Enhanced B-Cell Differentiation and Increased Production of Antibodies Targeting Surface Membrane Antigens in Patients with Chronic Graft-Versus-Host Disease
Authors: Wang Kathy S. , Cutler Corey S. , Nikiforow Sarah , Ho Vincent T. , Koreth John , Alyea Edwin P. , Armand Philippe , Soiffer Robert J. , Antin Joseph H. , Ritz Jerome 
Published: October 2019

The mechanisms of chronic graft-versus-host disease (cGVHD) are complex and involve multiple elements of the immune system. Previous studies have indicated that donor B cells and the antibodies they produce play an important role in the development of cGVHD. T cells that facilitate B-cell differentiation, such as T Follicular Helper cells (Tfh), were also shown to exhibit enhanced function in patients with cGVHD. IL-21, a cytokine produced by Tfh cells plays an important role in the B-cell differentiation process. Activation of IL-21R expressed on B cells promotes B-cell differentiation into plasmablasts and plasma cells.
To examine the responsiveness of B cells to this Tfh cytokine and antibody production in the setting of cGVHD, we analyzed B cells from 107 patients after allogeneic HSCT: 31 with no cGVHD, 44 with persistent stable cGVHD, and 22 patients with cGVHD receiving high-dose steroid therapy. Samples were collected from 9 months on after transplant. We also analyzed samples from 32 healthy donors as controls. The total CD19+CD20+ B-cell numbers in patients with stable cGVHD were comparable to those of healthy donors and slightly higher than normal in patients without cGVHD, suggesting normal recovery of B cells in these patients by the time the samples were collected. In patients with cGVHD, the number of IL-21R expressing B cells (CD19+CD20+IL21R+) was significantly increased compared to patients without cGVHD (p=0.046) or healthy donors (p=0.0036). The number of plasmablasts (CD19+CD20-CD38Hi) in patients with cGVHD was also significantly higher than in healthy donors (p=0.0079). Plasmablasts levels were higher in patients with cGVHD compared to those without cGVHD, but this difference was not statistically significant (p=0.3702). High-dose steroid therapy reduced both the number of IL-21R positive B cells and the number of plasmablasts, but neither reduction was statistically significant. We also examined IL-21 levels in plasma using multiplex cytokine bead arrays. Levels of circulating IL-21 were significantly higher in patients with cGVHD compared with healthy donors or patients without cGVHD. Increased levels of IL-21 and increased numbers of IL-21R+ B cells suggest that both homeostatic stimulation and inherent B-cell susceptibility promote B-cell differentiation in patients with cGVHD.
Previous studies have shown that male transplant patients who received sex-mismatched grafts developed significantly higher levels of anti-H-Y IgG antibodies and the development of H-Y antibodies is highly correlated with development of cGVHD. To examine the impact of the observed enhanced B-cell differentiation on production of allo- and autoantibodies, we developed a fluorescent-linked immuno-assay to detect antibodies capable of targeting cell-surface proteins. Membrane protein extracts were prepared from a human skin fibroblast cell line (Detroit 551) and coated onto 96-well plates. Plasma samples from the above cohort of patients and healthy donors were incubated on the plate, and IgG antibodies reactive with plate-bound antigens were detected with fluorescence-conjugated anti-human IgG. Compared with patients who did not develop cGVHD and healthy donors, patients who developed cGVHD showed significantly higher levels of IgG reactive against cell-surface antigens (cGVHD vs HD, p=0.0124; cGVHD vs no GVHD, p=0.0027). High-dose steroid therapy significantly reduced the level of these antibodies (cGVHD vs cGVHD-T, p=0.0009). We also examined the presence of these antibodies at various times following HSCT. While patients who did not develop cGVHD had persistently low levels of these antibodies over a 3-year period, patients who developed cGVHD during this time had persistently high levels of these antibodies.
These results indicate that in patients who developed cGVHD after transplant, B-cell differentiation and the production of antibodies that target living cells were enhanced. These data further support a role for B cells and antibodies in the development of cGVHD. Further characterization of the specificity of these antibodies will enhance our understanding of the role played by donor B cells in the development of cGVHD and provide new potential targets for therapy.

Disclosures
Koreth: prometheus labs inc: Research Funding; kadmon corp: Membership on an entity's Board of Directors or advisory committees; takeda pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; amgen inc: Consultancy; LLS: Research Funding; millennium pharmaceuticals: Research Funding. Armand:Roche: Research Funding; Pfizer: Research Funding; Infinity Pharmaceuticals: Consultancy; Sequenta Inc: Research Funding; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding. Soiffer:GentiumSpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees.