ALLOGENEIC stem cell transplantation is the treatment of choice for many hematologic malignancies or inherited disorders. In the partially incompatible HLA setting ex vivo T-cell depletion of the graft and posttransplantation immunosuppression prevent development of graft-versus-host disease (GVHD), but lead in turn to a delay in immune reconstitution and a concordant increase in the incidence of opportunistic infections and disease relapses. To deplete the T cells responsible for GVHD but retain T cells responsible for the anti-leukemic and antiinfectious activities, we have developed an in vitro procedure to allo-activate donor T cells responsible for GVHD and eliminate them with an immunotoxin that is reactive with the cell surface activation antigen CD25. We infused the remaining allodepleted T cells between day 15 and 47 into pediatric patients with acquired or congenital hematopoietic disorders who received hematopoietic stem cell transplants (HSCT) on day 0. We evaluated the rates of GVHD and opportunistic infections as well as the time to immune reconstitution. Sixteen infusions of allodepleted T cells, ranging from 1 to 8 10 cells/kg were administered to pediatric patients between March 1999 and April 2001, none of whom received prophylactic therapy for GVHD. Despite the risk of transfusing donor T cells activated against recipient alloantigens ex vivo, GVHD greater than grade II was not observed. Two patients (13%) developed grade 1 aGVHD limited to skin, and two others (13%) developed grade II aGVHD with one case involving both the skin and gastrointestinal tract. All cases resolved with treatment. Interestingly, all cases correlated with a residual alloproliferation of 1% (P .001) (data not shown). Three patients displayed an active cytomegalovirus (CMV) infection at the time of HSCT that required antiCMV treatment. In addition, one patient (#5) experienced pulmonary and cerebral EBV-induced lymphoproliferation and a varicella zoster virus infection. After the infusion of allodepleted T cells, two types of situations were observed. Patients who did not show major infectious complications or GVHD displayed a progressive increase in the CD4 T-cell compartment, particularly of naive CD45RA phenotype cells, which were detectable by week 14. In contrast, patients who were infected at time of T-cell infusion demonstrated a rapid expansion in the CD4 T-cell compartment with clinically significant numbers of cells, all expressing CD45RO by week 8. In an attempt to assess the ability of an allodepleted T-cell “add-back” to exert an anti-viral infection effect, immunity to EBV and CMV infections was tested at 1 month after infusion in two infected patients, included the one presenting with cerebral and pulmonary lesions. In these two cases, circulating T cells were highly responsive to CMV and EBV as shown by their capacity to secrete -interferon in the presence of an autologous EBV cell line (transfected or not with CMV pp65 antigen) and their capacity to kill CMV pp65-expressing target cells without in vitro restimulation. As shown by CT scan, pulmonary and central nervous system lesions in patient 5 caused by the EBV infection disappeared 11 weeks after the T-cell infusion. For the other patient, CMV antigen was negative 15 weeks post-HSCT (10 weeks postinfusion). Infused allodepleted T cells seem to not recirculate in noninfected patients, an observation that may depend upon the low numbers of infused cells. As illustrated by the three cases of relapse among the five leukemic patients included in the clinical trial, cellular therapy for viral infections is achieved with doses of lymphocytes within the range of the