Ch17 transplant immunology (2)

Clinical Immunology & SerologyClinical Immunology & Serology
A Laboratory Perspective, Third EditionA Laboratory Perspective, Third Edition
Copyright © 2010 F.A. Davis CompanyCopyright © 2010 F.A. Davis Company
Transplantation ImmunologyTransplantation Immunology
Chapter Seventeen

Copyright © 2010 F.A. Davis Company
 Transplantation is a potentially lifesaving
treatment for endstage organ failure, cancers,
autoimmune diseases, immune deficiencies,
and a variety of other diseases.
 An allogeneic response within the recipient
may result in graft rejection in solid-organ and
stem cell transplantation or graft-versus-host
disease in stem cell transplantation.

 The classical (transplant) HLA antigens,
also known as major histocompatibility
antigens, include the class I (HLA-A, B, and
C) and class II (HLA-DR, DQ, and DP)
proteins.
 HLA proteins are encoded by a set of closely
linked genes on the short arm of chromosome
6 in the major histocompatibility complex
(MHC).

 The HLA genes are inherited as haplotypes
from parental chromosomes (see Fig. 17-1).
 Offspring receive one maternal and one
paternal HLA haplotype.

Figure 17-1

 HLA proteins are heterodimeric molecules.
 Class I proteins are the product of the HLA-A,
B, and C genes and are expressed on the cell
surface covalently bound to beta-2-
microglobulin.
 Class I heterodimers are codominantly
expressed on virtually all nucleated cells.

 Class II heterodimers are the products of the
HLA-DRB1 + DRA1, HLA-DQB1 + DQA1, and
DPB1 + DPA1 genes.
 Class II heterodimers are codominantly
expressed primarily on antigen-presenting
cells (e.g., dendritic cells, monocytes,
macrophages, B lymphocytes).

 The HLA system is the most polymorphic
genetic system in humans (see Table 17-1).
 While this has successfully enabled
populations to survive infectious challenges, it
severely restricts the ability to transplant
foreign tissues or cells between any two
individuals.

 A second set of transplantation antigens was
identified based on studies in mice and
humans demonstrating tissue rejection in
MHC-identical transplants and based on
outcomes of human stem cell transplants
between HLA-identical siblings in whom graft-
versus-host disease has developed.

 Early experimental studies documented a
“slower” rejection pace mediated by these
transplantation antigens.
 Thus the name—minor histocompatibility
antigens (mHAs).

 The MHC class I–related chain A (MICA)
encodes a cell surface protein that is involved
in gamma/delta T-cell responses.
 MIC proteins are expressed on endothelial
cells, keratinocytes, fibroblasts, epithelial cells,
dendritic cells, and monocytes, but they are
not expressed on T or B lymphocytes.

 MICA antibodies have been associated with
rejection episodes and decreased graft
survival in as many as 11 percent of kidney
transplant patients.

 ABO blood group incompatibility is a barrier
to solid-organ transplantation, because these
antibodies can bind corresponding antigens
expressed on the vascular endothelium.
 Binding activates the complement cascade,
which can lead to hyperacute rejection of the
transplanted organ.
 As a result, recipient–donor pairs must be
ABO identical or compatible.

 Another polymorphic genetic system that
impacts allogeneic transplantation is the killer
immunoglobulin-like receptor (KIR) system.
 KIRs are one of several types of cell surface
molecules that regulate the activity of
natural killer (NK) lymphocytes.

 Within the KIRs are activating and inhibitory
receptors that vary in number and type on any
individual NK cell.
 The balance of signals received by the
activating and inhibitory receptors governs the
activity of the NK cell (see Fig. 2-12).

 Transplantation of cells or tissues between
two individuals is classified by the genetic
relatedness of the donor and the recipient.
 An autograft is the transfer of tissue from one
area of the body to another of the same
individual.
 A syngeneic graft is the transfer of cells or
tissues between identical twins.

 An allograft is the transfer of cells or tissue
between two individuals of the same species
(accounting for most transplantation).
 A xenograft is the transfer of tissue between
two individuals of different species.
 The recipient immune system recognizes
foreign HLA proteins via two distinct
mechanisms—direct and indirect
allorecognition (see Fig. 17-2).

Figure 17-2

 In direct allorecognition, recipient T cells
bind and respond directly to foreign (allo) HLA
proteins on graft cells (Fig. 17-2A).
 Indirect allorecognition is the second
pathway by which the immune system
recognizes foreign HLA protein (Fig. 17-2B).

Transplantation Immunology; 17-2Transplantation Immunology; 17-2
Figure 17-2

 The effector responses against
transplanted allogeneic tissue include direct
cytotoxicity, delayed-type hypersensitivity
responses, and antibody-mediated
mechanisms.
 Rejection episodes vary in the time of
occurrence and the effector mechanism that is
operative.

 Hyperacute rejection occurs within minutes
to hours after the vascular supply to the
transplanted organ is established.
 This type of rejection is mediated by
preformed antibody that reacts with donor
vascular endothelium, leading to clotting.
 ABO, HLA, and certain endothelial antigens
may elicit hyperacute rejection.
 This type of rejection is seldom encountered.

 Acute cellular rejection may develop days to
weeks after transplant.
 This is a cellular-type rejection but may also
involve antibodies.
 Antibody may be involved in acute graft
rejection by binding to vessel walls, activating
complement, and inducing transmural necrosis
and inflammation as opposed to the
thrombosis typical of hyperacute rejection.

 Chronic rejection results from a process of
graft arteriosclerosis characterized by
progressive fibrosis and scarring with
narrowing of the vessel lumen due to
proliferation of smooth muscle cells.
 May be due to minor MHC antigens.

 Stem cell transplants (and, less commonly,
lung and liver transplants) are complicated by
a unique allogeneic response—graft-
versus-host disease (GVHD).
 Recipients of stem cell transplants for
hematologic malignancies typically have
depleted bone marrow prior to transplantation.

 Donor bone marrow or, more commonly,
peripheral blood stem cells are infused.
 The infused products often contain some
mature T cells.
 Beneficial effects of these T cells include
promotion of engraftment, reconstitution of
immunity, and mediation of a graft-versus-
leukemic cell effect.

 However, these mature T cells may also
mediate GVHD by a massive release of
cytokines due to large-scale activation of the
donor cells by MHC mismatched proteins and
by infiltration and destruction of tissue .
 Acute GVHD occurs during the first 100 days
postinfusion and targets the skin,
gastrointestinal tract, and liver.

 Immunosuppressive agents are used in
several ways, including induction and
maintenance of immune suppression and
treatment of rejection.
 Combinations of different agents are
frequently used to prevent graft rejection.

 The immunosuppressed state (and graft
survival) comes at a price of increased
susceptibility to infection, malignancies, and
other associated toxic side effects.
 Immunosuppressive agents include
corticosteroids, anti-metabolic agents,
calcineurin Inhibitors, monoclonal antibodies,
and polyclonal antibodies.

 Two main activities carried out by
histocompatibility laboratories in support
of transplantation include HLA typing and
HLA antibody screening/identification.
 HLA typing is the phenotypic or genotypic
definition of the HLA antigens or genes in a
transplant candidate or donor.

 The classic procedure for determining the
HLA phenotype is the complement-
dependent cytotoxicity (CDC) test.
 Panels of antisera or monoclonal antibodies
that define individual or groups of
immunologically related HLA antigens are
incubated with lymphocytes from the individual
to be HLA typed.

 Purified T lymphocytes are used for HLA class
I typing, while purified B lymphocytes are used
for HLA class II typing.
 After incubation with the antisera, complement
is added.
 A vital dye is then added that distinguishes live
cells from dead when they are viewed
microscopically.

 Using this assay, an extensive array of HLA
antigens can be defined (see Table 17-1).
 There are several limitations to the CDC
method for HLA typing.
 For unrelated stem cell transplantation, a
higher level of resolution is required, such as
that offered by DNA-based (molecular) HLA
typing methods.

 Molecular-based HLA genotyping methods
use polymerase chain reaction (PCR)–based
amplification of HLA genes followed by
analysis of the amplified DNA to identify the
specific HLA allele or group of alleles.
 The most common approaches for analysis
include PCR amplification of HLA genes with
panels of primer pairs, each of which amplifies
specific alleles or related allele groups.

 Amplification is detected by agarose gel
electrophoresis (see Fig. 17-3).

Figure 17-3

 HLA genotyping overcomes the limitation of
CDC-based HLA phenotyping.
 HLA genotyping can provide varying levels of
resolution that can be tailored to the specific
clinical need.

 DNA-based typing can provide results at a
level of resolution comparable to CDC-based
typing (antigen equivalent) or can provide
allele-level results (required for matching of
unrelated stem cell donors and recipients)
(see Table 17-1).

 Antibodies to HLA antigens can be
detected in candidates and recipients of
solid-organ transplants.
 If detected, the specificity (which HLA proteins
they bind) of the antibodies is then determined
so that donors possessing those HLA antigens
can be eliminated from consideration for
donation to that patient.

 The CDC method used for HLA typing is
also used for HLA antibody detection and
identification.
 In this case, panels of lymphocytes with
defined HLA phenotypes are incubated with
the patient’s serum.
 If the serum contains HLA antibodies, they will
bind to those lymphocytes in the panel that
express the cognate HLA antigen.

 Binding is detected by addition of complement
and a vital dye to assess cell death
microscopically.
 The proportion of lymphocytes in the panel
(usually 30–60 unique lymphocyte
preparations are included in the panel) that
are killed by the patient’s serum is referred to
as the percent panel reactive antibody
(%PRA).

 In addition, the specificity of the antibodies can
be determined by evaluating the phenotype of
the panel cells.
 Enzyme-linked immunosorbent assay
(ELISA) has been developed in recent years
as a substitute for CDC-based HLA antibody
testing.

 ELISA assays utilize purified HLA antigens
bound to the wells of microtiter plates.
 Patient serum is added to the wells of the
plate, and if HLA-specific antibody is present,
it will bind to the antigen.
 Bound antibody is detected by addition of an
enzyme-labeled anti-immunoglobulin reagent.
 Addition of substrate results in a color change
in the wells that have bound antibody.

 Another approach for antibody detection
and identification is flow cytometry.
 Antibody in patient serum can be incubated
with latex beads that are coated with HLA
antigens, either from a single donor or a single
purified HLA protein.

 Patient serum is incubated with the beads,
and bound antibody is detected by adding an
FITC-labeled anti-IgG reagent (see Fig. 17-4).

Figure 17-4

 Flow cytometric methods are the most
sensitive technology for detecting HLA
antibodies.
 In addition, they can provide the most specific
determination of the specificity of HLA
antibodies when beads coated with a single
HLA antigenic type are used.

 Once a donor has been identified for a
particular patient, a donor–recipient
crossmatch test is performed to confirm
the absence of donor-specific antibody.
 Donor lymphocytes are incubated with
recipient serum in a CDC assay to verify a
lack of binding as detected by microscopic
analysis after addition of a vital dye.

 Alternatively, binding of antibody can be
detected by flow cytometry using an FITC-
labeled anti- IgG reagent.
 As for antibody screening and identification,
the flow cytometric crossmatch is the most
sensitive method for detecting donor-specific
antibody.

Ch17 transplant immunology (2)

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