Shotgun Immunoproteomics for Identification of Nonhuman Leukocyte Antigens Associated With Cellular Dysfunction in Heart Transplant Rejection

Abstract

Background. The International Society for Heart and Lung Transplant consensus panel notes that too little data exist regarding the role of non-HLA in allograft rejection. We developed a novel shotgun immunoproteomic approach to determine the identities and potential roles non-HLA play in antibody-mediated rejection (AMR) in heart transplant recipients. Methods. Serum was collected longitudinally from heart transplant recipients experiencing AMR in the absence of donor-specific anti-HLA antibodies (n = 6) and matched no rejection controls (n = 7). Antidonor heart affinity chromatography columns were formed by recipient immunoglobulin G immobilization at transplantation, acute rejection, and chronic postrejection time points. Affinity chromatography columns were used to capture antigens from individual patient’s donor heart biopsies collected at transplantation. Captured proteins were subjected to quantitative proteomic analysis and the longitudinal response was calculated. Results. Overlap in antigen-specific response between AMR and non-AMR patients was only 8.3%. In AMR patients, a total of 155 non-HLAs were identified, with responses toward 43 high prevalence antigens found in ≥50% of patients. Immunofluorescence staining for representative high prevalence antigens demonstrated that their abundance increased at acute rejection, correlating with their respective non-HLA antibody response. Physiological changes in cardiomyocyte and endothelial cell function, following in vitro culture with patient immunoglobulin G, correlated with response toward several high prevalence antigens. Conclusions. This work demonstrates a novel high-throughput strategy to identify clinically relevant non-HLA from donor endomyocardial biopsy. Such a technique has the potential to improve understanding of longitudinal timing of antigen-specific responses and their cause and effect relationship in graft rejection.

Proteomics: Its Promise and Pitfalls in Shaping Precision Medicine in Solid Organ Transplantation

“…While also investigating the non-HLA antibody repertoires, Gates et al 135 used immunoproteomics in adult heart transplantation. Sera were collected at different time points from heart transplant recipients with AMR but without donor-specific anti-HLA antibodies and matched controls without rejection.…”

A Well-armed Approach Drives Relevant Non-HLA Antibody Targets Into the Open

“…As illustrated by the latter study, genomics and proteomics offer exciting and powerful new approaches to better analyze non-HLA Ab repertoires. In this issue of Transplantation , Gates et al 10 describe a novel, elegant, affinity chromatography approach (shotgun immunoproteomics) for high throughput non-HLA Ag discovery, that they have applied in adult heart transplantation, but which they contend has potential utility in all transplant settings. The authors used this powerful approach to identify, from the sera of patients without DSA, but with or without AMR, Ag targets from individual patient donor heart biopsies obtained at the time of transplantation.…”

“…Equally significantly, immunofluorescence staining for 4 selected, high prevalence Ag targets in donor endomyocardial biopsies indicated that, as the non-HLA response increased at the time of acute rejection, so did the abundance of the corresponding Ag (junctional plakoglobin, troponin T, prohibitin, and ATP synthase) in graft tissue. Gates et al 10 deduced that the non-HLA Abs examined can inhibit the normal functions of their target Ags and potentially increase their expression in response to loss of function. In chronic AMR, or in patients without AMR, IgG reactivity was more commonly directed against intracellular Ag targets that were likely made accessible by initial graft injury/acute rejection.…”