Comprehensive B Cell Phenotyping Profile for Chronic Graft-versus-Host Disease Diagnosis

Previous studies have reported single B cell-related chronic graft-versus-host disease diagnostic (cGVHD) biomarkers, such as B cell-activating factor (BAFF), CD21 ^low , and immature B cells, but research on the performance of biomarker combinations and the covariate effect of steroids is lacking. The primary objective of this study was to determine the most accurate combination of B cell populations using cell surface staining flow cytometry in an independent cohort of patients with cGVHD. Secondary objectives included assessing the effect of corticosteroid use at sample collection on the makeup and accuracy of the diagnostic panel and identifying the mechanism underlying low surface expression of BAFF receptor (BAFF-R) on B cells in cGVHD. Flow cytometry analysis was performed in an adult cohort of post-HCT patients with cGVHD onset (n = 44) and time-matched recipients without cGVHD (n = 63). We confirmed that the onset of cGVHD was associated with higher soluble BAFF (sBAFF) levels, elevated CD27 ⁻ CD10 ⁻ CD21 ^low CD19 ⁺ B cell and classical switched memory B cell counts, and reduced transitional and naïve B cell counts. The highest single B cell population area under the receiver operating characteristic (ROC) curve (AUC) was.72 for transitional type 1 CD21 ^low B cells. We also showed a significant inverse relationship between sBAFF and surface BAFF-R expression caused by sBAFF modulation of BAFF-R. Steroid use at sample collection influenced the significance of the sBAFF:B cell ratio, naïve and marginal zone-like B cells. The optimal combination of B cell subsets most significantly associated with cGVHD onset with or without concurrent corticosteroid use resulted in ROC AUCs of.87 and.84, respectively. Transitional and CD21 ^low B cells were the only populations present in both panels; however, analyzing only these populations resulted in ROC AUCs of.79 and.78, respectively. This suggests that the inclusion of other populations and use of different panels depending on steroid use is necessary to achieve better accuracy. sBAFF was not a component of either panel. These novel B cell profiles could be tested prospectively in patients post-HSCT and could lead to focused mechanistic studies.