Transplantation immunology part 1

Transplantation
Immunology
Part I
Topic Review
February 5th, 2021
Rapisa Nantanee, M.D.
Pediatric Allergy and Immunology Unit
King Chulalongkorn Memorial Hospital

Outline
• Definition
• Introduction
• General Principles
• Adaptive Immune Responses to Allografts
• Patterns and Mechanisms of
Allograft Rejection
• Prevention and Treatment of
Allograft Rejection

Definition
• Transplantation is the process of taking cells, tissues, or
organs, called a graft, from one individual and placing them into
a (usually) different individual.
• The individual who provides the graft is called the donor, and the
individual who receives the graft is called either the recipient or the
host.
• If the graft is placed into its normal anatomic location, the procedure is
called orthotopic transplantation; if the graft is placed in a different
site, the procedure is called heterotopic transplantation.
A.K. Abbas, et al. Cellular and Molecular Immunology. 9th ed. Chapter 17.

Definition
Transplantation
Donor Graft Recipient
or Host

• Transplantation of
hematopoietic stem cells
(HSCs), kidneys, livers,
and hearts is now
common practice in
clinical medicine, and
transplantation of other
organs such as lung and
pancreas is becoming
more frequent.

Transplantation in Thailand
729
31
91
Kidney Heart Liver
Number
of
Transplants
Number of Transplants in 2562
สมาคมปลูกถ่ายอวัยวะแห่งประเทศไทย. รายงานข้อมูลการปลูกถ่ายอวัยวะ ประจาปี พ.ศ. 2562.

Introduction
• The immune response against grafted tissues is the major
barrier to survival of transplanted tissues or organs.
• Conversely, controlling this immune response is key to
successful transplantation.
• Led to the development of transplantation immunology

General Principles
• Transplantation of cells or tissues from one individual to a
genetically nonidentical individual invariably leads to rejection
of the transplant due to an adaptive immune response.
• This problem was first appreciated when attempts to replace damaged skin
on burn patients with skin from unrelated donors , within 1 to 2 weeks, the
transplanted skin would undergo necrosis and fall off.
• Peter Medawar and other investigators studied skin transplantation in animal
models, established that the failure of skin grafting was caused by an
inflammatory reaction, which they called rejection.

• Graft rejection is the
result of an adaptive
immune response.
• The process had
characteristics of
memory and
specificity and was
mediated by
lymphocytes.

General Principles
• Autologous graft: a graft transplanted from one individual to the same individual.
• Syngeneic graft: a graft transplanted between two genetically identical individuals.
• Allogeneic graft (or allograft): A graft transplanted between two genetically different
individuals of the same species
• Xenogeneic graft (or xenograft): a graft transplanted between individuals of different
species.
• The molecules that are recognized as foreign in allografts are called alloantigens, and
those in xenografts are called xenoantigens.
• The lymphocytes and antibodies that react with alloantigens or xenoantigens are
described as being alloreactive or xenoreactive, respectively.

General Principles
• Innate immunity
• The interruption of blood supply to tissue and organs during the time between
removal from a donor and placement in a host usually cause some ischemic
damage. This can result in expression of damage-associated molecular patterns
(DAMPs) in the graft, which simulate innate responses mediated by both host innate
cells within the graft and the donor innate immune system.
• Host natural killer (NK) cells can respond to the absence of syngeneic
histocompatibility molecules on donor graft cells and therefore contribute to graft
rejection.
• These innate responses can directly cause graft injury, but they are also believed to
enhance adaptive responses by activating antigen-presenting cells (APCs).

Adaptive Immune Responses to
Allografts
• The Nature of Alloantigens
• Recognition of Alloantigens by T Cells
• Activation and Effector Functions of Alloreactive T Lymphocytes
• Activation of Alloreactive B Cells and Production and Functions
of Alloantibodies

Allografts
• Most of the antigens that stimulate adaptive immune responses against
allografts are proteins encoded by polymorphic genes that differ among
individuals.
• Histocompatibility molecules

A. Cells or organs transplanted between
genetically identical individuals (identical
twins or members of the same inbred strain
of animals) are not rejected.
B. Cells or organs transplanted between
genetically nonidentical people or
members of two different inbred strains of a
species are almost always rejected.
C. The offspring of a mating between two
different inbred strains of animal will not
reject grafts from either parent.
• An (A × B) F1 animal will not reject grafts from an A or B
strain animal. (This rule is violated by hematopoietic
stem cell (HSC) transplantation, when NK cells in an (A ×
B) F1 recipient do reject HSCs from either parent.)
D. A graft derived from the offspring of a
mating between two different inbred strains
of animal will be rejected by either parent.
• A graft from an (A × B) F1 animal will be rejected by
either an A or a B strain animal.

Allografts
• Most of the antigens that stimulate adaptive immune responses against
allografts are proteins encoded by polymorphic genes that differ among
individuals.
• Histocompatibility molecules
• Polymorphic: These graft antigens differ among the individuals of a species.
(other than identical twins)
• Codominant expression: Every individual inherits genes encoding these
molecules from both parents, and both parental alleles are expressed.

Allografts
• The molecules responsible for strong and rapid rejection reactions are major
histocompatibility complex (MHC) molecules that bind and present
peptides to T cells.
• Transplants of most tissues between any pair of individuals, except identical
twins, will be rejected because MHC molecules are so polymorphic that no
two individuals inherit the same ones.
• Human MHC molecules are called human leukocyte antigens (HLAs).
• In the context of human transplantation, the terms MHC and HLA are used
interchangeably.

Allografts
• The molecules responsible for strong and rapid rejection reactions are major
histocompatibility complex (MHC) molecules that bind and present peptides to T
cells.
• Minor histocompatibility antigens: Polymorphic antigens other than MHC
molecules against which the recipient may mount an immune response
• Typically induce weak or slower (more gradual) rejection reactions than do MHC molecules.
• The relevance of minor histocompatibility antigens in clinical solid organ transplantation is
uncertain.
• Play a more significant role in stimulating graft-versus-host responses after HSC
transplantation.

Allografts
• Allogeneic MHC molecules of a graft can be presented for recognition by the
recipient’s T cells in two different ways, called direct and indirect.
• Direct presentation (or direct recognition) of alloantigens: T cells of a graft
recipient recognize intact, unprocessed MHC molecules in the graft.
• Indirect presentation (or indirect recognition): The recipient T cells recognize graft
(donor) MHC molecules only in the context of the recipient’s MHC molecules.
• Implying that the recipient’s MHC molecules must be presenting peptides derived from
allogeneic donor MHC proteins to recipient T cells.
• The initial T cell response to MHC alloantigens most likely occurs in lymph nodes
draining the graft.

Direct alloantigen recognition occurs when alloreactive T cells bind directly to an intact
allogeneic MHC molecule with bound peptide on a graft (donor) dendritic cell or other APC,
within lymph nodes.
Recipient CD4+ or CD8+ T cells can directly recognize donor Class II or Class I MHC
molecules, respectively, and will differentiate into helper T cells or CTL.
The CTL will directly recognize the same donor MHC-peptide complex displayed on graft
tissue cells and kill these cells.

Molecular basis of direct recognition of allogeneic MHC molecules.
Direct recognition of allogeneic MHC molecules may be thought of as a cross-reaction in which a T
cell–specific for a self MHC molecule–foreign peptide complex (A) also recognizes an
allogeneic MHC molecule (B and C). Peptides that bind to MHC molecules in the graft may
contribute to allorecognition (B) or they may not (C).

Allografts
• Direct Recognition of MHC Alloantigens on Donor Cells
• T cell responses to directly presented allogeneic MHC molecules are
very strong because there is a high frequency of T cells that can
directly recognize any single allogeneic MHC protein.
• Estimated 1% to 10% of all T cells in an individual will directly recognize and
react against an allogeneic MHC molecule on a donor cell.
• Direct allorecognition can generate both CD4+ and CD8+ T cells that
recognize graft antigens and contribute to rejection.

Allografts
• There are several explanations for the high frequency of T cells
that can directly recognize allogeneic MHC molecules.
• Many different peptides derived from donor cellular proteins may combine
with a single allogeneic MHC molecule, and each of these peptide-MHC
combinations can theoretically activate a different clone of recipient T cells.
• The range of peptide-MHC complexes that can activate T cells is much
greater if the MHC is allogeneic.
• Many of the T cells that respond to an allogeneic MHC molecule, even on first
exposure, are memory T cells.

Allografts
• Indirect Recognition of Alloantigens
• Donor (allogeneic) MHC molecules are captured and processed by recipient APCs, and
peptides derived from the allogeneic MHC molecules are presented in association with
self MHC molecules.
• Like conventional foreign protein antigens
• Each allogeneic MHC molecule may give rise to multiple peptides that are foreign for the
host, each recognized by different clones of T cells.
• Cross-presentation or cross-priming
• Allorecognition by CD4+ T cells, some are indirectly recognized by CD8+ T cells.
• May contribute to chronic rejection of human allografts.
• CD4+ T cells from heart and liver allograft recipients recognize and are activated by peptides derived
from donor MHC when presented by the patient’s own APCs.

Indirect alloantigen recognition occurs when allogeneic MHC molecules from graft cells are taken up and processed
by recipient APCs and peptide fragments of the allogeneic MHC molecules containing polymorphic amino acid
residues are bound and presented by recipient (self) MHC molecules.
Donor-MHC–specific helper T cells that are generated in this way can help B cells to produce donor- MHC–specific
antibodies that can damage graft cells.
The helper T cells can also be activated in the graft by recipient macrophages presenting the same donor MHC-
derived peptides, leading to inflammatory damage to the graft.

Allografts
• Activation of Alloreactive T Lymphocytes
• The T cell response to an organ graft may be initiated in the lymph nodes that drain the
graft.
• The connection between lymphatic vessels in allografts and the recipient’s lymph
nodes is surgically disrupted during the process of transplantation, and it is likely
reestablished by growth of new lymphatic channels in response to inflammatory stimuli
produced during grafting.
• Sensitization to alloantigens: Naive CD4+ and CD8+ lymphocytes that normally traffic
through the lymph node encounter these alloantigens and are induced to proliferate and
differentiate into effector helper T cells and cytotoxic T lymphocytes (CTLs).
• The effector cells migrate back into the graft and mediate rejection.

Allografts
• Activation and Effector Functions of Alloreactive T
Lymphocytes
• Unlike naive T cells, memory T cells may not need to see antigens
presented by dendritic cells in lymph nodes in order to be activated,
and they may migrate directly into grafts where they can be activated
by APCs or tissue cells displaying alloantigen.

Allografts
Lymphocytes
• Mixed lymphocyte reaction (MLR)
• In vitro test of the response of alloreactive T cells to foreign MHC molecules
• Lymphocytes from two genetically distinct individuals are mixed together in cell
culture. The T cells from one individual become activated by recognition of allogeneic
MHC molecules on the cells of the other.
• Used clinically in the past as a predictive test of T cell–mediated graft rejection, and
as an in vitro model to study the mechanisms of alloreactivity

Allografts
Lymphocytes
• Role of Costimulation in T Cell Responses to Alloantigens
• In addition to recognition of alloantigen, costimulation of T cells primarily
by B7 molecules on APCs is important for activating alloreactive T cells.
• Costimulation is likely most important to activate naive alloreactive T cells,
but even alloreactive memory T cell responses can be enhanced by
costimulation.
• One possibility is that the innate response to ischemic damage of some
cells in the graft, results in increased expression of costimulators on APCs.

Allografts
Lymphocytes
• Effector Functions of Alloreactive T Cells
• Alloreactive CD4+ and CD8+ T cells that are activated by graft alloantigens
cause rejection by distinct mechanisms.
• The CD4+ helper T cells differentiate into cytokine-producing effector cells
that damage grafts by cytokine-mediated inflammation, similar to a delayed-
type hypersensitivity (DTH) reaction.
• CD8+ T cells differentiate into CTLs, which kill graft cells.

Allografts
• Effector Functions of Alloreactive T Cells
• Only CTLs that are generated by direct allorecognition can kill graft cells, whereas both CTLs
and helper T cells generated by either direct or indirect alloantigen recognition can cause cytokine-
mediated damage to grafts.
• CD8+ CTLs that are generated by direct allorecognition of donor MHC molecules on donor APCs can recognize
the same MHC molecules on parenchymal cells in the graft and kill those cells. These T cells can also secrete
cytokines that cause damaging inflammation.
• In contrast, any CD8+ CTLs that are generated in response to indirect recognition of allogeneic MHC are restricted
to recognition of peptides from these allogeneic MHC molecules bound to recipient (self) MHC molecules, and
therefore the T cells will not be able to kill the foreign graft cells because the graft does not express recipient MHC
molecules. The principal mechanism of rejection is inflammation caused by the cytokines produced by the effector T
cells.
• CD4+ effector T cells are generated by direct or indirect recognition of allogeneic MHC, the principal mechanism
of rejection is inflammation caused by the cytokines produced by the effector T cells.

Allografts
• Activation of Alloreactive B Cells and Production and Functions of
Alloantibodies
• Antibodies against graft antigens, called donor-specific antibodies, also contribute to rejection.
• These high-affinity alloantibodies are mostly produced by helper T cell–dependent activation of
alloreactive B cells, much like antibodies against other protein antigens.
• Naive B lymphocytes recognize the allogenic MHC molecules, internalize and process these proteins, and present
peptides derived from them to helper T cells that were previously activated by the same peptides presented by
dendritic cells. (Indirect presentation of alloantigens)
• The antigens most frequently recognized by alloantibodies are donor MHC molecules, including both class I
and class II MHC proteins.
• Donor-specific antibodies against non-HLA alloantigens also contribute to rejection.
• The same effector mechanisms that antibodies use to combat infections.
• Complement activation, and Fc receptor-mediated binding and activation of neutrophils, macrophages, and NK cells.
• Because MHC antigens are expressed on endothelial cells, much of the alloantibody-mediated damage is targeted at
the graft vasculature.

Patterns and Mechanisms of
Allograft Rejection
• For historical reasons, graft rejection is classified on the basis
of histopathologic features and the time course of rejection
after transplantation rather than on the basis of immune effector
mechanisms.
• Hyperacute Rejection
• Acute Rejection
• Chronic Rejection and Graft Vasculopathy

Allograft Rejection
• Characterized by thrombotic occlusion of the graft vasculature that begins within minutes to
hours after host blood vessels are anastomosed to graft vessels and is mediated by preexisting
antibodies in the host circulation that bind to donor endothelial antigens.
• Complement activation leads to endothelial cell injury and exposure of subendothelial basement
membrane proteins that activate platelets.
• The endothelial cells are stimulated to secrete high–molecular-weight forms of von Willebrand factor, which
causes platelet adhesion and aggregation.
• Both endothelial cells and platelets undergo membrane vesiculation, leading to shedding of lipid particles
that promote coagulation.
• Endothelial cells lose the cell surface heparan sulfate proteoglycans that normally interact with antithrombin
III to inhibit coagulation.
• These processes contribute to thrombosis and vascular occlusion, and the grafted organ suffers irreversible
ischemic necrosis.

Hyperacute
Rejection

Allograft Rejection
• In the early days of transplantation: preexisting IgM alloantibodies specific for the carbohydrate
ABO blood group antigens that are expressed on red cells and endothelial cells.
• Hyperacute rejection by anti-ABO antibodies is extremely rare now because all donor and recipient pairs have compatible
ABO types.
• Natural antibodies, specific for a variety of antigens that differ among species, is a major barrier to
xenotransplantation.
• Currently, the rare instances of hyperacute rejection of allografts are mediated by IgG antibodies
directed against protein alloantigens, such as donor MHC molecules, or against less defined
alloantigens expressed on vascular endothelial cells.
• Such antibodies generally arise as a result of previous exposure to alloantigens through blood transfusion, previous
transplantation, or multiple pregnancies.
• If the level of these alloreactive antibodies is low, hyperacute rejection may develop slowly, during several days, but the onset
is still earlier than that typical for acute rejection.
• Patients in need of allografts are routinely screened before grafting for the presence of antibodies that bind to cells of a
potential organ donor to avoid hyperacute rejection.

Allograft Rejection
• In unusual cases in which grafts have to be done between ABO-incompatible donors
and recipients, graft survival may be improved by rigorous depletion of antibodies and
B cells.
• Sometimes, if the graft is not rapidly rejected, it survives even in the presence of anti-
graft antibody.
• One possible mechanism of this resistance to hyperacute rejection is increased
expression of complement regulatory proteins on graft endothelial cells, a beneficial
adaptation of the tissue called accommodation.

Allograft Rejection
• Acute Rejection
• Acute rejection is a process of injury to the graft parenchyma and blood vessels
mediated by alloreactive T cells and antibodies.
• Often begin several days to a few weeks after transplantation
• Reflects the time needed to generate alloreactive effector T cells and antibodies in response to
the graft
• In current clinical practice, episodes of acute rejection may occur at much later times,
even years after transplantation, if immunosuppression is reduced.
• Acute Cellular Rejection (mediated by T cells)
• Acute Antibody-Mediated Rejection
• Both typically coexist in an organ undergoing acute rejection.

Allograft Rejection
• Acute Cellular Rejection
• The principal mechanisms of acute cellular rejection are CTL-mediated
killing of graft parenchymal cells and endothelial cells and
inflammation caused by cytokines produced by helper T cells.

Acute
cellular
rejection
On histologic
examination of
kidney allografts,
there are infiltrates of
lymphocytes and
macrophages.
In kidney allografts, the
infiltrates may involve the
tubules (called tubulitis),
with associated tubular
necrosis, and blood
vessels (called
endotheliitis), with
necrosis of the walls of
capillaries and small
arteries.
The helper T cells
include IFN-γ– and
TNF-secreting Th1
cells and IL-17–
secreting Th17 cells.

Allograft Rejection
• Acute Antibody-Mediated Rejection
• Alloantibodies cause acute rejection by binding to alloantigens, mainly HLA
molecules, on vascular endothelial cells, leading to endothelial injury and
intravascular thrombosis that results in graft destruction.
• The binding of the alloantibodies to the endothelial cell surface triggers local complement
activation, which causes lysis of the cells, recruitment and activation of neutrophils, and
thrombus formation.
• Alloantibodies may also engage Fc receptors on neutrophils and NK cells, which then kill the
endothelial cells.
• Alloantibody binding to the endothelial surface may directly alter endothelial function by
inducing intracellular signals that enhance surface expression of proinflammatory and
procoagulant molecules.

Acute
antibody
mediated
rejection
The histologic
hallmarks of acute
antibody-mediated
rejection of renal
allografts are acute
inflammation of
glomeruli and
peritubular capillaries
with focal capillary
thrombosis.
Immunohistochemical
identification of the
C4d complement
fragment in
capillaries of renal
allografts is used
clinically as an
indicator of activation
of the classical
complement pathway
and humoral rejection
(brown staining).

Allograft Rejection
• As therapy for acute rejection has improved, the major cause of the failure of
vascularized organ allografts has become chronic rejection.
• Develops insidiously during months or years
• May or may not be preceded by clinically recognized episodes of acute rejection
• Chronic rejection of different transplanted organs is associated with distinct
pathologic changes.
• In the kidney and heart, chronic rejection results in vascular occlusion and interstitial fibrosis.
• Lung transplants undergoing chronic rejection show thickened small airways (called
bronchiolitis obliterans).
• Liver transplants show fibrotic and nonfunctional bile ducts.

Allograft Rejection
• A dominant lesion of chronic rejection in vascularized grafts is arterial occlusion as
a result of the proliferation of intimal smooth muscle cells, and the grafts eventually
fail mainly because of the resulting ischemic damage.
• The arterial changes are called graft vasculopathy or accelerated graft
arteriosclerosis.
• Frequently seen in failed cardiac and renal allografts
• Can develop in any vascularized organ transplant within 6 months to a year after
transplantation.
• The likely mechanisms are activation of alloreactive T cells and secretion of IFN-γ and
other cytokines that stimulate proliferation of vascular smooth muscle cells.

Allograft Rejection
• As the arterial lesions of graft arteriosclerosis progress, blood flow to the
graft parenchyma is compromised, and the parenchyma is slowly replaced
by nonfunctioning fibrous tissue.
• The interstitial fibrosis seen in chronic rejection may also be a repair response to
parenchymal cell damage caused by repeated bouts of acute antibody-mediated or
cellular rejection, perioperative ischemia, toxic effects of immunosuppressive drugs,
and even chronic viral infections.
• Chronic rejection leads to congestive heart failure or arrhythmias in cardiac
transplant patients or loss of glomerular and tubular function and renal failure
in kidney transplant patients.

Chronic
rejection
Chronic rejection
in a kidney
allograft with graft
arteriosclerosis.
The vascular lumen
is replaced by an
accumulation of
smooth muscle cells
and connective
tissue in the vessel
intima.
Fibrosis and loss of tubules
in a kidney with chronic
rejection (lower left) adjacent to
relatively normal kidney (upper
right). The blue area shows
fibrosis, and an artery with graft
arteriosclerosis is present
(bottom right).

Prevention and Treatment of
Allograft Rejection
• The strategies used in clinical practice and in experimental models to avoid or to
delay rejection are general immunosuppression and minimizing the strength of
the specific allogeneic reaction.
• An important goal of transplantation research is to find ways of inducing
donor-specific tolerance, which would allow grafts to survive without nonspecific
immunosuppression.
• Methods to Reduce the Immunogenicity of Allografts
• Immunosuppression to Prevent or to Treat Allograft Rejection
• Methods to Induce Donor-Specific Tolerance

Allograft Rejection
• Solid organs used in transplantation come from both living and deceased donors,
and graft survival after transplantation varies depending on the source.
• Living donors can donate one kidney, a lobe of a lung, and parts of liver, pancreas,
or intestine.
• Related donors will share more alleles of polymorphic genes, including MHC genes,
than unrelated donors, and this will reduce the incidence and severity of rejection
episodes.
• Because MHC genes are inherited as linked haplotypes, there is a 25% chance that two
siblings will have identical MHC genes, whereas the chance of an unrelated donor and
recipient having identical MHC genes is extremely low.

Allograft Rejection
• Solid organs used in transplantation come from both living and deceased donors,
and graft survival after transplantation varies depending on the source.
• Deceased donors, called cadaveric donors, are sources of any transplantable
organ and the only source of organs such as hearts.
• The survival of grafts from deceased donors is on average lower than from either
related or unrelated living donors because
• There is more ischemic damage to organs removed after death of the donor.
• Grafts from unrelated donors usually express more antigens that differ from the recipient and
can simulate stronger rejection responses than those from living donors.

Allograft Rejection
• In human transplantation, the major strategy to reduce graft
immunogenicity has been to minimize alloantigenic differences between the
donor and recipient.
• ABO blood typing
• Tissue typing: The determination of HLA alleles expressed on donor and recipient
cells.
• The detection of preformed antibodies in the recipient that recognize HLA and other
antigens representative of the donor population; and
• The detection of preformed antibodies in the recipient that bind to antigens of an
identified donor’s cells, called crossmatching.
• Not all of these tests are done in all types of transplantation.

Allograft Rejection
• To avoid hyperacute rejection, the ABO blood group antigens of the
graft donor are selected to be compatible with the recipient.
• Renal and cardiac transplantation
• Natural IgM antibodies specific for allogeneic ABO blood group antigens will
cause hyperacute rejection.

Allograft Rejection
• In kidney transplantation, the larger the number of MHC alleles that
are matched between the donor and recipient, the better the graft
survival.
• Of all class I and class II MHC loci, matching at HLA-A, HLA-B, and
HLA-DR is most important for predicting survival of kidney allografts.
• HLA-C is not as polymorphic as HLA-A or HLA-B, and HLA-DR and HLA-DQ are
in linkage disequilibrium, so matching at the DR locus often also matches at the
DQ locus.

Influence of MHC matching
on graft survival.
• Matching of MHC alleles
between the donor and
recipient significantly
improves renal allograft
survival.
• The data shown are for
deceased donor (cadaver)
grafts.
• HLA matching has less of an
impact on survival of renal
allografts from live donors,
and some MHC alleles are
more important than others in
determining outcome.

Allograft Rejection
• Possible to have 0 to 6 HLA mismatches of these three loci between the
donor and recipient.
• Because 2 codominantly expressed alleles are inherited for each of these HLA genes.
• Zero-antigen mismatches predict the best survival of living related donor
grafts, and grafts with one-antigen mismatches do slightly worse.
• The survival of grafts with 2 – 6 HLA mismatches is significantly worse than that of
grafts with zero- and one-antigen mismatches, even greater impact on nonliving
(unrelated) donor renal allografts.

Allograft Rejection
• Therefore, attempts are made to reduce the number of differences in
HLA alleles expressed on donor and recipient cells, which will have
a modest effect in reducing the chance of rejection.
• HLA matching in renal transplantation is possible because
• Donor kidneys can be stored for up to 72 hours before being transplanted.
• Patients needing a kidney allograft can be maintained on dialysis until a well-
matched organ is available.

Allograft Rejection
• Therefore, attempts are made to reduce the number of differences in HLA
alleles expressed on donor and recipient cells, which will have a modest
effect in reducing the chance of rejection.
• HLA matching in renal transplantation is possible because
• Donor kidneys can be stored for up to 72 hours before being transplanted.
• Patients needing a kidney allograft can be maintained on dialysis until a well-matched
organ is available.
• In hematopoietic stem cell transplantation, HLA matching is essential to
reduce the risk of graft-versus-host disease (GVHD).

Allograft Rejection
• In the case of heart and liver transplantation, organ preservation is more
difficult, and potential recipients are often in critical condition.
• HLA typing is not considered in pairing of potential donors and recipients, and
the choice of donor and recipient is based on
• ABO blood group matching, other measures of immunologic compatibility, and
anatomic compatibility.
• The paucity of heart donors, the emergent need for transplantation, and the
success of immunosuppression override any benefit of reducing HLA
mismatches between donor and recipient.

Allograft Rejection
• HLA haplotype determinations are now performed by polymerase
chain reaction (PCR), replacing older serologic methods.
• The actual nucleotide sequence, and therefore, the predicted amino acid
sequence, can be directly determined for the MHC alleles of any cell, providing
precise molecular tissue typing.
• The nomenclature of HLA alleles: Each allele defined by sequence
has at least a four-digit number, but some alleles require six or eight
digits for precise definition.

Allograft Rejection
• Patients in need of allografts are also tested for the presence of
preformed antibodies against donor MHC molecules or other cell
surface antigens.
• 2 types of tests
• Panel reactive antibody (PRA) test: screened for the presence of preformed
antibodies reactive with allogeneic HLA molecules prevalent in the population.

Allograft Rejection
• Panel reactive antibody (PRA) test: screened for the presence of preformed
antibodies reactive with allogeneic HLA molecules prevalent in the population.
• May be produced as a result of previous pregnancies, transfusions, or transplantation
• Increases risk for hyperacute or acute vascular rejection
• Small amounts of the patient’s serum are mixed with multiple fluorescently labeled
beads coated with defined MHC molecules. Binding of the patient’s antibodies to
beads is determined by flow cytometry.
• The results are reported as PRA: the percentage of the MHC allele panel with which
the patient’s serum reacts.
• Determined on multiple occasions while a patient waits for an organ allograft.

Allograft Rejection
• Cross-matching test: if the patient has antibodies that react
specifically with that donor’s cells.
• A potential donor is identified.
• Mixing the recipient’s serum with the donor’s blood lymphocytes (a convenient
source of cells, some of which express both class I and class II MHC proteins).
• Complement-mediated cytotoxicity tests or flow cytometric assays can then
be used to determine if antibodies in the recipient serum have bound to the donor
cells.

Allograft Rejection
• Immunosuppressive drugs that inhibit or kill T lymphocytes are the principal
agents used to treat or prevent graft rejection.
• Inhibitors of T Cell Signaling Pathways
• Antimetabolites
• Function-Blocking or Depleting Anti-Lymphocyte Antibodies
• Costimulatory Blockade
• Drugs Targeting Alloantibodies and Alloreactive B Cells
• Antiinflammatory Drugs

Mechanisms of
action of
immunosuppressive
drugs.
• Each major category
of drugs used to
prevent or to treat
allograft rejection is
shown along with the
molecular targets of
the drugs.

A. C. Wiseman. Clin J Am Soc Nephrol 11: 332–343, 2016.

P. F. Halloran. N Engl J Med 2004;351:2715-29.

Allograft Rejection
• The calcineurin inhibitors cyclosporine and tacrolimus (FK506) inhibit transcription
of certain genes in T cells, most notably genes encoding cytokines such as IL-2.
• Cyclosporine is a fungal peptide that binds with high affinity to a ubiquitous cellular protein
called cyclophilin.
• The complex of cyclosporine and cyclophilin binds to and inhibits the enzymatic activity of the
calcium/calmodulin-activated serine/threonine phosphatase calcineurin.
• Calcineurin is required to activate the transcription factor NFAT (nuclear factor of activated T cells).
• Cyclosporine inhibits NFAT activation and the transcription of IL-2 and other cytokine genes.
• The net result is that cyclosporine blocks the IL-2–dependent proliferation and
differentiation of T cells.

Allograft Rejection
• The calcineurin inhibitors cyclosporine and tacrolimus (FK506) inhibit
transcription of certain genes in T cells, most notably genes encoding
cytokines such as IL-2.
• Tacrolimus is a macrolide made by a bacterium that functions like cyclosporine.
• Tacrolimus binds to FK506 binding protein (FKBP) and the complex shares with
the cyclosporine-cyclophilin complex the ability to bind calcineurin and inhibit its
activity.

Influence of
cyclosporine on graft
survival.
• Five-year survival
rates for patients
receiving cardiac
allografts increased
significantly beginning
when cyclosporine
was introduced in
1983.

Allograft Rejection
• The calcineurin inhibitors cyclosporine and tacrolimus (FK506)
• Limitations
• At doses needed for optimal immunosuppression, cyclosporine causes kidney
damage, and some rejection episodes are refractory to cyclosporine treatment.
• Tacrolimus was initially used for liver transplant recipients, but it is now used
widely for immunosuppression of kidney allograft recipients, including those who
are not adequately controlled by cyclosporine.

Allograft Rejection
• The immunosuppressive drug rapamycin (sirolimus) inhibits growth
factor–mediated T cell proliferation.
• Rapamycin binds to FKBP, like tacrolimus, but the rapamycin-FKBP complex
does not inhibit calcineurin. Instead, this complex binds to and inhibits a cellular
enzyme called mammalian target of rapamycin (mTOR), which is a
serine/threonine protein kinase required for translation of proteins that promote
cell survival and proliferation.
• mTOR is negatively regulated by a protein complex called tuberous sclerosis
complex 1 (TSC1)–TSC2 complex.

Allograft Rejection
• The immunosuppressive drug rapamycin (sirolimus) inhibits growth
factor–mediated T cell proliferation.
• Phosphatidylinositol 3-kinase (PI3K)–Akt signaling results in phosphorylation
of TSC2 and release of mTOR inhibition.
• Several growth factor receptor signaling pathways, including the IL-2
receptor pathway in T cells, as well as TCR and CD28 signals, activate mTOR
through PI3K-Akt, leading to translation of proteins needed for cell cycle
progression.
• By inhibiting mTOR function, rapamycin blocks T cell proliferation.

Allograft Rejection
• Rapamycin (sirolimus)
• Inhibits the generation of effector T cells but does not impair the survival and
functions of regulatory T cells (Tregs) as much, which may promote immune
suppression of allograft rejection.
• mTOR is involved in
• Dendritic cell functions -- may suppress T cell responses by its effects on dendritic
• B cell proliferation and antibody responses -- may also be effective in preventing or
treating antibody-mediated rejection.
• Combinations of cyclosporine (which blocks IL-2 synthesis) and rapamycin
(which blocks IL-2–driven proliferation) potently inhibit T cell responses.

Allograft Rejection
• Other molecules involved in cytokine and TCR signaling are also
targets of immunosuppressive drugs that are in trials for treatment or
prevention of allograft rejection.
• One of these target molecules is the tyrosine kinase JAK3, which is
involved in signaling by various cytokine receptors, including IL-2, and
protein kinase C, an essential kinase in TCR signaling.

Allograft Rejection
• Antimetabolites
• Metabolic toxins that kill proliferating T cells are used in combination
with other drugs to treat graft rejection.
• Inhibit the proliferation of lymphocyte precursors during their maturation and
also kill proliferating mature T cells that have been stimulated by alloantigens.
• Azathioprine
• The first drug to be developed for the prevention and treatment of rejection.
• Still used, but it is toxic to precursors of leukocytes in the bone marrow and
enterocytes in the gut.

Allograft Rejection
• Antimetabolites
• Metabolic toxins that kill proliferating T cells are used in combination with
other drugs to treat graft rejection.
• Mycophenolate mofetil (MMF): The most widely used drug in this class.
• Metabolized to mycophenolic acid, which blocks the activity of inosine monophosphate
dehydrogenase, an enzyme required for de novo synthesis of guanine nucleotides.
• Because proliferating lymphocytes are particularly dependent on de novo synthesis of
purines, MMF targets lymphocytes in a relatively specific manner.
• MMF is now routinely used, often in combination with cyclosporine or tacrolimus to
prevent acute allograft rejection.

Post-2017 INN
-mab
P. Mayrhofer and R. Kunert. Human Antibodies 27 (2019) 37–51.

Allograft Rejection
• Antibodies that react with T cell surface structures and deplete or inhibit T cells are
used to treat acute rejection episodes.
• The first anti-T cell antibody used in transplant patients was a mouse monoclonal
antibody called OKT3 that is specific for human CD3.
• OKT3 was the first monoclonal antibody used as a drug in humans, but it is no longer being
produced.
• Anti-thymocyte globulin: Polyclonal rabbit or horse antibodies specific for a mixture
of human T cell surface proteins
• Treat acute allograft rejection
• Deplete circulating T cells either by activating the complement system to eliminate
T cells or by opsonizing them for phagocytosis.

Allograft Rejection
• Monoclonal antibodies specific for CD25, the α subunit of the IL-2 receptor
• Prevent T cell activation by blocking IL-2 binding to activated T cells and IL-2
signaling.
• Anti-CD52 (called alemtuzumab): A cell surface protein expressed widely
on most mature B and T cells whose function is not understood.
• Administered just before and early after transplantation, with the hope that it may
induce a prolonged state of graft tolerance as new lymphocytes develop in the
presence of the allograft.

Allograft Rejection
• Function-Blocking or Depleting Anti-Lymphocyte
Antibodies
• The major limitation to the use of monoclonal or polyclonal antibodies
from other species is that
• Humans given these agents produce anti-Ig antibodies that neutralize the injected
foreign Ig.
• Human-mouse chimeric (humanized) antibodies (e.g., against CD3
and CD25), which are less immunogenic, have been developed.

Allograft Rejection
• Drugs that block T cell costimulatory pathways reduce acute allograft rejection.
• To prevent the delivery of costimulatory signals required for activation of T cells.
• Belatacept: A high-affinity form of CTLA4-Ig
• CTLA4-Ig is a recombinant protein composed of the extracellular portion of CTLA4 fused to an IgG Fc
domain.
• Binds to B7 molecules on APCs and prevents them from interacting with T cell CD28.
• As effective as cyclosporine in preventing acute rejection, but its high cost and other factors have
limited widespread use
• Anti-CD40L antibody: An antibody that binds to T cell CD40 ligand (CD40L) and
prevents its interactions with CD40 on APCs.
• Thrombotic complications, apparently related to the expression of CD40L on platelets.

Allograft Rejection
• Costimulation Blockade by CD154:CD40
Targeting (Anti-CD40 mAb)
• The CD154 (also known as CD40L; present on activated
T cells): CD40 (on APCs) interaction is a critical step in T
cell costimulatory signaling, because this interaction leads
to the upregulation of CD80/86 on APCs.
• A number of mAbs against CD40 are in development, with
a fully human anti-CD40 (ASKP1240; Astellas) under study
in phase 2 clinical trials in kidney transplantation.

Allograft Rejection
• Intravenous immunoglobulin (IVIG)
• Pooled IgG from normal donors is injected intravenously into a patient.
• The mechanisms of action are not fully understood but likely involve binding of
the injected IgG to the patient’s Fc receptors on various cell types, thereby
reducing alloantibody production and blocking effector functions of the patient’s
own antibodies.
• Enhances degradation of the patient’s antibodies by competitively inhibiting their
binding to the neonatal Fc receptor.

Allograft Rejection
• Rituximab: An anti-CD20 antibody
• B cell depletion
• Approved for treatment of B cell lymphomas and for autoimmune diseases
• Used in some cases of acute antibody-mediated rejection
• Chimeric anti-CD20 mAb (30% murine and 70% human)
• Leads to side effects attributable to cytokine release, such as fever,
bronchospasm, and hypotension.

Allograft Rejection
• Anti-CD20 Targeting: Rituximab, Ocrelizumab, Ofatumumab, and
Veltuzumab
• Agents that are humanized (ocrelizumab) or fully humanized
(ofatumumab) have been developed to minimize these untoward
infusion reactions.
• However, ocrelizumab development in RA has been discontinued because of an
increased risk of serious infections.
• A phase 1/2 trial of ofatumumab in RA has shown preliminary efficacy, with
mild/moderate infusion reactions still prevalent.
• All anti-CD20 therapy carries a risk of hepatitis B reactivation.
• Therefore, before starting treatment, patients should be screened for hepatitis B
surface antigen and hepatitis B core antibody.

Allograft Rejection
• Anti-CD22 Targeting: Epratuzumab
• CD22 is expressed on B cells during B cell maturation and loss of CD20
expression.
• B cell receptor signaling is modulated by phosphorylation of CD22, which
regulates B cell activation.
• Humanized anti-CD22 mAb that inhibits B cell activation and has a more
modest depleting effect on B cells than rituximab.
• It is currently in phase 3 trials in patients with moderate to severe SLE after
phase 2 trials suggested a low rate of adverse events, similar to placebo

Allograft Rejection
• Targeting B Cell Differentiation: Belimumab and Atacicept
• A key pathway for differentiation of B cells is the binding of the cytokine B cell–
activating factor (BAFF; also referred to BlyS) to its B cell receptors [(BAFF-R, B
cell maturation (BCMA), and transmembrane activator and CAML interactor
(TACI)] and the binding of the cytokine proliferation–inducing ligand to its B cell
receptors (BCMA and TACI).
• Lead to increases in NF-κβ, which in turn, promote B cell differentiation and
inhibit apoptosis.
• Belimumab is a humanized anti-BAFF/BlyS mAb that interferes with
ligand/receptor binding and inhibits this maturation.
• Atacicept is a recombinant fusion protein that inhibits both BlyS and
proliferation-inducing ligand.

Allograft Rejection
• Drugs Targeting Alloantibodies and
Alloreactive B Cells
• Bortezomib: proteasome inhibitor
• Kills plasma cells
• Approved to treat multiple myeloma
• Sometimes used to treat antibody-mediated allograft
rejection.

Allograft Rejection
• Antiinflammatory Drugs
• Antiinflammatory agents, specifically corticosteroids, are frequently
used to reduce the inflammatory reaction to organ allografts.
• To block the synthesis and secretion of cytokines, including
• TNF and IL-1, and other inflammatory mediators, such as prostaglandins, reactive
oxygen species, and nitric oxide, produced by macrophages and other
inflammatory cells.
• The net result of this therapy is reduced leukocyte recruitment, inflammation,
and graft damage.

Allograft Rejection
• Current immunosuppressive protocols have dramatically improved graft survival.
• Induction therapy: Strong immunosuppression is usually started in allograft recipients at the
time of transplantation with a combination of drugs.
• After a few days, the drugs are changed for long-term maintenance of immunosuppression.
• Acute rejection is managed by rapidly intensifying immunosuppressive therapy.
• Chronic rejection is more insidious than acute rejection and is much less responsive to
immunosuppression than is acute rejection.
• Immunosuppressive therapy leads to increased susceptibility to various types of
infections and virus-associated tumors.

Allograft Rejection
• Current immunosuppressive protocols have dramatically improved graft survival.
• Immunosuppressive therapy leads to increased susceptibility to various types of infections
and virus-associated tumors.
• Defense against viruses and other intracellular pathogens, the physiologic function of T cells, is
compromised in immunosuppressed transplant recipients.
• Frequent reactivation of latent herpesviruses: CMV, HSV, VZV, EBV
• Prophylactic antiviral therapy for herpesvirus infections are now given.
• Greater risk for opportunistic infections
• Higher risk for development of cancer
• Uterine cervical carcinoma (HPV), lymphomas (EBV)
• The lymphomas found in allograft recipients as a group are called post-transplantation lymphoproliferative
disorders (PTLDs), and most are derived from EBV-infected B lymphocytes.

Allograft Rejection
• Allograft rejection may be prevented by making the host tolerant to the alloantigens
of the graft.
• Tolerance: The host immune system does not injure the graft despite the withdrawal of
immunosuppressive agents.
• Alloantigen specific and will therefore avoid the major problems associated with nonspecific
immunosuppression, may reduce chronic rejection.
• Renal transplant patients who have received blood transfusions containing allogeneic
leukocytes have a lower incidence of acute rejection episodes.
• The postulated explanation for this effect is that the introduction of allogeneic leukocytes by transfusion
produces tolerance to alloantigens. One underlying mechanism for tolerance induction may be that the
transfused donor cells contain immature dendritic cells, which induce unresponsiveness to donor
alloantigens.

Allograft Rejection
• Several strategies are being tested to induce donor-specific tolerance
in allograft recipients.
• Costimulatory blockade
• Hematopoietic chimerism
• Transfer or induction of Tregs

Allograft Rejection
• Recognition of alloantigens in the absence of costimulation would lead to T cell
tolerance.
• The clinical experience is that they suppress immune responses to the allograft
but do not induce long-lived tolerance, and patients have to be maintained on the
therapy.

Allograft Rejection
• Long-term allograft tolerance by hematopoietic chimerism has been achieved
in a small number of renal allograft recipients who received a hematopoietic stem
cell transplant from the donor at the same time as the organ allograft.
• But the risks of hematopoietic stem cell transplantation and the availability of
appropriate donors may limit the applicability of this approach.

Allograft Rejection
• Attempts to generate donor-specific Tregs in culture and to transfer these into
graft recipients are ongoing.
• There has been some success reported in recipients of hematopoietic stem cell
transplants, in whom infusions of Tregs reduce GVHD.

Allograft Rejection
• Liver transplants frequently survive and function even with little or no
immunosuppressive therapy.
• “Operational tolerance”
• It is not clear in most cases if alloreactive T cell responses are reduced or
extinguished. It is also not known why the liver is unique among transplanted organs
in its ability to resist rejection.

Hematopoietic Stem Cell (HSC)
Transplantation
• Indications, Methods, and Immune Barriers in Hematopoietic
Stem Cell Transplantation
• Immunologic Complication of Hematopoietic Stem Cell
Transplantation

Transplantation immunology part 1

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