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Created by:
Emily Israeli, Vivian Fong,
Sarah Mitchell, Jill Harrison, Ashley Schomer
Three Main Types of Rejection



Hyperacute rejection – caused by
preexisting antibodies and activation of
complement system – occurs
immediately
Acute Rejection – caused by mismatched
HLA antigens – influx of T-Cells and
cytokines – occurs 7-10 days out
Chronic rejection – caused by
proliferation of SMCs and excess
collagen production that occludes the
vasculature over time – occurs over long
periods
Healthy Islets
surrounded by
non-insulin
producing
pancreatic cells
Islets
suffering
from
hyperacute
rejection
Chronic rejection still
remains a major
problem up to 3 years
out. At this point up to
40% of allografts have
been rejected
Basics of Molecular Mechanism of Rejection
• Major histocompatibility complex (MHC) – locus on Chromosome 6
that encodes MHC molecules – polymorphic and codominant
• Called human lymphocyte angtigen (HLA) molecules in humans
• Mismatched HLA molecules present bound peptides
• That MHC-peptide complex is presented by antigen presenting cells
(APCs) to host T-cells
• Two main methods of presentation: direct and indirect
Indirect Presentation
• Host APCs digest foreign MHCpeptide complexes
• Then present the peptides from
the digested complex on its own
MHC molecules to T-cells
• In this case the foreign MHC
molecules are handled as if they
were any other foreign antigen
Direct Presentation
• Donor APCs present donor
MHC-peptide complexes
• Host T-cells still recognize
these complexes due to
similarities
• Most rejection mechanisms are
elicited through direct
presentation
• Many different host T-cells can
recognize a single foreign MHC
molecule
Indirect vs. Direct Presentation
The direct and indirect pathways of antigen presentation. In the direct pathway, MHC
molecules on donor APCs from the graft tissue present graft-derived peptides to host T-cells. In
the indirect pathway, host APCs take up graft proteins and present donor-derived processed
peptides on host MHC molecules to host T-cells.
Immunosuppressive Agents
"There are few examples of clinical procedures
that have moved from complete failure to outstanding success
in such a short space of time (Parrot)."
Organ transplantation therapy is one of those examples. The human HLA antigen proved such a barrier to
organ grafting,
"that prior to the late 1950's, transplantation yielded uniformly dismal and
consistently fatal results (Lysaght)."
It was only through the discovery, evolution, and routing use of immunosuppressant agents that this
barrier has finally been overcome. In fact, many organ transplantations are now routine clinical
procedures - the kidney transplant is a prime example, with 15,122 procedures performed in 2003. Organ
transplantation therapy is highly dependent on the success of pharmacotherapy to suppress recipient
immune responses to the foreign organ; allograft rejection remains as the major barrier to long-term graft
survival in patients. In fact, transplant patients require lifelong immunosuppressive drug therapy to
prevent this rejection.
Clinical Science currently has a
comprehensive understanding of
the cellular mechanisms behind
graft rejection. Rejection follows a
certain path with steps that
include graft recognition,
cytokine signaling, lymphocyte
proliferation, and attack.
Different immunosuppressive
agents work by blocking this path
at various steps.
Corticosteroid
Prednisone (Deltasone®), Methylprednisolone (SoluMedrol®)
s
Corticosteriods are used in maintenance immunosuppression, and the treatment of acute rejection.
A corticosteroid is a medication that works principally to block T cell and
APC derived cytokine and cytokine-receptor expression.
The major elements blocked are IL-1, IL-6, IL-2, INF-γ , and TNF-α . These elements, notably IL-1, are
essential for lymphocyte-APC communication.
A decrease in production effectively obstructs an APC's capacity to activate allograft-specific lymphocytes.
As a result, threat of acute rejection is reduced.
Corticosteroids have a hydrophobic structure that allows them to easily diffuse into cells and bind to specific
cytoplasmic receptors. The resulting complexes progress to the nucleus, where they are able to inhibit the
transcription of the genes of the cytokines named above.
Corticosteroids are also able to inhibit cytokine production in macrophages. This subsequently inhibits the
macrophage phagocytosis and chemotaxis properties. Corticosteroids are also potent non-specific antiinflammatory agents - administration results in an acute reduction of circulating lymphocytes and monocytes.
Calcineurine Inhibitors
Cyclosporine (Sandimmune®, Neoral®, Gengraf®), Tacrolimus (Prograf®, FK506)
Calcineurine Inhibitors are used in maintenance immunosuppression.
The mechanisms of Calcineurine Inhibitors converge at the inhibition of the calcineurin. This
inhibition ultimately inhibits the production and secretion of IL-2. The interaction between IL-2
and the IL-2 Receptor is crucial in the activation, differentiation, and proliferation of B and T
cells. Therefore, halting the rejection process at this step is highly effective at combating rejection.
Cyclosporine is a small fungal cyclic peptide. Cyclosporine works by binding a
protein found in the cytosol: cyclophilin. This complex inhibits calcineurin. The
rest of the mechanism is outlined above.
Cyclosporine is a highly effective immunsuppressant. In fact, cyclosporine is generally hailed as
the cornerstone of immunosuppression. The clinical introduction of Cyclosporine significantly
increased graft survival and significantly reduced the occurrence of acute rejection in patients.
Tacrolimus is a microlide antibiotic that works in a mechanism similar to that of cyclosporine. Tacrolimus
binds the cytosolic protein FKPB-121. This complex inhibits calcineurin in a manner parallel to cyclosporine.
Antiproliferative Agents
Mycophenolate Mofetil (CellCept®), Azathioprine (Imuran®)
Sirolimus (Rapamune®)
Antiproliferative Agents are used in maintenance immunosuppression and treatment of rejection.
Antiproliferative Agents are drugs that work to block the proliferative
phase of acute cellular rejection. They are an integral part of most
immunosuppression regimens.
Mycophenolate Mofetil
Mycophenolate Mofetil (MMF) is absorbed and rapidly hydrolyzed in the blood to its active form: MPA.
MPA inhibits the key enzyme in the de novo pathway of purine biosynthesis, IMPDH. Rapidly dividing
cells, such as activated lymphocytes, depend on the de novo pathway for the production of purines necessary
for RNA and DNA synthesis. In this way, activated lymphocytes are selectively inhibited since they are not
allowed to proliferate once activated.
Antiproliferative Agents
Azathioprine
Azathioprine is rapidly hydrolyzed in the blood to 6-mercaptopurine. In this form (a
purine analog and antimetabolite), it incorporates into the DNA, inhibiting nucleotide
synthesis by causing feedback inhibition in the early stages of purine metabolism.
This ultimately prevents mitosis and proliferation of rapidly dividing cells, such as
activated B and T lymphocytes. Through this action, Azathioprine is able to block most Tcell functions and inhibit primary antibody synthesis. Azathioprine has little effect on
established immune responses, and is therefore effective only in the prevention (not
treatment) of acute rejection.
Sirolimus
Sirolimus is a highly potent macrolide antibiotic that has a chemical structure similar to Tacrolimus. Sirolimus
binds to the same protein as Tacrolimus: FKBP-12. Instead of inhibiting Calcineurin as Tacrolimus does, this
complex inhibits mTOR. This inhibition prevents the progression of T cells from the G1 to the S phase of
the cell cycle by blocking signaling downstream of the IL-2 receptor. It therefore is able to block DTH
immune reactions, CTL activity, and humoral responses directed against a transplanted organ. Because
Cyclosporine and Sirolimus have different mechanisms, the combination of clinical combination of the two in an
immunosuppression regimen results in effects that are synergistic.
Monoclonal Antibodies
Muromonab-CD3 (Orthoclone OKT3®), Daclizumab (Zenapax®)
Interleukin-2 Receptor Antagonist (Basiliximab, Simulect®)
Monoclonal Antibodies are used in early rejection prophylaxis and treatment of
rejection.
Monoclonal antibodies are antigen-specific immunosuppressants that
will reduce immune response to alloantigens of the graft while
preserving the response to alloantigens to unrelated antigens.
These agents are specific to blocking T-cell activation, resulting in rapid
depletion of T cells from the circulation by binding of antibody coated T cells
to Fc receptors on phagocytic cells. The most recently FDA approved
monoclonal antibodies are the IL-2 receptor antagonists genetically engineered
to possess both human and murine antibody sequences. The chimerization
of these antibodies is an attempt to decrease the immunogenicity of the agent.
Other monoclonal antibody-based drugs are still in clinical trials for FDA
approval.
Monoclonal Antibodies
Muromonab – CD3
Muromonab-CD3 is the first type of murine monoclonal antibody directed against the ε chain of the CD3
molecule (an integral part of the T Cell Receptor complex) and modulates the receptor and inactivates T-cell
function blocking both naïve T cells and CTLs. This results in rapid depletion of T cells from circulation
and cytokine release. This antibody is used to treat acute rejection and steroid resistant rejection.
Baxiliximab
Basiliximab is a chimeric (70% human and 30% murine) monoclonal antibody utilized in the prevention
of acute organ rejection. This monoclonal antibody has a specificity and high affinity for the α subunit of
the interleukin (IL)-2 receptor (IL-2Rα, also known as CD25 or Tac) preventing IL-2 from binding to the
receptor on the surface of activated T cells. By acting as an IL-2Ra antagonist, Basiliximab inhibits IL-2mediated activation and proliferation of T cells, the critical step in the cascade of cellular immune
response of allograft rejection. Therefore, Basiliximab has a long half-life of approximately 7-12 days and
saturates the IL-2 receptor for up to 59 days. Due to its high percentage of humanization in its antibody
sequences the occurrence and acuteness of adverse effects is significantly lower when used with standard
immunotherapy.
Monoclonal Antibodies
Muromonab – CD3
Daclizumab is a similar agent to Basiliximab, but is a more humanized IgG monoclonal antibody (90%
human and 10% murine). It also binds to and inhibits the α-subunit of IL-2 receptor and thus works in a
manner similar to Basiliximab. Daclizumab is able to saturate the IL-2 receptor twice as long as Basiliximab.
Because it is more humanized, there are less side effects associated with Daclizumab.
Side Effects - HAMA
One of the major problems affecting monoclonal therapy is the side effects associated with the HAMA
(Human anti-Murine Antibody) response and serum sickness. These are both directly caused by the
structure of the antibodies. Laboratories routinely use murine antibodies as a starting point for monoclonal
antibodies.
This poses a problem: human immune system is able to identify the murine antibody as non-self
and eliminate the treatment from circulation. This renders the monoclonal therapy ineffective.
A counteractive measure includes humanizing (or chimerizing) the antibody. Because the only
immunologically offensive portion of the antibody is the constant region, recombinant techniques can be
used to splice and replace the constant region of the murine antibody with that of a characteristic
human antibody. Current drugs have been developed that are more favorably humanized. They are also
shown to be more effective with less adverse side effects. Further chimerization and other improvements in
antibody therapy are active fronts of current research.
Polyclonal Antibodies
Antithymocyte globulin-equine (Atgam®)
Antithymocyte globulin-rabbit (RATG, Thymoglobulin®)
Polyclonal Antibodies are used in early rejection prophylaxis, treatment of rejection.
Polyclonal antibodies are directed against lymphocyte antigens;
instead of the single-specificity of the monoclonal antibodies, these anitlymphocyte
antibodies are directed against multiple epitopes.
Antithymocyte globulin is a polyclonal antibody derived from either horses (Atgam®) or rabbits
(Thymoglobulin®). The agents contain antibodies specific for many common T cell surface antigens including
CD2, CD3, CD4, CD8, CD11a, CD18. The antithymocyte globulin binds lymphocytes that display the surface
antigens previously listed. This effectively depletes T-cell concentration in the body through complementdependent cytolysis and cell mediated opsonization following with T cell clearance from the circulation by
the reticuloendothelial system (RES).
Despite the glamorous advances of immunosuppressants, it is important to
bear in mind the mechanism behind immunosuppression:
Immunosuppressants dampen the body's immune system.
With current therapy, there are adverse side-effects that include, among others, a
high incidence of opportunistic infection and transplant-related malignancies in
patients. These are the unfortunate consequences of overimmunosuppression.
Accordingly, a major goal of immunosuppression is to identify the optimal
balance of therapy such that there is effective prevention of allograft rejection,
while drug-related adverse effects, infection, and malignancies are minimized.
Because this compromise is largely unsatisfactory, there is a constant search for
more effective and specific immunosuppressive agents and strategies.
Side Effects Associated with Immunosuppressants:
alopecia - loss of hair, baldness
hypertricosis - excessive hair growth
anemia - a pathological deficiency in the oxygen carrying compound of the
blood
hypertriglyceridemia - a disorder due to disturbances in synthesis and/or
degradation of triglycerides-rich plasma lipoprotiens
arthralgias - pain experienced in the joints
hyperuricemia - the presence of excess uric acid in the blood
bone marrow depletion - a depletion of bone marrow (a soft, fatty, vascular
tissue that fills most bone cavities - it is the source of blood cells)
hypomagnesemia - a deficiency of magnesium in the blood
coronary artery disease - a stage of arteriosclerosis involving fatty deposits
inside the artery walls that feed the heart
cushingoid appearance - moon face, buffalo hump, centripetal obesity
gastrointestinal upsets - discomfort spurring from the stomach and/or
intestines
gingival hyperplasia - an increase in the amount of gum tissue in the mouth
glaucoma - eye diseases characterized by high intraocular fluid pressure,
damaged optic disk, hardening of the eyeball, and loss of vision
hepatoxicity - damage to the liver
hirsutism - excess facial and body hair
hypercholesterolemia - the presence of excess cholesterol in blood
hyperglycemia - the presence of excess sugar in the blood
leucopenia - a decrease in the total number of white blood cells in
circulating blood
malignancy - presence of a tumor (cancer)
nausea - extreme disgust with an urge to vomit
nephrotoxicity - damage or poisoning to the kidneys
neoplasia - the formation of tumors
opportunistic infection - any infection caused by a microorganism that does
not normally cause disease in humans
osteoporosis - a condition characterized by decrease in bone mass and bone
density
pancreatitis - inflammation of the pancreas
pruritis - a sensation of itchiness on the skin
hyperkalemia - a condition where potassium levels are too high in the body
thrombocytopenia - decrease in the number of blood platelets that is often
associated with hemorrhage conditions
hyperlipidemia - the presence of excess fat or lipids in the blood
tremor - trembling or shaking, usually from physical weakness and disease
hypertension - abnormally high blood pressure - especially arterial blood
pressure
ulcer formation - development of a break in the skin or mucous membrane
with loss of surface tissue
Drug Efficacy
Immunosuppression Usage by
Organ in 2001 and 2002
Drug Efficacy
Tacrolimus vs. Cyclosporine
Polyclonal
vs. Monoclonal
Results from a randomized control trial of a Monoclonal Antibody (against the
IL-2R) as compared with RATG for prophylaxis against rejection against renal
allographs (New England Journal of Medicine)
Inductive Therapy
Inductive therapy refers to the prophylactic application of perioperative
antibodies in addition to baseline immunosuppression.
The goal of the employment of these drugs is to induce
hyporesponsiveness in the organ recipient toward the
transplanted organ in order to prevent early post-transplant
rejection.
Ideally, only T cells that respond to the donor antigen are inhibited and the rest
of the patient's immune system would remain fully functional.
Unfortunately, modern medication is not yet so specific, and so the general
inhibition of the immune system causes the patient to be more susceptible to
infection. The drugs must be strong enough to prevent rejection while
protecting the patient from infection at the same time. This balance is
complicated by the various interactions of multi-drug therapy; reactions can
often be synergistic or lead to the up/down-regulation of drug metabolism.
Initial efforts at inductive therapy utilized polyclonal antibodies, which are
nonspecific and can cause allergic reactions, release of pro-inflammatory
cytokines, neutropenia, and hemolysis. Today, however, murine monoclonal
anti-lymphocyte antibodies, such as OKT3, are often used. To combat the side
effects and generally increased risk of infection (caused by relatively
nonspecific suppression of the immune system), antimicrobial agents are a
necessary component of inductive therapy.
Maintenance Therapy
Maintenance immunosuppression refers to the classic combination therapy (known clinically as Triple
Therapy) to which transplant recipients usually adhere for the rest of their lives. The combination includes:
 a corticosteroid
 a calcineurine inhibitor
 an antiproliferative
The concurrent administration of these three drugs have distinct combined effects on each individual. The
balance of dosages can be altered to enhance the efficacy of the immunosuppression for each patient.
As with inductive therapy, the goal of maintenance immunotherapy is to balance between
underimmunosuppression (which result in graft rejection) and overimmunosuppression (which expose the
patient to high risks of infection and other potentially fatal side effects). The various side effects of each drug
must be considered, as well as potential interactions between drugs, especially those that cumulatively present
significant risk factors to certain patients.
Thus, although the regimen of triple therapy is conventionally standardized, there is much room to
improve immunosuppressive therapy to maximize efficacy and safety for the thousands of patients
permanently on this treatment.
Functional Tolerance
The reduction of dosages in maintenance immunotherapy without
graft rejection consequences is a phenomenon that has been observed
by many researchers (as well as many patients who routinely
disregard their doctors' prescriptions). Most notably, Dr. Tom Starzl
has studied the induction of functional tolerance in transplant
patients on maintenance immunotherapy. By mechanisms not entirely
understood, over time some patients require less
immunosuppression in order to prevent graft rejection. The
medical benefits to such a reduction of therapy, as well as the cost
benefits for those spending as much as $25,000 per year on
immunosuppression, are outstanding. Functional tolerance is certainly
a focus for future research and a realistic goal for maintenance
immunotherapy treatment.
Dr. Tom Starzl presented some of his findings on functional tolerance at the 19th International
Congress of the Transplantation Society. A Tolerance Video available on the group webpage
features his insightful explanations of his recent studies.
Episodic Treatment
Immunotherapy is truly a
treatment that delays the inevitability of
graft rejection.
However, when an acute rejection episode does finally occur,
transplant patients still have good therapy options.
Rejection Episodes can be treated by the one of the following
treatments:
 Corticosteroid
 Polyclonal and Monoclonal Antibodies
 Antiproliferatives
Current Areas of Research
Despite enhancements in immunosuppressant pharmacological therapies,
doctors still struggle to balance the adverse reactions of
overimmunosuppression and organ rejection from
underimmunosuppression. Additionally, organ transplantation is severely
limited by availability of donor organs; 75% on the organ waiting list who
are in desperate need of an allograft die empty-handed. The individuals
lucky enough to receive donor organs are resigned to a life of
immunosuppressant therapy and its long list of associated side effects.
Overall, there is an apparent problem with the current
methods of immunosuppression.
There are two main potential answers to this dilemma. Either immunosuppressant drugs need to become more
specific and effective in dealing with transplant rejection and side effects must be significantly diminished,
or immunosuppressant therapy needs to step away from drug use as a whole with alternative therapies
stepping in. Both approaches represent the forefront of medical research. New drugs are being developed that
are increasingly specific and effective in combating transplant rejection. Drug Efficacy is also discussed. These
topics are supplemented by research into alternate means of immunosuppression through tissue engineering,
stem cell research, and induction of tolerance. Xenotransplantation is also being worked out as a possible
alternative to allogenic transplantation.
As stated earlier, an important current area of research includes improving current drugs, and developing concepts
for new approaches for pharmacotherapy. There are many aspects of current drug immunosuppressant therapy that
can be improved. A first point of concern is the many side affects associated with current medications. Drugs can
also be improve in effectiveness, as most transplants ultimately fail. This section highlights some of the drugs being
researched, but is in no way meant to represent a complete list of new drugs.
Drugs Under
Development Include:
 Basiliximab (Simulect)
 Anti-CD20
 CP-690,550
 Antibodies Targeting Costimulatory Proteins
Alternative Therapies
Xenotransplantation
Tissue Engineering
Stem Cell Research
Induction of Tolerance
Tolerance refers to a state where adverse immune response to transplant antigens is eliminated while the rest of the immune system
remains intact. Tolerance can be achieved through lymphocyte anergy. Anergy is a situation in which the lymphocytes specific for
transplant antigens are unable to respond effectively or potential transplant-specific lymphocytes are deleted before having a
chance to mature and circulate.
Tolerance can be achieved in the following methods
suppression of appropriate APC derived co-stimulatory signals and obstruction of pathways
through which rejection occurs
clonal deletion of cells reactive to the foreign graft - irradiating the thymus and
administering anti-CD4 and anti-CD8, theoretically collapse the immune system
suppressor T cell (or Ts cell)