B cells from patients and mice with active cGVHD have increased TLR7 expression and show heightened proliferative responses to low-level BCR and TLR7 stimulation. Post-HSCT B cells from patients with active (n = 6-9) or no/inactive (n = 7-9) cGVHD at the time of sample collection were isolated by negative magnetic selection from peripheral blood mononuclear cell (PBMCs). Cells from healthy donors are shown for reference. Plated B cells were stimulated with the (A) TLR7 agonist R848 (1 μg/mL) or (B) TLR9 agonist CpG (1 μg/mL) in the presence of low-dose surrogate BCR antigen (anti-IgM, 0.625 μg/mL) for 48 hours and assessed for cell cycle entry (Ki-67 expression). Data represent frequency of Ki-67⁺ B cells of total CD19⁺ B cells. (C-D) RNA was isolated from B cells and quantitative polymerase chain reaction (qPCR) was performed to quantify (C) TLR7 and (D) TLR9 gene expression. Levels are expressed as a fold change when compared with normalized levels in individuals with no/inactive cGVHD. (E-H) TLR7 protein levels in B cells (n = 10 per group) from mice with cGVHD (BM + Sp) or control mice (BM only) were quantified via intracellular flow cytometry in (E-F) blood at day 33 after transplantation or (G-H) in lung tissue at days 46 to 47 after transplantation. Half-overlayed flow cytometry histograms from representative BM and BM + Sp mice are shown. Levels were expressed as geometric mean fluorescence intensity (MFI) in the diseased mice (BM + Sp) when compared with levels in the nondiseased (BM-only) group. All samples were run on the same day. Data represent median ± range. ∗P < .05 and ∗∗P < .01 between sample groups. No, no cGVHD; In, inactive cGVHD; Act, active cGVHD; H, healthy donor; BM only, BM (control group); BM + Sp, BM + spleen (cGVHD group).