Abstract
Antibody-mediated allograft rejection (AMR) causes more kidney transplant failure than any other single cause. AMR is mediated by antibodies recognizing antigens expressed by the graft, and antibodies generated against major histocompatibility complex (MHC) mismatches are especially problematic. Most research directed towards the management of clinical AMR has focused on identifying and characterizing circulating donor-specific HLA antibody (DSA) and optimizing therapies that reduce B-cell activation and/or block antibody secretion by inhibiting plasmacyte survival. Here we describe a novel set of reagents and techniques to allow more specific measurements of MHC sensitization across different animal transplant models. Additionally, we have used these approaches to isolate and clone individual HLA-specific B cells from patients sensitized by pregnancy or transplantation. We have identified and characterized the phenotypes of individual HLA-specific B cells, determined the V(D)J rearrangements of their paired H and L chains, and generated recombinant antibodies to determine affinity and specificity. Knowledge of the BCR genes of individual HLA-specific B cells will allow identification of clonally related B cells by high-throughput sequence analysis of peripheral blood mononuclear cells and permit us to re-construct the origins of HLA-specific B cells and follow their somatic evolution by mutation and selection.
Highlights
HLA sensitization remains a barrier to transplantation of all organ types [1, 2]
We recently developed the K530 cell line, a CD32Adeficient derivative of K562 cells, which was subsequently used as the parental cell line for endogenous barcoding by fluorescent proteins (FPs) and cell surface antigen expression
By the generation of representative recombinant antibodies (rAbs), we demonstrate the utility of single antigen bead (SAB) Luminex® panels and HLA eplet analysis to define HLA conformational epitopes recognized by individual B cells and the trajectory of their evolution by somatic mutation
Summary
HLA sensitization remains a barrier to transplantation of all organ types [1, 2]. The etiology of alloantibodies stem from exposure to foreign HLA through pregnancy, transfusion, and previous transplantation and is driven by the high polymorphism among HLA genes. Screening for HLA antibodies is routinely performed prior to transplantation to avoid DSA and the increased risk for AMR and graft failure. In this way, donor selection and transplant outcomes are optimized. HLA single antigen bead (SAB) Luminex® assays allow sensitive screening for antibodies specific for hundreds of HLA alleles and are used widely in clinical practice. These assays, are prohibitively expensive for non-clinical studies and unavailable for animal species frequently used to study humoral responses to MHC antigens, notably macaques and swine. In a series of recent studies [6, 7], swine SLA class II single antigen expressing HEK293 cell lines were generated to monitor xenoreactive antibody responses with human serum samples, albeit, with singleplex capability and limited MHC allele coverage
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