Summary:The success of allogeneic hematopoietic stem cell transplantation from HLA-disparate donors depends on the development of new strategies for graft-versus-host disease prevention able to target specifically donor antihost alloreactivity, while preserving GVL and antiviral immune surveillance. Recent experimental and clinical work has shown the feasibility of an approach based on induction of anergy to host alloantigens through blockade of B7/CD28 costimulatory signal in donor T cells, but data on the impact of this strategy on the recovery of the immune system are still lacking. We devised an ex vivo method for induction of host alloantigen-specific unresponsiveness based on treatment with the B7/CD28 blocking agent CTLA4-Ig associated with CsA. We then proceeded to assess the maintenance of an effective immune response towards viral pathogens and tumor cells after CTLA4-Ig/CsA treatment, by measuring the frequency of CTL precursors directed against CMVand EBV-infected targets, and against autologous leukemic blasts. We demonstrated that (1) CTLA4-Ig and CsA can act synergistically in inducing a state of unresponsiveness to alloantigens; (2) the number of leukemia-reactive, EBV-specific and CMV-specific CTLp is not impaired by CTLA4-Ig/CsA treatment. Our data provide the first direct in vitro evidence that it is possible to preserve antiviral and antileukemia effector cells after blockade of CD28/B7 interaction during MLR. Bone Marrow Transplantation (2001) 27, 1263-1273. Keywords: T lymphocytes; anergy; infectious immunityvirus; tumour immunity; transplantation Allogeneic hematopoietic stem cell transplantation (HSCT) offers the chance of a cure to patients with hematological and immunological disorders. Unfortunately, no more than two-thirds of patients eligible for this procedure have an HLA-matched donor, either related or unrelated. Attempts to augment the size of the donor pool by transplanting unmanipulated hematopoietic progenitors from HLA-haploidentical related or mismatched unrelated donors resulted in a considerable increase in the incidence and severity of GVHD. 1,2 The feasibility of HSCT from HLA-haploidentical related or mismatched unrelated donors, achieved through GVHD prevention by means of T cell depletion prior to infusion of stem cells, was first demonstrated in pediatric patients transplanted for primary immunodeficiencies, 3 and more recently confirmed in both adult and pediatric patients given HSCT for malignancy. 4,5 Notwithstanding the remarkable improvements in the achievement of engraftment without GVHD, infections and leukemia relapse are still a major complication of the delayed immune recovery experienced by patients given T cell-depleted HSCT from HLA-partially matched donors. 4,5 The development of new approaches to T cell manipulation aimed at selective induction of unresponsiveness 6-10 or depletion of alloreactive T cells, 11-13 could allow prevention of GVHD, while drastically reducing the risk of infection and leukemia relapse by accelerating immunological recovery a...