Transplant Immunobiology

Phuong-Thu T. Pham

Qiuheng J. Zhang

Michelle J. Hickey

ABO BLOOD GROUP ANTIGENS

The ABO blood group antigens are expressed on the surface of red blood cells as well as in the kidneys, gastrointestinal, respiratory, and other organ systems. The ABO blood group is the most important tissue barrier to successful kidney transplantation, followed by the major histocompatibility complex (MHC) antigens. Non-MHC molecules, referred to as minor histocompatibility complex antigens, can also mediate rejection. Whereas transplant from an identical twin requires no immunosuppression, 2-haplotype-matched siblings (fraternal twins or non-twin siblings) require immunosuppressive therapy to prevent graft rejection
ABO-incompatible kidney transplantation results in hyperacute rejection and graft loss.
A number of variant A antigens are known, with the A1 antigen providing more potent antigenicity than the A2 antigens. Successful transplantation can be performed using A2 kidneys into O recipients and A2 and A2B kidneys into B recipients.¹
Within the last 10 to 15 years, various desensitization protocols have allowed successful ABO-incompatible kidney transplantation.² Discussion is beyond the scope of this chapter.

MAJOR HISTOCOMPATIBILITY COMPLEX, HUMAN LEUKOCYTE ANTIGEN MOLECULES

The MHC genes are located on the short arm of chromosome 6 and represent the most polymorphic genes in human genome. The extensive polymorphism in the MHC, driven mainly by recombination between genes, leads to wide-ranging human genetic variability.³
Human leukocyte antigens (HLAs) are glycoproteins encoded by the MHC genes. In humans, the MHC molecule was first discovered in leukocytes; therefore, it is also called the human leukocyte antigen.
In kidney transplantation HLAs are the predominant antigens that form the targets for the immune response.

Over 17,600 distinct HLA alleles have been defined through DNA sequencing.⁴ Despite significant diversity at the level of DNA, the majority of polymorphisms that stimulate alloactivation of the recipient’s immune system are located in the α1 and α2 chains of HLA class I, and α1 and β1 chains of class II (Figure 1-1).

Figure 1-1 Schematic Structures of Class I and II Human Leukocyte Antigen Molecules The human leukocyte antigen (HLA) class I antigen is composed of a polymorphic α chain comprising α₁, α₂, and α₃ domains and a nonpolymorphic light chain, β₂-microglobulin. Class II antigen is composed of polymorphic α and β chains covalently bound to each other.

Polymorphisms in noncoding regions of the gene, as well as nucleotide differences that are synonymous at the protein level, or mutations in regions of the mature protein that are inaccessible to immune recognition (transmembrane and intracellular regions) are unlikely be recognized by the immune system. Alloantibody of the immunoglobulin G (IgG) subclass is generated by the patient in response to recognition of mismatched donor HLA antigens. Solid phase antibody detection methods (described below) identify antibodies to ˜90 HLA class I or II antigens.

HLA class I (see Figure 1-1)

The classical HLA class I antigens (HLA-A, HLA-B, and HLA-C) are composed of a polymorphic α or heavy chain of 44 kDa and a nonpolymorphic light chain, β₂-microglobulin of 12 kDa.³
They are encoded by the HLA-A, HLA-B, and HLA-Cw genes.
They are expressed on all nucleated cells and generally present peptides derived from intracellular proteins (eg, viral proteins) to cytotoxic CD8⁺ T cells (Figure 1-2).

Figure 1-2 Antigen Presentation

The endogenous pathway: Endogenous antigens are digested into peptides and loaded into class I major histocompatibility complex (MHC). The MHC-peptide complex is assembled within the cell’s endoplasmic reticulum, transported through the Golgi apparatus and expressed on the cell surface where it is recognized by CD8⁺ T-cell receptor (TCR), leading to T-cell activation. Exogenous pathway: Exogenous antigens are degraded within endosomes and loaded into class II MHC. The MHC-peptide complex is ultimately expressed on the cell surface where it is recognized by CD4⁺ TCR, leading to T-cell activation.

HLA class II (see Figure 1-1)
- The classical class II antigens (HLA-DP, HLA-DQ, and HLA-DR) are composed of polymorphic α and β chains of similar molecular weight (32 kDa), covalently bound to each other. These antigens are encoded by the DPA, DPB, DQA, DQB, DRA, DRB genes.³
- Note: All HLA-DR types have the DRB1 gene, but some contain an additional functional DRB gene, namely, the DRB3, DRB4, or DRB5 genes. These genes code for the HLA-DRB3, HLA-DRB4, and HLA-DRB5 antigens, respectively.⁵
- HLA class II are constitutively expressed only on professional antigen-presenting cells (APCs) including dendritic cells, macrophages, and B lymphocytes. Their expression may be upregulated on epithelial and vascular endothelial cells after exposure to proinflammatory cytokines.³
Class II molecules present larger peptides derived from extracellular proteins (eg, bacterial proteins) to CD4⁺ T cells (see Figure 1-2).
Each parental chromosome 6 provides a linked set of MHC genes (called a haplotype) to the offspring in mendelian codominance inheritance. Statistically, there is a 25% chance that siblings share the same 2 haplotypes (2-haplotype match), a 50% chance they share 1 same haplotype (1-haplotype match), and a 25% chance they do not share any of their parental haplotypes (0-haplotype match or 2-haplotype mismatch).³ By definition, a child is a 1-haplotype match to each parent unless recombination has occurred (Figure 1-3).
The degree of HLA mismatch between donor and recipient plays an important role in rejection risk and graft loss.⁶
Currently, kidney donors and recipients in the United States are typed for HLA-A, HLA-B, HLA-Cw, HLA-DRB1, HLA-DRB3/4/5, HLA-DQB1, HLA-DQA1, and HLA-DPB1. The United Network for Organ Sharing (UNOS) uses HLA-A, HLA-B, and HLA-DRB1 matching as part of the donor allocation algorithm. In the setting of kidney transplant, fewer HLA mismatches correlates to better graft survival.⁷

Terms used for HLA match (or mismatch)

Kidney from parent (father or mother): 1-haplotype match
Kidney from siblings: 2-haplotype match, 1-haplotype match, or 2-haplotype mismatch (see Figure 1-3)

Kidney from deceased donor when considering HLA-A, HLA-B, and HLA-DR

1 of 6 HLA match (or 5 HLA mismatch)

2 of 6 HLA match (or 4 HLA mismatch)

Figure 1-3 Inheritance of Haplotypes and Human Leukocyte Antigen Profile in Four Theoretical Siblings

Sibling 1 is a 1-haplotype match to sibling 2, a 2-haplotype match to sibling 3, and a 2-haplotype mismatch to sibling 4.

3 of 6 HLA match (or 3 HLA mismatch)
4 of 6 HLA match (or 2 HLA mismatch)
5 of 6 HLA match (or 1 HLA mismatch)
6 of 6 HLA match (or 0 HLA mismatch)
Example: Consider the HLA phenotype of the following recipient/donor pair:
- Recipient: A 1, 2; B 18, 7; DR 4, 17
- Donor: A 1, 3; B 18, 41; DR 11, 9
- The donor is a 4 HLA-A, HLA-B, HLA-DR antigen mismatch from the recipient.

One-year graft survival is more related to HLA class II mismatching than to class I mismatching.

HUMAN LEUKOCYTE ANTIGEN-TYPING TECHNIQUES

Serotyping

HLA typing by serologic methods was previously performed using the complementdependent microlymphocytotoxicity test; however, more accurate and higher resolution molecular typing of DNA (described in the following text) has replaced these methods.⁸
- The test is performed in a microtiter plate with multiple small wells.
- Each well is loaded with a selected antiserum, and lymphocytes from the individual to be typed are added.
- After an appropriate incubation period, complement is added.
- Binding of specific anti-HLA antibody to its specific HLA target antigen on the cell surface activates the complement cascade, leading to cytotoxic injury. A vital dye is added to permit visualization of the proportion of dead cells in each well when the tray is examined under phase-contrast microscopy.
- The HLA-typing antiserum does not recognize all antigens and is considered low resolution.

DNA typing

DNA-based tissue typing uses standardized synthetic probes and primers, or sequencing, to determine an individual’s HLA tissue type.⁸
DNA probes hybridize to the complementary DNA nucleotide sequence that is unique to an HLA locus, allele, or groups of alleles. DNA hybridization probe techniques allow identification at the “antigen level” with varying levels of resolution based on the method used (low to intermediate resolution), whereas sequencing provides highresolution “allele level” HLA typing.
Molecular-based HLA typing reveals a much greater degree of polymorphism of the individual HLA than that detected by serologic tests.
Generally, DNA isolated from blood anticoagulated with acid citrate dextrose (ACD) is preferred for DNA-based HLA-typing methods; however, any source of cells can serve as a sample for molecular-based tissue typing including samples isolated from biopsy.

Human leukocyte antigen nomenclature

The level of resolution provided by various molecular HLA-typing methods leads to complex HLA nomenclature. Methods that produce low-resolution typing results distinguish an antigen such as “A2,” whereas high-resolution probes make it possible to distinguish alleles of that antigen such as “A*02:01:01:02” (Figure 1-4). Intermediate-resolution HLA-typing results may include a “string” of alleles that cannot be ruled out by the method such as A*02:01/03/09/212.³,⁴

Figure 1-4 Human Leukocyte Antigen Nomenclature

Low-resolution human leukocyte antigen (HLA) typing, performed by serology during the early days of tissue typing, defines the HLA locus (eg, A, B, C, DR) and antigen, shown here as “2.” HLA typing performed by state-of-the-art molecular methods is denoted by an “*”. Intermediate-resolution HLA typing defines the locus, antigen, and a string of alleles that cannot be ruled out by the method “:01/03/09/212,” whereas high-resolution methods define the allele as well as nucleotide substitutions in exons resulting in silent mutations and intronic mutations. Intermediate-resolution HLA typing is generally sufficient for solid organ transplant patients and donors, whereas high-resolution typing is required for bone marrow and peripheral blood stem cell transplant.

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