Invasive aspergillosis is a major cause of morbidity and mortality in patients undergoing allogeneic hematopoietic SCT. There is a growing body of evidence that T cells are important in the host defense against Aspergillus, and adoptively transferred anti-Aspergillus T-helper 1 (T(H)) 1 cells might reduce infectious mortality in hematopoietic transplant recipients. Here we present for the first time a simple and rapid method for the clinical-scale generation of functionally active anti-Aspergillus T cells according to good manufacturing practice conditions. A total of 1.1 x 10(9) WBCs derived from a leukapheresis product were incubated with Aspergillus antigens. Stimulated cells were selected by means of the IFN-gamma secretion assay and expanded. In three independent experiments, a median number of 2 x 10(7) CD3+CD4+cells (range, 0.9-3.2 x 10(7)) were obtained within 13 days. The cultured CD3+CD4+ cells exhibited almost exclusively a memory activated T-helper cell phenotype. Upon restimulation, the generated T cells produced IFN-gamma, but no IL-4 or IL-10, indicating a T(H)1-cell population. Additionally, the cells proliferated upon restimulation and showed reduced alloreactivity compared to unselected CD4+ cells. This method of generating is suitable for future prospective trials designed to evaluate the effect of adoptive immunotherapy in hematopoietic transplant recipients with invasive aspergillosis.