Efficient depletion of alloreactive donor T lymphocytes based on expression of two activation‐induced antigens (CD25 and CD69)

T lymphocytes play an important role in allogeneic bone marrow/stem cell transplantation by supporting engraftment and immune recovery. Moreover, donor T cells have been shown to mediate the so‐called graft‐versus‐leukaemia effect and are, therefore, increasingly used for adoptive immunotherapy. However, T‐cell infusions are associated with the risk of a graft‐versus‐host reaction, which may lead to a life‐threatening disease. To overcome this problem, we followed a new strategy for the exclusive depletion of alloreactive cells. We activated allogeneic T cells by cultivation on an adherent cell layer derived from peripheral blood. We then depleted activated cells based on the expression of CD25, CD69 or both activation‐induced antigens using magnetic cell sorting. Mixed lymphocyte culture (MLC) reactions and helper T‐lymphocyte precursor cell frequency (HTLP‐f) assays demonstrated that this technique led to a significant decrease in alloreactivity of ‘donor’ cells, which at the same time preserved reactivity against third‐party cells. The lowest level of alloreactivity was found when CD25 and CD69 antibodies were used together for depletion. This corresponds with our observation that expression of CD25 or CD69 may partially represent different activation pathways. We conclude that ex vivo depletion of CD25‐ and CD69‐expressing alloreactive cells may help to overcome limitations of adoptive immunotherapy.