LSel^hi, but not LSel^lo, Tregs protect against GVHD-associated mortality and morbidity by interfering with the activation and expansion of GVHD effector T cells. Lethally irradiated B6 mice were infused with BALB/c BM. Cohorts received 5 × 10⁶ BALB/c CD25-depleted effector T cells alone or with ex vivo activated and expanded BALB/c LSel^hi or LSel^lo Tregs at the indicated T/reg ratio. Experiments illustrated in panels C-E used a ratio of 1 T cell to 3 Tregs. (A) Survival is indicated (n = 8/group;^*P = .008 compared to T cells). (B) Average weight in grams of mice from panel A is indicated. ^*Only one mouse survived after this time point. (C) Spleens were harvested and enumerated 6 days after transplantation. Use of BALB/c Thy1 congenics allowed Thy1.1⁺ effectors to be distinguished from Thy1.2⁺ Tregs. The average total number of effector CD4⁺ and CD8⁺ T cells per spleen is shown. Error bars indicate standard deviation (n = 4 separate pools of 2 spleens/pool for each group; ^*P < .05). Data for panels C-E were taken from one of 2 replicate experiments. (D) Splenocytes from panel C were phenotyped. Shown is LSel expression on gated GVHD effector thy1.1⁺ CD4⁺ and CD8⁺ T cells. Shown are data from one of 4 representative splenic pools per treatment group. Percentage of LSel⁺ cells is indicated in the top right quadrant. (E) Splenocytes from panels C and D were stimulated in vitro with irradiated host-type splenic stimulators. Shown are peak secondary antihost proliferative responses of 4 separate pools of 2 spleens/pool from GVHD control mice, LSel^hi and LSel^lo Treg-treated mice. Cultures consisted of 3 × 10⁴ effector Thy1.1⁺ CD4⁺ T cells restimulated with 10⁵ irradiated B6 splenocytes, plated in replicates of 6. Note that infused Tregs are not depleted from the culture.