Chronic Graft-versus-Host Disease (GVHD) is a major limitation to successful allogeneic bone marrow transplantation (BMT). Lipopolysaccharide (LPS), a cell-wall component of gram negative bacteria that signals through Toll-like receptor 4 (TLR4), is an established contributor to acute GVHD. Our lab has previously shown that splenocytes from mice with acute GVHD were hyper-responsive to CpG, unmethylated DNA with C-G repeats from bacteria and viruses, which induce rapid responses by dendritic cells, B cells, and monocytes through TLR9. As part of Children’s Oncology Group phase III randomized trial ASCT0031 evaluating the efficacy of hydroxychloroquine in the treatment of newly diagnosed chronic GVHD, peripheral blood was drawn for immune phenotype and functional studies in patients and control (no GVHD, 80–120 days post allogeneic transplant). The non-BMT group were healthy adult volunteers. After Ficoll isolation, lymphocytes were cultured with or without synthetic human immunostimulatory CpG 2006 (6μg/mL) or LPS (1μg/mL) for 48 hours and analyzed by flow cytometry. Cell surface marker expression was evaluated as fold difference between stimulated and unstimulated cells. In some samples, B cells were purified by negative selection to determine proliferative response and/or for the quantitation of TLR9 mRNA. Chronic GVHD samples had a 3.7 +/− 1.2 fold (n=11) increase in B cell CD40 mean channel fluorescence after CpG stimulation which was significantly greater than in the control group at 1.0 +/− 0.6 fold (n=3, Mann-Whitney U test p=0.02) and the non-BMT group at 1.5 +/− 0.9 fold (n=3, p=0.01). Similarly, chronic GVHD samples had a 9.1 +/− 5.7 fold (n=10) increase in the percentage of B cells expressing CD86 after CpG stimulation which was significantly greater than the control group at 2.1 +/− 2.1 fold (n=4, p=0.008) and the non-BMT group at 2.7 +/− 0.9 fold (n=5, p=0.008). Responses to LPS stimulation or culture without stimulation were not significantly different between the three groups. Purified B cells from GVHD samples (n=3) had enhanced mitogenic response to CpG compared to the control group (n=1), non-BMT group (n=2), and to non-stimulated cells. CpG response appears to be closely correlated with TLR9 mRNA expression (R2=0.82). These findings suggest that CpG responsiveness or TLR9 mRNA expression may potentially be useful as a biomarker for diagnosis or staging of chronic GVHD. Further evaluation on larger numbers of samples is required to confirm these observations.

Author notes

Corresponding author

Sign in via your Institution