Tools to Identify Organ Rejection and Immune Quiescence for Biological Understanding and Personalized Medical Care
2010; Future Medicine; Volume: 4; Issue: 1 Linguagem: Inglês
10.2217/bmm.09.73
ISSN1752-0371
AutoresPaul Keown, W. Robert McMaster, Bruce M. McManus,
Tópico(s)Cytomegalovirus and herpesvirus research
ResumoBiomarkers in MedicineVol. 4, No. 1 EditorialFree AccessTools to identify organ rejection and immune quiescence for biological understanding and personalized medical carePaul A Keown, W Robert McMaster & Bruce M McManusPaul A Keown† Author for correspondencePROOF Centre of Excellence, University of British Columbia, Vancouver, BC, CanadaDivision of Nephrology, University of British Columbia, 855 W 12th Ave, Vancouver, BC V5Z 1M9, Canada. Immunology Laboratory, University of British Columbia, Vancouver, BC, CanadaInfection & Immunity Research Centre, University of British Columbia, Vancouver, BC, CanadaDepartment of Medicine, University of British Columbia, Vancouver, BC, CanadaPathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada, W Robert McMasterPROOF Centre of Excellence, University of British Columbia, Vancouver, BC, CanadaInfection & Immunity Research Centre, University of British Columbia, Vancouver, BC, CanadaMedical Genetics, University of British Columbia, Vancouver, BC, Canada & Bruce M McManusPROOF Centre of Excellence, University of British Columbia, Vancouver, BC, CanadaJames Hogg iCAPTURE Centre, Heart & Lung Institute, University of British Columbia, Vancouver, BC, CanadaPathology & Laboratory Medicine, University of British Columbia, Vancouver, BC, CanadaPublished Online:2 Feb 2010https://doi.org/10.2217/bmm.09.73AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInRedditEmail Over the past two decades, the success of organ transplantation has markedly increased, owing to advances in biology, surgery and medicine [101–103]. Patient and graft survival now exceed 90% during the first year for most solid organs, acute rejection and infectious complications have diminished in frequency and severity, and there has been corresponding improvement in both quality of life and overall cost–effectiveness [1–4]. Potent immunosuppression, while yielding significant benefits during this period, has also led to significant complications, and attention has focused on individualizing therapy to optimize function and survival while minimizing toxicity. This focus, often described as 'personalized medicine', requires indepth understanding of biological mechanisms of graft injury, precise estimation of recipient risk, selection of appropriate prophylaxis, accurate and early diagnosis of rejection, and speedy intervention to prevent graft damage. Current diagnostic approaches do not fulfill these goals. Routine laboratory tests employed for graft monitoring are largely nonspecific, do not discriminate between mechanisms of injury [5,6] and do not provide a clear measure of immunological risk or accommodation between graft and host. Despite its many limitations, graft biopsy therefore remains the primary diagnostic tool for monitoring graft status [7–9].Mechanisms of graft injury & accommodationUnderstanding of the molecular and cellular basis of allograft recognition and response has advanced quickly with the introduction of novel diagnostic technologies. Sophisticated studies of molecular genetics in mouse and man have uncovered the sequence, regulation, function and expression of the major histocompatibility genes and the corresponding protein structure, functional interactions and antigenic epitopes of the cell-surface molecules that serve as both the principal immunogens and targets for the graft response [10]. Minor transplantation antigens have also been defined on several chromosomes, and recent research now reveals the distinct possibility that organ-specific epitopes, unveiled following tissue injury, may serve as autoantigens and potentiate immunological injury to the transplanted organ.The response to the allograft is increasingly recognized as a sophisticated concert of coordinated cognate interactions involving the innate and adaptive immune systems in which either cellular or antibody-mediated events may dominate. In the former, CD4+ and CD8+ mononuclear cells with increased cytotoxic transcripts for granzyme B, perforin, Fas-L and T-bet infiltrate the interstitial space within the graft accompanied by increased production of inflammatory cytokines, including IFN-γ, TNF-β, TNF-α and chemokines CXCL10, CCL2–5 and MIP-1α, leading to basement membrane injury, cellular destruction (through apoptosis and necrosis) and progressive organ injury. Antibodies to major histocompatibility antigens, present prior to implantation or developing afterwards, further compound this injury, and are both highly destructive and resistant to therapy. Complement activation is signaled by deposition of C4d, endothelial cell lysis and detachment from the basement membrane, and activation of the coagulation cascade leading to microthrombotic injury. Continuous or repeated response leads to chronic mesenchymal change, vascular luminal narrowing and organ fibrosis characteristic of chronic rejection [11]. This process is resistant to current therapy and is the leading cause of allograft loss.However, not all immunological responses lead to graft injury. True tolerance occurs in autografts or transplantation between identical twins, whereas immunological accommodation or immune regulation may occur in other settings [11]. The molecular mechanisms of these phenomena are slowly being uncovered. Classical tolerance is characterized by the lack of alloantibody production or cytotoxic cell responses directed against graft antigens, and is mediated by central and peripheral mechanisms. Accommodation may reflect an adaptation of the host response, such as a change in antibody titer, or type, or an adaptation of the graft, such as a reduced expression of target antigens, so that a balance is achieved, resulting in sustained quiescence. This phenomenon is seen most clearly in ABO incompatible transplantation where the graft is tolerated without injury despite the presence of circulating donor-specific A or B isoagglutinins. Immune regulation is observed particularly in liver transplantation, where up to 25% of grafts survive long term without a requirement for immunosuppression. The continued presence of alloantigen is required to maintain active regulation of the immune system by T-regulatory cells.Measures of the immune responseOver the past decade, concerted research and technological development over the past decade has produced a growing number of measures to define immunological risk or injury, several of which are gradually being incorporated into the clinical and laboratory practice of transplantation. The majority of these innovations have evolved outside a formal framework for biomarker discovery and validation, and consequently lack precision in roles, reproducibility, sensitivity or specificity.Genetic prediction of transplant outcome is a compelling goal, and the relationship between functional polymorphisms in genes related to the immune response and other critical physiological pathways has been examined by many investigators [12,13]. Numerous polymorphisms in cytokine, growth factor, adhesion molecule and complement genes including IFN-γ, TNF-α, IL-2, IL-10, IL-6, CCR2, CCR5, MCP-1, ICAM-1, VEGF and C3, have been explored. However, with rare exceptions such as carriage of the high-producer TNF-α-308 single nucleotide polymorphisms, few single or combinatorial polymorphisms have been consistently associated with graft outcome [12–15]. Many of these studies have failed in the design phase: examining small cohorts with poorly defined outcomes or short follow-ups and not probing satisfactorily for linkage disequilibrium [16]. Variants the in genes controlling immunosuppressive drug absorption, distribution, metabolism and elimination appear more biologically important, and the CYP3A5 genotype appears to be intimately related to blood concentrations of tacrolimus and sirolimus, while membrane transporter gene variants may be related to both the concentrations and adverse effects of calcineurin inhibitors and mycophenolic acid [17]. Variants in the renin–angiotensin genes may contribute to chronic renal injury, but no role for vitamin D receptor genes (FokI and BsmI) has yet been confirmed despite the enticing potential of vitamin D in immunocompetence [18].Direct measurement of cell phenotype and function, including cytokine production or protein concentration, has offered greater potential for biomarker use. Flow cytometric quantitation of CD3+ T cells or CD19/CD20+ B cells are simple and precise measures of the effectiveness of biological agents employed for the induction of immunosuppression or for treatment of recurrent rejection later in the transplant course. Additional screening of CD38+/CD138+ plasma cells is increasingly valuable for evaluation of antibody-producing cells in guiding the use of a proteasome blockade or other inhibitors of antibody production. Expression of CD25 has been proposed as a marker of T-cell activation and combined with CD95 or CD11a and CD154 may provide information on functional status, although the clinical relevance remains to be determined [19]. Recognition of circulating T-regulatory cells expressing CD4/CD25/FoxP3 has rekindled interest in autologous immune modulation and provided an opportunity to monitor the regulatory balance post-transplantation [20,21]. Quantitative inhibition of IL-2 and -4 offers a pharmacodynamic marker of immunosuppression and the use of an IL-2 reporter gene construct provides a biomarker of dynamic drug effect [22]. Combination of these approaches to simultaneously measure intracellular production of IL-2, IFN-γ, TNF-α or FoxP3 and surface expression of differentiation markers, such as CD86, CD95 and CD69, is now being explored [19].Detection of antibodies to HLA antigens is perhaps the single most important measure of alloimmune activity currently available. HLA class I antigens HLA-A, -B and -C consist of a single heavy chain linked to the invariant B2-microglobulin, while the more complex class II antigens HLA-DR, -DQ and -DP comprise both α- and β-chains [10]. Amino acid variation within the antigen-binding regions confers extensive heterogeneity at the molecular and cellular level, critical to the immunological repertoire of each individual. Antibodies to HLA antigens may be formed following antigenic stimulation by infection, pregnancy, transfusion or transplantation, and may persist for months or years. Donor-specific antibody is the primary cause of hyperacute rejection, and an important mechanism of injury in acute or chronic rejection [23,24]. Precise and sensitive assays for accurate detection of HLA antibody are now routinely performed pre- and increasingly post-transplant using solid-phase technology to identify circulating antibodies, and to guide the selection of donors and immunosuppression [24]. Important uncertainties remain regarding the exact influence of antibody target, titer, timing and type (IgG/IgM, subtype), and are the focus of active research in many centers.The search for protein markers of immunological activity has, to this point, been focused largely in urine for technical and methodological reasons. Urine offers a sample of more than 1000 proteins and peptides, with increased proportions of low-molecular-weight protein and peptide components compared with plasma, but presents some challenges as a diagnostic specimen. Studies using SELDI mass spectrometry have identified a number of proteins of interest showing differential expression during renal rejection or quiescence [25,26]. More detailed analysis of these peaks has shown B2-microglobulin to be a key component, reflecting increased HLA class I antigen expression and shedding, as well as altered glomerular filtration and decreased catabolism at the level of the tubular epithelial cell [27,28]. Although increased B2-microglobulin has been reported in cardiac allograft rejection, this remains a nonspecific marker, and has not been shown alone to differentiate well between graft rejection and other inflammatory events. The development of new technologies and the opportunity for realistic exploration of plasma proteomics will provide a new canvas for research in this field.Biomarker discovery & developmentIn light of the extreme complexity of the alloimmune response, it is unlikely that any single blood test will provide the necessary diagnostic information or mechanistic understanding to enable comprehensive diagnosis and effective management of organ transplantation throughout the lifetime of the graft. Rather, a combination of distinct markers will offer the opportunity to identify the activity or quiescence of each key biological process, and enable the construction of a picture of the allograft response at a level that is not possible using the current measures of histological injury or physiological dysfunction. This challenge is even greater in light of the differences observed in organ-specific response to alloimmunity; for example, resistance of the liver to humoral injury and the propensity of comorbid events, such as recurrent hepatitis C in the liver or polyomavirus infection in the kidney, alter function and structure, thereby confounding the phenotypic diagnosis. It is therefore recognized that the biomarkers identified, even for similar immunological events such as acute rejection, may differ from organ to organ, further adding to the complexity of the challenge.The biomarker development processes currently underway in several centers comprise both discovery and validation phases [29–31]. Single or multiple genomic and proteomic platforms are being used to find candidate biomarkers in small populations of carefully selected patients with clear clinical phenotypes and multiple time points, and bioinformatical methods used to identify differentially expressed genes, proteins and/or metabolites and to define biologic functions, pathways or networks. Combinatorial analysis methods are then applied to assess different data types for their additive or geometric influence on the separation of patient groups and clinical states over time. Importantly, clear clinical phenotypes are employed in this combinatorial approach to maintain their independent discriminative value. The result of the tiered analysis is a number of plausible predictive, diagnostic or prognostic markers of potential utility in a biomarker panel that may embrace clinical, genomic, proteomic and/or metabolomic markers, numbering from a few to perhaps a hundred.The development of high-throughput microarray and proteomics technologies, permitting simultaneous measurement of changes in expression of multiple genes or proteins within the peripheral blood, provides the opportunity for rapidly acquired, novel insights into disease processes and molecular pathways of tissue injury [32–34]. Recent advances have improved the diagnostic sensitivity, specificity, precision and accuracy of histological diagnosis using this technology [35–37], and both biomarker panels and individual biomarkers have been identified within the allograft to improve the diagnostic, prognostic and, potentially, therapeutic categorization of acute rejection [38–41]. The use of this technology to identify biomarkers in peripheral blood is, as yet, in its infancy, but is advancing rapidly [29,31,42]. Studies in renal transplantation have shown that genes differentially expressed during rejection encompass major biological categories of processes related to immune signal transduction, cytoskeletal reorganization and apoptosis, and emphasize the participation of the cytokine-activated Jak–Stat pathway, interferon signaling, and lymphocyte activation, proliferation, chemotaxis and adhesion [31]. Gene clusters can be identified within these families to serve as classifiers for rejection. Similar results have been obtained in cardiac transplantation, where classifier families comprising upregulated genes linked to innate and humoral immunity, and response to wounding and hypoxia were highly related to clinical rejection [30]. As anticipated, while reflecting common biological categories, classifiers may differ specifically between organs or reports, reflecting organ-based, therapeutic influence and microarray technology specificities [43]. Within our own studies, for example, acute renal allograft rejection is typified by upregulation of genes such as TncRNA, FKSG49 and AVIL, and downregulation of EGFR, while in the heart TFR2 and FGFR1OP2 were upregulated and KLF4 and BID were downregulated [30,31].Over the last two decades, there has been rapid evolution of quantitative proteomic technologies [44–48], which have enabled protein expression profiling of many human diseases [49–52]. To date, proteomic analysis in human renal transplantation has focused principally on urine for both technological and biological reasons [48,53,54]. Several potential urinary markers of acute graft rejection have been identified in these studies, but few have been successfully validated [53]. The use of urine as a starting matrix is also complicated since the varying pH of urine can lead to the degradation of proteins of interest [55].The peripheral blood is rapidly emerging as a viable matrix for proteomic measurement. The plasma proteome corresponds closely with dynamic gene expression, and the field of convergent functional biology is consequently a focus of intense investigation in many disease states [34,56]. Characterization, identification and quantification of plasma protein content have progressively improved the understanding of the plasma proteome [57–59], although exploration of biomarkers within this matrix has been extremely challenging. This is due both to the extreme dynamic range of protein concentrations, extending from 10-6 to 103 µg/ml [57,60], and to the fact that a small number of abundant plasma proteins constitute 99% of the total protein mass, with many proteins of potential interest existing at very low concentrations [57]. Quantitative proteomic analysis in a dynamic biological process, such as transplantation, is particularly complex due to variability between individuals and even within individuals, reflecting differences in graft and patient recovery, immunosuppressive treatments, infection and other patient-specific events following implantation of the new organ. Experimental animal studies indicate that the peripheral blood proteome may offer important diagnostic information in detecting graft rejection, but until recently, no studies had detected potential biomarkers in human transplantation [48,54]. We have employed iTRAQ-MALDI-TOF/TOF methodology, in light of its reliability, reproducibility, relative sensitivity and substantial sampling of the dynamic range [Cohen Freue G, Bergman A, Ohlung L et al.: Bias, reproducibility and variation in a large-scale quantitative proteomics analysis of plasma (2010), Submitted], to examine the differential patterns of expression in the human plasma proteome in patients with or without early graft rejection after renal or heart transplantation, and have identified classifiers of protein group codes, whose concentrations differ significantly between patients with or without acute graft rejection. These groups represent a range of biologic processes involved in inflammation, complement activation, blood coagulation and wound repair, consistent with the current understanding and pathogenesis of acute rejection injury. In addition to these established pathways, our analysis has revealed several novel proteins with unknown roles in acute rejection, which may provide insight into new mechanisms of rejection as well as new targets for therapeutic intervention. Tiered analysis shows that these can be combined with genomic and clinical information to enhance the discriminant value of classifiers.Biomarkers & personalized medicineThe growing understanding of biological mechanisms, coupled with the rapid evolution of genetic predictors and molecular markers of tissue injury, offers the enticing potential for individualized management of the transplant course in a manner not previously feasible. Reliable markers of recipient immunological risk coupled with pharmacogenetic indicators of drug kinetics and dynamics will permit knowledgeable selection of therapeutic strategies for each individual, while monitoring of peripheral blood or other matrices for signals of immunological quiescence or rejection will enable the prophylactic adjustment of therapy to avoid tissue injury and functional deterioration in the transplanted organ [61]. The PROOF Centre of Excellence was established to pursue this expanding discipline of personalized medicine and to move the focus of care upstream, beyond the horizon of clinical detection, to the earliest stages of molecular interactions, initiating events and baseline risk. This unique partnership of academia, government, healthcare, patients, industry and the public is devoted to the discovery, development, commercialization and implementation of biomarkers to prevent, predict, diagnose, manage and treat injury in the native and transplanted organ.The challenges in developing robust and reliable biomarkers are formidable [62]. While a growing number of potential biomarkers to diagnose acute rejection, immunological quiescence or chronic injury have been proposed, few have yet withstood the rigorous evaluation required for clinical application [63,64,104]. Several translational teams are now moving to the second, and perhaps more demanding, validation phase of biomarker development, performed to confirm original observations and to evaluate the sensitivity, specificity and predictive values of the candidate biomarkers. This is normally conducted on a second patient cohort from the same institution, using the same platforms and statistical/informatical analyses, and consensus analysis is performed between the discovery and internal validation cohorts to present a modified biomarker panel suitable for external validation (qualification) in a broader and more heterogeneous multicenter observational study.We are optimistic that this process will rapidly generate an increasing array of robust markers, translated to simple and cost-effective diagnostic platforms, and exhibiting the sensitivity, specificity and predictive values necessary for widespread clinical use. Simple blood tests will then be routinely available to monitor the transplant course, to identify organ rejection or quiescence with accuracy and precision, and will enhance our understanding of the complex biology of the graft response. However, if we are to reap the enormous potential of these technologies, the introduction of personalized medicine requires a transformative approach in all disciplines and across the healthcare spectrum [65]. While the initial drive may come from molecular scientists and the biotechnology sector, close collaboration with regulatory agencies will be crucial in introducing these new tests into clinical use with health providers and payers to clarify their cost benefits and clinical utility.Financial & competing interests disclosureThe authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. 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