Mechanistic Aspects of Cell-Mediated Rejection

Transplantation Pathology

Contents Mechanistic Aspects of Cell-Mediated Rejection Allograft Tolerance Xenotransplantation Liver Transplantation Pathology of Lung Transplantation Pancreas and Islet Transplantation for the Treatment of Diabetes Mellitus Pathophysiology of the Skin and Oral Squamous Mucosa in Allogeneic Hematopoietic Stem Cell Transplantation Immunosuppression in Transplantation

Mechanistic Aspects of Cell-Mediated Rejection P Randhawa, University of Pittsburgh, Pittsburgh, PA, USA ã 2014 Elsevier Inc. All rights reserved.

Introduction

Macrophages bear pattern recognition receptors (PRRs) that can detect repeating structural units expressed by pathogens referred to as pathogen-associated molecular patterns (PAMPs). PRRs also detect tissue damage-associated molecular patterns (DAMPs) on a broad range of molecules including heat-shock proteins, heparin sulfate, fibrinogen, and highmobility group box-1 (Figure 1). PRRs are a rather diverse family of molecules and include (a) transmembrane proteins such as Toll-like receptors (TLRs) and C-type lectin receptors,

(b) intracellular proteins such as nucleotide-binding oligomerization domain receptors and retinoic acid-inducible gene-I-like receptors, and (c) secreted molecules such as mannose-binding lectin. Recognition of DAMPs by PRRs results in enhanced gene transcription, and synthesis of mRNAs leads to the production of proinflammatory and chemoattractant cytokines and growth factors including interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF), type I interferons, MIG/CXCL9, RANTES/CCL5, endothelial P-selectin (CD62P), transforming growth factor (TGF), platelet-derived growth factor, and insulin-like growth factor-1. Increased chemokine receptor expression by macrophages (also neutrophils, NK cells, T cells, and B cells) enables these cells to migrate along a chemoattractant gradient into the transplanted organ. Production of reactive oxygen species and protein degradative enzymes by activated macrophages injures the vascular endothelium and other anatomical compartments of the graft parenchyma. DAMPs can also activate the alternate and lectin pathways of complement. The complement system is primarily a proinflammatory proteolytic cascade in the plasma. Some complement components can be synthesized in the kidney and liver. The physical process of harvesting, transporting, reimplanting, and reperfusing donor organs during transplantation can activate complement. For deceased organ donors, an additional proinflammatory element is a cytokine release syndrome triggered directly by brain or cardiac death. The resulting graft injury amplifies the T-cell response of the recipient to alloantigens, thus providing a link between the innate and cellmediated immune systems. Several mechanistic links also exist between the innate and antibody limbs of antidonor immunity:

Pathobiology of Human Disease: A Dynamic Encyclopedia of Disease Mechanisms

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Rejection of allograft organs can be mediated by cells and antibodies. The term cell-mediated rejection is generally used to refer to T-cell-mediated allograft rejection. Here, we will interpret the term in a broader context and cover the role of a broad range of cells that belong to the innate and acquired immune systems. The innate immune system is important because it is present since birth and its function is independent of prior sensitization by foreign antigens (Figure 1). The principal constituent cell types include macrophages, dendritic cells (DCs), neutrophils, mast cells, and natural killer (NK) cells. In contrast, the acquired or adaptive immune system consists of T cells and B cells that must first be primed by foreign antigens, and only a subsequent reexposure to those antigens elicits an inflammatory reaction in the tissue. As a general rule, innate mechanisms are not sufficient to elicit rejection per se, but they do interact with, augment, and perpetuate tissue damage caused by the acquired immune system. Antibody-mediated rejection is discussed elsewhere in this encyclopedia.

Macrophages

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Infection

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Inflammation tissue repair adaptive immunity Figure 1 Role of the innate immune system in eliciting inflammation and tissue injury. Infectious agents express repeating structural units called pathogen-associated molecular patterns (PAMPs). Likewise, tissue damage due to ischemic or chemical injury results in expression of damageassociated molecular patterns (DAMPs). PAMPs interact with cell surface Toll-like receptors (TLRs) 1, 2, 4, 5, and 6 and with intracellular nucleotidebinding oligomerization domain (NOD) receptors 1 and 2. In contrast, DAMPs are recognized by intracellular TLRs 7, 8, and 9. Both PAMPs and DAMPs ultimately result in increased production of NF-kB, which leads to upregulation of major histocompatibility complex (MHC) and costimulatory molecules. Ensuing tissue inflammation and cellular injury releases neoantigens, which can stimulate the adaptive immune system and further enhance alloimmune injury. Reproduced from Brennan, T.V., Lunsford, K.E., Kuo, P.C., 2010. Innate pathways of immune activation in transplantation. J. Transplant. 2010, with permission. http://www.hindawi.com/journals/jtran/2010/826240/.

1. Binding of alloantibody to a cell surface or the formation of immune complexes activates the classical pathway of complement. This results in degradation of C4 into C4a and C4b. C4b and C3b can bind covalently to target cells. The coated targets are engulfed by macrophages that express complement receptors (CRs) in a process that is referred to as opsonization. 2. Binding of CRs on leukocytes to target cell-bound C3b and C4b can facilitate antigen presentation and T-cell activation. 3. Cleavage of C3, C4, and C5 during complement activation results in the release of C3a, C4a, and C5a. These soluble peptides act as anaphylatoxins leading to vasodilation and chemotaxis, thus further augmenting the immune response to the graft. 4. Generation of the terminal components of the complement cascade (C5b-9) punches holes in cell membranes and causes target cell lysis. 5. In a process called antibody-dependent cellular cytotoxicity, macrophages ingest antibody-coated cells by utilizing Fc receptors.

Dendritic Cells DCs are now classified into the myeloid and plasmacytoid types. Together, these cells comprise < 0.5% of circulating white cells. Myeloid DCs stain for CD11c and CD45RO antigens, while plasmacytoid DCs express BDCA-2, BDCA-4, and

CD45RA antigens. The exact pathogenetic role of DC subsets in clinical transplantation has not yet been clarified. However, in general, DCs are extremely potent antigen-presenting cells (APCs). Through this function, they mediate tissue injury caused by both T cells and B cells.

Neutrophils Neutrophils are the most abundant population in circulating white blood cell compartment. They are recruited to the graft as part of the innate immune response and also as a reaction to T-cell- or antibody-mediated injury. Transport across the vessel wall involves binding to endothelial cells as a consequence of leukocyte integrins, interacting with the adhesion molecule ICAM-1 (intercellular adhesion molecule-1). Mechanisms whereby neutrophils can induce tissue injury include secretion of chemokines (CXCL1, 2, 3, and 8), heparin-binding protein, and generation of reactive oxygen species that result in vascular leakage.

Mast Cells Mast cells are the tissue counterpart of circulating basophils. A small proportion, about 1%, can be seen in all inflammatory infiltrates. It is now recognized that IL-9-secreting T cells (Th9 cells) facilitate recruitment of mast cells into inflamed tissue. Increased frequencies are abnormal and can have detrimental effect. In one recent microarray analysis-based study,

Transplantation Pathology | Mechanistic Aspects of Cell-Mediated Rejection

high expression of mast cell transcripts was associated with renal scarring, reduced graft survival, and poor functional recovery.

T Cells T-cell-mediated rejection is one of the best known mechanisms of alloimmune injury. It is the end result of a carefully orchestrated sequence of events that are outlined below: (a) Antigen presentation: Donor-specific peptides derived from allogeneic major histocompatibility complex (MHC) molecules are presented to T-cell receptors (TCRs) by APCs, namely, DCs, macrophages, and B cells. Class I MHC molecules present allopeptides originating in the

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intracellular compartment to cytotoxic CD8 þ T cells (Figure 2). On the other hand, class II MHC molecules present allopeptides that are released into the extracellular space to CD4þ T cells. As one might expect, grafts from HLA-identical siblings survive much longer than HLAmismatched grafts from siblings or unrelated donors. Yet, it is recognized that transplants between siblings with identical MHC molecules are also not completely protected from rejection. Rejection in this latter setting occurs via T-cell recognition of other polymorphic non-MHC molecules called minor histocompatibility antigens. (b) Allorecognition pathways: APCs within the graft carry donor antigens from the transplanted organ to the recipient’s draining lymph nodes and spleen. In turn, recipient APCs residing in lymph nodes or spleen circulate through

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Figure 2 Processing of endogenous and exogenous antigens by class I and class II MHC pathways. Endogenous antigens are digested into peptides by proteosomes and are loaded into class I MHC (left). In contrast, exogenous antigens are degraded within endosomes and are loaded into class II MHC (right). Assembly of the MHC within the cell’s endoplasmic reticulum precedes its transport through the Golgi apparatus and its ultimate expression on the cell surface along with peptide, where the MHC–peptide complex interacts with CD8þ or CD4þ T cells. b2m, b2-microglobulin; CLIP, class II-associated invariant-chain peptide; HLA-DM, an intracellular protein involved in peptide presentation; TAP, transporter associated with antigen processing. Reproduced from Schwartz, R., 2010. Rejection of the kidney allograft. N. Engl. J. Med. 363, 1451–1462, with permission.

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the graft and then home to lymphoid organs where they may effect activation of additional T cells. Priming of recipient T cells with antigen can occur by three distinct pathways designated as direct, indirect, and semidirect (Figure 3). In direct allorecognition, recipient TCRs interact with intact allogeneic MHC–peptide complexes presented by donor-derived APCs. Indirect allorecognition refers to a process wherein peptides derived from donor MHC are degraded and presented by recipient APCs. The term semidirect allorecognition refers to transfer of cell membrane fragments from allogeneic cells expressing donor MHC to recipient APCs. The direct pathway of allorecognition plays a dominant role in the early posttransplant period. As the number of migratory donor APC in a transplanted organ is limited, the role of the direct pathway in allograft rejection diminishes with time.

Therefore, the indirect pathway becomes the primary mechanism of allorecognition at later time points after transplantation. (c) Costimulation: Allorecognition results in antigen-specific signals being delivered to the T cell through the TCR– CD3 complex. However, complete activation of naive T cells requires a second essential signal provided by the interaction of costimulatory molecules with their ligands. Costimulatory molecules can be classified into two families. The B7 family is exemplified by CD28, CD80, CD86, and CD152 (CTLA-4) (Figure 4). The TNF/TNF receptor family includes CD40 and CD154 (CD40L) as the best known receptor–ligand pair. CD28 is constitutively expressed by T cells and binds CD80 and CD86 on APCs. Signaling through CD28 lowers the threshold for T-cell activation and promotes T-cell proliferation and resistance

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Recipient T cells Figure 3 Direct and indirect antigen presentation pathways. In direct allorecognition, recipient (color-coded green) T-cell receptors (TCRs) interact with intact allogeneic MHC–peptide complexes presented by donor-derived (color-coded yellow) antigen-presenting cells (APCs). In contrast, indirect allorecognition involves presentation of donor-derived MHC peptides by recipient APCs to recipient TCRs. Reproduced from Benichou, G., 1999. Direct and indirect antigen recognition: the pathways to allograft immune rejection. Front. Biosci. 4, D476–D480, with permission.

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Figure 4 Costimulation pathways in professional APCs and T cells. Allorecognition results in antigen-specific signals being delivered to the activated T cell through interaction between the MHC and the CD3 TCR. However, complete activation of T cells cannot proceed without a second set of signals provided by the so-called costimulatory molecules and their corresponding ligands. For example, the costimulatory molecules B7-1 and B7-2 on APC interact with CD28 on activated T cells to enhance T-cell activation and proliferation. In contrast, interaction of B7-1 and B7-2 molecules with CTLA-4 generates an inhibitory signal that acts as a negative regulator of immune responses. Reproduced from Karandikar, N.J., Vanderlugt, C.L., Bluestone, J.A., Miller, S.D., 1998. Targeting the B7/CD28:CTLA-4 costimulatory system in CNS autoimmune disease. J. Neuroimmunol. 89 (1–2), 10–18, with permission.

Transplantation Pathology | Mechanistic Aspects of Cell-Mediated Rejection

to apoptosis. CD28 signaling during T-cell activation also upregulates CD154 (CD40L), which is a ligand for CD40 expressed by APCs. CD40–CD154 ligation activates APCs, causes increased expression of B7 family molecules, and amplifies T-cell activation. In contrast, upregulated expression of CD152 (CTLA-4) inhibits immune responses by competing with CD28 for ligation of CD80 and CD86. The importance of CD152 as a negative regulator of immune responses is highlighted by studies of CD152 knockout mice: these animals develop a fatal disorder characterized by massive proliferation of lymphocytes in response to normal environmental antigens. Thus, the outcome of T cell–APC interaction is determined by the balance of positive and negative signals delivered by the costimulatory molecules. (d) Signal transduction: The biphospholipid layer of cell membranes in activated T cells contains cholesterol-rich regions referred to as ‘lipid rafts.’ Lipid rafts are not seen in resting T cells. During antigen recognition by T cells, multiple TCRs binding to MHC–peptide on the surface of the APC cause clustering of costimulation, signaling, and adhesion molecules, resulting in the formation of a supramolecular activation complex. The cell membrane in the vicinity of these interactions reorganizes and allows TCR–CD3 complexes to integrate into lipid rafts. This reorganization facilitates downstream signaling by placing TCR–CD3 complexes near signal transduction molecules. TCR–MHC–peptide engagement triggers phosphorylation of several signaling molecules, which in turn results in activation of the Ras- and Rac-mitogen-activated protein kinase pathways. Hydrolysis of membrane phosphatidylinositol 4,5-biphosphate generates the secondary messengers inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 releases stored calcium from the endoplasmic reticulum and activates the phosphatase calcineurin, which in turn dephosphorylates a transcription factor called nuclear factor of activated T cells (NFAT). Generation of DAG also results in the activation of transcription factors nuclear factor-kB (NF-kB) and AP-1. These transcription factors cause upregulation of IL-2 and IL-2 receptor a -chain (CD25). IL-2 behaves like an autocrine and paracrine proliferative cytokine, which delivers the so-called signal 3 for the clonal expansion and differentiation of activated T cells. (e) Helper T-cell differentiation: Activation of T cells in response to antigenic stimulation is followed by their differentiation into cells with specific cytokine signatures and function. CD4þ class II-restricted T cells usually acquire helper function (Th), while CD8þ class I-restricted T cells usually evolve into cytotoxic T lymphocytes (CTLs). Factors that influence the direction of T-cell differentiation include immune status of the recipient, extent of ischemia– reperfusion injury, degree of donor recipient mismatch, antigen load, and immunosuppressive regimen. In essence, these factors determine the cellular and cytokine milieu and hence the exact signals that drive T-cell differentiation. Thus, ischemia–reperfusion injury typically results in the production of DAMPs, which stimulate TLR signaling in APC. IL-12 secreted by APCs promotes differentiation of Th1 cells that express the transcription factor

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Tbet and secrete interferon-g (Figure 5). While the generation of Th1 cells is triggered in an antigen-specific manner, the effector mechanisms that lead to the destruction of the graft by the so-called delayed-type hypersensitivity (DTH) response are nonspecific. DTH reactions are characterized by the release of multiple proinflammatory cytokines that trigger infiltration by monocytes, macrophages, neutrophils, and eosinophils. These inflammatory cells produce mediators, such as nitric oxide, reactive oxygen species, and arachidonic acid derivatives (prostaglandin E2, thromboxane, and leukotrienes), which play a role in both acute and chronic allograft rejection. Several other helper T-cell differentiation pathways have been defined. Thus, a cellular microenvironment rich in IL-4 is known to promote generation of Th2 cells, which can express GATA-3 and secrete IL-4 themselves to attract eosinophils into rejection-associated inflammatory infiltrates (Figure 5). In different experimental models, TGF-b, IL-6, IL-1-b, IL-21, or IL-23 can facilitate emergence of T cells that secrete IL-17 (Th17 cells). IL-17 is a proinflammatory cytokine that can promote granulopoiesis and neutrophil migration to the graft. Generation of IL-9secreting T cells (Th9 cells), which facilitate recruitment of mast cells, is dependent on the presence of TGF-b and IL-4. Finally follicular helper T cells (Tfh cells) require IL21 for differentiation, express the transcription factor bcl6, and play a role in B-cell maturation and antibody production. (f) CTL differentiation: As mentioned earlier, it is CD8 þ class Irestricted T cells that get earmarked for differentiation toward the CTL lineage. Generation of CTLs occurs in the context of three-cell clusters comprised of CD8-positive T cells, CD4-positive T cells, and APC. Activated CD4 þ T-helper cells can also ‘license’ APC to activate CTLs directly. CTL differentiation is aided by CD40–CD154 costimulatory signals. Once generated, CTLs migrate to the graft and identify their target cells in the context of allopeptides presented by class I MHC molecules. Mechanisms of target cell injury include release of granules containing cytotoxic molecules (perforin and granzyme B), soluble mediators such a TNF-a, and upregulating cell surface expression of Fas ligand (FasL). Perforin punches holes in the target cell membrane to facilitate the entry of granzyme B, which is a protease that initiates apoptosis by activation of caspase cascades. Binding of FasL to Fas on the target cell surface provides an alternate pathway of caspase-mediated apoptosis. mRNAs of some of the aforementioned molecules, notably perforin and granzyme B, have been exploited as noninvasive markers for monitoring rejection in the urine of kidney transplant patients. (g) Memory T-cell differentiation: Primary antigen exposure generates long-lived antigen-specific memory T cells, which can deliver a more rapid and intense immune response if the same antigen is encountered again. This is possible since memory cells have a reduced activation threshold and are less dependent on costimulation than naive cells. The existence of memory cells explains why previous sensitization to donor antigens leads to increased risk of acute rejection and immunologic graft failure. Typical sensitization events include blood

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TGFb + IL-2

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Figure 5 CD4 T-cell differentiation. Activation of T cells in response to antigenic stimulation is followed by their differentiation along several different pathways depending on the cytokine environment to which they are exposed. The principal cell types that result are T-regulatory cells (Treg), IL-17producing cells (Th17), and T-helper cells (Th1 and Th2). The cytokine milieu required for differentiation, cellular phenotype, and function for each of these cell types is illustrated in this diagram. For example, a milieu rich in TGF-b and IL-2 results in differentiation of CD4-positive T cells into Tregs, which have a Stat5 þ FOXP3þ phenotype. Tregs are involved in maintaining immune system balance and have been implicated in both allograft tolerance and pathogenesis of autoimmune disease. These different cellular lineages may be identified and differentiated from each other by expression of RAR-related orphan receptor gt (RORgt), stat3, stat4, stat5, and Gata3. Reproduced from IT Center for Science Ltd, Espoo, Finland. http:// www.csc.fi/english/csc/publications/cscnews/2009/1/allergy_research, with permission.

transfusion, pregnancy, previous transplant, or infection by microorganisms with antigen cross-reactivity (heterologous immunity). It is now appreciated that homeostatic mechanisms control the size of the peripheral T-cell pool and relative proportions of CD4 þ/CD8 þ and naive memory cells. Consequently, reduction of T-cell counts during illness or after lymphocytic depletion therapy can induce residual T cells to proliferate and replenish the memory T-cell pool. Memory cells show upregulation of CD44, an adhesion molecule that binds to hyaluronic acid and enables memory T cells to leave the vascular system and reside in peripheral lymphoid organs. In contrast, these cells show downregulation of CD62L (L-selectin), an adhesion molecule that is necessary for migration across high endothelial venules.

Natural Killer Cells NK cells have been classically described as large granular lymphocytes that express a variety of surface markers, particularly CD56. These cells have been designated as ‘natural’ since their presence does not require a prior sensitizing event. NK cells can kill infected, neoplastic, or allogeneic targets by mechanisms similar to those employed by cytotoxic CD8-positive T cells. In addition, these cells are endowed with a unique system of activating and inhibitory receptors that enables them to recognize their targets. Inhibitory NK cell receptors include killer-cell immunoglobulin-like receptors and NKG2A/CD94, which bind to self-MHC class I

molecules. Interestingly, NK cells can recognize absence of self-antigen in target cells and respond by activation and killing. It has been suggested that gene polymorphisms in NK cell receptor targets can generate alloreactive NK cells that can be injurious to graft tissue. NK cells have been shown to be present in transplanted organs with both acute and chronic rejection. NK cells isolated from grafts have been demonstrated to kill target cells ex vivo, suggesting that they do indeed participate in the pathogenesis of both acute and chronic rejection, particularly antibody-mediated rejection.

B Cells The role of B cells as antibody-secreting cells is well known and discussed elsewhere in this encyclopedia. A number of other important B-cell functions are now recognized. Thus, B cells can act as APCs and express major histocompatibility molecules and the costimulatory molecule CD40. The antigen presentation function requires interaction with TCRs, with the resulting secretion of T-cell cytokines affecting both B-cell activation and differentiation. It is apparent that B cells can contribute to the rejection process via transcripts other than those related to immunoglobulins. Some studies suggest that the presence of B-cell clusters in allograft biopsies portends a bad prognosis, but others find no correlation other than time posttransplant. Regulatory B cells that produce IL-10 have been detected in tolerant patients and are believed to dampen the alloimmune response.

Transplantation Pathology | Mechanistic Aspects of Cell-Mediated Rejection

Endothelial Cells Endothelial cells participate actively in cell-mediated rejection as evidenced by morphological changes that include hypertrophy of the cytoplasm and a vesicular chromatin pattern. Ultrastructural examination reveals externalization of preformed granules called Weibel–Palade bodies that contain the adhesion molecule P-selectin. These manifestations of endothelial activation are caused by cytokines, chemokines, and complement components produced within the graft during rejection. Chemokines released in the graft bind to the endothelium and attract leukocytes circulating through the capillaries. The migration of inflammatory cells across the endothelial cell and capillary wall into the interstitial compartment is an intricate process. Normally, leukocytes are part of a fast laminar flow at the center of blood vessels, but once activated leukocytes reach postcapillary venules in a damaged graft, they move toward the periphery of the vascular lumen in response to the local chemokine gradient. The next step is the development of low-affinity interactions between endothelial P-selectin and sialyl-LewisX moieties present on the surface of activated leukocytes. If chemokines are present, conformational changes in leukocyte integrin molecule result in binding to other endothelial adhesion molecules, particularly ICAM-1. This high-affinity interaction causes arrest of the leukocyte on the endothelial surface, allowing it to commence extravasation and migrate along a chemokine gradient into the graft. T cells use a related adhesion molecule called leukocyte-functionassociated antigen-1 (LFA-1) to traverse the capillary wall.

Animal Models The importance of animal models in the development of clinical transplantation cannot be overstated. Surgical techniques for all organ transplants were perfected in animals before being applied to human beings. All of the pathophysiological concepts formulated in this article have been confirmed in animal experiments. In general, four complementary methods are available to study the pathogenesis of rejection in vivo. The first method consists of injecting the chemical or cell type of interest into an animal. A potential caveat of this approach is that the mode of experimental administration may not recapitulate normal physiology. The second method is based on studying transgenic mice in which a molecule of interest is overexpressed systemically or locally. Again, there is concern that a persistent overexpression could lead to nonphysiological effects. The third method uses antibody or receptor antagonists to see the effect of eliminating specific molecules on the evolution of rejection. The main disadvantage of this method is that the intended block of function may be incomplete. This problem can be circumvented by a fourth method in which gene-knockout technology is used to completely disrupt the production of the molecule under study. Unfortunately, gene inactivation in the embryonic stem cells may result in multiple abnormalities, which may confound data interpretation. Notwithstanding these limitations, the complementary application of these four approaches has greatly advanced our current understanding of allograft rejection. Representative examples of animal studies that have been particularly instructive are provided in the following sections.

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Within the innate immune system, participation of pathogen-associated PRRs and PAMPs in the development of rejection-associated lesions has been clarified by experimental transplantation. Thus, mice deficient in the TLR adaptor protein MyD88 are protected from both acute and chronic allograft rejection. The affected animals showed defective development of Th17 immune responses to alloantigen. Interaction of PRRs with PAMPs results in the generation of multiple chemokines, which excite an inflammatory response that leads to tissue injury. Accordingly, the induced deficiency of chemokines, their receptors, or both modulates the rejection of allografts. For example, deletion of genes for anti-inflammatory cytokines such as IL-10 and TGF-b accelerates graft rejection in mice. The role of adaptive immunity in rejection is highlighted by experiments demonstrating that animals lacking T cells are unable to reject fully mismatched transplants, whereas adoptive transfer of purified wild-type T cells to these animals is able to restore allograft rejection. Animal models have also played a crucial role in defining MHC restriction with respect to antigen presentation, elucidating pathways of allorecognition and costimulation, and enhancing our understanding of signal transduction and differentiation of T-helper and T-cytotoxic cells. Experiments have shown that T-cell-mediated rejection is associated with the release of multiple cytokines, but taken individually, these are not necessarily a prerequisite for mounting the rejection response. Thus, interferon-g has been considered a crucial mediator of acute allograft rejection because it upregulates MHC class II expression on APC and activates NK cells, CTL, and macrophages. Yet, cardiac graft survival is not prolonged in untreated interferon-g knockout (IFN-g /) mice. Likewise, IL-2 gene-knockout (IL-2 /) mice are capable of rejecting murine vascularized cardiac allografts. Along the same lines, IL-4-deficient mice (IL-4 /) and double geneknockout mice deficient in both IL-2 and IL-4 reject pancreatic islet cell and cardiac allografts quite effectively. These findings indicate that other cytokines that signal through the g-chain of the IL-2 receptor may compensate for the absence of IL-2 and/or IL-4. The g-chain is shared by IL-2, IL-4, IL-7, IL-9, and IL-I 5 receptors and is responsible for signal transduction following binding of these cytokines to their receptors on T cells. Another practical implication of these studies is that for cytokine-based immunosuppressive regimens to succeed, it will be necessary to target multiple cytokines. Animals have been indispensable in defining the natural history and pathogenesis of lesions that define rejection in transplanted organs. In kidney transplants, the temporal sequence of events has been determined to begin with interstitial inflammation, followed by MHC induction, venulitis by day 5, tubulitis, arteritis, and glomerulitis developing later and progressing through day 21. It appears that alloimmune effector T cells mediate tubular epithelial injury by contactindependent mechanisms related to DTH, followed by invasion of the altered epithelium to produce tubulitis. The earliest form of tubular injury is increased expression of solute carrier transcripts and reduction of E-cadherin and Ksp-cadherin. This is followed by redistribution of cadherin to the apical membrane, indicating loss of polarity. The loss of cadherins is independent of perforin and granzymes A and B. Animal models have also been useful in studying the link between viral infection, heterologous immunity, and graft

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arteriosclerosis. The availability of humanized mice provides a unique tool to analyze human immunology in vivo. Finally, there is a lot of current interest in the diagnosis of human rejection using DNA microarray analysis of cellular transcripts. These studies have been greatly facilitated by initial transcriptome analysis of mouse kidney allografts.

Principles of Therapy This section is intended to illustrate the principles of pharmacological intervention directed to the control of cellmediated rejection. The emphasis will be on outlining the mechanisms of action of drugs currently in use to suppress T-cell function. Non-T-cell actions of these drugs will be pointed out as applicable. Selected small molecules that look promising in vitro but are still in the pharmacology developmental pipeline and others that did not pass muster will also be briefly mentioned. Additional information related to the clinical use of these drugs can be found elsewhere in this encyclopedia. Calcineurin inhibitors: Cyclosporine (CsA), the first calcineurin inhibitor to be approved, virtually revolutionized the field of transplantation and is still widely used as a primary immunosuppressive agent. Tacrolimus is a potent alternative to CsA that was developed in Pittsburgh and now preferred in many medical centers. The mechanism of action is similar for tacrolimus and CsA in that it requires binding to cytoplasmic immunophilins. Tacrolimus inhibits lymphocyte activation up to 100 times more potently than CsA, and this seems to be related to the higher-binding affinity of tacrolimus to FK506binding protein (FKPB) compared to the binding of CsA to cyclophilin. The FK506–FKPB binding complex inhibits the activity of calcineurin, a serine–threonine phosphatase that regulates IL-2 promoter induction after T-cell activation. Inhibition of calcineurin impedes calcium-dependent signal transduction and inactivates transcription factors of the NFAT family that promote cytokine gene activation. Specifically, there is reduced synthesis of IL-2, IL-3, IL-4, IL-5, interferon-g, TNF-a, and granulocyte-macrophage colonystimulating factor, and IL-2 and IL-7 receptors. In vitro immunosuppressive effects of tacrolimus include suppression of immunoglobulin production and proliferation of stimulated B cells. However, in clinical use, calcineurin inhibitors primarily inhibit proliferative, cytotoxic, and primary antibody responses to T-cell-dependent antigens, while antibodydependent functions are not significantly affected. Sirolimus (rapamycin, SRL) resembles tacrolimus structurally, but the mechanism of action is distinct. SRL binds to FKBP but does not inhibit calcineurin phosphatase. The SRL–FKBP complex binds to the mammalian target of rapamycin (mTOR). The mTOR complex consists of two distinct signaling complexes mTORC1 and mTORC2, containing the scaffolding proteins raptor and rictor, respectively. The mTORC1 signaling pathway includes RAFT1/FRAP, p70 s6 kinase, and ribosomal proteins, which allow cell cycle progression through G1 to the S phase. The mTORC2 pathway regulates cell polarity via its action on the cell skeleton. SRL also inhibits IL-2-induced binding of transcription factors to the promoter of proliferating cell nuclear antigen. SRL suppresses B-cell immunoglobulin synthesis, antibody-dependent cellular

cytotoxicity, and lymphocyte-activated and NK cell activity. Another characteristic property is an antiproliferative effect on fibroblasts, endothelial cells, and smooth muscle cells, which may be relevant for prevention of chronic rejection. SRL and CsA show synergism in immunosuppression. Clinically, SRL is used as a part of immunosuppressive regimens designed to reduce nephrotoxicity associated with calcineurin inhibitors. However, SRL can itself cause proteinuria. Everolimus is a derivative of sirolimus and shares its mechanism of action. It is more hydrophilic, which results in greater bioavailability. Clinical use has identified similar side effects including hyperlipidemia and exacerbation of CsA-associated nephrotoxicity. These side effects are manageable if everolimus is administered in combination with reduced CsA doses. The availability of this drug will increase the number of mTOR inhibitor options available to transplant recipients. Mycophenolate mofetil (MMF): MMF is a commonly used immunosuppressant for the prophylaxis of acute rejection. Typically, it is given with an induction agent, a calcineurin inhibitor, and steroids but is now also a component of steroidfree regimens. It is an oral ester prodrug that is rapidly hydrolyzed into its active ingredient mycophenolic acid (MPA) by plasma esterases. MPA is in turn metabolized to MPA glucuronide by uridine diphosphate-glucuronosyltransferase in the liver. MPA secreted into the bile undergoes substantial enterohepatic circulation, which explains its gastrointestinal toxicity and a second pharmacokinetic peak up to 24 h after an oral dose. MPA is a noncompetitive and reversible inhibitor of inosine monophosphate dehydrogenase, a key enzyme in the de novo biosynthesis of guanosine. The resulting depletion of guanine nucleotides inhibits proliferation of both T and B lymphocytes in response to mitogenic and allospecific stimulation. The specificity of MPA for proliferating lymphocytes reflects the critical dependence of these cells on de novo pathway of purine biosynthesis. Most other cells can function with the salvage pathway alone. Guanosine nucleotides are necessary for glycosylation of lymphocyte and monocyte glycoproteins. Therefore, MPA can inhibit glycosylation of adhesion molecules, which may result in impaired migration of white cells to the sites of rejection. Leflunomide is a synthetic isoxazole derivative that is approved for use in rheumatoid arthritis. It is metabolized in the gut and liver to a malononitriloamide metabolite referred to as A77126. The principal mechanism of action of A77126 is inhibition of protein tyrosine kinases and dihydroorotate dehydrogenase (DHODH0), an enzyme that is essential for de novo pyrimidine synthesis. Leflunomide suppresses T-cell and B-cell proliferation and also inhibits the proliferation of smooth muscle cells. These latter properties may be relevant to the prevention of chronic rejection, but no controlled clinical trials addressing this issue have been performed. Leflunomide also inhibits replication of cytomegalovirus (CMV) and polyomavirus BK. Therefore, it has been used empirically as an adjunct antiviral agent in transplant recipients who do not respond to first-line therapy. FK778: FK778 is a synthetic malononitriloamide similar in chemistry, mechanism of action, and antiviral properties to leflunomide. It was explored in a number of trials in solid organ transplant recipients, but further clinical development was not pursued.

Transplantation Pathology | Mechanistic Aspects of Cell-Mediated Rejection

Janus kinase inhibitors (JAKs): JAKs are cytoplasmic tyrosine kinases that participate in the signaling of cell surface receptors that contain the cytokine receptor common g-chain, which is shared by tissue receptors for IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Ligand–receptor-induced activation of JAKs initiates signaling by phosphorylating signal transducers and activators of transcription (STATs). STAT phosphorylation facilitates STAT dimerization, transport to the nucleus, and regulation of the corresponding cytokine genes, particularly those associated with the IL-2 family. Mammals have several members of the JAK family such as JAK1, JAK2, JAK3, and tyrosine kinase 2. JAK3 is a particularly attractive target for immunosuppression, since it has a tissue distribution restricted primarily to hematopoietic cells. Therefore, blocking JAK3 is expected to suppress the immune response by selectively inhibiting T-cell activation without interrupting T-cell signaling. A JAK3 inhibitor, CP690550, is currently in clinical trials and appears to be effective for the prevention of acute rejection, although patients in the high-dose arm of this study had more infections, CMV disease, and BK-associated nephropathy. Other JAK inhibitors are being developed with a view to increasing efficacy and lowering the incidence of side effects. Protein kinase C (PKC) inhibition: PKC isoforms play a key role in signaling pathways downstream of the TCR (signal 1) and CD28 (signal 2) and thereby modulate early T-cell activation. Three PKC isoforms (a, b, and y) are particularly relevant to T- and B-cell signaling. PKCy is largely restricted to T cells and mediates activation of the transcription factors activator protein-1 and NF-kB, leading to downstream IL-2 production. AEB071 (AEB) is an oral PKC inhibitor that has been explored in clinical trials based on the reasoning that it would block early T-cell activation and IL-2 production, thereby providing a mechanism of action that is distinct from calcineurin inhibitors. However, trials in which calcineurin inhibitor withdrawal was attempted had to be halted due to an increased rate of acute rejection. Nevertheless, AEB may be a satisfactory substitute for MMF in triple or quadruple drug-based therapeutic regimens. FTY720: FTY720 (fingolimod) is a structural analog of sphingosine that gets phosphorylated by sphingosine kinases in the cell. It is now approved for use in multiple sclerosis (as Gilenya™; Novartis). As an immunosuppressant, it has a unique mechanism of action that targets lymphocyte recirculation. FTY720 entraps lymphocytes in secondary lymphoid organs, thus reducing their availability for cell-mediated immune responses in the allograft organ. The drug also acts on endothelial cells where it helps preserve vascular integrity by enhancing adherens junction assembly and endothelial barrier function. Unfortunately, a phase III clinical trial had to be halted due to a higher incidence of bradycardia and increase in airway resistance. Polyclonal antibodies: Polyclonal anti-T-cell antibody preparations raised in animals were introduced several decades ago and represented a breakthrough in the quest to prevent acute rejection. For example, antithymocyte globulin (thymoglobulin) has been used as an induction agent and steroid-sparing agent and as primary treatment agent for steroid-resistant acute rejection. The mechanism of action extends beyond T-cell-depleting activity and includes modulation of lymphocyte surface antigens and function of B cells,

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DCs, NK cells, T cells, and regulatory T cells. Immunosuppression is nonspecific and accompanied by significant adverse events including cytokine release syndrome, thrombocytopenia, and lymphopenia. Advances in our knowledge of T-cell function have led to the development of more specific monoclonal T-cell antibodies that are discussed later. OKT3 monoclonal antibody: This was the first commercially available monoclonal antibody and became available in 1981. It was raised in mice against human CD3-positive T cells and used successfully as an induction agent and for therapy of steroid-resistant rejection. As a relatively nonspecific agent that interacted with all T cells, it had significant side effects such as cytokine release syndrome, CMV, and herpes infection, acceleration of hepatitis C, and posttransplant lymphoproliferative disease. This stimulated an effort to develop more specific monoclonal antibodies directed against specific antigens in the T-cell activation pathway. A second major issue with OKT3 was its heterologous nature: clinical use led to rapid development of neutralizing antibodies that reduced its efficacy. An additional concern with this murine monoclonal antibody was potential inability to efficiently interact with the human complement system and cause lysis of target cells. The solution to these problems was the development of chimeric and humanized monoclonal antibodies (vide infra). IL-2 receptor monoclonal antibodies: The IL-2/IL-2 receptor system plays an important part in antigen-driven clonal Tcell proliferation. Moreover, the a-chain of the IL-2 receptor (CD25) is selectively expressed on activated T cells. Therefore, antibodies to CD25 block binding of IL-2 to its receptor and also lead to downregulation of this receptor. This leads to marked attenuation of the so-called signal 3 for Tcell activation and subsequent proliferation. Two murine antibodies, anti-Tac and BT563, did not perform well in clinical trials. This led to the development of a humanized antibody, daclizumab (humanized anti-Tac or Zenapax®) and a human–mouse chimeric antibody, basiliximab (Simulect®), both of which have been used as induction agents to reduce the incidence of acute rejection in kidney transplant patients. The antibodies are well tolerated with no evidence of cytokine release syndrome and no increase in the rate of infection or malignancy. Alemtuzumab (Campath-1H): Alemtuzumab is a humanized, monoclonal antibody to CD52, an antigen found on B cells, T cells, monocytes, and NK cells. It was used widely for chronic B-cell lymphocytic leukemia, other hematopoietic malignancies, rheumatoid arthritis, and multiple sclerosis before being introduced to renal transplantation by Roy Calne. Administration of Campath-1H results in a profound and prolonged depletion of its target cells from the circulating blood. Complete recovery of B-cell and T-cell function can take more than 12 and 36 months, respectively. Not surprisingly, use of Campath as an induction agent leads to very low rejection rates in the first year, even in the context of tacrolimus monotherapy and minimal immunosuppression. However, there is a rebound increase in late rejection, which may compromise long-term graft survival. Costimulation blockade: Costimulation pathways have been targeted for development of anti-T-cell therapy. On theoretical grounds, an ideal drug would block the CD28 receptor,

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Transplantation Pathology | Mechanistic Aspects of Cell-Mediated Rejection

without any effect on the counter-receptor CD152 (CTLA-4), which ligates CD80/CD86 on APCs to generate an inhibitory signal. Monoclonal antibodies to CD28 developed by two different pharmaceutical companies (TGN1412 by TeGeneroAG and FK734 by Astellas) could not attain their clinical potential on account of agonistic actions and precipitation of a cytokine storm that led to multiorgan failure in healthy volunteers. Another disappointment was the failure of the anti-CD154 monoclonal antibody (Biogen Idec) due to thromboembolic complications. The first success was achieved with Abatacept (Orencia, Bristol Myers Squibb), which is a soluble fusion protein consisting of the extracellular domain of human CTLA-4 (CD40L) and a fragment of the Fc portion of human IgG1. This antibody binds human B7 (CD80/86) more strongly than CD28 and blocks the so-called second signal required for T-cell activation. It is an approved medication for the treatment of rheumatoid arthritis. Belatacept (LEA29Y, Nulojix™) has since been developed as a higher-affinity and more potent second-generation abatacept and was approved for adult renal transplantation by the FDA in June 2011. This humanized soluble fusion protein and monoclonal antibody prolongs graft survival in combination therapies that include prednisone and basiliximab or MMF. Its principal advantage is freedom from calcineurin inhibitor toxicity. Potential drawbacks include the need for intravenous infusions every 4–8 weeks, a higher rate of subclinical rejection, and the need to carefully monitor for posttransplant lymphoproliferative disease. Blockade of cell adhesion molecules: The most promising therapeutic target is lymphocyte-associated function-1 (LFA-1) protein. LFA-1 is a member of the heterodimeric b-2 integrin family and each molecule is comprised of a unique a-chain (CD11a) and a b-chain (CD18) that is common to other b-2 integrins. LFA-1 is expressed by neutrophils, monocytes, macrophages, and lymphocytes and binds to ICAMs expressed on APCs and endothelial cells. LFA-1–ligand interactions not only recruit leukocytes to the site of inflammation but also stabilize the interaction between T cells and APCs, thereby providing coactivation signals. A humanized CD11a-specific IgG1 (efalizumab) has been approved for the treatment of psoriasis and is now being developed as a subcutaneously administered maintenance agent in renal transplant recipients on calcineurin inhibitor-free therapeutic regimens. A related developmental compound is alefacept (Amevive): this is a dimeric, humanized LFA-3Ig fusion protein that blocks T-cell activation by antagonizing the CD2 receptor–LFA-3 interaction. This drug has also been approved for psoriasis and is now being developed for clinical transplantation. CD2 (also referred to as LFA-2 or LFA-3 receptor) functions as a cell adhesion molecule and a costimulatory molecule on T and NK cells.

Summary In summary, this article has reviewed the role of cellular elements in the pathogenesis of organ rejection. While T cells are certainly the most important player in this process, there is active participation by a broad range of cells belonging to both the innate and acquired immune systems. It is now recognized

that innate mechanisms can play an important role in augmenting and perpetuating tissue damage caused by the acquired immunity. The principal steps in T-cell-mediated rejection are (i) antigen presentation primarily by DCs; (ii) allorecognition by the direct or indirect pathways early and late posttransplant, respectively; (iii) delivery of costimulatory or inhibitory signals; (iv) signal transduction; (v) helper T-cell differentiation into Th1 cells, Th2, Th9, Th17, and Tfh cells; and (vi) CTL differentiation with release of granzyme B, perforin, and activation of caspases that promote apoptosis. In addition to the aforementioned effector cells, primary antigen exposure generates long-lived antigen-specific memory T cells, which can deliver a more rapid and intense immune response if the same antigen is encountered again. The existence of memory cells explains why previous sensitization to donor antigens leads to increased risk of acute rejection and immunologic graft failure. Typical sensitization events include blood transfusion, pregnancy, previous transplant, or infection by microorganisms with antigen cross-reactivity (heterologous immunity). A broad armamentarium of drugs is now available to prevent and treat T-cell-mediated rejection. The excellent efficacy of these drugs makes it increasingly difficult to show a statistically significant benefit of newer agents that are still in the developmental pipeline. Unfortunately, none of the available agents have demonstrated significant effect on controlling the low-grade smoldering T-cell- and antibody-mediated injury that leads to chronic rejection.

Further Reading Abele-Ohl, S., Leis, M., Wollin, M., et al., 2012. Human cytomegalovirus infection leads to elevated levels of transplant arteriosclerosis in a humanized mouse aortic xenograft model. Am. J. Transplant. 12 (7), 1720–1729. Alessiani, M., Spada, M., Vaccarisi, S., et al., 1996. Combined FK 506 and mycophenolate mofetil immunosuppression prolongs survival after small bowel transplantation in pigs. Transplant. Proc. 28 (5), 2499–2500. Anil Kumar, M.S., Heifets, M., Fyfe, B., et al., 2005. Comparison of steroid avoidance in tacrolimus/mycophenolate mofetil and tacrolimus/sirolimus combination in kidney transplantation monitored by surveillance biopsy. Transplantation 80 (6), 807–814. Aranda-Dios, A., Lage, E., Sobrino, J.M., et al., 2006. Sirolimus experience in heart transplantation. Transplant. Proc. 38 (8), 2547–2549. Arinsoy, T., Uslu, A., Mir, S., et al., 2011. Evaluation of efficacy and safety of mycophenolate sodium in patients with de novo and maintenance renal transplantation: results of a multicenter, prospective, observational study. Transplant. Proc. 43 (3), 826–832. Benichou, G., 1999. Direct and indirect antigen recognition: the pathways to allograft immune rejection. Front. Biosci. 4, D476–D480. Brennan, T.V., Lunsford, K.E., Kuo, P.C., 2010. Innate pathways of immune activation in transplantation. J. Transplant. 2010, 826240. http://dx.doi.org/10.1155/2010/ 826240. Epub 2010 Aug 31. Calne, R.Y., 2009. Transplantation: current developments and future directions. Rev. Neurosci. 20 (3–4), 267–273. Coelho, T., Tredger, M., Dhawan, A., 2012. Current status of immunosuppressive agents for solid organ transplantation in children. Pediatr. Transplant. 16 (2), 106–122. Denton, M.D., Davis, S.F., Baum, M.A., et al., 2000. The role of the graft endothelium in transplant rejection: evidence that endothelial activation may serve as a clinical marker for the development of chronic rejection. Pediatr. Transplant. 4 (4), 252–260. Deuse, T., Schrepfer, S., Reichenspurner, H., 2003. Immunosuppression with FK778 and mycophenolate mofetil in a rat cardiac transplantation model. Transplantation 76 (11), 1627–1629.

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Durrbach, A., Pestana, J.M., Pearson, T., et al., 2010. A phase III study of belatacept versus cyclosporine in kidney transplants from extended criteria donors (BENEFITEXT study). Am. J. Transplant. 10 (3), 547–557. Einecke, G., Fairhead, T., Hidalgo, L.G., et al., 2006. Tubulitis and epithelial cell alterations in mouse kidney transplant rejection are independent of CD103, perforin or granzymes A/B. Am. J. Transplant. 6 (9), 2109–2120. Einecke, G., Broderick, G., Sis, B., Halloran, P.F., 2007. Early loss of renal transcripts in kidney allografts: relationship to the development of histologic lesions and alloimmune effector mechanisms. Am. J. Transplant. 7 (5), 1121–1130. Einecke, G., Reeve, J., Mengel, M., et al., 2008. Expression of B cell and immunoglobulin transcripts is a feature of inflammation in late allografts. Am. J. Transplant. 8 (7), 1434–1443. Famulski, K.S., Einecke, G., Reeve, J., et al., 2006. Changes in the transcriptome in allograft rejection: IFN-gamma-induced transcripts in mouse kidney allografts. Am. J. Transplant. 6 (6), 1342–1354. Famulski, K.S., Broderick, G., Einecke, G., et al., 2007. Transcriptome analysis reveals heterogeneity in the injury response of kidney transplants. Am. J. Transplant. 7 (11), 2483–2495. Fung, J.J., 2004. Tacrolimus and transplantation: a decade in review. Transplantation 77 (9 Suppl.), S41–S43. Gurk-Turner, C., Manitpisitkul, W., Cooper, M., 2012. A comprehensive review of everolimus clinical reports: a new mammalian target of rapamycin inhibitor. Transplantation 94 (7), 659–668. Hidalgo, L.G., Sellares, J., Sis, B., Mengel, M., Chang, J., Halloran, P.F., 2012. Interpreting NK cell transcripts versus T cell transcripts in renal transplant biopsies. Am. J. Transplant. 12 (5), 1180–1191. Ito, R., Takahashi, T., Katano, I., Ito, M., 2012. Current advances in humanized mouse models. Cell. Mol. Immunol. 9 (3), 208–214. Jabs, W.J., Sedlmeyer, A., Ramassar, V., et al., 2003. Heterogeneity in the evolution and mechanisms of the lesions of kidney allograft rejection in mice. Am. J. Transplant. 3 (12), 1501–1509. Jorgensen, K.A., Koefoed-Nielsen, P.B., Karamperis, N., 2003. Calcineurin phosphatase activity and immunosuppression. A review on the role of calcineurin phosphatase activity and the immunosuppressive effect of cyclosporin A and tacrolimus. Scand. J. Immunol. 57 (2), 93–98. Karandikar, N.J., Vanderlugt, C.L., Bluestone, J.A., Miller, S.D., 1998. Targeting the B7/ CD28:CTLA-4 costimulatory system in CNS autoimmune disease. J. Neuroimmunol. 89 (1–2), 10–18. LaCorcia, G., Swistak, M., Lawendowski, C., et al., 2009. Polyclonal rabbit antithymocyte globulin exhibits consistent immunosuppressive capabilities beyond cell depletion. Transplantation 87 (7), 966–974. Lakkis, F.G., 1998. Role of cytokines in transplantation tolerance: lessons learned from gene-knockout mice. J. Am. Soc. Nephrol. 9 (12), 2361–2367. Lieberthal, W., Levine, J.S., 2009. The role of the mammalian target of rapamycin (mTOR) in renal disease. J. Am. Soc. Nephrol. 20 (12), 2493–2502. Mengel, M., Reeve, J., Bunnag, S., et al., 2009. Molecular correlates of scarring in kidney transplants: the emergence of mast cell transcripts. Am. J. Transplant. 9 (1), 169–178.

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Nankivell, B.J., Alexander, S.I., 2010. Rejection of the kidney allograft. N. Engl. J. Med. 363 (15), 1451–1462. Nicolls, M.R., Gill, R.G., 2006. LFA-1 (CD11a) as a therapeutic target. Am. J. Transplant. 6 (1), 27–36. Padiyar, A., Bodziak, K.A., Hricik, D.E., Augustine, J.J., 2010. Clinical predictors of proteinuria after conversion to sirolimus in kidney transplant recipients. Am. J. Transplant. 10 (2), 310–314. Pearson, P.J., Wei, C.M., Lin, P.J., et al., 2004. Endothelium-dependent vasodilation during acute rejection in dogs. J. Surg. Res. 121 (1), 56–61. Ponticelli, C., 2012. The mechanisms of acute transplant rejection revisited. J. Nephrol. 25 (2), 150–158. Rogers, N.M., Matthews, T.J., Kausman, J.Y., Kitching, A.R., Coates, P.T., 2009. Review article: kidney dendritic cells: their role in homeostasis, inflammation and transplantation. Nephrology (Carlton) 14 (7), 625–635. Rose, M.L., 2004. Long-term effects of damage to the endothelium and chronic rejection. J. Heart Lung Transplant. 23 (9 Suppl.), S240–S243. Rowshani, A.T., Vereyken, E.J., 2012. The role of macrophage lineage cells in kidney graft rejection and survival. Transplantation 94 (4), 309–318. Tang, D., Kang, R., Coyne, C.B., Zeh, H.J., Lotze, M.T., 2012. PAMPs and DAMPs: signal 0 s that spur autophagy and immunity. Immunol. Rev. 249 (1), 158–175. Tedesco, D., Haragsim, L., 2012. Cyclosporine: a review. J. Transplant. 2012, 230386. http://dx.doi.org/10.1155/2012/230386. Epub 2012 Jan 4. Tedesco-Silva, H., Szakaly, P., Shoker, A., et al., 2007. FTY720 versus mycophenolate mofetil in de novo renal transplantation: six-month results of a double-blind study. Transplantation 84 (7), 885–892. Teschner, S., Burst, V., 2010. Leflunomide: a drug with a potential beyond rheumatology. Immunotherapy 2 (5), 637–650. Vincenti, F., Kirk, A.D., 2008. What’s next in the pipeline. Am. J. Transplant. 8 (10), 1972–1981. Wearne, N., Swanepoel, C.R., Boulle, A., Duffield, M.S., Rayner, B.L., 2012. The spectrum of renal histologies seen in HIV with outcomes, prognostic indicators and clinical correlations. Nephrol. Dial. Transplant. 27 (11), 4109–4118. Wood, K.J., Goto, R., 2012. Mechanisms of rejection: current perspectives. Transplantation 93 (1), 1–10. Wu, H., Noordmans, G.A., O’Brien, M.R., et al., 2012. Absence of MyD88 signaling induces donor-specific kidney allograft tolerance. J. Am. Soc. Nephrol. 23 (10), 1701–1716. Relevant Websites http://www.cellsignal.com/reference/pathway/T_Cell_Receptor.html – T-cell receptor signaling. http://www.hindawi.com/journals/jtran/2010/826240/ – Innate pathways of immune activation in transplantation. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840233/ – Antibody immunosuppressive therapy in transplantation.