*Purpose: Kidney transplant recipients with sensitization to HLA defined by a high percentage of Panel Reactive Antibodies (PRA) or Donor-specific anti-HLA antibodies (DSA) are at higher immunologic risk for poor graft outcomes. Little is known about the prevalence and impact of circulating Donor-specific B cells (DSB) on graft outcomes. Utilizing a novel multiplex bead-based assay to identify DSB, we studied the circulating DSB in kidney transplant recipients and correlated with Graft failure (GF).

*Methods: PBMC from kidney transplant recipients (n=24; 19 with no GF, 5 with GF) were incubated with single antigen HLA-coated multiplexed beads (One Lambda) for HLA Class I and II. Using multi-parameter flow cytometry, HLA-specific B cells (HSB) were identified by the formation of HLA Bead-B-cell Rosettes (BBR) (Blue dots, Fig A blue box) by plotting beads on the Y-axis and CD19 on the X-axis (Fig A). The frequency of events in the blue box out of the red box (Fig A) represents HSB frequency out of CD19⁺ cells. The events in a gate around beads alone and BBR (Orange box, Fig A) are used to create a dot plot to reveal the matrix of HLA bead regions (orange areas, Fig B), where each orange area represents a set of beads with a single HLA specificity. The blue dots in the bead regions (overlying the orange areas, Fig B) represent BBR with the specificity of the bead region. HSB with specificities of the donor were defined as DSB and their frequency among CD19⁺ cells was calculated.

*Results: DSB frequencies at 12 months post-tx were higher (Class I: 0.1700 ± 0.09550% vs 0.05579 ± 0.009313%, p=0.0304, Fig D; Class II: 0.1200 ± 0.04494% vs 0.05895 ± 0.01006%, p=0.0493, Fig E) in recipients who went on to have GF in 2 to 5 yrs post-transplant compared to those with no GF. The Pearson Correlation was r=0.442, p<0.05 for Class I and r=0.405, p<0.05 for Class II.

*Conclusions: B cell sensitization, as assessed by Donor-specific B cell (DSB) frequencies, for both HLA Class I and II, 12 months post-transplantation, predicted graft failure. These data suggest that circulating B cells specific for donor HLA antigens assessed by this novel assay serve as potential biomarkers for risk stratification of kidney transplant recipients.

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