Analysis of CD8⁺ T cells infiltrating rAAVeGFP-transduced quadriceps muscle. (A) (Left column) H-2K^d eGFP_200-208 tetramer staining of lymphocytes isolated from spleen, liver, and muscle of mice injected with rAAVeGFP 56 days earlier. Numbers represent the percentage of CD8⁺ T cells that costain with the eGFP tetramer. Plots were gated as in Figure 1D. (Right column) Annexin V binding by H-2K^d eGFP_200-208 tetramer⁺ CD8⁺ T lymphocytes isolated from spleen, liver, and muscle of mice injected with rAAVeGFP 56 days earlier. Numbers represent the percentage of tetramer⁺ cells that are negative (top left quadrant) and positive (top right quadrant) for annexin V binding. Plots were gated on FSC/SSC appropriate for lymphocytes, PI⁻CD4⁻B220⁻F4/80⁻CD3⁺CD8⁺ cells. (B) Side-by-side IFN-γ ELISpot analysis of eGFP_200-208-specific CD8⁺ T lymphocytes harvested from spleen, liver, and quadriceps muscle 56 days after transduction with rAAVeGFP (performed in parallel with experiment depicted in panel A). Numbers represent the number of spot forming cells per 100 eGFP-tetramer⁺ cells. Data represent 2 independent experiments each with tissues pooled from 5 mice. The relationship between IFN-γ secretion and annexin V binding was assessed using the standard correlation equation, where r = −.95, P < .001; n = 6.