Outcome-based Risk Assessment of Non-HLA Antibodies in Heart Transplantation: A Systematic Review.

BACKGROUND: Current monitoring after heart transplantation (HT) employs repeated invasive endomyocardial biopsies (EMB). Although positive EMB confirms rejection, EMB fails to predict impending, subclinical, or EMB-negative rejection events. While non-human leukocyte antigen (non-HLA) antibodies have emerged as important risk factors for antibody-mediated rejection after HT, their use in clinical risk stratification has been limited. A systematic review of the role of non-HLA antibodies in rejection pathologies has the potential to guide efforts to overcome deficiencies of EMB in rejection monitoring. METHODS: Databases were searched to include studies on non-HLA antibodies in HT recipients. Data collected included the number of patients, type of rejection, non-HLA antigen studied, association of non-HLA antibodies with rejection, and evidence for synergistic interaction between non-HLA antibodies and donor-specific anti-human leukocyte antigen antibody (HLA-DSA) responses. RESULTS: A total of 56 studies met the inclusion criteria. Strength of evidence for each non-HLA antibody was evaluated based on the number of articles and patients in support versus against their role in mediating rejection. Importantly, despite previous intense focus on the role of anti-major histocompatibility complex class I chain-related gene A (MICA) and anti-angiotensin II type I receptor antibodies (AT1R) in HT rejection, evidence for their involvement was equivocal. Conversely, the strength of evidence for other non-HLA antibodies supports that differing rejection pathologies are driven by differing non-HLA antibodies. CONCLUSIONS: This systematic review underscores the importance of identifying peri-HT non-HLA antibodies. Current evidence supports the role of non-HLA antibodies in all forms of HT rejection. Further investigations are required to define the mechanisms of action of non-HLA antibodies in HT rejection.

Optimization of a high-throughput shotgun immunoproteomics pipeline for antigen identification.

Identification of proteins which initiate and/or perpetuate adaptive immune responses has potential to greatly impact pre-clinical and clinical work across numerous fields. To date, however, the methodologies available to identify antigens responsible for driving adaptive immune responses have been plagued by numerous issues which have drastically limited their widespread adoption. Therefore, in this study, we sought to optimize a shotgun immunoproteomics approach to alleviate these persistent issues and create a high-throughput, quantitative methodology for antigen identification. Three individual components of a previously published approach, namely the protein extraction, antigen elution, and LC-MS/MS analysis steps, were optimized in a systematic manner. These studies determined that preparation of protein extracts using a one-step tissue disruption method in immunoprecipitation (IP) buffer, eluting antigens from affinity chromatography columns with 1% trifluoroacetic acid (TFA), and TMT-labeling & multiplexing equal volumes of eluted samples for LC-MS/MS analysis, resulted in quantitative longitudinal antigen identification, with reduced variability between replicates and increased total number of antigens identified. This optimized pipeline provides a multiplexed, highly reproducible, and fully quantitative approach to antigen identification which is broadly applicable to determine the role of antigenic proteins in inciting (i.e., primary antigens) and perpetuating (i.e., secondary antigens) a wide range of diseases. SIGNIFICANCE: Using a systematic, hypothesis-driven approach, we identified potential improvements for three individual steps of a previously published approach for antigen-identification. Optimization of each step created a methodology which resolved many of the persistent issues associated with previous antigen identification approaches. The optimized high-throughput shotgun immunoproteomics approach described herein identifies more than five times as many unique antigens as the previously published method, greatly reduces protocol cost and mass spectrometry time per experiment, minimizes both inter- and intra-experimental variability, and ensures each experiment is fully quantitative. Ultimately, this optimized antigen identification approach has the potential to facilitate novel antigen identification studies, allowing evaluation of the adaptive immune response in a longitudinal manner and encourage innovations in a wide array of fields.

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

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.