Effects of IFN-γ neutralization on autoantibody production and deletion of donor CD4⁺ T cells and B cells. (A-D) cGVHD was induced by transferring 8 × 10⁷ DBA/2 parental cells into BDF₁ mice, and 3H3 or control Ig was administered immediately afterward. (A) Three days later, splenocytes were harvested and stimulated with PMA/ionomycin. Twenty-four hours thereafter, the concentration of IFN-γ in culture supernatants was measured. (B-D) Serum was harvested from cGVHD mice at days 3 (B), 7 (C), and 14 (D) after disease induction. Serum IFN-γ levels were measured by ELISA. ND indicates not detectable. Data are presented as means ± SD of n = 3-4 per group. ^*P < .05 between the 2 groups. (E-I) Starting from day 2, 500 μg anti–IFN-γ mAb was administered to cGVHD mice every 4 days. On day 14, serum and splenocytes were harvested from the cGVHD mice. (E) OD values for anti-DNA IgG₁ levels are shown as means ± SD at 10-fold dilution of samples. (F-G) Donor CD4⁺ T-cell and B-cell numbers were counted by staining splenocytes with anti–H-2K^b plus anti-CD4 (F) or anti-B220 (G), respectively. (H) Autoreactive B cells producing anti-DNA IgG₁ were counted using ELISPOT. (I) Splenocytes were triple-stained with anti–H-2K^b, anti-CD4, and anti-CD62L, and H-2K^b–negative CD4⁺ T cells were gated and analyzed for CD62L expression. Representative FACS plots are shown in the left column, and percentages of CD62L^low versus CD62L^high donor CD4⁺ T cells are summarized in the right column. Data are presented as means ± SD of n = 3-5 per group. ^*P < .05 between the control Ig–treated group without neutralization and the indicated group (E, F, H), or between the indicated groups (G).