ch17 transplant immunology (2)

Clinical Immunology & SerologyClinical Immunology & SerologyA 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 ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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).



Transplantation ImmunologyTransplantation Immunology The HLA genes are inherited as haplotypes

from parental chromosomes (see Fig. 17-1). Offspring receive one maternal and one

paternal HLA haplotype.



Transplantation ImmunologyTransplantation ImmunologyFigure 17-1



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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).



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology Early experimental studies documented a

“slower” rejection pace mediated by these transplantation antigens.

Thus the name—minor histocompatibility antigens (mHAs).



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology MICA antibodies have been associated with

rejection episodes and decreased graft survival in as many as 11 percent of kidney transplant patients.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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 ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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).



Transplantation ImmunologyTransplantation Immunology 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; 17-2Figure 17-2



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology Amplification is detected by agarose gel

electrophoresis (see Fig. 17-3).



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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).



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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).



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology Patient serum is incubated with the beads,

and bound antibody is detected by adding an FITC-labeled anti-IgG reagent (see Fig. 17-4).



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.



Transplantation ImmunologyTransplantation Immunology 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.

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