Expansions of HY-specific T cells from PBLs and spleen. (A) Tetramer-positive T cells clonally expand with repeated in vitro restimulation. Panels i to iii show clonal expansion in vitro of tetramer-positive T cells from tolerant, memory, and naive mice. PBLs from 3 groups of mice, rejected (i; n = 4), tolerant (ii; n = 11), and naive (iii; n = 6), were stimulated with irradiated syngeneic male spleen cells plus IL-2. Cultures were sampled on day 7 for tetramer analysis, and the remaining cells restimulated on day 14, with sampling and restimulation repeated for 2 further rounds. Each symbol represents an individual mouse. The y-axes of panels ii and iii differ to facilitate identification of individual mice. Panel iv shows clonal expansions in vitro from spleen cells of long-term tolerant mice. T-cell lines were generated by repeated restimulation every 4 weeks of spleen cells from 6 tolerant mice (shown in Figure 4Bii) with irradiated male B6 splenocytes plus IL-2. Seven days after each restimulation, T cells were analyzed for the frequency of HY^DbUty tetramer–positive CD8⁺ T cells (each symbol representing an individual mouse). (B) In vivo cytotoxic capacity of tolerant, naive, and sensitized mice. Five groups of B6 recipient mice (n = 3 or 4), naive males (i,vi), naive females (ii,vii), graft-rejected (iii,viii), and long-term (> 100 days) tolerant females following high-dose (3 × 100 μg; iv,ix), or low-dose (3 × 3 μg; v,x) intranasal HY^AbDby peptide, were given intravenous injections on day 0 with a mixture of 1 × 107 female B6 splenocytes (labeled with 5 μM CFSE) and 1 × 107 male B6 splenocytes (labeled with 0.5 μM CFSE). PBLs taken between days 2 and 24 were analyzed for CFSE-labeled cells and HY^DbUty tetramer–positive CD8⁺ T cells. The ratio of male to female cells is shown in panels i to v, the frequency of HY^DbUty tetramer–positive cells within CD8 T-cell population in panels vi to x. Each symbol represents an individual mouse.