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Prognostic assays for rejection and tolerance in organ transplantation
Transplant Immunology 14 (2005) 193 – 201 www.elsevier.com/locate/trim
Prognostic assays for rejection and tolerance in organ transplantation Benjamin A. BradleyT The East Barn, The Pound, Lower Almondsbury, Bristol BS32 4EF, England, United Kingdom Accepted 14 March 2005
Abstract In this review, I have summarised our understanding of acute rejection of organ transplants, and for convenience I have identified three processes, recognition, rejection and regulation. In stark contrast to this text-book picture of acute rejection, I have drawn attention to some of the clinical realities, where processes are altered by powerful immunosuppressive drugs, and where many transplant recipients are presensitised to transplantation antigens prior to engraftment. The ultimate goal is to encourage the emergence of a utopian immunological state, wherein patients tolerate organ transplants for life after being weaned from all immunosuppressive drugs. Assays that may be used in the future to reliably monitor this process are still at a very exciting stage of development. D 2005 Elsevier B.V. All rights reserved. Keywords: Organ transplantation; Acute rejection; Tolerance; Regulation; Immunosuppression; Monitoring
1. Introduction Acute rejection is defined here as a response by the recipient’s adaptive immune system to non-self HLA and non-HLA molecules on donor tissue. In the clinical context acute rejection occurs more frequently and is more severe during the early post transplant period. It wanes, but does not completely resolve 6 to 12 months after the transplant. Organ transplant recipients usually receive continuous immunosuppressive therapy for life in an attempt to prevent ongoing rejection and to preserve the function of the graft, but newer protocols attempt to wean recipients off these drugs in the hope that durable tolerance will be established [1]. As a result recipients are immunosuppressed and may suffer from recurrent infection and a high probability of malignancy. Episodes of acute rejection remain frequent in organ graft recipients, implying an overall lack of efficacy of clinical immunosuppression. For some years investigators have worked on developing laboratory tests to predict and monitor rejection risk in the clinical setting. The aim is firstly to prevent rejection altogether, and secondly to identify recipients in whom immunosuppressive therapy T Tel.: +44 1454 201077 or +44 7803165327. E-mail address: [email protected]. 0966-3274/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2005.03.022
can be reduced to low non-toxic doses, or discontinued because immunological tolerance has occurred. Unfortunately, the mechanisms involved in the development of peripheral tolerance involve active processes that differ only subtly from acute rejection, but are most likely to be equally susceptible immunosuppressive drug therapy. To date, none of the laboratory tests have performed well in the clinical setting, so more attention should be given to developing tests that identify the initial phases of both acute rejection and tolerance induction.
2. Acute rejection and tolerance In this review the acute rejection response has been divided into three sequential processes: recognition, during which the recipient’s immune system first becomes aware of foreign antigens in the graft; rejection, during which the recipient’s anti-donor T- and B-cell clones expand and attack the graft; and regulation, during which the recipient’s regulatory immune system dampens down the anti-donor Tcell responses. Regulation is the essential first step towards establishing durable transplantation tolerance. These three processes are transient and overlapping events in an evolving reaction and are briefly describe below.
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2.1. Recognition When an allogeneic organ is transplanted, the arterial and venous circulation, but not lymphatic system, is reestablished, whereupon resting dendritic-cells (DC) of donor origin, resident in the transplanted tissue, migrate to the recipient’s spleen [2] (Fig. 1a). The HLA peptides,
expressed naturally on the now-activated donor DC’s surface offer a highly potent mitogenic stimulus to circulating recipient T-cells in transit through the spleen. Recipient T-cells expressing complimentary T-cell receptors (TCR) to non-self donor peptides engage the DC and become activated. Within a few days the supply of donor DC wanes and their function is taken over by the recipient’s DC. These
a)
Donor Kidney
Resting Recipient DC
Activated Recipient DC + HLA Peptide
Activated Donor DC + HLA Peptide
CD4 T-cells
CD8 T-cells
Recipient Splenic Lymphoid Tissue
b) Resting Recipient DC
B Cells Anti inflammatory Cytokines
Donor Kidney
Activated Recipient DC + HLA Peptide
CD4 T-cells
CD8 T-cells
CD4 T-cells
Pro -inflammatory Cytokines
CD8 T-cells
Recipient Splenic Lymphoid Tissue
Fig. 1. Illustrates the three sequential processes that are envisaged to occur in a kidney transplant; see text for description. (a) Recognition process. (b) Rejection process. (c) Regulation process.
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c) Resting Recipient DC
Regulatory DC
Donor Kidney
Plasma cells secreting anti-donor HLA antibodies
Activated Recipient DC + HLA Peptide
CD4 T-cells
Anti inflammatory Cytokines
CD8 T-cells
Regulatory T-cells Anti inflammatory Cytokines
Memory CD4 Memory CD8
Recipient Splenic Lymphoid Tissue
Fig. 1 (continued).
migrate from the recipient’s arterial circulation into donor tissue, take up donor HLA molecules and digest them into peptides. Peptide-loaded recipient DC passes via the circulation to spleen and lymph nodes. They enter the para-cortical areas, where they engage an extended range of T-cell clones with TCR directed to donor HLA-peptides. Surveillance of the donor’s tissue by the recipient’s DC continues for as long as the circulation to the transplant remains intact. Eventually, the immune system adapts to the continuing presence of foreign antigen, and the predominant functional activity of the recipient’s DC changes from an activation to a regulatory function. In addition to cell-bound donor material, soluble donor HLA molecules, shed by transplanted tissue, pass to the germinal centres in spleen and lymph nodes where they interact with B-cells. A tiny fraction of B-cells binds to these HLA molecules by virtue of specific B-cell receptors (BCR) and differentiate into plasma cells. The BCR precedes and mirrors specificity of antibody subsequently synthesised by the plasma-cells. The BCR-bound donor HLA is internalised, partially digested into peptides, and the peptides re-assembled with newly synthesised recipient HLA Class II molecules which it presents to T-cells. The close anatomical proximity of the B-cells in the cortical areas to the T-cells in the para-cortical areas of lymph nodes and spleen allows interaction of these B-cells with CD4 positive T-helper cells specific for the same peptide. This interaction between CD4 T-cells and B-cells leads to the release of cytokines (e.g. IL-10) by the T-cell, resulting in Bcell activation, differentiation, clonal-expansion; and then differentiation into anti-HLA antibody producing plasma cells.
2.2. Rejection Upon entering the para-cortical areas in spleen and lymph nodes, activated DC are confronted with the entire array of T-cells directed to non-self peptides (Fig. 1b). The T-cells that carry a TCR that fits the peptide bound to the DC undergo activation, differentiation and clonal expansion. This process is driven by cytokines, such as IL-12 secreted by the DC, and autocrines, such as IL-2 secreted by T-cells. The T-cells may respond in a naı¨ve or an accelerated manner depending on previous experience of the peptide. If, prior to this moment, a T-cell has never engaged its specific peptide, it will respond to the DC in an immature manner. If, as frequently occurs in clinical practice, T-cells have experienced the peptide through pregnancy, blood transfusions or former transplants, they will respond in a more committed and accelerated manner. Unlike B-cells, the clonal progeny resulting from T-cell activation retain the unique specificity of their TCR for a particular peptide, in perpetuity. The expression of co-receptors, identified as cluster differentiation (CD) molecules, CD4 and CD8, is determined during the early stages of T-cell maturation in the thymus. One sub-set of naı¨ve T-cells expresses the CD4, which binds exclusively to HLA Class II molecules; hence, it is only activated by peptide presented on the HLA-Class II molecules of DC. The other sub-set of naı¨ve T-cells expresses CD8, which binds exclusively to HLA Class I molecules; hence, it is only activated by peptide presented on the HLA-Class I molecules of DC. Since peptides presented by HLA Class I or II, differ in their origin specificity and size, these two sub-sets differ in their range of TCR peptide-binding specificities.
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The strength of the signal delivered by the DC via the TCR to the T-cell is determined by the duration of cell –cell contact. The weakest (shortest) signal results in the development of a memory T-cell. The strongest (longest) signal results in apoptotic or dead T-cells. An intermediate signal allows migratory T-cells to home to inflamed tissue or recently transplanted organs, the migration pathway being determined by the differential expression of chemokine receptors (e.g. CCR7). During the interaction between CD4 T-cells and B-cells within lymphoid follicles, an anti-inflammatory cytokine environment facilitates the process of somatic hypermutation of the BCR genes. Somatic hyper-mutation, driven by soluble donor HLA antigen, yields clonal progeny of increasingly high avidity. Eventually, these highly avid mature B-cells differentiate into plasma cells that secrete specific antibody directed to unique molecular shapes (epitopes) on the donor’s HLA antigens. Mature migratory T-cells, B-cells and plasma cells, all pass from the circulation into inflamed tissue, in this case the recently transplanted organ. The process of extravasation, whereby circulating cells pass from capillary blood into the tissues, is facilitated by activation of the vascular endothelium: an inevitable consequence of ischaemic damage in transplanted organs. Endothelial activation is further enhanced by humoral factors released in brain-dead heart-beating cadaveric donors. This phenomenon accounts for the difference in graft survival observed between transplants derived from cadaveric donors and living donors. Graft injury, resulting from entry of mature T- and B-cells into the donor tissue, is poorly understood, but at least three mechanisms have been implicated, delayed-type hypersensitivity, T-cell cytotoxicity, and anti-HLA antibodies, especially those that activate the complement cascade. 2.3. Regulation Much of the immune system is dedicated to downregulating activation processes by curtailing clonal expansion and restraining auto-aggression with a view to maintaining homeostasis (Fig. 1c). Knowledge of these immuno-regulatory mechanisms is incomplete, but four broad categories, that could be relevant in transplantation, can be described. Clonal-shrinkage through programmed cell death. Clonal shrinkage is the sequel to clonal expansion. It results from several processes including: the acquisition of cellsurface death-receptors, such as Fas-ligand; blocking responsiveness to IL-2; and the establishment of signaltransduction pathways that induce death by apoptosis, such as Caspase 3. Non-specific suppression of activation and differentiation, through the release of anti-inflammatory cytokines. Non-specific release of anti-inflammatory cytokines into the microenvironment is attributed to sub-sets of T-cells
(including CD4 + CD25 + subsets) and Natural Killer Tcells (NK T-cells). Thus, IL-4, IL-10 and TGF-h produced by these cells, suppress functional activity of aggressor Tcells and dampen down acute rejection and graft-versushost disease. Under normal circumstances, the main roles of NK T-cells would be to protect vital organs from the damaging effects of pro-inflammatory cytokines and escalating autoimmunity. Deletion or suppression of peptide-specific clones by Tregulator cells. T-regulator cells, which specifically suppress a particular immune response, are generated as a distinct population within the thymus. In concept, they are thought to undergo clonal expansion during evolution of the alloimmune response, and eventually target and kill the nonself HLA-peptide-specific T-cells responsible for acute rejection. The most attractive idea is that their TCR specificities are directed to peptides derived from the TCRs utilized by the effector T-cells involved in acute rejection. Thus, a negative feedback mechanism involving TCR from apoptotic T-cells digested into peptides by activated DC, then offered to T-regulators specific for the TCR peptides would eventually arrest the clonal expansion. The Tregulator cells undergo clonal expansion in the usual way and eventually suppress or destroy those particular aggressor T-cells that carry the specific TCR. Replicative senescence or clonal exhaustion (Hayflick phenomenon). Clonal-exhaustion or replicative-senescence is the end stage of many cycles of T-cell replication. With each mitosis, the telomeres responsible for the DNA repair after replication are shortened. Eventually, telomere shortening is such that repair of the genes vital for cell division is impossible and the cell cycle is arrested. At this point the cells remain viable, but cease to replicate, leaving holes in the T-cell repertoire, as evidenced by deletion of certain families of TCR-Vh genes. Such holes in the T-cell repertoire are manifestations of normal ageing, resulting from a lifetime of chronic antigenic stimulation with certain microorganisms. Insofar as an organ transplant is a source of chronic antigenic stimulation, a similar process could be envisaged leading to weakening of acute rejection with time.
3. Relevant aspects of clinical transplantation 3.1. Manifestations of acute rejection The clinical diagnosis of acute rejection is based on changes in function and changes in the histological appearance of transplant biopsy material. Biopsies taken during the early stages after renal transplantation show cellular infiltrates consisting of: T-cells expressing activation markers, FasL, granzyme, perforin, and chemokine receptors; activated macrophages; polyclonal B-cells and plasma cells; and, when anti-donor HLA antibody is present, neutrophils. In mild rejection, cellular infiltrates are confined to the interstitial tissue (70 – 85% of cases), but
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in more severe cases inflammatory vascular changes occur (25 –30% cases). These histological changes are graded according to an internationally agreed system called the Banff Scheme of Allograft Pathology, which delineates three types of active rejection: Type I Rejection, with tubulo-interstitial mononuclear cell infiltration and tubulitis; Type II Rejection, with moderate to severe intimal arteritis; and Type III Rejection, with transmural arteritis, fibrinoid change and necrosis of medial smooth muscle cells. Rejection due to anti-donor antibodies is categorised as a separate group in the Banff Scheme. The prognostic value of this scheme is that it correlates with early loss of function, steroid responsiveness and graft survival at 1 year. 3.2. Impact of immunosuppressive drugs. The effect of these drugs on acute rejection and tolerance depends on the type of drug given and its timing in relation to transplantation. Thus, induction therapy is given before, during and shortly after transplantation; initial drug therapy starts immediately after the operation and is tapered over the ensuing 6 months; maintenance therapy is given thereafter for the lifetime of the transplant; and anti-rejection therapy is given transiently to reverse episodes of acute rejection. The recent trend has been to investigate the effect of induction therapy prior to transplantation. A variety of agents have been used including anti-thymocyte globulin (ATG), which reacts with all re-circulating T-cells, especially memory T-cells; monoclonal antibody to the T-cell receptor (OKT3), which prevents T-cell activation by inhibiting co-stimulation; monoclonal antibody to the IL-2-receptor alpha-chain (IL-2Ra), which inhibits cytokine-driven clonal expansion of T-cells, NK-cells and NKT-cells; or a monoclonal antibody to CD52 (Campath1H), a glycoprotein antigen expressed by T-cells, B-cell, monocytes and certain sub-sets of activated DC. Campath1H is a lytic antibody that prevents T-cell activation during the recognition phase. Induction therapy can significantly reduce the incidence and severity of acute rejection, but may concurrently delay the emergence of regulator cells. Initial drug therapy begins immediately after re-establishing the blood supply to the transplant, but may also be started prior to this moment. Drug dosage is initially high to suppress acute rejection, but is tapered over the first 6 to 12 months to maintenance levels that are continued for the lifetime of the transplant. The most widely used drugs are inhibitors of purine synthesis such as azathiaprine and the more recently introduced micophenolate mofetil (MMF). Corticosteroids inhibit a wide range of cellular functions including the NF-nB signal transduction pathway and are used extensively for initial and maintenance therapy and in large doses for anti-rejection therapy. The calcineurin inhibitors such as cyclosporin and tacrolimus inhibit the NFAT signal transduction pathway, and rapamycin, which inhibits the expression of the IL-2 receptor. These drugs
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influence different intracellular pathways and can therefore be used in synergistic combinations to minimize toxicity and maximize therapeutic efficacy. Whereas, the purine inhibitors and corticosteroids tend to affect all components of the immune system, the calcineurin inhibitors and Rapamycin affect mainly T-cells.
4. Principles and attributes of prognostic assays The search for the ideal prognostic assay dates back to the 1960s when HLA typing, HLA-matching, serum crossmatching, and the one-way mixed lymphocyte reaction (MLR) were invented. For many years the MLR and assays derived from it, such as the cell mediated lympholysis (CML) assay, were explored as donor-specific in-vitro models for studying rejection, but they are time consuming and expensive and have failed to gain wide acceptance [3]. A wide range of alternative assays have been explored and advocated as a means of predicting and diagnosing acute rejection or tolerance. For the purposes of this review, these assays have been grouped with reference to the part of the rejection response for which the assay is most informative, namely: recognition, rejection and regulation. 4.1. Recognition The prognostic value of anti-HLA antibody detected in patients awaiting transplantation, and the exclusion of prospective donors who are cross-match positive as a result of these antibodies, is widely accepted. This topic has been discussed extensively elsewhere. Nonetheless, most methods of detecting antibodies to-date have relied on the capacity of those antibodies to fix complement. Noncomplement fixing antibodies detectable by ELISA techniques are additional indicators of impending acute rejection if directed to both the donor’s HLA Class I and Class II antigenic mismatches. The inclusion of ELISA testing for non-complement fixing antibodies is recommended to make antibody-detection techniques prior to transplantation more rigorous [4]. In contrast, the prognostic value of anti-donor HLA antibodies arising de-novo after transplantation seems to correlate with the onset of acute rejection in several types of transplant [5– 7]. In addition, the available evidence suggests that these and other antibodies may lead to chronic allograft vasculopathy by activating donor vascular endothelium [8,9]. However, the result of this interaction seems to depend on the molecule targeted. Whereas endothelial cells proliferate in response to anti-HLA Class I antibodies, they also evade damage by anti-donor HLA Class II antibodies. Other antibodies directed to vascular endothelial antigens of the donor promote apoptosis [10 –12]. Antibodies directed to the HLA-linked MICA antigens, which arise after transplantation, are directed to donor endothelial cells, and are also suspected of triggering vascular damage [13].
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The cytokine microenvironment within the lymphoid tissues determines the differentiation pathway adopted by activated T-cells. If pro-inflammatory cytokines (e.g. TNFa) predominate the risk of acute rejection may be higher; whereas, if anti-inflammatory cytokines predominate (e.g. IL-10), the risk may be reduced. Since the level of synthesis of many cytokines varies according to genetic polymorphisms in their promotor regions (up to seven-fold increase in the case of the TNF-a-308 SNP), these producergenotypes have prognostic value for acute rejection. A single nucleotide polymorphism (SNP) at the position 308 in the TNF-a promotor predicts a significant increase in risk of steroid resistant acute rejection episodes in recipients of renal and liver transplants [14,15]. Using meta-analysis to combine several underpowered studies, prognostic significance has also been attached to the IL-10 SNP at position 1082, in liver transplantation [16]. At present, this area of immunogenetics is handicapped by a lack of understanding of the functional haplotypes associated with these genes. Haplotypes consist of several SNPs, any one of which may, or may not, be associated with acute rejection or its suppression. Certain haplotypes are more frequent in some populations than others through the process of linkage disequilibrium that has arisen between widely separated SNP. An analysis of commonly occurring haplotypes gives more accurate prognostic information for transplant outcomes. Hence, in lung transplantation the IL-10 highproducer haplotype predicts a reduced risk of acute rejection [17]. Functional assays for certain cytokines conducted on peripheral blood mononuclear cells (PBMC) taken prior to transplantation may also be of prognostic value. Thus PBMC from prospective liver transplant recipients, cultured in-vitro with the mitogen lipopolysaccharide (LPS), were shown to synthesize higher levels TNF-a in those who developed acute rejection compared with those who developed no rejection; but, in contrast to the genetic evidence already cited above, no prognostic changes were found for IL-10 synthesis [18]. Since different mitogens activate different PBMC subsets, and use different signal transduction pathways, other studies in which staphylococcal A protein mitogen (SACI) was used, showed that IL-10 synthesis levels, when low, did indeed predict acute rejection [19]. Pre-operative serum levels of certain cytokine may of prognostic value. In prospective kidney transplant recipients a marked difference was observed in serum IFN-g levels in rejecting patients (1826 T 2424 pg/ml), compared to stable patients (316 T 373 pg/ml) [20]. Unfortunately, the standard deviation of these mean values indicates wide variations between patients, possibly attributable to differences in the turnover of these molecules in blood and the presence of inhibitors. Intriguingly, high plasma levels of a soluble form of a cell surface marker, sCD30, is an excellent prognostic indicator for acute rejection in kidney transplant recipients
[21,22]. This sCD30 is a little studied member of the TNFR receptor super-family shed by a subset of T-cells that have been activated by allogeneic DC. This subset has been identified as the predominant proliferating T-cell population in alloimmune responses, and is characterised by expression of CD30, CD25 and CD45RO. T-cells within this subset synthesise IFN-g and IL-5 [23]. High levels of sCD30 appear to increase the vulnerability of the graft to the effects of prior sensitisation, identified as elevated levels of panel reactive antibody, and to the effects of HLA Class I and Class II mismatching, such that the two assays in combination (sCD30 levels and PRA) give excellent prediction of kidney transplant outcome [24]. It is also fascinating to know that sCD30 levels decline significantly with increasing age; an observation in keeping with the declining incidence of acute rejection with increasing age [25,26]. Quantitative functional assays for measuring the frequency of donor-specific helper T-cell (HTLpf assay), are based on the principle of limiting dilutions analysis and the traditional MLR. In prospective kidney transplant recipients, the HTLpf assay appears to correlate with the risk of developing acute rejection after transplantation. However, the cytotoxic T-cell precursor frequency (CTLpf) failed to correlate with acute rejection [27]. Such assays may be of value in tailoring immunosuppressive drug therapy. 4.2. Rejection Prognostic indicators of early, ongoing and established acute rejection may be found in blood samples, graft biopsies and excretory products such as urine. Since the result of a particular test may indicate that immunosuppressive drug dosage be increased, it is important that changes attributed to acute rejection can be differentiated from those brought about by infection. Post-operative blood levels of TNF-a and IL-6 are increased in acute rejection of kidney transplants [28]. But these changes are also observed in cytomegalovirus infections, and urinary tract infections in male transplant recipients [29,30]. IFN-g induces HLA expression in graft and other tissues, and although it appears to have a protective role against acute rejection in certain murine models [31], the human story is different. Changes in serum levels are uninformative, but increased mRNA expression is found both in the CD4+ T-cells in blood, and in-situ, in biopsies from rejecting cardiac transplants [32,33]. Furthermore, certain IFN-g inducible factors, (IP10, I-TAC, and Mig) are elevated in biopsies of acutely rejecting cardiac grafts [34]. Increased gene expression of IL-18 and perforin in the blood, measured by real-time PCR, closely correlates with acute rejection in renal transplants [35]. In liver transplants, acute rejection is accompanied by elevated levels of the soluble form of the IL-2 receptor (sIL-2R) [36].
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Peripheral blood T-cell subsets show increased expression of activation markers during acute rejection of kidney transplants, as exemplified by increased expression of CD40 in CD4+T-cells, and CD69 in CD8+T-cells [37,38]. Since the increased CD40 expression is mirrored by increased CD40 ligand (CD40L) gene expression in biopsy material from rejecting renal transplants, the net result of their interaction may be to amplify production of anti-donor HLA antibodies by B-cells within the graft [39]. Within the graft, evidence of damage by anti-donor HLA and other antibodies is in the form of deposits of the complement breakdown product, C4d [40 – 42]. Immune cells expressing chemokine ligands (e.g. CXCR3) are attracted into rejecting graft tissues that express chemokines. Thus biopsies from rejecting kidney, cardiac and lung transplants show increased expression of chemokine receptors and their ligands [34,43 – 49]. Since these and other molecules (e.g. IP-10) are detectable in the urine, acute rejection can also be diagnosed non-invasively [50,51]. 4.3. Regulation Detection of a state of transplant tolerance begs the question as to the multiple mechanisms involved. Data from murine models suggests that it is an active process involving IL-2 signalling, particularly of CD4+CD25+ T-regulator cells [52,53]. At present, there are no foolproof assays, which have been fully tested in drug weaning studies, although this is the ultimate goal [54]. Nonetheless, several correlates of stable rejection-free transplant function exist, and many of these will prove valuable as clinical indicators for the withdrawal of immunosuppressive therapy in future. In brief, these assays involve measuring: soluble serum factors (e.g. HLA-G levels) [55]; anti-inflammatory cytokine profiles generated in donor specific MLRs [56]; ratios of monocytoid to plasmacytoid DC in recipient’s blood [57]; hypo-responsiveness and reduced frequencies of donor reactive helper T-cells and cytotoxic T-cells [58 – 63]; reduced levels of mRNA for IL-2 and IFN-g in peripheral blood [64]; reduced expression of CD40L on activated Tcells[65]; increased percentages of CD25+CD4+T-regulator cells [66,67]; increased levels of donor specific CD8+CD28-CD27+T-suppressor cells in peripheral blood [68 –70]; and graft infiltrates of CD4+T-cells secreting TGF-h [71].
5. Summary Over the past 10 years much progress has been made in understanding the cellular and molecular basis of allogeneic organ graft rejection. There have been parallel advances in the efficiency of therapeutic immunosuppression of organ transplant patients with a remarkable reduction the severity and frequency of rejection episodes. Some innovative and
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encouraging research techniques have been described above; however, there is still a need for simple, costeffective tests routinely run by clinical laboratories in hospitals to monitor organ transplant patients. The intensity of immunosuppressive regimens now in routine clinical use has lead to a narrow therapeutic window between effective immunosuppression and toxicity. Better monitoring will not only further reduce the probability of rejection, but will also decrease morbidity and mortality due to powerful immunosuppressive drugs, eventually leading to the ultimate goal of drug free graft tolerance.
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[15] Sankaran D, Asderakis A, Ashraf S, Roberts IS, Short CD, Dyer PA, et al. Cytokine gene polymorphisms predict acute graft rejection following renal transplantation. Kidney Int 1999;56(1):281 – 8. [16] Warle MC, Metselaar HJ, Hop WC, Tilanus HW. Cytokine gene polymorphisms and acute liver graft rejection: a meta-analysis. Liver Transpl 2005;11(1):19 – 26. [17] Zheng HX, Burckart GJ, McCurry K, Webber S, Ristich J, Iacono A, et al. Interleukin-10 production genotype protects against acute persistent rejection after lung transplantation. J Heart Lung Transplant 2004;23(5):541 – 6. [18] Bathgate AJ, Lee P, Hayes PC, Simpson KJ. Pretransplantation tumor necrosis factor-alpha production predicts acute rejection after liver transplantation. Liver Transpl 2000;6(6):721 – 7. [19] Weimer R, Zipperle S, Daniel V, Carl S, Staehler G, Opelz G. Superior 3-year kidney graft function in patients with impaired pretransplant Th2 responses. Transpl Int 1998;11(Suppl. 1):S350 – 6. [20] Sadeghi M, Daniel V, Weimer R, Wiesel M, Hergesell O, Opelz G. Pre-transplant Th1 and post-transplant Th2 cytokine patterns are associated with early acute rejection in renal transplant recipients. Clin Transplant 2003;17(2):151 – 7. [21] Susal C, Pelzl S, Simon T, Opelz G. Advances in pre- and posttransplant immunologic testing in kidney transplantation. Transplant Proc 2004;36(1):29 – 34. [22] Pelzl S, Opelz G, Wiesel M, Schnulle P, Schonemann C, Dohler B, et al. Soluble CD30 as a predictor of kidney graft outcome. Transplantation 2002;73(1):3 – 6. [23] Chan KW, Hopke CD, Krams SM, Martinez OM. CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses. J Immunol 2002;169(4):1784 – 91. [24] Susal C, Pelzl S, Opelz G. Strong human leukocyte antigen matching effect in nonsensitized kidney recipients with high pretransplant soluble CD30. Transplantation 2003;76(8):1231 – 2. [25] Susal C, Pelzl S, Opelz G. High pretransplant serum sCD30 content as a possible explanation for the high rejection rate in young kidney graft recipients. Pediatr Transplant 2003;7(48):2003. [26] Bradley BA. Rejection and recipient age. Transpl Immunol 2002; 10(2 – 3):125 – 32. [27] van der Mast BJ, van Besouw NM, de Kuiper P, Vaessen LM, Gregoor PJ, van Gelder T, et al. Pretransplant donor-specific helper T cell reactivity as a tool for tailoring the individual need for immunosuppression. Transplantation 2001;72(5):873 – 80. [28] Malan Borel I, Racca A, Garcia MI, Bailat A, Quiroga F, Soutullo A, et al. Gammadelta T cells and interleukin-6 levels could provide information regarding the progression of human renal allograft. Scand J Immunol 2003;58(1):99 – 105. [29] Sadeghi M, Daniel V, Naujokat C, Wiesel M, Hergesell O, Opelz G. Strong inflammatory cytokine response in male and strong antiinflammatory response in female kidney transplant recipients with urinary tract infection. Transpl Int 2005;18(2):177 – 85. [30] Tong CY, Bakran A, Williams H, Cuevas LE, Peiris JS, Hart CA. Association of tumour necrosis factor alpha and interleukin 6 levels with cytomegalovirus DNA detection and disease after renal transplantation. J Med Virol 2001;64(1):29 – 34. [31] Hidalgo LG, Halloran PF. Role of IFN-gamma in allograft rejection. Crit Rev Immunol 2002;22(4):317 – 49. [32] Shulzhenko N, Morgun A, Franco M, Souza MM, Almeida DR, Diniz RV, et al. Expression of CD40 ligand, interferon-gamma and Fas ligand genes in endomyocardial biopsies of human cardiac allografts: correlation with acute rejection. Braz J Med Biol Res 2001;34(6): 779 – 84. [33] Wang YL, Tang ZQ, Gao W, Jiang Y, Zhang XH, Peng L. Influence of Th1, Th2, and Th3 cytokines during the early phase after liver transplantation. Transplant Proc 2003;35(8):3024 – 5. [34] Zhao DX, Hu Y, Miller GG, Luster AD, Mitchell RN, Libby P. Differential expression of the IFN-gamma-inducible CXCR3-binding chemokines, IFN-inducible protein 10, monokine induced by IFN, and IFN-inducible T cell alpha chemoattractant in human cardiac allog-
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B.A. Bradley / Transplant Immunology 14 (2005) 193 – 201 [55] Le Rond S, Le Maoult J, Creput C, Menier C, Deschamps M, Le Friec G, et al. Alloreactive CD4+ and CD8+ T cells express the immunotolerant HLA-G molecule in mixed lymphocyte reactions: in vivo implications in transplanted patients. Eur J Immunol 2004;34(3): 649 – 60. [56] Chung SW, Yoshida EM, Cattral MS, Hu Y, Gorczynski RM. Donorspecific stimulation of peripheral blood mononuclear cells from recipients of orthotopic liver transplants is associated, in the absence of rejection, with type-2 cytokine production. Immunol Lett 1998; 63(2):91 – 6. [57] Mazariegos GV, Zahorchak AF, Reyes J, Chapman H, Zeevi A, Thomson AW. Dendritic cell subset ratio in tolerant, weaning and nontolerant liver recipients is not affected by extent of immunosuppression. Am J Transplant 2005;5(2):314 – 22. [58] Reinsmoen NL. Immunologic monitoring of T helper cell reactivity. Transplant Proc 2001;33(1 – 2):302 – 3. [59] Weimar W, Rischen-Vos J, de Kuiper P, Gregoor PJ, JN IJ, van Besouw NM, et al. Tapering immunosuppression in recipients of living donor kidney transplants. Nephrol Dial Transplant 2004;19(Suppl. 4): iv61 – 3. [60] van Besouw NM, van der Mast BJ, de Kuiper P, Smak regoor PJ, Vaessen LM, Ijzermans JN, et al. Down-regulated donor-specific Tcell reactivity during successful tapering of immunosuppression after kidney transplantation. Clin Exp Immunol 2002;128(2):388 – 93. [61] Hornick PI, Mason PD, Yacoub MH, Rose ML, Batchelor R, Lechler RI. Assessment of the contribution that direct allorecognition makes to the progression of chronic cardiac transplant rejection in humans. Circulation 1998;97(13):1257 – 63. [62] Claas FH. Towards clinical transplantation tolerance. Lancet 2003; 361(9368):1489 – 90. [63] de Haan A, van der Gun I, Hepkema BG, de Boer WJ, van der Bij W, de Leij LF, et al. Decreased donor-specific cytotoxic T cell precursor frequencies one year after clinical lung transplantation do not reflect
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Prognostic assays for rejection and tolerance in organ transplantation Benjamin A. BradleyT The East Barn, The Pound, Lower Almondsbury, Bristol BS32 4EF, England, United Kingdom Accepted 14 March 2005
Abstract In this review, I have summarised our understanding of acute rejection of organ transplants, and for convenience I have identified three processes, recognition, rejection and regulation. In stark contrast to this text-book picture of acute rejection, I have drawn attention to some of the clinical realities, where processes are altered by powerful immunosuppressive drugs, and where many transplant recipients are presensitised to transplantation antigens prior to engraftment. The ultimate goal is to encourage the emergence of a utopian immunological state, wherein patients tolerate organ transplants for life after being weaned from all immunosuppressive drugs. Assays that may be used in the future to reliably monitor this process are still at a very exciting stage of development. D 2005 Elsevier B.V. All rights reserved. Keywords: Organ transplantation; Acute rejection; Tolerance; Regulation; Immunosuppression; Monitoring
1. Introduction Acute rejection is defined here as a response by the recipient’s adaptive immune system to non-self HLA and non-HLA molecules on donor tissue. In the clinical context acute rejection occurs more frequently and is more severe during the early post transplant period. It wanes, but does not completely resolve 6 to 12 months after the transplant. Organ transplant recipients usually receive continuous immunosuppressive therapy for life in an attempt to prevent ongoing rejection and to preserve the function of the graft, but newer protocols attempt to wean recipients off these drugs in the hope that durable tolerance will be established [1]. As a result recipients are immunosuppressed and may suffer from recurrent infection and a high probability of malignancy. Episodes of acute rejection remain frequent in organ graft recipients, implying an overall lack of efficacy of clinical immunosuppression. For some years investigators have worked on developing laboratory tests to predict and monitor rejection risk in the clinical setting. The aim is firstly to prevent rejection altogether, and secondly to identify recipients in whom immunosuppressive therapy T Tel.: +44 1454 201077 or +44 7803165327. E-mail address: [email protected]. 0966-3274/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.trim.2005.03.022
can be reduced to low non-toxic doses, or discontinued because immunological tolerance has occurred. Unfortunately, the mechanisms involved in the development of peripheral tolerance involve active processes that differ only subtly from acute rejection, but are most likely to be equally susceptible immunosuppressive drug therapy. To date, none of the laboratory tests have performed well in the clinical setting, so more attention should be given to developing tests that identify the initial phases of both acute rejection and tolerance induction.
2. Acute rejection and tolerance In this review the acute rejection response has been divided into three sequential processes: recognition, during which the recipient’s immune system first becomes aware of foreign antigens in the graft; rejection, during which the recipient’s anti-donor T- and B-cell clones expand and attack the graft; and regulation, during which the recipient’s regulatory immune system dampens down the anti-donor Tcell responses. Regulation is the essential first step towards establishing durable transplantation tolerance. These three processes are transient and overlapping events in an evolving reaction and are briefly describe below.
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2.1. Recognition When an allogeneic organ is transplanted, the arterial and venous circulation, but not lymphatic system, is reestablished, whereupon resting dendritic-cells (DC) of donor origin, resident in the transplanted tissue, migrate to the recipient’s spleen [2] (Fig. 1a). The HLA peptides,
expressed naturally on the now-activated donor DC’s surface offer a highly potent mitogenic stimulus to circulating recipient T-cells in transit through the spleen. Recipient T-cells expressing complimentary T-cell receptors (TCR) to non-self donor peptides engage the DC and become activated. Within a few days the supply of donor DC wanes and their function is taken over by the recipient’s DC. These
a)
Donor Kidney
Resting Recipient DC
Activated Recipient DC + HLA Peptide
Activated Donor DC + HLA Peptide
CD4 T-cells
CD8 T-cells
Recipient Splenic Lymphoid Tissue
b) Resting Recipient DC
B Cells Anti inflammatory Cytokines
Donor Kidney
Activated Recipient DC + HLA Peptide
CD4 T-cells
CD8 T-cells
CD4 T-cells
Pro -inflammatory Cytokines
CD8 T-cells
Recipient Splenic Lymphoid Tissue
Fig. 1. Illustrates the three sequential processes that are envisaged to occur in a kidney transplant; see text for description. (a) Recognition process. (b) Rejection process. (c) Regulation process.
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c) Resting Recipient DC
Regulatory DC
Donor Kidney
Plasma cells secreting anti-donor HLA antibodies
Activated Recipient DC + HLA Peptide
CD4 T-cells
Anti inflammatory Cytokines
CD8 T-cells
Regulatory T-cells Anti inflammatory Cytokines
Memory CD4 Memory CD8
Recipient Splenic Lymphoid Tissue
Fig. 1 (continued).
migrate from the recipient’s arterial circulation into donor tissue, take up donor HLA molecules and digest them into peptides. Peptide-loaded recipient DC passes via the circulation to spleen and lymph nodes. They enter the para-cortical areas, where they engage an extended range of T-cell clones with TCR directed to donor HLA-peptides. Surveillance of the donor’s tissue by the recipient’s DC continues for as long as the circulation to the transplant remains intact. Eventually, the immune system adapts to the continuing presence of foreign antigen, and the predominant functional activity of the recipient’s DC changes from an activation to a regulatory function. In addition to cell-bound donor material, soluble donor HLA molecules, shed by transplanted tissue, pass to the germinal centres in spleen and lymph nodes where they interact with B-cells. A tiny fraction of B-cells binds to these HLA molecules by virtue of specific B-cell receptors (BCR) and differentiate into plasma cells. The BCR precedes and mirrors specificity of antibody subsequently synthesised by the plasma-cells. The BCR-bound donor HLA is internalised, partially digested into peptides, and the peptides re-assembled with newly synthesised recipient HLA Class II molecules which it presents to T-cells. The close anatomical proximity of the B-cells in the cortical areas to the T-cells in the para-cortical areas of lymph nodes and spleen allows interaction of these B-cells with CD4 positive T-helper cells specific for the same peptide. This interaction between CD4 T-cells and B-cells leads to the release of cytokines (e.g. IL-10) by the T-cell, resulting in Bcell activation, differentiation, clonal-expansion; and then differentiation into anti-HLA antibody producing plasma cells.
2.2. Rejection Upon entering the para-cortical areas in spleen and lymph nodes, activated DC are confronted with the entire array of T-cells directed to non-self peptides (Fig. 1b). The T-cells that carry a TCR that fits the peptide bound to the DC undergo activation, differentiation and clonal expansion. This process is driven by cytokines, such as IL-12 secreted by the DC, and autocrines, such as IL-2 secreted by T-cells. The T-cells may respond in a naı¨ve or an accelerated manner depending on previous experience of the peptide. If, prior to this moment, a T-cell has never engaged its specific peptide, it will respond to the DC in an immature manner. If, as frequently occurs in clinical practice, T-cells have experienced the peptide through pregnancy, blood transfusions or former transplants, they will respond in a more committed and accelerated manner. Unlike B-cells, the clonal progeny resulting from T-cell activation retain the unique specificity of their TCR for a particular peptide, in perpetuity. The expression of co-receptors, identified as cluster differentiation (CD) molecules, CD4 and CD8, is determined during the early stages of T-cell maturation in the thymus. One sub-set of naı¨ve T-cells expresses the CD4, which binds exclusively to HLA Class II molecules; hence, it is only activated by peptide presented on the HLA-Class II molecules of DC. The other sub-set of naı¨ve T-cells expresses CD8, which binds exclusively to HLA Class I molecules; hence, it is only activated by peptide presented on the HLA-Class I molecules of DC. Since peptides presented by HLA Class I or II, differ in their origin specificity and size, these two sub-sets differ in their range of TCR peptide-binding specificities.
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The strength of the signal delivered by the DC via the TCR to the T-cell is determined by the duration of cell –cell contact. The weakest (shortest) signal results in the development of a memory T-cell. The strongest (longest) signal results in apoptotic or dead T-cells. An intermediate signal allows migratory T-cells to home to inflamed tissue or recently transplanted organs, the migration pathway being determined by the differential expression of chemokine receptors (e.g. CCR7). During the interaction between CD4 T-cells and B-cells within lymphoid follicles, an anti-inflammatory cytokine environment facilitates the process of somatic hypermutation of the BCR genes. Somatic hyper-mutation, driven by soluble donor HLA antigen, yields clonal progeny of increasingly high avidity. Eventually, these highly avid mature B-cells differentiate into plasma cells that secrete specific antibody directed to unique molecular shapes (epitopes) on the donor’s HLA antigens. Mature migratory T-cells, B-cells and plasma cells, all pass from the circulation into inflamed tissue, in this case the recently transplanted organ. The process of extravasation, whereby circulating cells pass from capillary blood into the tissues, is facilitated by activation of the vascular endothelium: an inevitable consequence of ischaemic damage in transplanted organs. Endothelial activation is further enhanced by humoral factors released in brain-dead heart-beating cadaveric donors. This phenomenon accounts for the difference in graft survival observed between transplants derived from cadaveric donors and living donors. Graft injury, resulting from entry of mature T- and B-cells into the donor tissue, is poorly understood, but at least three mechanisms have been implicated, delayed-type hypersensitivity, T-cell cytotoxicity, and anti-HLA antibodies, especially those that activate the complement cascade. 2.3. Regulation Much of the immune system is dedicated to downregulating activation processes by curtailing clonal expansion and restraining auto-aggression with a view to maintaining homeostasis (Fig. 1c). Knowledge of these immuno-regulatory mechanisms is incomplete, but four broad categories, that could be relevant in transplantation, can be described. Clonal-shrinkage through programmed cell death. Clonal shrinkage is the sequel to clonal expansion. It results from several processes including: the acquisition of cellsurface death-receptors, such as Fas-ligand; blocking responsiveness to IL-2; and the establishment of signaltransduction pathways that induce death by apoptosis, such as Caspase 3. Non-specific suppression of activation and differentiation, through the release of anti-inflammatory cytokines. Non-specific release of anti-inflammatory cytokines into the microenvironment is attributed to sub-sets of T-cells
(including CD4 + CD25 + subsets) and Natural Killer Tcells (NK T-cells). Thus, IL-4, IL-10 and TGF-h produced by these cells, suppress functional activity of aggressor Tcells and dampen down acute rejection and graft-versushost disease. Under normal circumstances, the main roles of NK T-cells would be to protect vital organs from the damaging effects of pro-inflammatory cytokines and escalating autoimmunity. Deletion or suppression of peptide-specific clones by Tregulator cells. T-regulator cells, which specifically suppress a particular immune response, are generated as a distinct population within the thymus. In concept, they are thought to undergo clonal expansion during evolution of the alloimmune response, and eventually target and kill the nonself HLA-peptide-specific T-cells responsible for acute rejection. The most attractive idea is that their TCR specificities are directed to peptides derived from the TCRs utilized by the effector T-cells involved in acute rejection. Thus, a negative feedback mechanism involving TCR from apoptotic T-cells digested into peptides by activated DC, then offered to T-regulators specific for the TCR peptides would eventually arrest the clonal expansion. The Tregulator cells undergo clonal expansion in the usual way and eventually suppress or destroy those particular aggressor T-cells that carry the specific TCR. Replicative senescence or clonal exhaustion (Hayflick phenomenon). Clonal-exhaustion or replicative-senescence is the end stage of many cycles of T-cell replication. With each mitosis, the telomeres responsible for the DNA repair after replication are shortened. Eventually, telomere shortening is such that repair of the genes vital for cell division is impossible and the cell cycle is arrested. At this point the cells remain viable, but cease to replicate, leaving holes in the T-cell repertoire, as evidenced by deletion of certain families of TCR-Vh genes. Such holes in the T-cell repertoire are manifestations of normal ageing, resulting from a lifetime of chronic antigenic stimulation with certain microorganisms. Insofar as an organ transplant is a source of chronic antigenic stimulation, a similar process could be envisaged leading to weakening of acute rejection with time.
3. Relevant aspects of clinical transplantation 3.1. Manifestations of acute rejection The clinical diagnosis of acute rejection is based on changes in function and changes in the histological appearance of transplant biopsy material. Biopsies taken during the early stages after renal transplantation show cellular infiltrates consisting of: T-cells expressing activation markers, FasL, granzyme, perforin, and chemokine receptors; activated macrophages; polyclonal B-cells and plasma cells; and, when anti-donor HLA antibody is present, neutrophils. In mild rejection, cellular infiltrates are confined to the interstitial tissue (70 – 85% of cases), but
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in more severe cases inflammatory vascular changes occur (25 –30% cases). These histological changes are graded according to an internationally agreed system called the Banff Scheme of Allograft Pathology, which delineates three types of active rejection: Type I Rejection, with tubulo-interstitial mononuclear cell infiltration and tubulitis; Type II Rejection, with moderate to severe intimal arteritis; and Type III Rejection, with transmural arteritis, fibrinoid change and necrosis of medial smooth muscle cells. Rejection due to anti-donor antibodies is categorised as a separate group in the Banff Scheme. The prognostic value of this scheme is that it correlates with early loss of function, steroid responsiveness and graft survival at 1 year. 3.2. Impact of immunosuppressive drugs. The effect of these drugs on acute rejection and tolerance depends on the type of drug given and its timing in relation to transplantation. Thus, induction therapy is given before, during and shortly after transplantation; initial drug therapy starts immediately after the operation and is tapered over the ensuing 6 months; maintenance therapy is given thereafter for the lifetime of the transplant; and anti-rejection therapy is given transiently to reverse episodes of acute rejection. The recent trend has been to investigate the effect of induction therapy prior to transplantation. A variety of agents have been used including anti-thymocyte globulin (ATG), which reacts with all re-circulating T-cells, especially memory T-cells; monoclonal antibody to the T-cell receptor (OKT3), which prevents T-cell activation by inhibiting co-stimulation; monoclonal antibody to the IL-2-receptor alpha-chain (IL-2Ra), which inhibits cytokine-driven clonal expansion of T-cells, NK-cells and NKT-cells; or a monoclonal antibody to CD52 (Campath1H), a glycoprotein antigen expressed by T-cells, B-cell, monocytes and certain sub-sets of activated DC. Campath1H is a lytic antibody that prevents T-cell activation during the recognition phase. Induction therapy can significantly reduce the incidence and severity of acute rejection, but may concurrently delay the emergence of regulator cells. Initial drug therapy begins immediately after re-establishing the blood supply to the transplant, but may also be started prior to this moment. Drug dosage is initially high to suppress acute rejection, but is tapered over the first 6 to 12 months to maintenance levels that are continued for the lifetime of the transplant. The most widely used drugs are inhibitors of purine synthesis such as azathiaprine and the more recently introduced micophenolate mofetil (MMF). Corticosteroids inhibit a wide range of cellular functions including the NF-nB signal transduction pathway and are used extensively for initial and maintenance therapy and in large doses for anti-rejection therapy. The calcineurin inhibitors such as cyclosporin and tacrolimus inhibit the NFAT signal transduction pathway, and rapamycin, which inhibits the expression of the IL-2 receptor. These drugs
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influence different intracellular pathways and can therefore be used in synergistic combinations to minimize toxicity and maximize therapeutic efficacy. Whereas, the purine inhibitors and corticosteroids tend to affect all components of the immune system, the calcineurin inhibitors and Rapamycin affect mainly T-cells.
4. Principles and attributes of prognostic assays The search for the ideal prognostic assay dates back to the 1960s when HLA typing, HLA-matching, serum crossmatching, and the one-way mixed lymphocyte reaction (MLR) were invented. For many years the MLR and assays derived from it, such as the cell mediated lympholysis (CML) assay, were explored as donor-specific in-vitro models for studying rejection, but they are time consuming and expensive and have failed to gain wide acceptance [3]. A wide range of alternative assays have been explored and advocated as a means of predicting and diagnosing acute rejection or tolerance. For the purposes of this review, these assays have been grouped with reference to the part of the rejection response for which the assay is most informative, namely: recognition, rejection and regulation. 4.1. Recognition The prognostic value of anti-HLA antibody detected in patients awaiting transplantation, and the exclusion of prospective donors who are cross-match positive as a result of these antibodies, is widely accepted. This topic has been discussed extensively elsewhere. Nonetheless, most methods of detecting antibodies to-date have relied on the capacity of those antibodies to fix complement. Noncomplement fixing antibodies detectable by ELISA techniques are additional indicators of impending acute rejection if directed to both the donor’s HLA Class I and Class II antigenic mismatches. The inclusion of ELISA testing for non-complement fixing antibodies is recommended to make antibody-detection techniques prior to transplantation more rigorous [4]. In contrast, the prognostic value of anti-donor HLA antibodies arising de-novo after transplantation seems to correlate with the onset of acute rejection in several types of transplant [5– 7]. In addition, the available evidence suggests that these and other antibodies may lead to chronic allograft vasculopathy by activating donor vascular endothelium [8,9]. However, the result of this interaction seems to depend on the molecule targeted. Whereas endothelial cells proliferate in response to anti-HLA Class I antibodies, they also evade damage by anti-donor HLA Class II antibodies. Other antibodies directed to vascular endothelial antigens of the donor promote apoptosis [10 –12]. Antibodies directed to the HLA-linked MICA antigens, which arise after transplantation, are directed to donor endothelial cells, and are also suspected of triggering vascular damage [13].
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The cytokine microenvironment within the lymphoid tissues determines the differentiation pathway adopted by activated T-cells. If pro-inflammatory cytokines (e.g. TNFa) predominate the risk of acute rejection may be higher; whereas, if anti-inflammatory cytokines predominate (e.g. IL-10), the risk may be reduced. Since the level of synthesis of many cytokines varies according to genetic polymorphisms in their promotor regions (up to seven-fold increase in the case of the TNF-a-308 SNP), these producergenotypes have prognostic value for acute rejection. A single nucleotide polymorphism (SNP) at the position 308 in the TNF-a promotor predicts a significant increase in risk of steroid resistant acute rejection episodes in recipients of renal and liver transplants [14,15]. Using meta-analysis to combine several underpowered studies, prognostic significance has also been attached to the IL-10 SNP at position 1082, in liver transplantation [16]. At present, this area of immunogenetics is handicapped by a lack of understanding of the functional haplotypes associated with these genes. Haplotypes consist of several SNPs, any one of which may, or may not, be associated with acute rejection or its suppression. Certain haplotypes are more frequent in some populations than others through the process of linkage disequilibrium that has arisen between widely separated SNP. An analysis of commonly occurring haplotypes gives more accurate prognostic information for transplant outcomes. Hence, in lung transplantation the IL-10 highproducer haplotype predicts a reduced risk of acute rejection [17]. Functional assays for certain cytokines conducted on peripheral blood mononuclear cells (PBMC) taken prior to transplantation may also be of prognostic value. Thus PBMC from prospective liver transplant recipients, cultured in-vitro with the mitogen lipopolysaccharide (LPS), were shown to synthesize higher levels TNF-a in those who developed acute rejection compared with those who developed no rejection; but, in contrast to the genetic evidence already cited above, no prognostic changes were found for IL-10 synthesis [18]. Since different mitogens activate different PBMC subsets, and use different signal transduction pathways, other studies in which staphylococcal A protein mitogen (SACI) was used, showed that IL-10 synthesis levels, when low, did indeed predict acute rejection [19]. Pre-operative serum levels of certain cytokine may of prognostic value. In prospective kidney transplant recipients a marked difference was observed in serum IFN-g levels in rejecting patients (1826 T 2424 pg/ml), compared to stable patients (316 T 373 pg/ml) [20]. Unfortunately, the standard deviation of these mean values indicates wide variations between patients, possibly attributable to differences in the turnover of these molecules in blood and the presence of inhibitors. Intriguingly, high plasma levels of a soluble form of a cell surface marker, sCD30, is an excellent prognostic indicator for acute rejection in kidney transplant recipients
[21,22]. This sCD30 is a little studied member of the TNFR receptor super-family shed by a subset of T-cells that have been activated by allogeneic DC. This subset has been identified as the predominant proliferating T-cell population in alloimmune responses, and is characterised by expression of CD30, CD25 and CD45RO. T-cells within this subset synthesise IFN-g and IL-5 [23]. High levels of sCD30 appear to increase the vulnerability of the graft to the effects of prior sensitisation, identified as elevated levels of panel reactive antibody, and to the effects of HLA Class I and Class II mismatching, such that the two assays in combination (sCD30 levels and PRA) give excellent prediction of kidney transplant outcome [24]. It is also fascinating to know that sCD30 levels decline significantly with increasing age; an observation in keeping with the declining incidence of acute rejection with increasing age [25,26]. Quantitative functional assays for measuring the frequency of donor-specific helper T-cell (HTLpf assay), are based on the principle of limiting dilutions analysis and the traditional MLR. In prospective kidney transplant recipients, the HTLpf assay appears to correlate with the risk of developing acute rejection after transplantation. However, the cytotoxic T-cell precursor frequency (CTLpf) failed to correlate with acute rejection [27]. Such assays may be of value in tailoring immunosuppressive drug therapy. 4.2. Rejection Prognostic indicators of early, ongoing and established acute rejection may be found in blood samples, graft biopsies and excretory products such as urine. Since the result of a particular test may indicate that immunosuppressive drug dosage be increased, it is important that changes attributed to acute rejection can be differentiated from those brought about by infection. Post-operative blood levels of TNF-a and IL-6 are increased in acute rejection of kidney transplants [28]. But these changes are also observed in cytomegalovirus infections, and urinary tract infections in male transplant recipients [29,30]. IFN-g induces HLA expression in graft and other tissues, and although it appears to have a protective role against acute rejection in certain murine models [31], the human story is different. Changes in serum levels are uninformative, but increased mRNA expression is found both in the CD4+ T-cells in blood, and in-situ, in biopsies from rejecting cardiac transplants [32,33]. Furthermore, certain IFN-g inducible factors, (IP10, I-TAC, and Mig) are elevated in biopsies of acutely rejecting cardiac grafts [34]. Increased gene expression of IL-18 and perforin in the blood, measured by real-time PCR, closely correlates with acute rejection in renal transplants [35]. In liver transplants, acute rejection is accompanied by elevated levels of the soluble form of the IL-2 receptor (sIL-2R) [36].
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Peripheral blood T-cell subsets show increased expression of activation markers during acute rejection of kidney transplants, as exemplified by increased expression of CD40 in CD4+T-cells, and CD69 in CD8+T-cells [37,38]. Since the increased CD40 expression is mirrored by increased CD40 ligand (CD40L) gene expression in biopsy material from rejecting renal transplants, the net result of their interaction may be to amplify production of anti-donor HLA antibodies by B-cells within the graft [39]. Within the graft, evidence of damage by anti-donor HLA and other antibodies is in the form of deposits of the complement breakdown product, C4d [40 – 42]. Immune cells expressing chemokine ligands (e.g. CXCR3) are attracted into rejecting graft tissues that express chemokines. Thus biopsies from rejecting kidney, cardiac and lung transplants show increased expression of chemokine receptors and their ligands [34,43 – 49]. Since these and other molecules (e.g. IP-10) are detectable in the urine, acute rejection can also be diagnosed non-invasively [50,51]. 4.3. Regulation Detection of a state of transplant tolerance begs the question as to the multiple mechanisms involved. Data from murine models suggests that it is an active process involving IL-2 signalling, particularly of CD4+CD25+ T-regulator cells [52,53]. At present, there are no foolproof assays, which have been fully tested in drug weaning studies, although this is the ultimate goal [54]. Nonetheless, several correlates of stable rejection-free transplant function exist, and many of these will prove valuable as clinical indicators for the withdrawal of immunosuppressive therapy in future. In brief, these assays involve measuring: soluble serum factors (e.g. HLA-G levels) [55]; anti-inflammatory cytokine profiles generated in donor specific MLRs [56]; ratios of monocytoid to plasmacytoid DC in recipient’s blood [57]; hypo-responsiveness and reduced frequencies of donor reactive helper T-cells and cytotoxic T-cells [58 – 63]; reduced levels of mRNA for IL-2 and IFN-g in peripheral blood [64]; reduced expression of CD40L on activated Tcells[65]; increased percentages of CD25+CD4+T-regulator cells [66,67]; increased levels of donor specific CD8+CD28-CD27+T-suppressor cells in peripheral blood [68 –70]; and graft infiltrates of CD4+T-cells secreting TGF-h [71].
5. Summary Over the past 10 years much progress has been made in understanding the cellular and molecular basis of allogeneic organ graft rejection. There have been parallel advances in the efficiency of therapeutic immunosuppression of organ transplant patients with a remarkable reduction the severity and frequency of rejection episodes. Some innovative and
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encouraging research techniques have been described above; however, there is still a need for simple, costeffective tests routinely run by clinical laboratories in hospitals to monitor organ transplant patients. The intensity of immunosuppressive regimens now in routine clinical use has lead to a narrow therapeutic window between effective immunosuppression and toxicity. Better monitoring will not only further reduce the probability of rejection, but will also decrease morbidity and mortality due to powerful immunosuppressive drugs, eventually leading to the ultimate goal of drug free graft tolerance.
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