25/04/2011 Immunology of Transplant Rejection

Immunology of Transplant Rejection

Author: Prashant Malhotra, MBBS; Chief Editor: Ron Shapiro, MD more...

Updated: Apr 20, 2011

Overview
Transplantation is the act of transferring cells, tissues, or organs from one site to another. The malfunction of an
organ system can be corrected with transplantation of an organ (eg, kidney, liver, heart, lung, or pancreas) from a
donor. However, the immune system remains the most formidable barrier to transplantation as a routine medical
treatment. The immune system has developed elaborate and effective mechanisms to combat foreign agents.
These mechanisms are also involved in the rejection of transplanted organs, which are recognized as foreign by
the recipient's immune system.

Understanding these mechanisms is important, as it aids in understanding the clinical features of rejection and,
hence, in making an early diagnosis and delivering appropriate treatment. Knowledge of these mechanisms is also
critical in developing strategies to minimize rejection and in developing new drugs and treatments that blunt the
effects of the immune system on transplanted organs, thereby ensuring longer survival of these organs.

For more information on various transplantation procedures, see eMedicine’s Transplantation journal and the
Medscape resource centers for Heart & Lung Transplant, Kidney & Pancreas Transplant, and Liver & Intestine
Transplant.

History
In 1944, Medawar showed that skin allograft rejection is a host versus graft response. Mitchison later
demonstrated the cell-mediated features of this response. The first successful identical twin transplant of a human
kidney was performed by Joseph E. Murray in 1954 in Boston, followed by the first successful liver transplant by
Dr. Thomas E. Starzl in 1967, the first heart transplantation by Christian Barnard in 1967, and the first successful
bone marrow transplant by E. Donnall Thomas in 1968. Schwartz and Dameshek, in 1959, showed that 6-
mercaptopurine was immunosuppressive in rats, ushering in the era of immunosuppressive drug treatment. Since
then, many new and progressively more selective immunosuppressive agents have been developed. These
therapies have enabled the transplantation of and improved the survival of transplanted organs.

Types of Grafts
The degree of immune response to a graft depends partly on the degree of genetic disparity between the grafted
organ and the host. Xenografts, which are grafts between members of different species, have the most disparity
and elicit the maximal immune response, undergoing rapid rejection. Autografts, which are grafts from one part of
the body to another (eg, skin grafts), are not foreign tissue and, therefore, do not elicit rejection. Isografts, which
are grafts between genetically identical individuals (eg, monozygotic twins), also undergo no rejection.

Allografts are grafts between members of the same species that differ genetically. This is the most common form
of transplantation. The degree to which allografts undergo rejection depends partly on the degree of similarity or
histocompatibility between the donor and the recipient.

The degree and type of response also vary with the type of the transplant. Some sites, such as the eye and the
brain, are immunologically privileged (ie, they have minimal or no immune system cells and can tolerate even
mismatched grafts). Skin grafts are not initially vascularized and so do not manifest rejection until the blood supply
develops. The heart, kidneys, and liver are highly vascular organs and lead to a vigorous cell mediated response in
the host.

Immunobiology of Rejection

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Genetic Background
The antigens responsible for rejection of genetically disparate tissues are called histocompatibility antigens; they
are products of histocompatibility genes. Histocompatibility antigens are encoded on more than 40 loci, but the
loci responsible for the most vigorous allograft rejection reactions are located on the major histocompatibility
complex (MHC).

In humans, the MHC is called the human leukocyte antigen (HLA) system and is located on the short arm of
chromosome 6, near the complement genes. Other antigens cause only weaker reactions, but combinations of
several minor antigens can elicit strong rejection responses. The MHC genes are codominantly expressed, which
means that each individual expresses these genes from both the alleles on the cell surface. Furthermore, they are
inherited as haplotypes or 2 half sets (one from each parent). This makes a person half identical to each of his or
her parents with respect to the MHC complex. This also leads to a 25% chance that an individual might have a
sibling who is HLA identical.

The MHC molecules are divided into 2 classes. The class I molecules are normally expressed on all nucleated
cells, whereas the class II molecules are expressed only on the professional antigen-presenting cells (APCs),
such as dendritic cells, activated macrophages, and B cells. The physiological function of the MHC molecules is
to present antigenic peptides to T cells, since the T lymphocytes only recognize antigen when presented in a
complex with an MHC molecule. The class I molecules are responsible for presenting antigenic peptides from
within the cell (eg, antigens from the intracellular viruses, tumor antigens, self-antigens) to CD8 T cells. The class
II molecules present extracellular antigens such as extracellular bacteria to CD4 T cells.

Mechanisms of Rejection
The immune response to a transplanted organ consists of both cellular (lymphocyte mediated) and humoral
(antibody mediated) mechanisms. Although other cell types are also involved, the T cells are central in the
rejection of grafts. The rejection reaction consists of the sensitization stage and the effector stage.

Sensitization stage
In this stage, the CD4 and CD8 T cells, via their T-cell receptors, recognize the alloantigens expressed on the
cells of the foreign graft. Two signals are needed for recognition of an antigen; the first is provided by the
interaction of the T cell receptor with the antigen presented by MHC molecules, the second by a costimulatory
receptor/ligand interaction on the T cell/APC surface. Of the numerous costimulatory pathways, the interaction of
CD28 on the T cell surface with its APC surface ligands, B7-1 or B7-2 (commonly known as CD80 or CD86,
respectively), has been studied the most. [1] In addition, cytotoxic T lymphocyte–associated antigen-4 (CTLA4)
also binds to these ligands and provides an inhibitory signal. Other costimulatory molecules include the CD40 and
its ligand CD40L (CD154).

Typically, helices of the MHC molecules form the peptide-binding groove and are occupied by peptides derived
from normal cellular proteins. Thymic or central tolerance mechanisms (clonal deletion) and peripheral tolerance
mechanisms (eg, anergy) ensure that these self-peptide MHC complexes are not recognized by the T cells,
thereby preventing autoimmune responses.

At least 2 distinct, but not necessarily mutually exclusive, pathways of allorecognition exist, the direct and indirect
pathways. Each leads to the generation of different sets of allospecific T cell clones.

Direct pathway
In the direct pathway, host T cells recognize intact allo-MHC molecules on the surface of the donor or stimulator
cell. Mechanistically, host T cells see allo-MHC molecule + allo-peptide as being equivalent in shape to self-MHC
+ foreign peptide and, hence, recognize the donor tissue as foreign. This pathway is presumably the dominant
pathway involved in the early alloimmune response.

The transplanted organ carries a variable number of passenger APCs in the form of interstitial dendritic cells. Such
APCs have a high density of allo-MHC molecules, and are capable of directly stimulating the recipient's T cells.
The relative number of T cells that proliferate on contact with allogeneic or donor cells is extraordinarily high as
compared with the number of clones that target antigen presented by self-APC. Thus, this pathway is important in
acute allorejection.

Indirect pathway

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In the indirect pathway, T cells recognize processed alloantigen presented as peptides by self-APCs. Secondary
responses such as those that occur in chronic or late acute rejection are associated with T cell proliferative
responses to a more variable repertoire, including peptides that were previously immunologically silent. Such a
change in the pattern of T cell responses has been termed epitope switching or spreading.

A link between self-MHC + allopeptide-primed T cells and the development of acute vascular type rejection has
been demonstrated to be mediated in part by accelerated alloantibody production. In addition, chronic allograft
vasculopathy may be mediated by T cells primed by the indirect pathway.

Molecular mechanisms of T cell activation

During T cell activation, membrane-bound inositol phospholipid is hydrolyzed into diacylglycerol (DAG) and IP3.
This increases the cytoplasmic calcium. The elevation in calcium promotes the formation of calcium-calmodulin
complexes that activate a number of kinases as well as protein phosphatase IIB or calcineurin. Calcineurin
dephosphorylates cytoplasmic nuclear factor of activated T cells (NFAT), permitting its translocation to the
nucleus, where it binds to the IL-2 promoter sequence and then stimulates transcription of IL-2 mRNA. Numerous
other intracellular events, including protein kinase C (PKC) activation by DAG and activation of nuclear factor
kappa B (NFkB) also occur at the molecular level.

Effector stage
Alloantigen-dependent and independent factors contribute to the effector mechanisms. Initially, nonimmunologic
"injury responses" (ischemia) induce a nonspecific inflammatory response. Because of this, antigen presentation
to T cells is increased as the expression of adhesion molecules, class II MHC, chemokines, and cytokines is
upregulated. It also promotes the shedding of intact, soluble MHC molecules that may activate the indirect
allorecognition pathway. After activation, CD4-positive T cells initiate macrophage-mediated delayed type
hypersensitivity (DTH) responses and provide help to B cells for antibody production.

Various T cells and T cell-derived cytokines such as IL-2 and IFN-γ are upregulated early after transplantation.
Later, ß-chemokines like RANTES (regulated upon activation, normal T cell expressed and secreted), IP-10, and
MCP-1 are expressed, and this promotes intense macrophage infiltration of the allograft. IL-6, TNF-α, inducible
nitric oxide synthase (iNOS) and growth factors, also play a role in this process. The growth factors, including
TGF-ß and endothelin, cause smooth muscle proliferation, intimal thickening, interstitial fibrosis, and, in the case
of the kidney, glomerulosclerosis.

Endothelial cells activated by T cell–derived cytokines and macrophages express class II MHC, adhesion
molecules, and costimulatory molecules. These can present antigen and thereby recruit more T cells, amplifying
the rejection process. CD8-positive T cells mediate cell-mediated cytotoxicity reactions either by delivering a
"lethal hit" or, alternatively, by inducing apoptosis.

Apoptosis

The final common pathway for the cytolytic processes is triggering of apoptosis in the target cell. [2] After activation
of the CTLs, they form cytotoxic granules that contain perforin and granzymes. [2] At the time of target cell
identification and engagement, these granules fuse with the effector cell membrane and extrude the content into
the immunological synapse. By a yet unknown mechanism, the granzymes are inserted into the target cell
cytoplasm where granzyme B can trigger apoptosis through several different mechanisms, including direct
cleavage of procaspase-3 and indirect activation of procaspase-9. This has been shown to play the dominant role
in apoptosis induction in allograft rejection.

Alternatively, CD8-positive CTLs can also use the Fas-dependent pathway to induce cytolysis and apoptosis. The
Fas pathway is also important in limiting T cell proliferation in response to antigenic stimulation; this is known as
fratricide between activated CTLs. Cell-mediated cytotoxicity has been shown to play an important role in acute,
although not chronic, allograft rejection.

Role of natural killer cells

The natural killer (NK) cells are important in transplantation because of their ability to distinguish allogenic cells
from self and their potent cytolytic effector mechanisms. [3] These cells can mount a maximal effector response
without any prior immune sensitization. Unlike T and B cells, NK cells are activated by the absence of MHC
molecules on the surface of target cells (“missing self” hypothesis). The recognition is mediated by various NK
inhibitory receptors triggered by specific alleles of MHC class I antigens on cell surfaces.

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In addition, they also possess stimulatory receptors, which are triggered by antigens on nonself cells. These
effector responses include both cytokine release and direct toxicity mediated through perforin, granzymes, Fas
ligand (FasL), and TNF-related apoptosis-inducing ligand (TRAIL). Through this “double negative” mode of
activation, they are thought to play a role in the rejection of both bone marrow and transplantable lymphomas in
animal models.

NK cells also provide help to CD28-positive host T cells, thereby promoting allograft rejection. [4] Their importance
in the field of bone marrow transplants has been recognized for years. In humans, their graft-versus-host
alloresponse has been used for its potent graft-versus-leukemia effect and has contributed to an increase in the
rate of sustained remission in patient with acute myelogenous leukemia.

NK cells are now being recognized as active participants in the acute and chronic rejection of solid tissue grafts. [3]
Recent studies have indicated that NK cells are present and activated following infiltration into solid organ
allografts.[3] They may regulate cardiac allograft outcomes. Studies have also shown that humans with killer cell
immunoglobulin-like receptors that are inhibited by donor MHC have a decreased risk of liver transplant rejection.
In cases of renal transplantation, these cells are not suppressed by the current immunosuppressive regimens.

Role of innate immunity

Although T cells have a critical role in acute rejection, the up-regulation of proinflammatory mediators in the
allograft is now recognized to occur before the T cell response; this early inflammation following engraftment is due
to the innate response to tissue injury independent of the adaptive immune system. Several recent studies have
examined the role of Toll-like receptor (TLR) agonists and TLR signals in allorecognition and rejection.

These innate mechanisms alone do not appear sufficient to lead to graft rejection itself. However, they are
important for optimal adaptive immune responses to the graft and may play a major role in resistance to tolerance
induction. The development of methods to blunt innate immune responses, which has potential implications for a
wide variety of diseases, is likely to have a significant impact on transplantation, as well.

Clinical Stages of Rejection

Hyperacute Rejection
In hyperacute rejection, the transplanted tissue is rejected within minutes to hours because vascularization is
rapidly destroyed. Hyperacute rejection is humorally mediated and occurs because the recipient has preexisting
antibodies against the graft, which can be induced by prior blood transfusions, multiple pregnancies, prior
transplantation, or xenografts against which humans already have antibodies. The antigen-antibody complexes
activate the complement system, causing massive thrombosis in the capillaries, which prevents the
vascularization of the graft. The kidney is most susceptible to hyperacute rejection; the liver is relatively resistant,
possibly because of its dual blood supply, but more likely because of incompletely understood immunologic
properties.

Acute Rejection
Acute rejection manifests commonly in the first 6 months after transplantation.

Acute cellular rejection

Acute cellular rejection is mediated by lymphocytes that have been activated against donor antigens, primarily in
the lymphoid tissues of the recipient. The donor dendritic cells (also called passenger leukocytes) enter the
circulation and function as antigen-presenting cells (APCs).

Humoral rejection
Humoral rejection is form of allograft injury and subsequent dysfunction, primarily mediated by antibody and
complement. It can occur immediately posttransplantation (hyperacute) or during the first week. The antibodies are
either preformed antibodies or represent antidonor antibodies that develop after transplantation.

The presence of even low levels of donor-specific antibodies that may not be detected by complement-dependent
cytotoxic and flow cytometry crossmatches have been shown to be associated with inferior renal allograft
outcomes. [5] These patients may require augmented immunosuppression.

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The classic pathway inactive product C4d has been shown to be deposited in the peritubular capillaries (PTC), and
immune detection of this product in renal allograft biopsies is used in diagnosis of antibody-mediated rejection.
However, one study has demonstrated that there is a substantial fluctuation in the C4d Banoff scores in the first
year posttransplant, and this may reflect the dynamic and indolent nature of the humoral process.[6] Thus, C4d by
itself may not be a sufficiently sensitive indicator, and microvascular inflammation with detection of donor-specific
antibodies may be more useful in diagnosing humoral rejection.

Chronic Rejection
Chronic rejection develops months to years after acute rejection episodes have subsided. Chronic rejections are
both antibody- and cell-mediated. The use of immunosuppressive drugs and tissue-typing methods has increased
the survival of allografts in the first year, but chronic rejection is not prevented in most cases.

Chronic rejection appears as fibrosis and scarring in all transplanted organs, but the specific histopathological
picture depends on the organ transplanted. In heart transplants, chronic rejection manifests as accelerated
coronary artery atherosclerosis. In transplanted lungs, it manifests as bronchiolitis obliterans. In liver transplants,
chronic rejection is characterized by the vanishing bile duct syndrome. In kidney recipients, chronic rejection
(called chronic allograft nephropathy) manifests as fibrosis and glomerulopathy. The following factors increase the
risk of chronic rejection:

Previous episode of acute rejection

Inadequate immunosuppression
Initial delayed graft function
Donor-related factors (eg, old age, hypertension)
Reperfusion injury to organ
Long cold ischemia time
Recipient-related factors (eg, diabetes, hypertension, hyperlipidemia)
Posttransplant infection (eg, cytomegalovirus [CMV])

Transplant Tolerance and Minimizing Rejection

Rejection cannot be completely prevented; however, a degree of immune tolerance to the transplant does develop.
Several concepts have been postulated to explain the development of partial tolerance. They include clonal
deletion and the development of anergy in donor specific lymphocytes, development of suppressor lymphocytes, or
factors that down-regulate the immune response against the graft. Other hypotheses include the persistence of
donor-derived dendritic cells in the recipient that promote an immunologically mediated chimeric state between the
recipient and the transplanted organ.

Tissue typing or crossmatching is performed prior to transplantation to assess donor-recipient compatibility for
human leukocyte antigen (HLA) and ABO blood group. These tests include the following:

The ABO blood group compatibility is tested first because incompatibility between the blood groups leads
to rapid rejection.
In the lymphocytotoxicity assay, patient sera are tested for reactivity with donor lymphocytes. A positive
crossmatch is a contraindication to transplantation because of the risk of hyperacute rejection. This is used
mainly in kidney transplantation.
Panel-reactive antibody (PRA) screens the serum of a patient for lymphocytic antibodies against a random
cell panel. Patients with prior transfusions, transplants, or pregnancies may have a high degree of
sensitization and are less likely to have a negative crossmatch with a donor. A reduced risk of sensitization
at the time of second transplant has been observed when using more potent immunosuppression with rabbit
antithymocyte globulin, tacrolimus, and mycophenolate mofetil/sodium for nonsensitized primary kidney or
kidney/pancreas transplant patients. [7]
Mixed lymphocyte reaction (MLR) can be used to assess the degree of major histocompatibility complex
(MHC) class I and class II compatibility. However, it is not a rapid test and can be used only in cases
involving living related donors. It is rarely used at present.

Immunosuppression
Initially, radiation and chemicals were used as nonselective immunosuppressive agents. In the late 1950s and
1960s, the agents 6-mercaptopurine and azathioprine were used in conjunction with steroids. Newer
immunosuppressive agents have since been developed; they are more effective, more selective, and less toxic and

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have made possible the advances in the field of transplantation. Immunosuppressive drugs are used in 2 phases:
the initial induction phase, which requires much higher doses of these drugs, and the later maintenance phase.
Immunosuppressive agents in current use include the following:

Immunophilin-binding agents
The available immunophilin-binding agents are cyclosporine and tacrolimus. These agents are calcineurin
inhibitors; they primarily suppress the activation of T lymphocytes by inhibiting the production of cytokines,
specifically IL-2. They are associated with numerous toxicities that are often dose-dependent. Nephrotoxicity
occurs with both the drugs. Hirsutism, gingival hypertrophy, hypertension, and hyperlipidemia develop more often
with cyclosporine than tacrolimus. (Click here to complete a Medscape CME activity on hirsutism.) Potential drug
interactions are also important to recognize.

Tacrolimus is a macrolide lactone antibiotic produced by the soil fungus Streptomyces tsuk ubaensis. It binds to a
different intracellular protein (FKBP-12) than cyclosporine but has the same mechanism of action. Neurotoxicity,
alopecia, and posttransplant diabetes mellitus develop more frequently with tacrolimus than with cyclosporine.

Mammalian target of rapamycin (mTOR) inhibitors

Sirolimus is a macrocyclic antibiotic produced by fermentation of Streptomyces hygroscopicus. It binds to FKBP-
12 and presumably modulates the activity of the mTOR inhibitor, which inhibits IL-2–mediated signal transduction
and results in T- and B-cell cycle arrest in the G1-S phase. Sirolimus is associated with numerous adverse
effects, such as leukopenia, thrombocytopenia, anemia, hypercholesterolemia, and hypertriglyceridemia. It has
also been associated with mucositis, delayed wound healing, lymphocele formation, pneumonitis, and prolonged
delayed graft function.

Antiproliferative agents
Azathioprine and mycophenolate mofetil (MMF) are the agents commonly used in this category. Other
antiproliferative agents, such as cyclophosphamide and, more recently, leflunomide, have also been used.

Antiproliferative agents inhibit DNA replication and suppress B- and T-cell proliferation. MMF is an organic
synthetic derivative of the natural fermentation product mycophenolic acid (MPA) that causes noncompetitive
reversible inhibition of inosine monophosphate dehydrogenase. This interferes with purine synthesis. Adverse
effects of MMF are nausea, diarrhea, leukopenia, and thrombocytopenia. Invasive CMV infection has been
sometimes associated with MMF.

Antibodies
Two antibodies that are IL-2 receptor antagonists (basiliximab and daclizumab) are FDA-approved for kidney
transplantation induction. Antilymphocyte globulin, such as the monoclonal antibody muromonab-CD3, and the
polyclonal antibodies, antithymocyte globulins derived from either equine or rabbit sources, are approved for the
treatment of rejection. They also have been used as induction agents at some transplantation centers.

Antibodies interact with lymphocyte surface antigens, depleting circulating thymus-derived lymphocytes and
interfering with cell-mediated and humoral immune responses. Lymphocyte depletion also occurs either by
complement-dependent lysis in the intravascular space or by opsonization and subsequent phagocytosis by
macrophages. Adverse effects such as fever, chills, thrombocytopenia, leukopenia, and headache typically occur
with the first few doses.

Corticosteroids
Steroids have been the cornerstone of immunosuppression and are still used. However, the newer regimens are
trying to minimize the use of steroids and thereby avoid the adverse effects that are associated with them.
Steroids are still important in treating episodes of acute rejection.

Future Therapies
Many new agents are designed to interfere with secondary signaling, and this may aid in induction of tolerance.

Efalizumab is a humanized monoclonal antibody that targets the T-cell lymphocyte function-associated antigen-1
(LFA-1) receptor through the CD11a side chain. Efalizumab (Raptiva), a drug indicated for psoriasis, is being

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withdrawn from the US market and will no longer be available after June 8, 2009, because of potential risk for
progressive multifocal leukoencephalopathy (PML). PML is a rapidly progressive infection of the central nervous
system caused by the JC virus that leads to death or severe disability. Demyelination associated with PML is a
result from the JC virus infection. JC virus belongs to the genus Polyomavirus of the Papovaviridae. PML should be
considered in any patient presenting with new-onset neurologic manifestations who have taken efalizumab. For
more information, see the Food and Drug Administration MedWatch Safety Alert. [8]

Monoclonal antibodies to B7-1 (CD80) and B7-2 (CD86) have been developed to block T-cell CD28 activation and
proliferation responses. In a recent trial, one of these antibodies, belatacept, did not appear to be inferior to
cyclosporine as a means of preventing acute rejection after renal transplantation.

Cytotoxic T lymphocyte antigen 4 immunoglobulin (CTLA4Ig) can simultaneously inhibit B7-1 and B7-2 interaction
with CD28 and has been used successfully in animal models, demonstrating a beneficial effect on chronic allograft
rejection.

Other antibodies targeting CD28 are also in development.

Monoclonal anti-CD45-RB, leflunomide, FK778, FTY720, alemtuzumab (anti-CD52 antibody), and rituximab are
some of the other agents in different phases of evaluation.

Natural killer (NK) cell inactivation or depletion also harbors the promise that it may improve the long-term outcome
of transplanted organs.

The use of any immunosuppressive drug requires a balance between the risk of loss of transplanted organ and the
toxicity of the agent. The goal is to balance an appropriate level of immunosuppression with the long-term risks,
which include development of infections, cancer, and metabolic complications.

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