Figure 3.

The transcription factor IRF5 downstream of TLR7 is significantly increased in B cells of humans and mice with active cGVHD. (A-C) B cells from patients who had received HSCT with active or inactive/no cGVHD at the time of sample collection were isolated by negative magnetic selection from PBMCs. Cells from healthy donors are shown for reference. (A) RNA was isolated from B cells and qPCR was performed to quantify IRF5 gene expression. Levels were expressed as a fold change of active cGVHD (n = 7) when compared with normalized levels in no/inactive subjects (n = 11). (B-C) B cells from patients who had received HSCT with no cGVHD (open circles, n = 5) or active cGVHD (closed circles, n = 4) were left unstimulated or were stimulated with low-dose anti-IgM (0.625 μg/mL), R848 (1 μg/mL) or both R848 and anti-IgM. After 24 hours, cells were harvested, and intracellular flow cytometry was performed to measure IRF5 expression. (B) Half-overlayed flow cytometry histograms from representative active or no patients with cGVHD are shown. (C) Geometric MFI of IRF5 in B cells under each stimulatory condition was determined. (D-F) B cells were isolated via negative magnetic selection from the spleens taken from mice with (BM + Sp) vs mice without (BM only) cGVHD manifestations. (D) RNA was isolated and qPCR was performed to quantify IRF5 gene expression. Levels were expressed as a fold change of BM + Sp (n = 5) when compared with normalized levels in BM only (n = 7) mice. (E-F) IRF5 protein levels in B cells (n = 10 per group) were quantified via intracellular flow cytometry. (E) Half-overlayed flow cytometry histograms from representative BM and BM + Sp mice run on the same day are shown. (F) Protein levels were expressed as a fold change of MFI in the diseased mice (BM + Sp) when compared with levels in the nondiseased (BM only) group to accommodate for the fact that samples were run on multiple days. (G) Representative peribronchiolar B220 and IRF5 staining from diseased (BM + Sp) and nondiseased (BM only) lungs. Images were captured on a Zeiss Axio Imager Z2 upright microscope (Carl Zeiss, Oberkochen, Germany) with the Axiocam 506 color camera. (H-I) The area of positive staining per lung bronchus was quantified for (H) B220 and (I) IRF5 from 3 different images of each individual mouse using Image J software; n = 4 (BM only) and n = 5 (BM + Sp). Statistical analysis was performed using the Mann-Whitney U test; ∗P < .05 and ∗∗P < .01 between sample groups. Data represent median ± range. No, no cGVHD; In, inactive cGVHD; Act, active cGVHD; H, healthy donor; BM only, BM (control group); BM + Sp, BM + spleen (cGVHD group).