Immunology of
Transplantation
Allan D. Kirk and Eric A. Elster
General Considerations and Terminology 1705
A Brief History of Transplant Immunology 1706
Physiological Immunity 1708
Transplant Immunity 1717
Mechanisms of Allograft Rejection 1720
Clinical Rejection Syndromes 1721
T
issues transferred between genetically nonidentical
individuals are destroyed through a process known
broadly as refection. It has been apparent throughout
most of medical history that these tissues could provide relief
from disease if they were not rejected. Thus, the field of
transplantation has grown in tandem with the understanding
of the biology of rejection and, to the extent that rejection
is an immune-mediated process, of the immune system in
general. This close relationship between immunological
science and clinical transplantation has fueled remarkable
progress in our understanding of immune function and of the
fundamental nature of our existence as individuals. The com-
ponents of the immune system that have been defined in this
context are now widely recognized not only for their impor-
tance in graft rejection but also for their roles in infection
control, shock, tumor growth, autoimmune disease, and the
systemic response to trauma. As such, the understanding of
immunology that has been born of the study of transplanta-
tion has become key to the thorough understanding of the
biology of modem medicine and surgery.
It is important to underscore that everything involved in
the rejection response evolved under selective pressure to
maintain the integrity of the individual, not to prevent the
relatively recent and artificial practice of organ sharing. Rejec-
tion is a fluke of nature-one that is the result of physiologi-
cal immunity in a nonphysiological state. The student should
remain acutely aware that immune responses to foreign
tissues are both similar to and distinct from immune responses
to environmental pathogens. Thus, while all maneuvers that
interrupt the rejection response are likely to have an impact
on natural host defenses, the differences between pathogens
and foreign tissues can be exploited to prevent rejection while
minimizing the risk of infection.
Immunosuppression 1723
New Immunosuppressive Agents 1727
Other Agents 1728
Tolerance 1728
Xenotransplantation 1730
References 1731
General Considerations and Terminology
At the most basic level, rejection involves recognition of
a tissue that is foreign in a context that is perceived to be
appropriate for a defensive response . Put another way, all
rejection responses involve something on the graft that is
recognized as foreign, some component of the immune system
that recognizes it, and something that defines the context of
the foreign object as worthy of the immune system's atten-
tion. To begin to describe these fundamental aspects of rejec-
tion, a rudimentary vocabulary is required. This is followed
by a review of physiological immune function and finally a
discussion of the biology of transplant rejection.
The word antigen is used to describe a molecule or tissue
that can be recognized by the immune system. An epitope is
the portion of the antigen, generally a carbohydrate or peptide
moiety, that actually serves as the binding site for a receptor
of the immune system. Thus, antigens contain one or many
epitopes. Each is bound by one of two types of lymphocyte
receptors: the T-cell receptor ITCR) of T cells or the antibody
(or immunoglobulins of B cells. In general, a TCR or antibody
binds to one epitope, and each cell expresses a single type of
antigen receptor. These receptors allow a given lymphocyte
to "see" and respond to one epitope and thus establish the
specificity of an immune response . The signal from these
receptors to the lymphocyte on which they reside defines
immune recognition. As discussed below, this general concept
of specificity applies well to physiological immunity but is
now recognized as imprecise for transplantation immunity.
Considerable attention is now directed toward cross-reactive
antigen receptors as important mediators of rejection.
The context or appropriateness of an immune response is
governed by another set of receptors on lymphocytes broadly
1 705
1706 CHAPTER 80
referred to as costimulation receptors. These receptors bind
irrespective of the epitope and allow the lymphocyte to deter-
mine whether the specific signal generated by the antigen
receptor should evoke a response. By having separate signals
for specificity and appropriateness, the immune system can
carefully regulate its response to be active when a pathogenic
threat is present and inactive as the threat subsides. In general,
the ligands for costimulation receptors are expressed most
prominently on cells with the job to initiate immune responses,
so-called antigen-presenting cells (APCs). Thus, immune
responses are typically not initiated unless the antigen is pre-
sented to lymphocytes by APCs in a lymphoid organ such as
lymph nodes or the spleen. In this way, new immune responses
are tightly regulated to avoid autoimmune reactions.
Given the myriad surface receptors involved in lympho-
cyte function, the descriptive names that are frequently given
to a newly discovered molecule are unwieldy. Thus, as new
molecules are characterized, they are assigned a cluster of
differentiation (CD) number. This nomenclature is vital to
any discussion of complex cellular interactions.
Organs transplanted between genetically nonidentical
individuals of the same species are termed allografts. Anti-
gens from these grafts are thus alloantigens, and immunity
toward these antigens is known as alloreactivity. The word
homograft was used in earlier literature to describe allografts.
The degree to which an allograft shares antigens with the
recipient is referred to as the histocompatibility of the graft.
This term generally refers to the similarity of a cluster of
genes on chromosome 6 known as the major histocompatibil-
ity complex (MHC, also known as human leukocyte antigen
[HLA] in humans). Thus, transplant antigens are unique,
genetically encoded characteristics of an individual. The
structure of the MHC is described in detail in the following
section.
Basically, two different classes of MHC gene products are
produced, termed class I and class II. The importance to
transplantation of MHC gene products stems from their poly-
morphism. Unlike most genes, which are identical within a
given species, polymorphic gene products differ in detail
while still conforming to the same basic structure. Thus,
polymorphic MHC proteins from one individual are foreign
alloantigens to another individual. Allografts that are matched
to their recipient at HLA are referred to as RLA-identical
allografts, and those matched at half of the HLA loci are
termed haplo-identical.
Note that HLA-identical allografts still differ genetically
and are to be distinguished from isogiafts. Isografts are organs
transplanted between identical twins, are immunologically
inconsequential, and thus do not reject. Xenografts are organs
transplanted from one species to another and were formerly
described as heterografts. Xenografts are classified based on
their genetic similarity to humans. Concordant xenografts
are closely related species (e.g., Old World monkeys and apes),
while discordant xenografts are distantly related species (e.g.,
New World monkeys and pigs).
A Brief History of Transplant Immunology
Many investigators, beginning with Carrel and Guthrie in
the early 1900s, recognized that transplanted tissues failed
for reasons that were nontechnical in nature.P Alexis Carrel
used organ transplantation as a method for perfecting the
technique of vascular anastomosis and was awarded the
Nobel Prize in 1912 for his methods.
Murphy was the first to observe that lymphocytes were
critical to the process of transplant failure," but it was not
until the 1940s that Peter Medawar systematically approached
the problem of transplant rejection.' His classic studies using
skin-grafted rabbits demonstrated that rejection was a gen-
etically controlled, donor-specific event governed by multiple
transplant-related antigens, and that it was mediated by lym-
phocytes and monocytes.v" He went on to show that immu-
nity could be invoked by prior exposure to donor tissues other
than the transplanted tissue, that the immunity was remem-
bered by the host immune system, and that active cell divi-
sion was required for amplification of the host response.'
At the time of these studies, Ray Owen demonstrated that
freemartin cattle, nonidentical cattle with fused placentas
and a shared fetal circulation, could accept skin grafts from
their dizygotic twins." Expanding on Owen's studies, Medawar
and his colleagues showed that transplant immunity was
acquired during ontogeny rather than being an innate prop-
erty of the host, and thus the barrier to transplantation was
not absolute. In other words, he found that tolerance to self-
tissues is an acquired trait and as such should be able to be
developed for nonself-tissues.? In 1960, the Nobel Committee
recognized Medawar's advances. Mitchison galvanized the
importance of lymphocyte-derived immunity in 1954 with
his demonstration that leukocyte transfusion could transfer
transplant immunity.'?
It is remarkable to note that, within a period of 10 years,
from 1945 to 1955, Medawar and his contemporaries defined
the basic barriers to allotransplantation and demonstrated
that a biological solution could exist. Their investigations
have served as the basis for all subsequent work in the field.
The next two decades were marked by extraordinary prog-
ress in the understanding of the genetic basis of immune
recognition. This work was guided by a search for the surface
alloantigens. Landmark investigations on the immunogenet-
ics of the MHC began with Gorer and Snell's description
of the H-2 system in mice, a genetic locus that segregated
with transplanted tumor survival.l":" This erythrocyte-based
system was soon shown by Amos" to exist on leukocytes and
to elicit an antibody response toward the antigens encoded by
H-2. This antibody response became critical for establishing
the correlate of H-2 in man. Dausset used this finding together
with his observation that antibodies could also be generated
by antigen exposure via transfusion or pregnancy to establish
the first serologically based typing system for human trans-
plant antigens, the Mac antigens.P:" In keeping with the
fundamental nature of discoveries in transplant immunobiol-
ogy, Snell and Dausset shared a Nobel Prize in 1980 for their
initial observations.
Class II MHC polymorphism was also defined in this
period. Bach demonstrated that lymphocytes would prolifer-
ate in response to contact with lymphocytes of a different
genetic background. I? This phenomenon, known as the mixed
lymphocyte culture (MLC), was found to be based on differ-
ences in class II MHC molecules and was defined as HLA-D
(eventually shown to be the same molecule as HLA-DR).
Thus, both serological and cellular assays had been defined
that could determine the degree of similarity between indi-
viduals.
 IMMUNOLOGY OF TRANSPLANTATION 1707

These findings set the stage for a series of extraordinary
workshops held between 1964 and 1970 to bring the growing
number of methods for defining transplant antigens together
under one unified system." This effort resulted in the current
definition of HLA as six loci on chromosome 6, encoding two
related cell surface molecules: class I (HLA-A, -B, and -C) and
class II (HLA-DR, -DP, and -DQ) (Fig. 80.1). Stimulated by the
increasingly rapid pace in defining HLA polymorphisms,
methods for rapid serological definition of HLA were devel-
oped and broadly applied to the clinic. The primary methods
adopted were the lymphocytotoxicity assay of Terasaki,18 in
which antibodies with known specificities against HLA anti-
gens are reacted with unknown cells to determine the HLA
antigens on the unknown sample, and refinements of the
MLC of Bach."
In the 1970s, subsequent advancements in molecular
biological techniques allowed the genetic basis of HLA poly-
morphism to be established, eventually leading to the dis-
covery by Bjorkman19,20 and Brown 21,22 of the three-dimensional
structure of HLA antigens. In addition, these methods allowed
for the fundamental aspects of antigen recognition to be
defined. Particularly important to this effort were the 1987
Nobel Prize-winning contributions of Susumu Tonegawa,
which showed that antigen receptors were generated by
complex rearrangements of the somatic genes in T and B
cells." This principle, described next, offered an explanation
of how the immune system could learn not to react toward
itself during development and has allowed for further exami-
nation of the fundamental nature of neonatal acquired toler-
ance first described by Medawar. The biological role of HLA
as an antigen-presenting molecule was also defined during
this period through the work of many investigators, including
the 1996 Nobel Prize-winning contributions of Zinkemagel
and Doherty.r':"
The characterization of HLA also catalyzed numerous
fundamental investigations in immunology in the 1980s.
With the transplant antigens largely defined, the antigen
receptors were quickly characterized. The function and struc-
ture of the TCR, the importance of adhesion molecules, and
the elucidation of the soluble mediators of cell activation,
cytokines, were all products of this period.
The issue of context has been the final major component
of the immune response to be elucidated. Bretscher and
Cohn were first to propose that antigen recognition was not
sufficient to initiate an immune response.i" Their theory
was generalized in the 1970s by Lafferty and Cunningham
as the two-signal model for lymphocyte activation, with
signal one an antigen-specific signal and signal two a non-

ORB Gene Organization

DR1, 10, 103
(DR1 group)
DR15,16
(DR51 group)
-----
DRB1 DRB6

-----
DRB1 DRB6 DRB5 DRB9
DRB9?

DR3,
14, 11, 12,
1403, 140413,
--- -
DRB1 DRB2 DRB3 ----1_.-
DRB9?

-----
(DR52 group)
DRB1 DRB7 DRB8 DRB4 DRB9
DR4, 7,9

- -
(DR53 group)
DR8 DRB1 DRB9?
(DRBgroup) ? Thepresence of DRB9in these
haplolypes requires to be confirmed

I
o
i
100
i
200
,,
DRB9
I,oRA

1000
CLASS II
REGION

Complement genes HSP70 TNFandLTB

I I n2 nLTB
Bf
I f2 II/Hom II,
TNF

1'-'
C4B C4A AB
I I
CLASS III
III REGION
I i i i i i i i i "I i I
0 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

Class I genes
I I
90
17B C X E 3092 J A 16H G 1,75F
II I I I II I I II III I CLASS I
Iii i i i i iii i REmON
o 2200 2400 2600 2800 3000 3200 3400 3600 3800 4000
FIGURE 80.1. The human major histocompatibility complex (HLA). -B, -C (class I region), HLA-DR, -DP, and -DQ (class II region). Inset:
An abridged map of the human MHC locus on chromosome 6. One The DR ~-chain loci polymorphisms. (From Kirk and Sollinger,"!
copy of this locus is inherited from each parent, each of which with permission.)
encodes the sequences for the major transplantation antigens HLA-A,
1708 CHAPTER 80
specific contextual signal." The components of this second
signal, now known as costimulation, have been defined by
June, Ledbetter, Linsley, Nadler, and others and are dis-
cussed in subsequent sections." The current theory impli-
cating context as a critical element of immune recognition,
put forth recently by Matzinger as the "danger" theory of
lymphocyte activation, has provided the needed link between
an active disease state and antigen recognition. Tissue injury
promotes costimulation and makes lymphocyte activation
more likely to occur with the appropriate antigen recog-
nition." Together, the studies of the past decade have led
to a reasonably comprehensive picture of the mechanism
of allograft rejection and of the physiological functions of
lymphocytes.
A final historical consideration is particularly germane for
students of clinical surgery. The pace of discovery in immu-
nology has been accelerated by a close link between the basic
scientific community and surgeons. This in tum has led to
the rapid application of new discoveries to the problem of
rejection and to clinical observations that have aided greatly
in the understanding of basic laboratory studies. There are
several particularly notable examples of this symbiosis. Joseph
Murray applied the concept that transplant antigens were
genetically determined shortly after it was proposed by suc-
cessfully transplanting a kidney between identical twins."
The importance of lymphocyte division was also quickly rec-
ognized and exploited. Gertrude Elion and George Hitchings
developed a 6-mercaptopurine analogue, azathioprine (Aza),
that inhibited lymphocyte nucleic acid metabolism.l'r" It was
applied clinically by Murray to allow for the first successful
human allografts." Elion and Hitchings were honored by the
Nobel committee in 1988 and Murray in 1990.
The most influential advance in immunosuppression has
been the 1976 discovery of cyclosporine by Borel.34,35 This
relatively selective inhibitor of T-cell activation was quickly
recognized as important by White and Calne and applied to
renal transplantation.f-" The general release of this drug in
1983 made clinical transplantation routinely successful not
only for kidneys but also for extrarenal organs. The success
of cyclosporine has reciprocated a benefit to T-cell biology
in general as it has stimulated much of the investigation
necessary for understanding overall transmembrane receptor
signaling.
As a result of the studies since 1964, it is now rare for an
allograft to be lost to acute rejection. Future advances must
now focus on the chronic diseases of a growing transplant
population, including chronic rejection (CR) and the chronic
toxicity associated with lifelong immunosuppression. It is
likely that these challenges will be met as were those of the
past decades: by cooperation between basic investigators and
surgeon scientists.
Physiological Immunity
The mediators of rejection exist not to prevent the well-
intended efforts of transplant surgeons, but rather to prevent
our bodies from being invaded by foreign pathogens or suc-
cumbing to malignant disease. Thus, to understand rejection,
and particularly to appreciate the consequences of the phar-
macological suppression of rejection, a general understanding
of immunity as it functions in a physiological setting is
required. This section outlines the constituents of normal
immune responses. Subsequent sections discuss these ele-
ments of immunity as they relate to transplantation.
Two complementary arms of the immune system have
evolved in vertebrates to combat disease: the innate and
acquired immune systems. They differ in their fundamental
responsibilities but are now recognized to influence one
another to achieve overall homeostasis. Broadly speaking, the
innate immune system recognizes general motifs that have,
through selective evolutionary pressure, come to represent
universally pathological states to our species (ischemia,
necrosis, trauma, and certain nonhuman cell surfaces]."
Innate recognition leads to prompt and direct attempts to
remove the offending entity. Innate mechanisms of defense
are thus direct and are not steeped in regulation. The likeli-
hood that self-reactive innate immunity will occur is low
because the molecules that trigger innate processes have been
defined by their stark differences from normal tissues.
However, the more important role of innate immunity is in
recruiting acquired responses to areas where there is clear
evidence of a pathological process. Thus, innate responses are
frequently precursors of acquired responses rather than pro-
tective responses in and of themselves.
In contrast, the acquired immune system recognizes spe-
cific pathogens through antigen binding. Antigen binding
leads to carefully regulated destruction of the antigen-express-
ing tissue. Obviously, a large number of receptors are required
to specifically distinguish the seemingly endless array of
pathogens. Highly specific receptors must also respond to
very minor differences in antigen structure. With a large and
varied assemblage of receptors, the potential for cross-reactiv-
ity with self is high. This system must therefore be under
constant tight regulation to prevent autoimmunity. Acquired
responses are therefore characterized by many regulatory
steps designed to prevent autoimmune attack and uncon-
trolled lymphocyte proliferation. Clearly, an immune system
tailor made for one individual will be perturbed when it
encounters tissues from another individual. This perturbation
is the fundamental cause of allograft rejection.
Physiological Innate Immunity
The innate immune system uses protein receptors encoded
in the germ line (passed from one individual to its offspring)
to identify foreign or aberrant/damaged tissues." These recep-
tors can exist on cells, such as macrophages, neutrophils, and
natural killer (NK) cells, or free in the circulation, as is the
case for complement.P"" They are limited in specificity but
are broadly reactive against common components of patho-
genic organisms, for example, lipopolysaccharides on gram-
negative organisms or other glycoconjugates. Thus, the
receptors of innate immunity are the same from one indi-
vidual to another within a species and, in general, do not play
a role in the recognition of a foreign graft per see They do,
however, come into play when an injured tissue (e.g., one that
has been made ischemic and moved from one individual to
another) is present.
Once activated, the innate system performs two vital
functions. It initiates cytolytic pathways for the destruction
of the offending organism, primarily through the complement
cascade" (Fig. 80.2). It also communicates the encounter to
the acquired immune system for a more specific response
 IMMUNOLOGY OF TRANSPLANTATION
IALTERNATIVE I
~~
Virus
C3b Bb)C~
)--C4b,"
C2b tl ~ r' Sa
I TERMINAL I ca C3
C3a _ ~ \J(O C5 ~ (detailed below) for distinguishing infected and transformed
C3b ~ ,,~
C3bC5
C5b
~ rapid response to future encounters, a phenomenon known as
FIGURE 80.2. Complement plays a central role in many innate
responses . In a process known as alternative pathway complement
activation, C3, the central activating enzyme of the complement
cascade, can be activated when carbohydrates lacking sialic acid (a
sugar moiety that is not found in most bacteria but is common on
human cells) are encountered. One of the cleaved fragments of C3,
C3b, is released and binds to the invader , flagging it as foreign, a
process known as opsonization. C3a, another product of C3 activa-
tion, acts to recruit neutrophils to the site, whil e C3d enhances the
immunogenicity of the organism, making it more likely to stimulate
a dendritic cell to present the antigen to T cells. C3 activation also
leads to formation of the membrane attack complex (MAC). This
product is the result of activated C3 catalyzing the activation of CS,
which in tum catalyzes the polymerization of C6, C ], C8, and C9,
forming the MAC, a pore embedded in the foreign cell that results in
disruption of the membrane and lysis.
through by-products of complement activation and through
the function of phagocytic cells . Because this system leads to
a vigorous and relatively direct response, there can be no
room for error; recognition must be based on absolute differ-
ences that cannot be present on normal cells.
Macrophages and dendritic cells (DCs) engulf not only
foreign cells that have been bound by complement, but also
cells identified through receptors for foreign carbohydrates
[e.g., mannose receptorsl." A recently described and highly
evolutionarily conserved family of proteins known as Toll -
like receptors are now recognized as important activation
molecules for innate APCs. They bind to pathogen-associated
molecular pattern (PAMP) motifs common to pathogenic
organisms." Engulfed cells or tissues are reduced to peptide
fragments and presented to the acquired immune system
embedded in MHC molecules so that T cells specific for these
peptides can be activated. Acqui red immunity can also recip-
1709
rocally activate the innate system. In a pathway known as
the classical complement activation cascade, antigen-bound
antibody can bind to the complement molecule Clq, which
in tum becomes activated and activates C3. The C3 activa-
tion products then proceed toward the membrane attack
complex (MAC) and serve chemoattractant functions, as
described for the alternative pathway. One breakdown product
of this pathway (C4d) is now used diagnostically to detect
antibody-mediated allograft rejection."
Physiological Acquired Immunity
The hallmark of acquired immunity is specific recognition
and elimination of cells. Highly specialized antigen receptors
cells from normal tissues have evolved to facilitate this goal.
The altered cell is recognized as a specific entity, not just as
nonself, and a record of that encounter is retained for more
im m unological memory." As discussed previously, the spe-
cialized response makes the system more prone to error; thus,
the innate response aids the acquired system in determining
whether an antigen is worthy of its attention."
THE GENETICS AND STRUCTURAL CHARACTERISTICS OF
ANTIGEN RECEPTORS
Two cell types have evolved with the ability to specifically
bind to antigen: T cells and B cells (Fig. 80.3). Their receptors
are similar in genetic development but differ in the types of
antigens they bind . The T-cell antigen receptors bind peptide
antigens that have been processed by cells and combined with
MHC molecules, while B-cell antibodies bind antigens in
their native conformation on an invading pathogen or free in
the extracellular fluid. The TCRs are fixed, while antibodies
can be secreted and act at locations remote from the cell.
Va
FIGURE 80.3. The general structure of antigen receptors. The two
receptor types used by cells of the acquired immune system for spe-
cific recognition of antigen. The Bvcell antigen receptor (left) is an
antibody molecule made up of two identical light chains disulfide
bonded to two identical heavy chains, thus forming two identical
sites for binding soluble antigen . The T-cell antigen receptor (right)
is associated with a five-chained signal transduction unit called CD3
(shown as the subunits S2€2gdl. It has a single ant igen-binding site for
recognition of a processed peptide antigen bound to an MHC mole-
cule. Striped areas represent the regions of most structural variabil-
ity . (From Kirk and Sollinger,"! with permission.]
1710 CHAPTER 80
THE T-CELL RECEPTOR
The formation of the TCR is fundamental to the understand-
ing of its function." :" The T cells are formed in the fetal liver
and bone marrow and migrate to the thymus during the first
trimester of fetal development. At this stage, they have no
TCR or accessory molecules. On entering the thymus, T cells
undergo a remarkable rearrangement of the DNA that encodes
the two chains of the TCR (a and ~, or y and 15) (Fig. 80.4).49
The order of genetic rearrangement recapitulates the evolu-
tion of the TCR. The T cells first attempt to recombine the
y and 15 TCR genes and then resort to the more diverse a and
~ TCR genes. The y15 configuration is typically not successful,
and thus most T cells are a~ T cells. The T cells expressing
the y15 TCR have more primitive functions, including recogni-
tion of heat-shock proteins and activity similar to NK cells
as well as MHC recognition, while a~ T cells are more typi-
cally limited to recognition of MHC-presented peptide."
Thus, y15 T cells represent an evolutionary link between the
innate and acquired immune systems, possessing the ability
to recognize generally injured tissue and specific antigen.
It is important to note that, regardless of the genes used,
individual cells recombine to express a TCR with a single
specificity. The rearrangements occur randomly, resulting
in a population of T cells capable of binding 109 different
specificities, essentially all combinations of MHC and
peptide. These developing T cells also express both CD4 and
CD8, accessory molecules that strengthen the TCR binding
to MHC (described in more detail following). These acces-
sory molecules further increase the binding repertoire of
the population to include either class I or class II MHC
molecules.
If these T cells were released unmodified, they would
quickly bind to self-cells and destroy the individual. To avoid
the release of autoreactive T cells, developing cells undergo
a process following recombination known as thymic selec-
tion 51,52 (Fig. 80.5). Cells initially interact with the MHC-
expressing cortical thymic epithelium. If binding does not
occur to self-MHC, the cells are useless to the individual as
v~
JDV
c~
PChain of TCR
they would be unable to bind to and survey cells in the periph-
ery for foreign antigen. Thus, all nonbinding cells undergo
apoptosis, or programmed self-destruction, a process called
positive selection. Cells surviving positive selection then
move to the thymic medulla and lose either CD4 or CD8. If
binding to self-MHC in the medulla occurs with an unaccept-
ably high affinity, apoptosis again results; this is called nega-
tive selection.
The precise nature of this affinity threshold remains a
matter of intense investigation and involves interaction with
hematopoietic cells that reside in the thymus." The only
cells released into the periphery are those that can both bind
self-MHC and avoid activation. Any foreign peptide encoun-
tered will alter the affinity that has been preordained in the
thymus, resulting in the initial events of T-cell activation.
Likewise, MHC molecules that were not part of the T cell's
thymic education will bind the TCR with unacceptable affin-
ity. This phenomenon defines alloreactivity. The end result
of TCR formation is a heterodimeric transmembrane receptor
with a site for binding to a peptide-MHC combination." The
receptor is combined with an accessory-binding molecule
(CD4 or CD8) and a transmembrane-signaling complex known
as CD3 (Fig. 80.3).
In addition to thymic selection, it is now clear that mech-
anisms exist for peripheral modification of the T-cell reper-
toire ." Much of this is in place for removal of T cells
following an immune response and downregulation of acti-
vated clones. A molecule known as Fas (CD98) is expressed
on activated T cells." Under appropriate conditions, binding
of this molecule to its ligand leads to apoptosis. This method
is dependent on TCR binding and the activation state of the
T cell. In addition to its role in downregulation, the Fas ligand
can serve as a molecular barrier to T-cell invasion of certain
immunologically privileged sites, for example, testes." Com-
plementing this deletional method to TCR repertoire control
are nondeletional mechanisms that selectively anergize (make
unreactive) specific T-cell clones. One prominent receptor
group mediating this function is the CD28:B7 pair (described
in detail later] ," Binding of the TCR only leads to activation
FIGURE 80.4. The genetic rearrangement leading to the for-
mation of a diverse repertoire of T-cell antigen receptors .
Genomic DNA is spliced under the direction of specific enzy-
matic regulation in the T cell during intrathymic T-cell
maturation. Random segments from regions termed variable
(V), joining In, diversity IDI, and constant ICl are brought
together to form a unique gene responsible for transcription
of a unique TCR chain. The TCR ~ chain is represented here.
Similar rearrangements are required for formation of the a,
y, and 0 chains of the TCR, as well as for the heavy and light
chains of the B-cell antigen receptor (antibody). Two recom -
bination-activating genes IRAq, RAG-l and RAG-2, drive
this series of genetic deletions and splicing events .50,51 Four
distinct loci la and 0 on chromosome 14 and ~ and y on
chromosome 7l, each made up of a highly polymorphic vari-
able (V), junctional, or diversity lJ and D, respectively) region,
and a well-conserved constant ICl region, are involved . The
recombination event randomly joins C, V, D, and J regions
together to form a functional a, ~, y, or 0 chain. The y and 0
loci recombine first, and if recombination is successful, a yo
TCR is formed. If this event is not successful, the a and ~
regions recombine to form an a~ TCR. Approximately 95%
of cells progress to express an a~ TCR. (From Kirk and
Sollinger.i" with permission.]
 IMM U NOLOGY OF TRAN SPLANTATION
Double Negative
L.....- II
Positive selection
FIGURE 80.5. The T-cell precursors that arrive in the thymus express
neither CD4 nor CD8 and are referred to as double-negative cells.
This cell population acquires CD4 and CD8, becoming double posi-
tive, as well as expression of the <X~ TCR and CD3 . These double-
positive cells initially undergo positive selection, in which cells that
fail to recognize self-peptide and MHC (class I or II) are deleted via
if the costimulatory molecule B7 is bound to its ligand CD28,
generally found on APCs. In the absence of binding, the cell
is turned off until exogenous interleukin (IL) 2 is added. Thus,
TCR binding that occurs to self in the absence of appropriate
antigen presentation or active inflammation fails to lead to
self-reactivity. The manipulation of the TCR repertoire by
central (thymic) and peripheral mechanisms is responsible for
the phenomenon described by Medawar as tolerance. Its
understanding will undoubtedly yield the methods required
for specific allograft acceptance.
T-CELL ACTIVATION
The T cells recognize and destroy cells of the body that make
peptide products of mutation or viral infection. They do not
recognize these peptide antigens unless they are presented by
a self-cell. Molecules of the MHC (described later) perform
this presentation. By requiring that T cells only respond to
antigen encountered when it is physically embedded in self-
1711
Thymic dend rit ic
APC-MNCClassII
+Self Ag
Mat ure (04+ T-cell
Decreased autoreactivit y
Allor eactivity maintained
CLONAL
DELETION
Thymic dendri tic
APC-MHC ClassI
+Self-Ag
Mature (08+ T-cell
Low
affinity
_
Negat ive selection
apoptosis . Those remaining cells then undergo negative selection, in
which T cells with high affinity for self-peptides bound to self-MHC
are also deleted via apoptosis. This two-step process ensures that the
mature T-cell population retains alloreactivity while decreasing auto-
reactivity, thereby allowing for a competent immune response .
cells, the body avoids having its T cells constantly activated
by soluble molecules.
Because the number of potential antigens is high, and the
likelihood is that self-antigens vary minimally from foreign
antigens, the nature of the TCR-binding event has evolved
such that a single interaction with an MHC molecule is not
sufficient to cause activation. In fact, a T cell must register a
signal from approximately 8000 TCR-ligand interactions with
the same antigen before a threshold of activation is reached." :
S9 Each event results in the internalization of the TCR . Because
resting T cells have low TCR density, sequential binding and
internalization over several hours is required. Transient
encounters are not sufficient. This threshold is reduced con-
siderably by appropriate costimulation signals (see following) .
The binding between the TCR and MHC is governed by
accessory molecules that improve the TCR-b inding affinity
and regulate the type of cell that the T cell can "see ."60,6 1
Parenchymal cells express class I MHC molecules. These
class I molecules display peptides from within (e.g., peptides
1712
from normal cellular processes or from internal viral replica-
tion).62,63 The T cells responsible for surveying parenchymal
cells express the accessory molecule CD8, a molecule that
binds to class I, and will only stabilize a TCR interaction
with a class l-presented antigen. Thus, CD8+T cells evaluate
parenchymal cells and mediate most of the destruction of
altered cells. They have been termed cytotoxic T cells.
Hematopoietic cells express class II MHC molecules in
addition to class 1. Class II molecules display peptides that
have been phagocytized from surrounding extracellular spaces
and are thus more appropriate for the presentation of newly
acquired antigen. 62,63 Cells in itiating an immune event need
to have access to this newly processed antigen. The accessory
molecule stabilizing the TCR-class II interaction is called
CD4. Thus, under physiological conditions, CD4+T cells are
first alerted to an invasion of the body by hematopoietic
APCs, which present their newly devoured antigen in a class
II molecule (Fig. 80.6). These cells are free to present antigen
Antigen-Presenting Cell
Adhesion
molecule
Helper T-Cell
FIGURE 80.6. The T-cell interactions with an antigen-presenting
cell (APe). The T-cell receptor ITCR) binds to an MHC molecule
(class Il is shown). This event is stabilized by an accessory molecule
(CD4). The costimulatory molecule CD40 upregulates the expression
of the APC costimulation molecules, inducing many critical APC
functions. Other costimulatory molecules include PD-l and ICOS.
Costimulatory molecule binding is shifted away from the negative
CHAPTER 80
without evoking a cytotoxic response from cytotoxic CD8+T
cells (remember that the new antigen will not be presented
in class I because it is not internally derived). The CD4+cells
recognize DCs and macrophages that have an antigen. They
then signal back to the APC to activate CD8+cells to search
the body for cells that have been infected by this invader.
Thus, APCs alert CD4+T cells, and CD4+T cells then endow
APCs with the ability to martial CDW T cells and "license
them to kill. "64,65Thi s process is mediated through upregula-
tion of costimulation molecules (described later). The CD8+
T cells then survey parenchymal cells for the offending
antigen in the context of MHC class I and kill tho se cells
found to be expressing the antigen from within.
When the TCR is bound to an MHC molecule and the
proper accessory molecule stabilizes its binding, it transmits
its signal to the cell by initiating the activity of intracytoplas-
mic protein tyrosine kinases (PTKs) (Fig. 80.71.66,67 These
PTKs include p56 lck (on CD4 or CD8), p59Fyn , and ZAP70 i the
Cytokines released ;f' Cytotoxic T-cell
maturation
CD 134L
MHC o 0
o 0 0
o 0 0
o INF- y 0
o 0
o (}OO
°o()
o
o
IL-2 recepto r
regulation by CTLA4 to positive regulation by CD28 . Thi s potenti-
ates signal transduction by the TCR through CD3 and in tum induce s
IL-2 and interferon-y synthesis. Interleukin 2 works in an autocrine
loop to force the cell into a division cycle. Interferon-y works in
concert with APC-derived cytokines to facilitate cytotoxic T-cell
maturation.
 IMMUNOLOGY OF TRANSPLANTATION
Signa/2
Co-Stimulation
blockade
CTLA4
(CD 152)
Gene encoding for
inflammatory produ cts
IK- Ba mR NA
FIGURE 80.7. The T-cell receptor activation signal transduction and
the sites of action of various immunosuppressants. The TCR binding
facilitates kinase activity by CD3 and CD4(or CD8). The costimula-
tory molecules CD28, CD152, and CD154 determine the relative
potency of these signals. The TCR signal transduction proceeds via
a calcium-dependent dephosphorylation of NF-AT, which enters the
nucleus and acts in concert with NF-kB to facilitate cytokine gene
expression . Interleukin 2 works in an autocrine loop to force the cell
into a division cycle. Cyclosporine A (CyA) and tacrolimus IFK506)
both block this signal transduction by blocking the calcineurin/
last two are associated with the TCR-associated transmem-
brane protein complex called CD3. Repetitive binding signals
combined with the appropriate costimulation eventually acti-
vate phosphokinase-y (PLC-y I J, which in tum hydrolyzes the
membrane lipid phosphatidyl inositol biphosphate (PIP2 I,
thereby releasing inositol triphosphate (IP3 ) and diacylglycerol
(DAGJ.68- 70 The IP3 binds to the endoplasmic reticulum, caus-
ing a release of calcium that induces calmodulin to bind to
and activate calcineurin. Calcineurin dephosphorylates the
critical cytokine transcription factor nuclear factor of acti-
vated T cells (NF-ATJ, prompting it, with the transcription
factor NF-kB, to initiate transcription of cytokines, including
IL-2. Then, IL-2 is released and binds to the T cell in an auto-
crine loop, potentiating DAG activation of protein kinase C
(PKCl, which is important in activating many gene regulatory
steps critical for cell division.
1713
Signa/3
OKT3 Anti-tac
A~
IL-2
calmodulin-potentiating proteins cyclophilin and FK-BP, respec-
tively. This step limits the access of NF-AT to the nucleus. Rapamy-
cin IRAP) blocks the IL-2 receptor signal transduction by blocking
the interaction of RAFT and FKBP. Steroids increase IkBa synthesis
and limit the ability of NF-kB to enter the nucleus. Azathioprine
(Aza) and mycophenolate mofetil (MMF) interrupt the cell cycle by
interfering with nucleic acid metabolism. Monoclonal antibodies
(OKT3, anti-IL-2 receptor, and anti-CD40L) interrupt key surface
int eractions required for T-cell function.
T -CELL REGULATION
The ability of the immune system to act with a "measured"
response is thought in part to be due to the activity of regula-
tory T cells (TREd. A subset of lymphocytes, T REG can down-
regulate the immune response by acting on either effector
cells or APCs. These cells not only have the ability to sup-
press cytokines, adhesion molecules, and costimulatory
signals, they are also able to focus this response by expression
of integrins that allow T REG to home to the location of immune
engagement. The most extensively studied" population of
T REG are those CD4+ T cells that express CD25 . These
CD4+CD25+T cells have been the target of numerous attempts
to alter immune function . Other molecules that have been
suggested to be unique to regulatory populations include
glucocorticoid-induced tumor necrosis factor receptor family-
1714 CHAPTER 80
related gene (GITR) and forkhead box P3 (FoxP3).71,72 While
the physiological import of T REG remains unclear, several
animal models have suggested that they are vital in control
of day-to-day immune activation, and absence of these cells
leads to lymphoproliferative syndromes in several animal
models.I':"
T -CELL-MEDIATED CYTOTOXICITY
Physiological cytotoxicity is mediated by CD8+T cells because
they bind to the class I MHC of all nucleated cells. Killing
occurs either by a Ca 2+-dependent secretory mechanism or
a Ca 2+-independent mechanism that requires direct cell con-
tact." The Ca 2+ influx that occurs with activation causes
exocytosis of cytolytic granules. These granules contain a
lytic protein called perforin and serine proteases called gran-
zymes. Perforin polymerization in the presence of extracel-
lular Ca 2+ forms defects in the target cell's membrane,
allowing granzyme activity to lyse the cell. In the absence of
Ca 2+, T cells can induce apoptosis of a target cell. Apoptosis
is a programmed death that involves fragmentation of the
nuclear contents. It occurs when surface fas is crosslinked by
its ligand. Cytotoxic T cells upregulate fas ligand on activa-
tion, which can then bind to fas on the target cell, leading to
the target's death.
ANTIBODY
Antibody, also called immunoglobulin (Ig), is formed in B
cells much the same way the TCR is in T cells, although
maturation occurs in the bone marrow, and not in the thymus,
and continues in the periphery.P:" Five different heavy-chain
loci (Jl, Y, a, £, and 0) on chromosome 14, and two light-chain
loci (x: and A on chromosome 2, each with V, D or J, and C
regions, are brought together randomly by the RAG-l and
RAG-2 recombination-activating genes (RAG) apparatus to
form a functional antigen receptor. Antibodies have a basic
structure of four chains, two of which are identical heavy
chains and two of which are identical light chains (see Fig.
80.3). The heavy-chain usage defines the Ig type as IgM, IgG,
IgA, IgE, or IgD. This structure forms two identical antigen-
binding sites brought together on a common region known as
the Fc portion of the antibody. The Fc portion is critical for
opsonization, a process by which Fc receptors on phagocytic
cells of the innate immune system bind to antibody, facilitat-
ing phagocytic destruction of the antigen to which the anti-
body is bound and facilitating antigenic peptide processing.
The Fc portion of IgM and some classes of IgG also serve to
activate complement. The mechanism for regulating B-cell
tolerance remains a subject of intense investigation."
Unlike the TCR, the Ig loci undergo continued alteration
after B-cell stimulation to improve the affinity and function-
ality of the secreted antibody. In an alteration known as
isotype switching, Ig genes change their initial heavy-chain
gene usage from the IgM type (used for initial and baseline
responses against common carbohydrate antigens) to one of
four types, each of which provides heightened specialization
for a given purpose." Immunoglobulin G becomes the most
significant soluble mediator of opsonization and is clearly the
dominant antibody resulting from allostimulation. Immuno-
globulin A is formed for mucosal immune responses, IgE for
mast cell-mediated immunity, and IgD as a primary cell-
bound antibody form. Once a clone of cells is activated, the
D and J regions of the utilized gene undergo random additions
through the action of terminal deoxynucleotide transferase to
slightly alter the gene coding for the antigen-binding site.
This process, called affinity maturation, results in clones that
have altered antigen affinity." Those with increased antigen
affinity are retained for a more vigorous response in the event
that antigen is reencountered. Thus, individuals who have
prior exposure to an alloantigen are likely to have clones of
B cells that have mutated to form a gene complex expressing
an IgG with extremely high affinity. One must screen for
these antibodies before transplantation to avoid a vigorous
humoral rejection of the graft (see following).
B-CELL ACTIVATION
B cells recognize antigen in its native form without the
requirement for processing and presentation on MHC mole-
cules." When antigen is bound by two surface antibodies (or
a multimeric form of antibody), the antibodies are brought
together on the cell surface in a process known as crosslink-
ing. This is the event that stimulates B-cell activation, pro-
liferation, and differentiation into a plasma cell. Like the T
cell, the threshold for B-cell activation is high. This can be
lowered 100-fold by costimulation signals received by the
transmembrane complex CD19-CD2l. 79 The B cells also can
internalize antigens bound to surface antibodies and process
them for presentation to T cells. They can receive signals
from T cells via CD40 by binding to the T cell CD154. 80 This
signal upregulates expression of B7 molecules on B cells and
facilitates antigen presentation and T-cell costimulation
(described later). As such, B cells can bind antigen in circula-
tion and initiate a T-cell response to respond to antigen incor-
porated into tissues of the body. Plasma cells (activated B
cells) are distinguished histologically by their hypertrophied
Golgi apparatus. They secrete large amounts of monoclonal
antibody (antibody with a single specificity). During the acti-
vation process, the specificity of the antibody is altered by
affinity maturation, as detailed earlier.
In addition to secretion following exposure to an antigen,
antibody can be present as part of a natural repertoire in cir-
culation for initial response to common pathogens." Antigen
exposure generally leads to B-cell affinity maturation and
isotype switching and produces high-affinity IgG antibodies.
Naturally occurring antibodies, however, are generally IgM
antibodies with low affinity and are generally thought to
respond to a broad array of carbohydrate epitopes found on
many common bacterial pathogens. Natural antibody is
responsible for ABO blood group antigen responses and dis-
cordant xenograft rejection (see following).
ANTIBODY-MEDIATED CYTOTOXICITY
Antibody facilitates both the destruction and removal of
antigenic cells. Once bound to an antigen, antibody serves
as an anchoring site for the complement component Clq,
as described." Antibody can also serve as an opsonin
directly. Most phagocytic cells have receptors for the Fc
portion of IgG and actively engulf antibody-coated targets in
a process known as antibody-dependent cellular cytotoxicity
(ADCC).
 IMMUNOLOGY OF TRANSPLANTATION 1715

CYTOKINES CD28 or CD152, but their affinity is much greater for CD152.
Generally, B7 is not found on resting parenchymal cells.
The entire immune process is dependent on cell-to-cell com-
Rather, it is expressed on cells with potent APC function (so-
munication. While receptor interactions can serve this pur-
called professional APCs, such as DCs and macrophages). In
pose between two cells, soluble mediators of communication
this way, T-cell interactions with self-MHC class I on norm~l
are required to facilitate the amplification of the response.
tissues do not induce a response but rather reinforce the qUI-
Cytokines (also known as interleukins [ILs]) (Table 80.1.' are
escence of autoreactive T cells that have escaped thymic
polypeptides that are released by many cell types and activate
selection. When B7 is limiting, CD152 has the dominant
or suppress adjacent cells." They are particularly fundamen-
effect by virtue of its higher affinity. Thus, in the absence of
tal to the interactions between CD4+ T cells and APCs.
APC activity actively driving an immune response, the default
The prototypical cytokine of T-cell activation is IL-2.83
is for T-cell activity to wane.
The T cells that have bound to their properly presented
An additional costimulation molecule pair is CD40, a
antigen along with the appropriate costimulation release IL-2
molecule found on endothelium, DCs, and other APCs, and
and other cytokines to influence their maturation and that of
its T-cell-based ligand CD154 (CD40L)95,96 (Fig. 80.6). Binding
adjacent cells." They also up regulate a high-affinity chain for
of CD40 is required for APCs to stimulate a cytotoxic T-cell
the IL-2 receptor (IL-2R), CD25, so that the effects of IL-2 on
response. It leads to the release of activating cytokines, par-
the activating cell can be amplified without recruiting non-
ticularly IL-12, and the upregulation of B7 molecules.":" It
specific cells into the cycle." The pattern of cytokine expres-
also stimulates innate functions of APCs, including nitric
sion is thought to influence the type of T-cell response that
oxide synthesis and phagocytosis. Its ligand CD40L is upreg-
results.":"
ulated on T cells after the TCR binds antigen and provides
It has been suggested that T cells, once activated, develop
positive feedback for the APC during its interaction with the
into one of two phenotypes based on cytokine expression. The
T cell. Also, CD40L may directly influence the T cell. Inter-
T cells mediating cytotoxic responses, such as delayed-type
estingly, CD40L is also found in and released in soluble for~
hypersensitivity, express IL-2, IL-12, IL-15, and interferon
by activated platelets." Thus, sites of trauma that recruit
(IFN)-yand have been called Th1 cells. T cells supporting the
activated platelets simultaneously recruit the ligand required
development of humoral or eosinophilic responses express
to activate tissue-based APCs, providing a link between
IL-4, IL-5, IL-10, and IL-13 and have been called Th2 cells.
innate and acquired immunity.l'"
This dichotomy is supported by the fact that many Th1 cyto-
The mechanism of costimulation remains an area of
kines have a common receptor chain called y that is not used
active investigation. Binding of CD28 directly potentiates the
by Th2-type cytokines." Cytokine receptors are now known
TCR-initiated tyrosine phosphorylation." allowing more effi-
to function through the Janus Kinase (Jak) signal transduction
cient signal transduction and lowering the number of binding
proteins. They convey signals to the signal transducers and
events required for T-cell activation from 8000 to about
activators of transcriptions (STATs), DNA-binding proteins
1500. 57,58 In contrast, when CTLA4 is bound to B7, the T cell
that translocate to the nucleus to influence gene transcrip-
becomes unable to make IL-2 during the encounter and,
tion. Resulting transcripts known as suppressors of cytokine . encounters antigen
.
remarkably, w h en It at a Ia tert 'nne. 101
signaling (SaCS) proteins feed back onto Iak proteins to
Clearly, CD28 signaling is a fundamental part of T-cell
control their response to cytokines. The pattern of Jak/STAT/
responses to antigen, and CTLA4 function is critical to the
sacs activity is known to influence the Th1/Th2 phenotype
downregulation of T cells after the antigen has been elimi-
of activated cells. The Th1 cytokines tend to encourage cyto-
nated. 102,103 Also, CTLA4 is likely to playa role in preventing
toxic CD8+ T-cell activity." Although these groupings seem
autoimmunity. Temporal changes in expression and differ-
to apply to the responses to many environmental pathogens,
ences in binding kinetics control the function of costimula-
the response to alloantigens is more typically heteroge-
tion molecules.I?'
neous. 90-92 In addition to cytokines, many other soluble chem-
Evidence from small animal models also suggests that
icals released during an immune response or other types of
activation is dependent on the presence of a "danger"
inflammation increase blood flow to the area and improve the
signal.":'?' Spontaneous T-cell activation is prevented by
exposure of the area to lymphocytes and the innate immune
requiring that there is some indication that the antigen-
system.
binding event is occurring in a location undergoing injury.
Dendritic cells activated by injured or opsonized material or
COSTIMULATION
by complement may give this signal. While thought to play
As mentioned, TCR binding is not usually sufficient to cause a key role in immune activation, allograft rejection has been
a new T-cell response. Rather, receptor-ligand pairs known demonstrated in the absence of "danger," reinforcing the fact
as costimulation molecules determine how the T cell will that multiple mechanisms are involved in the immune
respond'V" (Fig. 80.8). Depending on the type of costimula- response. 105,106
tion that is given to the T cell, TCR signal transduction can Several other molecules have been characterized that
lead to activation or a state in which the T cell becomes demonstrate costimulatory activity. Inducible costimulator
dormant. Costimulatory molecules include the T-cell-based (ICaS), expressed on T cells, and its ligand (ICaSL or B7-H2),
molecules CD28 and CD152 (CTLA4) (Fig. 80.6). In general expressed on APCs, interact with one another on activated T
terms, CD28 binding permits the TCR signal to lead to acti- cells.l'" While CD154 and Icas are positive regulators of the
vation, while CTLA4 binding directs the TCR signal to induce immune response, the cell surface receptor PD-1 (programmed
anergy. The ligands for CD28 and CTLA4 are the B7 mole- cell death) works in a similar fashion to CTLA4 in providing
cules (CD80, CD86). Both B7 molecules can bind to either negative regulation of immune interaction.l'" In addition,
TABLE 80.1. Properties of Some Human Cytokines.
Cytokine Alternative name Source(s) Target cell types Actions
IFN-a and IFN-~ Activated T cells, Activated T and B cells, NK Induces antiviral state, antitumor activity,
endothelial cells, and LAK cells fever; increases class I and II MHC expression;
macrophages, stimulates activated B-cell differentiation and
fibroblasts proliferation and NK activity; inhibits T and
LAK cell activity
IFN-y Activated T cells, Activated and resting Band Induces antiviral state, antitumor activity,
LAK cells plasma cells; NK, fever; increases class I and II MHC expression;
endothelial, and LAK cells; stimulates activated B-cell differentiation and
macrophages proliferation and NK and LAK activity;
activates macrophages and endothelial cells,
stimulates IgG2a isotype switch
TGF-y T cells, Monocytes, fibroblasts Chemotactic for fibroblasts and monocytes,
macrophages, NK induces extracellular matrix remodeling,
cells repair, and fibrosis; induces B-cell
differentiation and isotype switching, T-cell
proliferation, and angiogenesis
TNF Activated T cells, Resting T, activated T and B Induces antiviral state, antitumor activity,
LAK cells, cells; plasma, stem, and fever; increases class I MHC expression;
macrophages endothelial cells; activates macrophages, granulocytes,
eosinophils, fibroblasts, eosinophils, and endothelial cells, chemotactic
macrophages and angiogenic activity
IL-I Endogenous Activated T and Resting T and B cells; Induces antiviral state, antitumor activity,
pyrogen B cells, LAK activated T and B cells; fever; stimulates activated B-cell
cells, endothelial plasma, stem, and differentiation and proliferation; activates and
cells, endothelial cells; stimulates proliferation of T cells; activates
macrophages, eosinophils, fibroblasts, granulocytes and endothelial cells; stimulates
fibroblasts macrophages hematopoiesis
IL-2 T-cell growth Activated T cells, Activated T cells; activated Activates macrophages, T, NK, and LAK cells;
factor LAK cells and resting B cells; NK and stimulates differentiation of activated B cells;
LAK cells, macrophages stimulates proliferation of activated Band T
cells; induces fever
IL-3 Multi-CSF Activated T cells Stem cells, activated B cells, Stimulates hematopoiesis; activates B-cell
eosinophils proliferation and eosinophil activity
IL-4 B-cell- Activated T cells Activated T cells; activated Activates macrophages, T and B cells;
stimulating and resting B cells; plasma stimulates differentiation of activated B cells;
factor-l LAK cells; macrophages stimulates proliferation of activated Band T
cells; induces IgE receptors on B cells;
stimulates IgE and IgGI isotype switch
IL-S B-cell growth Activated T cells Activated and resting B cells, Stimulates 19A isotype switch and eosinophil
factor-2 plasma cells, eosinophils activity
IL-6 B-cell- Activated T cells, Activated T, resting B, and Activates T cells; stimulates activated B-cell
stimulating endothelial cells, stem cells differentiation and activated T- and B-cell
factor-2, B-cell- fibroblasts, proliferation
differentiating macrophages
factor,
interferon -~2
IL-7 Activated T cells Activated T and resting B Stimulates activated T-cell and resting B-cell
cells proliferation
IL-8 Activated T cells Granulocytes Stimulates granulocyte activity, chemotactic
activity
IL-9 Activated T cells T cells Stimulates T-cell proliferation
IL-IO Macrophages, B Macrophages, Band T cells Inhibits macrophage cytokine release; induces
and T cells B-cell differentiation and isotype switching;
induces class II expression, T-cell stimulation
IL-II Bone marrow Hematopoietic stem cells Stimulates megakaryocyte and B lineage stem
stromal cells cell maturation
IL-I2 NK cells and T cells Induces T-cell maturation and cytotoxic
macrophages activity
G-CSF Endothelial cells, Granulocytes Stimulates granulocyte activity and
fibroblasts, hematopoiesis
macrophages
M-CSF Macrophages Macrophages Activates macrophages
GM-CSF Endothelial cells, Stem cells, granulocytes, Activates macrophages, stimulates
fibroblasts, macrophages, eosinophils granulocyte and eosinophil activity and
activated T cells hematopoiesis
CSF, colony-stimulating factor; IL, interleukin, INF, interferon; LAK, Iymphokine-activated killer; NK, natural killer; TGF, transforming growth factor; TNF, tumor
necrosis factor.
Cytokines are secreted polypeptides that mediate autocrine and paracrine cellular communication but do not bind antigen. They include those compounds previ-
ously termed interleukins and lymphokines.
Source: Based on the consensus cytokine chart of the British Cytokine Croup.F"
 IMMUNOLO GY O F TRANSPLANTATION
APC
T-Cell
FIGURE 80.8. Costimulation of T-cells.
The T-cell responses depend on two signals,
signal one is given by antigen through the
TCR, and signal two is given by costimula-
1 y
tory receptors. Both are required for activa- Activation
tion. Signal one alone leads to T-cell anergy & or
or death. Signal two has no independent Expansion
effect.
many other adhesion molecules (intercellular adhesion mol-
ecule [ICAMsl, selectins, integrins, etc.] control the move-
ment of immune cells through the body, monitor their
trafficking to specific areas of inflammation, and nonspecifi-
cally strengthen the TCR-MHC binding interactton.Pv'?'
They differ from costimulation molecules in that they
enhance the interaction of the T cell with its antigen without
influencing the quality of the TCR response. Almost all are
upregulated by cytokines released during T cell and endothe-
lial activation.
Transplant Immunity
The Genetics and Structural Characteristics of
Transplant Antigens
The antigens primarily responsible for human allograft rejec-
tion are those encoded by the HLA region of chromosome 6
(see Fig. 80.1).108 The polymorphic proteins encoded by this
locus include class I molecules (HLA-A, -B, and -C) and class
II molecules (HLA-DR, -DP, and -DQ). Class I genes with
limited polymorphism (E, F, G, H, and J) are not currently
typed and are not considered here. Other genes encoded by
HLA are the tumor necrosis factors C( and ~, components of
the complement cascade (class III molecules I, the heat-shock
protein HSP 70 (which may be involved in danger signaling],
and genes necessary for class I and class II presentation of
peptides to the body's T cells (peptide transporter proteins
TAP-l and TAP-2 and proteosome proteases LMP-2 and LMP-
7). Although other polymorphic genes, referred to as minor
histocompatibility antigens, exist in the genome, they are not
covered in this section. It is, however, important to point out
that even HLA-identical individuals are subject to rejection
on the basis of these minor differences. The blood group anti-
gens of the ABO system must also be considered polymorphic
transplant antigens, and their biology is critical to humoral
rejection.
1717
APC APC
T-Cell T-Cell
1
No Response
Anergy
Apoptosis
Each class I molecule is encoded by a single polymorphic
gene that is combined with the nonpolymorphic protein ~r
microglobulin (~2M, from chromosome lSI for expression'Y"
(Fig. 80.9). The polymorphism of each class I molecule is
extreme, with 30 to 50 alleles per locus. Class II molecules
are made up of two chains, C( and ~, and individuals differ not
only in the alleles represented at each locus, but also in the
number of loci present in the HLA class II region ." The poly-
morphism of class II is thus increased by combinations of C(
and ~ chains as well as of hybrid assembly of chains from one
class II locus to another. !" As the HLA sequence varies, the
ability of various peptides to bind to the molecule and be
presented for T-cell recognition changes . Teleologically, this
extreme diversity is thought to improve the likelihood that a
given pathogenic peptide will fit into the binding site of these
antigen-presenting molecules, thus preventing a single viral
agent from evading detection by T cells of an entire popula-
tion. Il O,1l 1 The importance of class I and class II structure is
also underscored by the relationship of HLA allotypes to
many viral and autoimmune diseases.
While the structure of HLA is complex, the clinical impor-
tance of the region with respect to transplantation is easily
understood by simple Mendelian genetics. Recombination
within the locus is uncommon, occurring in approximately
1% of molecules. The HLA type of the offspring is therefore
predictable. The unit of inheritance is the haplotype, which
consists of one chromosome 6 and therefore one copy of each
class I and class II locus (HLA-A, -B, -C, -DR, -DP, and -DQ).
The genetics of HLA is particularly important in understand-
ing clinical living-related donor (LRD) transplantation. Each
child inherits one haplotype from each parent, therefore, the
chance of siblings being HLA-identical is 25 %. Haploidenti-
cal siblings occur 50 % of the time and completely nonidenti-
calor HLA-distinct siblings occur 25% of the time. Biological
parents are haploidentical with their children unless there has
been a rare recombination event. The degree of HLA match
can also improve if the parents are homozygous for a given
allele, thus giving the same allele to all children. Likewise, if
1718
- CD8
Binding
Site
.............................. .
.............................. ..
CLASS I CLASS II
the parents share the same allele, the likelihood of that allele
being inherited improves to 50%. The inheritance of HLA is
also affected by linkage disequilibrium, or a propensity of
certain class I and class II genes to be located on the same
chromosome.
The three-dimensional structure of class I molecules
(HLA-A, -B, and -C) was first elucidated in 1987 and is shown
in Figure 80.9.19,20 Class I is expressed as a single MHC-
encoded, transmembrane a chain, in combination with ~2M.
The a chain has three domains, ai , ell, and aa. The critical
structural feature of class I molecules is the presence of a
groove formed by two a helices mounted on a ~-pleated sheet
in the al and ell domains. Within this groove, a nine-amino-
acid peptide, formed from fragments of proteins being synthe-
sized in the cell's endoplasmic reticulum, is mounted for
presentation to the body's T cells. Almost all the significant
sequence polymorphism of class I is located in the region of
the pept ide-binding groove and in areas that directly contact
T cells. It is this variation in sequence at the HLA-TCR
interface that is the essence of alloreactivity. The TCR rep-
ertoire formed in the thymus is formed on the basis of its
ability to bind to these a helices without becoming activated.
When the amino acid structure of the a helices on MHC
molecules is different from that presented in the thymus, the
TCR is likely to interpret this interaction as one with a
foreign antigen. Organs reject because recipient T cells were
not educated with donor MHC in the thymus.
The three-dimensional structure of class II molecules
(HLA-DR, -DP, and -DQ) was inferred in 1988 by sequence
homology to class 121 and eventually proven in 1993 by x-ray
crystallography.f The structural features of class II molecules
are strikingly similar to those of class I molecules (see Fig.
80.9). Class II molecules are found primarily on cells of the
innate immune system, particularly phagocytes, such as
DCs, macrophages, and monocytes, but can be upregulated to
appear on other parenchymal cells by cytokines released
during an immune response or injury.
Another important sequence-related difference between
class I and class II molecules is located on the supporting
CHAPTER 80
Exogenous
peptide
FIGURE 80.9. The three-dimensional structure of
MHC class I and class IT molecules. The structure
of the two major classes of transplantation antigens
is shown . Note that while class I molecules are
made of a single chain combined with ~2-micro­
globulin (~2Ml and class II molecules are two sepa-
rate chains , the general structure is very similar.
Two <X helices form a groove on top of a ~-pleated
sheet to present peptide antigens for TCR binding .
The peptide presented by class I is short (9 amino
acids) and derived from endogenous proteins. The
- CD4 peptide presented by class IT varies in length (up to
Bindin g 22 amino acids) and is derived from exogenous pro-
Site teins engulfed by the cell and is substantially larger
(up to 23 amino acids long) than the peptide found
in class I. Like class I, the sequence polymorphism
of class II is located at this TCR interface region.
The binding sites for the T-cell accessory molecules
CD4+and CDs> are also shown. They regulat e the
type of T cell that can bind to each MHC molecule .
(From Kirk and Sollinger,"! with perrnission.]
subunits laa domain for class I and the ~2 domain for class II).
A loop extends outward and serves as a binding site for acces-
sory molecules associated with the TCR . The TCR accessory
molecule CD8 selectively binds the loop of class I,Il2 while
the accessory molecule CD4 binds the loop of class II.113 In
this way, T cells geared toward the initial recognition of
intruders and subsequent amplification of the immune
response (CD4+ helper T cells) are targeted to bind the cells
with the ability to capture and present these antigens. Simi-
larly, T cells that survey the body's parenchyma for signs of
entrenched intracellular pathogens and destroy infected cells
(CD8+cytotoxic T cells) are outfitted to perform this duty .
The Biology of Transplant Antigens
The physiological role of MHC molecules is twofold : to
provide a mechanism for T-cell inspection of parenchymal
cells and to provide an interface between APCs and T cells.
For the structural reasons just detailed, class I molecules
serve the first role, while class II molecules serve the second .
It is important to reiterate, however, that organ transplanta-
tion is not a physiological process, but rather an artificial
situation. Thus, T-cell responses to either class of MHC mol-
ecule can generate a rejection episode.
The assembly of class I is dependent on association of the
a chain with ~2M and occupation of the peptide groove with
a native peptide. Incomplete molecules are not expressed .'!'
In general, all peptides made by a cell are candidates for pre-
sentation, although sequence alterations in this region favor
certain sequences over others. Human class I presentation
occurs on all nucleated cells in contact with blood, thus
allowing the acquired immune system to inspect and approve
ongoing protein synthesis. Deviation of the peptide content
from that present during thymic maturation can induce T-cell
activation if it is presented in the proper context. In the case
of transplantation, this alteration is possible not only with
the peptide but also with the presenting molecule itself or
with the allo-MHC engulfed by APCs and presented remote
from the allograft .
 IMMUNOLOGY OF TRANSPLANTATION
Class II molecule assembly requires association of an a
chain and a ~ chain in combination with a temporary protein
called the invariant chain.115 This third protein covers the
peptide-binding groove until the class II molecule is out of
the endoplasmic reticulum and is sequestered in an endo-
some. Proteins that are engulfed by a phagocytic cell are
degraded at the same time as the invariant chain is removed,
allowing peptides of external sources to be associated with
and presented by class II. In this way, the acquired immune
system can inspect and approve of proteins that are present
free in circulation. The T cells that bind class II molecules
are CD4+ and have enhanced abilities for directing the APC-
mediated activation of CD8+ T cells and antibody-producing
B cells.
When an inappropriate peptide is detected in the appropri-
ate setting, an alloreactive response is generated in which
CD4+ T cells release cytokines to activate APCs to recruit
CD8+ cells into the area to inspect nearby cells for intracyto-
plasmic presence of the offending peptide. As alloreactive T
cells have a much higher precursor frequency than naive cells,
the stage is set for clonal expansion of this population and an
aggressive response. This is in contrast to a physiologic
response to antigens (such as viral peptides), by which naive
cells have a much lower precursor frequency and expansion
proceeds in a more leisurely fashion. The high frequency of
alloreactive thymic precursors is thought to be a consequence
of the collective immunologic memory of viral (and other
antigen) peptides and resulting cross-reactivity, a phenome-
non entitled heterologous immunityi"
Once the immune response has been engaged, B cells are
also stimulated to release antibody to bind the offending
peptide (in its native form) and aid in its clearance by the
innate immune system. The cytokines released by CD4+
cells, particularly IFN-y, also induce expression of class II
molecules on local cells and increase expression of class I
molecules locally.':" This reaction increases the chance that
infected cells will be detected. In the case of transplanted
organs, ischemic injury at the time of transplantation accen-
tuates the potential for T-cell activation by upregulation of
both class I and class II molecules.!" Surgical trauma and
ischemia also upregulate class II (as well as B7 and adhesion
molecules) on all cells of an allograft, making antigen more
abundant.
If rejection can be prevented until the cells of the graft
can return to their baseline state of antigen expression, the
chance of a T cell encountering foreign antigen in sufficient
density to become activated is greatly reduced. This altera-
tion is the primary reason that immunosuppression is heavily
administered in the immediate postoperative period and
tapered to lower, less-toxic levels over time.
Clinical Definition of Transplant Antigens
For the reasons already discussed, closely matched transplants
are less likely to be recognized and rejected than are similar
grafts differing at the MHC. Historically, an MHC match has
been defined using two assays: the lymphocytotoxicity assay
and the MLC. 17,18 Both assays define MHC epitopes but do
not comprehensively define the entire antigen or the exact
genetic disparity involved. Techniques now exist for precise
genotyping that distinguishes the nucleotide sequence of an
individual's MHC.
1719
The lymphocytotoxicity assay is performed by taking
serum from individuals with anti-MHC antibodies of known
specificity and mixing it with lymphocytes from the indi-
vidual in question. Rabbit complement is added, as is a vital
dye such as trypan blue, which is not taken up by intact cells.
If the antibody binds to MHC, it activates the complement,
leads to cell membrane disruption, and stains the cell. Micro-
scopic examination of the cells can then determine if the
MHC antigen exists on the cells. Although this assay has
been extremely valuable, it is limited by the nature of the
reagents. Antibodies bind to epitopes. The presence of one
epitope does not preclude the presence of other epitopes that
may differ from the desired MHC type. Thus, many antibod-
ies cross-react with MHC antigens other than the one to
which they were raised. Fortunately, the pattern of these
cross-reactivities is reasonably well established such that use
of a large panel of antibodies allows for reasonable definition
of the genetic locus in question.
The MLC is performed by incubating recipient T cells
with irradiated donor cells in the presence of 3H-thymidine.
If the cells differ at the class II MHC locus, the recipient cells
proliferate and incorporate the radionucleotide into the new
cells. This incorporation can be detected and quantified.
While class II polymorphism is detected by this assay, it takes
several days to complete one assay (unlike the lymphocyto-
toxicity assay, which takes 4-6h). Thus, use of MLC as a
prospective typing assay is limited to LRDs. Again, the
antigen receptor is the biological reagent of this assay, and
the genetic basis for the reaction can only be inferred from a
series of reactions.
The sequencing of the class I and class II HLA loci has
allowed several genetic-based techniques to be used for his-
tocompatibility testing.l'v"? These methods include restric-
tion fragment length polymorphism (RFLP),121 oligonucleotide
hybridization, 122 and polymorphism-specific amplification
using the polymerase chain reaction and sequence-specific
primers (PCR_SSPS).123,124 Of these methods, the PCR-SSP
technique is most commonly employed for class II typing.
Serological techniques are still the predominant method for
class I typing because of the complexity of class I sequence
polymorphism. It is important to point out that sequence
polymorphisms that do not alter the TCR-MHC interface are
unlikely to affect allograft survival; thus, the enhanced preci-
sion of molecular typing may provide more information than
is clinically relevant.
While HLA matching donors and recipients has clearly
been shown to improve outcome following kidney, heart, and
pancreas transplantation, no such correlation exists with liver
transplantation. Indeed, matching may in fact reduce overall
survival. 124,125 The reasons for this lie in the dualistic nature
of HLA in the pathophysiology of liver disease. The T-cell-
mediated rejection of the liver is mechanistically the same as
with other organs; therefore, rejection is reduced with
improved HLA compatibility. However, the physiological
role of HLA is the presentation of viral peptides to T cells to
initiate destruction of virally infected cells. Thus, HLA com-
patibility potentiates the inflammation during viral reinfec-
tion following transplantation for viral hepatitis and increases
the chance for clinical recurrence of the original disease.
Similarly, T-cell-mediated autoimmune diseases, such as
primary biliary cirrhosis, are etiologically based on T-cell
recognition of HLA-presented peptides. As such, recurrence
1720 CHAPTER 80
TABLE 80.2. Degree of Mismatch Critically Influences Graft
Survival.
Degree of match (mismatch for CAD) Graft half-life (years)
HLA identical (siblings) 23.1
LRD (siblings) 14
LURD (spousal) 14.5
o antigen mismatch CAD 13
1 antigen mismatch CAD 11
2 antigen mismatch CAD 9.3
3 antigen mismatch CAD 9.5
4 antigen mismatch CAD 8.6
5 antigen mismatch CAD 8.4
6 antigen mismatch CAD 8.2
Source: Data from Refs. 127 and 128.
of autoimmune diseases may be potentiated as well. Further
knowledge regarding specific disease states worsened by
certain HLA matches may be useful for selective typing in
the future.
Matching is only temporally feasible before cadaveric
renal transplantation and living-related allografts. While of
paramount importance in predicting long-term success of
cadaveric grafts, the degree of HLA matching plays a limited
role in living kidney donation 126-128 (Table 80.2). It seems
apparent that the quality of the allograft (from healthy donors)
and the amount of associated ischemic injury are key factors
that dictate long-term renal allograft survival.!" For nonrenal
transplants, some centers now use HLA typing to dictate
pancreas allocation as well. Other organs are MHC typed
retrospectively. As would be predicted, the incidence of acute
rejection declines with decreasing MHC disparity. 127,128
However, any mismatch puts the patient at risk for antibody
or T-cell-mediated graft destruction and mandates T-cell-
specific immunosuppression. As immunosuppression has
improved, the relative importance of MHC matching, even
for renal allotransplantation, has decreased, even allowing for
transplantation in the face of a positive crossmatch or ABO
incompatabiliry.F'{" Now, when determining the destina-
tion of an organ, significant emphasis is also placed on the
recipient's physical condition and time on the waiting list.
Mechanisms of Allograft Rejection
T-Cell-Mediated Rejection
The T cells are the primary mediators of acute allograft reiec-
tion.131-132 They can respond to transplant antigens either
directly, through TCR binding to foreign MHC molecules
expressed on transplanted tissues in the presence of donor
costimulation, or indirectly, by encountering self-APCs that
have phagocytosed alloantigens and processed them for pre-
sentation on self-MHC. It is also probable that recipient T
cells can bind to donor APCs, particularly DCs, activating
them and allowing them to direct recipient cytotoxic T-cell
maturation. Regardless of the source of the activating MHC,
however, the ensuing internal T-cell events proceed as
described for physiological T-cell function. Knowledge of this
process has been exploited at almost every critical step along
the T-cell activation pathway to prevent acute rejection, and
its understanding is key to the rational use of immunophar-
maceuticals (see Fig. 80.7).
Initial T-cell binding to a donor APC or endothelial cell
is nonspecific and mediated by adhesion molecules.l" These
molecules, including ICAM-I, VCAM-I, LFA-I, and other
integrin family molecules, are all upregulated on donor cell
activation. This reaction is most critical on donor endothe-
lium and APCs and is mediated at least in part by activation
through the CD40 surface molecule.P' CD40 is activated
when bound by CDl54, a molecule expressed by platelets and
T cells. Thus, both activated T cells and activated platelets
likely have the ability to drive allograft rejection.
Once nonspecific adhesion has formed, MHC recognition
can occur. The costimulatory environment of the donor tissue
is affected by many factors, most of which are related to the
mechanics of transplantation. Ischemia and surgical trauma
induce B7 expression on the endothelium.i" Platelet interac-
tions and C3d deposition greatly improve the antigen-present-
ing ability of the donor tissues'f MHC class IT is upregulated,
as are many nonspecific adhesion molecules.!"
Once T-cell activation occurs, cytokines, particularly IL-2
and IFN-y from T cells and IL-12 from APCs, create a potent
milieu recruiting other T cells into the response and potenti-
ating clonal expansion. 82,91,92,136 Interleukin 2 works on the
cell that is secreting it, so-called autocrine secretion, and
leads to upregulation of CD25, the high-affinity receptor for
IL-2. To amplify the effect of local IL-2, CD25 combines with
two additional constitutively expressed IL-2R chains." The
central roles of IL-2 and the IL-2R have been exploited to
prevent allospecific T-cell activation by drugs such as cyclo-
sporine, tacrolimus, and anti-CD25 monoclonal antibodies
(see following). Also, IFN-yinduces nonlymphoid graft tissues
to express class II molecules and to upregulate expression
of class I molecules, making alloantigen more prevalent.!"
It also fosters cytotoxic T-cell maturation. Mediation of B-
cell activation also occurs through cytokine secretion. Cyto-
kines are responsible for many of the systemic symptoms of
fever and malaise that can be associated with severe graft
rejection.
As discussed, many physiological responses lead to pre-
dictable differential cytokine expression that clearly alters
the character of the effector response. More recent study has
shown that the response to alloantigen is not easily catego-
rized into Th l versus Th2 responses. 82,9G-92,136,137 Rather,
responses during transplant rejection are characterized by
both Thl and Th2 cytokines, including IFN-y, TNF-a, IL-2,
IL-5, IL-6, IL-IO, IL-12, and IL-15. Immunosuppression also
produces artificial patterns of gene expression. Responses also
vary based on the organ targeted for rejection. Kidneys are
infiltrated by T cells, leading to a Thl-type milieu plus IL-IO,
but livers have a significant eosinophil infiltration and a strik-
ing presence of IL-5. Thus, the patterns of inflammation seen
during allograft rejection result from cytokine-mediated
amplification but vary considerably based on many other
factors.
In the late phases of rejection, the inflammatory response
recruits cells with nonspecific cytotoxic activity to the organ.
The T cells expressing the yo TCR as well as other T-lineage
cells, such as lymphokine-activated killer cells, are activated
to destroy surrounding tissue in an MHC-unrestricted
fashion." Interleukin 8, released by activated macrophages
 IMMUNOLOGY OF TRANSPLANTATION
and T cells, also recruits neutrophils to remove necrotic
tissue."
Although cytotoxicity is best mediated by CD8+ cells, in
the artificial situation presented by allotransplantation, both
CD4+ and CD8+ cells have been shown to mediate cytotoxic-
ity.132 Both perforinjgranzyme-dependent mechanisms and
fas-dependent mechanisms for graft destruction are involved. 137
It is now clear that, following activation, non-MHC-restricted,
T-cell-mediated cytotoxicity occurs in addition to MHC-
directed killing." This reaction allows the utilization of T
cells activated in the area of inflammation but without a TCR
specific to the antigen to take part in protective or, in the case
of transplant rejection, detrimental immunity. It is likely that
this additional promiscuous killing is spurred forward by a
failure to eliminate the antigen. The mechanisms of direct
T-cell-mediated destruction of microorganisms or non-MHC-
expressing tissue remain poorly defined.!" It is, however,
clear that T cells expressing the y'fJ TCR are prominent effec-
tors of non-MHC-restricted cytotoxicity. This type of activity
is induced by high concentrations of IL-2, which may be rel-
evant in the local milieu of a rejecting allograft.
Antibody-Mediated Rejection
Although acute rejection is always the result of T-cell recog-
nition and activation, most acute rejections are accompanied
by an antibody response.!" In addition, antibody can be the
primary mediator of two types of rejection: hyperacute rejec-
tion (HAR) and acute vascular rejection. Furthermore, anti-
body formed during the course of an allograft rejection remains
in the circulation even after the acute event has been success-
fully controlled. Many investigators believe that chronic
exposure to donor-specific antibody can have progressively
damaging effects on the graft and contribute significantly
to CR.
Donor-specific antibody has multiple effects on the graft.
Direct cell lysis occurs as a result of classical pathway com-
plement activation. Antibody opsonization increases the
phagocytic uptake of donor antigen and as such increases the
antigen presentation to the recipient immune system. Anti-
body also directly alters the activation status of the endothe-
lial cell. This change leads to cellular retraction and exposure
of the underlying matrix, which in tum potentiates platelet
activation and aggregation. Endothelial activation also alters
its usually anticoagulant environment in favor of a procoagu-
lant one. 140,141 Heparan sulfate is shed, as is thrombomodulin,
this prevents thrombomodulin-mediated activation of protein
C and the interaction of activated protein C with protein
s. The result is microvascular thrombosis, a hallmark for
antibody-mediated graft rejections.
Clinical Rejection Syndromes
There are three major types of rejection: hyperacute, acute,
and chronic. They differ in their general pathophysiology and
in the effector arm of the immune system involved with
current immunosuppression. Of these, only acute rejection
can be successfully reversed once it is established. As the
transplant community has become more sophisticated in its
use of immunopharmaceuticals, graft loss from acute rejec-
tion has become increasingly rare. Although untreatable,
1721
HAR can be prevented more than 990/0 of the time with proper
use of the lymphocytotoxic crossmatch before transplanta-
tion. Thus, most graft loss is now the result of CR, a disease
that is only now being defined.
Hyperacute Rejection
Hyperacute rejection (HAR) is caused by donor-specific anti-
body present in the recipient's serum at the time of transplan-
tation."" The antibody is not a result of the transplant but
rather is a result of prior sensitization to donor antigens or to
antigens that are sufficiently similar to those of the donor to
elicit cross-reactivity. Presensitization is usually the result of
prior transplant, transfusion, or pregnancy. Hyperacute rejec-
tion develops in the first minutes to hours following graft
reperfusion. Antibodies bind to the donor tissue, initiating
complement-mediated lysis, endothelial cell activation, a
procoagulant state, and immediate graft thrombosis.
Although there is no proven treatment for HAR, a thor-
ough understanding of its cause has resulted in its avoidance
through the use of two preoperative screening tests, namely,
the lymphocytotoxic crossmatch (described earlier) and
ABO blood group typing. These two tests identify donor-to-
recipient combinations in which HAR would likely occur.
The crossmatch is performed by mixing cells (generally
nonactivated T cells that express class I but not class II MHC
antigens on their surface) from the donor with serum from
the recipient. Lysis of the donor cells indicates that anti-
bodies directed against the donor are present in the recipient
serum, this is called a positive test result. A positive test can
result from IgG or IgM antibodies. The IgM antibodies are
frequently not directed at HLA antigens and are considered
false-positive results. By adding dithiothreitol, IgM molecules
can be broken down. If the lysis is due to IgM, it will not
occur in the presence of dithiothreitol. In general, only detec-
tion of IgG antibodies directed against class I MHC molecules
represents a positive test and an absolute contraindication to
transplantation. Preoperative verification of proper ABO
matching and a negative crossmatch effectively prevent HAR
in 99.50/0 of transplants.
The overall purpose of the crossmatch is to detect clini-
cally relevant antibodies and to prevent antibody mediated-
rejection. As such, many variations of the crossmatch
exise 42,143 j these include crossmatch studies performed with
class II-expressing B cells. As graft cells can express class II
molecules, particularly during rejection or following an is-
chemic insult, antidonor class II antibodies can initiate graft
injury. Noncytotoxic assays can also be performed. These
studies involve flow cytometry, a technique in which anti-
bodies that bind to a target can be stained using fluorescent
dyes and detected using specialized equipment. Although this
assay is very sensitive at detecting and characterizing antido-
nor antibodies, some of the antibodies that are detected rep-
resent autoantibodies or IgM HLA antibodies and have little
functional consequence (770/0 of positive B-cell crossmatches
are not from HLA antibodiesl.!" Other techniques involve
bead-based screening assays, which increase the specificity of
flow cytometry by removing interference from non-HLA
antibodies. When relevant antibodies are detected and the
flow crossmatch is positive, graft survival is significantly
decreased.r" Thus, a positive flow cytometric crossmatch for
donor T- or B-cell reactive IgG antibodies represents a high
1722 CHAPTER 8 0

risk of humoral rejection and a relative contraindication to

transplantation.
As one might expect, the sensitization status of a patient
can change over time. New exposures through transfusions
can lead to new antibody formation. Existing sensitivities
can wane with prolonged time on the waiting list. Thus,
serum from patients awaiting transplantation is frequently
screened against a battery of random donor cells to assess
their level of sensitization. The screening assay is known as
the panel reactive antibody assay, or PRA. A nonsensitized
patient has a PRA of 0%; in other words, that patient's serum
lyses 0% of randomly selected cells. The chance of that
patient having a positive crossmatch is very low. Patients
who have had multiple exposures to other human tissues
have higher reactivity to random cells. As their PRA rises,
the likelihood of their receiving an organ that elicits a nega-
tive crossmatch diminishes. Clinical desensitization proto-
cols exist that use plasmapheresis or intravenous immune
globulin (IVIG) to reduce circulating antibody (see following
sections) .
A delayed variant of HAR known as vascular rejection is
also mediated by humoral factors.!" Vascular rejection occurs
when offending alloantibodies exist in circulation at levels
undetectable by the crossmatch assay, even though presensi-
tization has taken place. Frequently, this is seen in a patient
with a high PRA that decreases with time. Serum tested at
the time of the transplant is negative, but serum from an
archived sample is positive. Reexposure leads to restimula-
tion of the memory B cells responsible for the donor -specific
antibodies. The result is initial graft function, followed by
rapid deterioration on or about postoperative day 3. Enhanced
immunosuppression with steroids, combined with nonspe-
cific antibody depletion with plasmapheresis, or administra-
tion of nonspecific Ig (see following sections) is occasionally
successful in reversing vascular rejection.
Because of these preoperative screening techniques, HAR
is rarely seen clinically. As a result of our inability to treat
this disease and the resulting extended waiting times for
transplantation, several protocols have been developed for
transplanting across ABO barriers or a positive crossmatch.
Using a combination of plasmapheresis, IVIG, rituximab (see
below), or splenectomy, several groups have gained signifi-
cant experience and good outcomes in this difficult patient
population.Fv'" As a result of these efforts, this group of
patients who otherwise would likely never receive a suitable
organ and would die without being transplanted now have a
potential option.
Acute Rejection
The T cells cause acute rejection.131, 132 Acute rejection can
occur at any time after the first 4 days postoperatively but is
most common in the first 6 months posttransplant. It evolves
over a period of days to weeks and is the inevitable result of
an allotransplant unless immunosuppression directed against
T cells is employed. To initiate acute rejection, T cells bind
alloantigen via their TCR directly or following phagocytosis
of donor tissue and representation of MHC peptides by self-
APe. This then leads to cell activation, as described. The
result is a massive infiltration of the graft of T cells (Fig.
80.10), with destruction of the organ through direct cytolysis
and endothelial perturbation leading to thrombosis.
Knowledge regarding the pathophysiology of acute rejec-
tion and the normal physiology of T cells has grown substan-
tially in the past 20 years. This understanding has led to the
development of relatively specific treatments, described next,
that are capable of counteracting this disease . In approxi-
mately 70% of allotransplants, Tvcell-specific treatment leads
to prevention of acute rejection. When it does occur, it can
be reversed in most cases.
Prompt recognition of acute rejection is imperative
because prolonged rejection leads to recruitment of multiple
arms of the immune system that do not respond to T-cell-
specific therapies. In addition, graft damage, particularly for
kidney, pancreas, and heart, is generally accompanied by a
permanent loss of function that is proportional to the mag-
nitude of involvement. Most acute rejection episodes for
patients on modem immunosuppression are asymptomatic
until the secondary effects of organ dysfunction occur. By this
time, the rejection is well entrenched and difficult to reverse.
For this reason, monitoring for acute rejection must be
intense, particularly during the first year following transplan-
tation. Generally, unexplained graft dysfunction should
prompt biopsy and evaluation for the lymphocytic infiltra-
tion and graft parenchymal necrosis characteristic of acute
rejection.
Like HAR resulting from humoral presensitization, T-cell
presensitization will result in an accelerated form of cellular
rejection mediated by memory T cells. It occurs within 3 days
of transplant and is usually accompanied by a significant
humoral response.
Chronic Rejection
Unlike acute rejection and HAR, chronic rejection (CR)
remains poorly understood.Y!" In many instances, it is
likely a combination of immune and nonimmune factors and
represents the aggregate effect of many offending pathways.
Thus, the term chronic rejection is falling out of favor in lieu
of terms acknowledging the multifactorial etiology!": chronic
allograft nephropathy for kidneys, chronic coronary vascu-
lopathy for hearts, vanishing bile duct syndrome for livers,
 IMMUNOL O GY OF TRAN SPLANTATION 1723

and bronchiolitis obliterans for lungs . Chronic graft disease
is insidious, occurring over a period of months to years, and
because the pathophysiology is not well defined, it remains
untreatable. Heightened immunosuppression is not effective
in reversing or retarding its progression. Thus, its distinction
from acute rejection by biopsy is important.
Chronic rejection tends to be poorly defined even by
histological criteria. Regardless of the organ involved, it is
characterized by parenchymal replacement by fibrous tissue
and a relatively sparse lymphocytic infiltrate. Infiltrates may
contain macrophages and DCs . Those organs with epithe-
lium show a dropout of the epithelial cells as well as endo-
thelial destruction. Chronic rejection is clearly related to the
events associated with transplantation, including allorecog-
nition and ischemic injury, which set the stage for tissue
remodeling. These effects set the stage for expression of
various soluble factors, including TGF-~ and the remodeling
of the organ's parenchyma and its replacement by fibrous
scar.
Immunosuppression
The redundancy and plasticity of the immune system has to
date prevented any single agent from specifically preventing
graft destruction. In addition, as allograft recognition is medi -
ated by an immune system formed for the detection and
elimination of pathogens, manipulations altering this system
do so at the expense of a vital defense network. Thus, no
immunosuppressive intervention is allograft -specific, or put
another way, all drugs preventing allograft loss put the recip-
ient at increased risk for infection and malignancy. Rational,
selective use of several immunosuppressive agents acting
through different synergistic mechanisms is required to suc-
cessfully prevent rejection without completely removing the
body's defense.
For all organs, it is clear that the events occurring at the
time of the initial antigen exposure are the most critical in
establishing a lasting state of immune unresponsiveness. For
this reason, immunosuppression is extremely intense in the
early postoperative period and tapered thereafter. This initial
conditioning of the recipient's immune system is known as
induction immunosuppression. It usually involves deletion
of the T-cell response completely and as such cannot be
maintained indefinitely without lethal consequences. Medi-
cations used to prevent acute rejection for the life of the
patient are called maintenance immunosuppressants. These
agents tend to be well tolerated acutely if dosed appropriately,
but all have chronic side effects. Immunosuppressants used
to reverse an acute rejection episode are called rescue agents.
They are generally the same as those agents used for induc-
tion therapy. The mechanisms of immunosuppressants are
described next , and the pivotal clinical trials demonstrating
their efficacy and mechanism are outlined in Table 80.3; the
clinical use of these agents is detailed in the chapter on rejec-
tion (see chapter 81 ).150-1 56
Corticosteroids
Corticosteroids, particularly glucocorticosteroids, remain a
central tool in the prevention and treatment of allograft rejec-
tion. Used alone at maintenance doses, they are ineffective
in preventing allograft rejection, but used in combination
with other agents, they significantly improve graft survival.
When used in higher doses, they effectively rescue acute cel-
lular rejection. Although steroids have a desirable immuno-

TABLE 80.3.
Immunosuppression Approaches.

Reference Year Approach Optimized regimen Organ Patien ts Results Comment

150 2001 Calcineurin Tacrolimus + MMF Kidney 223 23 % increase in allograft 2-year follow -up;
inhibition survival in DGF compared with CsA +
MMF and TAC + AZA
(all with steroids)
151 1998 Ca1cineurin Tac rolimus + AZA Kidney 412 Reduction in AR versus All patient s wi th
inhibition CyA (30.7% vs. 46.6%) induction therapy; high
an d nee d for antibody percen tage of African
th erapy Americans
152 1998 Antiproliferative CyA+MMF Kidney 503 50% reduct ion in AR No differenc e at 3 year s
when compared wit h AZA
153 1998 T-cell deplet ion rATG Kidney 163 88% AR reversal and 17% Increased and prol onged
for AR recurrent AR (vs. ATGAM depletion with rATG
at 76% and 36%) without increase in side
effects
154 2000 TOR inhibition CyA + predn isone Kidn ey 719 Dec reased rate of AR Sim ilar 12-mont h patient
+ sirolimus (12%) versus AZA (32.3%) and graft survival
ISS 1998 Anti -IL2 Daclizima b Kidney 260 Decreased rate of AR Induc tion with trip le
(22%) versus placebo therapy (CyA, AZA,
(35%) predn isone)
156 2004 T-cell depletion rATG Kidney 72 Increased graft surv ival S-year follow -up
for induction (77%) and freedom from
rejection (92%) versus
ATGAM (55 and 66%)
1724 CHAPTER 80
suppressive effect, they can contribute significantly to the
morbidity of transplantation.
The mechanism of the immunosuppressive effect of glu-
cocorticosteroids has only recently been elucidated.P''!" Glu-
cocorticosteroids bind to an intracellular receptor after
nonspecific uptake into the cytoplasm (see Fig. 80.7). The
receptor-ligand complex then enters the nucleus, where it
acts as a DNA-binding protein and increases the transcription
of several genes. The most important gene affected is probably
the gene for IkBa. This protein binds to and prevents the
function of NF-kB, a key activator of proinflammatory cyto-
kines and an important transcription factor used in T-cell
activation. By increasing IkBa in the cell, steroids prevent the
primary mechanism by which lymphocytes amplify their
responsiveness. The resulting effects are predictably diverse.
Steroids block transcription of IL-l and TNF-a by APCs. They
also block IFN-y production by T cells and migration and
lysosomal enzyme release by neutrophils. Phospholipase A2,
and consequently the entire arachidonic acid cascade, is
inhibited. Steroids also mute the upregulation of MHC. By
blocking the response of leukocytes to chemotactins and by
inhibiting vasodilators such as histamine and prostacyclin,
steroids dampen the inflammatory response and decrease the
costimulation environment. Steroids do not have a significant
influence on antibody production. The most commonly used
form of steroid is prednisone or its intravenous substitute,
methylprednisolone.
Antiproliferative Agents
AZATHIOPRINE
The antimetabolite azathioprine (Aza) was the first immuno-
suppressive pharmaceutical used in organ transplantation and
remains a part of many maintenance protocols.t":" Azathio-
prine undergoes hepatic conversion first to 6-mercaptopurine
(6-MP) and then to 6-thio-inosine monophosphate (6-tIMP).
These derivatives in tum inhibit DNA synthesis by alkylat-
ing DNA precursors and inducing chromosomal breaks
through interference with DNA repair mechanisms. In addi-
tion, they inhibit the enzymatic conversion of IMP to adenos-
ine monophosphate (AMP) and guanosine monophosphate
(GMP). The primary effect is to deplete the cell of adenosine.
The effects of Aza are relatively nonspecific. It acts not only
on proliferating lymphocytes and polymorphonuclear cells
(PMNs), but also on all rapidly dividing cells. As such, its
primary toxicity is directed at the bone marrow, gut mucosa,
and liver. Azathioprine is relatively ineffective alone and has
no efficacy as a rescue agent. It does effectively inhibit rejec-
tion when given as a maintenance agent in combination with
steroids and a calcineurin inhibitor.
MYCOPHENOLATE MOFETIL
Mycophenolate mofetil (MMF) is an immunosuppressive
agent approved in 1995 for use in adults.l'" It is a morpho-
linoethyl ester of mycophenolic acid (MPA), an established
noncompetitive, reversible inhibitor of IMP dehydrogenase.
This modification improves the bioavailability of MPA.
Physiological purine metabolism requires that GMP be
synthesized for subsequent synthesis of guanosine triphos-
phate (GTP) and deoxyguanosine monophosphate (dGTP). For
RNA synthesis, GTP is required, and dGTP is required for
DNA synthesis. Guanosine monophosphate is formed from
IMP by IMP dehydrogenase. Therefore, MMF prevents a crit-
ical step in RNA and DNA synthesis. Of major importance,
however, is the presence of a "salvage pathway" for GMP
production in most cells except lymphocytes (hypoxanthine-
guanine phosphoribosyl transferase-catalyzed GMP produc-
tion directly from guanosine). Thus, MMF exploits a critical
difference between lymphocytes and other body tissues,
including PMNs, to produce relatively lymphocyte-specific
immunosuppressive effects. Mycophenolate mofetil blocks
the proliferative response of both T and B cells, inhibits anti-
body formation, and prevents the clonal expansion of cyto-
toxic T cells.
Mycophenolate mofetil decreases the rate of biopsy-
proven rejection and the need for antilymphocyte agents in
rescue therapy compared to Aza. 160,161 As such, MMF has
replaced Aza in most patients and is used in combination
with either a calcineurin inhibitor or sirolimus by many
centers in steroid-sparing protocols. Subsequent study has
shown that MMF also has some effect as a rescue agent when
used with steroids. In addition, treatment with MMF has been
suggested to slow the progression of chronic allograft nephrop-
athy. Despite these improvements, MMF is not effective
enough to use without either steroids or calcineurin inhibi-
tors. Tacrolimus may interact with MMF, potentiating the
effect and toxicity of both drugs.
Calcineurin Inhibitors
CYCLOSPORINE
The T-cell-specific immunosuppressive drug cyclosporine
A (CyA) is a cyclic endecapeptide isolated from the fun-
gus Tolypocladium inflatum gams. 34,162 Its mechanism of
action is mediated primarily through its ability to bind
to cytoplasmic protein cyclophilin (Cn)l54 (see Fig. 80.7).
The CyA-Cn complex binds with high affinity to the
calcineurin-calmodulin complex and, in doing so, blocks its
role in the calcium-dependent phosphorylation and activation
of the transcription-regulating factor NF-AT. This prevents
the transcription of the IL-2 gene. Cellular cytotoxicity is also
probably interrupted through calcineurin inhibition. The
transcription of other genes critical for T-cell activation is
also altered. Cyclosporine A increases TGF-~ transcrip-
tion. 163,164 This cytokine is part of the natural downregulatory
milieu present after injury. It effectively inhibits T-cell acti-
vation, reduces regional blood flow, and activates many path-
ways critical in tissue remodeling and wound repair. The
toxicity of CyA may be related to the last two of these three
TGF-~-mediated effects. Also, CyA blocks Cn function as a
cis-trans peptidyl-prolyl isomerase, which is critical for the
proper folding of proteins. This function is not critical to the
immunosuppressive effects of CyA but may relate to the toxic
side effects of the drug.
Cyclosporine A blocks TCR signal transduction but does
not inhibit costimulation signals.l'" As such, on withdrawal
of CyA, the T cell is not rendered anergic but rather is capable
of mounting an attack on its intended antigen. The effects of
CyA can be overcome with exogenous (or in the case of an
ongoing rejection episode, ambient) IL-2. For this reason, once
IL-2 is present in the graft cytokine milieu, CyA is ineffective.
 IMMUNOLOGY OF TRANSPLANTATION
Cyclosporine therefore works solely as a maintenance agent
and is ineffective as a rescue agent.
Cyclosporine is insoluble in aqueous solutions but soluble
in lipids and organic solvents. Because of this, the gastro-
intestinal absorption of CyA is dependent on bile flow (a
significant concern in liver transplantation). A newer
microemulsion form is less bile-dependent. Cyclosporine is
metabolized by the hepatic cytochrome P-450 enzymes, and
blood levels are therefore increased by inhibitors of cyto-
chrome P-450 (ketoconazole, erythromycin, calcium channel
blockers) and decreased by cytochrome P-450 inducers
(rifampin, phenobarbital, phenytoin). Liver failure slows the
clearance of CyA because 900/0 of its metabolites are cleared
in the bile. Renal failure only minimally alters the clearance
of CyA.
Cyclosporine has much toxicity that governs its use.
It has a significant vasoconstrictor effect on proximal renal
arterioles that decreases the renal blood flow by approxi-
mately 30%. This action is most likely mediated through the
induction of TGF-~, which in addition to suppressing T-cell
activation, increases the transcription of endothelin.l'<'?'
Endothelin-mediated vasoconstriction activates the renin-
angiotensin pathway, promoting hypertension. The remodel-
ing effects of TGF-~ also induce fibrin deposition, which may
promote the fibrosis typically seen during CR. The vascular
effects of CyA tend to increase vascular resistance in the
kidney and delay the resolution of acute tubular necrosis
(ATN) or hepatorenal insufficiency. An additional renal effect
is an idiosyncratic reaction producing hemolytic uremic syn-
drome. Hyperkalemia may also result from its effects on both
the proximal and distal renal tubules.
Discontinuing the drug can reverse most of the toxic
effects of CyA. Cyclosporine frequently causes neurological
side effects consisting of tremors, paresthesias, headache,
depression, confusion, somnolence, and, rarely, seizures.
Hypertrichosis of the face, arms, and back is seen in about
500/0 of patients. Gingival hyperplasia may also occur. CyA
also may promote malignant transformation of some cell
types.l'"
TACROLIMUS
Tacrolimus, previously described investigationally as FK506,
is a macrolide produced by Streptomyces tsukubaensis that
was discovered in 1984 in Japan during a search for new
immunosuppressive agents. In 1987, Kino and coworkers first
demonstrated its in vitro immunosuppressive properties.!"
Tacrolimus, like CyA, blocks the effects of NF-AT, prevents
cytokine transcription, and arrests T-cell activation" (Fig.
80.7). The intracellular target is an immunophilin protein
distinct from cyclophilin known as FK-binding protein (FKBP);
thus, the effect is additive to that of CyA. As such, the use
of tacrolimus with CyA produces prohibitive toxicity. Tacro-
limus increases TGF-~ transcription, leading to both the ben-
eficial and toxic effects of this cytokine.l'<'?' It is 100 times
more potent in blocking IL-2 and IFN-yproduction than CyA,
but its toxicity limits its dose to approximately 1 % of Cy A.
Like CyA, the effects of tacrolimus are relatively T-cell-
specific, but in addition to its role as a maintenance agent,
tacrolimus has also shown promise as a rescue agent.I'"
The side-effect profile for tacrolimus is similar to that of
CyA with regard to renal toxicity. As compared with CyA,
1725
neurotoxicity, in the form of tremors and mental status
changes, is somewhat more pronounced, as is its diabetogenic
effect. In addition, as tacrolimus and CyA are both metabo-
lized by cytochrome P-450 enzymes, they share similar drug
interactions. Cosmetic side effects are substantially reduced.
Tacrolimus has been shown to be extremely effective for liver
transplantation and has become the drug of choice for most
centers.
Antilymphocyte Preparations
ANTITHYMOCYTE GLOBULIN
Antilymphocyte globulin (ALG) is produced by inoculating
heterologous species with human lymphocytes, collecting the
plasma, and purifying the IgG fraction. The resulting prepara-
tion, known as a polyclonal antibody preparation, contains
antibodies directed against many antigens on lymphocytes.
Thymocytes rather than lymphocytes are sometimes used as
the immunogen, and antibodies formed from this process are
known as antithymocyte globulin (ATG). The most com-
monly used preparation in the United States is a rabbit ATG
(Thymoglubulin, Sangstat).
Antibodies in ATG coat multiple epitopes on the T
cell. l 70,l 7l This has many effects, including promoting T-cell
clearance through complement-mediated lysis and opsonin-
induced phagocytosis, as well as limiting the ability of the T
cell to generate an effective TCR signal by internalization of
key surface receptors. In addition, the antibodies crosslink
several key molecules, including adhesion and costimulation
molecules", crosslinking has the effect of impairing or alter-
ing signals generated by these receptors and either preventing
activation or possibly leading to an anergic phenotype. The
overall result is to reduce the precursor frequency of the
primary effector cells below that required for acute rejec-
tion and allow for slow repopulation during the critical time
posttransplantation.
When used as an induction agent, the profound and long-
term T-cell depletion reduces the possibility that T-cell-
mediated antigen recognition will occur when the graft is in
its most vulnerable state. Cell surface crosslinking prevents
appropriate costimulation for those cells that do encounter
antigen. The costimulatory milieu produced by organ preser-
vation and postischemic reperfusion can subside before the
introduction of T cells, which lessens the possibility that T
cells will encounter alloantigen in an environment that will
foster a positive TCR signal. When ATG is used as a rescue
agent, antigen recognition has already occurred, and the ben-
eficial effects of ATG are likely related solely to its ability to
destroy cytotoxic T cells.
Antithymocyte globulin is most commonly used as part
of a multidrug induction immunosuppression protocol with
CyA, Aza or MMF, and prednisone and more recently as a
key depletional agent in minimization protocols.F"!" Often,
ATG is used sequentially with CyA or tacrolimus in the early
postoperative period following renal transplantation to avoid
the nephrotoxicity of these drugs early in the transplant
course. In addition, ATG is an effective rescue agent in cases
of steroid-resistant acute rejection.
Most of the side effects of ATG stem from its heterolo-
gous origin and heterogeneous composition. Because ATG is
polyclonal, antibodies specific for antigens on both T - and B
1726 CHAPTER 80
cells, as well as to other cells, are present. In fact, only a small
fraction of the serum is estimated to be biologically active
against T cells. One prominent side effect related to this
promiscuous specificity is thrombocytopenia resulting from
cross-reactivity with platelets. In addition, leukopenia and
anemia may also occur. Despite this, major side effects are
rare, and the drug is well tolerated by most transplant recipi-
ents. The most common symptoms are the result of transient
cytokine release following antibody binding. Chills and fevers
occur in up to 20% of patients and are easily treated with
antipyretics and antihistamines. The use of ATG has been
associated 'with an increase in the reactivation and develop-
ment of primary viral disease caused by cytomegalovirus
(CMV), herpes simplex virus (HSV), Epstein-Barr virus (EBV),
and varicella.
MUROMONAB (OKT3)
Although polyclonal antibody preparations have many binding
specificities, monoclonal antibodies have a single specific
target antigen. The first, and for years only, commercially
available monoclonal antibody preparation for use in organ
transplantation was muromonab (OKT3, Orthoclone, Ortho
Pharmaceuticals, Raritan, NJ). This murine monoclonal anti-
body is directed at the signal transduction subunit on human
T cells (CD3)l75 (see Fig. 80.7).
There are several ways in which OKT3 is thought to have
its effect. 175,176 The CD3 determinant, as described earlier, is
a cluster of transmembrane proteins found on the surface of
all mature T lymphocytes. When OKT3 binds to CD3, it leads
to internalization of the TCR complex, thus preventing
antigen recognition and TCR signal transduction. In addition,
T-cell opsonization and clearance by the reticuloendothelial
system occur. Following the administration of OKT3, there
is a rapid decrease in the number of circulating CD3+ lym-
phocytes. Effect is minimal on T cells residing in the thymus,
lymph nodes, or spleen. Following several days of administra-
tion, there is a return of T cells expressing the accessory
binding molecules CD4+ and CD8+ but lacking the TCR.
These "blind" T cells remain incapable of binding to antigen.
In addition to interfering with the generation of cytotoxic T
cells and the modulation of cell surface proteins, OKT3 blocks
the cytotoxic activity of already activated T cells; this is the
result of inappropriate activation and degranulation that
results when the CD3 is bound by OKT3. This reaction is
perhaps its most important function but leads to its substan-
tial side-effect profile.
The T-cell-derived cytokines have evolved as activators
of adjacent cells."? Their release is strongly polarized to the
side of the cell actively engaged in cell-to-cell contact. Indeed,
the most potent T-cell activator, IL-2, exerts most of its effect
in an autocrine loop. Pan-activation of the body's T cells leads
to transient activation and systemic cytokine release, similar
to the effect of the superantigen staphylococcal exotoxin, the
etiological agent for toxic shock syndrome. As such, admin-
istration of OKT3leads to profound, systemic cytokine release
syndrome that can result in hypotension, pulmonary edema,
and fatal cardiac myodepression. In approximately 2 % of
patients, the inflammatory response manifests itself as aseptic
meningeal inflammation. Administration of high-dose meth-
ylprednisolone before OKT3 administration is required to
blunt this adverse response, but it is rarely avoided altogether.
The syndrome abates with subsequent dosages as the target
cells available for degranulation become consumed or
exhausted.
Measurement of serum levels of OKT3 is possible but is
not done routinely.l" More commonly, the percentage of
CD3+ cells is determined by flow cytometry. The presence of
less than 10% CD3+cells is usually associated with therapeu-
tic efficacy. Greater than 10% CD3+ cells usually indicates
the presence of anti-OKT3 antibodies, which may limit the
desired therapeutic effect. Because OKT3 is a foreign protein,
it is itself an antigen that will elicit an immune response.
Antimurine antibodies may be directed against structural
regions of the antibody (constant or variable regions) or the
actual binding site (idiotypic) of the OKT3 antibody; they
usually arise after a prolonged course of OKT3 or following
the cessation of therapy. As would be expected, the use of
MMF or other immunosuppressants with anti-B-cell activity
during treatment with OKT3 may reduce the formation of
anti-OKT3 antibodies.
Much like ATG, OKT3 was first used as a rescue agent to
treat acute renal allograft rejection. 179,180 It is greatly superior
to conventional steroid therapy in reversing rejection and,
consequently, in improving allograft survival. However, its
side effects and the limiting nature of the antimurine anti-
body response have limited its use to steroid-resistant rejec-
tion, during which maintenance immunosuppression is
reduced. Use of OKT3 in induction immunosuppressive pro-
tocols for kidney transplants is now less frequent due to the
availability of both ATG and alemtuzumab. As with other
antilymphocyte preparations, OKT3 has been shown to cause
a very high reactivation rate of CMV and other viruses.
ANTI-IL-2 STRATEGIES
Two other monoclonal antibodies are currently approved
for use in transplantation induction protocols: daclizumab
[Zenapax, Roche Pharmaceuticals, Nutley, NJ) and basilix-
imab [Simulect, Novartis Pharmaceuticals, Basel, Switzer-
land).155,181,182 They are either humanized (daclizumab) or
chimeric (basiliximab), and both are directed against CD25,
the high-affinity chain of the IL-2R (Fig. 80.7). As discussed
in previous sections, a high-affinity form of the IL-2R is
required for T-cell clonal expansion. This form of the recep-
tor is dependent on the upregulation of CD25, a 55-kDa a
chain that combines with a ~ and 'Y chain to form a hetero-
trimer. Targeting this receptor has many potential advan-
tages. It is only present on activated T cells, and thus the
effects are limited to those T cells that have become acti-
vated in the face of an allotransplant. Theoretically, only
allograft-specific cells are affected, leaving T cells with phys-
iological specificity undisturbed. It should be pointed out,
however, that because alloreactivity is the result of cross-
reactivity, even removal of alloreactive cells might remove
cells with important specificity against pathogenic antigens.
An additional benefit of anti-CD25 therapy is that it is mono-
clonal, and the binding of its target does not precipitate a
T-cell signal that induces cytokine release. Thus, no acute
side effects occur. Finally, both these antibodies are the
products of genetic engineering. While the antigen-binding
regions of their parent murine antibodies have been retained,
the structural components have been replaced with human
IgG. Thus, they are mostly human proteins, and their pro-
 IMMUNOLOGY OF TRANSPLANTATION
pensity to generate a neutralizing immune response is greatly
reduced.
Both anti-CD25 antibodies function as induction agents
only. The prevention of IL-2R function efficiently prevents
the activation of nonactivated T cells. However, IL-2 is only
required very early in the activation process. Once a rejection
episode has been initiated, cytotoxic T cells can function
without high-affinity IL-2R signaling. As such, these agents
do not readily reverse rejection.
Experience in the form of several experimental and clini-
cal trials with anti-CD25 monoclonal antibodies has been
encouraging. 155,181,182 Induction with anti-CD25 has been
shown to prevent or reduce the frequency of early rejection
episodes when used in combination with a calcineurin inhib-
itor, an antiproliferative agent, and steroids. In addition,
several trials have shown success with the incorporation of
anti-IL-2 agents in steroid or calcineurin avoidance protocols
with the added benefit of reduced toxicity as compared with
other induction regimens.!"
New Immunosuppressive Agents
Sirolimus/Everolimus
Sirolimus and everolimus are macrolide antibiotics derived
from Streptomyces hygroscopicus. 184-186 The effects of these
agents depend on binding to the immunophyllin FKBPI2,
which is also the intracellular target for tacrolimus. How-
ever, these agents do not affect calcineurin activity.P"!" As
a result, sirolimus and everolimus do not inhibit the expres-
sion of NF-AT or IL-2 expression. Rather, they impair signal
transduction by the IL-2R through interaction of the rapamy-
cin-FKBP (FRAP) complex with the cytoplasmic protein
RAFT-I, a critical kinase in IL-2R-associated activation (Fig.
80.7). In doing so, the p70 S6 kinase cascade is arrested, and
T cells are prevented from progressing from G 1 to the S phase
of cell replication", this has the advantage of interrupting
the T-cell activation pathway even if IL-2 is present from an
exogenous source or ongoing rejection. Other receptors are
also affected, including those for IL-4, IL-6, and platelet-
derived growth factor (PDGF). An additional benefit may lie
in the ability of sirolimus to antagonize B-cell lymphomas
and the ability of everolimus to prevent the growth of
EBV. 191,192 As such, their use may potentially prevent the
development of posttransplant lymphoproliferative disease.
Both agents have been shown to dramatically prolong
allograft survival in multiple animal models. This effect has
been carried to the clinic, where sirolimus has become an
alternative to calcineurin inhibition in selected patients. In
addition, both have demonstrated synergy with cyclospo-
rine,154 tacrolimus.!" and steroids, thus allowing reduction in
their dose.'?"
As with calcineurin inhibition, the use of both of these
agents has been limited by their side effects. The increased
incidence of hypercholesterolemia and hypertriglyceridemia
seen with both agents often mandates treatment with a cho-
lesterol-lowering agent or changing to a calcineurin inhibitor.
In addition, thrombcytopenia is a common side effect of both
agents, and impaired wound healing and oral ulcers remain a
frequent problem with sirolimus. Overall, both agents offer
an adjunct or alternative to calcineurin inhibition, albeit with
1727
a different side-effect profile that is generally manageable
with dose adjustment.
Alemtuzumab
While lymphocyte depletion with ATG utilizes a polyclonal
preparation, other depletion strategies focus on monoclonal
antibodies directed against specific cell populations. Alemtu-
zumab (Campath, Berlex Laboratories, Seattle, WA) is a
humanized monoclonal antibody directed against CD52,
which is present on lymphocytes and monocytes but not on
leukocyte precursors in the bone marrow. As with ATG,
treatment with alemtuzumab results in a profound reduction
in the number of central and peripheral T lymphocytes at the
time of transplantation, therefore decreasing the frequency of
alloreactive cells at a time of increased immune susceptibil-
ity. Alemtuzumab also depletes B cells and monocytes to a
lesser degree. Alemtuzumab is approved for use in the treat-
ment of hematogenous malignancies.
Initial groundbreaking work by Calne!" using alemtu-
zumab and subsequent drug minimization'P'!" with cyclo-
sporine has set the stage for several studies demonstrating the
efficacy of this drug as an induction agent in both minimiza-
tion strategies and attempts at tolerance (Table 80.4). Both of
these approaches (depletion with either ATG or alemtu-
zumab) should be considered complementary and usage
should be directed by patient considerations.
Deoxyspergualin
The antitumor antibiotic spergualin was isolated from Bacil-
lus laterosporus in 1981. Its derivative deoxyspergualin (DSG)
was shown to have strong antiproliferative and immunosup-
pressant properties. 198 Although the mechanism of DSG is not
completely understood, there is evidence that it is immuno-
suppressive via a predominantly anti-APC effect."? It does
prevent the nuclear translocation of NF-kB, perhaps through
its association with the heat-shock proteins HSP 70 and HSP
90. This interaction inhibits the release of IkB, perhaps mim-
icking a critical part of the steroid mechanism of action. The
close relationship with HSPs, combined with evidence that
the inhibitory effect of DSG on cytotoxic T cells can be abol-
ished by the MHC-upregulating cytokine IFN-y, suggests that
the primary effect of DSG is to inhibit antigen presentation
or the costimulatory function of APCs. Deoxyspergualin has
been shown to effectively prolong allograft survival in many
animal models.'?" Early clinical studies have not been as
promising, and it is thought that DSG will have a minor role
in the transplantation armamentarium.P"
FTY720
FTY720 (2-amino-2-(2-[4-octylphenyI ]ethyI )-1,3-propanediol)
is a chemical derivative of the fungus Isaria sinclairii and
functions to produce a profound, yet reversible, state of lym-
phopenia by sequestering lymphocytes in lymphatic tissues.P'
This agent traps lymphocytes (T and B cells) in lymphoid
organs by rendering them unresponsive to the egress signal
provided by the lysophospholipid sphingosine l-phosphate
(SIP). In addition, FTY720 decreases vascular permeability
associated with ischemic injury via a similar mechanism,
thereby enhancing its effect. The major reported side effect of
1728 C H A P T E R 80

TABLE 80.4.
Results of Recent Tolerance and Minimization Trials.

Reference Year Approach Optimized regimen Organ Patients Results Comment

207 2001 Costimulation Anti-CD 154 Kidney 7 7 AR 3 thromboembolic events;
blockade all grafts rescued
195 1998 T-cell depletion Alemtuzumab and Kidney 13 2AR Prope tolerance; low-dose
low-dose CYA CYA as maintenance
208 1999 T-cell depletion Alemtuzumab and Kidney 31 6 AR (steroid- Follow-up of above study;
low-dose CYA responsive I 29 functioning grafts
(21 months)
196 2003 T-cell depletion Alemtuzumab Kidney 7 7 AR Placed on monotherapy
sirolimus after AR
209 2003 T-cell depletion Alemtuzumab and Kidney 5 5AR Placed on monotherapy
DSG sirolimus after AR
211 2000 Chimerism TLI, RATG, steroid Kidney 25 3 patients off > 10 years after
withdrawal immunosuppression tra nsplantation
212 2002 Chimerism TLI, RATG, ste m Kidney 4 2 patients weaned off HLA nonidentical
cells immunosuppression,
bot h with AR
210 1999 Chimerism Cyclophosphamide, Kidney 2 No AR (2 to 4 years) HLA identical patients
ATGAM, TLI, BM (Multiple off with multiple myeloma;
Myeloma) immunosuppression no GVHD and minimal
disease recurrence
ATGAM, antithymocyte globulin; AR, acute rejecton, BM, bon e marrow; CYA, cyclosporin e A; DSG, deoxyspergualin, RATG, rabbit anti thymocy te globulin;
TLI, total lymphoid irrad iati on.

FTY720 administration has been bradycardia. Currently,
these two properties of reversible lymphopenia and reduction
of ischemia reperfusion injury are being evaluated in renal
transplantation clinical trials.
Other Agents
Rituximab
Rituximab is a murine antihuman anti-CD20 chimeric anti-
body that has been used successfully in the treatment of
non-Hodgkin's lymphoma and posttransplant lymphoprolif-
erative disorder (PTLD). In addition, this agent has been used
in lieu of splenectomy in renal transplantation across ABO
and HLA boundaries (see hyperacute rejection section) . Ritux-
imab inhibits B-cell proliferation by apoptosis, Fc receptor-
mediated ADCC, and complement-dependent cytotoxicity
(CDC) and therefore limits antibody production. Recent
reports have highlighted the use of rituximab in highly
sensitized patients in whom humoral-mediated rejection
ensues.I" The success of this approach in certain circum-
stances helps illustrate the importance of B-cell responses in
the immune response.
Intravenous Immune Globulin
Intravenous immune globulin (MG) is a clinically available
blood product and is currently used for the treatment of
various diseases, such as immune thrombocytopenia, humoral-
mediated rejection, Cuillian-Barre syndrome, uveitis, and
myasthenia gravis. It is prepared from the pooled plasma of
50,000 to 100,000 human plasma donors and is comprised of
more than 90% IgG antibodies directed against the multitude
of antigens present in such a large population.
The roles of MG in transplantation have focused mainly
on pretreatment of the highly sensitized patient and on the
treatment of humoral-mediated rejection and hemolytic
uremic syndrome associated with calcineurin use. The mech-
anisms behind MG's actions are varied but focus on inhibi-
tion of antibody formation via Fe-mediated effects, direct
antiidiotypic antibodies present in MG, and antiinflamma-
tory activities mediated by complement. Of these, the satura-
tion of the neonatal Fe receptor (FeRn) on MG administration
and the subsequent catabolism of IgG, including alloantibod-
ies, seems the most compelling regarding the elimination of
alloantibody.203,204 Currently, treatment with high-dose MG
(1-2gjkg) is the cornerstone of attempts at antibody removal
either before transplantation in highly sensitized individuals
to reduce the PRA and potential positive crossmatch or after
transplantation in the treatment of antibody-mediated rejec-
tion .20s In addition, MG is also used in ABO-incompatible
renal transplants with reasonabl e success.P?
Tolerance
Physicians have long dreamed of a method that would allow
for organs to be transplanted and specifically accepted as self-
organs. Since the middle of the twentieth century, it has been
clear that one acquires the ability to respond to certain anti-
gens and not respond to others. More recently, it has become
apparent that tolerance to self-tissues is rarely absolute, is
closely regulated, and is probably maintained by repetitive
exposure to self-antigen in a context that fosters T-cell anergy
rather than T-cell activation. Several investigators are now
aggressively pursuing means to apply the body's natural ability
to acquire and maintain tolerance to transplanted organs
rather than relying on toxic immunosuppression for graft
survival. Extraordinary success has been achieved in animals
 IMMU N O L O GY O F TRAN SPLANTATI ON 1729

TABLE 80.5.
Preclinical Tol erance.

Reference Year Approach Optimized regimen Animal Organ MST (day s) MaxST Comment

213 1997 Costimulation Anti-CD 154 an d Rhesus Kidney 20-30 >6 months
blockade CTLAlg
214 1999 Costimulation Anti-CDl 54 Rhesus Kidney 750 >5 years 20mg/kg dose, 6-mo nth
blockad e treatment, conv entional
agents decrea sed
survival
215 200 1 Cost im ul ation Anti-CD154 and Rhesus Skin 236 >365 days No difference wit h DST
blockade DST
218 200 1 Costi mulatio n Anti-CD80/86 Rhesus Kidney 191
blockade
217 2002 Costimulation Anti-CD80/86 and Rhesus Kidne y 565 Delayed anti donor Ab,
blockade anti-CDl 54 no difference versus
anti-CD l 54 alone
216 2002 Costimulat ion Anti·CD40 Rhesus Kidney 49
blockade
219 1998 T-cell depletion FN I8-CRM9 Rhesus Kidney >100 (8 of 5 of 6 animals
immunotoxin 14 animals) m aintained repeat
challenge skin grafts
220 2003 T-cell depletio n Immunoto xin and Rhesus Kidney 1495 Tolerant group with
DSG high IL-lO/IFN-y ratio
221,222 1999, Chimerism TBI, th ym ic XRT, Cynom olgus Kidney >200 Transient
200 1 ALG, BM, CYA ± macrochimerism (less
splenectomy or than 2 m onths)
anti-CD l54
ALG, antilymphocyte globulin ; BM, bone marrow; CYA, cyclosporine A; DSG, deoxyspergualin, XRT, X irradiat ion.

using several techniques.f" Indeed, transplantation of kidneys
and hearts without any chronic immunosuppression is now
routinely successful in nonhuman primates, and early human
trials have been initiated (although none with definitive
success as yet). Several of the more promising approaches are
briefly outlined next, and both preclinical and clinical trials
are highlighted in Tables 80.4 and 80.5. 195, 196,207- 222
T-Cell Ablation
It is becoming increasingly clear that postischemic reper-
fusion and surgical trauma significantly increase immune
recognition following transplantation.I'S!" This concept has
been the rationale for the use of induction therapy. Current
antilymphocyte preparations successfully remove T cells
from the circulation for several days and render those that are
present inert, but they do not have a significant impact on a
subset of memory T cells, which appear to be sensitive to
calcineurin inhibition with tacrolimus.f" It has been demon-
strated that tolerance can be achieved by complete ablation
of all mature T cells at the time of engraftment.P' In nonhu-
man primates, a T-cell-specific immunotoxin has been used
to deplete all T cells from the body. Reconstitution does not
occur for approximately I month. When the cells return, cells
reactive to the graft are not found in the circulation, and acute
rejection does not occur. The animal is, however, capable of
rejecting a subsequent graft or fighting infection. Because the
T-cell ablation is nonspecific, the specific effect must be the
result of regulation occurring in the repopulated thymus or
in the peripheral lymphoid tissues.
Based on these preclinical data, depletion has been studied
with both globulin and alemtuzumab in an attempt to insti-
tute tolerance or minimize immunosuppression. As demon-
strated in Table 80.4, although depletion alone has not been
able to establish tolerance, the minimization of immunosup-
pression to single-agent therapy has been a tremendous step
toward achieving tolerance. The success of lymphocyte deple-
tion suggests the reduction in the number of alloreactive cells
available to institute graft rejection allows for a decreased
level of maintenance immunosuppression.
Mixed Chimerism
Donor antigen is clearly required for any specific immune
event to occur, be it rejection or tolerance. It has become
increasingly clear that the character of that antigen and the
means by which it is administered are important factors in
determining the direction the immune system will take.
Several investigators have had success achieving tolerance by
supplementing the transplanted organ antigen with additional
antigen in the form of bone marrow. 225- 227 Using a variety of
conditioning regimens, donor bone marrow is transplanted
and engrafts throughout the body. This situation is known as
mixed chimerism because donor and recipient hematopoietic
elements coexist. The mechanism by which this promotes
graft acceptance remains unclear. Thymic reeducation in the
face of the new "self"-antigen has been proposed, and some
investigators have improved their experimental success by
directly injecting donor antigen into the recipient thymus.? "
There is also evidence that the marrow cells establish a self-
regulating network that maintains a state of unresponsive-
ness to the graft.229 There is growing speculation that the
identification of mixed chimerism in a patient may in fact
denote a state of potential tolerance.r"
1730 CHAPTER 80
Despite promising results in preclinical models, the only
successful deliberate attempts at tolerance induction with
this strategy have been in human renal transplant recipients
with multiple myeloma and end-stage renal failure (Tables
80.4 and 80.5). In these patients, mixed chimerism was tran-
siently established after either total lymphoid irradiation
(TLI) or nonmyeloablative conditioning, and immunosuppres-
sion was withdrawn without graft 10ss.211,212 The applicability
of this approach to the broader transplant population is cur-
rently under investigation.
Costimulation Blockade
Current immunosuppressive regimens have relied on agents
that dramatically curtail normal T-cell functions, including
TCR signal transduction. As specificity is mediated through
the TCR, blocking this signal ensures that a nonspecific effect
is elicited. With a growing awareness that T cells can be
important mediators of tolerance, there has been growing
interest in using the T-cell response in the absence of activat-
ing costimulation to foster persistent T-cell tolerance.
As has been discussed, once the TCR is engaged, the T
cell can follow one of two paths: activation, through CD28
binding to B7, or anergy, in the absence of CD28 binding or
in the presence of CTLA4 binding (see Fig. 80.7). A toleragenic
phenotype will also result if the APCs cannot be appropriately
activated to foster helper T-cell function and induce cytotoxic
T-cell maturation. Interruption of the CD28 or CD40 path-
ways at the time of transplantation should thus selectively
anergize only those cells undergoing binding to the allograft,
leaving nonreactive cells unaffected. Preexisting immunity
and innate immunity should be unaffected by this approach.
There are an increasing number of reports in the literature
that verify this approach using specific biological blockade
of the CD28 or CD40 pathways.96,213,231 Most encouraging are
data showing, in rodents and primates, that simultaneous
blockade of CD28 and CD40 at the time of transplantation
can allow for prolonged survival of cardiac and renal allografts
without the need for any subsequent immunosuppression and
without any infectious or malignant side effects.
The extrapolation of these results to clinical practice has
been disappointing thus far. In the only human tolerance trial
of costimulation blockade, huSC8, a humanized anti-CD1S4
monoclonal antibody, demonstrated limited efficacy and was
associated with potential thromboembolic toxicity''" (see
Table 80.4). Currently, these agents are being reevaluated in
preclinical models and will most likely be added to the arma-
mentarium of transplant therapeutics.
Xenotransplantation
Transplantation has now become the treatment of choice for
most end-stage diseases of most solid organs. Unfortunately,
the indications for organ replacement have expanded at a rate
far exceeding the available donor supply. As such, most people
who could benefit from a transplanted organ are unable to
receive one. Those who do receive one generally must wait a
significant amount of time, during which they undergo dete-
rioration in their fitness for surgery. Many investigators are
now exammmg the feasibility of xenotransplantation to
improve the availability of organs.P? In addition to increasing
the supply of transplantable organs, xenotransplantation also
offers some of the same benefits achieved with living donors,
including decreased ischemic injury to the organ and optimi-
zation of recipient health status, albeit at the cost of potential
zoonotic viral transmission. In general, there are two types of
xenografts, discordant and concordant. The immunology of
these two types of xenografts differs markedly.
Concordant Xenografts
Concordant grafts are derived from closely related species. For
humans, these include Old World monkeys and apes. The
critical element defining an animal as concordant is the
assembly of carbohydrate antigens on the surface of their
cells. In particular, the enzyme galactosyl transferase is absent
in these animals, and as a result their carbohydrates are the
typical blood group antigens of the ABH system and lack the
N-linked disaccharide galactose a (1-3) galactose [GALa(l-
3)GAL].233,234 Thus, the antibodies present in circulation of
potential human recipients can be predicted by straightfor-
ward ABH typing, thereby avoiding the problem of HAR.
With HAR removed as a threat, the typical mechanisms of
graft rejection are left, including acute cellular rejection,
acute vascular rejection, and, presumably, CR.
It is now clear that most of the critical molecular
elements responsible for antigen presentation and T-cell-
mediated rejection are evolutionarily conserved in mammals.
Xenogeneic MHC polymorphism is, for the most part,
restricted to the antigen-binding region.?" The areas respon-
sible for accessory molecule binding are also conserved. In
addition, costimulatory and adhesion molecules required for
adequate T-cell interaction function well across the species
barrier. 236,237 For these reasons, cellular rejection and T-cell-
dependent humoral rejection proceed as they do for an allograft
completely mismatched at the HLA locus.
Several experimental models of concordant xenograft
rejection, as well as occasional ventures into the clinical
arena, have clearly demonstrated that concordant xenotrans-
plantation is feasible given currently available immunosup-
pressive pharmaceuticals.P'rt" Prevention of concordant
xenograft rejection would require induction with a T-cell-
specific biological agent and chronic cellular suppression,
including calcineurin inhibition, an antiproliferative agent
such as MMF, and steroids. Results matching those seen for
poorly matched allografts could be expected, including the
complications from the immunosuppression required for
engraftment.
With the possible exception of neonatal cardiac transplan-
tation, however, the use of endangered, intelligent species
with little ability to be bred or genetically altered could not
meet the need for readily available, size-matched organs.
Widespread application of concordant xenografts would
quickly deplete the supply of nonhuman primates, particu-
larly when a loss rate extrapolated from poorly matched
allografts is taken into consideration. In addition, there is
significant concern that zoonotic transfer of disease, in par-
ticular retroviral illness, will put the patient and the public
at undue risk. It is therefore anticipated that concordant xeno-
transplantation will not remedy the organ shortage.
 IMMUN OL O GY O F TRAN SPLANTATION
Al,.._ TABLE 80.6.
Xenotransplantation.
Optimized
Reference Year Approach regimen Animal
247 2000 Transgenic hDAF porcine Cynomolgus
248 1999 Transgenic hDAF porcine Baboon
249 2000 Transgenic hDAF porcine Baboon
hDAF, human decay-accelerating factor.
Discordant Xenografts
Discordant grafts are derived from distantly related species
that express galactosyl transferase. For the purposes of human
application, these include New World monkeys and nonpri-
mates such as pigs. When transplanted into humans, these
organs rapidly undergo HAR.241 The primary cause of the
rejection varies from species to species, but generally involves
preformed IgM antibodies directed against atypical carbohy-
drate residues, such as GALa(I-3)GAL, acting in concert with
complement.P'<" These preformed antibodies serve an innate
protective role by binding to GAL residues on bacteria. Vigor-
ous T-cell-mediated rejection can also occur as human T cells
can still be bound and activated by discordant MHC and
costimulated by discordant B7.236,237 Induced antibody
responses similar to a vigorous vascular rejection are seen . In
addition to the typical patterns of cellular rejection, there
may also be a substantial detrimental effect of NK cells and
other innate cellular elements.236,242
Given the spectrum of effectors involved in discordant
rejection, intervention at all levels of both innate and acquired
immunity will be required for successful engraftment. As
such, a multimodal approach will be required. Despite this
formidable challenge, significant headway has been made,
particularly in the pig, toward establishing a xenogeneic
source of donor organs.242- 244 Several groups have now trans-
genically altered pigs to express human complement regula-
tor proteins such as CDS9, decay-accelerating factor (DAF),
and membrane cofactor protein (MCP). Others have altered
the balance of carbohydrate processing to produce a( 1-3)
galactosylantransferase knockout animals, which would
eliminate the expression of GALa(1-3 )GAL in homozygotes,
removing the major target of complement activation.24s ,246
Using such an approach with transgenic swine that express
human DAF, prolonged cardiac allograft survival has been
demonsrrated'V?" (Table 80.6). Added to conventional immu-
nosuppression, these interventions, as well as several inhibi-
tors of complement, including anti-CS monoclonal antibodies,
cobra venom factor, and soluble complement receptor type I,
have been successful in abrogating HAR and extending sur-
vival in primates from minutes to weeks . While clinical appli-
cation remains several years away, it is clear that the
flexibility afforded by genetic engineering will one day allow
for custom organs available as necessary to combat end-organ
failure .
1731
No. of
Organ Animals MST(days) MaxST Comment
Kidney 14 >50 days (4/9 78 days Splenectomy and
animals) triple therapy
Heart 9 26 99 days Heterotopic heart;
triple therapy
Heart N/A 39 days Orthotopic; triple
the rapy
References
1. Carrel A. La technique operatoire des anastomoses vasculaires
et la transplantation des visceres. Lyon Med 1902;98:859.
2. Carrel A. Results of the transplantation of blood vessels, organs
and limbs . JAMA 1908;51:1662.
3. Converse JM, Casson PRo The historical background of trans-
plantation. In: Rapaport F, Dausset J, eds. Human Transplanta-
tion. New York: Grune and Stratton; 1968.
4. Terasaki PI, ed. History of Transplantation: Thirty-Five Recol-
lections. Los Angeles: UCLA Tissue Typing Laboratory;
1991.
5. Medawar PB. The behaviour and fate of skin autografts and skin
homograft s in rabbits . JAnat 1944;78:176-199.
6. Medawar PB. A second study of the behaviour and fate of skin
homografts in rabbits . JAnat 1945;79:157-176.
7. Medawar PE. Immunity to homologous graft skin , I: the suppres-
sion of cell division in grafts transplanted to immunized animals.
Br J Exp Pathol 1946;27:9.
8. Owen RD. Immunogenetic consequences of vascular anastomo-
ses between bovine twins. Science 1945;102:4D0-401.
9. Billingham RE, Brent L, Medawar PB. Actively acquired toler -
ance of foreign cells. Nature 1953;172:603-606.
10. Mitchison NA. Passive transfer of transplantation immunity.
Proc R Soc Lond B Biol Sci 1954;142:72.
11. Gorer PA. The antigenic basis of tumour transplantation.
J Pathol Bacteriol 1938;47:231-252.
12. Gorer PA, Lyman S, Snell GD. Studies on the genetic and anti-
genic basis of tumour transplantation: linkage betw een a histo-
compatibility gene and "fused" in mice . Proc Soc Lond B Bioi
Sci 1948;135:499.
13. Snell GD. Methods for the study of histocompatibility genes.
J Genet 1948-1949;49:87-108.
14. Amos DB, Gorer PA, Mikulska ZB. The antigenic structure
and genetic behavior of a transplanted leukosis. Br J Cancer
1955;9:209.
15. Dausset J, Nenna A, Presence d'une leuco-agglutinine dans le
serum d'un cas d'agranulocytose chronique [Presence of leuko-
agglutinin in the serum of a case of chronic agranulocytosis].
Compt Rendus Soc Bioi (Paris) 1952;146:1539.
16. Dausset J. Iso-leuco-anticorps . Acta Haematol 1958;20:156-
166.
17. Bach F, Hirschhorn K. Lymphocyte interaction: a potential his-
tocompatibility test in vitro . Science 1964;143:813-814.
18. Terasaki PI, McClelland ]D. Microdroplet assay of human serum
cytotoxins. Nature 1964;204:998-1000.
19. Bjorkman PJ, Saper MA, Samraoui B, et a1. Structure of the
human class I histocompatibility antigen, HLA-A2. Nature
1987;329:506-511.
1732 CHAPTER 80
20. Bjorkman PJ, Saper MA, Samraoui B, et al. The foreign antigen
binding site and T cell recognition regions of class I histocom-
patibility antigen. Nature 1987;329:512-518.
21. Brown JH, [ardetzky T, Saper MA, et al. A hypothetical model
of the foreign antigen binding site of class II histocompatibility
molecules. Nature 1988;332:845-850.
22. Brown JH, [ardetzky T, Gorga JC, et al. Three-dimensional struc-
ture of the human class II histocompatibility antigen HLA-DR1.
Nature 1993;364:33-39.
23. Hozumi N, Tonegawa S. Evidence for somatic rearrangement of
immunoglobulin genes coding for variable and constant regions.
Proc Nat! Acad Sci USA 1976;73:3628-3632.
24. Zinkernagel RM, Doherty PC. Restriction of in vitro T cell-
mediated cytotoxicity in lymphocytic choriomeningitis within
a syngeneic or semiallogeneic system. Nature 1974;248:701-
702.
25. Doherty PC, Zinkernagel RM. T cell-mediated immunopathol-
ogy in viral infections. Transplant Rev 1974;19:89-120.
26. Bretscher P, Cohn M. A theory of self-non-self discrimination.
Science 1970;169:1042-1049.
27. Lafferty KJ, Cunningham AJ. A new analysis of allogeneic inter-
actions. Aust J Exp Biol Med Sci 1975;53:27-42.
28. June CH, Bluestone JA, Nadler LM, Thompson CB. The B7 and
CD28 receptor families. Immunol Today 1994;15:321-331.
29. Ridge JP, Fuchs EJ, Matzinger P. Neonatal tolerance revisited:
turning on newborn T cells with dendritic cells. Science 1996;
271:1723-1726.
30. Murray JE, Merrill JP, Harrison JH. Renal homotransplantation
in identical twins. Surg Forum 1955;6:432.
31. Hitchings GH, Elion GB, Falco EA, et al. Antagonists of nucleic
acid derivatives, I: the Lactobacillus casei model. J Biol Chern
1950;183:1.
32. Hitchings GH, Elion GB. Chemical suppression of the immune
response. Pharmacol Rev 1963;15:365.
33. Calne RY, Murray JE. Inhibition of rejection of renal homografts
in dogs by Burroughs-Wellcome 57-322. Surg Forum 1961;12:
118-120.
34. Borel JF, Feurer C, Gubler HU. Biological effects of cyclosporine
A: a new antilymphatic agent. Actions Agents 1976;6:468-475.
35. Borel JF. Comparative study of in vitro and in vivo drug effects
on cell-mediated cytotoxicity. Immunology 1976;31:631-641.
36. Calne RY, White DJ, Rolles K, et al. Prolonged survival of pig
orthotopic heart grafts treated with cyclosporin A. Lancet 1978;
1:1183-1185.
37. Calne RY, White DJ, Thiru S, et al. Cyclosporin A in patients
receiving renal allografts from cadaver donors. Lancet 1978;2:
1323-1387.
38. Dempsey PW, Allison MED, Akkaraju S, et al. C3d of comple-
ment as a molecular adjuvant: bridging innate and acquired
immunity. Science 1996;271:348-350.
39. Fearon DT, Locksley RM. The instructive role of innate immu-
nity in the acquired immune response. Science 1996;272:50-
53.
40. Wright SD, Ramos RA, Tobias PS, et al. CD14, a receptor for
complexes of lipopolysaccharide (LPS) and LPS binding protein.
Science 1990;249:1431-1433.
41. Baldwon WM, Pruitt SK, Brauer RB, et al. Complement in organ
transplantation. Transplantation 1995;59:797-808.
42. Hart DNJ. Dendritic cells: unique leukocyte populations which
control the primary immune response. Blood 1997;90:3245-
3287.
43. AkiraS, TakedaK. Toll-like receptor signaling. Nat Rev Immunol
2004;4:499-511.
44. Koo, DDH, Roberts, lSD, et al. C4d deposition in early renal
allograft protocol biopsies. Transplantation 2004;78:398-403.
45. Ahmed R, Gray D. Immunological memory and protective
immunity: understanding their relation. Science 1996;272:54-
60.
46. Fearon DT. Seeking wisdom in innate immunity. Nature
1997;388:323-324.
47. Davis MM, Bjorkman PJ. T cell antigen receptor genes and T
cell recognition. Nature 1988;334:395-402.
48. Cooper MD. B lymphocytes: normal development and function.
N Engl J Med 1987;317:1452-1457.
49. Gill JI, Gulley ML. Immunoglobulin and T cell receptor gene
rearrangement. Hematol Oncol Clin North Am 1994;8:751-
770.
50. Kirk AD, Ibrahim S, Dawson DV, et al. Characterization of T
cells expressing the 1/'0 antigen receptor in human renal allografts.
Hum ImmunoI1993;36:11-19.
51. Kappler JW, Marrack P. T cell tolerance by clonal elimination
in the thymus. Cell 1987;49:273-280.
52. Bevan MJ. In thymic selection, peptide diversity gives and takes
away. Immunity 1997;7:175-178.
53. Fowlkes BJ, Ramsdell F. T cell tolerance. Curr Opin Immunol
1993;5:873-879.
54. Itoh N, Yonehara S, Ishii A, et al. The polypeptide encoded by
the eDNA for human cell surface antigen Fas can mediate apop-
tosis. Cell 1991;66:233-243.
55. Griffith TS, Brunner T, Fletcher SM, et al. Fas ligand-induced
apoptosis as a mechanism of immune privilege. Science 1995;
270:1189-1192.
56. Wilson lA, Garcia KC. T cell receptor structure and TCR com-
plexes. Curr Opin Struct Biol 1997;7:839-848.
57. Rothenberg EV. How T cells count. Science 1996;273:78-79.
58. Viola A, Lanzavecchia A. T cell activation determined by T cell
receptor number and tunable thresholds. Science 1996;273:104-
106.
59. Kumagai N, Benedict SH, Mills GB, et al. Requirements for the
simultaneous presence of phorbol esters and calcium ionophores
in the expression of human T lymphocyte proliferation-related
genes. J ImmunoI1987;139:1393-1399.
60. Saizawa K, Rojo J, Janeway CA Jr. Evidence for a physical asso-
ciation of CD4 and the CD3: alpha:beta T cell receptor. Nature
1987;328:260-263.
61. Leahy DJ, Axel R, Hendrickson WA. Crystal structure of a
soluble form of the human T cell coreceptor CD8 at 2.6 A reso-
lution. Cell 1992;68:1145-1162.
62. Germain RN. MHC-dependent antigen processing and peptide
presentation: providing ligands for T lymphocyte activation.
Cell 1994;76:287-299.
63. Monaco JJ. Structure and function of genes in the MHC class II
region. Curr Opin ImmunoI1993;5:17-20.
64. Ridge JP, Di Rosa F, Matzinger P. A conditioned dendritic cell
can be a temporal bridge between a CD4+ T-helper and aT-killer
cell. Nature 1998;393:474-478.
65. Lanzavecchia A. License to kill. Nature 1998;393:413-414.
66. Plas DR. Johnson R, Pingel JT, et al. Direct regulation of ZAP-70
by SHP-l in T cell antigen receptor signaling. Science 1996;
272:1173-1176.
67. Marengere LEM, Waterhouse P, Duncan GS, et al. Regulation of
T cell receptor signaling by tyrosine phosphatase SYP associated
with CTLA-4. Science 1996;272:1170-1173.
68. Crabtree GR. Contingent genetic regulatory events in T lympho-
cyte activation. Science 1989;243:355-361.
69. Ullman KS, Northrop JP, Verweij CJ, Crabtree GR. Transmis-
sion of signals from the T lymphocyte antigen receptor to the
genes responsible for cell proliferation and immune function:
the missing link. Annu Rev ImmunoI1990;8:421-452.
70. Siegel JN, June CR. Signal transduction in T cell activation and
tolerance. In: Gupta S, Griscelli C, eds. New Concepts in Immu-
nodeficiency Diseases. New York: Wiley; 1993;85-129.
71. Karthryn Wood and Shimon Sakaguchi. Regulatory T cells in
transplant tolerance. Nat Rev Immunol 2003;3:199-210.
72. Shimizu J, Yamazaki S, Takahashi T, Ishida Y, Sakaguchi S.
Stimulation of CD25(+)CD4(+) regulatory T cells through GITR
 IMMUNOLOGY OF TRANSPLANTATION
breaks immunological self-tolerance. Nat Immunol 2002;3:135-
142.
73. Baecher-Allan at al. CD4+CD25+ highly regulatory cells in
human peripheral blood. J ImmunoI2001;167:1245-1253.
74. Berke G. The CTL's kiss of death. Cell 1995;81:9-12.
75. Zouali M. B cell superantigens: implications for selection of
the human antibody repertoire. Immunol Today 1995;16:399-
405.
76. lung S, Rajewsky K, Radbruch A. Shutdown of class switch
recombination by deletion of a switch region control element.
Science 1993;259:984-987.
77. Griffiths GM, Berek C, Kaartinen M, Milstein C. Somatic muta-
tion and the maturation of immune response to 2-phenyloxazo-
lone. Nature 1984;312:271-275.
78. Cambier JC, Pleiman CM, Clark MR. Signal transduction by the
B cell antigen receptor and its coreceptors. Annu Rev Immunol
1994;12:457-486.
79. Tedder TF, Zhou LJ, Engel P. The CD19/CD21 signal transduc-
tion complex of B lymphocytes. Immunol Today 1994;15:437-
442.
80. Lederman S, Yellin MJ, Inghirami G, et al. Molecular interac-
tions mediating T-B lymphocyte collaboration in human lym-
phoid follicles: roles of the T cell-B cell activating molecule (5c8
antigen) and CD40 in contact dependent help. J Immunol 1992;
149:3817-3826.
81. Takeuchi Y, Porter CD, Strahan KM, et al. Sensitization of cells
and retroviruses to human serum by (al-3) galactosyltransferase.
Nature 1996;379:85-88.
82. Arai KI, Lee F, Miyajima A, et al. Cytokines: coordinators of
immune and inflammatory responses. Annu Rev Biochem 1990;
59:783-836.
83. Waldmann T, Tagaya Y, Bamford R. Interleukin-2, interleukin-
15, and their receptors. Int Rev Immunol 1998;16:205-
226.
84. Feldman M, Londei M, Haworth C. T cells and lymphokines. Br
Med Bull 1989;45:361-370.
85. Leonard WJ, Depper JM, Robb RJ, et al. Characterization of the
human receptor for T cell growth factor. Proc Nat! Acad Sci
USA 1983;8:6957-6961.
86. Mosmann TR, Cherwinski H, Bond MW, et al. Two types of
murine helper T cell clone, I: definition according to profiles of
lymphokine activities and secreted proteins. J Immunol
1986;136:2348-2357.
87. Mosmann TR. Cytokines: is there biological meaning? Curr
Opin ImmunoI1991;3:311-314.
88. Sugamura K, Asao H, Kondo M, et al. The common gamma-
chainformultiplecytokinereceptors.Advlmmunol1995;59:225-
277.
89. Aaronson DS, Horvath CM. A road map for those who don't
know JAK-STAT. Science 2002;296:1653-1655.
90. Kelso A. Th1 and Th2 subsets: paradigms lost? Immunol Today
1995;16:374-380.
91. Krams SM, Falco DA, Villaneuva JC, et al. Cytokine and T cell
receptor gene expression at the site of allograft rejection. Trans-
plantation 1992;53:151-156.
92. Kirk AD, Bollinger RR, Finn OJ. Rapid, comprehensive analysis
of human cytokine mRNA and its application to the study of
acute renal allograft rejection. Hum Immunol 1995;43:113-
128.
93. Allison JP, Krummel MF. The yin and yang of T cell costimula-
tion. Science 1995;270:932-933.
94. Chambers CA, Allison JP. Co-stimulation in T cell responses.
Curr Opin Immunol 1997;9:396-404.
95. Larsen CP, Pearson TC. The CD40 pathway in allograft rejec-
tion, acceptance, and tolerance. Curr Opin ImmunoI1997;9:641-
647.
96. Harlan DM, Kirk AD. Anti-CD154 therapy to prevent graft rejec-
tion. Graft 1998;1:63-70.
1733
97. Bennett SRM, Carbone FR, Karamalis F, et al. Help for cytotoxic
T cell responses is mediated by CD40 signalling. Nature 1998;
393:478-480.
98. Schoenberger SP, Toes REM, van der Voort EIH, et al. T cell help
for cytotoxic T lymphocytes is mediated by CD40-CD40L inter-
actions. Nature 1998;393:480-483.
99. Henn V, Slupsky JR, et al. CD40 ligand on activated platelets
triggers an inflammatory reaction of endothelial cells. Nature
1998;391:591-594.
100. Czapiga M, Kirk AD, Lekstrom-Himes J. Platelets deliver
costimulatory signals to antigen-presenting cells: a potential
bridge between injury and immune activation. Exp Hematol
2004;32:135-139.
101. Blair PJ, Riley JL, Levine BL, et al. CTLA-4 ligation delivers a
unique signal to resting human CD4 T cells that inhibits inter-
leukin-2 secretion but allows Bcl-XL induction. J Immunol1998;
160:12-15.
102. Waterhouse P, Penninger JM, Timms E, et al. Lymphoprolifera-
tive disorders with early lethality in mice deficient in CTLA-4.
Science 1995;270:985-988.
103. Walunas TL, Lenschow DJ, Bakker CY, et al. CTLA-4 can func-
tion as a negative regulator of T cell activation. Immunity
1994;1:405-413.
104. Linsley PS, Greene JL, Brady W, et al. Human B7-1 (CD80) and
B7-2 (CD86) bind with similar avidities but distinct kinetics to
CD28 and CTLA-4 receptors. Immunity 1994;1:793-801.
105. Pennisi E. Teetering on the brink of danger. Science 1996;271:
1665-1667.
106. Bingaman AW, Ha J, et al. Vigorous allograft rejection in the
absence of danger. J ImmunoI2000;164:3065-3071.
107. Okazaki T, Iwai Y, Honjo T. New regulatory co-receptors: induc-
ible co-stimulator and PD-1. Curr Opin Immunol 2002; 14:779-
82.
108. Campbell RD, Trowsdale J. Map of the major histocompatibility
complex. Immunol Today 1993;14:349-352.
109. Lotteau V, Teyton L, Borroghs D, Charron D. A novel HLA class
II molecule (DRalpha-DQbeta) created by mismatched isotype
pairing. Nature 1987;329:339-341.
110. Parham P, Ohta T. Population biology of antigen presentation
by MHC class I molecules. Science 1996;272:67-74.
111. Nowak MA, Bangham CRM. Population dynamics of immune
responses to persistent viruses. Science 1996;272:74-79.
112. Salter RD, Benjamin RJ, Wesley PK, et al. A binding site for the
T cell co-receptor CD8 on the a3 domain of HLA-A2. Nature
1990;345:41-46.
113. Doyle C, Strominger JL. Interaction between CD4 and class II
MHC molecules mediates cell adhesion. Nature 1987;330:256-
259.
114. Williams DB, Barber BH, Flavell RA, et al. Role of b2-microglo-
bin in the intracellular transport and surface expression of
murine class I histocompatibility molecules. J Immunol 1989;
142:2796-2806.
115. Teyton L, O'Sullivan D, Dickson PW, et al. Invariant chain
distinguishes between the exogenous and endogenous antigen
presenting pathways. Nature 1988;348:39-44.
116. Adams AB, Pearson TC, Larsen CPo Heterologous immunity:
an overlooked barrier to tolerance. Immunol Rev 2003;196:
147.
117. Halloran PF, Madrenas J. Regulation of MHC transcription.
Transplantation 1990;50:725-738.
118. Gerritsen ME, Bloor CM. Endothelial cell gene expression in
response to injury. FASEB 1993;7:523-533.
119. Marsh SGE, Bodmer JG. HLA class II nucleotide sequences,
1992. Immunogenetics 1993;37:79-94.
120. Zemmour J, Parham P. HLA class I nucleotide sequences, 1992.
Immunogenetics 1993;37:239-250.
121. Bidwell J. DNA-RFLP analysis and genotyping of HLA-DR and
DQ antigens. Immunol Today 1998;9:18-23.
1734 CHAPTER 80
122. Nevinny-Stickel C, Bettinotti MP, Andreas A, et al. Nonradioac-
tive HLA class II typing using polymerase chain reaction and
digoxigenin-11-29-39-didesoxy-uridinetriphosphate labeled oli-
gonucleotide probes. Hum Immunol1991;31:7-13.
123. Nevinny-Stickel C, Hinzpter M, Andreas A, et al. Nonradioac-
tive oligotyping for HLA-DR1-DRw10 using polymerase chain
reaction, digoxigenin-labeled oligonucleotides and chemilumi-
nescence detection. Eur J Immunogenet 1991;18:323-332.
124. Olerup 0, Zetterquist H. HLA DR typing by PCR amplification
with sequence-specific primers (PCR-SSP) in 2 hours: an alterna-
tive to serological typing in clinical practice including donor-
recipient matching in cadaveric transplantations. Tissue
Antigens 1992;39:225-235.
125. Markus BH, Duquesnoy RJ, Gordon RD, et al. Histocompatibil-
ity and liver transplantation. Does HLA exert a dualistic effect?
Transplantation (Baltimore) 1988;46:372-377.
126. Steinhoff G. HLA/ABO matching. In Neuberger J, Adams D, eds.
Immunology of Liver Transplantation. London: Edward Arnold;
1993:261-266.
127. Terasaki PI, Cecka JM, Gjertson DW, Takemoto S. High survival
rates of kidney transplants from spousal and living unrelated
donors. N Engl J Med 1995;333:333-336.
128. Terasaki PI, Cecka JM, Gjertson DW, Cho YW. Spousal and
Other Living Renal Donor Transplants. Los Angeles: UCLA
Tissue Typing Laboratory, Clinical Transplants; 1997.
129. Gloor JM, DeGoey SR, et al. Overcoming a positive crossmatch
in living-donor kidney transplantation. AJT 3:1017, 2003:1017-
1023.
130.Takahashi K, Saito K, et al. Excellent long-term outcome of ABO-
incompatible living donor kidney transplantation in Japan. Am
J Transplant 2004;4:1089-1096.
131. Burdick JF. An anatomy of rejection. Transplant Rev 1991;5:81-
90.
132. Kirk AD, Ibrahim MA, Bollinger RR, et al. Renal allograft infil-
trating lymphocytes: a prospective analysis of in vitro growth
characteristics and clinical relevance. Transplantation 1992;
53:329-338.
133. Fuggle SV, Koo DDH. Cell adhesion molecules in clinical renal
transplantation. Transplantation 1998;65:763-769.
134. Henn V, Slupsky JR, Grafe M, et al. CD40 ligand on activated
platelets triggers an inflammatory reaction of endothelial cells.
Nature 1998;391:591-594.
135. Takada M, Chandraker A, Nadeau KC, et al. The role of the B7
costimulatory pathway in experimental cold ischemia/reperfu-
sion injury. J Clin Invest 1997;100:1199-1203.
136. Dallman MJ, Clark GJ. Cytokines and their receptors in trans-
plantation. Curr Opin ImmunoI1991;3:729-734.
137. Strehlau J, Pavlakis M, Lipman M, et al. Quantitative detection
of immune activation transcripts as a diagnostic tool in kidney
transplantation. Proc Nat! Acad Sci USA 1997;94:695-
700.
138. Levitz SM, Mathews HL, Murphy JW. Direct antimicrobial
activity of T cells. Immunol Today 1995;16:387-391.
139. Baldwin WM ill, Pruitt SK, Sanfilippo F, et al. Alloanti-
bodies: basic and clinical concepts. Transplant Rev 1991;5:100-
119.
140. Saadi S, Platt JL. Transient perturbation of endothelial integrity
induced by natural antibodies and complement. J Exp Med
1995;181:21-31.
141. Bach FH, Winkler H, Ferran C, et al. Delayed xenograft rejection.
Immunol Today 1996;17:379-384.
142. Gebel HM, Lebeck LK. Crossmatch procedures used in organ
transplantation. Clin Lab Med 1991;11:603-620.
143. Talbot D. The flow cytometric crossmatch in perspective.
Transplant ImmunoI1993;1:155-162.
144. Le Bas-Bernardet S, Hourmant M. Identification of the antibod-
ies involved in B-cell crossmatch positivity in renal transplanta-
tion. Transplantation 2003;75:477-482.
145. Noreen HJ, McKinley DM, et al. Positive remote crossmatch:
impact on short-term and long-term outcome in cadaver renal
transplantation. Transplantation 2003;75:501-505.
146. Colvin RB. The pathogenesis of vascular rejection. Transplant
Proc 1991;23:2052-2055.
147. Paul LC. Chronic renal transplant loss. Kidney Int 1995;47:1491-
1499.
148. Almond PS, Matas A, Gillingham KJ, et al. Risk factors for
chronic rejection in renal allograft recipients. Transplantation
1993;55:752-757.
149. Gourishankar S, Halloran PF. Late deterioration of organ trans-
plants: a problem in injury and homeostasis. Curr Opin Immunol
2002;14:576-583.
150. Ahsan N, Johnson C, et al. Randomized trial of tacrolimus plus
mycophenolate mofetil or azathioprine versus cyclosporine oral
solution (modified) plus mycophenolate mofetil after cadaveric
kidney transplantation: results at 2 years. Transplantation. 2001;
72:245-250.
151. Jensick SC. Tacrolimus in kidney transplantation: 3-year sur-
vival results of the US multicenter, randomized, comparative
trial. FK506 Kidney Transplant Study Group. Transplant Proc
1998;30:1216-1218.
152. Mathew TH. A blinded, long-term, randomized multicenter
study of mycophenolate mofetil in cadaveric renal transplanta-
tion: results at 3 years. Tricontinental Mycophenolate Mofetil
Renal Transplantation Study Group. Transplantation 1998;65:
1450-1454.
153. Gaber A. Results of the double-blind, randomized, multicenter,
phase III clinical trial of thymoglobulin versus ATGAM in the
treatment of acute graft rejection episodes after renal transplan-
tation. Clin Transplant 1998;66:29-37.
154. Kahan BD. Efficacy of sirolimus compared with azathioprine for
reduction of acute renal allograft rejection: a randomized multi-
centre study. Lancet 2000;356:194-202.
155. Vincenti F. Interleukin-2-receptor blockade with daclizimab to
prevent acute rejection in renal transplantation. N Engl J Med
1998;338:161-165.
156. Hardinger KL, Schnitzler MA, et al. Five-year follow up of thy-
moglobulin versus ATGAM induction in adult renal transplan-
tation. Transplantation 2004;78:136-141.
157. Auphan N, DiDonato JA, Rosette C, et al. Immunosuppression
by glucocorticoids: inhibition of NF-kB activity through induc-
tion of IkB synthesis. Science 1995;270:286-290.
158. Scheinman RI, Cogswell PC, Lofquist AK, et al. Role of tran-
scriptional activation of IkBa in mediation of immunosuppres-
sion by glucocorticoids. Science 1995;283:283-286.
159. Plaz KP, Sollinger HW, Hullet DA, et al. RS-61443, a new,
potent immunosuppressive agent. Transplantation 1991;51:27-
31.
160. Sollinger HW, Deierhoi MH, Belzer FO, et al. RS-61443: a phase
I clinical trial and pilot rescue study. Transplantation 1992;53:
428-432.
161. Sollinger HW. US Renal Transplant Mycophenolate Mofetil
Study Group. Mycophenolate mofetil for the prevention of acute
rejection in primary cadaveric renal allograft recipients. Trans-
plantation 1995;60:225-232.
162. Kahan BD. Role of cyclosporine: present and future. Transplant
Proc 1994;26:3082-3087.
163. Khanna A, Sharma VK, Suthanthiran M. Immunoregulatory and
fibrogenic activities of cyclosporine: a unifying hypothesis based
on transforming growth factor-b expression. Transplant Proc
1996;28:2015-2019.
164. Kirk AD, Jacobson LM, Heisey DM, et al. Post-transplant dia-
stolic hypertension: associations with intragraft TGF-~, endo-
thelin and renin transcription. Transplantation 1997;64:
1716-1720.
165. June CH, Ledbetter JA, Gillespie MM, et al. T cell proliferation
involving the CD28 pathway is associated with cyclosporine-
 IMMUNOLOGY OF TRANSPLANTATION
resistant interleukin-2 gene expression. Mol Cell Biol 1987;
7:4472-4481.
166. Hojo M, Morimoto T, Maluccio M, et al. Cyclosporine induces
cancer progression by a cell-autonomous mechanism. Nature
1999;397:530-534.
167. Kino T, Hatanaka H, Miyata S, et al. FK-506, a novel immuno-
suppressant isolated from streptomyces, II: immunosuppressive
effect of FK-506 in vitro. J Antibiot 1987;40:1256-1265.
168. Fruman DA, Klee CB, Bierer BE, Burakoff SJ. Calcineurin phos-
phatase activity in T lymphocytes is inhibited by FK506 and
cyclosporin A. Proc Natl Acad Sci USA 1992;89:3686-3690.
169. Starzl TE, Fung H, Venkataramanan, et al. FK-506 for liver,
kidney, and pancreas transplantation. Lancet 1989;334:1000-
1004.
170. Gaber AO, First MR, Tesi RJ, et al. Results of the double-blind,
randomized, multicenter, phase III clinical trial of thymoglobu-
lin versus ATGAM in the treatment of acute graft rejection
episodes after renal transplantation. Transplantation 1998;66:29-
37.
171. Merion RM, Howell T, Bromberg JS. Partial T cell activation and
energy induction by polyclonal antithymocyte globulin. Trans-
plantation 1998;65:1481-1489.
172. Swanson SJ, Hale DA, Mannon RB, et al. Kidney transplantation
with rabbit antithymocyte globulin induction and sirolimus
monotherapy. Lancet 2002;360: 1662.
173. Starzl TE, Murase N, et al. Tolerogenic immunosuppression for
organ transplantation. Lancet 2003;361:1502-1510.
174. Brennan DC, Flavin K, Lowell JA, et al. A randomized, double-
blinded comparison of Thymoglobulin versus Atgam for induc-
tion immunosuppressive therapy in adult renal transplant
recipients. Transplantation 1999;67:1011-1018.
175. Wilde MI, Goa KL. Muromonab CD3: a reappraisal of its phar-
macology and use as prophylaxis of solid organ transplant rejec-
tion. Drugs 1996;51:865-894.
176. Delmonico FL, Cosimi AB. Monoclonal antibody treatment of
human allograft recipients. Surg Obst GynecoI1988;166:89-98.
177. Kupfer A, Mosmann TR, Kupfer H, et al. Polarized expression
of cytokines in cell conjugates of helper T cells and splenic B
cells. Proc Natl Acad Sci USA 1991;88:775-779.
178. Shield CF III, Norman DJ. Immunological monitoring during
and after OKT3 therapy. Am J Kidney Dis 1988;11:120-124.
179. Ortho Multicenter Transplant Study Group. A randomized clin-
ical trial of OKT3 monoclonal antibody for acute rejection of
cadaveric renal transplants. N Engl J Med 1985;313:337-342.
180. Light JA, Khawand N, Aquino A, et al. Quadruple immunosup-
pression: comparison of OKT3 and Minnesota antilymphocyte
globulin. Am J Kidney Dis 1989;14:10-13.
181. Soulillou JP, Le Mauff B, Olive D, et al. Prevention of rejection
of kidney transplants by a monoclonal antibody directed against
interleukin 2. Lancet 1987;1:1339-1342.
182. Nashan B, Moore B, Amlot P, et al. Randomised trial of basilix-
imab versus placebo for control of acute cellular rejection in
renal allograft recipients. Lancet 1997;350:1193-1198.
183. Flechner M, Goldfarb D, Modlin C, et al. Kidney transplantation
without calcineurin inhibitor drugs: A prospective, randomized
trial of sirolimus versus cyclosporine. Transplantation 2002;74:
1070-1076.
184. Segal SN, Baker H, Vezina C, et al. Rapamycin (AY-22,989), a
new antifungal antibiotic, II: fermentation, isolation and char-
acterization. J Antibiot (Tokyo) 1975;28:727-732.
185. Martel RR, Klicius J, Galet S. Inhibition of the immune response
by rapamycin, a new antifungal antibiotic. Can J Physiol Phar-
macol 1977;55:48-51.
186. Baker H, Sidorowicz A, Sehgal SN, Vezina C. Rapamycin (AY-
22,989), a new antifungal antibiotic, III: In vitro and in vivo
evaluation. J Antibiot 1978;31:539-545.
187. Molnar-Kimber KL. Mechanism of action of rapamycin (siroli-
mus, rapamune). Transplant Proc 1996;26:964-969.
1735
188. Dumont FJ, Melino MR, Staruch MJ, et al. The immunosuppres-
sive macrolides FK-506 and rapamycin act as reciprocal antago-
nists in murine T cells. J ImmunoI1990;144:1418-1424.
189. Dumont FJ, Staruch MJ, Koprak SL, et al. Distinct mechanisms
of suppression of murine T cell activation by the related macro-
lides FK-506 and rapamycin. J ImmunoI1990;144:251-258.
190. Kuo CJ, Chung J, Fiorentino DF, et al. Rapamycin selectively
inhibits interleukin 2 activation of p70 S6 kinase. Nature
1992;358:70-73.
191. Muthukkumar S, Ramesh TM, Bondada S. Rapamycin, a potent
immunosuppressive drug, causes programmed cell death in B
lymphoma cells. Transplantation 1995;60:264-270.
192. Bjorn Nashan. Review of the proliferation inhibitor everolimus.
Exper Opin Invest Drug 2002;11:1845-1857.
193. McAlister VC, Gao Z, et al. Sirolimus-tacrolimus combination
immunosuppression. Lancet 2000;355:376-377.
194. Morris RE. Rapamycins: antifungal, antitumor, antiproliferative
and immunosuppressive macrolides. Transplant Rev 1992;6:39-
87.
195. Calne RY, Friend P, Moffatt S, et al. Prope tolerance, periopera-
tive campath IH, and low-dose cyclosporin monotherapy in
renal allograft recipients. Lancet 1998;351:1701.
196. Kirk AD, Hale DA, Mannon RB, et al. Results from a human
renal allograft tolerance trial evaluating the humanized CD52-
specific monoclonal antibody alemtuzumab (CAMPATH-IH).
Transplantation 2003;76:120.
197. Knechtle SJ, Pirsch JD, Fechner H, et al. Campath-l H induction
plus rapamycin monotherapy for renal transplantation: results
of a pilot study. Am J Transplant 2003;3:722.
198. Kaufman DB. 15-Deoxyspergualin in experimental transplant
models: a review. Transplant Proc 1996;28:868-870.
199. Ramos EL, Nadler SG, Grasela DM, Kelly SL. Deoxyspergualin:
mechanism of action and pharmacokinetics. Transplant Proc
1996;28:873-875.
200. Kirk A. Results from a human tolerance trial using alemtu-
zumab (campath-lH) with deoxyspergualin (DSG). Am J Trans-
plant 2003;3(S5):S310.
201. Brinkmann V, Cyster JG, Hla T. FTY720: sphingosine I-phos-
phate receptor-l in the control of lymphocyte egress and endo-
thelial barrier function. Am J Transplant 2004;4:1019.
202. Becker YT, Becker BN, et al. Rituximab as treatment for refrac-
tory kidney transplant rejection. Am J Transplant 2004;4:996.
203. Samuelsson A, Towers TL, Ravetch JV. Anti-inflammatory
activity of MG mediated through the inhibitory Fe receptor.
Science 2001;291:484.
204. Bleeker WK, Teeling JL, Hack CEo Accelerated autoantibody
clearance by intravenous immunoglobulin therapy: studies in
experimental models to determine the magnitude and time
course of the effect. Blood 2001;98:3136-3142.
205. Takemoto SK, Zeevi A, et al. National Conference to Assess
Antibody-Mediated Rejection in Solid Organ Transplantation.
Am J Transplant 2004;4:1033-1041.
206. Kirk AD. Transplant tolerance: a look at the non-human primate
literature in the view of modem tolerance theories. Crit Rev
Immunol 1999;19:349-388.
207. Kirk AD, Knechtle SJ, Sollinger H, Vincenti FG, Stecher S,
Nadeau K. Preliminary results of the use of humanized anti-
CD 154 in human renal allotransplantation. Am J Transplant
2001;1:S191.
208. Calne R, Moffatt SD, Friend PJ, et al. Campath IH allows low-
dose cyclosporine monotherapy in 31 cadaveric renal allograft
recipients. Transplantation 1999;68:1613-1616.
209. Kirk A. Results from a human tolerance trial using alemtu-
zumab (campath-1H) with deoxyspergualin (DSG). Am J Trans-
plant 2003;3(S5):S310.
210. Spitzer TR, Delmonico F, Tolkoff-Rubin N, et al. Combined
histocompatibility leukocyte antigen-matched donor bone
marrow and renal transplantation for multiple myeloma with
1736 CHAPTER 80
end stage renal disease: the induction of allograft tolerance
through mixed lymphohematopoietic chimerism. Transplanta-
tion 1999;68:480.
211. Strober S, Benike C, Krishnaswamy S, Engleman EG, Grumet
FC. Clinical transplantation tolerance 12 years after prospective
withdrawal of immunosuppressive drugs: studies of chimerism
and anti-donor reactivity. Transplantation 2000;69:1549.
212. Millan MT, Shizuru JA, Hoffmann P, et al. Mixed chimerism
and immunosuppressive drug withdrawal after HLA-mismatched
kidney and hematopoietic progenitor transplantation. Trans-
plantation 2002 j 73:1386.
213. Kirk AD, Harlan DM, Armstrong NN, et al. CTLA4-lg and anti-
CD40 ligand prevent renal allograft rejection in primates. Proc
Natl Acad Sci USA 1997j94:8789.
214. Kirk AD, Burkly LC, Batty DS, et al. Treatment with humanized
monoclonal antibody against CD 154 prevents acute renal
allograft rejection in nonhuman primates. Nat Med 1999 j
5:686.
215. Elster EA, Xu H, Tadaki DK, et al. Treatment with the human-
ized CDl54-specific monoclonal antibody, hu5C8, prevents
acute rejection of primary skin allografts in nonhuman primates.
Transplantation 2001;72:1473.
216. Pearson TC, Trambley J, Odom K, et al. Anti-CD40 therapy
extends renal allograft survival in rhesus macaques. Transplan-
tation 2002;74:933.
217. Montgomery SP, Xu H, Tadaki DK, et al. Combination induc-
tion therapy with monoclonal antibodies specific for CD80,
CD86, and CD154 in nonhuman primate renal transplantation.
Transplantation 2002 j 74:1365.
218. Kirk AD, Tadaki DK, Celniker A, et al. Induction therapy with
monoclonal antibodies specific for CD80 and CD86 delays the
onset of acute renal allograft rejection in non-human primates.
Transplantation 2001 j 72:377-384.
219. Knechtle SJ, Fechner JH Jr, Dong Y, et al. Primate renal trans-
plants using immunotoxin. Surgery 1998j 124:438.
220. Hutchings A, Wu J, Asiedu C, et al. The immune decision
toward allograft tolerance in non-human primates requires early
inhibition of innate immunity and induction of immune regula-
tion. Transpl ImmunoI2003 jfl:335.
221. Kawai T, Poncelet A, Sachs DH, et al. Long-term outcome and
alloantibody production in a non-myeloablative regimen for
induction of renal allograft tolerance. Transplantation 1999;
68:1767.
222. Kawai T, Abrahamian G, Sogawa H, et al. Costimulatory
blockade for induction of mixed chimerism and renal allograft
tolerance in nonhuman primates. Transplant Proc 2001;33:
221.
223. Pearl JP, Parris J, Hale DA, et al. Immunocompetent T-cells with
a memory-like phenotype are the dominant cell type following
antibody-mediated T-cell depletion. Am JTransplant 2005;5:465-
474.
224. Knechtle SJ, Vargo D, Fechner J, et al. FNI8-CRM9 immuno-
toxin promotes tolerance in primate renal allografts. Transplan-
tation 1997;63:1-6.
225. Sykes M, Sachs DH. Mixed allogeneic chimerism as an approach
to transplant tolerance. Immunol Today 1998;9:23-27.
226. Sharabi Y, Sachs DH. Mixed chimerism and permanent specific
transplantation tolerance induced by a nonlethal preparative
regimen. J Exp Med 1989jI69:493-502.
227. Kaufman CL, Ildstad ST. Induction of donor-specific tolerance
by transplantation of bone marrow. Ther ImmunoI1994;1:101-
111.
228. Odorico JS, O'Connor T, Campos L, et al. Examination of the
mechanisms responsible for tolerance induction after intrathy-
mic inoculation of allogeneic bone marrow. Ann Surg 1993 j
218:525-531.
229. Qin S, Cobbold SP, Pope H, et al. Infectious transplant tolerance.
Science 1993j259:974-977.
230. Starzl TE, Demetris AJ, Murase N, et al. Cell migration, chime-
rism and graft acceptance. Lancet 1992;339:1579-1582.
231. Larsen CP, Elwood ET, Alexander DZ, et al. Long-term accep-
tance of skin and cardiac allografts after blocking CD40 and
CD28 pathways. Nature 1996;381:434-438.
232. Kirk AD, Harlan DM, Armstrong NN, et al. CTLA4-lg and anti-
CD40 ligand prevent renal allograft rejection in primates. Proc
Natl Acad Sci USA 1997 j94:8789-8794.
233. Auchincloss H [r, Sachs DH. Xenogeneic transplantation. Annu
Rev ImmunoI1998;16:433-470.
234. Sandrin MS, Vaughan HA, et al. Anti-pig IgM antibodies in
human serum react predominantly with gal(al-3)gal epitopes.
Proc Natl Acad Sci USA 1993j90: 11391-11395.
235. Cooper DKC, Good AH, et al. Identification of alpha-galactosyl
and other carbohydrate epitopes that are bound by human anti-
pig antibodies: relevance to discordant xenografting in man.
TranspllmmunoI1993 jl:198-205.
236. Prilliman K, Lawlor D, Ellexson M. Characterization of baboon
class I major histocompatibility molecules. Transplantation
1996;61:989-996.
237. Kirk AD, Li RA, Kinch MS, et al. The human antiporcine cel-
lular repertoire. In vitro studies of acquired and innate cellular
responsiveness. Transplantation 1993;55:924-931.
238. Tadaki D, Saini A, Craighead N, et al. Costimulatory pathways
are active in xenogeneic immune responses [abstract]. Trans-
plantation 1998;65:87.
239. Reemtsma K, McCracken BH, et al. Renal heterotransplantation
in man. Ann Surg 1964;160:384.
240. Starzl TE, Marchioro TL, et al. Renal heterotransplantation from
baboon to man: experience with six cases. Transplantation
1964;2:752-759.
241. Bailey LL, Nehlsen-Cannarella SL, et al. Baboon-to-human
cardiac xenotransplantation in a neonate. JAMA 1985;254:3321-
3329.
242. Platt JL, Fischel RJ, et al. Immunopathology of hyperacute xeno-
graft rejection in a swine-to-primate model. Transplantation
1991;52:214-220.
243. Goodman DJ, von Albertini M, et al. Direct activation of porcine
endothelial cells by human natural killer cells. Transplantation
1996;61:763-771.
244. McCurry KR, Kooyman DL, et al. Human complement regula-
tory proteins protect swine-to-primate cardiac xenografts from
humoral injury. Nat Med 1995;1:423-427.
245. Sandrin MS, Fodor WL, et al. Enzymatic remodeling of the car-
bohydrate surface of a xenogeneic cell substantially reduces
human antibody binding and complement-mediated cytolysis.
Nat Med 1996;1:1261-1267.
246. Lai L, Kolber-Simonds D, et al. Production of a-1,3-galactosyl-
transferase knockout pigs by nuclear transfer cloning. Science
2002:295: 1089-1092.
247. Cozzi E, Bhatti FNK, Schmoeckel M, et al. Long-term survival
of nonhuman primates receiving life-supporting transgenic por-
cine kidney xenografts. Transplantation 2000;70:12-21.
248. Bhatti FNK, Schmoeckel M, Zaidi A, et al. Three-month sur-
vival of HDAFF transgenic pig hearts transplanted into primates.
Transplant Proc 1999;31:958.
249. Vial CM, Ostlie DJ, Bhatti FNK, et al. Life supporting function
for over one month of a transgenic porcine heart in a baboon. J
Heart Lung Transplant 2000;19:224-229.
250. Burke F, Naylor MS, et al. The cytokine wall chart. Immunol
Today 1993;14:165.
251. Kirk AD, Sollinger HW. Transplant immunology and immuno-
suppression. In: Schwartz S, ed. Principles of Surgery, 7th Ed.
New York; McGraw-Hill, 1998.