Principles in transplantation: immunology

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Principles in transplantation: immunology

Key developments in transplant immunology 1900 1938 1954 1953

Discovery of blood groups Antigeneantibody binding hypothesis First renal transplant between identical twins Medawar describes concept of ‘actively acquired tolerance’ 1958 e Description of human leucocyte antigen 1966 e Recognition that positive cross-matching causes hyperacute rejection 1973 e Discovery of dendritic cells 1978 e Ciclosporin used in clinical setting 1978 e Application of human leucocyte antigen matching in renal transplantation 1981 e Discovery of IL2 receptor 1983 e Discovery of T cell receptor 1986 e Th1 e Th2 T helper cell function 1987 e Introduction of new immunosuppressants such as tacrolimus

Murali Somasundaran Isabel Quiroga

Abstract The immune system has evolved to protect the body from diseasecausing agents by using innate and adaptive systems. The innate system acts rapidly using non-specific processes to target a large variety of pathogens and prevent the spread of disease. The adaptive system works more slowly and uses lymphocytes to target specific pathogens. Prevention and treatment of rejection post-transplantation is achieved by controlling both these immune mechanisms pharmacologically.

Keywords Immunology; immunosuppression; transplantation

Transplantation may involve the use of grafts from the ‘self’ (autograft), identical twins (isograft), or other animal species (xenograft) but the majority of clinical applications involve grafts from other human donors (allografts), either living or deceased. All transplantation results in an immunological response and it remains a challenge to overcome the body’s natural defence mechanisms to prevent organ rejection, whilst minimizing the undesirable side effects of the immunosuppression.

Box 1

response can also be divided into cellular and non-cellular (i.e. molecules soluble in plasma e humoural e components). Innate immune system If physical barriers such as the skin and mucous membranes are breached, the innate immune system forms the second line of defence. Pathogens attacking a host may be extracellular and may not cross the cell membrane (e.g. parasites) or may become intracellular and attack the inner-workings of the cells (e.g. viruses). The innate immune system recognizes molecular motifs common to many groups of pathogens and not present in the host, rather than specific pathogens. Molecules such as lipopolysaccharides on the cell wall of bacteria or mannans present in yeasts are collectively denominated ‘pathogen-associated molecular patterns’ (PAMPs). The recognition of PAMPs by receptors of the immune system results in a rapid destruction of pathogens by the effector mechanisms of the innate system. The process of acute inflammation (involving complement pathways, phagocytosis by macrophages and neutrophil migration) occurs as a result of activation and mobilization of the innate immune system. The system is nonspecific but highly sensitive enabling the most minimal threat to be recognized and intercepted early. In addition to preventing ongoing damage from the invading pathogen, innate immunity also provides alerting signals to activate the adaptive immune system.

Historical background The first successful experimental kidney transplant was performed in 1902 and early progress in the field was primarily related to the development of techniques in vascular surgery. By 1914, Carrell stated in a landmark lecture that whilst the surgical aspects of transplantation had been mastered, all future efforts in the field should be ‘...directed toward the biological methods which will prevent the reaction of the organism against foreign tissue.’. Some of the key advances in immunology in relation to transplantation are summarized in Box 1.

Basic immunology The simplest accepted classification of the immune system involves division into two separate parts; the innate (nonspecific/antigen-independent) response and the adaptive (acquired/ specific/ antigen-dependent) response. There is close cooperation between the two responses and each consists of two phases; recognition and effector. The different components of

Complement: the complement system/cascade involves the activation of a number of proteins to promote opsonization, chemotaxis and lysis of invading pathogens. The proteins are primarily synthesized by the liver and found in high concentrations in the blood and tissues. The complement system can be activated by three pathways; classical, alternative and lectin activation. The activation of these pathways is a common effector mechanism of both the innate and adaptive immune system.

Murali Somasundaran BSc MBBS MRCS is studying for a DPhil in Tissue Engineering in Transplanatation at the University of Oxford, UK. Conflicts of interest: none declared. Isabel Quiroga FRCS (Gen Surg) DPhil is a Consultant Surgeon in Renal and Pancreas Transplantation and Vascular Access and Endocrine Surgery in Oxford, UK. Conflicts of interest: none declared.

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e e e e

Cytokines: cytokines are proteins secreted by various cells of the immune system and include molecules such as interleukin 2 (IL-2)

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and interferon-g (IFN-g). They have a variety of functions in amplifying the immune response and can be viewed as the messengers of the immune system enabling communication between cells. Chemokines are specific types of cytokines that recruit cells of the immune system to a site of inflammation.

receptors (TCRs) that bind to presented antigen. Each T cell has a unique TCR. There are a number of subtypes of T cells: Helper T cells (Th cells) e express T cell receptor and CD4 protein on the cell surface and activate macrophages and cytotoxic T cells to accelerate B-cell maturation. Th cells secrete cytokines (e.g. IL2) which are essential for activation of the adaptive immune response. Cytotoxic T cells (CTL) e express T cell receptor and CD8 proteins on their cell surface and are also known as CD8þ T cells. CTLs are involved primarily in recognition of virus-infected cells and tumour cells and cause their destruction. Memory T cells e these cells have encountered antigens previously and on a second exposure can mount a faster immune response. They can be CD4þ or CD8þ T cells. Regulatory T cells e also called suppressor T cells. Their main function is to suppress the response of other immune cells. T cells are the main drivers of the adaptive immune system at the recognition and the effector stage. Antigens presented to T lymphocytes result in activation and proliferation of T cells (antigen presentation). Typically, invading antigens are located either within cells (e.g. viral antigens) or externally (e.g. bacterial). Antigen presentation involves the presentation of protein fragments by major histocompatibility complex (MHC) molecules on the antigen-presenting cell to the T cell receptor on T cells. The MHC presents antigen and is a complex structure expressed on the surface of cells. MHC is common to numerous species and in humans it is also known as human leucocyte antigen (HLA). MHC can be divided into two groups: MHC class I and II. MHC class I is expressed on the cell surface of all nucleated cells. In contrast, MHC class II is only displayed by dendritic cells, macrophages and B cells. These molecules have a similar three dimensional structure with both exhibiting a peptide (antigen) binding groove. The amino acids flanking the groove demonstrate the highest degree of variability (polymorphism). The origin of the peptides loaded on the class I and II molecules is one of the major differences between these molecules. MHC class I molecules mainly load peptide from the intracellular environment. In contrast, MHC class II molecules load peptide acquired from extracellular antigens. There is a degree of crossover during antigen processing and presentation in the MHC class I and II pathways and therefore exogenous and endogenous antigen can also be loaded onto class I and II respectively (Figure 1). Under normal conditions, the peptides presented by the MHC molecules are self-peptides mostly derived from MHC proteins themselves and so are recognized by T cells as ‘self’ and do not generate an immune response. When a pathogen is present, the MHC molecules are loaded with foreign peptide and upon recognition by the T cell receptor on T cells, an immune response can be mounted. MHC molecules exhibit considerable polymorphism. With two alleles at each locus, most people can express six different MHC class I proteins and six different MHC class II proteins. It is therefore extremely difficult to find perfectly matched unrelated donors for the recipients of transplants. Co-receptors e play an important role in T cell activation. CD4 molecules expressed on the surface of Th cells strengthen the bind between the TCR and the MHC Class II molecules. Similarly, the CD8 molecule on CTLs strengthen the bind

Macrophages: these are forms of white blood cells (leucocytes), derived from pluripotent stem cells in the bone marrow, giving rise to myeloid and lymphoid stem cells in the bone marrow. The myeloid lineage includes the mononuclear leucocytes (monocytes, macrophages and dendritic cells) and the polymorphonuclear leucocytes (neutrophils, eosinophils, basophils and mast cells). Monocytes represent immature forms of macrophages circulating in the blood stream. Monocytes exit the blood, mature and differentiate in the tissues where they become macrophages, which scavenge cellular debris. When activated by PAMPs they secrete cytokines and engulf and digest invading pathogens. In addition, they act as antigen-presenting cells (APC) to the adaptive immune system. Neutrophils: these cells play an essential role in the innate immune response, focused specifically on pathogen destruction. Expression of endothelial cell proteins such as selectin (SEL) is upregulated by cytokines such as interleukin-1 (IL1) and promotes rolling of the leucocytes and interaction with adhesion molecules such as integrin (INT). Finally chemoattractants and their receptors on the neutrophil enable the leucocyte to leave the endothelium and exit the blood. Natural killer (NK) cells: NK cells are leucocytes derived from the lymphoid lineage (others include T lymphocytes e cytotoxic and helper e and B lymphocytes) and are able to secrete cytokines and destroy some virus-infected cells. Adaptive immune system The adaptive immune system acts as the third line of defence with much interplay (i.e. there is no discrete stage at which the adaptive system begins to predominate). Adaptive immunity is a specific process that targets disease causing agents and creates an amplified response in order to prevent the spread of disease. Following a first exposure to a pathogen the adaptive immune system ‘remembers’ specific antigens and on re-exposure to the same antigen it can mount a more rapid and effective response. Antigens: an antigen is any molecule that can be recognized by the immune system. Antigens are typically proteins or polysaccharides that form part of the structure of invading pathogens. Antigens are recognized by receptors on the surface of cells of the immune system and by antibody. Antibodies: antibodies are proteins (g globulins) found in blood or other body fluids, consisting of light and heavy chains and the variability of these dictates the specific type and binding specificity. They are secreted by lymphocytes and bind to antigens to generate and augment an immune response. T lymphocytes: T cells are produced in the bone marrow and mature in the thymus to play a crucial role in cell-mediated immunity. T cells have surface receptors known as T cell

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Killer T cells

Antigens within cells MHC I

ThC

Antigens in plasma

MHC II

Figure 1 Antigen processing and presentation. Antigens within the cells are divided into smaller peptides, loaded onto MHC class I molecules and transported to the cell surface. MHC class I can present antigen to the TCR on the surface of cytotoxic T cells. Extracellular antigen is engulfed and processed by APCs, loaded onto MHC class II molecules and transported to the cell surface. MHC class II can present antigen to the T cell receptor of T helper cells. APC, antigen presenting cell; MHC, major histocompatibility complex; CTL, cytotoxic T lymphocyte; ThC, T helper cell.

Transplant immunology

between the TCR and MHC Class I molecules. A further requirement is the process of cross-linking which brings together numerous signalling molecules within a small region of the T cell surface in order to promote activation (no true chemical cross-linking occurs). Cross-linking without antigen presentation is also insufficient for complete activation to occur. Co-stimulation involves signals that are not antigen specific and differ according to the T cells involved. An example of this is B7 proteins on APC that plug into CD28 proteins on the surface of T cells (important example due to its pharmacological significance). These mechanisms of T cell activation are summarized in Figure 2. The process of antigen presentation can be performed by various cells, but certain cells have the ability to express both class I and II MHC molecules and provide co-stimulation. Such cells are designated antigen-presenting cells (APCs) and include dendritic cells, macrophages and activated B cells.

Preoperative considerations i) Pre-formed antibodies Transplantation of an organ into a recipient who has preformed antibodies to antigens displayed by the transplanted organ will likely result in rapid and vigorous destruction of the graft (hyperacute rejection). This is now rare. The most relevant pre-formed antibodies are ABO blood group antibodies and HLAspecific antibodies. The HLA system describes a group of antigens encoded from the short arm of chromosome 6. HLA antibodies develop after exposure to allogenic antigen by blood transfusions, pregnancy or previous transplants. HLA antigens are divided into Class I and Class II (Class III are also described but are less important). Class I encodes the classic antigens; HLA-A, HLA-B and HLA-C amongst others. Class II encodes for HLA-DR, HLA-DQ and HLA-DP. When a clinician refers to the HLA ‘mismatch’, it is the mismatch between donor and recipient HLAA, HLA-B and HLA-DR that is being considered as these are the most polymorphic and antigenic loci and therefore, most relevant in transplantation. A high number of mismatches between these HLA antigens are associated with a worse outcome in renal transplantation. For example, the best possible outcome would be observed where there were no mismatches between donor and recipient for HLA-A, HLA-B and HLA-DR (0-0-0), whereas the worst would occur if there were two mismatches for each of the three antigens (2-2-2). Whilst current practice in renal and to a lesser extent pancreas transplantation, is to select donors with favourable HLA matching, this practice if often not applied to other

B lymphocytes: B cells mature in the bone marrow and (like T cells) possess a surface receptor (BCR) which consists of an antibody like molecule capable of binding to antigen. B cells produce antibodies capable of recognizing any organic molecule whereas T cell receptors recognize primarily protein antigens. Following activation, the B-cell can differentiate towards plasma cells (which secrete antibodies) or memory B cells. B cells are capable of recognizing antigens independently via the BCR, but T cells require the specific form of antigen presentation described above. In addition, B cells can function as antigen-presenting cells when mature. Unlike T cells, B cells produce only moderate amounts of cytokines.

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Direct

Indirect

Donor APC

Donor

Host TC

Recipient

Host APC

Recipient

Host TC

Recipient

Figure 2 T cell activation. T cell activation needs the interaction of the major histocompatibility complex-peptide complex on the surface of antigenpresenting cells (APCs) with the T cell receptors on T cells. The CD8 molecule on cytotoxic T cells and the CD4 molecule on the surface of T helper cells strengthen the bond between APC and T cell. A second signal is essential for T cell activation and is provided but a number of co-stimulatory molecules such CD28 on the T cell which can interact with B7 on the APC.

organs (e.g. liver, heart and lung) and remains controversial. HLAcompatibility seems to be important in cardiac transplantation but waiting for a well-matched organ is not practical given the great level of polymorphism of these genes and the shortage of suitable organs. Adopting a matching strategy would therefore result in many potential recipients dying on the waiting list. However, even in a well-HLA-matched transplant, there may be problems due to the minor histocompatibility complex (MiHC) and while the presence of antibodies to differences encoded by this system is not as significant as the HLA system, they may contribute to the development of rejection. ii) Detection of alloantibody e tissue crossmatch test Tissue crossmatching is performed before most kidney and pancreas transplants. With complement-dependent cytotoxicity, donor leucocytes and recipient serum are incubated with complement. In a positive test, donor cells are destroyed by antibodies present in the serum of the recipient. In this case the transplant cannot proceed. Flow cytometry: a more sensitive method to detect recipient antibody binding to donor leucocytes. iii) Organ selection Organs retrieved from donors where the organs have been exposed to a more traumatic and or prolonged dying process will have suffered greater tissue injury. In addition to this the retrieval process with prolonged warm ischaemic times suffer greater ischaemic and inflammatory injury.

recruitment of macrophages and neutrophils into the graft and migration of dendritic cells from the graft to the peripheral lymph nodes where they mature and increase the expression of MHC and co-stimulatory molecules. ii) Presentation of antigen to recipient T cells Following transplantation, allogenic donor antigens are recognized by the recipient’s T cells by two distinct but not exclusive pathways: direct and indirect allorecognition. During direct recognition, donor APCs expressing donor allogeneic-MHC presenting donor peptide, activate recipient T cells. The direct recognition pathway dominates the acute rejection response during the early post-transplant period but seems to be less important at later times, following the departure of donor dendritic cells from the graft. In contrast, the indirect recognition pathway is characterized by the stimulation of T cells by donor allopeptides processed and presented on self-MHC expressed by recipient APC. This pathway is a major contributor to acute rejection and the immunological processes involved in chronic rejection of organs such as the kidney, heart and lung. These pathways are illustrated in Figure 3. Circulatory T cells via their T cell receptor are primed by the process of positive clonal selection in the thymus to recognize selfMHC. Therefore, it seems paradoxical that they can react to alloMHC in the context of transplantation. Furthermore, the frequency of T cells that directly recognize intact allo-MHC molecules on the surface of target cells is at least 100-fold higher than that of T cells recognizing allopeptide processed and presented by self-MHC. iii) Activation of recipient T cells Following antigen presentation to T cells via direct and indirect mechanisms, sufficient T cell activation must occur to allow the immune response to proceed. Co-stimulation and crosslinking are important factors in this process, just as in non-transplant immunology. Such processes provide pharmacological targets in transplant patients.

Postoperative considerations Following transplantation, a series of immunological changes occur over the course of the life of the graft: i) Trauma of transplantation The greater the preceding injury to the donor organ, the greater the resultant inflammation on reperfusion. Introduction of vascular flow onto the donor endothelium causes secretion of cytokines (e.g. IL1, IL6) and activation of complement with

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APC

iv) The controlling influences of acute rejection Acute rejection can be regarded as the effector (efferent) stage of the immune response to an allograft and it is mainly T-cell dependent. T cells recognize the graft as ‘non-self’ and can then cause graft damage by inflammation and delayed-type hypersensitivity in a non-specific manner and by specific cytotoxic T cells and antibodies. The exact mechanisms of how this occurs are poorly understood but many factors have been shown to be contributory. A role for B cells in the development of acute rejection has been postulated and investigated, but the effects of T cells are currently far better characterized. The cells driving the immune response to an allograft are the CD4þ T helper cells and have been grouped into Th1 and Th2 populations. It is believed that the Th1 populations are largely responsible for the cell-mediated response and that Th2 subsets are mainly involved with promoting the B cell dependent humoural response to transplanted organs. Th1 cells secrete the cytokines IFN-g and IL2, resulting in recruitment and proliferation of specific CTLs, NK cells and activated macrophages. Th1 cells are linked to transplant graft damage and rejection. Th2 cells secrete the cytokines IL4 to IL6 amongst others and create a bias towards the activation and proliferation of B cells. Both cell subsets are illustrated in Figure 4. Although the different groups of T cells and their cytokines are known, manipulation of these effects remains difficult. Research has indicated that a Th2 driven immune response may be involved in generating transplant tolerance rather than

T-cell MHC I MHC II

Ag presentation

MHC I

CD8 MHC II Co receptor CD4 B7

CD28

Co-stimulation

Figure 3 Direct and indirect mechanisms of allorecognition. Direct allorecognition involves the recognition of donor major histocompatibility complex (MHC) on donor antigen-presenting cells (APCs) by the recipient T cell. Indirect allorecognition takes place when donor MHC is processed and presented to the recipient T cells by the recipient APCs. Note that the direct pathway of antigen presentation is specific to transplantation.

CELL LYSIS APOPTOSIS IFN-γγ CD8+Tcell

IL2 IFN-γ IFN-γ IL2

IL2 IFN-γ

Th1

NK cell

IFN-γ

IFN-γ

IL4 IL10

CD4+Tcell

Macrophage

IL1 TNF-α α FREE RADICALS

GRAFT DAMAGE REJECTION

IL2

Th2

IL4 IL6

ANTIBODIES B cell

Figure 4 Mechanisms of graft damage. CD4þ cells are the drivers of the allo-immune response. Th1 and Th2 cells cytokine profiles and their effector mechanisms are shown. IL, interleukin; IFN, interferon; NK cell, natural killer cell; TNF, tumour necrotic factor.

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rejection. The obvious approach would be to upregulate the activities of Th2 cells and reduce those of Th1 to promote a state of tolerance to the transplanted tissue. v) Migration of activated cells into donor graft Recipient leucocytes must cross the endothelium to enter the graft by the mechanisms described above. Therefore, the manipulation of the endothelial cells to inhibit the migration of leucocytes into the graft would be an effective way to prevent acute rejection. vi) Destruction of donor graft The previous stages described in the classical transplant immune response all account for early reactions that occur towards any donor graft. Experiments have shown that such immunological reaction is highly specific and usually targets the graft accurately with minimal collateral damage to the recipient tissues. This is surprising, given that both antigen e specific and non-specific mechanisms of immunity are in operation. Exactly how antigen non-specific mechanisms are prevented from causing damage to the self is unknown. It is known however, that cellular and humoural mechanisms of the immune system are involved in acute rejection and long-term graft damage. a) Cell-mediated graft damage Activated T helper CD4þ cells secrete interferon-g which increases the expression of adhesion molecules in the graft endothelium and promote the adhesion and migration of leucocytes. IFN-g also activates macrophages which in turn will release cytokines such as IL1 and tumour necrotic factor 1, free radicals and other enzymes that can cause significant graft damage. Furthermore, IFN-g increases the cellular expression of MHC class II in graft cells and endothelium and thus facilitating activation of T cells by APC. IL2 released by T cells is a potent growth factor for B and T cells and a major player in graft rejection. CD8þ cytotoxic T cells can cause graft damage by two mechanisms. On activation after recognition of allo-antigen these cells can release perforin that punches holes on the membrane of cells and gramize that enters the cells through the holes created by perforin and starts lysis of the target cell. Cytotoxic cells can also cause cell death by apoptosis using FaseFas ligand interaction. b) Antibody-mediated graft damage Antibody-dependant cellular cytotoxicity is one mechanism of damage where the antibody serves as a bridge between effector cell and target tissue. Antibodies are also involved in the fixation of complement which results in the recruitment of macrophages

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and neutrophils and injury to the endothelium. This is often used to diagnose antibody-mediated rejection by C4d staining which identifies the presence of the fixed complement component C4d. The importance of antibodies in rejection has led to the development of targeted immunosuppressants.

Immunosuppression Immunosuppressive drugs are used in patients to provide protection from rejection and preserve graft function. Steroids inhibit T cell and macrophage activation, reduce expression of adhesion molecules and MHC molecules and inhibit IL1 and IL6 production. Anti-proliferative agents (mycophenolate mofetil, MMF, azathioprine) inhibit DNA replication and suppress B and T cell proliferation. Calcineurin inhibitors (tacrolimus, cyclosporine) bind to intracellular proteins to inhibit IL-2 and therefore reduce T cell activation. mTOR inhibitors (sirolimus, everolimus) lead to an arrest of the B and T cell, cell cycles in the G1-S phase. Polyclonal antibodies are derived by harvesting and purifying antibodies produced in response to injection of human lymphoid cells into animals. Antibodies can mask, remove or kill the cells expressing lymphoid cell receptors, cause complementdependent lysis and promote lymphocyte uptake back into the reticulo-endothelial system. Examples include anti-thymocyte globulin (ATG) derived from rabbit or horse. This treatment primarily targets T cells and may be used to treat rejection, graft versus host disease or as an induction agent. Similar to this are monoclonal antibodies, but these are derived from a single genetic template. The commonly used basiliximab targets the CD25 molecule of the IL2 receptor on T cells. Muromonab e CD3 (OKT3) targets the CD3 receptor on the surface of T cells. More recently, other agents, such alemtuzumab targets the CD52 receptor, a protein found on B cells, T cells and monocytes. Rituximab targets CD20, a protein on the cell surface of B cells. Not all of these preparations are licensed for use in transplant patients but may form a treatment option in certain cases. A

FURTHER READING Forsythe JLR, ed. Transplantation: a companion to specialist surgical practice. Elsevier Saunders, London 2009. Rose ML, Hutchinson IV. Transplant immunology I: immunological mechanisms of graft injury. Surgery 2006; 24: 47e52.

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