Brief communicationImmune complexome analysis of antigens in circulating immune complexes from patients with acute cellular rejection after living donor liver transplantation
Introduction
Liver transplantation has become a life-saving and effective procedure for many end-stage liver diseases; however, rejection is still a major cause of early graft loss and a risk factor for graft prognosis [1]. Rejection is divided into two major types: T cell-related acute cellular rejection (ACR) and antibody-mediated rejection; the most common form of rejection is ACR [2].
To understand the mechanism of rejection, it is necessary to identify what a recipient's immune system considers “non-self”. Previous studies on rejection mechanism have focused on immune responses against donor major histocompatibility complex (MHC) molecules expressed on graft cells, or alloantigens in the form of peptides presented by the recipient's antigen-presenting cells [3,4]. Screening and identifying these antigenic proteins may provide insights into the mechanism underlying rejection; however, such proteins related to rejection remain undiscovered.
Several approaches have been used to identify antigens based on recognition by T-cell receptors (TCRs) or antibodies for use in clinical studies. TCRs detect antigens in the form of peptides bound to MHC molecules on the surface of antigen-presenting cells. Several methods have been employed to identify antigen-specific TCRs, especially neo-antigens for cancer; however, these techniques are tedious and require the development of antigen-specific T cells clones for several months, because T cells expressing high-affinity TCRs are difficult to detect in peripheral blood [5,6]. In addition, TCRs bind monomeric peptide-MHC with very low binding affinity (1000- to 200,000-fold weaker than a typical antibody-antigen interaction) [7]. Contrastingly, antibodies recognize their corresponding antigens with very high binding affinity and much highly amount of circulating antibodies are present in blood. Since the development of B cell immunity depends upon concordant T cell immunity, antibody identification can lead to the identification of relevant T cell antigens, and in fact, has directly led to the mapping of CD4+ and CD8+ T lymphocyte epitopes [6,8], which has formed the basis for using antibody responses to identify T cell antigens [9]. Thus, the identification of antigens recognized by antibodies is important for understanding antigen recognition in both B cell and T cell immunity [10].
Therefore, we hypothesized that the immune target responsible for ACR could be identified from antibodies and focused on circulating immune complexes (CICs) that are products of antigen-antibody reaction. To comprehensively identify and profile constituent antigens in CICs (CIC-antigens), we developed a proteomic strategy, designated immune complexome analysis, in which CICs are separated from whole serum and then subjected to direct tryptic digestion and identification of CIC-antigens by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS). We successfully used this method to determine that the CIC-antigens were specifically and frequently detected for established and early rheumatoid arthritis [11,12] or lung cancer [10].
In this report, we applied immune complexome analysis to sera from recipients who experienced ACR (rejection group) and did not experience ACR (non-rejection group) after liver transplantation to find CIC-antigens specific for ACR by comparing the CIC-antigens between the two patient groups.
Section snippets
Patients
Serum samples were collected from Nagasaki University Hospital. We examined 32 living donor liver transplant (LDLT) recipients (24–72 years; 21 female) in this paper. Ten recipients experienced ACR (rejection group) and the others (22 recipients) did not experience ACR (non-rejection group). We analyzed 32 serum sample collected before transplantation for both groups, 22 samples collected 4-weeks after transplantation (non-rejection group) and 10 samples collected when ACR occurred (rejection
Results
We analyzed 64 serum samples from 32 recipients by immune complexome analysis, and identified 47 CIC-antigens from all of the recipients (Table S1). In addition to these antigen candidates, the list also includes seven complement-related proteins that could possibly bind to CICs. The CIC-antigens were compared between rejection and non-rejection groups to elucidate those that were only detected in the rejection group; 11 proteins met this criterion (Table 1). Among the 11 antigens found in the
Discussion
In this study, we compared CIC-antigens in sera between liver transplant recipients with or without rejection to find the CIC-antigens occurring at ACR. Also, since we compared CIC antigens between before and after rejection in the same patient, several factors rather than immune response can be ignored and the CIC-antigens specifically detected after rejection can be thought to associate with rejection-related immune response. Among the 11 proteins that were specifically detected in the
Conflict of interest
The authors have no conflicts of interest to declare.
Funding
This work was supported by a Grant-in-Aid for Young Scientists (B), a Grant-in-aid for Scientific Research (C) and challenging Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (24790158, 23591439, 15K15373), a Grant-Aid for Scientific Research from Nagasaki University, the Joint Research Promotion Project of Nagasaki University Graduate School of Biomedical Sciences in 2013, The Research Foundation for Pharmaceutical Sciences.
Authorship
N.A., K.O., M.H., and N.K. participated in the research design. N.A, K.O., M.H., and M.T. participated in writing the paper. K.O., M.H., N.K., Y.M., M.T., S.E., and N.K. provided feedback on the analytic methods and interpretation of the results. N.A. conducted the analysis. All of the authors approved the final manuscript.
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