*Purpose: Antibody-mediated rejection is recognized as an important mediator of graft loss in the clinic, and the induction of donor-specific B cell tolerance may contribute to long-term allograft acceptance. Studies from our laboratory used donor MHC Class I and II tetramers to track donor-specific B cells and demonstrate that they differentiate into germinal center (GC) B cells and antibody-secreting cells (ASC) in rejection, and donor MHC-specific B cells were prevented from differentiating into GC B cells and ASC during transplant tolerance. In this study, we confirmed the specificity of MHC tetramer capture approach and tested whether high-avidity of donor MHC-specific B cells was preferentially deleted in tolerant recipients.

*Methods: Using donor MHC Class II I-E_d specific tetramers to flow-sort individual B cells, we cloned the heavy and light chains of the B cell receptor (BCR) and expressed them in HEK293 human embryonic kidney cells as mouse IgG1 mAb.

*Results: Affinities were determined (KD₅₀) by displacement of antibody binding or surface plasmon resonance, and that low and high-affinity mAbs were present at comparable frequencies in naïve, tolerant and acute rejecting mice. Furthermore, there was no significant difference in mutation rates of heavy chains from high or low-affinity clones, suggesting that high-affinity non-mutated anti-donor MHC B cell clones spontaneously arise. Interestingly, a feature of high-affinity clones was significantly shorter CDR3, a common feature of autoreactive B cells.

*Conclusions: Taken together, these studies confirm the specificity of MHC tetramers for identifying donor-specific B cells, and the absence of deletion of high-affinity clones in mice made tolerant to allografts with co-stimulation blockade.

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