In vivo T-cell response after adoptive transfer of hexon-specific T cells after allogeneic HSCT. Antigen-specific T cells were analyzed by flow cytometry after stimulation of blood samples with hexon antigen, followed by intracellular cytokine staining. (A) Fourteen of 23 evaluable patients developed successful in vivo T-cell expansion after ACT. The threshold of positive antigen-specific T-cell responses was defined as 0.01% IFN-γ⁺ T cells of viable T cells. In vivo expansion could be detected until a mean of 72 ± 47 days (range, 11 to 177 days) after ACT and was found within both the CD4⁺ and CD8⁺ T-cell compartments in 13 (93%) of the 14 patients with detectable IFN-γ⁺ T cells. Exclusive expansion in the CD4⁺ T-cell compartment was found in patient 7 (and in patient 30 after first ACT). Detailed information on in vivo T-cell expansion of the patients is shown in supplemental Figure 2. (B) Time course of in vivo T-cell response and virologic response of patient (Pat.) 31 is shown. In vivo expansion of AdV-specific T cells after adoptive transfer was associated with clearance of the viral load. Patient 31 shows a straight and fast virologic response with clearance of viremia <6 weeks after second ACT. (C) Immune reconstitution status prior to and after ACT. Number of leukocytes (white blood cell count [WBC]) and CD3⁺ T cells are shown prior to and 1 week and 1 month after ACT. WBC counts did not change after ACT, but CD3 counts significantly increased after ACT, confirming that the response was not related to an overall reconstitution of WBCs. ns, not significant.