clinical-scale SD process, which involves co-culture of donor lymphocytes and irradiated recipient cells, followed by the addition of an immunotoxin (IT) directed against the f -chain of the IL-2 receptor (CD25), expressed on activated donor T cells. Methods : Stimulator cells were generated from immunomagnetically selected and expanded recipient T lymphocytes. Donor PBMCs from G-CSF-mobilized peripheral blood were co-cultured for 72 h with irradiated stimulator cells. Alloreactive T cells were targeted for elimination by the addition of the anti-CD25 IT, RFT5-SMPT-dgA, and the IT enhancer, NH4Cl. Results : Stimulator-cell selection/expansion yielded > 2 2 1010 highly enriched CD3+ cells (98.9 - 2.2%). After SD, cell recovery was 68.5 - 23.3% and viability was 84.6 - 6.4%. This permitted a potential T-cell dose S 1 2 108 CD3+ cells kg-1 to transplant recipients. Although SD donor lymphocytes retained little proliferative capacity against the original stimulator cells (2.6 - 0.6%), responses were conserved against third party cells (107.6 - 18.6%), the bacterial superantigen staphylococcus enterotoxin B (108.2 - 4.2%), and CMV Ag (72.1 - 3.8%). Discussion : We have demonstrated that ex vivo SD is feasible in clinical-scale culture conditions. The ability of this strategy to prevent GvHD is the subject of an ongoing clinical trial, in which the SD lymphocyte product is transplanted in conjunction with a T cell-depleted PBSC allograft.