Functional assessment of tetramer-positive cells from multiparous and memory mice. (A) Spleen cells from memory B6 mice that had rejected male skin grafts, and from multiparous B6 mice, were stimulated with irradiated syngeneic male spleen cells plus IL-2 for 7 days. The cells were analyzed for IFNγ production by intracellular staining, as described in detail in “Materials and methods.” In brief, after removing the dead cells, the viable cells were restimulated with unpulsed APCs or with HY peptide—pulsed APCs for 2 hours in the presence of monensin. After harvesting, the cells were stained with anti-CD4, anti-CD8, and HY^DbUty tetramer. After fixation and permeabilization, the cells were stained with anti-IFNγ prior to flow cytometric analysis. Representative results are shown from a memory and a multiparous mouse. The percentages shown in 2 of the panels in the left column indicate tetramer-positive cells within the CD8⁺ T-cell populations of each mouse. (B) IFNγ production by gated CD8⁺ tetramer⁺ T cells is represented as histograms (filled, APC+HY peptide; unfilled, APCs alone). The percentage represents the frequency of IFNγ-producing cells within the gated tetramer⁺ CD8⁺ T-cell population.