(A) Anti-K^d alloresponses were estimated using IFN-γ ELISpot. C57BL/6 mice were primed against K^d by rejection of a B6.Kd skin graft. Thirty to 35 days later, some mice were injected with 5 × 10¹¹ vgc AAV-K^d or K^d-D227K. After a further 7 days, splenocytes from inoculated mice, primed mice that were not inoculated with vector, or unprimed C57BL/6 controls (n = 9–10/group) were prestimulated in a 1-way multiple linear regression (MLR) with irradiated B6.Kd splenocytes, and IFN-γ–producing cells were enumerated using ELISpot. (B) Statistical analysis was performed using 1-way ANOVA in conjunction with Sidak’s multiple comparisons test. The number of responder cells producing IFN-γ in response to K^d-bearing stimulators increased from 41.78 ± 16.45 spot-forming cells/million unfractionated splenocytes (SFC/1 × 10⁶) in naive C57BL/6 to 336.1 ± 71.67 SFC/1 × 10⁶ in primed mice (P = 0.0009). Inoculation of primed C57BL/6 mice with AAV-K^d significantly decreased IFN-γ production (117.4 ± 32.63 SFC/1 × 10⁶, P = 0.015 compared with primed mice not receiving vector), while treatment with AAV-K^d-D227K did not reduce the anti-K^d response (247.4 ± 66.33 SFC/1 × 10⁶, P = 0.55). Parallel results were obtained when pooled, CD8-enriched splenocytes from primed mice were used as responders (n = 3/group). Results were analyzed as for A. CD8-enriched splenocytes from unprimed C57BL/6 mice contained 807.6 ± 23.84 SFC/1 × 10⁶, while 2,324 ± 525.8 SFC/1 × 10⁶ were detected in the primed animals (P = 0.0265). In primed mice transduced with AAV-K^d, the number of IFN-γ–producing cells fell to 384.4 ± 122 SFC/1 × 10⁶ (P = 0.007), whereas a nonsignificant reduction was noted when primed mice received AAV-K^d-D227K (1,014 ± 313.5 SFC/1 × 10⁶, P = 0.053). Box shows minimum to maximum with a line at the mean. All data are described as mean ± SEM.