*Purpose: While B-cells contribute to allograft rejection by presenting antigens to T-cells and by producing donor-specific antibodies (DSAs) and various cytokines, their pathophysiology in transplant rejection is poorly understood. The purpose of this study is to characterize B-cell subsets in the blood of stable and rejecting patients after intestinal transplantation (ITx), as well as, to associate B-cell changes with DSAs. We hypothesize rejecting patients as having lower transitional B-cells (Bregs) and higher plasmablasts when compared with stable patients; we also predict changes in B-cell phenotypes to correlate with development of de-novo DSA.

*Methods: Peripheral blood from 17 stable patients (without any complications) and 17 rejecting patients (with acute rejection) after ITx at two timepoints, pre-transplant (day 0) and at acute rejection episode (AR), was stained for B-cell phenotyping (IgD, CD21, CD19, CD27, CD24, CD38, IgM, CD45). Data were analyzed using flow cytometry. DSA levels were obtained from electronic medical records.

*Results: Already at day 0, we found significantly less percentages of Bregs (8% v. 19%) and higher plasmablasts (18% vs. 16%) in those who acutely rejected compared to stable controls (p = 0.014, 0.042, respectively). At AR, we found increases in percentages of total CD19+ B-cells percentages (24% vs. 14%; p = 0.1) in rejecting patients compared to stable patients. We then compared rejecting patients to stable patients > 6 months from native ITx, and found significant decreases in Bregs in rejecting patients compared to stable patients (2.4% vs. 17.6%; p = 0.001). Other subsets of B-cells, including marginal zone B-cells, class-switched memory B-cells, and pathogenic memory B-cells did not confer statistical significance. Next, we investigated the relationship between B-cells and DSA. 82% of rejecting patients (14/17) developed de-novo DSA after ITX when compared with 23% (4/17) of stable patients (p=0.0016). Interestingly, there was a trend towards less Bregs in the rejectors with DSA compared with stable controls without DSA (2.9% vs. 8.6%; p = 0.2).

*Conclusions: We demonstrated decreases in Bregs and increases in plasmablasts in ITx allograft rejection. We also found that DSAs are associated with ITx rejection. In conjunction with DSA monitoring, a more robust characterization of B-cell phenotypes may offer an adjunct for a non-invasive biomarker for detecting rejection in ITx.

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