The success of transplantation medicine is closely associated with the development and introduction of potent immunosuppressive drugs. The individualization of this therapy is carried out for the calcineurin inhibitors cyclosporine and tacrolimus as well as the mTOR (mammalian target of rapamycin) inhibitors sirolimus and everolimus via therapeutic drug monitoring (TDM). For drugs with a narrow therapeutic index, TDM enables dose adjustment, which is superior to treatment control by way of a fixed dose . Especially in immunosuppressive therapy, however, it has been shown that patients may respond very differently to the treatment, although concentrations of the drugs are in the therapeutic range. One must therefore ask whether the dose adjustment via TDM is sufficient to individualize the therapy correctly. Pharmacokinetic monitoring can always only be a surrogate for the pharmacodynamic effect of therapy, and the therapeutic ranges are statistically determined target values on the basis of a large number of patients. Pharmacodynamic biomarkers, which specifically detect the effect of the drug on its pharmacological target molecule or, non-specifically, the general effect on the patient’s immune system, therefore, are an attractive addition to TDM to achieve immunosuppression that is better adapted individually . An assessment of the immunological status prior to the transplantation would be helpful in order to be able to adapt the therapy optimally immediately after the transplantation. Patients with a strongly pre-activated immune system would benefit from a more robust therapy, while patients with a moderate immune status, or even a tendency to develop tolerance, would be better served with a weaker therapy. In combination therapies with various medications, one also observes synergistic and antagonistic effects. This can result in chronic over- or under-immunosuppression. In fact, the long-term outcome of the transplantation, despite the great success in the early stage to reduce the incidence of acute rejection, is not encouraging. Chronic humoral rejection, the long-term side effects of immunosuppressive drugs that manifest themselves in kidney damage, cardiovascular diseases, infections and malignant neoplasms cause the graft to be lost or the premature death of organ recipients. Suitable biomarkers could help avoid these complications through better individualization of therapy. Biomarkers are defined as a characteristic that is objectively measured and evaluated and used as an indicator for normal or pathogenic biological processes or pharmacological responses to therapeutic interventions .
Non-specific markers of immune response
Biomarkers of immune activation
ATP concentration in CD4 T-cells
The intracellular ATP (iATP) concentration increases as a result of the activation of the T-cells. A commercial test kit is available for measurement in CD4 T-cells which is approved by the FDA for diagnostic purposes (ImmuKnow®, Viracor IBT Laboratoriesc., Lee’s Summit, MO, USA). The test requires an incubation of whole blood overnight in the presence of the mitogen phytohemagglutinin (PHA) and an isolation of CD4 T-cells with magnetic particles. The ATP is then measured by luminometry . Whole blood anticoagulated with heparin can be stored up to 30 h at room temperature . The result of the ATP measurement is not dependent on the number of CD4 cells, and there is no dependency on the concentrations of immunosuppressive drugs . A meta-analysis has shown that the iATP concentrations in stable kidney-transplant patients with rejection were higher than in those without rejection, and lower in patients with infections than in patients without infections . According to a multicenter study, iATP concentrations >375 ng/mL before the transplantation were linked to a higher risk of acute rejection , whereas a recent study with 583 transplant recipients did not find any correlation between rejection or infection and a one-time determination of the ATP concentration . Although a statistically determined target range is recommended, it can be assumed that strong, inter-individual variations may occur in the immune response, which is why it is also recommended that individual patients be monitored individually over time, but this is very time-consuming and expensive. Similar results to kidney transplantation have also been obtained in the field of heart transplantation [10, 11].
Patients after a lung transplantation with infections also had lower iATP levels than those without infection; with reduction of immunosuppressive therapy and the healing of the infections, the levels increased . Low iATP concentrations were found especially in infections with CMV, other viral infections and pneumonia caused by bacteria, whereas this was less the case for fungal infections . For liver transplants as well, low iATP levels were associated with a high incidence of infections . To date, such clear results have not been published in connection with any organ regarding the association between elevated iATP levels and rejection as a sign of under-immunosuppression, which is why the assay seems to be better suited to indicate over-immunosuppression and the risk of developing infections. Studies that have prospectively examined whether an adjustment of immunosuppression based on iATP levels is reasonable, however, have not been published to date.
Interferon-γ release from alloreactive T-cells (ELISPOT)
Activated T-cells synthesize cytokines, and CD4 cells can be divided into subgroups based on the cytokine pattern. These are the type 1 (Th1) and type 2 helper cells (Th2). Th1 cells produce IL-2, IFN-γ and TNF-α, while IL-4, IL-5, IL-6 and IL-10 are cytokines typical of Th2 cells . An elegant way to track the production of cytokine is the ELISPOT assay (enzyme-linked immunosorbent spot assay). It has been used particularly for the detection of IFN-γ-producing lymphocytes. For this purpose, mononuclear leukocytes are incubated for 18–24 h with donor-specific or non-specific antigens on a membrane, and the cytokine-producing cells are spot-stained with a monoclonal antibody against the cytokine and then counted . This way, Augustine and colleagues have been able to show for kidney transplant recipients that there are memory cells, which can be activated by the alloantigen, even before the transplantation. These memory cells correlate with a higher incidence of rejection after transplantation . This is especially important, because it is believed today that these cells in particular may have a negative impact on the long-term outcome of the transplantation . Hricik et al. have demonstrated that there is, after the transplantation, an association between the activation of T-cells by both donor-specific and non-specific antigens and renal function . To be able to also predict the immunological risk to the organ recipient prior to a post-mortem donation, a panel of donor-foreign cells with major HLA antigens has been developed [20, 21]. However, the results were more convincing in the case of the living donation with donor-specific antigens . A weaker IFN-γ formation was associated with better renal function, allowing a milder immunosuppression . Similar to the ImmuKnow® test, the ELISPOT assay can be standardized and controlled very well [24, 25], which significantly improves the comparability of results between different laboratories. This allows for multicenter studies and increases the chances of routine use if the relevant clinical data are available. In that respect, Bestard et al. have recently demonstrated that INF-γ ELISPOT approaches with donor-specific cells can be usefully employed for the control of immunosuppression . A disadvantage of the method is the long duration of the assay, as it always requires an incubation phase of several hours. For the donor-specific IFN-γ ELISPOT, storage of mononuclear leukocytes of the organ donor is also a problem if follow-up tests are to be conducted following the transplantation.
T-cell surface markers
T-cell activation leads not only to ATP synthesis and cytokine production, but also to the expression of specific surface molecules . These molecules are, for example, receptors, co-stimulatory molecules, adhesion molecules and HLA molecules. Examples of receptors are CD25 (IL-2 receptor) and CD71 (transferrin receptor). Co-stimulatory molecules include CD26, CD28, CD30 and CD154. LFA-1 (lymphocyte function-associated antigen-1) and ICAM-1 (intercellular adhesion molecule 1), also known as CD54, are adhesion molecules that are expressed in larger numbers on activated T-cells. CXCR3 and CCR5 are chemokine receptors that are also found on activated T-cells in larger numbers. The MHC class II molecules include all isotypes . For immune monitoring in transplantation medicine, mainly receptor proteins and the expression of co-stimulatory molecules have been studied. Surface markers can be determined by means of flow cytometry using fluorescence-labeled monoclonal antibodies. This often involves cell function tests, in which either anticoagulated and diluted whole blood or isolated mononuclear leukocytes are stimulated with mitogens or antibodies. Alternatively, a mixed lymphocyte culture with donor-specific or non-specific cells is used. The effect of immunosuppressive drugs can be examined in these incubations through ex vivo additions. In such an approach, as has been demonstrated by Böhler et al., the expression of CD25 and CD71 on CD3 cells (T-cells) can be inhibited dose-dependently by cyclosporine, tacrolimus, mycophenolic acid, sirolimus and methylprednisolone . The imprecision of the assay with cells from a healthy donor in series was <10%, and between different donor cells <21%. The expression of CD25 and CD71 in cells of kidney transplant patients under immunosuppression was also lower than in cells from healthy controls and patients on dialysis [30, 31]. An inverse correlation between the mycophenolic acid concentration in plasma and the expression of CD25 and CD71 was observed in kidney and liver transplant patients [32, 33]. In another study, the change from cyclosporine to tacrolimus produced a decrease in the expression of CD28, CD54 and CD154 . This observation was interpreted as a more potent immunosuppression by means of tacrolimus.
There are only very few data on the connection between surface markers and clinical events. In heart transplant patients, the CD25 and CD71 expressions on CD3 and CD4 cells could support a distinction between organ dysfunction and rejection, while fewer cells with CD25 and CD71 were interpreted as a sign of over-immunosuppression with subsequent organ dysfunction [35, 36]. Our own studies involving kidney transplant recipients suggest that a low expression of CD26 on T-cells is associated with a low risk of acute rejection, while a reduction of the CD71 expression could be a sign of over-immunosuppression because this observation was made especially in patients with infections . CD154 expression has been shown to be associated with acute rejection in pediatric small bowel transplantation in a donor antigen specific cell function assay  and is commercially offered in the US as Pleximmune™ test by a central laboratory (Plexision, Pittsburgh, PA, USA). In contrast to the complicated cell function assays using ex vivo stimulation, the measurement of CD26 on CD3 cells directly in whole blood seems to allow for a statement about the degree of activation of the T-cells in vivo. Boleslawski et al. report, in connection with liver transplantation, that CD28 on CD8 cells can be associated with both rejections in the early phase after transplantation (many CD28/CD8 cells) and tumor diseases over the long term (few CD28/CD8 cells) [39, 40].
Soluble CD30 in serum (sCD30)
CD30 is a co-stimulatory molecule that is released into the blood by activated T-cells . It can therefore be measured as a marker of T-cell activation in serum or plasma, for which commercial ELISA methods are available. . In the meantime, there is also a Luminex method with beads that carry antibodies against CD30 . The source of sCD30 seems to be particularly memory T-cells . In recent years, a number of studies have been carried out on the benefit of this biomarker in transplantation medicine [45–58].
In kidney transplantation, it has been found that a concentration >100 U/mL before surgery is associated with an increased incidence of rejection responses in the first year and an organ loss in the first 5 years . If the sCD30 levels remain high even after the transplantation (day 3–5), it poses an additional risk for acute rejection . A significantly worse graft survival has been observed when there are concentrations >40 U/mL on day 30 after the transplantation . It was not always possible to reproduce the promising results with sCD30 [51–53], which could be partly due to the high intra-individual variation of the marker . The sCD30 concentrations fluctuated by about 20% in renal failure patients on the waiting list, which suggests that sequential determination, for example, on a quarterly basis, would be useful. For other organs and tissues (lung, liver, heart, islet cells), the results are discrepant [55–58]. Unfortunately, there have not been any studies to examine the effect of different immunosuppressive regimens on sCD30.
The proliferation of lymphocytes can be tracked using various techniques. These include, for example, the use of labeled DNA building blocks such as 3H-thymidine or bromodeoxyuridine (BrdU), which are incorporated into the newly synthesized DNA and which can be measured in a scintillation counter or by means of ELISA [59, 60]. The distribution of the fluorescent dye carboxyfluorescein diacetate succinimidyl ester (CFSE) in connection with cell division to the daughter cells can be monitored by flow cytometry . The proliferating cell nuclear antigen (PCNA), as an intracellular protein, can be measured using flow cytometry, or its mRNA expression by quantitative PCR (qPCR) [62, 63].
Increased cell proliferation in stable liver transplant patients has been associated with increased proliferation of CD8 cells when rejection responses were observed . In a small study of 55 kidney recipients, a low PCNA with mRNA expression was associated with the risk of infections . According to the CFSE dilution assay, increased proliferation was linked to an increased rejection rate in 28 children after a small bowel transplantation . The incorporation of BrdU in lymphocytes was lower in kidney transplant patients with leukopenia .
Antibody-mediated rejection (humoral rejection) plays an important role, alongside cellular rejection, especially when it comes to chronic damage of the graft. The antibodies produced by B-cells can be directed very specifically against HLA class I and II characteristics of the organ donor or other antigens, such as major histocompatibility complex class I-related chain A (MICA) or the angiotensin II type 1 receptor. These are called donor-specific antibodies (DSA). DSA may already be present before the transplantation or occur after the surgery. Emerging post-transplant DSA, so-called de novo DSA, are associated with a worse prognosis . The measurement is usually performed today using solid phase assays. In the Luminex technology, microparticles are used to which HLA antigens or other non-HLA antigens are fixed . Antibodies from the serum of the transplant recipient are detected by fluorescence signals. Complement activating antibodies in kidney recipients, in particular, are associated with a significantly increased risk of rejection . The occurrence of de novo DSA can be interpreted as a sign of under-immunosuppression. A study on dose reduction of immunosuppressive drugs yielded an inverse relationship between the drug levels in the blood and the occurrence of de novo DSA . Immunosuppressive drugs may, perhaps, differ in terms of the risk of developing DSA, because during treatment with the mTOR inhibitor everolimus, there were more DSA and humoral rejections than those observed with cyclosporine . Also, in heart and liver transplantations, the incidence of de novo DSA is associated with a poorer graft survival [70, 71]. An antibody-mediated rejection was the most common cause of organ failure in kidney transplant patients in a study performed by Sellarés et al. . The unreliable taking of immunosuppressive drugs was, in half of the cases, associated with the occurrence of DSA, and this lack of compliance could possibly have been uncovered via TDM, but this would not have affected the other half. This emphasizes the notion that the determination of DSA as biomarkers of immune activation may be useful in addition to TDM to better identify vulnerable patients and adjust their immunosuppression. A recently published consensus guideline describes a proposed procedure for routine monitoring, which is summarized here in a simplified manner. The recommendation calls for DSA to be measured in patients at high immunological risk over the first 3 months after transplantation; in patients with a medium risk, only in the first month, and in patients with a low risk, once between the third and 12th months .
Biomarkers indicating damage to the graft
Under- or over-immunosuppression and toxic drug effects can damage transplanted organs. To measure the damage organ-specifically, the usual parameters, such as transaminase, bilirubin, LDH, creatinine, cystatin C or troponins, are analyzed in the blood. For the kidney, urine-based biomarkers can also be used. These include, for example, neutrophil gelatinase-associated lipocalin (NGAL) or the kidney injury molecule-1 (KIM-1). The widespread use of these biomarkers is due to the fact that there are well-validated CE-certified tests. Newer biomarkers that are based on the detection of mRNA or protein patterns have been published. Ling et al. have described a panel of 40 peptides in the urine of kidney transplant patients, which was typical of acute rejection. Six genetic biomarkers in the blood (COL1A2, COL3A1, UMOD, MMP-7, SERPING1, TIMP1) were highly significant for acute rejection . Using mass spectrometry, it was possible to identify in the blood genomic and proteome-based biomarkers of nephropathy, which were validated by means of biopsy findings . In 2009, Moreira et al. reported on the possibility of diagnosing an acute rejection in kidney transplant recipients on the basis of free DNA circulating in the blood of the recipient that is released from the transplanted organ . The release of DNA from the donor organ also increases in heart transplant patients during rejection events . New technical developments such as the digital droplet PCR allow for a rapid and relatively inexpensive way of detecting and quantifying this circulating DNA from the graft . A relatively new publication shows that the measurement of cell-free DNA from the donor organ could also help control the immunosuppression .
Biomarkers for the development of tolerance
In order to identify patients who may be able to cope with weak immunosuppression, biomarkers are sought that indicate the development of tolerance. Regulatory T-cells (Tregs), which can prevent rejection of the new organ, have been known for years. The detection of such cells could serve as an indicator of tolerance. These cells can be identified by their surface markers (e.g., CD4/CD25/Foxp3) by means of flow cytometry. A better alternative is the PCR-based determination of the demethylated locus on the Foxp3 gene, also called TSDR (Treg-specific demethylated region) . In fact, the determination of the amount of such Tregs was used to examine the tolerance-inducing potential of various immunosuppressive protocols. While the mTOR inhibitors sirolimus and everolimus were able to be linked to the development of tolerance, calcineurin inhibitors led to a smaller number of Tregs . In a recent study, Bouvy et al. demonstrated the different effect of induction therapy with anti-thymocyte globulin (ATG) and the IL-2 receptor antibody basiliximab on the repopulation of Tregs, which was better under ATG .
A new direction in the study of the development of tolerance puts the focus on the role played by B-cells in this process . Through typical patterns of gene expression in B-cells, it was possible to detect tolerant patients [83, 84]. Biomarkers of tolerance, for example, are currently being studied by the RISET (Reprogramming the Immune System for Establishment of Tolerance) consortium, which is supported by the European Commission (www.risetfp6.org).
Specific pharmacodynamic biomarkers
Enzymes as a pharmacological target of immunosuppressive drugs
The calcineurin inhibitors cyclosporine and tacrolimus inhibit the enzyme calcineurin phosphatase in lymphocytes. Halloran et al.  have been able to show that the inhibition of the enzyme activity in lymphocytes is associated with the cyclosporine concentrations in whole blood. The greatest inhibition 1 and 2 h after dosing corresponded with the highest concentration in the blood. Although these experiments demonstrate the feasibility of such an approach, the method is technically very demanding and, in principle, not suitable for routine use . It has therefore not yet come into widespread use in clinical studies.
The enzyme protein 70S6 kinase (p70S6 kinase) is phosphorylated by the kinase mTOR. The decrease in phosphorylation of the enzyme by the inhibition of mTOR is an indicator of the effect of the mTOR inhibitors sirolimus and everolimus . A small study on kidney transplant patients has shown that there is no correlation between the phosphorylation of p70S6 kinase and the concentrations of sirolimus in the blood . A little-decreased phosphorylation was observed in the Western blot of five patients who experienced an acute rejection, while patients with a greatly weakened phospho-p70S6 kinase band did not experience rejection. The assay is time-consuming and requires a specialized laboratory, which is why routine use is doubtful. However, an ELISA has been developed for the detection of phospho-p70S6 kinase , but it has not been validated in clinical trials yet. An alternative method is the so-called phospho-flow technique, in which intracellular phosphorylated proteins are detected by means of a flow cytometer. Methods for the detection of the phosphorylated p70S6 kinase and of the phosphorylated ribosomal S6 protein (rS6P) have been published [90, 91]. Hoerning et al. have been able to demonstrate that phosphorylation of p70S6 kinase is reduced significantly in kidney transplant patients undergoing mTOR therapy compared to controls without treatment or under calcineurin inhibitor therapy. There was no correlation between the pharmacodynamic biomarkers and the whole blood levels of sirolimus or everolimus .
The enzyme inosine monophosphate dehydrogenase (IMPDH) is inhibited in mononuclear leukocytes by the immunosuppressant mycophenolic acid. In 79 hemodialysis patients, the activity of the enzyme exhibited a high degree of inter-individual variability before the kidney transplantation . Patients with activity above a certain threshold, for whom the mycophenolic acid dose was reduced after the transplantation, showed the highest rates of rejection. The determination of enzyme activity prior to the transplantation could therefore be used to individualize the dose after the transplantation. After the transplantation, the IMPDH activity was inversely associated with the mycophenolic acid concentration in plasma, which is why it was hypothesized that the combination of concentration measurements of the immunosuppressant in plasma and enzyme activity in leukocytes could be useful for controlling the treatment with mycophenolic acid . To date, however, there is no sufficient evidence from clinical studies to apply this approach to routine patient care. In contrast to a point measurement of IMPDH activity, enzyme activity was measured in a study during the first 4 h after administration of the drug, and the area under the concentration time curve (AUEC) was calculated . It was shown that in the first 2 weeks after the transplantation, the IMPDH inhibition was significantly lower in patients with rejection than in patients without rejection.
The relationship between genetic polymorphisms in type I and type II IMPDH and clinical events was examined in two large studies involving kidney transplant recipients . It was observed that a polymorphism in the IMPDH I gene was associated with a lower risk of acute rejection and leukopenia in the first year after the transplantation.
Interleukin-2 in CD8 T-cells
Interleukin-2 (IL-2) is essential for the activation of T-cells. As such, T-cells produce IL-2 themselves and stimulate themselves or paracrinally via the IL-2 receptor. The calcineurin inhibitors cyclosporine and tacrolimus inhibit the IL-2 effect and thus prevent the activation and proliferation of effector T-cells (CD8). The percentage of CD8 cells producing IL-2 was increased in patients with rejection after a liver transplant already before the transplantation , indicating activation of the immune system. These results were confirmed in two studies [64, 97]. The measurement of IL-2-producing T-cells is relatively easy and reproducible by means of flow cytometry, which is why this biomarker seems suitable for use in clinical trials with calcineurin inhibitors, which, however, have not been conducted yet.
Expression of NFAT-regulated genes
The inhibition of the enzyme calcineurin phosphatase through cyclosporine and tacrolimus results in the prevention of dephosphorylation of the so-called nuclear factor of activated T lymphocytes (NFAT). Dephosphorylated NFAT translocates into the nucleus and switches on certain genes, such as IL-2, INF-γ and GM-CSF, which are required for T-cell activation. The group around Zeier has studied intensively the expression of these genes in recent years as pharmacodynamic biomarkers of the calcineurin inhibitor effect . Using qPCR, the expression of the three genes is measured in stimulated leukocytes by means of the LightCycler instrument (Roche Diagnostics, Mannheim, Germany) before and 2 h or 1.5 h after taking cyclosporine or tacrolimus. There is a commercially available kit for this. The gene expression was inversely associated with the cyclosporine or tacrolimus concentration in the blood. A very low expression of the three genes (<15%) was observed in patients with recurrent infections and tumors while receiving cyclosporine and interpreted as a sign of over-immunosuppression . There was no association between clinical events and the concentrations of the calcineurin inhibitor in the blood. Dose reduction was accompanied by an increase in the gene expression, and as long as a cut-off of 30% was not exceeded, there were no rejection responses . These results were reproducible in other studies involving stable pediatric patients and patients following liver or heart transplants [101–104]. Reports in the literature on treatment with tacrolimus have been less convincing [105, 106].
The control of immunosuppressive therapy via TDM has made the treatment safer and above all has helped avoid toxic side effects. However, it has also been found that some patients respond very differently to therapy and that the achievements in the early phase after the transplantation are clouded by modest long-term results. Individualization of therapy seems sensible. As an addition to TDM, the determination of biomarkers in the blood or other body fluids could represent an approach that better reflects the individual responses of the immune system to the treatment . Although pharmacodynamic biomarkers have been developed that can capture, directly and specifically, the pharmacological effect on a target molecule or, more generally and non-specifically, the effect on the immune system, these have not really been validated in larger prospective clinical trials and have rarely entered clinical routine. A major drawback of most biomarkers is the complicated and time-consuming analysis. Many in-house procedures are also not well validated analytically and certainly not standardized, making it difficult to compare results from different laboratories and studies. The greatest potential for clinical application is found in assays that are commercially available as kits or which are performed in central laboratories, such as the ImmuKnow® test, the Pelximmune™ test, the NFAT-regulated gene expression, the IFN-γ ELISPOT or the determination of sCD30 with commercially available ELISA tests and the measurement of DSA on the basis of the Luminex technology. The determination of the DNA of the donor organ in the blood of the recipient by way of the digital droplet-PCR analysis is also promising in analytical terms. However, it seems unlikely that a biomarker has the necessary diagnostic sensitivity and specificity for all test situations. Rather, it is to be expected that a combination of analytically robust and clinically validated biomarkers could be a complementary diagnostic tool for the diagnosis of complications, therapy management and prognosis for recipients of solid organs. At this point, it is impossible to say which combinations are useful, but having a group of markers for immune activation (T-cells and B-cells), development of tolerance and organ damage seems to be the right approach.
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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Original German online version at: http://www.degruyter.com/view/j/labm.2014.38.issue-6/labmed-2014-0043/labmed-2014-0043.xml?format=INT. The German article was translated by Compuscript Ltd. and authorized by the authors.