A comparison of an anti-CD25 immunotoxin, Ontak and anti-CD25 microbeads for their ability to deplete alloreactive T cells in vitro

Ex vivo depletion of alloreactive CD25⁺ T cells from a stem cell transplant (SCT) can reduce the incidence of graft-versus-host disease (GVHD) while preserving antimicrobial and perhaps antileukemia activity. However, the most effective methods for allodepleting T cells prior to transplant have not been determined. In this study, we have compared three agents that deplete CD25⁺ activated, alloreactive T cells. These included Ontak (Denileukin Diftitox), an IL-2 fusion toxin, anti-CD25 microbeads (MACS), an anti-CD25 immunotoxin (IT) and a combination of the IT and MACS. Peripheral blood mononuclear cells (PBMCs) activated in a primary mixed lymphocyte reaction (MLR) were allodepleted using optimal amounts of each agent, and the cells were then analyzed by flow cytometry. The treated cells were examined both for remaining alloreactivity and for the preservation of third party reactivity by testing them in a secondary MLR. Our data demonstrate that both the anti-CD25 IT and the anti-CD25 MACS were equally effective in depleting CD4⁺CD25⁺ cells and in sparing T cells that were reactive with third party cells. The anti-CD25 IT was, however, superior in depleting alloreactive CD8⁺CD25⁺ cells. In contrast, Ontak did not eliminate alloreactive cells and the Ontak-treated cells retained significant reactivity against the original stimulator cells.