Tcms display low veto activity in vitro, but upon reactivation acquire an effector phenotype, which is associated with potent and specific veto activity. (A) 2c splenocytes were stimulated with irradiated BALB/c (H-2^d) splenocytes in the presence or absence of CB6 (H-2^bd)–derived purified Tcms, CTLs, or purified Tcms reactivated in vitro with their cognate third-party FVB stimulators for 60 hours (R′ Tcms). The veto cells were added at the indicated veto-effector ratios. Veto activity was analyzed by FACS analysis 3 days after the initiation of the mixed lymphocyte reaction (MLR), to monitor the inhibition of CD8⁺1B2⁺ 2c cell expansion. Results are presented as mean ± SD of percentage inhibition from 5 independent experiments. (B) FACS analysis of annexin V levels on living (7AAD⁻) CD8⁺1B2⁺ 2c cells, at the end of the MLR, plated with or without veto cells at a veto-effector ratio of 0.02. Results are presented as mean ± SD of percentage annexin V in 7 independent experiments. (C) MLR was established as in panel A. The inhibition of 2c cell expansion was evaluated when veto cells derived from specific CB6 (H-2^bd, spe′) or nonspecific C3B6F1 (H-2^bk, nonspe′) mice were added at a 0.02 veto-effector ratio. Results are presented as mean ± SD of percentage inhibition from 4 independent experiments. The 2c cells were also analyzed for annexin V levels (D). (E) Lethally irradiated (8 Gy) BALB/c mice received 4 × 10⁶ C57BL/6-NUDE BM cells and 1.25 × 10⁴ syngeneic T cells. Mice then received a transplant of 2 to 10 × 10⁶ purified CB6 CD8⁺ Tcms, which were analyzed for CD62L phenotype before the adoptive transfer (left panel, Tcms). Mice were killed 4 days after adoptive transfer, and LNs were harvested and mashed; CD62L expression on CB6 CD8⁺ T cells, isolated from the LNs, was analyzed by FACS (right panel, LN Tcms). Representative result of Tcms isolated from LNs of 1 of 20 mice tested in 4 independent experiments is displayed. (A-E) ***P < .001.