Optimized clinical-scale culture conditions for ex vivo selective depletion of host-reactive donor lymphocytes: A strategy for GvHD prophylaxis in allogeneic PBSC transplantation

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 α-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 > 2×10¹⁰ 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 ≥ 1 × 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.