The diagnosis of rejection in a kidney transplant recipient is suspected when a patient presents with allograft dysfunction (elevated serum creatinine or proteinuria) and requires confirmation on a kidney biopsy. However, neither elevated serum creatinine or proteinuria is sensitive or specific. The kidney biopsy is logistically challenging, costly, inconvenient, invasive, risky, subject to sampling errors due to focal characteristic of the rejection, and has poor reproducibility in interpretation. The ‘for-cause’ biopsies may be indeterminate in as many as 30% cases. Surveillance biopsies similarly have low yield with approximately 80% of these procedures revealing normal histology. Moreover, surveillance biopsies have not been validated to actually improve outcomes. Consequently, there has been an unmet need for immune biomarkers that would rapidly, accurately, inexpensively, and noninvasively identify subjects with or at risk for incipient allograft injury (e.g., rejection), discern the type of injury (e.g., antibody mediated versus cellular rejection), differentiate rejection from other causes of graft damage (e.g., infection), and assess recipient's immune status to allow individualized immunosuppression [1▪]. An ideal biomarker is supposed to have high sensitivity, specificity, positive predictive value, negative predictive value, and receiver-operating characteristic curves .
The last decade has seen a rapid progress in development of new biomarkers that hold promise for safe, noninvasive, frequent, and quantitative assessment of the immune injury to graft. In this review, we will focus on the new assays of the biomarkers of rejection, some of which have received U.S. Food and Drug Administration (FDA) approval and have moved from research arena to clinical practice.
BIOMARKERS OF ANTIBODY-MEDIATED REJECTION
The diagnosis of antibody mediated rejection (AMR) requires acute tissue histological injury, detection of circulating donor-specific antibodies (DSA) (human leukocyte antigen [HLA] or non-HLA) and evidence of antibody interaction with the vascular endothelium either by C4d positivity or moderate microvascular inflammation or demonstration of AMR-specific gene transcripts .
The preformed or de-novo DSAs can be considered as early markers of acute antibody-mediated rejection. However, not all DSAs may be necessarily bad or equally pathogenic, and pathogenicity of DSAs may depend on characteristics like isotype (IgM versus IgG), class specificity (HLA class I versus class II), antigenic specificity, strength, IgG subclass, and complement binding capacity . Although, class I and class II antibodies are considered equally pathogenic, there are some notable differences. Both pretransplant class I and II DSA may mediate acute AMR early posttransplant. Posttransplant, class II de-novo DSA develop more commonly and are associated with chronic AMR and transplant glomerulopathy . C3d binding is reported to be a property of class II DSA predominantly, whereas C1q binding is expressed equally by both classes . Higher DSA strength [as assessed by titer or mean fluorescence intensity (MFI)] is required to activate complements [6▪,7,8] and carries a higher risk of AMR and graft loss [9,10]. Different DSA subtypes are associated with different abilities to bind complement and distinct patterns of antibody-mediated injury . IgG1 and IgG3 antibodies are strong complement binding, whereas IgG2 and IgG4 antibodies are weak or noncomplement-binding. IgG1 represents the predominant anti-HLA DSA subclass and is variably associated with the other IgG subclasses. AMR-free patients had mostly IgG1 alone or no subclass detected. Presence of IgG3-positive DSA has a high correlation with active AMR with intense microvascular inflammation and higher C4d deposition and a greater risk of graft loss [11,12]. IgG4-containing DSA is present more in patients with features of subclinical or chronic AMR with features of transplant glomerulopathy and IF/TA . Hence, IgG DSA subtyping trigger the need for allograft biopsy and may enhance the diagnosis of the type of antibody-mediated injury. DSA-mediated rejection typically happens due to activation of classic complement pathway. However, complement activation is not necessary and some DSAs may cause graft damage due to antibody-dependent cellular cytotoxicity or direct endothelial cell proliferation [13▪▪]. As C4d deposition could be missing in almost 50% biopsies with AMR , direct assessment of the capacity of anti-HLA antibodies to bind the complement components, C4d and C3d using flow beads have come in foray. The detection of complement binding de novo DSA (using C1q binding assay) after transplantation are associated with higher rate of AMR and higher risk of graft loss compared with noncomplement binding HLA antibodies . In patients with AMR, the ability of DSA to bind C3d (a cleavage product of C3 positioned downstream the complement cascade) is associated with higher DSA MFI, worse estimated glomerular filtration rate (eGFR) and higher risk of graft loss as compared with C1q-positive DSAs . Even C3d-positive DSAs with low MFI carry a high risk for graft loss . Hence, C3d-binding assay at the time of AMR diagnosis allows for identification of patients at risk for allograft loss.
Posttransplant DSA monitoring may allow for the early diagnosis of AMR and subsequent specific treatment and adjustment of immunosuppressive therapy , especially in sensitized patients . However, the value of DSA monitoring is not clearly established in predicting AMR or graft loss. Addition of DSA IgG3 positivity or C1q binding capacity may improve the risk stratification for allograft loss posttransplant . The consensus guidelines published by Transplantation Society recommend posttransplant DSA monitoring as follows  high-risk patients (i.e., desensitized or DSA positive/crossmatch negative) should be monitored by measurement of DSA and protocol biopsies in the first 3 months after transplantation. Intermediate-risk patients (history of DSA but currently negative) should be monitored for DSA within the first month. If DSA is present, a biopsy should be performed. Low-risk patients (nonsensitized first transplantation) should be screened for DSA at least once 3–12 months after transplantation. If DSA is detected, a biopsy should be performed. In all three categories, the recommendations for subsequent treatment are based on the biopsy results.
The AlloSure test (CareDx, Inc, Brisbane, California, USA) measures circulating donor-derived cell-free deoxyribonucleic acid (dd-cfDNA) in transplant recipients. The test was recently validated as a noninvasive marker for diagnosis of graft rejection in adult kidney transplant recipients at least 2 weeks posttransplant in a multicenter prospective, observational study named, Circulating Donor-Derived Cell-Free DNA in Blood for Diagnosing Acute Rejection in Kidney Transplant Recipients study [19▪]. The premise for quantitative interpretation of this biomarker is that rejection entails injury, including increased cell death in the allograft, leading to increased dd-cfDNA released into the bloodstream. The dd-cfDNA levels less than 1% reflect the absence of active rejection (T-cell-mediated type ≥1B grade of rejection or AMR) and levels more than 1% indicate a probability of active rejection with positive and negative predictive values (PPV and NPV) for active rejection of 61% and 84%, respectively. The dd-cfDNA discriminates AMR from no AMR allograft status (receiver operative curve-area under curve [ROC-AUC] 0.87, NPV 96%, PPV 44%, cutoff of 1.0% dd-cfDNA). The PPV for AMR is increased (85%) in DSA-positive patients. The dd-cfDNA levels tend to be higher in AMR than in T-cell mediated rejection (TCMR). The dd-cfDNA, by itself, may not be able to distinguish BK virus nephropathy from rejection. The elevated dd-cfDNA levels could increase the prebiopsy probability of detecting treatable injury, so that biopsy could be made an even more effective diagnostic tool, whereas a negative result may obviate the need for biopsy in some cases of elevated creatinine, especially when patients are on anticoagulation therapy or have other reasons to avoid biopsy. The dd-cfDNA levels decrease after rejection treatment. The serial monitoring of dd-cfDNA for surveillance has been recommended but the optimal time interval and utility remains to be defined. Results may be inaccurate in case of whole blood or other blood transfusions containing white blood cells within 1 month prior or falsely positive within 24 h after a renal biopsy.
The chemokines and their receptors play a critical role in leukocyte trafficking and their transcripts are significantly increased in renal allografts during rejection . The urinary chemokines CXCL-9 and CXCL-10 can diagnose rejection of renal allografts early on. Positive urinary CXCL9 is reported to predate clinical detection of acute rejection by a median of 15 days . Combining the urinary CXCL10-to-creatinine ratio with DSA monitoring has been shown to significantly improve the noninvasive diagnosis of AMR and the stratification of patients at high risk for graft loss .
Complement C3 gene polymorphism
The single nucleotide polymorphisms (SNPs) in complement C3 gene, especially two SNPs (rs10411506 and rs2230205) have been found to correlate with AMR in kidney transplantation .
Allogenic B-cell and B-cell activating factor assay
A memory B-cell enzyme-linked immunospot (ELISPOT) assay allows quantification of donor HLA-specific memory B cells in the peripheral blood of HLA-sensitized individuals and has been found to predict high risk of AMR postkidney transplantation [24,25]. Circulating plasmablasts (as identified by their expression of CD38 and CD27 using flow cytometry) induce the differentiation of human T follicular helper cells via interleukin (IL)-6 production and that could spread antibody-driven inflammation . The serum B-cell activating factor level drawn on postoperative day 7 has been shown to predict posttransplant AMR, and the prediction improves further when combined with DSA testing .
Peripheral blood gene expression assay
A study done to evaluate the expression of genes related to AMR in the peripheral blood of renal allograft recipients found that patients with AMR had higher levels of mRNA transcripts of the CD20, vWF, and FOXP3 genes as compared with patients of the no-rejection group (P < 0.01) but not significantly higher compared with acute cellular rejection (ACR) .
The kidney biopsy gene expression assay
In a study evaluating the microarray-based RNA expression of endothelial cells, natural killer cells, or interferon-γ (IFN-γ)-inducible genes in allograft biopsies predicted the risk of AMR without knowledge of HLA antibody status, histology, or C4d staining . Increased expression of endothelial-associated transcripts in biopsies from patients with DSA can identify kidneys undergoing antibody-mediated damage irrespective of C4d stain . In nonhuman primate model, the increased expression of human AMR-related endothelial genes (VWF, DARC, and CAV1) RNA transcripts has been shown to precede AMR and provides excellent discrimination between AMR and non-AMR (area under the curve = 0.92) . The molecular microscope diagnostic system (MMDx) which has been introduced recently in the clinical practice is a central real-time diagnostic system and measure changes in mRNA expression in transplant kidney biopsies using microarrays to assess the probability of rejection. The MMDx predicts the risk of graft failure better than conventional biopsy assessment . Hence, the kidney allograft transcripts as biomarkers can increase the diagnostic yield of biopsy and help prognosticate allograft injury with greater precision than histology alone.
Plasma endothelial microparticles
A study is currently under way to assess the role of plasma endothelial microparticles measured using flow cytometric assay as an early diagnostic biomarker of AMR in renal transplantation.
(Endothelial Microparticules and Antibody-mediated Rejection and Kidney Transplantation: Biomarker of Antibody-mediated Rejection in Kidney Transplantation (MICROMARK RJ). https://clinicaltrials.gov/ct2/show/NCT03098238).
BIOMARKERS OF T-CELL-MEDIATED REJECTION
ImmuKnow assay (Cylex assay; Viracor-IBT Laboratories Inc, Lee's Summit, Missouri, USA)) is an FDA-approved test that measures ATP generation by mitogen-stimulated CD4 lymphocytes . Cylex guidelines classify weak immune response as less than 224 ng/ml (suggesting overimmunosuppression), moderate 224–524 ng/ml, and strong more than 525 ng/ml (suggesting underimmunosuppression). In a study by Myslik et al., preoperative ATP values were found to be correlating well with predicating rejection episodes than postoperative ATP values. In summary, they suggested that ImmuKnow assay as a one-time test during pretransplant evaluation may help in determining the need for induction therapy, and development of personalized protocols for postrenal transplant immunotherapy and rejection monitoring . Others have also demonstrated that immune responses with ATP levels below 130 ng/ml and above 450 ng/ml are at higher risk of rejection, suggesting optimal therapeutic target ranges . The test needs further validation with randomized controlled data.
Peripheral blood and leukocytes gene expression assay
A Kidney Solid Organ Response Test (kSORT; Immucor DX, Grand Rapids, Michigan, USA) is a peripheral blood RNA expression assay measured by quantitative, real-time PCR that was developed using a 17-gene set shown to be associated with acute rejection. The raw data generated by kSORT is analyzed using a proprietary centroid-based algorithm (kSAS), providing a qualitative Immune Risk Index Score of High, Low, or Indeterminate that can be reported to clinicians. The assay has sensitivity of 100% and specificity of 96% after excluding 11% indeterminate results. In a multicenter study called ‘Assessment of Acute Rejection in Renal Transplantation study’, the test was able to predict acute rejection up to 3 months prior to detection by the current gold standard (biopsy) . In another study, combining kSORT and IFN-γ ELISPOT assay provided better prediction for subclinical rejection and the ability to distinguish between subclinical TCMR and AMR in patients with stable graft function [37▪].
The increased peripheral blood messenger RNA (mRNA) expression of molecules T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and perforin [both expressed by cytotoxic T lymphocytes (CTLs)] in the of kidney transplant recipients has also been reported to predict the acute rejection, with decrease in mRNA transcripts after rejection treatment [38▪▪]. Similarly, peripheral blood gene expression assay of circulating lymphocytes may diagnose rejection and avoid the need for a kidney biopsy [39,40].
Allograft gene expression assay
The MMDx discussed earlier provides rejection-related scores for both TCMR and AMR which concur with histological diagnosis of rejection in high (77%) percentage of cases with clinicians agreeing with MMDx score more often than with their local biopsy assessment [41▪▪].
Allogeneic circulating T-cell assays
The donor-specific IFN-γ ELISPOT is a highly sensitive assay measures IFN-γ secretion by recipient T cells in response to donor antigens at single-cell level. In this test, cells are cultured on a surface with specific capture antibody and cytokines like IFN-γ secreted by cells which are captured by specific antibody in the presence or absence of stimuli. Each spot suggests individual cytokine secreting cell. In a retrospective study of 53 renal transplant recipients using the IFN-γ ELISPOT assay by Koscielska-Kasprzak et al. higher donor-specific T-cell reactivity as indicated by an increased spot number (P < 0.05), median spot size (P < 0.05), and intensity (P < 0.05) was found in patients who experienced a biopsy-proven rejection episode within the 1st year after transplant. Significantly, 14 of the 16 patients with an acute rejection had a positive ELISPOT result compared with only one of the 16 patients that had elevated panel reactive antibody (PRA) alone, suggesting that the predictive power of the donor-specific ELISPOT was greater than PRA status . The pretransplant T-cell alloreactivity as measured by the test may be associated with a higher posttransplant risk of rejection and lower estimated glomerular filtration rate (eGFR), although not in patients given rabbit antithymocyte globulin for induction . The test can also assess risk for developing subclinical TCMR and antidonor HLA antibodies, potentially limiting the need for surveillance biopsies . In addition, the donor-specific memory CD4 T cells in the recipient have been linked to AMR of renal allografts . However, the ELISPOT procedure requires donor cells and takes 24–36 h, which limits application in deceased donor kidney recipients.
Single nucleotide polymorphisms
Inheritance of specific genetic variants by some individuals has been postulated to be associated with increased risk for acute rejection. Most of these genetic variants are in the form of SNPs. The protein products coded for by many of the genes containing these variants are involved in the regulation and responsiveness of the immune system such as IL10, transforming growth factor-beta-1 (TGF-β) and tumor necrosis factor-alpha (TNF-α). There are different platforms for SNP genotyping, for example, Affymetrix Gene Chip and others . The recipient's TNF-α and IL-10 gene polymorphisms are associated with rejection episodes and severity following kidney transplantation . In a systematic review by Oetting et al., authors analyzed 23 genetic variants, previously reported to have a significant association with acute rejection, using a cohort of 969 clinically well defined kidney transplant recipients. Only one SNP, rs6025 (FactorV Leiden gene mutation), showed a significant association (P value ≤0.011) and an additional SNP, rs11706052 in inosine monophosphate dehydrogenase 2 gene gave a modest P value of 0.044, using multiple variable analysis, which became insignificant when multiple testing was is taken into consideration . A major limitation to using these SNPs in clinical care has been the lack of further validation studies.
Peripheral blood microRNAs
Quantification of microRNAs (miRNAs) in peripheral blood may represent a noninvasive biomarker for T-cell-dependent acute rejection. In a study by Matz et al. , combined measurement of five miRNAs (miR-15B, miR-16, miR-103A, miR-106A, and miR-107) in peripheral blood samples enhanced the sensitivity and specificity for the diagnosis of severe T-cell-mediated vascular rejection. Measurement of peripheral blood miRNAs still needs future development in more precise and clinically applicable way.
Urinary cell mRNA
During rejection, CTL can release perforin, which perforates cell membranes, causing direct cell damage. CTL also release granzymes A and B, which cause cell death via caspase-dependent and independent apoptosis. Li et al.  determined that urinary perforin and granzyme B mRNA were significantly elevated in patients with acute clinical rejection. In the CTOT-04 study, Suthanthiran et al. studied urinary-cell mRNA as potential noninvasive diagnostic markers for acute cellular rejection in a large prospective cohort of 485 patients. They showed that urinary CD3ε, CXCL10, perforin, and granzyme B mRNA were elevated in acute cellular rejection and subsequently developed a three-gene signature utilizing CD3ε mRNA, CXCL10 mRNA, and18s rRNA (AUC 0.85) for detecting acute rejection. Though the three-gene signature was externally validated in other cohorts for biopsy proven rejection, it is unclear if it can detect subclinical rejection. Also, the study reported technical limitations for translating this assay from bench-to-bedside as extracting sufficient quality urinary mRNA for analysis was difficult (only 83% passed quality control in CTOT-04) .
Quantification of urine miRNA has also evaluated as innovative method to assess renal graft function. Preliminary studies showed combination of miRNA profiling (miR-142-3p, miR-204, miR-107, miR-211, and miR-32) of biopsy and urine samples could be used to monitor graft function and predict progression to allograft dysfunction . However, these findings need to be validated in future studies to assess the diagnostic and/or prognostic accuracy. In addition, the ideal medium to be used for urine miRNA profiling is still uncertain.
Urinary CXCL9 is significantly elevated in acute rejection. It rises prior to an episode of biopsy-proven acute clinical rejection and decreases in response to therapy. Furthermore, urinary CXCL9 is also reported to distinguish subclinical tubulitis from normal and borderline histology (AUC 0.78) . In CTOT-01 study, Hircik et al. prospectively evaluated multiple novel biomarkers using different methodologies on 280 kidney transplant recipients. They found CXCL9 as one of the few biomarkers found to be significant predictors of clinical acute rejection. CXCL9 diagnosed acute rejection with an AUC 0.86, rose up to 30 days prior to clinical rejection and had a strong NPV of 92% . Urinary CXCL10 is a sensitive marker for inflammation, and now there is enough data showing urinary CXCL10 is associated with acute rejection and its rise precede the rise in serum creatinine [55,56]. In future, these urine proteins (CXCL9 and CXCL 10) may rationalize a noninvasive chemokine-directed monitoring strategy instead of use of surveillance biopsies to detect acute graft rejection .
Several proteins and peptides like collagens fragments, beta-2-microglobulin, alpha-1-antichymotrypsin, and uromodulin are differentially expressed in patients with acute rejection. There are high-throughput methods developed to identify these proteomic signatures of acute rejection in urine and blood samples . However, implementing these markers into widespread clinical application will need standardization of procedures for sample preparation and development of simplified test systems.
TruGraf is a blood test developed by Transplant Genomics Inc. (TGI, Mansfield, Massachusetts, USA) that measures gene expression signatures using DNA microarrays in kidney transplant recipients and is intended for use in subjects with stable renal function as an alternative to protocol biopsies. The test can differentiate a state of immune quiescence, indicating an adequate state of immunosuppression, referred to as Transplant eXcellence from not-Transplant eXcellence, an indication of suboptimal immunosuppression or immune activation. The test was validated using surveillance biopsy data in 24 months, multicenter observational study [Clinical Trials in Organ Transplantation (CTOT-08)] [59▪▪]. At a predefined threshold, 72–75% of kidney transplant recipients achieved a negative biomarker test correlating with the absence of subclinical acute rejection (negative predictive value: 78–88%), whereas a positive test was obtained in 25–28% correlating with the presence of subclinical acute rejection (positive predictive value: 47–61%). The study showed that the test reduces the need for the indiscriminate use of invasive surveillance biopsies and could be used to monitor kidney transplant recipients with stable renal function, including after treatment for subclinical acute rejection, potentially improving kidney transplant outcomes.
PleximarkTx (Plexision, Inc., Pittsburg, Pennsylvania, USA) is a T-cytotoxic memory cell functional assay, which measures the immune response of recipient lymphocytes to donor lymphocytes in cell culture. Recipient T-cytotoxic memory cells, which express the inflammatory marker, CD40 ligand or CD154 are measured with flow cytometry. Results are expressed as an index of rejection, which is a measure of the likelihood of rejection. The test identifies renal transplant recipients experiencing rejection with sensitivity of 88% of specificity of 86% (https://www.plexision.com/pleximarktx). Allospecific CD154+ T-cytotoxic memory cell response correlates with histological severity of ACR and has proposed to be useful in minimizing protocol biopsies among recipients at reduced rejection risk . Although the test is FDA approved, further validation and multicenter studies are required.
The development of and further studies of noninvasive biomarkers are necessary to fulfill an unmet need for personalized prediction of acute rejection in the organ transplant recipients. The additional risks of undetected false positive and false negative results are minimized by using test results as an adjunct with all available clinical and laboratory information, in a manner concurrent with current clinical practice.
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Conflicts of interest
There are no conflicts of interest.
REFERENCES AND RECOMMENDED READING
Papers of particular interest, published within the annual period of review, have been highlighted as:
- ▪ of special interest
- ▪▪ of outstanding interest
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