In vivo evidence of a T-cell-mediated B6 anti-LYL8 response. (A) Top panels: 1 × 10⁶ BCZ 108-2 cells (positive control for LYL8 tetramers) were coincubated with fluorescein isothiocyanate (FITC)-anti-CD8α and PE-LYL8 (left panel) or HSV8 (right panel) H2K^b tetramers. Dot plots of the cells are shown as acquired. Bottom panels: Splenocytes from immunized B6 mice were stained in the same fashion as the BCZ 108-2 cells. Prior to acquisition, 7-AAD was added to each sample to allow gating on live cells. Dot plots gated on live CD8⁺ cells are shown; the percentage of tetramer-positive CD8⁺ cells is shown in the top right quadrant. (B) IFNγ ELISPOT assay on CD8⁺ splenocytes from immunized B6 (imm B6) mice or naive mice with APCs from naive B6 mice (left) or BALB.B mice (right). APCs, listed along the x-axis, were pulsed with nothing (“imm B6”), H2K^b peptides LYL8 or HSV8 (nonspecific control), or H2K^d peptide J1B (irrelevant control). NIL indicates negative controls without APCs. Shown are the data from one representative experiment of 2 experiments total. Bars indicate the mean number of spots plus or minus the standard error of the mean (SEM).