Abstract
Background:
Although donor-specific antibody pre- and posttransplantation is routinely assessed, accurate quantification of memory alloreactive B cells that mediate recall antibody response remains challenging. Major histocompatibility complex (MHC) tetramers have been used to identify alloreactive B cells in mice and humans, but the specificity of this approach has not been rigorously assessed.
Methods:
B-cell receptors from MHC tetramer-binding single B cells were expressed as mouse recombinant immunoglobulin G1 (rIgG1) monoclonal antibodies, and the specificity was assessed with a multiplex bead assay. Relative binding avidity of rIgG1 was measured by modified dilution series technique and surface plasmon resonance. Additionally, immunoglobulin heavy chain variable regions of 50 individual B-cell receptors were sequenced to analyze the rate of somatic hypermutation.
Results:
The multiplex bead assay confirmed that expressed rIgG1 monoclonal antibodies were preferentially bound to bait MHC class II I-E d over control I-A d and I-A b tetramers. Furthermore, the dissociation constant 50 binding avidities of the rIgG1 ranged from 10 mM to 7 nM. The majority of tetramer-binding B cells were low avidity, and ~12.8% to 15.2% from naive and tolerant mice and 30.9% from acute rejecting mice were higher avidity (dissociation constant 50 <1 mM).
Conclusions:
Collectively, these studies demonstrate that donor MHC tetramers, under stringent binding conditions with decoy self-MHC tetramers, can specifically identify a broad repertoire of donor-specific B cells under conditions of rejection and tolerance.