Alloantigen-induced CD8⁺Tregs are less potent than CD4⁺Tregs for the suppression of GVHD. (A) Sorted CD4⁺EGFP^─ and CD8⁺EGFP^─ T cells (1 × 10⁵) from Foxp3^EGFP mice were cultured with Balb/c CD11c⁺ dendritic cells (5 × 10⁴) in the presence of all-trans retinoic acid (10 nM), IL-2 (100 U/mL), and TGF-β (10 ng/mL) for 5 days. Representative histogram depicting cell surface expression of CD25, CD62L, CD103, GITR, CD44, and CTLA-4 on in vitro-differentiated alloantigen-induced CD4⁺ and CD8⁺ Tregs vs staining with an isotype control antibody. (B) Purified B6.PL Thy1.1⁺ T cells (1 × 10⁵) were cultured with Balb/c CD11c⁺ dendritic cells (5 × 10⁴) in the presence of varying ratios of Thy1.2⁺ CD4⁺ Tregs (green bars), or CD8⁺ Tregs (blue bars) that were generated against either Balb/c (Balb/c→ Bab/c) or FVB (Balb/c→FVB) dendritic cells after 5 days in culture. Control wells are depicted as red bars. Data are presented as the mean percentage of triplicate wells of proliferating Thy1.1⁺ T cells ± 1 SD and represent cumulative results of 3 to 6 experiments. (C-D) Lethally irradiated Balb/c mice were transplanted with B6.PL BM and spleen cells (adjusted to yield a T-cell dose of 0.6 × 10⁶ αβ T cells). Cohorts of animals then received an equivalent number of CD4⁺ or CD8⁺ in vitro-differentiated Thy1.2⁺ Tregs. Representative dot plots showing the percentage of H-2K^b+ Thy1.2⁺ CD4⁺ or CD8⁺ iTregs in the spleen, liver, lung, or colon of recipient mice 10 days posttransplantation are shown in (C). The absolute number and percentage of donor-derived CD4⁺ (red bars) and CD8⁺ (blue bars) Thy1.2⁺ EGFP⁺ T cells in the specified tissue sites on day 10 are depicted in (D). Data are from 8 to 12 mice per tissue from 3 experiments. (E) Lethally irradiated Balb/c mice were transplanted with 8 × 10⁶ B6.PL (Thy1.1⁺) BM alone (red circles, n = 17) or together with spleen cells (adjusted to yield a dose of 0.6 × 10⁶ αβ T cells). Animals transplanted with adjunctive spleen cells received no other cells (blue squares, n = 24), 0.6 × 10⁶ in vitro-generated alloantigen-induced CD4⁺ Tregs (green triangles, n = 23), or 0.6 × 10⁶ alloantigen-induced CD8⁺ Tregs (purple triangles, n = 25). Survival is shown. Data are the cumulative results of 5 experiments. *P < .05; **P < .01; ***P < .001; ****P < .0001.