Figure 3.
XBP-1 mediates B-cell activation and cGVHD pathogenicity. Lethally irradiated BALB/c mice were transplanted with TCD-BM (5 × 106 per mouse) from XBP-1flox/floxCD19Cre− (n = 21), XBP-1flox/floxCD19Cre+ (n = 15), or TBCD-BM B6Ly5.1+ (n = 10) donors on a B6 background with (n = 40) or without (n = 6) between 0.5 and 1 × 106 whole splenocytes. Mice were monitored for cGVHD clinical scores until day 60 after allo-HCT, and statistics were performed using a Mann-Whitney U test of the entire time course (A). Subsets of recipient mice were killed on day 30, and spleens were excised and processed into single-cell suspensions for flow cytometric analysis of B220+ B cells (B), CD86-expressing B cells (C), GC B cells (Fas+GL7+) (D-E), and B-cell surface IgM (D,F). Serum from peripheral blood was collected on day 30 and assayed for anti-dsDNA autoantibodies using ELISA (G). On day 30, 2 × 106 recipient splenocytes from each mouse were stimulated with lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), and Ionomycin, and B cells were intracellularly stained and analyzed for IL-12p35 and IFN-γ cytokine production (H-J). Data shown in panel A are representative clinical scoring of 3 replicate experiments. Data in panels B-D and F-J were collected from 1 subset of mice (n = 10) out of n = 40 recipients. Data in panel E are pooled from 2 independent experiments. P < .05 indicates statistical significance. MFI, mean fluorescence intensity; O.D., optical density.