The use of nonspecific immunosuppressive drugs has significantly reduced the incidence of acute kidney graft rejection (Sayegh & Carpenter, 2004). This led to a significant improvement in first-year graft survival rates that are “almost close to perfect”, as mentioned in a recent review (Lamb et al., 2011). However, the benefits of such immunosuppressive therapies on chronic rejection and overall long-term graft survival are uncertain (Meier-Kriescher et al., 2004a; McDonald et al., 2007). Thus, long term graft survival remains unchanged over decades (Meier-Kriescher et al., 2004a; Meier-Kriescher et al., 2004b). Persistent excessive immunosuppression −related to these immunosuppressive drugs− exposes renal transplant recipients to long-term toxicities including: increased incidence of cancers, severe infectious complications and “metabolic” diseases (for instance, diabetes, and accelerated atherosclerosis leading to cardiovascular diseases).
An excess risk of cancer after renal transplantation has been increasingly recognized over recent decades (Penn et al., 1979; Kasiske et al., 2004; Grulich et al., 2007; Villeneuve et al., 2007; Webster et al., 2007; van Leeuwen et al., 2009), as advances in medicine have extended the life of renal transplant recipients. A meta-analysis including five studies of cancer risks in organ transplant recipients, including 31’977 organ transplant recipients −among which 97% have received a kidney graft− from Denmark, Finland, Sweden, Australia, and Canada shows an increase in the incidence of cancers related to infection with Epstein-Barr virus (EBV), human herpesvirus 8 (HHV8), hepatitis viruses B and C (HBV and HCV), and
The other main cause of patient’s death with functional graft is cardiovascular diseases (Kahwaji et al., 2011) related to accelerated atherosclerosis associated with kidney transplantation (Sarnak et al., 2003; Ojo, 2006). The incidence of cardiovascular diseases is at least 3 to 5 times higher than in the general population (Sarnak et al., 2003). For instance, while left ventricular hypertrophy prevalence in the general population is estimated to 20%, this prevalence increases to 50 to 70% in renal transplant recipients (Sarnak et al., 2003). Depending on the considered reports, cardiovascular disease is reported to be the most common cause of death in patients with functional graft ranging from 24% to 55% (Kasiske et al., 1996; Ojo et al., 2000; Sarnak et al., 2003; Kahwaji et al., 2011). Risk factors for cardiovascular diseases in renal transplant recipients are multiple. They include traditional cardiovascular disease risk factors (e.g., tobacco use, exercise, hypertension, diabetes, or hyperlipidemia), which are highly prevalent, as well as nontraditional risk factors related to a long history of poor kidney function (e.g., hyperhomocysteinemia, chronic inflammation or anemia) (Kasiske et al., 1996; Ducloux et al., 2000; Sarnak et al., 2003; Liefeldt & Budde, 2010; Kahwaji et al., 2011). Moreover, factors related to transplantation itself, including the direct effects of immunosuppression or rejection episodes as well as new-onset diabetes after transplant (NODAT), impact on cardiovascular disease occurrence after kidney transplantation (Kasiske et al., 1996; Sarnak et al., 2003; Ducloux et al., 2005a; Liefeldt & Budde, 2010; Kahwaji et al., 2011).
Thus, it appears that excessive immunosuppression is involved in both increased cancer and cardiovascular disease incidence observed after kidney transplantation. A greater understanding of risk factors leading to excessive immunosuppression may help physicians to determine high-risk recipient profiles and optimize pre- and post-transplantation treatment strategies. In other words, identification of biomarkers predictive of immunosuppression-associated complications may improve late kidney transplantation outcome. In this chapter, we will report efforts of our laboratory to identify immunological factors that can predict the two main complications associated with kidney transplantation, namely cancer and accelerated atherosclerosis that leads to cardiovascular diseases. We focus on: i) CD4+ T cell lymphopenia, a consequence of anti-thymocyte globulin (ATG) administration and ii) recipient innate immune genetic factors appreciated by single nucleotide polymorphism (SNP) analysis. The analysis of these biomarkers was considered only in the settings of transplantation from deceased donors in a Caucasian population. Identification of biomarkers predicting chronic allograft dysfunction is beyond the scope of this review, despite significant advances reported recently in this field (please see a recent commentary in the
2. CD4+ T cell lymphopenia as a biomarker for immunosuppression-associated complications
CD4+ T cell lymphopenia in renal transplant recipients results mainly from ATG administration. Despite a limited treatment duration (until 4 days), CD4+ T cell lymphopenia persists for several years in some transplanted patients (Muller et al., 1997; Louis et al., 2007). We previously demonstrated that persistent CD4+ T cell lymphopenia is correlated with enhanced risks of cancers −including skin cancers (Ducloux et al., 1998a), monoclonal gammapathies (Ducloux et al., 1999), lymphomas as well as other non skin cancers such as colon or lung cancers (Ducloux et al., 2002a)−, of opportunistic infections (Ducloux et al., 1998b) and atherosclerotic events (Ducloux et al., 2003) in renal transplant recipients. In contrast, CD4+ T cell lymphopenia seems not to be associated with
2.1. A role for an altered immune reconstitution on persistent CD4+ T cell lymphopenia after anti-thymocyte globulin administration?
We will describe below factors identified as participating to CD4+ T cell reconstitution (
2.1.1. The role of the thymic activity on immune reconstitution after T cell depletion
Diseases (e.g., human immunodeficiency virus [HIV] infection), but also treatments (total body irradiation, high dose anti-cancer chemotherapy or depleting antibodies) may be responsible for profound lymphopenia. Studying immune reconstitution after hematopoietic cell transplantation or following anti-HIV therapy has caught the attention of many teams (see below). Understanding factors involved in accelerated −or in contrast delayed− immune reconstitution may limit side effects of these therapies. Based on studies performed in animal models, Mackall and colleagues proposed several years ago that immune reconstitution after T cell depletion arises from two main pathways: thymopoiesis and homeostatic proliferation expansion of residual host lymphocytes or, in the context of hematopoietic cell transplantation, of graft-derived mature T cells (Fig. 1; Mackall et al., 1997). The latter pathway remains the major pathway early after hematopoietic cell transplantation, until donor-derived prothymocytes migrate to the recipient thymus, where they undergo maturation (Moss & Rickinson, 2005). Several evidences in human settings support today the hypothesis sustained by Mackall et al. (1997) due to the development of innovative tools allowing discrimination of recent thymic emigrants (RTE, a reflect of thymic activity/output) from other lymphopenia-induced expanded T cells (
A critical issue during T cell recovery is the reconstitution of a most diverse polyclonal T cell repertoire. While thymopoiesis generates “new” T cells with a polyclonal TCR repertoire, homeostatic proliferation expansion results in a very limited TCR repertoire diversity (Fig. 1). Thus, patients exhibiting impaired immune reconstitution due to altered thymic function are less equipped to respond to pathogens or even to control tumors than patients presenting an efficient T cell reconstitution with a fully diverse TCR repertoire (for a review, Williams et al., 2007).
A last concern is that the thymus involutes with age and injury, but keeps its capacity for renewal. This is well illustrated in clinical settings associated with T cell recovery (Dion et al., 2004) where the thymus expands and may become greater than the normal size with intense cellular density, as attested by computerized tomography (Williams et al., 2007). Radiographic measurement of thymus by computer tomographs correlates with circulating TREC levels (Harris et al., 2005). However, thymus renewal capacity declines with age (for a review, Williams et al., 2007). In consequence, circulating TREC levels are inversely correlated with age (Gruver et al., 2007). Over the age of 45-50, thymic activity/output is reduced and naive T cell recovery may take until 5 years after severe iatrogenic lymphopenia (Williams et al., 2007).
Few data are available to date concerning the human thymic function and CD4+ T cell recovery after kidney transplantation. Nickel et al. (2005) reported stable frequencies of RTE –assessed by CD31, CD45RA CD4 phenotype− 6 months after transplantation. These authors concluded that uremia due to past history of chronic renal dysfunction has no impact on thymic activity (Nickel et al., 2005). However, only 7 patients among 48 received depleting induction therapy (Nickel et al., 2005). This renders difficult to interpret the effects of thymic activity in the context of lymphopenia. In contrast, Scarsi et al. (2010) reported a massive reduction of RTE one year post-transplantation after Campath-1H administration. This supports that naive CD4+ T cells −including RTE− may be highly sensitive to ATG (Louis et al., 2007; Gurkan et al., 2010)(see also below, paragraph §2.1.2) and that time is necessary for RTE “replenishment” after T cell depletion. The role of the thymic function at the time of kidney transplantation was not assessed in human. Several years ago, Monaco
2.1.2. The role of homeostatic proliferation expansion and homeostatic cytokines on immune reconstitution after T cell depletion
The second pathway of immune reconstitution after induction therapy-induced lymphopenia is the homeostatic proliferation of residual T cells. This process, also called lymphopenia-induced proliferation, has been extensively studied in mice (for a review, Boyman et al., 2009). In murine models, this homeostatic proliferation expansion requires homeostatic cytokines (e.g., IL-7) and sometimes cognate antigen-driven interactions (Fig.1; Boyman et al., 2009). Several features with clinical consequences for lymphopenic patients are associated with homeostatic proliferation expansion: a limited TCR repertoire diversity, a shift from naive to memory/activated phenotype in the proliferating cells, a competition for limiting levels of homeostatic cytokines (increasing TCR repertoire skewing, hence decreasing the capacity of the host to respond to antigen challenge), a more delayed T cell recovery (Williams et al., 2007), a possibility to lose transplantation tolerance (Wu et al., 2004) or to favor autoimmunity by expanding autoreactive memory T cells (Monti et al., 2008).
Homeostatic proliferation expansion is the first pathway to be triggered when peripheral T cells decline acutely. This decrease in circulating T cell counts reduces IL-7 consumption, hence leads to enhanced levels of IL-7. This cytokine is then available for residual T cell expansion. High serum levels of IL-7 were found in allografted patients with severe lymphopenia after treatment depletion (Bolotin et al., 1999). However, IL-7 levels decrease rapidly with lymphocyte recovery (Bolotin et al., 1999). Interleukin-7 can be considered as a true regulator of the naive T cell pool size, driving homeostatic proliferation of CD4+ CD31+ RTE with sustained CD31 expression (Azevedo et al., 2009). Memory CD4+ T cells express high levels of CD127 (Boyman et al., 2009), then compete with RTE for IL-7. Dependency on IL-15 or IL-21 for homeostatic proliferation expansion is less marked for CD4+ T cells than CD8+ T cells. Thus, IL-7 levels after lymphopenia are a critical factor to be considered after depletion therapy. Cox
2.2. CD4+ T cell subsets, sensitivity to anti-thymocyte globulin administration and immunosuppression-associated complications: the example of accelerated atherosclerosis
Anti-thymocyte globulins are a complex mixture of antibodies with multiple specificities directed against different molecules expressed by T cells, but also non T cells (Bonnefoy-Berard et al., 1991; Rebellato et al., 1994). Several authors believe that ATG exerts its effects through depletion as well as depletion-independent mechanisms. It has been reported that the different CD4+ T cell subsets were not equally sensitive to ATG-induced depletion. Initial works in mice showed that regulatory T cells (Treg) –playing a key role in the control and maintenance of tolerance (Fig.2)− were spared by anti-lymphocyte serum (ALS) (Minamimura et al., 2006), by a mechanism dependent of OX40 signaling pathway present in Treg with a memory phenotype (Kroemer et al., 2007). Moreover, in
A thoroughly study in non human primates reported that ATG treatment induced a dose-dependent T cell depletion in the peripheral blood, the spleen and in the lymph nodes. T cell apoptosis in secondary lymphoid organs was identified as the main depletion mechanism (Preville et al., 2001). This supports that lymphocyte depletion is the major mechanism by which ATG preparation exerts its immunosuppressive effect. Another study in mice
reported that all CD4+ T cell subsets are equally sensitive to mouse ATG, but that naive T cells expand very quickly after homeostatic proliferation with the acquisition of a memory phenotype (Sener et al., 2009). This may explain why initial studies reported that memory phenotype T cells are more resistant to ATG-induced death (see above). The hypothesis of a different susceptibility to ATG-induced death or an imbalance in CD4+ T cell subset reconstitution is tantalizing to explain the relationship between CD4+ T cell lymphopenia and accelerated atherosclerosis after kidney transplantation. Depending on the cytokine microenvironment in which naive CD4+ T cells are primed, different effector CD4+ T helper cell (Th) subsets have been described (Fig.3). Whether ATG or immune recovery following ATG-induced lymphopenia may differently affect CD4+ Th subsets remains to be determined in renal transplant recipients. A study in renal transplant recipients suggested that Th2 subsets were less sensitive than Th1 subsets to ATG treatment (Weimer et al., 2005). However, other Th subsets −such as Th17 (Betteli et al., 2006; Mangan et al., 2006), or the putative Th9 (Dardahlon et al., 2008; Veldhoen et al., 2008) or Th22 (Duhen et al., 2009; Trifari et al., 2009) subsets (Fig.3)− have not been explored. Experimental mouse models of atherosclerosis using atherosclerosis prone apolipoprotein-E deficient or low density lipoprotein (LDL) receptor deficient mice permitted to distinguish pro-atherogenic from anti-atherogenic CD4+ T cell subsets (for recent reviews, Taleb et al., 2010a; Hansson & Hermansson, 2011; Fig.3). One may hypothesize that ATG-induced CD4+ T cell lymphopenia may favor a preferential expansion of pro-atherogenic Th1 cells in detriment of anti-atherogenic Treg (
As mentioned above, atherosclerosis is a chronic immune-mediated disease implicating both adaptive and innate immunity (Yan & Hansson, 2007; Woollard & Geismann, 2010; Hansson & Hermansson, 2011). In this review, we focused on CD4+ T cells to explain the increased incidence of atherosclerotic events associated with ATG administration. However, accelerated atherosclerosis is not only associated with induction therapy responsible for persistent CD4+ T cell lymphopenia, since cardiovascular diseases are observed in most renal transplant recipients (please see paragraph §1). This is why we also explore innate immune genetic factors to explain accelerated atherosclerosis. Most of the widely used immunosuppressive drugs target T cells and sometimes B cells (Halloran, 2004), while innate cells or factors are not always affected. For instance, calcineurin inhibitors may prevent Treg suppressive functions (Zeiser et al., 2006; Bonnefoy et al., 2008). Thus, regulatory functions are certainly unpaired in renal transplant recipients receiving calcineurin inhibitors. It was shown in mice that Treg dysfunction or blockade increases innate cell activation through Toll-like receptor (TLR) ligands (De Wilde et al., 2008). If this occurs in atherosclerotic plaques, this will favor atherosclerosis progression (Fig.4).
3. Recipient innate immune genetic factors as biomarkers of immunosuppression-associated complications
Inflammation plays a major role in atherosclerosis processes (Hansson, 2005). The atherosclerotic lesions contain large numbers of immune cells, particularly macrophages (Stary et al., 1995) and CD4+ T cells (Zhou et al., 2001.). Macrophages are present in atherosclerotic lesions at early stages and play a critical role in lipid accumulation as well as in the plaque rupture (Hansson, 2005). The plaque rupture is responsible in coronary thrombosis (Hansson, 2005). Furthermore, atherosclerosis is associated with systemic immune responses and signs of inflammation. Histopathological and clinical investigations point at inflammatory activation of atherosclerotic plaques as a cause of acute coronary syndromes (Hansson, 2005), and sero-epidemiological studies have suggested links between atherosclerosis and microbial infections (Morre et al., 2000; Neumann et al., 2000). Moreover, several epidemiological studies and therapeutic trials have underlined the importance of inflammation in cardiovascular clinical end-points (Ridker, 2001; Ridker et al., 2009). The relevance of inflammation in atherosclerotic complications in humans is well illustrated by the JUPITER trial showing that achieving CRP levels under 2 mg/L with Rosusvastatin is associated with a 31% decrease in major cardiovascular events (Ridker et al., 2009).
In order to explore how inflammation and cells from the innate immunity may influence the complications associated with kidney transplantation, the analysis of different SNPs was performed in our laboratory. We followed the recommendations provided by Nature Genetics (1999) that are: a plausible/expected link between the analyzed protein or its gene promoter and the pathology, a functional characterization of the SNP, and a validation in independent cohorts. Moreover, the size of patient cohort has to be adapted to the frequency of mutated SNP. The following SNPs were analyzed in the settings of kidney transplantation outcome: TLR-4 (Ducloux et al., 2005b) and NOD2/CARD15 (Courivaud et al., 2006), as these two pattern recognition receptors (PRR) are involved in the recognition of pathogens and in the initiation of inflammatory immune responses (Fig.4). Sero-epidemiological studies have suggested a link between atherosclerosis and microbial infections (Morre et al., 2000; Neumann et al., 2000) and the atherosclerotic lesions contain large numbers of immune cells, particularly macrophages and dendritic cells (Woollard &Geissmann, 2010
; Fig.4). Thus, we hypothesized that an enhanced inflammatory response (including inflammatory cytokines, such as IL-6 or TNF-α) in the intima of the artery walls due to a “hyper-responsive” PRR (
Overall, the aim of this review is to report our experience on the identification of biomarkers (CD4+ T cell lymphopenia after ATG, TREC levels at the time of transplantation, and innate immune genetic factors) predicting transplantation-related complications (mainly atherosclerosis and cancer occurrence), and to propose to the use these biomarkers in patient follow up and/or in immunosuppressive strategy design. Furthermore, we propose other “tracks” to improve the clinical relevance of these biomarkers as well as to understand their implications in the occurrence of immunosuppression-associated complications. The efficacy of these identified biomarkers should be tested and validated in prospective clinical trials in order to select the most appropriate immunosuppressive strategy. In the future, one could imagine that these biomarkers may help physicians to manage risks of cancers and cardiovascular diseases in renal transplant recipients. The management of these risks is of great interest as attested by recent reviews (Webster et al., 2008; Wang & Kasiske, 2010).
We would like to thank Sarah Odrion for excellent editorial assistance and the Centre d’Investigation Clinique intégré en Biothérapies du CHU de Besançon (CBT-506) for its support. Our work in the fields was supported by grants from the Programme Hospitalier de Recherche Clinique 2011 (to D.D), the Fondation de France (Appel d’offre “Maladies cardiovasculaires” 2007, #2007 001859, to P.S.), the DHOS/INSERM/INCa (Appeld’offre Recherche Translationnelle 2008, to D.D. and P.S.), and the APICHU 2010 (SIGAL project to J.B.).