Priniciples of transplantation

Dr. Shahab Qadir

 Classification of grafts

* Autologous grafts
Grafts transplanted from one part of the body to another in the same
individual
* Syngeneic grafts (Isografts)
Grafts transplanted between two genetically identical individuals of
the same species
Allogeneic grafts (Allografts)
Grafts transplanted between two genetically different individuals of
the same species
Xenogeneic grafts (Xenografts)
Grafts transplanted between individuals of different species
2006-7year Immunology 3
 IMMUNE RESPONSES TO TRANSPLANTED TISSUES

* Transplant rejection caused by genetic differences between donor and

recipient
* HLA and blood group antigens

* Alloantigens
* Antigens which vary between members of same species

* Alloreaction
* Immune response to an alloantigen

* Alloreactions in transplantation
* Host-versus-graft (transplant rejection)
* Graft-versus-host
Figure 12-11
 Generation Of Alloimmune Response

* ANTIGEN PRESENTATION:
* Antigen Presenting Cells:
* Specialized cells capable Of activating T cells.
* Antigen is endocytosed by APCs and then displayed by MHC
molecules on their surface.
* DCs, macrophages, and B cells are considered “professional APCs,”
although DCs are the most potent at antigen presentation
* DCs of either donor or host origin become activated and move to T
cell areas of secondary lymphoid organs (SLOs).
* The movement of DCs and naïve T cells is coordinated to bring them
into contact in the T cell areas of SLOs
* Once activated, T cells leave SLOs via lymph vessels and enter the blood
and peripheral tissues, particularly into sites of inflammation
* B cells are activated when antigen engages their antigen receptors,
initially in the border of T and B cell areas of SLOs, where helper T cell
function is provided.
 Major Histocompatibility Complex Specificity

* Allografts induce alloimmune responses by the recognition of nonself antigens

(e.g., MHC) from the graft by recipient T cells.
* T cell ontogeny in embryogenesis occurs resulting in TCR recognition of self
and non-self antigens, so that they respond to multiple foreign antigens.
* The mature T cell repertoire is determined in the thymus by two processes,
positive and negative selection.
* Positive Selection:
* This process ensures that mature T cells will interact effectively with
MHC to allow recognition of foreign antigen in the context of self-MHC
* Negative Selection:
* Negative selection occurs by deletion of T cells with excessively high
affinity for self-peptide and MHC, thereby preventing release of high-
affinity T cells with autoimmune potential.
* Mature T cells expressing their clone-specific TCRs exit the thymus as either
CD4 or CD8 T cells.

* The TCRs of CD4 T cells (also called T helper cells) are selected to interact
with class II MHC molecules.
* The TCRs of CD8 T cells (precursors of cytotoxic T lymphocytes [CTLs])
interact with class I MHC molecules.
* Fundamental principle is that T cells do not recognize intact foreign proteins
directly, but instead as peptides presented by self-MHC on APCs.
* However, allelic variation among MHC molecules from individual to
individual is quite small, resulting in similarities between donor and
recipient MHC structure.
* recipient TCRs have a strong affinity for intact donor MHC
molecules and can recognize them directly
Pathways of Allorecognition
• Direct Antigen Presentation:
• dominant pathway involved in the early alloimmune response
• has not been demonstrated outside of alloimmunity
• Indirect Antigen Presentation:
• has not been demonstrated outside of alloimmunity
• Semi-Direct Antigen Presentation:
• semidirect antigen presentation can occur through the transfer of intact
donor MHC:peptide complexes contained in extracellular vesicles derived
from the surface of donor APCs or from the graft to the surface of
recipient APCs for presentation to recipient T cells, a process called cross-
dressing or cell nibbling.
 MHC complex

* Encode molecules crucial to the initiation and

propagation of immune response
* The HLA complex on chromosome 6 contains
over 200 genes, more than 40 of which encode
leukocyte antigens
* The HLA genes that are involved in the immune
response fall into two classes, I and II, which are
structurally and functionally different
Location and Organization of the HLA Complex on Chromosome 6
 Types of MHC

* There are three classes of MHC molecules.

* Class I- encodes glycoproteins expressed on the surface of nearly all nucleated cell; the major
function of the class I gene is presentation of peptide antigens to cytotoxic T-cells

* Class II- encodes glycoproteins expressed primarily on antigen-presenting cells, examples:

macrophages, dendritic cells and B-cells, where they present processed antigenic peptides to
T helper cells.

* Class III- encodes various secreted proteins that have immune function including components
of the complement system; C2,C4, Factor B, &TNF, and molecules involved in
inflammation.
 Nucleated cells

Class I MHC

RBCs

Class II MHC

APCs
 Function of MHC

* The function of both class I and class II molecules is

the presentation of short, pathogen-derived peptides to
T cells, a process that initiates the adaptive immune
response
* Class I - Sample cytosolic proteins and detect foreign
proteins that would indicate an intracellular pathogen
such as virus or intracellular bacteria
* Recognised by CD 8 T cells and provide a
surviellance mechanism to target infected cells for
destruction
Antigen Processing and Presentation
Biological functions of Class I and Class II molecules

* Class I
* Present peptides derived from
endogenously synthesized proteins
* Responding T cells express CD8+
* Class II MHC is designated to sample
extracellular proteins by extracellular proteins by
specialized APC’s
* Class II are recognized by CD 4 helper T cells and
allow for the generation of immune response to
invading pathogens
Antigen Processing and Presentation
Biological functions of Class I and Class II molecules

* Class II
* Present peptides derived from
exogenously synthesized proteins
* Responding T cells express CD4+
 Class I

* The class I genes code for the polypeptide chain of the

class I molecule; the ß chain of the class I molecule is
encoded by a gene on chromosome 15, the beta2-
microglobulin gene.
* There are some 20 class I genes in the HLA region; three
of these, HLA-A, B, and C, the so-called classic, or class Ia
genes, are the main actors in the immunologic theater
 Structure of Class I MHC

* Two polypeptide chains, a long α chain and a

short β (β2 microglobulin)

* Four regions
* Cytoplasmic region containing sites for
phosporylation and binding to
cytoskeletal elements

* Transmembrane region containing

hydrophobic amino acids
 Structure of Class I MHC

* Four regions
* A highly conserved α3 domain to
which CD8 binds
* A highly polymorphic peptide binding
region formed from the α1 and α2
domains
* Β2-microglobulin helps stabilize the
conformation
Structure of HLA Class I and Class II Molecules
 Class II

* The class II genes code for the alpha and ß polypeptide

chains of the class II molecules .
* The designation of their loci on chromosome 6 consists of
three letters: the first (D) indicates the class, the second
(M, O, P, Q, or R) the family, and the third (A or B) the
chain ( or ß, respectively).
* HLA-DRB, for example, stands for class II genes of the R
family coding for the ß chains.
 Structure of Class II MHC

* Two polypeptide chains,α and β, of

roughly equal length
* Four regions
* Cytoplasmic region containing sites
for phosporylation and binding to
cytoskeletal elements
 Structure of Class II MHC

* Four regions
* Transmembrane region containing
hydrophobic amino acids
* A highly conserved α2 and a highly
conserved β2 domains to which CD4
binds
* A highly polymorphic peptide binding
region formed from the α1 and β1
domains
Structure of HLA Class I and Class II Molecules
 Important aspects of MHC

* Normally, the proteins that undergo recycling are the organism's own, but in infected
cells, proteins originating from the pathogen are also routed into the processing
pathways.
* With the exception of jawed vertebrates, no organisms appear to make a distinction
between peptides derived from their own (self) proteins and those derived from foreign
(nonself) proteins.
* Jawed vertebrates, by contrast, use the peptides derived from foreign (usually microbial)
proteins to mark infected cells for destruction
 Important aspects of MHC

* Protein processing and loading of peptides onto class I molecules are taking place all the
time in most cells. There is always plenty of material to feed the processing machinery,
because worn-out, damaged, and misfolded proteins are continuously being degraded and
replaced by new ones.
* By contrast, the processing of exogenous proteins and the loading of peptides onto class
II molecules are normally restricted to B cells, macrophages, and dendritic cells, which
are very efficient in taking up material by endocytosis or phagocytosis.
 Important aspects of MHC

* The consequence of protein processing is that the surfaces of cells become adorned with
peptide-laden HLA molecules, amounting on a per cell basis to roughly 100,000 to
300,000 class I or class II products of each of the highly expressed HLA loci.
* Since each HLA molecule has one peptide bound to it, each uninfected cell displays
hundreds of thousands of self peptides on its surface.
* Each cell thus displays a heterogeneous collection of peptides, and the surface of a cell
resembles rows of well-stocked stalls at a bazaar, with bargain hunters scrutinizing the
wares.
* But if, in this metaphor, the vendors are the HLA molecules and the peptides the goods,
who are the potential buyers? They are a group of lymphocytes reared in the thymus and
then turned loose to roam the body — the T cells.
 Functions and Characteristics of HLA

* HLA’s are cell-surface proteins involved in the recognition of self and non-self by the
immune system

* HLA’s present foreign antigens to the immune system – resistance to viral and bacterial
pathogens

* HLA’s are codominantly expressed

* Highly polymorphic and polygenic

HLA genes are co dominant: A protein from
each parental gene is expresed on cell-
surfaces
 Polymorphism and polygeny

* MHC genes are polymorphic: that is, there

are large numbers of alleles for each gene

* MHC genes are polygenic: that is, there are

a number of different MHC genes.
 Crossing over
results in new haplotypes
 Structure of Class I MHC

Variability map of Class 1 MHC α Chain

 Class I polymorphism

Number of alleles
Locus (allotypes)
HLA - A 3657
HLA - B 4459
HLA - C 2208
There are also HLA - E, HLA Relatively few alleles
- F and HLA - G
 Structure of Class II MHC

Variability map of Class2 MHC β Chain

 Class II polymorphism

Number of alleles
Locus (allotypes)
HLA - DPA 3657
HLA - DPB
HLA - DQA 4459
HLA - DQB
HLA - DRA 2208
HLA - DRB1
HLA – DRB3
HLA – DRB4
HLA – DRB5

There are also HLA - DM and HLA - DO Relatively few alleles

 Polymorphism

* Multiple alleles of HLA in a population increases the

likelihood that the population will survive a pathogen
threat

* Unfortunately, it also cause histoincompatibility in organ

and tissue transplants
 Important Aspects of MHC

* Primary HLA products that contribute to rejection are the

most polymorphic including HLA- A, - B and DR
* Efforts are made to match HLA-A, - B and DR genes and
proteins in kidney transplantation
Response to antigenic stimulus
 T cell activation

* Activation of T cell is a crucial step in generation of immune response

to specific antigens
* Naive T cells restricted to SLOs
* They interact with DCs that have migrated from the periphery in
response to infection or injury
* Once naïve T cells encountered their cognate antigen presented on
mature DCs, they become activated.
* Following activation CD 4 T cells help B cells to convert to plasma
cells
* Plasma cells produce antibodies
 T Cell Activation

* T Cell Receptor :
* T cell bears about 30,000 identical antigen-receptor molecules
* receptor consists of two different polypeptide chains, termed the TCR α
and β chains
* The α:β heterodimers are very similar in structure to the Fab fragment of
an immunoglobulin molecule
* recognize a composite ligand of a peptide bound to an MHC molecule
 Structure of the T cell Receptor

* Heterodimer with one α

and one β chain of
roughly equal length
* A short cytoplamic tail
not capable of
transducing an activation
signal
* A transmembrane region
with hydrophobic amino
acids
 Structure of the T cell Receptor

* Both α and β chains

have a variable (V) and
constant (C) region
* V regions of the α and β
chains contain
hypervariable regions
that determine the
specificity for antigen
 Structure of the T cell Receptor

* Each T cell bears TCRs

of only one specificity
(allelic exclusion)
• CD4 and CD8
Coreceptors
• CD4 and CD8 binding
to MHC are required
to make an effective
response. Thus these
molecules are called
coreceptors
• T Cell Receptor
Engagement of
Antigen: Signal 1
• T Cell
Costimulation:
Signal 2
 TCR and CD3 Complex

* TCR is closely
associated with a
group of proteins
collectively called the
CD3 complex
* γ chain
* δ chain
* 2 ε chains
* 2 ξ chains
* CD3 proteins are
invariant
 Role of CD3 Complex

* CD3 complex
necessary for cell
surface expression of
TCR during T cell
development
* CD3 complex
transduces signals to
the interior of the cells
following interaction
of Ag with the TCR
 T Cell Co-stimulation

* The interaction between

the TCR and MHC
molecules are not strong
* Accessory molecules
stabilize the interaction
* CD4/Class II MHC or
CD8/Class I MHC
* CD2/LFA-3
* LFA-1/ICAM-1
 The “Immunological Synapse”

* Specificity for antigen

resides solely in the TCR
* The accessory molecules
are invariant
* Expression is increased
in response to cytokines
 The “Immunological Synapse”

* Engagement of TCR and Ag/MHC is

one signal needed for activation of T
cells
* Second signal comes from
costimulatory molecules
* Positive Co-stimulation
* CD28/ B7.1,B7.2
* iCOS/ iCOS-L
* CD40/ CD40-L
* Negative Co-stimulation
* CTLA-4/ B7
* BTLA/ HVEM
* PD-1/ PD-L1, PD-L2
* Costimulatory molecules are invariant
* “Immunological synapse”
 Costimulation is Necessary for T Cell Activation

* Engagement of TCR and

Ag/MHC in the absence of
costimulation can lead to anergy
* Engagement of costimulatory
molecules in the absenece of
TCR engagement results in no
response
* Activation only occurs when
both TCR and costimulatory
molecules are engaged with
their respective ligands
* Downregulation occurs if
CTLA-4 interacts with B7
* CTLA-4 send inhibitory signal
 Clonal expansion

* Signals 1 and 2 activate calcium calcineurin pathway,

MAP kinase pathway and NF-Kb pathway
* These pathways activate transcription factors that trigger
the expansion of many new molecules including IL-2,
CD 154 and CD 25.
* IL-2 and other cytokines activate the TOR pathway to
provide signal 3, the trigger for cell proliferation
* A subset of activated helper T cells migrate to the B region
of lymph nodes located in the cortex and help to
differentiate B cells while the remainder of the effector T
cells leave the lymph node and proceeds the inflamed site
* The activated T cells rapidly accumulate in the interstitium
of the allograft as the response mounts in the first few
days.
* CD4 T cells are cytokine secreting cells that express IL-2
and alter a variety of cytokines
* CD4 cells help B cells to enhance their antibody
production through CD40 ligand
* Alloantibody produced during rejection is mainly IG g and
primarily participates in the destruction of vascular
endothelium of the graft
* CD 8 T cells participate in rejection through DTH or
cytotoxicty
 Key Steps in T cell Activation

* APC must process and present peptides to T cells

* T cells must receive a costimulatory signal
* Usually from CD28/B7
* Accessory adhesion molecules help to stabilize binding of
T cell and APC
* CD4/MHC-class II or CD8/MHC class I
* LFA-1/ICAM-1
* CD2/LFA-3
* Signal from cell surface is transmitted to nucleus
* Second messengers
* Cytokines produced to help drive cell division
* IL-2 and others
Rejection
 1. Hyperacute rejection

* Occurrence time
* Occurs within minutes to hours after host blood vessels are
anastomosed to graft vessels
* Pathology
* Thrombotic occlusion of the graft vasculature
* Ischemia, denaturation, necrosis
 ORIGINS OF ANTIBODIES TO HLA AND ABO ANTIGENS IN HYPERACUTE
REJECTION

* Pregnancy
* Fetus is allograft in mothers body
* During birth, fetal cells can stimulate maternal immune response

* Blood transfusion
* HLA typing not performed for routine transfusion
* Leukocytes and platelets in whole blood

* Transplantation
* Persons with more than one transplant
Figure 12-15
 * Complement activation
* Endothelial cell damage
* Platelets activation
* Thrombosis, vascular occlusion, ischemic damage

2006-7year Immunology 73
 2. Allograft Rejection

* tissue injury produced by the effector mechanisms of the alloimmune

response, leading to deterioration of graft function.
* two types of rejection
* T cell–mediated rejection (TCMR)
* antibody-mediated rejection (AMR)

* caused by several cellular elements of the immune system, including T cells,

macrophages, B cells, plasma cells, eosinophils, and neutrophils
* endothelial and tubular cells are particularly affected by these mediators
 MECHANISM

* T cells serve as the main effectors and regulators of the alloimmune

response, and macrophages serve as possible effectors but also aid in
the removal of apoptotic cells
* B cells and plasma cells serve in the production of alloantibodies, and
neutrophils likely cause significant damage, particularly during AMR

* three-step model has been proposed to explain the entry of cellular

infiltrates into the allograft
* Tethering
* Adhesion
* transmigration
 MECHANISM

* The endothelium of postcapillary venules in the graft serves as the

entry point of recipient leukocytes from the bloodstream.
* Selectins cause leukocytes to roll along the vessel wall, a process
called tethering
* chemokines bind to receptors on leukocytes,
* integrin, LFA-1 cause the adhesion of leukocyte to endothelium via
ligands like ICAM-1,2,3 ( intracellular adhesion molecules)
* This adhesion contributes to antigen recognition, and, through a
process called haptotaxis
* after integrin engagement of their ligands, leukocytes flatten and then
undergo diapedesis through gaps between endothelial cells
 MECHANISM

* Protease degrade basement membrane.

* Once in the interstitium, secretion of matrix metalloproteinases allows
leukocytes to digest extracellular matrix to move through the tissue on
a chemokine gradient by a process called chemotaxis
* Once confronted with foreign antigen in the allograft, previously
activated T cells are capable of releasing proinflammatory cytokines
(helper T cells) or directly killing foreign cells (cytotoxic T cells)
 Acute T Cell–Mediated Rejection

* TUBULITIS
* Deterioration of renal function during TCMR correlates with tubulitis and
arterial inflammation (endothelialitis), which is much less common
* CTLs secrete GRANZYME, Perforins
* Apoptosis by Binding Fas on Target cells by FasL
* T-bet, a master transcription factor for effector Th1 lymphocytes, during
rejection episodes
* Tubulitis may involve a specific subset of CTLs expressing the integrin
CD103, which binds to its ligand E–cadherin on epithelial cells
* Rejection may also occur through Cytotoxicity and DTH
reactions
* Delayed Type Hypersensitivity Reactions:
* DTH responses involve the release of reactive oxygen species, proteolytic
enzymes, eicosanoids, and other products
* These products may act directly on tubular epithelium and interstitial
matrix or indirectly via effects on endothelium and the vascular supply.
* Endarteritis
* Found in minority of biopsies with TCMR
* Very responsive to T cell therapies.
* INTERSTITIAL INFLAMMATION is distinct
* Glomerulitis:
* Occasional Feature.
 Acute Antibody-Mediated Rejection

* Acute AMR may occur with or without a component of TCMR

* Typically a response to donor HLA antigens, ABO blood group antigens ,
endothelial alloantigens, angiotensin II type 1 receptors.
* kidney typically shows an accumulation of T cells, neutrophils, and monocytes
in peritubular and glomerular capillaries (microvascular inflammation)
* Hyperacute rejection, which is due to the presence of preformed antidonor
HLA antibody in sensitized recipients
* Alloantibodies are cytotoxic through their ability to activate complement
* C4d deposition is strongly associated with circulating antibody to donor HLA
class I or II antigens
* Effects of complement include
* Chemoattraction of neutrophils and macrophages via C3a and C5a
* Vasospasm through the release of prostaglandin E2 from macrophages
* Edema through the release of histamine from mast cells
C3a and C5a increase endothelial adhesion molecules and various
cytokines and chemokines
* membrane attack complex, C5b-9, causes lysis of endothelial cells
* Common feature of all types of AMR is the presence of microthrombi
* diagnostic criteria for acute/active AMR
* histologic evidence of acute tissue injury
* immunopathologic evidence of current/recent antibody interaction with
vascular endothelium eg c4d
* evidence of circulating antibody reactive to the donor
 Chronic Rejection

* Late allograft dysfunction is due to both alloimmune mechanisms (i.e.,

chronic rejection) and alloimmune-independent mechanisms .
* Chronic rejection may occur by either cellular or humoral
mechanisms, or both
* Histologic features characteristic of chronic rejection are
* transplant glomerulopathy
* peritubular capillaropathy
* tubular atrophy and interstitial fibrosis
* GBM duplication is caused by multiple insults to the allograft
glomerulus
* fluctuating donor-specific antibody levels are observed in some
patients
* Steps in Transplant Glomerulopathy:
* alloantibody production (I)
* antibody interaction with alloantigens resulting in the deposition of C4d
(II)
* pathologic changes (III)
finally graft dysfunction (IV)
*
 TRANSPLANTATION TOLERANCE

* Transplantation tolerance is a state characterized by the absence of a

destructive immune response in the recipient toward a well-functioning donor
allograft, with a fully intact immune system and no exogenous
immunosuppression
* transplantation tolerance is achieved through control of T cell reactivity by
both central and peripheral mechanisms
* Central tolerance involves thymic deletional mechanisms that eliminate T cells
with reactivity against donor antigens
* Reconstituted donor antigen “reeducates” the thymus to delete developing T
cells with antidonor reactivity, leading to a state of chimerism .
* Peripheral tolerance mechanisms include deletion, anergy, and regulation.
* With self-tolerance, these mechanisms prevent deleterious autoimmune
responses from T cells that escape central deletion
* Hurdle to the development of clinical tolerance strategies is the lack of
reproducible immune monitoring assays for tolerance .