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Subclinical Antibody-Mediated Rejection

Arias, Manuel1; Serón, Daniel2; Herrero, Ignacio3; Rush, David N.4; Wiebe, Chris4; Nickerson, Peter W.4; Ussetti, Piedad5; Rodrigo, Emilio1; de Cos, Maria-Angeles6

doi: 10.1097/TP.0000000000001735

1 Nephrology Department, Hospital Universitario Marqués de Valdecilla–IDIVAL, Universidad de Cantabria, Santander, Spain.

2 Department of Nephrology, Hospital Universitari Vall D´Hebrón, Universidad Autónoma de Barcelona, Barcelona, Spain.

3 Liver Unit, Clínica Universidad de Navarra, Biomedical Research Centre in the Liver and Digestive Diseases Network (CIBERehd), Instituto de Investigación Sanitaria de Navarra (IdiSNA), Navarra, Spain.

4 Section of Nephrology, Department of Internal Medicine, University of Manitoba, Winnipeg, Manitoba, Canada.

5 Department of Neumology, Clínica Puerta de Hierro, Madrid, Spain.

6 Clinical Pharmacology Service, Hospital Universitario Marqués de Valdecilla–IDIVAL, Santander, Spain.

Received 5 January 2017. Revision received 2 March 2017.

Accepted 14 March 2017.

The authors declare no conflicts of interest.

Correspondence: Manuel Arias, Hospital Universitario Marqués de Valdecilla, Avda, Valdecilla sn, 39008, Santander, Spain. (

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Manuel Arias

The role of anti-HLA antibodies and, thus, the importance of the humoral response have been relegated for decades in the pathogenesis of transplant rejection and graft loss due to the predominance of the cell theory (T-Centric). However, in recent years, epidemiological and clinical evidence has proven otherwise. In addition, neither the academy nor the industry has deepened in the investigation of new drugs directed to control antibody-mediated allogeneic response. Therefore, the immune management of organ transplant recipients is clearly unsatisfactory, especially in the long term. Beyond general dosing guidelines for immunosuppressive agents and clinical diagnostic tests for rejection, which are focused on organ function but not on immunological markers, there are few objective tools to determine the aggregate status of a patient's alloimmune response.

Recent studies indicate that antibody-mediated rejection (ABMR) is among the most important barriers to improving long-term outcomes. In recent years, understanding the roles of acute and chronic ABMR has improved because of major progress in the technical ability to detect and quantify recipient anti-HLA antibody production. Additionally, new knowledge of the immunobiology of B cells and plasma cells that pertains to allograft rejection and tolerance has emerged. Still, questions regarding the classification of ABMR, the accuracy of diagnostic approaches, and the efficacy of various strategies for managing affected patients abound.1

These and other lesser known reasons contribute to the disappointing attrition of grafts on a slowly progressive downward slope observed with impotence by the shortage of effective drugs.

Noninvasive biomarkers that could serve as predictive tools or surrogate end points for rejection might help clinicians to individualize immunosuppression (IS) and allow for early intervention, ideally before clinically evident organ dysfunction. Although the growing understanding of organ rejection has provided numerous candidate biomarkers, none has been confirmed in robust validation studies as sufficiently useful to guide clinical practice out of the boundaries of the traditional clinical methods.

The development of ABMR caused by donor-specific antibodies (DSA) is considered a risk factor for low graft survival after kidney transplantation. Although less studied, the deleterious consequence of ABMR is being increasingly seen in other solid organ transplantations, such as heart and lung.2 Recent reports have shown that the occurrence of DSA in the liver, classically considered a relatively resistant organ to DSA-mediated injury, can result in lower graft and patient survival.3-8 The diagnosis of ABMR after transplantation has been defined using a combination of clinical, histological, and immunological criteria.

Recently, a subclinical variety of ABMR which may also progress into chronic ABMR has been described.9 In these cases, and considering the lack of clinical evidence, the availability of more sensitive diagnostic tests allowing the early detection of low titers of DSA3 and the routine use of protocol biopsies10 could facilitate the early start of an appropriate immunosuppressive treatment.3

This review addresses the spectrum of ABMR after organ transplantation, including some aspects of its pathogenesis, risk factors, and outcomes. In this document, relevant professionals review and update different topics.

In the first chapter, Dr Serón reviews the influence of IS in surveillance biopsies and subclinical kidney rejection. In his chapter, early inflammation and its relation with fibrosis and the development of de novo DSA (dnDSA) is assessed, stressing the importance of its prevention.

In his chapter, a critical analysis of the role of DSA in liver graft loss is presented by Dr Herrero. Despite that liver transplantation has been considered poorly affected by humoral rejection, recent evidence shows a relationship between acute and chronic rejection and the presence of preformed and dnDSA. Considering the increased risk of graft fibrosis, graft loss, and lower patient survival, DSA should be carefully considered in liver transplantation.

Considering the difficulty of reversing the humoral rejection process once it has been established, Dr Rush focuses on the importance of developing strategies to prevent chronic humoral rejection at an early stage.

Furthermore, ABMR in lung transplantation is reviewed by Dr Ussetti. Like liver transplantation, humoral rejection has been considered infrequent in this area. Similarly, recent data reveal a role of ABMR in the advent of bronchiolitis obliterans syndrome (BOS), which is the most important cause of morbidity and mortality after lung transplantation. Because of the specific clinical, immunological, and histological characteristics of lung rejection, ABMR diagnosis should be considered from a multidisciplinary point of view.

Finally, high intrapatient variability (IPV) of TAC blood levels, linked to a poorer outcome, has been related to noncompliance, among other factors. Dr Rodrigo reviews the consequences of such IPV on graft outcomes—including the development of dnDSA and the need of a reliable test to detect nonadherence, as a cause of graft loss.

Despite the important role of ABMR in patient morbidity and mortality after renal transplantation, our current understanding of the pathogenesis and pathologic phenotypes of ABMR is limited. Evidence supports that DSA has an important role in acute and chronic ABMR. However, not all DSA detected by current assays results in allograft injury, and not all ABMR phenotypes cause rapid allograft failure. Similarly, C4d has significant limitations as a biomarker of ABMR. Therefore, risk stratification strategies for DSA, C4d, and ABMR phenotypes are needed to guide preventive and therapeutic approaches, including plasmapheresis, IVIG, and anticomplement and anti-B/plasma cell therapies.1

DETECTA is a scientific gathering of Spanish professionals who are experts in all the specialties related to organ transplantation. Its main objective is the continuous updating of the changing knowledge in organ transplantation, and above all, it tries to confront the challenge of ABMR. With the organization of Astellas Pharma Spain, it has become a training aid highly esteemed by Spanish transplantologists. This Supplement mainly presents relevant information provided at a recent DETECTA held in Madrid.

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Daniel Serón

The presence of inflammation in early surveillance biopsies is associated with an accelerated progression of interstitial fibrosis (IF) and tubular atrophy (TA). Inflammation has recently shown to have a poorer outcome when it occurs in patients with IF/TA (IF/TA + i) rather than in patients without IF/TA. Early inflammation has also been associated with a higher risk of dnDSA.

The prevalence and severity of inflammation is closely related with the type of IS and probably with the exposure to tacrolimus (TAC) trough levels so that patients who are more exposed to TAC have less severe inflammation. However, this relation is observed when inflammation is evaluated in healthy tissue (i-Banff punctuation). Some, but not all, clinical trials have shown that IS treatments associated with less severe inflammation soon after transplantation are also associated with less IF/TA progression. However, it is still unknown whether preventing early inflammation with treatment is also associated with the prevention of dnDSA.

In this chapter, 3 main topics regarding early inflammation will be reviewed: its relation with fibrosis and with dnDSA, and the consequences of its prevention by IS treatment.

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Early Inflammation and Fibrosis

More than one decade ago, Nankivell et al11 evaluated prospectively the natural history of renal allograft lesions in 961 surveillance biopsy specimens obtained at regular intervals for 10 years from the time of transplantation in recipients of kidney-pancreas transplant. They observed that the histological lesions followed a temporal sequence. They distinguished an early phase characterized by the presence of subclinical tubulointerstitial inflammation and the rapid progression of IF/TA and, a later phase, characterized by the progression of other chronic lesions like glomerulosclerosis or transplant glomerulopathy, 1 year after transplant. Subsequently, the same authors12 evaluated in pairs of surveillance biopsies whether the presence of inflammation, either borderline changes or subclinical rejection, was associated with an increased risk for the progression of IF/TA. They showed that the presence of inflammation in the first biopsy implied an increased risk for the progression of IF/TA in the second one as shown in Figure 1.



In the surveillance biopsies performed in the first year, tubulointerstitial inflammation and fibrosis constituted the most common histological lesions. Accordingly, various studies classified early lesions into 4 groups according to the presence or absence of tubulointerstitial lesions: a) normal histology, b) inflammation (i), c) IF/TA, and d) IF/TA associated with tubulointerstitial inflammation (IF/TA + i). It is worth reminding that according to Banff classification, inflammation is defined as the presence of inflammatory cells in healthy tissue, and inflammation in scarred areas is not taken into consideration. A number of studies showed that the presence of IF/TA + i was associated to a poorer renal allograft survival in comparison to patients with inflammation or fibrosis alone.13-16

IF/TA + i is a nonspecific histological pattern related to different factors. However, the question of why IF/TA + i is associated with a poor outcome has not been properly answered. From a theoretical point of view, we could enunciate 2 possible hypotheses: either inflammation may be more harmful in an already damaged kidney or the association of IF/TA + i may represent a more aggressive immune response.

In a recent study17 including 598 patients without pretransplant donor-specific antibodies and treated with calcineurin inhibitors (CNIs), histological diagnoses 6 weeks after transplantation were as follows: a) normal histology (34%), b) inflammation (5%), c) IF/TA (43%), and d) IF/TA + i (18%). Using patients with normal histology as the reference group, inflammation was observed to be associated with the number of HLA DR mismatches and the use of cyclosporine (CsA) in comparison to tacrolimus; IFTA was associated with donor age, male gender, and HLA B incompatibility, whereas IF/TA + i was associated with all the above-mentioned factors, that is, donor age, male gender, HLA B and DR incompatibility, and CsA versus TAC (Table 1).



In a study conducted by Heilman et al,18 the relationship between early inflammation in 1- and 4-month surveillance biopsies and the risk of IFTA ≥ 2 and IF/TA + i ≥ 2 at 1 year was assessed. They not only confirmed that the more severe the inflammation in early biopsies, the higher the probability of having IFTA ≥ 2 but also that the more severe the inflammation is in early biopsies, the higher the probability of showing IFTA + i, which suggests that the severity of inflammation soon after transplantation modulates the risk of having IF/TA + i at 1 year. The percentage of patients with IF/TA + i was minimal in patients with no previous inflammation, about half in those with previous borderline status and approximately 75% in patients with early subclinical acute rejection (AR) (Figure 2).



Furthermore, the presence of IFTA + i in 6-week biopsies was associated with a higher probability of showing IFTA ≥ 2 at 1 year compared with patients showing IF/TA at 6 weeks, despite that the severity of tubulointerstitial chronic lesions was similar in both groups at 6 weeks.17 These data confirm that early inflammation constitutes a driving force for the progression of tubulointerstitial chronic lesions (Figure 3).



The relation between inflammation and fibrosis has been evaluated in other studies. Park et al19 observed that the combination of fibrosis and inflammation in surveillance biopsies 1 year after transplantation is associated with reduced graft function and graft survival. In this study, the immunohistochemistry showed an increase of interstitial T cells and macrophages/dendritic cells in patients with both fibrosis and inflammation. Microarray profiles revealed an associated “rejection-like gene expression signature” even in patients without clinical risk factors for a poorer prognosis. Furthermore, Bestard et al20 monitored donor-specific alloimmune response with an IFN-γ ELISPOT assay in surveillance biopsies taken at 6 months. They found a correlation between donor T-cell alloreactivity and histological lesions of acute T cell-mediated rejection. These data suggest that inflammation in early surveillance biopsies could be partly considered a subordinated measure to the immune response.

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Early Inflammation and De Novo DSA

In recent studies, a relationship between early inflammation and an increased risk of dnDSA has been described. Moreso et al21 studied for-cause biopsies in patients with a previous surveillance biopsy. Indication biopsies were done, on average, 7 years after the surveillance biopsy. The most common diagnoses in the indication biopsy were ABMR and IF/TA. They searched for differences in the surveillance biopsies in patients who developed ABMR and in patients with IF/TA in the indication biopsy, and more severe inflammation in surveillance biopsies from patients with ABMR were seen. Their data suggest a link between the severity of early posttransplant inflammation and an increased risk for ABMR. Wiebe et al22 showed that the presence of inflammation in 6-month surveillance biopsies in patients without preformed DSA was associated with an increased risk of dnDSA and ABMR. Of note, this association could be explained by nonadherence that may be partly responsible for an enhanced posttransplant inflammation that, in turn, may trigger the humoral response. In this regard, El Ters et al23 examined the relation between AR during the first year diagnosed by means of surveillance biopsies and that diagnosed by indication biopsies and observed that patients with clinical rejection in comparison to patients without any evidence of AR had a higher probability of showing de novo anti-HLA II antibodies. Moreover, the presence of inflammation in otherwise normal biopsies or the presence of IFTA + i at 6 weeks have been shown to be associated with an increased risk of dnDSA at 1 year.17 Thus inflammation, with or without fibrosis, is associated with a higher risk of having DSA.

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Prevention of Early Inflammation

Because subclinical inflammation is indistinguishable from inflammatory infiltrates observed in patients with clinical episodes of AR, Rush et al10 conducted a clinical trial nearly 20 years ago to study the effect of steroid boluses on subclinical infiltrates. In this study, patients were treated with CsA, azathioprine, and prednisone and were randomly distributed to be biopsied at months 1, 2, or 3 and treated with steroid boluses if they presented subclinical inflammation. The control group was neither biopsied nor treated. The prevalence of subclinical inflammation was greater than 50% during the first 3 months. The authors showed that treatment of early subclinical inflammation prevented the progression of IF/TA at 6 months and preserved renal function at 24 months. This was the first study to show that treatment of subclinical inflammation prevents progression of IF/TA. The same authors conducted a similar study several years later.24 In this second study, subjects were treated with an immunosuppressive regimen based on TAC, mycophenolate mofetil (MMF), and prednisone. However, they could not reproduce the results of the previous clinical trial. They failed to show any significant difference between patients in the treatment group and those in the control group with regard to the progression of fibrosis or the preservation of renal function. Moreover, the multivariate analysis also showed that progression of fibrosis was significantly higher in patients in the treatment group. The reason for this result, which was opposed to that of the previous clinical trial, may be explained by a very low prevalence of subclinical inflammation that was less than 10% and could be attributed to the higher efficacy of a treatment with TAC and MMF compared with a CsA and azathioprine-based treatment. In fact, several studies have shown that the type of IS used may modulate the prevalence and severity of subclinical inflammation and IF/TA. In a case–control study, Moreso et al25 compared the histological lesions observed in surveillance biopsies in patients treated with TAC-MMF-P or CsA-MMF-P. They found less severe inflammation and transplant glomerulopathy in patients treated with TAC than in those treated with CsA. To elucidate whether this difference is related to the modulation of a specific cell immunophenotype, another case–control study was conducted in patients treated with TAC or CsA.26 In this study, the immunophenotype of infiltrating cells was evaluated with monoclonal antibodies against CD45 (all leukocytes), CD3 (T lymphocytes), CD68 (monocytes/macrophages), and CD20 (B lymphocytes), and the study found that, with the exception of CD20, the number of the other positive interstitial cells was lower in patients in the TAC group (Figure 4).



Later on, Thierry et al27 evaluated a subpopulation of the CONCEPT study.28 In this clinical trial, all patients received CsA, MMF, and prednisone and were randomized to either continue with this schedule or cross over to SRL and MMF. At 1 year, the incidence of subclinical inflammation was higher in the SRL group in which the renal function impairment at 30 months was significantly higher. Interstitial fibrosis/TA progression is also modulated by the type of immunosuppressive treatment. In a clinical trial conducted by Merville et al, it was shown that cyclosporine associated with mycophenolate in comparison to cyclosporine and azathioprine prevented IFTA progression at 1 year.29 Additionally, IFTA progression determined by 2 paired biopsies performed during the first year was associated with lower cyclosporine or tacrolimus exposure.30,31 However, in the clinical setting, it has been shown that AR depends not only on TAC trough levels but also on MMF exposure.32

Some studies have suggested that there is an interaction among the type of IS, the presence of subclinical inflammation, and the progression of fibrosis. A clinical trial comparing patients with TAC and MMF with patients who crossed over to a SRL-based regimen33 showed that TAC treatment was associated with lower progression of IF/TA at 1 year. Risk factors for the progression of fibrosis were the presence of subclinical inflammation and receiving an SRL-based treatment. Furthermore, in an open-label randomized trial comparing 4 different schedules: CsA + MMF, CsA + SRL, TAC + MMF, and TAC + SRL, it was shown that patients receiving a calcineurin-based regimen associated with SRL showed a lower incidence of biopsy-proven AR during the first year, a lower prevalence of subclinical rejection in the 1-year surveillance biopsy, and more importantly, a less severe IF/TA in the 5-year protocol biopsy.34 Furthermore, Gatault et al conducted a prospective randomized trial to compare 2 different doses of extended-release tacrolimus in kidney transplants between 4 and 12 months. Control group maintained standard levels between 7 and 12 μg/L, whereas in the study group, the dose was reduced by half provided that TAC levels were greater than 3 μg/L. The study group receiving low TAC exposure had a higher incidence of clinical rejection, higher prevalence of subclinical rejection in 1 year biopsies, and higher prevalence of 1-year de novo anti-HLA donor-specific antibodies. In this study, IF/TA and estimated glomerular filtration rate (eGFR) were not different between groups.35 Thus, early inflammation evaluated in surveillance biopsies may be related not only to the type of treatment but also to the degree of exposure to immunosuppressive drugs (Figure 5).



In summary, the presence of early subclinical inflammation is associated with an increased risk of IF/TA progression and dnDSA synthesis. Treatment with TAC combined either with MMF or sirolimus is associated with a lower prevalence and severity of inflammation in early surveillance biopsies. Immunosuppressive treatments decreasing the prevalence of inflammation in surveillance biopsies have been associated with decreased progression of IF/TA in some but not all studies. More information is needed to understand whether the prevention of early inflammation may constitute a strategy to prevent the appearance of dnDSA.

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Ignacio Herrero

Since the beginning of the transplantation era, liver transplant has been classically considered relatively resistant to humoral rejection. Several factors can be associated with this resistance, such as the higher vascular surface, enabling the absorption and clearance of circulating antibodies; the liver capacity of secreting soluble HLA that can bind and inactivate antibodies with the participation of the phagocytic Kupffer cells; and the high regenerative capacity of the liver even after humoral injury36,37 (Figure 6).



However, evidence published over the previous years show that preformed and dnDSA are associated with a higher incidence of acute and chronic rejection and graft fibrosis. Although infrequent, humoral rejection can lead to liver loss. Inadequate IS could lead to its development.

Five topics will be reviewed in this chapter: acute and chronic rejection, fibrosis, combined liver and kidney transplant, and survival.

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Acute Rejection

Early Rejection and Crossmatch Significance in Historical Studies

In the first studies done in the 80s, liver transplant recipients with preformed antibodies did not seem to have a worse posttransplant outcome.38 Despite this potential protection to humoral rejection, later studies showed a relative risk of early allograft injury in patients with positive crossmatches. Takaya et al, at the beginning of the 1990s, showed that patients with positive crossmatch (≥30%) had significantly lower survival rates at 1 and 3 months posttransplantation than those with negative (<10%) and weakly positive tests (10%-30%).39 In a subsequent study, Mañez et al40 followed patients with positive crossmatch at the time of transplant. They saw that nearly 90% of patients with persistently positive crossmatch had histologically confirmed rejection, resulting in 50% of allograft failure requiring retransplantation. Those patients with allograft failure were characterized by complement consumption and increased circulating immune complexes and refractory thrombocytopenia.

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Early Rejection and Preformed DSA: Role of the Induction Therapy

More recently, thanks to modern DSA detection techniques, a difference in the early rejection rates depending on the type of DSA has been observed. O'Leary et al41 retrospectively studied samples collected prospectively from recipients of primary liver transplantation between January 1, 2000, and May 31, 2009. Fourteen percent of the patients had preformed DSA (class I and/or II). Class I DSA persisted in 5% of patients and were not associated with rejection, whereas class II DSAs (persistent in 23% of patients) were associated with an increased risk of early rejection. In addition, preformed DSA, in comparison with no DSA, were independently correlated with the risk of death. Analyzing the potential role of induction therapy, the study showed a lower rate of early rejection in patients with preformed class II DSA who received induction therapy, although overall survival was not affected.

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Is Humoral Rejection Frequent? Is It Resistant?

A retrospective study42 reviewed 43 recipients of ABO-compatible donor livers who had indication liver biopsy stained for C4d and circulating DSA determination. Seventeen patients (40%) had significant circulating DSA and diffuse portal C4d, which was associated with higher frequency of acute cellular and steroid-resistant rejection. This result contrasts with that of Taner et al,43 who prospectively studied 90 consecutive liver transplant recipients for at least 1 year after transplant. One week after transplantation, 17 of the 20 recipients with preformed antibodies showed a marked decrease of DSA levels. The 3 patients with persistently high DSA levels (mean fluorescence intensity [MFI], >20,000) had acute graft rejection that responded to steroids and, thus, continued with normal allograft function 1 year after transplantation.

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Humoral Rejection and Graft Loss

The contribution of preformed DSA in the development of early allograft loss has been studied by O'Leary et al.44 They evaluated 60 patients with idiopathic allograft loss (defined as death or retransplantation at <90 days) from which serum samples and tissue specimens were available for a retrospective blinded review of histology and C4d staining. Four criteria were considered for the diagnosis of ABMR: circulating DSA, histological findings consistent with antibody-mediated injury (diffuse microvascular endothelial cell injury and micro-vasculitis), strong and diffuse C4d positive in tissue, and the exclusion of other causes of injury that might result in similar findings. They observed positive correlation between graft loss with histological signs of ABMR and high levels of DSA and between the presence of pretransplant DSA (with a high MFI despite dilution) and the development of ABMR leading to graft loss. However, these results have to be taken with caution, considering the weaknesses of such a retrospective study and the limited number of patients with this event (1% of all early graft losses and 5% of previously unexplained early graft losses).

Several factors related to graft quality may also influence the risk of graft failure induced by DSA. On the one hand, suboptimal grafts, when experiencing greater ischemia-reperfusion injury, can induce a greater activation of the immune system.45 On the other hand, grafts from living donors, although subject to less ischemia-reperfusion injury, have a smaller vascular bed, and they may be more sensitive to damage caused by DSA. A recent study has shown that both de novo and preformed DSAs are associated with an increased risk of graft failure not only in the transplant from a deceased donor but also in the graft from a living donor.46

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Humoral Rejection and De Novo DSA

Finally, Del Bello et al47 investigated the incidence of dnDSA, its risk factors and associated complications in patients without preformed anti-HLA DSA. Among the 152 patients studied, 21 (14%) developed dnDSA, of which, 9 (43%) experienced acute ABMR. Younger age, low exposure to CNI, and noncompliance were predictive factors for dnDSA onset. Nonetheless, there were no differences in graft survival rates between patients with and without humoral rejection.

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Chronic Rejection

Donor-specific antibodies have also been associated with chronic rejection. Kaneku et al48 studied 368 serum samples from 39 patients with biopsy-proven chronic rejection which were compared with 66 control patients, to investigate the potential role of DSA in chronic rejection and to understand whether immunoglobulin G (IgG) subclasses could play a different role in this process. Patients in the chronic rejection group were younger, and the frequency of CMV infection was higher; other characteristics (such as donor age; cold ischemia times; Model for End-Stage Liver Disease score at the time of transplant; median of HLA mismatches; or use of CNI, steroids, and antimetabolites at 1 month) were similar.

The study showed that the proportion of patients with DSA, both preformed and de novo, was higher in the chronic rejection group than in the control group (Figure 7). Regarding IgG classes, although the study does not clarify all the factors, it suggests that patients with DSA and normal function often have isolated IgG1, whereas patients with chronic rejection often have a combination of IgG subclasses, being IgG3 DSA frequently associated with the greatest risk of graft loss.



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We have seen the high incidence of preformed and dnDSA and its association with acute and chronic rejection. The potential relation of DSA with the development of fibrosis after transplantation has also been investigated. Miyagawa-Hayashino et al49 studied protocol liver biopsies of 79 pediatric recipients with good graft function, more than 5 years after transplantation. Donor-specific antibodies and C4d deposition were assessed in the last and previous biopsies. Donor-specific antibodies (mostly class II) were detected in 32 patients (48%) who experienced bridging fibrosis or cirrhosis with more frequency than DSA-negative patients (Figure 8). Also, DSA-positive patients had a significantly higher frequency of C4d staining and mild-to-indeterminate AR. Authors concluded that graft fibrosis and DSA (class II) in late protocol biopsies may be associated, suggesting the contribution of alloreactivity to the process of graft fibrosis after liver transplantation. These findings were obtained from the pediatric population and may not be the same in adults.



Donor-specific antibodies may also contribute to accelerated fibrosis progression in liver transplant patients with hepatitis C virus. O'Leary et al50 evaluated the impact of preformed and dnDSA on fibrosis progression after liver transplantation in HCV patients. Aiming to identify modifiable risk factors for fibrosis progression, the study performed a multivariate model, controlling the donor and the recipient characteristics. The results show that preformed DSA (class I and II) were independent predictors of progression to stage 2 to 4 fibrosis, whereas dnDSA had borderline significance. Preformed DSA were also significantly associated with an increased risk of death.

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Kidney and Liver Transplant

There is a belief that liver graft can protect kidney from rejection, even in cases of positive crossmatch. A study from 1986 by Gordon et al51 demonstrated a good evolution of a combined kidney-liver transplant. Renal allografts did not have hyperacute rejection, so they concluded that the liver could diminish circulating DSA. However, this has been questioned. The survival rates of patients and their grafts, when receiving combined liver-kidney transplantation with or without pretransplant positive crossmatch were compared.52 Patients and grafts with positive crossmatch had a much lower survival rate. The differences between both groups were statistically significant, despite the low number of patients (Figure 9).



In another work,53 it was suggested that the liver could be even more sensitive to rejection than the kidney when a combined transplantation was performed in patients with pretransplant DSA. In this study, patients with preformed class II DSA had an increased risk of early ABMR of the renal allograft and liver allograft rejection, whereas the risk of liver allograft rejection in those patients with preformed class II DSA receiving induction therapy was as low as in patients without preformed class II DSA.

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Patient Survival

Finally, we studied how DSA could influence patient survival. A Spanish work54 assessed whether the degree of donor-recipient compatibility and preformed antibodies could modify graft survival. They confirmed that pretransplant DSA were associated with allograft rejection and lower graft survival rates within the first year posttransplant. A few years later, other study6 confirmed that the development of dnDSA is an independent risk factor for patient death and graft loss. A multivariable modeling showed that factors increasing the risk of dnDSA onset were the use of CsA instead of TAC and low CNI levels, whereas the risk was reduced when the calculated Model for End-Stage Liver Disease score was higher than 15 at transplant and the recipient age was older than 60 years. Considering the IgG class, both preformed and de novo IgG3 patients had the highest risk for death.55 Regarding C4d staining, there is also an association, in terms of lower survival, in patients with endothelial C4d deposition.56Table 2 summarizes the most relevant aspects of this revision.



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Future Directions

Evidence available to date suggests that DSA may play a role in hepatic graft damage (probably less important than in renal and cardiac transplantation). To know the role of DSAs in liver transplantation, it is necessary to conduct prospective and multicenter trials studying DSAs before and after transplantation and liver per protocol biopsies to diagnose humoral rejection and detect early changes that precede rejection. These studies should evaluate not only de novo or preformed DSA levels that may cause chronic graft damage but also the specificity of DSA (anti HLA class I or II) or its ability to bind to the complement. If chronic graft damage caused by DSA is confirmed, studies evaluating the role of different immunosuppressive regimens in the prevention and treatment of humoral rejection should be conducted.

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David N Rush, Chris Wiebe, and Peter W Nickerson

Rejection mediated by DSA is frequently associated with renal allograft loss. Donor-specific antibodies can be present in the recipient before the transplant or develop de novo, often several years after surgery. Donor-specific antibodies directed against class II HLA antigens are associated with a worse prognosis than those directed against class I HLA antigens.

Risk factors for dnDSA development include pretransplant factors, such as HLA mismatching and immunological memory, and posttransplant factors, the most important of which are those related to insufficient IS, either because of patient nonadherence or physician-guided IS minimization, leading to cellular rejection and predisposing to DSA development. Because current treatment of DSA-mediated rejection has so far been ineffective, it seems clear that efforts should focus on its prevention.

We have followed a cohort of 560 patients for more than a decade with the aim of evaluating the risk factors at the time of dnDSA detection that predict progression to premature graft loss.57 After excluding patients with preformed DSA or primary dysfunction, 508 patients, with a median follow-up of 75 months, have been analyzed. Most of them (n = 388; 76.4%) neither developed dnDSA nor kidney dysfunction. Dysfunction without dnDSA was present in 56 patients (11%). Among the 64 patients (12.5%) who developed dnDSA, 45 (70%) had histological lesions compatible with humoral rejection, although without clinical criteria for kidney dysfunction (serum creatinine rise, ≤25%; and proteinuria, < 500 mg/d within 2 months of dnDSA detection); the remaining 30% presented clinical dysfunction.

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Risk Factors for DSA Development

Risk factors for the development of dnDSA are well known and include nonadherence to IS, HLA mismatch, younger age, and previous clinical rejection (Table 3).58



Nonadherence is a major problem. In our cohort, the risk of developing dnDSA was significantly higher in nonadherent patients.57 In fact, 72% of patients that were nonadherent had developed dnDSA by 12 years, whereas only 12% of patients who were adherent developed dnDSA in that time frame (P < 0.001), often in the setting of a decrease in IS.

Another important risk factor for dnDSA development is class II HLA epitope mismatching between the donor and recipient, which is synergistic with nonadherence in determining the risk of dnDSA development. Thus, patients with a high number of class II epitope mismatches should be followed closely; in such patients, IS minimization should be avoided as much as possible. Conventional class II mismatching for HLA DR and DQ is less precise than class II epitope mismatching, as one HLA class II molecule may have a wide range of epitopes; therefore, epitope matching is a more precise way of assessing immunological risk.59

In addition, early clinical T-cell–mediated rejection (TCMR) is linked to the development of dnDSA and ABMR.21,23 In a cohort studied by El Ters et al,23 15.2% of patients had AR during the first year, which was related to a significant reduction of graft survival. Half of the AR was subclinical (diagnosed by protocol biopsy), and 3 histological lesions were associated with a poorer outcome: moderate/severe graft IG (GIF), GIF associated with inflammation in nonscarred areas (GIF + i), and transplant glomerulopathy (TG). Moreover, the rate of dnDSA was twice as high in the patients with previous AR (21.2%) than in those with no previous AR (11.1%).

An increased risk for dnDSA has been reported after a switch from cyclosporine-based IS to everolimus-based IS.60

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ABMR Pathology

According to the 2013 Banff criteria,61 the diagnosis of acute/active ABMR requires the presence of 3 elements:

  1. Histological evidence of acute tissue injury, including one or more of the following:
    • Microvascular inflammation (g > 0 and/or ptc > 0)
    • Intimal or transmural arteritis (v > 0)
    • Acute thrombotic microangiopathy, in the absence of any other cause
    • Acute tubular injury, in the absence of any other apparent cause
  2. Evidence of current/recent antibody interaction with vascular endothelium, including at least one of the following:
    • Linear C4d staining in peritubular capillaries (C4d2 or C4d3 by IF on frozen sections, or C4d > 0 by IHC on paraffin sections)
    • At least moderate microvascular inflammation (g + ptc) ≥ 2
    • Increased expression of gene transcripts in the biopsy tissue indicative of endothelial injury, if thoroughly validated
  3. Serologic evidence of DSA (HLA or other antigens).

Although C4d deposition in the peritubular capillaries was considered a key finding for the diagnosis of ABMR, we now know that up to 50% of humoral rejections have no C4d tissue deposition. Also, it is important to know that cell rejection is common as it is present in a high percentage of protocol biopsies, when antibodies appear in the circulation. In our study,57 approximately 60% of patients had TCMR (borderline or grade 1-2) at the time of dnDSA detection.

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Prognosis and Evolution

The time to reach 50% of graft losses was 3.3 years in patients who presented with clinical dnDSA and 8.3 years in those patients with subclinical dnDSA.57 Furthermore, our group highlighted an interesting point regarding the progression of the fall in eGFR over the years of follow-up in our cohort. In stable adult patients, eGFR slope was close to the normal-life decline, suggesting no toxic effect attributable to CNI. On the other hand, in patients developing dnDSA, there is a negative slope of the eGFR which starts before DSA detection and is accelerated thereafter. This suggests that there is an alloimmune process previous to the DSA detection, and hence, DSA are not solely responsible for the graft injury. In dnDSA patients, clinical and histological factors considered predictors of graft loss include delayed graft function, nonadherence to IS, DSA titer (MFI), tubulitis, and chronic glomerulopathy.

Based on what we know today, our model for the graft loss is outlined in Figure 10.



Finally, as there is currently no effective therapy to reverse the humoral rejection process once it has commenced, the better strategy to improve graft outcomes is to begin at the beginning.62 That is,

  • Avoid transplanting patients with preexisting DSA to class II HLA antigens (DR and DQ primarily)
  • Limit allorecognition through class II HLA matching and adequate IS long term
  • Screen for and intervene early in cases of medication nonadherence
  • Screen for and treat subclinical TCMR
  • Avoid drug minimization in those patients at risk for dnDSAs (class II HLA mismatched or with early TCMR)
  • And, when deemed acceptable or necessary, use allograft monitoring to minimize IS (eg, protocol biopsies, urine chemokines, and serum DSA).

All these strategies need to be supported through clinical studies focused on the prevention of early cellular and humoral rejection and on the early treatment of inflammation associated with dnDSA development.

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Piedad Ussetti


Traditionally, humoral rejection has been considered infrequent in the lung transplant context. However, the application of more sensitive and specific techniques for the detection of DSA has allowed progress in the diagnosis of humoral rejection in the pulmonary graft (ABMR). In addition, a relationship between the development of DSA, chronic rejection, and mortality in lung transplant recipients has been observed.

Humoral response may cause several clinicopathological finding, but ABMR is defined by the presence of DSA, histological injury, and acute or chronic graft dysfunction (CLAD).

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Role of Antibodies in Lung Transplantation

In lung transplant, like in other solid organ transplants, antibodies to MHC class I or II can be donor-(DSA) or nondonor-specific (non-DSA) and can be present before or develop after surgery. In addition to HLA, other non-HLA molecules can be targeted by the immune system. These antibodies to tissue-restricted self-antigens (autoantibodies) generated before or after transplantation can interact with alloimmunity and influence posttransplant outcomes.

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Preformed Antibodies

Preformed antibodies are clearly associated to a poor prognosis in terms of survival. The UNOS database, in a large series of lung transplant patients, showed that panel reactive antibody level exceeding 25% is a predictor of death.63 Type of DSA and MFI (Luminex) have been related to posttransplant survival. In a study64 correlating HLA-specific antibodies and C4d deposition with graft survival, it was demonstrated that preformed DSA (particularly complement-fixing) and high MFI greater than 5000 are associated with poor survival within 1 year after transplantation.

Intervention before transplant is possible in 2 ways. On one side, the use of virtual mismatch allows avoiding the potential donor-reactive HLA.65 Also, it is possible to manage sensitized candidates through perioperative desensitization and maintenance immunotherapy.66 The latter consists of intraoperative plasma exchange (PLEX) in DSA-positive transplants, followed by subsequent postoperative PLEX over 2 weeks, and in the administration of intravenous immune globulin (IVIG) after final PLEX. This strategy, implemented at the time of transplant instead of while the patient is in the waiting list, reduces unnecessary treatments, costs, and complications in patients not proceeding to transplant and demonstrates that sensitized lung transplant patients may expect equivalent allograft survival and function than nonsensitized recipients.

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Lung-specific self-antigens may activate autoimmune response with the generation of autoantibodies before or after transplantation. Collagen V (Col V) forms the core component of the fibrillar extracellular matrix. K-alpha1 tubulin (Kα1T) is a cytoskeletal protein involved in intracellular locomotion. Before transplantation, these “cryptic” proteins may be exposed to the immune system in several lung conditions. Idiopathic pulmonary fibrosis and cystic fibrosis are the lung diseases with the highest prevalence of Col V and Kα1T.67

After transplantation, metalloproteases produced during isquemia-reperfusion or other lung injuries may impair fiber integrity, exposing them to the immune system. Autoantibodies to Col V and Kα1T have been associated with an increased risk of developing primary graft dysfunction, DSA, and chronic rejection.67

The interplay between alloimmune and autoimmune responses has been recently reinforced by Reinsmoen et al.68 They demonstrated that high levels of autoantibodies to AT1R exacerbate the immune response by increasing the susceptibility to the alloimmune response through the development of de novo donor HLA antibodies.

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Posttransplant DSA

In 1998, Sundaresan et al69 showed for the first time a relation between antihuman leukocyte antigen (HLA) antibodies detected by panel reactive antibody and chronic rejection. Years later, Girnita et al70,71 showed that the presence of HLA antibodies detected by enzyme-linked immunosorbent assay was associated with lymphocytic bronchiolitis; chronic allograft dysfunction; and acute, severe, or refractory rejection; among 54 receptors of lung transplantation, 10 developed antibodies in the first (n = 8), second (n = 1), and third (n = 1) month after surgery.

There is increasing evidence recognizing the development of HLA antibodies as an important risk factor for BOS after lung transplantation. Hachem et al72 evaluated prospectively 166 patients that had a negative direct crossmatch with the donor at the time of transplantation. Among them, 65 (56%) developed posttransplant DSA, mainly during the first 90 days after transplantation (80%), most of them class II or a combination of class I and II. However, not all antibodies are harmful and, in some patients, disappear spontaneously. Therefore, there is no consensus about the preemptive treatment of patients who develop DSA without graft dysfunction.

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Histological Injury

Regarding the presumed relation of antibodies and histological lesions in lung transplant rejection, the previously mentioned Classification and Grading of Pulmonary Allograft Rejection73 document published in 2007, only states that “the presence of serum anti-HLA antibodies and the deposition of complement in alveolar tissue after transplantation suggest a role for humoral immune responses in lung.”

To investigate whether there are specific histological lesions in lung transplant ABMR, Yousem et al74 studied lung biopsies of 23 patients developing HLA antibodies and lung dysfunction. Among them, 17 patients (74%) presented high-grade acute cellular rejection and 5 (22%) patchy acute lung injury. When comparing the 17 cases of acute cellular rejection with coexistent anti-HLA antibodies with a matched group of 26 patients with equivalent cellular rejection grade without anti-HLA antibodies, capillaritis and C4d deposition were more frequently seen in the HLA group. For the histopathological diagnostic, however, it has to be considered that the degree of concordance may not be optimal depending on the reviewer and the assay, as it was observed by Roden et al75 when evaluating C4d with either immunohistochemical or immunofluorescence techniques.

Recently, the International Society of Heart and Lung Transplantation76 published an update of findings and recommendations on pulmonary ABMR. Considering the histopathological findings as nonspecific for ABMR, the consensus group agreed that “the definitive diagnosis of pulmonary ABMR requires the combination of clinical dysfunction, circulating DSA and C4d immunoreactivity” and listed a number of histopathological patterns that should require further immunopathological and serologic studies (Table 3).

Indications for immunopathological evaluation76 (Table 3)

  • Neutrophilic capillaritis
  • Neutrophilic septal margination
  • High-grade acute cellular rejection (ZA3)
  • Persistent/recurrent acute cellular rejection (any A grade)
  • Acute lung injury pattern/diffuse alveolar damage
  • High-grade lymphocytic bronchiolitis (grade B2R)
  • Persistent low-grade lymphocytic bronchiolitis (grade B1R)
  • Obliterative bronchiolitis (grade C1)
  • Arteritis in the absence of infection or cellular rejection
  • Graft dysfunction without morphologic explanation
  • Any histological findings in the setting of dnDSA positivity
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Graft Dysfunction

In presensitized patients, hyperacute rejection is a rare but widely accepted form of ABMR immediately after transplantation.77 However, there is no clear definition of other clinical phenotypes of rejection.

The development of DSA after transplantation has been related to patient and graft outcomes. Hachem et al72 studied patients developing DSA after transplantation and preemptively treated with intravenous immune globulin alone or in combination with rituximab. They compared patients with persistent DSA with those with cleared DSA in terms of BOS and survival rates, finding that patients with persistent DSA were significantly more likely to develop BOS and had a significantly worse survival rate than those with cleared DSA.

To evaluate the clinical, immunological, and histological characteristics of ABMR, Witt et al77 studied 21 recipients with acute lung ABMR. All patients had clinical allograft dysfunction, DSA, histology of acute lung injury, and capillary endothelial C4d deposition. Outcomes were poor, with 6 patients dying of refractory ABMR and 13 developing chronic allograft dysfunction. Again, patients with persistent DSA had a worse prognostic. In a recent publication regarding chronic ABMR, DeNicola et al78 showed that patients with DSA were BOS-free for less time and had poorer survival. These outcomes worsened in patients presenting both DSA and histological ABMR characteristics and were even worse when 3 ABMR features (DSA + histology + dysfunction) were present.

Recently, Vandermeulen et al demonstrated an overlap between ABMR and restrictive phenotype of CLAD (restrictive allograft syndrome) evidenced by the presence of circulating DSA and increased levels of immunoglobulins and complement proteins in these patients.79 These finding are consistent with published data by Roux et al observing the development of restrictive allograft syndrome in the subgroup of patients with ABMR.80

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ABMR Evidence and Controversies

Considering the evidence, we know that a high percentage of receptors (25%-50%) will develop DSA. The persistence of DSA has been associated with poor outcomes. Donor-specific antibody is related to higher frequency and severity of rejection and lower long-term survival and is associated with compatible (although no specific) histological lesions. However, diagnostic criteria are not yet well defined, and regarding the tissue deposition of C4d, there is a considerable histological variability intercenter and interobserver dependent on the assay used (immunohistochemical vs immunofluorescence). Features among different clinical phenotypes are not clear.

For these reasons, the ISHLT81 has recently published a consensus report focused on lung ABMR. It considers that ABMR may be clinical (symptomatic or asymptomatic but with some evidence—eg, radiological or functional—of graft dysfunction) or subclinical (asymptomatic with no other evidence of dysfunction). Both main categories can be possible, probable, or definite depending on the degree of agreement with the diagnostic criteria (Figure 11).



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Summary and Conclusions

  • DSA detection is necessary but not indispensable for the diagnosis of ABMR.
  • There is no consensus regarding the timing of the antibody sample testing or the need of preemptive treatment of patients who develop DSA. However, close follow-up of these patients is warranted to initiate treatment when graft dysfunction is detected.
  • Histological lesions are characteristic but not specific of ABMR; thus, other causes need to be excluded.
  • The diagnostic of ABMR has to be multidisciplinary.
  • Clinical phenotypes are poorly defined, and subclinical forms of ABMR should be considered.
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Emilio Rodrigo, Maria-Angeles de Cos, and Manuel Arias

Progressive reduction in AR rates has led to an improvement of kidney graft survival throughout the first year. By contrast, graft attrition rates remain stable or with only minor reductions beyond this point.82 Alloimmune mechanisms, mainly ABMR, are the most frequent causes of graft loss.83 Lack of adherence to immunosuppressive therapy has been identified in almost half of kidney transplant recipients with rejection.83 Furthermore, nonadherence is one of the main risk factors for dnDSA development.22 Among other variables, lack of adherence influences the IPV of CNI blood levels. In this sense, Shemesh and Fine have proposed that the best way to evaluate nonadherence to medications in transplant recipients is by calculating the variability of CNI blood levels in each individual patient.84 Calcineurin inhibitor blood level variability can be easily estimated by the standard deviation (SD) or by the coefficient of variation (CV), calculated according to the following equation: CV (%) = (SD/μ) × 100, where μ is the mean CNI blood concentration of all available samples.85 Because of the fact that, currently, TAC is the most used immunosuppressive drug,86 we focus this review on the meaning of IPV of TAC blood levels (Tac-IPV) in the outcome of solid organ transplants.

Patients receiving CNI-based immunosuppressive therapy are at risk of over immunosuppresion or underimmunosuppresion because these drugs have a narrow therapeutic range. Thus, both TAC and CsA require blood level monitoring throughout all the transplant duration. Intraindividual variability of CsA was early identified as a potential factor affecting long-term outcome of kidney transplants.87,88 In 1996, Kahan et al reported that a high CV of average CsA concentration obtained from serial pharmacokinetic profiles was related to the occurrence of biopsy-proven chronic rejection.89 In 1997, a round table of 8 experts acknowledged inconsistent CsA exposure as a risk factor for chronic renal allograft rejection.90 The influence of CsA trough blood level variability was confirmed in a further study demonstrating that a high variability (≥36%) in trough CsA concentration increased the risk of chronic rejection without needing to perform a pharmacokinetic profile.91 These relationships between CsA level variability and various poor transplant outcomes, such as biopsy-proven AR, biopsy-proven chronic allograft nephropathy, reduced graft survival, and higher serum creatinine, were also independent of the mean CsA trough level.92,93

Initial studies about Tac-IPV were carried out in pediatric liver transplant recipients. Several authors reported that a high SD of TAC levels was a powerful predictor of late liver graft rejection and suggested using IPV of drug blood level as a surrogate marker of immunosuppressive drug adherence and adequacy, similar to HbA1c for diabetic control.84,94-97 Although most experts feel that TAC levels are fairly stable in most patients, the results of the Symphony study showed that a high percentage of patients treated with TAC do not remain within the target range even in a better-controlled-than-real-life environment such as a clinical trial. In fact, an analysis of individual patients showed that only 11% of the patients in the TAC group were within target range at all times during the first months after transplantation mainly because of IPV. In the Symphony trial, the intrapatient CV was 28% for patients receiving TAC.98 Besides, as Whalen et al suggested, we must expect that patients who are currently taking a low-dose tacrolimus-based regimen on a regular basis can show a higher TAC-IPV because the effect of a change in drug concentration is magnified in patients with low blood levels.99 Taking into account these findings, Borra et al reported for the first time that IPV in TAC levels was associated with a worse long-term outcome in adult kidney transplant recipients. In 297 patients, those with high IPV of TAC levels, measured 6 to 12 months posttransplantation, reached a primary composite end point consisting of graft loss, biopsy-proven chronic allograft nephropathy, and doubling of plasma creatinine concentration more frequently than those with low variability. This relationship was independent of other variables (P = 0.003). Of note, neither mean TAC concentration nor mycophenolic acid concentration variability relate to the composite end point.100

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High TAC Blood Level Variability Increases the Risk of Graft Failure and Renal Dysfunction

After Borra et al, there has been a growing interest in analyzing the consequences of a high IPV of TAC levels on kidney transplant outcome in adult and pediatric recipients.99,101-110 Most of these studies have shown that a high IPV is associated with a poorer transplant outcome which could be either death censored graft loss (DCGL)99,105,107 or a composite end point (Figure 12).100,104,106 Moreover, this relationship was independent of other variables including mean TAC level105-107 and remained significant after analyzing IPV, both as a dichotomous and as a continuous variable.106 Of interest, Sapir-Pichhadze et al reported a progressively increased risk of graft failure for each increment of variability estimated as a standard deviation.104



To highlight the independent relationship between Tac-IPV and DCGL, we added the CV of TAC levels between months 4 and 12 to the Kidney Transplant Failure Score (KTFS).111 This recently reported score takes into account 8 accepted variables at 1 year that predicts graft failure with an area under the receiver operating characteristic curve of 0.78.111 In our reported population of 310 patients, the KTFS had an area under the receiver operating characteristic curve of 0.628 (95% confidence interval [CI], 0.532-0.725; P = 0.005) for predicting DCGL. As shown in Figure 13, those patients with high CV have a worse outcome than those with low CV in each group of low and high risk for DCGL according to KTFS. In this sense, adding the IPV of TAC levels at first year in routine clinical practice seems useful to better predict the risk for DCGL and provides information independent of other variables such as mean TAC levels.



In addition to the risk of DCGL, we found that the CV of TAC levels was related to 1-year GFR. Estimated GFR was significantly lower in the group of patients with CV of 30% or greater (47 ± 17 mL/min per 1.73 m2 vs 52 ± 16 mL/min per 1.73 m2; P = 0.019).105 Similarly, Whalen et al reported that MDRD-estimated GFR was significantly worse in the group of patients with high Tac-IPV at 1, 2, 3, and 4 years of follow-up (P < 0.0001).99

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Causes of Graft Failure in Patients With High Variability

What are the reasons for the worse outcome of kidney transplants with high IPV? Patients with high IPV may experience episodes of overimmunosuppression and underimmunosuppression (Figure 14).



During underimmunosuppression lapses, alloimmune responses against the graft can be triggered. It is known that there is a 7.2% increased risk of AR in the first 6 months for each 1 ng/mL decrease in TAC trough levels.112 With respect to Tac-IPV, several studies have demonstrated that those patients with high IPV of TAC levels are at a higher risk of early and late AR.99,101-104,106,109Figure 15 shows a pooled analysis of the reported mean differences of CV of TAC blood levels between patients with and without AR (random effects, 2.22; 95% CI, 1.33-3.09).



Measuring CV up to 12 months posttransplantation relates to higher AR risk before and beyond the first year,99,102,103,106 suggesting that the influence of Tac-IPV remains longer than the period of measurement or that the patients will continuously experience a high Tac-IPV. Similarly, Huang et al reported that CV of TAC trough levels measured 6 months before a biopsy-proven AR was significantly higher (12.1% ± 7.9% vs 39% ± 15.6%, P < 0.001) than in a control group without AR. Receiver operating characteristic curve analysis showed that CV of previous TAC levels could predict AR with an AUC of 0.923 (P < 0.001) in 161 kidney transplant recipients.109 In this sense, it could be advisable to measure IPV as CV or SD at short (3-4 months) and long periods (6-12 months) to detect those patients at a higher risk of rejection based on their high IPV.

Together with the cellular alloimmune response, IPV has been related to antibody-mediated allograft injury. Sapir-Pichhadze et al and Shuker et al included transplant glomerulopathy in the composite end point related to high IPV.104,106 Wiebe et al reported a strong association between nonadherence and dnDSA development (odds ratio [OR], 8.75; P < 0.001). Nonadherence is defined as lack of adherence to medication reported by the patient and documented by clinicians and/or drug levels below the detectable limit and a repeated failure to attend clinic visits or perform laboratory tests.22 Nonadherence definition is always partly subjective and cannot be reported in clinical records in the absence of an appropriate search strategy. Because of this, we used CV of TAC levels from months 4 to 12 in 310 adult kidney transplant recipients without pretransplant DSA to analyze if Tac-IPV was related to dnDSA development. We found that a CV greater than 30% was independently related to dnDSA (hazard ratio [HR], 2.925; 95% CI, 1.473-5.807; P = 0.002).105 A similar finding has been recently reported in pediatric kidney transplant recipients.108

Apart from biopsy-proven AR, there is scarce information about the long-term influence of Tac-IPV on histological kidney damage. Vanhove et al analyzed a cohort of 220 renal transplant recipients with protocol biopsies performed at 3 months and 2 years posttransplantation. They reported that recipients who had the highest CV tertile of TAC levels between 6 and 12 months had a significantly increased risk of having moderate to severe fibrosis within 2 years with respect to the low CV tertile. Moreover, the chronicity score increased significantly in the high CV tertile recipients. On the other hand, CV was not associated with other Banff variables related to cellular (i, t, v) or antibody-mediated (g, ptc) subclinical inflammation in the 2-year biopsy. A tentative explanation for the lower than “expected” subclinical inflammation in high Tac-IPV patients may be that all patients included in the study had favorable baseline characteristics and survived for at least 2 years, with a low incidence of subclinical rejection (3.6%).110 Mayo Clinic reported similar results showing that high SD in trough TAC levels between 2 and 12 months were risk factors for unfavorable histology in the 1-year protocol biopsy, although only in univariate analysis.113

These studies have shown that underimmunosuppression related to high Tac-IPV is associated with a worse graft outcome. However, does overimmunosuppression have any role in high Tac-IPV? If overimmunosuppression had a relevant role in patients with high Tac-IPV, we would expect a lower survival rate in those patients because of TAC-associated side effects such as posttransplant diabetes mellitus, infection, cancer, and cardiovascular risk (Figure 15). No studies have been conducted to specifically examine whether this overimmunosuppression-related comorbidity is more frequent in patients with high Tac-IPV. In relation to TAC nephrotoxicity, only Vanhove et al reported that CV was an independent predictor of de novo arteriolar hyalinosis in the 2-year biopsy (OR, 2.29; 95% CI, 1.17-4.47; P = 0.015).110 Given these facts, it cannot be assured that the variability in TAC levels contributes to poorer graft outcomes because of overimmunosuppressive episodes. By contrast, all these data suggest that underimmunosupression has a greater influence on the graft outcome than overimmunosuppression, as it has been acknowledged.31,112 Considering that the relationship of Tac-IPV with graft survival, renal function, and AR is independent from the mean TAC levels, we can assume that by measuring Tac-IPV, we will obtain more information than by measuring individual blood levels.

A similar influence of Tac-IPV on the outcome of nonkidney solid organ transplants has been demonstrated. As previously mentioned, the relationship between Tac-IPV and transplant outcomes was initially reported in pediatric liver transplant recipients.94-97 A study including 144 heart, kidney, liver, and lung pediatric transplant recipients showed that an increased SD of TAC blood levels was an independent risk factor for late acute cellular rejection (OR, 1.6; 95% CI, 1.1-2.1; P = 0.02) and allograft loss (HR, 1.6; 95% CI, 1.2-2.0; P = 0.003).114 Some studies performed in adult liver and lung transplants have obtained similar results. First, Tac-IPV measured as SD was higher in liver transplant recipients who were going to develop biopsy-confirmed rejection,115 and the SD of TAC levels measured between months 6 and 18 was independently associated with liver graft failure.116 Second, for each SD unit, the risk of early rejection in 108 lung transplant recipients increased by 23% (HR, 1.23; 95% CI, 1.04-1.46), and a high TAC SD between 6 and 12 months independently increased the risk of chronic lung allograft dysfunction and death in 110 lung transplant recipients.117,118 In summary, both kidney and nonkidney transplant recipients with a high Tac-IPV are at risk of having not only early and late AR but also chronic allograft dysfunction and graft loss.

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Non-CNI Immunosuppressive Drug Variability

Apart from CNI, there is scarce information about the relationship between the drug variability of other immunosuppressants and the transplant outcome. For instance, although it is well known that reducing or discontinuing the dose of MMF increases the risk of AR and renal allograft loss,119,120 Borra et al and Hsiau et al did not find any relationship between the variability of mycophenolic acid levels and kidney graft outcome.100,101 On the other hand, a recent study carried out in 23 pediatric renal transplant recipients reported an association between high sirolimus CV and biopsy-proven rejection.108 The relationship between mTOR inhibitors and AR and graft loss must be confirmed in larger studies in adult transplant recipients.

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Is it Possible to Reduce Tac-IPV?

It is beyond the scope of this review to analyze the causes of Tac-IPV. Shuker et al pointed out that the type of TAC analytical assay, certain food and time of meals, diarrheal illnesses, drug-drug interactions, generic TAC substitution, and nonadherence can determine the magnitude in Tac-IPV.85 Although some genetic factors such as CYP3A5 genotype have a role in Tac interpatient variability, it seems that this genotype does not influence Tac IPV.121 Recently, it has been shown that even changes in gut microbiota have an influence on TAC dosing requirements.122 Physicians must be aware of Tac-IPV to identify patients at risk, and all the causes of the above-mentioned variability should be reviewed. In clinically stable patients with no changes in prescribed drugs, lack of adherence is thought to be one main determinant of a high Tac-IPV. Special care must be taken in improving adherence to drug therapies.85 Switching from twice-daily tacrolimus to once-daily formulations offers an opportunity to reduce Tac-IPV,123-127 although not all studies have shown a statistically significant IPV improvement.128-130 Besides, there are ongoing trials trying to demonstrate whether pharmacist-driven interventions can help to improve adherence and reduce Tac-IPV.131

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In conclusion, as Sapir-Pichhadze et al pointed out, no matter the causes for TAC variability, Tac-IPV predicts a worse prognosis for kidney and nonkidney allografts.104 High TAC variability increases the risk of AR, graft dysfunction, dnDSA development, and graft loss in kidney and nonkidney pediatric and adult solid organ transplants. Besides, this association is independent of other variables, mainly of the mean TAC levels. Being easy to calculate either as CV or SD from electronic records, we suggest that Tac-IPV should be added to routine clinical practice at least in 2 ways: first, using the last 5- to 10-level measurements or the samples drawn within 3 to 6 months before each visit to calculate the risk of AR; second, taking into account the outpatient or outpatient and inpatient blood levels between 3 or 6 to 12 months posttransplant to estimate the risk of graft loss beyond the first year.

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The authors thank Astellas and Eliana Mesa for providing sponsorship and medical writing assistance respectively.

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