Introduction to Transplantation Immunology
the past few decades, the field of transplantation has undergone a dramatic
Sophisticated surgical procedures have enabled the transplantation of almost
any type of vital organ or
tissue as an effective therapeutic modality. Since most transplants are
done from genetically different
donors, we must consider the immunological response of the recipient to the
presented by the donor graft. In general, this immune response is designed
to cause rejection of the
transplanted tissue and both cellular (or lymphocyte-mediated) and humoral
mechanisms are involved. However, under certain circumstances, a state of
develops which permits long-term normal function of a transplanted organ.
This overview is intended as
an introduction to the field of transplantation immunology. Since most
transplants are performed
between different members of the same species, we will focus on the basic
mechanisms of allogeneic
immune responses in transplant immunity.
immunity results after exposure of a recipient to different histocompatibility
These transplantation antigens have been grouped as major (i.e. HLA and ABO
blood groups) and
minor histocompatibility antigens (although the latter have been documented
in mice and other animals,
little is known about them in humans). HLA antigens are controlled by a
series of genes on
chromosome 6, referred to as the human Major Histocompatibility Complex
MHC). These genes
have been classified into major categories. HLA-A and HLA-B are examples
of Class I genes and
HLA-DR and HLA-DQ represent Class II genes. These genes are highly
polymorphic. For instance,
HLA-A has more than 20 alleles and HLA-B has more than 50 alleles.
Therefore, it is highly unlikely
that two unrelated persons have the same HLA type. Both HLA class I and
class II alloantigens can
induce transplant immunity at humoral (antibody) and cellular (T lymphocyte)
immune levels. ABO
incompatibility is important only in antibody-mediated injury of the
transplant immunity leads to rejection, it seems best that the patient and
matched for histocompatibility antigens. Although this is possible for
genetically identical twins
(syngeneic grafts) and to a lesser extent between HLA- and ABO-identical
siblings, the vast majority of
transplants come from incompatible unrelated (cadaveric) donors.
Immunosuppressive drugs are given
to control transplant immunity so that rejection can be prevented or
controlled and that the recipient
develops a long-term acceptance (or tolerance) of the transplant.
Rejection can be mediated by
antibodies, lymphocytes or both and can manifest itself in different ways:
hyperacute rejection (during
the early post-transplant period), acute rejection (may occur at any
time) and chronic rejection (a
slowly developing process causing a progressive decline in graft
organs (especially kidney and heart) are at risk for hyperacute rejection
patient has preformed donor-specific alloantibodies. This humoral
presensitization (or alloimmunization)
may be caused by a previous transplant, blood transfusions and pregnancy.
The primary target of the
donor-specific antibodies is the vascular endothelium of the transplanted
organ. The most common
target antigens are HLA class I, ABO but other less-well defined antigens
have also been considered.
HLA class II antigens seem less relevant in hyperacute rejection because
they are not stongly expressed
on the vascular endothelium.
mediating hyperacute rejection are almost always Complement-fixing and
be IgG or IgM type or both. Alloreactive T lymphocytes are occasionally
involved. A critical step in
alloantibody-mediated rejection is the initiation of the Complement
cascade. This involves binding and
activation of C1q, which requires at least two Complement-fixing sites
on the antigen/antibody complex.
This requires a close proximity between antigens and no Complement
activation will occur if the antigen
density is too low on the cell surface.
of the Complement cascade leads to the release of various inflammatory
and the initiation of the coagulation and fibrinolytic systems.
Hyperacute rejection in manifested by rapid
vascular constriction, edema and thrombotic occlusion. Endothelium
stimulation and exposure to
subendothelial basement membrane proteins activate platelets which then
adhere and aggregate in the
vasculature. Soon afterward, polymorphonuclear leukocytes (PMN) adhere
to the vascular endothelium.
histopathology of hyperacute rejection is manifested by thrombosis
and edematous changes
but no pronounced mononuclear cell infiltration. Immunostaining shows
deposits of immunoglobulin,
Complement component C3 and fibrinogen.
rejection may also occur without thrombotic occlusion. In this case the
histological pattern is a vasculitis which may lead to necrosis of
individual cells of graft blood vessels.
Because lymphocytes may be also involved, this process is referred to
as acute vascular rejection.
cellular rejection is mediated by lymphocytes which have become
alloactivated against donor
transplantation antigens. In vivo stimulation of alloreactive T cells
takes places primarily in the
peripheral lymphoid tissues of the recipient although intragraft
sensitization may occur. The strongest
stimulus is provided by donor dendritic cells (also referred to as
passenger leukocytes) in the allograft
which will enter the circulation and end up on the lymphoid tissues
of the recipient. These dendritic cells
function as antigen-presenting cells and they provide a strong stimulus
of HLA class II reactive CD4
cells, which can stimulate the growth and differentiation of HLA
class I reactive CD8+ cytolytic
lymphocytes. Alloreactive lymphocytes enter the circulation and
they will react with allograft vascular
endothelium, the primary target of the initial stage of cellular
lymphocyte-endothelial interactions depend on the expression of appropriate
on the endothelium. HLA class I antigens are constitutively expressed at
the cell surface whereas HLA
class II antigens expression must be induced by various cytokines,
especially gamma interferon.
Furthermore, adhesion molecules (ICAM, VCAM, integrins, selectins) play
an important role in the cell
surface interactions between lymphocytes and endothelium cells and their
activation and release of
cytokines and inflammatory mediators. These processes lead to migration
of lymphocytes through the vascular wall.
lymphocytes can mediate various effector mechanisms of allograft immunity.
Besides a direct cytotoxic effect on graft parenchymal cells, lymphocytes
may mediate a delayed type
hypersensitivity mechanism of graft rejection. The latter involves the
recruitment of macrophages and
the release of various cytokines. Other inflammatory cells may also be
seen during rejection, including
neutrophils, eosinophils, B lymphocytes and NK cells. Varying proportions
of CD4 and CD8
lymphocytes are found in cellular infiltrates of rejecting allografts.
Many of them express T cell
activation markers, like IL-2 receptors and probably through release of
gamma-interferon and other
cytokines, there is an increased expression of HLA antigens (especially
class II) on the vascular
endothelium and other target cells in the graft parenchyma.
must be given to additional immunological mechanisms of graft injury
to infection, especially by viruses. This means that other types of
lymphocytes may infiltrate the graft
and they could mediate immune effector responses against virus infected
cells in the graft. Another
possibility is that graft infiltrating lymphocytes mediate recurrent
autoimmune disease processes.
major complication, affecting most long-term transplant survivors, is not
understood. Chronic rejection is characterized by vasculopathy, fibrosis
and a progressive loss of organ function. Its
pathogenesis probably involves both humoral and cellular immune mechanisms.
Chronic rejection may
be mediated by a low-grade, persistent delayed type hypersensitivity
response in which activated
macrophages secrete mesenchymal cell growth factors. Of potential
importance are persistent viral
infections which induce cellular immune responses which may synergize
with donor-specific alloreactive
T cells within the allograft. Chronic rejection may also reflect chronic
ischemia secondary to injury of
blood vessels by antibody or cell-mediated mechanisms. Vascular occlusion
may occur as a result of
smooth muscle cell proliferation in the intima of arterial walls.
the goal of transplantation is to achieve a long-term acceptance of the
allograft, little is
known about the mechanisms responsible for this successful outcome.
Several concepts have been
forwarded to explain the immunological tolerance to the transplant.
One deals with the clonal deletion
of donor-specific lymphocytes in long-term transplant survivors. This
means that rejection mediating
donor-specific alloreactive T cells have selectively been eliminated in
these patients. Another possibility
is that donor-specific T lymphocytes have become anergic (or non-reactive)
so that they cannot function
anymore as effector cells mediating graft rejection. A third concept is
that long-term survivors have
developed suppressor lymphocytes or soluble suppressor factors which down
regulate the donor-specific immune effector responses against the allograft.
Relevant to this is the idiotypic network
concept whereby the immune response triggers anti-antibodies and anti-T
cells as regulatory
mechanisms of the immune response. The most recent hypothesis to explain
long-term graft acceptance
is the persistence in the recipient of donor-derived dendritic cells which
promote an immunologically-mediated chimeric state between the recipient
and the transplanted organ.
of the mechanism of transplant tolerance, it must be noted that most
transplant patients require continuous immunosuppressive treatment for
maintaining graft function
although some notable exceptions have been documented.
every transplant recipient requires treatment with immunosuppressive
drugs to control
graft rejection. During the past decade, improved immunosuppressive
strategies have increased the
success of organ transplantation. Although potent immunosuppressive
drugs may prevent graft
rejection, we must consider adverse side effects on the patient.
Immunosuppression was first done with
metabolic toxins that inhibit DNA replication of lymphocytes. Some
drugs are still in clinical use
especially azathioprine (or Imuran) and to a lesser extent,
cyclophosphamide. During the early 1980's,
Cyclosporine was introduced as a potent immunosuppressive drug.
This drug is a cyclic peptide
produced by a fungus. Its major site of action is the T cell in which
cyclosporine blocks transcription of
IL-2 and other genes, thereby inhibiting the production of lymphokines
which induce the activation and
differentiation of alloreactive T cells. Cyclosporine binds to a
small protein (12kD) called cyclophilin,
which functions as a peptidyl-prolyl-isomerase enzyme involved in
correct folding of the transcription
protein for the regulatory sequence of the IL-2 gene. During recent
years a new drug tacrolimus
(FK506) has been successfully used to treat transplant patients,
especially those with liver allografts.
Although tacrolimus is structurally unrelated to cyclosporine, it also
inhibits IL-2 synthesis by T
lymphocytes through binding to the FK506 binding protein (which is
different from cyclophilin) thereby
blocking lymphokine gene transcription.
are usually administered in combination with Cyclosporine or FK506.
steroids exert their immunosuppressive effects through lysis of certain
T cell subsets and especially by
blocking cytokine gene transcription in macrophages, especially for IL-1,
IL-6 and Tumor Necrosis
Factor (TNF) which are important costimulators of T cell activation.
recent years, other immunosuppressive drugs are being evaluated in
clinical trials with
organ transplant patients. These include mycophenylate mofetil,
rapamycin and deoxyspergulin.
chemical immunosuppression, transplant patients are often treated with
antibodies against T cell surface structures. These antibodies have been
produced as polyclonal antisera
in horses and rabbits immunized with human lymphocytes or thymocytes.
Mouse monoclonal antibodies
have been generated against specific T cell molecules like CD3, CD4,
IL-2 receptor, T cell receptor,
etc. Anti-CD3 antibodies (OKT3) are most widely used and their
immunosuppressive effect is likely
mediated through a modulating effect on the expression of CD3 through
a series of events involving
binding, aggregation, capping and endocytosis of the CD3 molecule.
A major limitation of monoclonal
antibodies is their immunogenicity which triggers anti-mouse Ig
antibodies in the recipient.
type of immunosuppressive drug has adverse side effects which
compromise the transplant
recipient. Immunosuppression is not limited to the transplant
response but also effects the immune
system as a whole and this increases the patient's susceptibility
to infection. Indeed, opportunistic
infections by a variety of microorganisms (including viruses) are
the most frequent complications in
transplant patients. Decreased immunosurveillance also increases
the risk for malignancy, in particular
post-transplant lymphoproliferative disease (PTLD). A major problem
is drug toxicity. The adverse
side effects of cyclosporine and tacrolimus include nephrotoxicity,
liver toxicity, hypertension (less for
tacrolimus) and neurological disorders. Metabolic complications are
associated with steroid treatment.
transplant patients receive a combination of immunosuppressive drugs.
treatments require a careful clinical management of the patient
which includes proper dosage strategies,
measurement of drug levels in the blood, monitoring of graft function
including biopsy histology and
assessment of potential side effects of the drug.
Tissue Typing for Clinical Transplantation
purpose of the tissue typing laboratory is to assess donor-recipient
compatibility for HLA
and ABO to analyze patient serum for lymphocytotoxic antibodies which
may be specific for the
potential transplant donor. Most relevant is the crossmatch assay
whereby patient sera are tested for
their reactivity with donor lymphocytes. This is usually done by
lymphocytotoxicity testing whereby
donor lymphocytes are first incubated with patient serum, then with
rabbit Complement and lysis of
lymphocytes is assessed by the uptake of an extravital dye like trypan
blue or eosin red. A positive
crossmatch is a contraindication for organ transplantation because of the
risk for antibody-mediated
(hyperacute) rejection. This applies particularly for kidney transplants
whereas the liver allograft shows
a relative resistance to antibody-mediated injury. In kidney
transplantation, several modifications of the
crossmatch assay have been used to increase its sensitivity including
antiglobulin augmentation and flow
cytometry. Serum treatment with dithiothreitol (DTT) is used to
distinguish clinically irrelevant IgM type antibodies.
candidates may become sensitized following a prior transplant, blood
previous pregnancies. Serum screening for lymphocytotoxic antibodies
against a random cell panel will
provide an assessment of the degree of sensitization expressed as the
percentage Panel Reactive
Antibody (PRA). The PRA can vary between 0% (non-sensitized) to 80-100%
indicating a high
degree of sensitization. Patients with high PRA values are less likely
to have crossmatch negative
donors and they must wait much longer for a transplant and some may never
receive a transplant.
Recent studies have provided insight about the HLA specificities of
antibodies in highly sensitized
patients. This is based on the concept that each HLA molecule has
multiple antigenic determinants
(epitopes) which can be grouped as private (e.g. HLA-A1, HLA-B7, etc.)
and public determinants
(sometimes referred to as crossreactive groups, CREGs). A public
determinant is an epitope shared by
molecules with different private specificities (e.g. A1+A3+A11,
A2+A9+A28, B5+15+35, B7+22+27+40, etc.). Most highly sensitized patients
show a persistence of the same pattern of
antibody specificity against one or a few public epitopes. A better
understanding of the antibody
specificity improves the selection of transplant donors with acceptable
evidence has been obtained that matching for HLA reduces allograft rejection
thereby promoting survival of kidney and heart transplant patients. Although
the HLA system comprises
multiple class I and especially class II genes, most matching strategies
consider only HLA-A, HLA-B
and HLA-DR. Because of the codominant expression of HLA genes, the
degree of compatibility
ranges from 0 to 6 matches (two for each locus). Survival statistics
from kidney and heart transplant
registries have shown the best survival rates of cadaveric transplants
with 6 matches, followed by 5
matches, etc. Although it make sense to find a perfect match for each
transplant patient, the reality of
practice dictates the selection of less well-matched donors because of the
limitations caused by
extensive polymorphisms of HLA, the limited availability of donors and the
urgency status of the patient.
However, recent experience has shown that transplanted kidneys with
"permissible" HLA mismatches have excellent graft survivals.
Post-Transplant Monitoring of Organ
methodologies have been developed to monitor various organ transplants.
transplant patients, the easiest way to assess renal function is by measuring
serum creatinine levels.
Elevations suggest rejection although cyclosporine induced nephrotoxicity
may also be responsible.
Histopathological examination of a renal biopsy may enable a differential
diagnosis between rejection
and cyclosporine toxicity. Immunostaining of renal tubular cells, a
primary target of infiltrating T cells,
shows increased expression of HLA class II antigens during rejection.
Heart transplant patients are
monitored by histopathological analysis of endomyocardial biopsies at
regular intervals. These biopsies
are obtained through a catheter passed into the right ventricle and the
histological rejection is assessed
by the degree of cellular infiltration and myocyte damage. In liver
transplants, histological analysis of
biopsies shows that the biliary epithelium is the primary target of
cellular rejection. Liver function is
monitored by measuring serum levels of bilirubin and liver enzymes
like alkaline phosphatase. Lung
transplant patients are monitored for rejection by X-ray analysis,
pulmonary function tests,
transbronchial biopsy histology and bronchoalveolar lavage analysis.
Since the transplanted lung is
susceptible to infection, a differential diagnosis between rejection and
infection is very important for the
proper management of these patients. Acute rejection episodes are treated
immunosuppression mostly in the form of a bolus treatment with steroids or
administration of OKT3 or other anti-lymphocyte antibodies.
major complication of any organ transplant is chronic rejection which is
associated with an obstructive vasculopathy, e.g. graft coronary disease
in heart transplant patients.
Chronic liver transplant rejection also leads to a progressive loss of
biliary epithelium, refeered to as
vanishing bile duct syndrome. In lung transplant patients, chronic
rejection is manifested by a
progressive obstructive disease of the airways, referred to as
obliterative bronchiolitis. An early
detection of chronic rejection is neccessary for effective immunotherapeutic
Rene J. Duquesnoy, Ph.D.
Professor of Pathology and Surgery
Division of Transplant Pathology
Thomas E. Starzl Transplant Institute
University of Pittsburgh Medical Center
June 18, 2009
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