HA-specific CD4⁺CD25+ cells suppress anti-HA B- and T-cell immune responses. At day 0, anesthetized BALB/c mice were injected into the tibialis anterior with 25 μL AAV-HA (6 × 10¹¹ physical particles (pp)/mL) and were either untreated (No cells) or intravenously injected twice at days 0 and 4 with 10⁶ CD4⁺CD25⁺ cells from TCR-HA mice (HA-Tregs). Their splenocytes were tested at day 14 in a standard IFNγ-ELISPOT assay against the HA512-520 epitope, and spot-forming units (SFU) are represented after subtraction of background spots obtained with unpulsed splenocytes (A). To monitor the specificity of the response, AAV-HA-transduced mice were untreated (No cells), or injected twice with 10⁶ CD4⁺CD25⁺ cells from TCR-HA mice (HA-Tregs), CD4⁺CD25-cells from TCR-HA mice (HA-Th), or CD4⁺CD25⁺ cells from BALB/c mice (Tregs). Splenocytes were tested at day 35 by IFNγ-ELISPOT assay (B), restimulated in vitro for 6 days with HA512-520, and tested in cytotoxic assay (C), and mouse sera were assayed for the presence of anti-HA IgG (D). For ELISPOT and ELISA assays, results represent the mean of 3 mice per group and are expressed as mean ± standard error of the mean (SEM). For cytotoxic assays, the percentage of specific lysis was calculated as the difference in lysis between HA512-520-pulsed (1 μM) and unpulsed P815 targets cells. Results from one representative mouse per group are shown. Comparison of SFU and IgG titers were performed using Mann-Whitney t test. Statistically significant P values less than .01 were found between HA-Tregs and each other group (^**), and less than .05 between HA-Th and No cells (^*).