Restimulation of generated functionally active anti-Aspergillus T cells and loss of alloreactivity after enrichment and expansion of anti-Aspergillus T cells. (A-B) CFSE-labeled generated anti-Aspergillus T cells were cocultured with autologous APCs, which were either unloaded (A) or preincubated with EC SAB (B). The CSFE staining and the expansion rate are shown in 1 representative donor after 4 days. The number of divisions and the percentages of lymphocytes that underwent at least 3 cell cycles are indicated. Multiple cell divisions can be detected in anti-Aspergillus T cells after activation with EC SAB-loaded APCs, as indicated by loss of intensity in the CFSE signal. In contrast, only a marginal number of anti-Aspergillus T cells expanded in the same time period when cocultured with unloaded APCs. (C-D) CFSE-labeled unselected CD4⁺ T-cells (C) or anti-Aspergillus CD4⁺ T cells (D) were coincubated with third-party APCs to assess alloreactivity. Whereas most unselected CD4⁺ T cells underwent multiple cell divisions, purified anti-Aspergillus CD4⁺ T cells show a weak proliferative response in the same time period (D).