Immunology of Transplant Rejection

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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.
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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.
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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.
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Immunobiology of Rejection

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 stageIn
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 pathwayIn 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 pathwayIn 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 activationDuring
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 stageAlloantigen-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. ApoptosisThe 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 cellsThe
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.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 immunityAlthough
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.
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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 rejectionAcute
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 rejectionHumoral
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. Proteinuria is associated
with donor-specific antibody detection and is likely to be an important
factor that determines rapid glomerular filtration rate decline and
earlier graft failure in patients developing de novo HLA antibodies.^[5] 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.^[6] These patients may require augmented immunosuppression.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.^[7] 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])
  

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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.^[8]
  
• 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.
  

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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 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 tsukubaensis.
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. The introduction of MMF has been shown to be associated with
improvement or stabilization of renal function, even several years after
transplantation.^[9]
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.
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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
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.^[10] 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.