CD4⁺CD25⁺ regulatory lymphocytes are not involved in 3H3-mediated inhibition of cGVHD. (A) cGVHD was induced by transferring 8 × 10⁷ DBA/2 parental cells into BDF₁ mice. 3H3 was administered immediately afterward. The mice were divided 6 weeks later into 2 groups (n = 5 per group); one group was infused with 8 × 10⁷ DBA/2 parental cells (▪) and the other remained without infusion (). Autoantibody production was measured from 2 weeks thereafter. OD values are shown as means ± SD of n = 5 per group at 10-fold dilution of samples. ^*P < .05 between the 2 groups. (B) Purified DBA/2 CD4⁺CD25⁺ T cells were stimulated in vitro by irradiated BDF₁ splenocytes in the presence of 1 μg/mL anti-CD3 plus anti-CD28 mAbs for 5 days. A quantity of 1 × 10⁶ of the allo-stimulated CD4⁺CD25⁺ T cells (> 90% purity) was infused into BDF₁ mice immediately after parental cell transfer. Autoantibody production was measured at day 14. OD values are shown as means plus or minus SD of n = 5 per group at 10-fold dilution of samples. ^*P < .05, between the 2 groups. (C-F) BDF₁ mice were depleted of CD4⁺CD25⁺ T cells as described in “Materials and methods.” On day 14, serum and splenocytes were harvested from cGVHD mice. (C) OD values are shown as means ± SD of n = 4 per group at 10-fold dilution of samples. ^*P < .05, between the control Ig–treated group without depletion and the indicated group. (D-E) Donor CD4⁺ T-cell and B-cell numbers were counted by staining splenocytes with anti–H-2K^b plus anti-CD4 (D) or anti-B220 (E), respectively. (F) Autoreactive B cells producing anti-DNA IgG₁ were counted using ELISPOT. Data are presented as means ± SD of n = 4 per group. ^*P < .05, between the control Ig–treated group without depletion and the indicated group.