Exacerbated inflammation partially contributes to GVHD in FA. (A) Expression of GVHD effective cytokines in FA Treg-transplanted mice. Lethally irradiated Balb/c recipients were transplanted with 5 × 10⁶ TCD cells from WT C57Bl/6 animals alone, or with sorted WT Teff cells (CD4⁺CD25⁻, 5 × 10⁵) plus or minus equal numbers of sorted CD4⁺CD25⁺ Treg cells from either WT C57BL/6, Fanca^−/− or Fancd2^−/− mice. Donor T cells isolated from the small intestine of the Balb/c recipients transplanted with the indicated donor cells were stimulated with 50 ng/mL PMA and 2 μg/mL Ionomycin for 1 hour, followed by 3 hours incubation in the presence of 1 μg/mL Brefeldin A. Treated cells were stained H-2^b+ and CD4⁺ antibodies before the treatment with Cytofix/Cytoperm reagent. Cytokines were intracellularly stained with antibodies specific for TNF-α, IFN-γ, and IL-6 followed by flow cytometric analysis gated on the H-2^b+CD4⁺ cell compartment. Each group includes 6 to 10 mice. (B) Levels of TNF-α, IFN-γ, and IL-6 in sera of recipient mice described in (A). (C) Increased NF-κB transcription activity in infiltrated FA T cells. H-2^b+CD4⁺ T cells isolated from liver (left) and small intestine (right) of the recipients described in (A) were used for flow cytometric analysis for CD154. Representative flow graphs are shown. (D) p65 deletion partially reduces FA GVHD mortality. Treg cells from p65^f/fFanca^+/+ or p65^f/fFanca^−/− mice plus TCD + Teff cells were transplanted to lethally irradiated Balb/c recipients followed by DMSO or Tamoxifen treatment of 3 days. Survival of recipients was monitored by Kaplan-Meier curve method. Each group includes 6 to 9 mice.