MHC Tetramers Specifically Identify High- and Low-avidity Donor-specific B Cells in Transplantation Tolerance and Rejection

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-Ed over control I-Ad and I-Ab 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.

Marginal Zone B Cells Are Necessary for the Formation of Anti-donor IgG After Allogeneic Sensitization

“…The use of MHC tetramers to identify antigenspecific B cells has been previously established in multiple models, 21,[40][41][42] and the specificity of allogeneic tetramer + B cells was recently demonstrated using biophysical methods. 43 However, given the abundance of naive B cells identified using tetramers by our group and others, 1 limitation to this approach is the potential for staining nonspecific or nonfunctional B cells. 21 In addition, B cells have known antigen-presentation functions in the setting of allogeneic antigens.…”