Ikaros deficiency in host APCs exacerbates GVHD. (A) WT B6 Ly5.2 animals were lethally irradiated with 11 Gy and infused with 5 × 10⁶ BM cells and 5 × 10⁶ splenocytes from syngeneic Ly5.1 WT B6 or Ik^−/− B6 donors. Four months later, these animals [B6 → B6Ly5.2] or [Ik^−/− → B6Ly5.2] chimeras received 9 Gy and 2 × 10⁶ CD90⁺ T cells along with 5 × 10⁶ BM cells from either syngeneic B6 or allogeneic MHC-matched, multiple miHA-mismatched C3H.sw donors and analyzed for survival. Shown are B6 → [B6 → B6Ly5.2] (n = 7) (■), B6 → [Ik^−/− → B6Ly5.2] (n = 6) (♦), C3H.sw → [B6 → B6 Ly5.2] (n = 12) (▴), and C3H.sw → [Ik^−/− → B6Ly5.2] (n = 11) (●). ***P < .001 for C3H.sw → [B6 → B6Ly5.2] compared with C3H.sw → [Ik^−/− → B6Ly5.2]. Data are representative of 2 independent experiments. (B) Representative hematoxylin and eosin–stained images and histopathologic score of gastrointestinal tracts (small and large bowels), liver, and skin on days +14 and +21 after allogeneic BMT. Each group includes 5-9 mice. (C) Serum IFN-γ and IL-17A levels at day 7. Each group includes 5-9 mice. (D) Donor T-effector and Treg expansion and ex vivo suppression. Donor CD8⁺ T-cell expansion was analyzed in splenocytes harvested on day +14 from allogeneic [B6 → Ly5.2] or [Ik^−/− → B6Ly5.2] chimeras (n = 5 animals/group) as shown by the black bars versus the gray bars, respectively (P < .05). Donor CD4⁺CD25⁺Foxp3⁺ Treg expansion was analyzed in the spleens harvested on days +14 and +21 and in the liver harvested on day +21 from the allogeneic [B6 → Ly5.2] or [Ik^−/− → B6Ly5.2] animals (n = 5 animals/group) as shown by the black bars versus the gray bars, respectively (P = not significant at both day +14 and day +21). The donor CD4⁺CD25⁺ and the CD4⁺CD25⁻ T cells were isolated from the livers (pooled from 5 mice/group) harvested on day +21 from the allogeneic [B6 → Ly5.2] or [Ik^−/− → B6Ly5.2] animals. The suppressive function of CD4⁺CD25⁺ T cells was analyzed by coculturing the isolated 2.5 × 10³ CD4⁺CD25⁺ T cells with 10⁴ CD4⁺CD25⁻ T cells in the presence of 5 × 10⁴ irradiated WT B6 spleen cells for 120 hours. Incorporation of ³H-thymidine (1 μCi/well) by proliferating cells was measured during the last 6 hours of culture. Data show means ± SEM. P = not significant. Data are from 1 of 2 similar experiments. (E) WT B6 Ly5.2 animals were lethally irradiated with 11 Gy and infused with 5 × 10⁶ BM cells and 5 × 10⁶ splenocytes from syngeneic Ly5.1 WT B6 or Ik^−/− B6 donors. Four months later, [B6 → B6Ly5.2] or [Ik^−/− → B6Ly5.2]) animals were irradiated with 9 Gy and transplanted with 2 × 10⁶ CD90⁺ T cells along with 5 × 10⁶ BM cells from either syngeneic B6 or allogeneic MHC-mismatched BALB/c donors and analyzed for survival. Shown are B6 → [B6 → B6Ly5.2] (n = 7) (■), BALB/c → [B6 → B6Ly5.2] (n = 10) (▴), and BALB/c → [Ik^−/− → B6Ly5.2] (n = 13) (●) animals. Data are representative of 2 independent experiments. (F) WT B6Ly5.2 animals were lethally irradiated with 11 Gy and infused with 5 × 10⁶ BM cells and 5 × 10⁶ splenocytes from syngeneic Ly5.1 WT B6 or IkDN^+/− B6 donors. Four months later, [B6 → B6Ly5.2] or [IkDN^+/− → B6Ly5.2]) animals were used as recipients, irradiated with 9 Gy, and transplanted with 2 × 10⁶ CD90⁺ T cells along with 5 × 10⁶ BM cells from either syngeneic B6 or allogeneic MHC-matched, miHA-mismatched C3H.sw donors, and the animals were evaluated for survival. Shown are B6 → [B6 → B6Ly5.2] (n = 6) (■), C3H.sw → [B6 → B6 Ly5.2] (n = 9) (▴), and C3H.sw → [IkDN^+/− → B6Ly5.2] (n = 11) ●. Data are representative of 2 independent experiments.