Promoter: Pr Daniel Abramowicz Co-Promoter: Pr Michel Toungouz Nevessignsky
Dr Anne Lemy
Clinique de Transplantation Rénale CUB-Hôpital Erasme
Université Libre de Bruxelles Faculté de Médecine
Do MICA antibodies impact on renal graft outcomes?
Table of contents
Résumé……….6
Summary………...8
Foreword……….10
Abbreviations………..11
1. Introduction……….14
1.1 Outcomes of kidney transplantation……….14
1.2 The allorecognition of the graft………..16
1.2.1 Direct allorecognition 1.2.2 Indirect allorecognition 1.2.3 The semi-direct pathway 1.3 The alloresponse to the graft……….18
1.3.1 Critical role of dendritic cells in the priming of alloreactive T cells 1.3.2 Differentiation of alloreactive CD4 T cells into Th1, Th2 or Th17 1.4 Effectors of graft destruction………..23
1.4.1 Alloreactive T cells 1.4.2 Natural Killer cells 1.4.3 Macrophage activation and delayed-type hypersensitivity reaction 1.4.4 Eosinophils and Th2 type alloreactive response 1.4.5 Neutrophils 1.4.6 Alloantibodies 1.4.6.1 Complement activation 1.4.6.2 Non complement-dependent mechanisms 1.5 Relationship of anti-HLA Abs with renal graft outcomes………...31 1.5.1 Methods for detection of anti-HLA antibodies in serum
1.5.1.1 Complement-dependent cytotoxicity methods 1.5.1.2 Flow cytometry
1.5.1.3 Solid-phase assays
1.5.2 HLA sensitization before transplantation
1.5.3 The relevance of donor-specific HLA antibodies after renal transplantation
1.6 Overview of the relationship between antibodies against non- MHC antigens and organ transplantation and special focus on MICA antibodies………38 1.6.1 Non-MHC targets of the humoral response in renal transplantation
1.6.1.1 Anti-endothelial cell antibodies (AECA) 1.6.1.2 Vimentin
1.6.1.3 Angiotensin 2 type 1receptor (AT1R)
1.6.1.4 Agrin and perixosomal-trans-2-enoyl-coA-reductase (PECR)
1.6.1.5 Glutathione S-transferase T1 (GSTT)
1.6.2 Major histocompatibility complex class I chain-related antigen A (MICA)
1.6.2.1 Cellular expression of MICA
1.6.2.2 Control of MICA cellular expression a) The transcriptional level b) The post-transcriptional level c) The post-translational level
1.6.2.3 Interaction of MICA with NKG2D receptor
1.6.2.4 NKG2D: link between innate and adaptative immunoresponse
a) NKG2D pathways of allorecognition by NK-DC crosstalk
b) Regulation of activated CD4+ T cells via the NKG2D receptor
c) Costimulator function of NKG2D in CD8+ T cell activation
1.6.2.5 MICA polymorphism 1.6.2.6 MICA and disease
1.6.2.7 MICA and the immune response to transplants 1.6.2.8 Routes of MICA sensitization
1.6.2.9 Methods for detection of MICA antibodies 1.6.2.10 MICA antibodies recognize epitopes
1.6.2.11 Are MICA antibodies developing after transplantation donor specific?
a) Acute rejection b) Chronic rejection c) Graft survival
- Kidney - Heart
d) Renal graft function
2. Objectives………...72
3. Results……….73
3.1 Preformed MICA antibodies………..73
3.2 Post-transplant MICA antibodies………..76
3.3 Influenza A/H1N1 vaccination and MICA immunisation…………79
4. General Discussion………...81
4.1 Minor antigens in renal transplantation………81
4.2 Epidemiology of MICA antibodies and risk factors for MICA sensitization………82
4.3 Impact of MICA antibodies on graft survival………83
4.4 How to provide evidence of a possible pathogenicity of MICA antibodies towards the renal graft? ...84
4.5 Influenza A/H1N1 vaccination and MICA sensitization………….90
5. Summary……….91
6. Perspectives………...92
7. References……….94
8. Secondary thesis……….134
9. Annexes………136
Résumé
La transplantation rénale représente le traitement de choix de l’insuffisance rénale terminale parce qu’il offre une espérance de vie plus longue et une meilleure qualité de vie.
Néanmoins, l’accès à la transplantation est limité par la pénurie d’organes et dans certains cas, par la présence d’anticorps anti-HLA avant la greffe.
Bien que la présence d’anticorps anti-HLA spécifiques du donneur avant ou après la greffe ait été associée au rejet aigu et à la perte chronique d’allogreffe, un rejet humoral tant aigu que chronique peut survenir sans que ces anticorps soient détectables dans le sérum, suggérant que des réponses autologues ou allo-immunes contre des antigènes dits « mineurs » pourraient jouer un rôle dans le rejet et la perte de greffe.
MICA, en raison de son polymorphisme important, est considéré aujourd’hui comme un des systèmes antigéniques mineurs les plus robustes par sa capacité à induire des allo-anticorps. Cependant, un effet pathogène des anticorps anti- MICA sur le greffon rénal demeure à ce jour, non formellement établi.
Le but de la présente recherche a été d’étudier l’épidémiologie des anticorps anti-MICA à partir d’une large cohorte de volontaires sains et de patients atteints d’insuffisance rénale chronique terminale, de déterminer les facteurs de risque d’immunisation contre MICA, de spécifier la nature autologue ou allogénique de ces anticorps et d’évaluer au sein des patients ultérieurement transplantés, l’impact de ces anticorps sur le rejet et la survie de greffe.
La méthode utilisée pour l’identification des anticorps anti-MICA est la technique Luminex, consistant à faire réagir du sérum avec des billes de polystyrène tapissées par un seul antigène MICA recombinant, l’intensité de la liaison antigène-anticorps étant révélée par un fluorocytomètre suite à l’adjonction d’un second anticorps anti-IgG couplé à une substance fluorescente.
Nous avons identifié la grossesse, les transfusions sanguines, la greffe préalable et également l’urémie comme étant des facteurs de risque indépendants d’immunisation contre MICA.
Nous n’avons pas observé d’effet délétère des anticorps anti-MICA sur la survie à long terme du greffon rénal alors que les anticorps anti-MICA ont été plus fréquents chez les patients dits «à haut risque immunologique» et en particulier chez les patients immunisés contre le HLA.
Nos résultats suggèrent que plutôt d’être pathogènes, les anticorps anti-MICA pourraient être simplement des marqueurs de haut risque immunologique. Ceci remet donc en question l’utilité d’un monitoring des anticorps anti-MICA par la technologie Luminex.
Summary
Renal transplantation represents the treatment of choice of stage V chronic kidney disease by offering a longer life expectancy and a better quality of life than dialysis. Nevertheless, the access to transplantation is limited by the shortage of organs and, in some cases, by the presence of HLA antibodies before transplantation.
While the presence of either preformed or post-transplant donor specific anti-HLA antibodies has been associated with acute rejection or chronic graft loss, acute or chronic antibody-mediated injury may also occur in the absence of detectable anti-HLA antibodies, suggesting that autologuous or allo-immune response to other relevant minor or non-HLA antigenic determinants might play a role in rejection and subsequent graft loss. Especially, MHC class I-related chain A (MICA), a highly polymorphic minor antigenic system, is now considered to be the most robust minor antigenic system capable of inducing allo-antibodies.
However, the possible deleterious effect of MICA antibodies has not been formerly established yet.
The goal of the following work was to determine the risk factors for MICA sensitization, to specify the autologuous or allogeneic nature of MICA antibodies and to assess the impact of preformed and 1 yr post-transplant MICA antibodies on defined renal graft outcomes in large cohorts of patients. The method employed for the identification of MICA antibodies was a Luminex single antigen beads assay. We found that pregnancy, previous blood transfusion, previous
graft as well as chronic kidney disease were independent risk factors for MICA sensitization.
We showed the absence of a deleterious effect of MICA antibodies on long-term renal graft outcomes while we found a higher frequency of MICA antibodies in patients with higher immunological risk and especially, a close association of MICA with HLA sensitization.
Our findings suggest that MICA antibodies are merely surrogate markers of high immunological risk and really question the monitoring of MICA antibodies by the presently available MICA Luminex assays.
Foreword
I want to thank Daniel Abramowicz for his enthusiasm, his availability and his unwavering support. I thank Michel Toungouz for welcoming me into his laboratory and also, Christian Noel, Myriam Labalette, Christian Hiesse, Caroline Suberbielle-Boissel, Martine De Meyer, Dominique Latinne and Michel Mourad for providing sera and patients’ clinical data.
A special thank also to Séverine Delsaut, Aurélie Vandersarren, Marc Andrien, Christine Heylen, Judith Racapé, Martin Wissing, Pierre Vereerstraeten, Nilufer Broeders, Nicole Lietar, Françoise Bernard †, Brigitte Borré, and Lidia Ghisdal for their invaluable help, their advice and assistance to carry out this work.
And finally, thank you to my parents, my husband and my kids.
Abbreviations
ADCC: antibody-dependent cell cytotoxicity AECA: anti-endothelial cell antibodies AMR: antibody-mediated rejection APC: antigen presenting cell AR: acute rejection
ATG: anti-thymocyte globulin
ATM: ataxia telangiectasia mutated
ATR: ataxia telangiectasia and Rad-3 related AT1R: angiotensin 2 type 1 receptor
BCR: B cell receptor
CDC: complement-dependent cytotoxicity CKD: chronic kidney disease
CR: chronic rejection
Crry: complement receptor-related protein CTL: cytotoxic lymphocyte
DAF: decay accelerating factor DAP: DNA activation protein DC: dendritic cell
DISC: death inducing signal complex DSA: donor-specific antibodies DTH: delayed-type hypersensitivity EC: endothelial cell
ECP: eosinophil cationic protein EPO: eosinophil peroxidase FC: flow cytometry
GSTT1: glutathione S-transferase T1 HIF-1: hypoxia-inducible factor-1 IVIG: intravenous immune globulins
KMEC: kidney micro-vascular endothelial cell MBP: major basic protein
MHC: major histocompatibility complex MICA: MHC class I-related chain A miRNA: microRNA
mTORC1: mammalian target of rapamycin complex 1 NDSA: non donor-specific antibodies
NK: natural killer NO: nitric oxide
PBMC: peripheral blood mononuclear cells PC: plasma cell
PCR: reverse transcriptase polymerase chain reaction PECR: perixosomal-trans-2-enoyl-coA-reductase PRA: panel reactive antigen
RAE-1: retinoic acid early inducible
ROR: retinoic acid-related orphan receptor
STAT: signal transducer and activator of transcription
TCR: T cell receptor TGF: tumor growth factor
TGP: transplant glomerulopathy TM: transmembrane
TNF: tumor necrosis factor UTR: untranslated region XM: crossmatch
1. Introduction
1.1 Outcomes of kidney transplantation
Renal failure is a worldwide major concern for health community. At present time, the incidence of stage V chronic kidney disease is reaching 190 patients per million population in Belgium (http://www.era-edta reg.org/files/annualreports/pdf/AnnRep2009.pdf) and the adjusted incident rate of end-stage renal disease in the U.S. was 351 per million population in 2008 (http://www.usrds.org/atlas.htm).
Renal transplantation represents the treatment of choice of stage V chronic kidney disease by offering a longer life expectancy and a better quality of life than dialysis (1, 2). However, the access to transplantation is limited primarily by the shortage of organs and HLA sensitization. In 2010, while 3705 patients had received a kidney from a deceased donor throughout Eurotransplant, 10768 patients remained on the waiting list by the end of the year (including 914
patients from Belgium)
(http://www.eurotransplant.org/cms/mediaobject.php?file=ar_2010.pdf).
Nowadays, pretransplant HLA sensitization is present in 25-30% of patients awaiting a kidney transplant according to current sensitive methods to detect anti-HLA antibodies.
While significant improvements over the past 3 decades regarding immunosuppression, HLA matching between donor and recipient and the medical care of the transplant recipients enhanced early outcomes of kidney transplantation by reducing the incidence of acute rejection (AR), these advances
have not resulted in dramatic improvements in long-term graft survival (3-5).
Moreover, kidney transplants still often progress to late failure. The reasons for this lack of improvement remain unclear and may be multifactorial. Firstly, patient death with a functioning graft continues to be the most common cause of graft failure (6, 7). In this category, the most frequent cause of graft is cardio-vascular.
However, in patients without diabetes or in older recipients, death is commonly associated with complications of immunosuppression such as infections and malignancies (7-10). Secondly, some important determinants of long-term graft survival may not have changed sufficiently to improve the overall results of kidney transplantation such as the cold ischemia time and the time duration of dialysis before patients are referred for transplantation. In addition, there has been little progress in the prevention or treatment of recurrent disease (11-13).
In the past 2 decades, some investigators have attempted to clarify the causes of graft loss in large cohorts of patients and to identify etiologies of graft failure on which it was possible to interfere with appropriate treatment (14, 15). However, those studies did not include detailed longitudinal and histological data particularly in the early post-transplant setting. These limitations have led to the concept that allografts were lost due to a common process involving interstitial fibrosis and tubular atrophy termed “chronic allograft nephropathy” (16). This histological term was further replaced by “interstitial fibrosis and tubular atrophy”
in the consensus Banff classification (17). However, the cause of chronic allograft dysfunction is multifactorial including immunologic and non-immunologic factors.
The latter include donor age and tissue quality, degree of reperfusion injury, BK
polyomavirus nephropathy and the effects of immunosuppressive drugs directly on the graft and systemically (hypertension, diabetes and dyslipidemia).
Evidence now suggests that allo-immunity also plays an important role in the development of this entity, making that antibody-mediated rejection has become the main cause of late chronic graft loss, raising the question about the possible patients’ non-adherence or the inappropriate minimization of immunosuppression by the attending clinicians (9, 18, 19).
1.2 The allorecognition of the graft
Allorecognition refers to the identification of tissues of allogeneic origin by the recipient’s immune system through engagement of a receptor-ligand system. T- cell (TCR) and B-cell receptors (BCRs) are key elements in the recognition of polymorphisms of cell-surface proteins encoded by genes on chromosome 6 between members of a same species. These polymorphisms, determining compatibility of tissues can be subdivided into major (class I and II) histocompatibility complex (MHC) and minor histocompatibility complex (non- MHC) antigens. The term major refers to the rapidity with which skin transplantation on mice models was rejected when MHC mismatches were present.
Three pathways of allorecognition have been described to date (Figure 1).
1.2.1 Direct allorecognition
The direct alloreactivity predominates in the immediate post-transplant period and direct pathway CD4+ T cells were demonstrated to be necessary and
sufficient to mediate allograft rejection in mice models (20, 21). In the direct pathway, recipient T cells recognize intact allogenic MHC-peptides complexes expressed on donor dendritic cells (DCs). The strength of alloresponses triggered by the direct pathway is partly due to the very high frequency of T cells with direct allospecificity (22). A large fraction of the direct alloresponse is derived from T cells with a memory phenotype that were previously primed against foreign antigens in the context of self-MHC molecules (crossreactivity) (23). The direct alloreactivity is based on two concepts: the “high determinant density” and the “multiple binary model” (24, 25). The high determinant density proposes that alloreactive TCRs are able to directly recognize the exposed amino acid polymorphisms on intact foreign MHC molecule while the multiple binary model refers to the specificity of the alloreactive TCR for recognition of peptide bound by allogenic MHC. Indeed, each peptide-allo-MHC complex is recognized by a different alloreactive T cell and any one mismatch will be able to stimulate a large number of diverse T cells responsive to different antigens.
1.2.2 Indirect allorecognition
The indirect pathway recognition proposes that recipient DCs traffic through the graft, take up soluble MHC or minor antigens to process them before presenting alloantigens as peptides on self MHC class II molecules to CD4+ T cells in local lymphoid tissue (20). It is generally assumed that indirect antigen presentation is more important for activating CD4+ T cells rather than CD8+ T cells because class II molecules present peptides from exogenous sources, unlike class I molecules which usually present peptides derived from endogenous sources.
Indirectly alloresponsive CD8+ T cells are activated through “cross-priming”, whereby antigen presenting cells (APCs) process donor proteins, including alloantigens, and present them in peptide form in the context of self-MHC class I molecules (26).
B cells, expressing many of the same co-stimulating molecules found on DCs, have also a special place in indirect alloresponses. They play the role of antigen presenting cells to T cells via the capture of alloantigen through their BCR. In return, the crosstalk between T cells and B cells is able to activate B cells. As a result, the activated B cells become either short-lived plasma cells (PCs) without somatic mutation or they enter a follicle in order to undergo gene rearrangement of immunoglobulin chains and a class-switching to produce Ig G rather than Ig M or Ig D (27, 28). The importance of the indirect pathway to transplant rejection was demonstrated by experimental systems in which immunization with peptides of allogeneic MHC was sufficient to mediate rejection (29).
1.2.3 The semi-direct pathway
In this pathway, intact surface donor MHC-peptide complexes are acquired by recipient APCs either through a cell-cell contact or in an exomal manner (30-32).
In this way, recipient DCs acquire and present intact donor MHC class I molecules to CD8+ T cells as well as, internalized and processed donor MHC molecules as peptides to CD4+ T cells.
1.3 The alloresponse to the graft
1.3.1 Critical role of dendritic cells in the priming of alloreactive T cells
The priming of naïve alloreactive T cells results from their interactions with DCs, which most likely occurs in secondary lymphoid tissues. DCs provide signals able to trigger T cell proliferation after TCR engagement and “costimulatory” signals, which are consecutive to interactions between co-stimulatory molecules present on activated DCs such as B7 (B7-1 and B7-2 corresponding to CD80 and CD86), CD40 and OX40 ligand and their respective counter-receptors CD28, CD40- ligand (CD154) and OX40 on T cell membranes (33). Following alloantigen recognition, in the absence of pharmacological intervention with immunosuppressive drugs, intense infiltration of lymphokine-secreting alloreactive T cells occurs in the graft, including the expression of MHC class II on endothelial and epithelial cells, conferring the ability to present antigen to CD4+ T cells (34, 35). All these interactions result in the activation of several transcription factors such as NFκB, which initiate the transcription of numerous genes coding for chemokines and cytokines, co-stimulatory molecules themselves resulting in T cell clonal expansion and protection from apoptosis.
Once activated by both TCR and co-stimulatory signals, T cell expansion further requires growth factors, mainly IL2, IL4 and IL15 (36-38).
1.3.2 Differentiation of alloreactive CD4 T cells into Th1, Th2 or Th17
CD4+ T cells play an essential role in rejection as shown by the inability of CD4- deficient mice to reject organ allografts (39). In the context of transplantation, CD4+ T cells can differentiate into 3 different subsets whose functional properties are characterized by the cytokines they secrete. Cytokines are one key factor
driving Th1, Th2 or Th17 expansion during the initial steps of CD4+ T cell activation. IL12 and IFN-γ promote Th1 differentiation. IL12 directly acts on CD4+
precursors where it stimulates IFN-γ synthesis and inhibits IL4 production. IFN-γ has anti-proliferative effects on emerging Th2, but not Th1 cells, as functional IFN-γ receptors are expressed on Th2 cells only. In addition, IFN-γ up-regulates the expression of IL12 receptor β2 chain on naive T cells and inhibits their IL4 production. IL4 favors Th2 responses by directly down-regulating the transcription factors promoting IFN-γ synthesis.
Th1 cells differentiation is mediated through IL-12 and the subsequent expression of STAT4. STAT4 collaborates with T-bet, a transcription factor that is induced by TCR signaling and strongly activated by activation of STAT1, which occurs in a positive feedback loop in response to auto/paracrine production of INFγ (40). Th1 cells secrete cytokines that include IFN-γ, IL2, IL12 and tumor necrosis factor (TNF) that will result in the context of rejection, in the activation of CD8+ cytotoxicity, macrophage-dependent delayed-type hypersensitivity, and the synthesis of complement-fixing IgG antibody by B cells. In addition, Th1 cells may become cytotoxic by the expression of Fas-ligand on their surface. Although Th1 response has been abundantly studied in murine models of active transplant rejection, the role of Th1 cells in human transplant rejection is questioned (41- 45). GATA-3, STAT6 and IL-4 govern Th2 cell differentiation. Th2 cells secrete IL4, IL5, IL-6, IL9 and IL13, which activate eosinophils (46). Although Th2 responses and IL-4, in particular, have traditionally been associated with antagonism of Th1 response and delayed-type hypersensitivity, there is now
evidence that in some circumstances IL-4 can drive Th1 responses and rejection.
Indeed, high level expression of IL-4 during initial antigen activation of DCs promotes their expression of IL-12 and development of Th1 responses (47, 48).
Several studies have shown that Th2 cytokines promote rejection in animal models by inducing intragraft recruitment and activation of eosinophils (49-51).
Contrary to Th1 cells, alloreactive Th2 cells do not express Fas-ligand and therefore do not mediate direct cytotoxicity. Th17 cells are considered as a distinct lineage of CD4+ helper cells that are regulated by the other Th1 and Th2 lineages. CD4+ T cell differentiation into Th17 and further expansion requires a mix of cytokines. While tumor growth factor beta (TGF-β) and IL6 are needed for the initial differentiation into Th17 subtype, IL6 and IL21, a cytokine up-regulated by IL6, activate STAT3. In addition, Th17 specifically express and require the presence of the transcription factor retinoic acid-related orphan receptor (RORγt).
Il 23, produced by APCs, is not required for the initial phase of Th17 differentiation, but it plays a role in the expansion and stabilization of Th17 cells.
Th17 response induces tissue damage through the secretion of IL17, which triggers the production of chemokines by endothelial and epithelial cells, the recruitment and the activation of neutrophils, as well as TNFα secretion (20, 52).
Th17 cells also act as excellent B-cell helpers through the secretion of IL17 and IL21, mediating the formation of germinal centers and isotype class switching (53). Conclusive demonstration that Th17 cells are capable and sufficient to mediate allograft rejection on their own has been recently reported in models of murine cardiac transplantation. T-bet knockout and T-bet INF-γ double-knockout
recipients of MHC class II-mismatched grafts showed accelerated graft rejection relative to wild type, as a result of profuse tissue infiltration of Th17 cells (54, 55).
More recently, Th17 cells were demonstrated to be critical for spontaneous rejection of minor but not major antigen-mismatched wild type mice by the lack of neutrophils infiltration following IL-17 neutralization (56). Th17/Treg ratio imbalance was found to play a central role in allograft rejection in this model but also in a wild-type murine cardiac transplantation model (57). It is hypothesized that Tregs could promote Th17 differentiation by several mechanisms. One of them might be the production of TGF-β by Tregs, which allows naive T cells to differentiate into Th17 or a phenotypic conversion of Tregs into Th17 cells after Foxp3 downregulation, both in the presence of IL-6, a key cytokine involved in Th17 differentiation (56).
Thereafter, alloreactive T cells primed in lymph node or spleen and circulating blood leukocytes such as monocytes and eosinophils are guided to the allograft by a chemoattractant gradient of chemokines released by the transplant itself (58). “Leukocyte homing” can be subdivided into 3 steps. First, endothelial cells (ECs) are activated, leading to the expression of selectins. The binding of selectins to their ligands on leukocytes slows their flow and the leukocytes start rolling on the endothelium. The second step involves the secretion of chemokines, which attract more leukocytes to the site of inflammation and leads to the firm attachment of leukocytes to endothelium. This adhesion is mediated by integrins on leukocytes binding to their ligand, either on the EC surface or the
extracellular matrix. The third step is the extravasation of leukocytes into surrounding tissues (59).
1.4 Effectors of graft destruction 1.4.1 Alloreactive T cells
Once activated, T cells acquire cytotoxic properties that enable them to kill their targets. The two main cytotoxic mechanisms are the perforin/granzyme and the Fas/Fas ligand (FasL) systems. The perforin/granzyme pathway is used foremost by CD8+ T cells and NK cells. The acquisition of cytotoxic properties by CD8+ T cell precursors requires the provision of Th1 cytokines, mainly IL2. When a cytotoxic lymphocyte (CTL) recognizes the allo-MHC molecule, it forms a tight junction with the allogeneic cell, allowing CTL granules to fuse with the target cell membrane. Perforin molecules insert within the allogeneic cell membrane and form polymers that create channels, through which granzymes A and B penetrate into the cytoplasm. There, granzymes can either directly enter within the nucleus, or they may cleave cytoplasmic procaspases into caspases which will then also move into the nucleus. Caspases are a family of cysteine proteases that cleave aspartate residues from many substrates. Granzyme B acts on the mitochondria to release cytochrome C, which can also trigger the caspase system. Caspase activation is responsible for DNA fragmentation and leads to apoptosis (60).
Fas/FasL interaction is the most important mechanism for CD4+ CTL-mediated cytotoxicity. While Fas, a member of the TNF family of death receptors, is constitutively expressed on most cell surfaces, FasL is essentially inducible. Its
expression becomes apparent 4 to 5 hours after T cell activation. FasL expression is restricted to Th1 alloreactive T cells subtypes. The interaction Fas- FasL results in the death-inducing signal complex (DISC) formation and the activation of caspase cascade that will ultimately induce target cell apoptosis similar to that induced by the perforin/granzyme system (60).
1.4.2 Natural Killer cells
Natural killer (NK) cells, a key cell type in the innate immune system, are frequently found in rejecting allografts (61-63).
But their exact role in solid organ transplantation remains not completely understood. Indeed, in various transplant models, NK cells have been shown to contribute to both allograft rejection and transplant tolerance (64-66).
NK cells have both stimulatory and inhibitory receptors on their cell surface that modulate NK functions (67). The inhibitory receptors include killer-cell immunoglobulin-like receptors in humans and Ly 49 in mice. In addition, NKG2A and CD94 usually form heterodimers on the cell surface and function as inhibitory NK receptors (68). An important feature of this system is that the ligands for such inhibitory receptors are self MHC class I molecules, and therefore, NK cells are in a state of dominant inhibition by constantly engaging the ubiquitously expressed self-MHC class I molecules.Activating receptors such as NKG2D recognize stress signal ligands or pathogen-derived ligands expressed on transformed cells or infected cells (e.g. H60, Rae-1, ULBP, MIC, Mult-1), making possible for NK cells to also kill stressed self target cells despite
the expression of inhibitory self MHC class I molecules (69). With regard to transplantation, NK cells are likely to induce allograft injury through interactions with other immune cells. First, NK cells may interact with donor DCs that expressed mismatched MHC class I molecules on their surface, promoting the secretion of INF-γ and the activation of the perforin/granzyme and Fas-Fas ligand pathways (70). IL-15 is also required for their expansion and maturation (71, 72).
Second, NK cells may also interact with T cells and directly enhance T cell alloreactivity, such as by producing INF-γ that promotes Th1-like response in a paracrine fashion or by direct contact with via OX40-OX40L interaction. Finally, NK cells are also able to mediate allograft injury through antibody-dependent cellular toxicity triggered by antibody Fc receptor binding and activation of host NK cells (73).
1.4.3 Macrophage activation and delayed-type hypersensitivity reaction
Delayed-type hypersensitivity (DTH) reactions are characterized by tissue swelling and induration consecutive to increased vascular permeability and the presence of an inflammatory infiltrate rich in T cells, macrophages and neutrophils. This reaction is delayed because, unlike immediate hypersensitivity mediated by preformed antibodies, some days are required to prime antigen- specific Th1 cells. Indeed, activated Th1 cells are at the heart of DTH through the release of IFN-γ and TNF-α. This in turn will trigger macrophages to produce toxic molecules such as nitric oxide (NO), oxygen intermediates, IL-1, INF- γ and TNF- α. NO, a highly reactive nitrogen metabolite, produced by the inducible form of NO synthase (iNOS), is cytotoxic at high concentrations. It also elicits the
vasodilation and oedema characteristic of DTH. TNF-α binds to TNF-receptors and induce target cell apoptosis or necrosis through caspase activation, as described for Fas/FasL and perforin/granzyme systems. Activated neutrophils release myeloperoxydase which will then generate toxic metabolites such as oxygen species and H2O2 (60).
1.4.4 Eosinophils and Th2 type alloreactive response
Eosinophils are recruited and activated within the allograft through the combined action of IL4, IL5, IL-9 and IL13 produced by alloreactive Th2 cells. IL5 plays an essential role in the differentiation and proliferation of eosinophils in the bone marrow (74, 75). IL4 and IL13 up-regulate the expression of VCAM-1 on ECs, a critical adhesion molecule for eosinophils which express the counter-receptor VLA-4 on their membrane (76-78). Furthermore, IL4 and IL13 stimulate the production of eotaxin, a CC chemokine, by several cell types including ECs (79- 82). IL5 and eotaxin then collaborate to recruit and activate eosinophils within inflamed tissues (83, 84).
Activated eosinophils release granules that contain highly noxious substances such as major basic protein (MBP), eosinophil-derived neurotoxin, eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO) (85). ECP and EDN are ribonucleases and ECP creates toxic pores into the target cell membranes. MBP induces smooth muscle cell hyperreactivity by causing dysfunction of vagal muscarinic M2 receptors. EPO is probably the most toxic molecule which leads
to the formation of brominating species responsible for oxidative damage of tissue proteins (86).
1.4.5 Neutrophils
In the early course of organ transplantation, oxidative stress induced by ischemia-reperfusion promotes the liberation of the pro-inflammatory cytokines TNF-α and IL-1 within the graft, which stimulate ECs to produce chemoattractants such CXCL8, CXCL1 and CXCL2 (58). The resulting influx of neutrophils is responsible for the early graft injury. Neutrophils induce tissue damage through several mechanisms, including the release of myeloperoxydase which will then generate toxic metabolites such as oxygen species and H2O2 (87). They also have direct cytotoxic effects by their expression of FasL and by antibody-dependent cytotoxic activity, involving perforin and granzym B (88).
Moreover, neutrophils were also seen to be recruited and activated following IL- 17 production by Th17 cells, CD8+ and CD4+ T cells during allograft rejection (54, 56, 89). In the kidney, IL-17 stimulates tubular epithelial cells to produce high levels of IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1) and complement component C3, which act as chemoattractants for neutrophils (90).
In addition, neutrophils themselves may facilitate rejection because they have the power to attract and activate immature DCs, thereby sensitizing naive T cells and also to induce DC maturation by increasing the expression of costimulatory molecules including CD40, CD80, CD86 (91). Neutrophils also act as APCs.
They can capture and present exogenous antigens via their class I molecules to
CD8+ T cells (92). In the context of transplantation, neutrophils were shown to promote cytotoxic T cell responses and Th1 polarization of CD4+ T cells alloreactive against alloantigens of the MHC class I and class II MHC, respectively (93).
1.4.6 Alloantibodies
It is established that antibodies can mediate EC injury through complement- dependent mechanisms (94) but data also indicate that antibodies also contribute to alterations in ECs through complement independent mechanisms by transducing proinflammatory and proliferative signals (95, 96).
1.4.6.1 Complement activation
Complement-fixing IgG1, IgG3 and IgM antibody bind to MHC present on the endothelium and are able to activate the complement cascade that leads to the formation of the “membrane attack complex” composed of C5b fragment bound to proteins C6-9 (Figure 2). The membrane attack complex causes the lysis of ECs, dependent on C6 and triggers the proliferation of ECs via the release of growth factors (platelet-derived growth factor, β-FGF) and chemokines (97). The cleavage products C3a and C5a are chemoattractants for macrophages and neutrophils by increasing adhesion molecules expression on ECs (98, 99). Both C5a and C5b-C9 also trigger the synthesis of tissue factor, which may be responsible for the thrombotic injury (100, 101). Moreover, C3a and C5a release prostaglandin E2 from macrophages and histamine from mast cells, respectively (102).
In addition, NK cells may bind through their Fcγ receptor to IgG1 or IgG3-coated cells. The cross-linking of NK Fc receptors triggers perforin/granzyme-mediated NK cytotoxicity, a process called antibody-dependent cell cytotoxicity (ADCC). As NK cells possess receptors for complement component C3b (CR3), NK activity in this setting is further stimulated by the activation of complement cascade.
Likewise, cells like macrophages, neutrophils and eosinophils are also activated by the cross-linking of their respective FcR, as well as by complement (103).
1.4.6.2 Non complement-dependent mechanisms
Antibody ligation of class I anti-HLA antibodies on ECs is able to stimulate proliferation by up-regulating the expression of FGF receptors on the cell surface in a dose-dependent fashion, increasing FGF binding and activating the ERK signaling pathway (104, 105). It also triggers activation of mammalian target of rapamycin complex 1 (mTORC1), resulting in concomitant activation of cell survival and proliferation signaling pathways (Figure 3). Activation of mTORC1 stimulates protein synthesis and therefore, cell proliferation by activating protein S6 kinase (S6K) which phosphorylates S6 ribosomial protein (S6RP) and 4E- BP1 protein (106). Up to now, little is known about signal transduction in response to class II ligation on ECs. Although the immune response to HLA antigens plays a central role in allograft destruction, recent reports show that alloimmune-mediated injury of the endothelium leads to the expression of self- antigens and subsequent generation of auto-antibodies (107). The lack of evidence for complement activation by non-HLA antibodies implies that they mediate graft injury via complement independent mechanisms. For example,
antibodies to the minor antigen vimentin, an intermediate filament protein mainly expressed in the intima and media of coronary arteries, as well as anti-HLA antibodies promote platelet and leukocyte recruitment by exocytosis of von Willebrand factor and externalization of P-selectin (108-110). Likewise, anti- angiotensin 2 type 1 (AT1) receptor antibodies transduce signals activating ERK and therefore, the transcription factor AP-1 and the nuclear factor NF-κB, resulting in an increased expression of proinflammatory cytokines, procoagulatory genes and cell proliferation (111).
Under certain circumstances, anti-EC antibodies may be beneficial to graft outcome by promoting accommodation. Accommodation is an acquired resistance of an organ allograft to antibody-mediated injury. This phenomenon was firstly observed in ABO-incompatible renal transplants and was extensively studied in vitro and in vivo in experimental xenotransplants (112).
Accommodation is induced by crosslinking proteins bearing carbohydrate epitopes such as ABO and Gal, which elicits survival signaling cascades (Figure 4). In experiments, accommodation could be achieved by lowering the titers of anti-donor antibodies, by inhibiting complement activation or by repeated injection of low-dose anti-donor antibodies (113-116). Treatment with low-dose antibodies up-regulated the expression of the complement regulatory protein decay accelerating factor (DAF), complement receptor-related protein (Crry) and CD59 in the graft endothelium, which in turn negatively regulated complement activation. Recently, the administration of cobra venom factor during 15 days in donor-skin presensitized rhesus monkeys who received the kidney graft from the
same donor, successfully prevented acute AMR, and resulted in accommodation and long-term allograft survival in most of sensitized non-human primates (115).
To date, how long accommodation can be maintained through persistent exposure of ECs to anti-HLA antibodies remains an important issue, even if they are at low titres. Indeed, data from mice and monkeys suggest that long-term exposure to anti-MHC antibodies leads to chronic allograft rejection, rather than accommodation (117-118).
1.5 Relationship of anti-HLA antibodies with renal graft outcomes 1.5.1 Methods for detection of anti-HLA antibodies in serum
Lymphocytotoxicity assays, as first described in 1969 by Patel and Terasaki, have formed the basis of antibody detection through decades. Even today, the presence of donor-specific anti-HLA antibodies (DSA), leading to a positive complement-dependent cytotoxicity (CDC) crossmatch (XM), remains a contraindication for transplantation since it has been associated for a long time with a high risk of graft loss due to hyperacute or earlyacute rejection (AR) (119).
In recent years, HLA antibodies screening assays, more sensitive than CDC, have become available. These assays fall into mainly three categories, namely, the flow-cytometry XM, ELISA-based methods and single HLA antigen-coated bead assaysused in a Luminexplatform (120-122).
1.5.1.1 Complement-dependent cytotoxicity methods
Lymphocytes from a single donor (XM) or a panel of donors (panel reactive antibody) are mixed with sera from a potential recipient. DSA, if present will bind
to their appropriate antigen. In the subsequent wash steps, any unbound antibody is removed from the reaction well. When complement is added, complement-activating antibodies will lead to the formation of the membrane attack complex and the cells to which the antibody is bound will be killed. The antibodies that are detected by cytotoxicity are usually against HLA antigen but occasionally maybe against non-HLA antigen also. They may be IgG or IgM.
A fairly widespread belief exists that antibodies of the IgM type detected during XM are often auto-antibodies and are harmless for transplantation (123).
However, it has been reported that donor-specific IgM antibodies have been detected in B-cell cultures from the peripheral blood of allograft recipients indicating the development of de novo immune responses against the transplanted organs (124). Their impact on graft outcome remains unclear although IgM antibodies against donor-specific HLA class I antigens were seen to be less detrimental than IgG antibodies (125).
1.5.1.2 Flow cytometry (FC)
Even low titer and non-complement fixing antibodies may be detected through this method but there is no false positive result for IgM antibody. Donor cells are mixed with recipient serum and washed to remove unbound antibody. Instead of complement, antibody to human IgG that has been conjugated with a fluorescent dye is added. This secondary antibody binds to lymphocyte-bound antibody. If a threshold of fluorescence is reached, then the test is considered to be positive for the detection of antibody.
1.5.1.3 Solid-phase assays
Elisa and Luminex single antigen beads assays are techniques that are able to detect non-complement-fixing antibodies but also Class I and Class II HLA antibodies or non-HLA antibodies such as MICA antibodies even of low titres.
Recipient serum is mixed with beads or an Elisa platform that bear purified recombinant HLA antigens. The addition of a human IgG conjugated with phycoerythrin permits to measure a degree of fluorescence that represents the amount of antibody present in the serum sample.
Up to now, donor-specific HLA antibodies defined by such methods have been considered a risk factor, rather than as a contraindication for transplantation.
All those techniques detect serum antibody levels, but not the potential activity of alloantibody producing cells. Very recently, a group from The Netherlands developed a novel HLA-specific B cell Elispot assay to detect and enumerate HLA-specific memory B cells that are able to produce antibodies (126). They used HLA monomers as targets in the Elispot assay.
This assay might be useful in several situations: (i) in case of an increment of B- cell numbers with antidonor specificity was observed, this result might be taken as a predictor of HLA antibody formation while HLA antibodies not yet detectable in recipient serum; (ii) to reveal the actual status of the humoral response, in cases where absorption of antibodies to the graft was suspected; (iii) if memory B cells with anti-donor specificity were present in transplant recipients, they could be a signal for not reducing immunosuppression. Up to now, the predictive value
for humoral rejection of this novel assay has not been established and therefore, deserves further investigation.
1.5.2 HLA sensitization before transplantation
Nowadays, ~15-30% of all patients awaiting a deceased donor kidney transplant are sensitized to HLA because of pregnancy, blood transfusion or previous graft.
Highly sensitized patients, defined by a panel reactive antigen (PRA) ≥85%, represent a minority of ~1% of patients on the waiting list. They have developed antibodies against a large variety of HLA antigens and have the lowest chance to receive a XM negative organ from standard allocation procedures. As a result, they often accumulate on the waiting list.
At the present time, two strategies have been developed to overcome this problem. Firstly, Eurotransplant has enrolled patients with a PRA ≥85% in the acceptable mismatch programme, which, by forbidding HLA antigens against which the patient has developed complement-fixing antibodies, allows for an improved identification of XM-negative donors and gives the highest priority to allocation of these donors to highly sensitized patients (126, 127).
Secondly, different desensitization protocols for the removal of anti-HLA antibodies have appeared in order to prevent antibody-mediated rejection (AMR) and therefore, to increase the probability of a successful transplantation. They include plasmapheresis, intravenous immune globulins (IVIG), rituximab, anti- thymocyte globulin (ATG). These agents either target B cells through different
mechanisms of action or will decrease the titres and/or activity of anti-HLA antibodies (128-136).
However, two major issues related to the use of current desensitization therapies remain unsolved. Firstly, in many cases, low titres of DSA remain present and these approaches are, therefore, still complicated by high rates of early humoral AR and late antibody-mediated injury, such as transplant glomerulopathy (TGP) (137-139). More and more, it appears that these depleting therapies incompletely prevent the histological lesions of humoral AR, giving rise to a new entity named subclinical AMR, likely contributing to chronic allograft injury (140). Secondly, a major pitfall with desensitization and the therapy of AMR is that most clinical trials are single-centre, non-randomized studies, with low number of patients, that eventually do not allow to clarify the hierarchical importance of plasmapheresis, IVIG, rituximab or other agents (141).
The importance of pretransplant anti-HLA antibodies in the outcome of kidney transplantation has been recognized for over 40 years (142). On the basis of the results obtained by CDC assays, it became clear that the presence of preformed HLA DSA was associated with a high incidence of hyperacute rejection (119, 143). Following this observation, testing transplant candidates for these antibodies became an essential component of pre-transplantation evaluation.
Nowadays, T cell CDC XM remains the gold-standard in the process-making to accept or deny an organ offer. Instead,the clinical relevance of a positive B cell XM, which often appears to be caused by irrelevant auto-antibodies, has been widely debated. However, a positive XM caused by IgG antibodies directed
against donor HLA-DR is associated with inferior graft and therefore, clinically relevant (144).
With the advent of more sensitive methods, the number of HLA sensitized patients increased sharply, making also the question of the clinical relevance of these antibodies. Many studies demonstrated an increase in AR episodes and a decrease in graft survival in kidney transplant recipients transplanted across a positive FCXM, when compared to a negative FCXM (120, 145). However, the significance of anti-donor antibodies detected by positive FCXM is still questionable and is therefore, likely to create a risk of excluding transplant patients who would not experience rejection or graft loss (146, 147). The recent development of solid-phase assays using fluorescent beads coated with purified HLA molecules or single antigen bead assays, has greatly improved the sensitivity of the identification of HLA DSA.
Actually, there was an initial wave of enthusiasm from centers to use this easy tool to determine the specificity of HLA antibodies present in the sera from their transplant recipients and some of them used the results obtained for the so- called virtual XM, which is the prediction of HLA mismatches that will give a positive serological XM reaction. However, many clinicians as well as tissue typers are very confused about the interpretation of the results generated by Luminex single antigen bead assays and its impact on decision making, especially in highly sensitized patients, for which it is difficult to find a compatible donor. Indeed, while HLA antibodies detected by Elisa or Luminex technology have been associated with an increased risk of AR graft loss in most reports,
some authors report that DSA were clinically irrelevant using single antigen beads assays only (148-156). Those discrepancies are mainly due to the lack of standardization of the interpretation of the assays.
Moreover, the predictive value of HLA antibodies detected by such methods remains low at the individual level, with up to more than 50% of patients with preformed HLA antibodies that will never develop rejection and keep a long-term functioning graft. It has been suggested that maybe the determination of subclass, titers, and the ability to fix complement might help to better stratify the risk of AR or chronic graft loss. However, the findings from the first studies that attempted to address this issue were variable and therefore, remain unconclusive (151-153, 156-159).
Major concerns about the high sensitivity of solid-phase assays, are that often HLA antibodies are found in sera, which are negative when testing in CDC or FC, even in patients with no sensitizing events. These so-called “natural” HLA antibodies appear to react with denatured HLA molecules. Indeed, by fixing solubilized HLA molecules to the solid phase, a proportion of molecules may loose their original conformation resulting in a mixture of intact HLA molecules consisting of heavy chain, β2-microglobulin and peptide, and denatured HLA molecules (160, 161).
1.5.3 The relevance of donor-specific HLA antibodies after renal transplantation Circulating HLA DSA after renal transplantation have been clearly associated with AR (162-164). Moreover, Banff classification defined the presence of DSA in
association with C4d deposition in peritubular capillaries and glomerulitis/peritubular capillary leukocytic infiltration as the landmark of acute AMR. However, the frequent report of the combination of DSA and histological features of humoral rejection in the absence of C4d deposition, made the Banff classification evolve to the diagnostic category of “suspicious for AMR” (165).
The presence of DSA at the time of diagnosis of any AR episode is predictive of worse graft outcome, especially in patients who do not experience a significant reduction in DSA levels after the rejection episode (166, 167). Recently, the increased expression of endothelial transcripts using microarrays in allograft biopsies from patients with DSA and allograft dysfunction was also predictive of active antibody-mediated graft damage and poor graft outcome (168).
A large amount of publications have demonstrated that post-transplant DSA were also a marker for worse long-term graft survival, and especially in patients who developed de novo antibodies related to a previous AR episode (169-175). But the precise role of circulating antibodies in the development of chronic rejection (CR) is less clear, particularly antibodies detectable by solid-phase assays only.
Indeed, stable patients and excellent graft function have been described with DSA, raising again the question of the predictive value of HLA antibodies (176).
Up to 25-50% of patients harboring post-transplant HLA antibodies have long- term functioning grafts without leading to CR (171, 172).
Absence of injury in the face of immunity is typical of accommodation. However, this accommodation seems to be an unstable state as described in cynomolgus monkeys treated with various immunosuppressive regimens (118). Moreover,
TGP, a feature of chronic AMR has been reported in the absence of DSA (177, 178).
1.6 Overview of the relationship between antibodies against non-MHC antigens and organ transplantation and special focus on MICA antibodies
Since several years, there exists a growing interest of the transplantation community to study the immune response to non-MHC antigens, which are expressed on ECs and epithelial cells, and its impact on graft outcomes.
The clinical importance of sensitization to non-MHC antigens was suggested by reports of hyperacute rejection and long-term graft loss of HLA-identical sibling grafts (179-181). In addition, antibodies against non-HLA antigens have also been recognized to contribute to the pathogenesis of acute AMR and have also been described in HLA negative patients with TGP (178, 182-185). Only the relationship between humoral sensitization to non-MHC antigens and graft outcomes has been investigated until now, while cellular sensitization has been obscured because of the lack of soluble products in most cases, raising the question about the role played by T cell response to non-MHC antigens in the development of allograft injury. In contrast, T cell sensitization to MHC antigens was determined in kidney transplant recipients using IFN-γ-enzyme-linked immunosorbent spot (ELISpot) assay identifying alloreactive donor-reactive effector/memory T cells. The pretransplant cellular allo-immunity measured by this method was correlated to AR and renal graft function impairment (186, 187).
1.6.1 Non-MHC targets of the humoral response in renal transplantation
1.6.1.1 Anti-endothelial cell antibodies (AECA)
AECA, comprising both IgM and IgG subclasses represent an extremely heterogeneous family,not only because of the variety of their target antigens, but also the subsequent diversity of their effects. The main effects of AECA are the induction of the up-regulation of adhesion molecules with consequent mononuclear cell adhesion and EC apoptosis (188-190).These antibodies have been reported before transplantation and in the post-transplant setting and were correlated to AR or CR in kidney, heart and liver (190-196). In addition to CDC, most studies have used Western Blot and Elisa to investigate the presence of AECA in the setting of transplantation. However, the knowledge of the antigenic specificity was lacking in those studies.
Classical lymphocyte crossmatching techniques fail to detect such antibodies.
Nevertheless, the development of a novel FCXM technique (XM-ONE) using peripheral blood endothelial progenitor cells as targets, has recently emerged, which was able to determine patients at risk for rejection and reduced graft function not identified by conventional lymphocyte XMs (197). Very recently, the longitudinal analysis of post-transplant sera for circulating allo-antibodies was performed by ECXM using EC cultures prospectively isolated from the transplant donor at the time of transplantation and from a third-party culture bank (198).
ECXM identified both DSA and NDSA reactive to ECs. Antibodies were mainly of IgG1 isotype. Of interest, this study provided evidence after gene transcription analysis that circulating DSA and NDSA reactive to ECs triggered selective regulatory pathways reflected by CCR4 and IL-1β up-regulation, respectively.
