Introduction

The immune system has evolved specifically to recognise and destroy hazardous infective agents such as bacteria and viruses. These same mechanisms function to reject non-self-allogenic tissues, which are an irritant to surgical transplantation. Successful organ transplants occur between monozygotic twins (syngeneic transplants) or between genetically related or unrelated individuals (allogeneic transplants) – but only when the immune system is hindered by effective immunosuppression. The degree of immunosuppression needed, which reflects the frequency and strength of the alloimmune response, will be determined by the immunogenetic disparity between the donor and the recipient. The ability of a recipient to respond to allogeneic tissue will reflect their own immunogenetic constitution – their immune responsiveness. There are two major genetic systems determining human allogenicity, each of which can convey a biological veto or a biological impediment to effective clinical transplantation.

Veto

• antibody to incompatible ABO blood group; or

• mismatched donor human leucocyte antigen (HLA) evidenced by IgG antibody present at the time of transplantation.

Impediment

• low-titre antibody specific for ABO blood group; or

• recipient previously exposed to HLA antigens present in the donor.

If transplantation proceeds, in the biological veto situation then hyperacute rejection of the transplant is highly probable.

In the situation of biological impediment, effective clinical transplantation can only be achieved through use of special protocols to remove circulating antibody (see Chapter 7).

In this chapter we explain the immunology and genetics of the HLA system in the context of clinical organ transplantation, current techniques used to identify the extensive HLA polymorphism at the gene (allele) and protein (specificity) levels, and will detail techniques used to establish recipient allosensitisation. The application of these techniques to attain effective clinical transplantation will be highlighted.

HLA genetics

The human leucocyte antigen (HLA) complex is located on the short arm of chromosome 6 at 6p21.3. This region is also known as the major histocompatibility complex (MHC). This collection of highly polymorphic genes codes for HLA molecules, which are cell-surface proteins that play a pivotal role in antigen presentation and recognition, and hence the survival of transplanted organs and tissues. For the purposes of this chapter the emphasis will be placed on elements of the HLA system that are characterised on a routine basis in the histocompatibility and immunogenetics laboratory.

There are two classes of HLA genes that are involved in the immune response to transplanted organs, class I and class II. These classes are structurally and functionally distinct. Class I molecules are involved in the processing and presentation of intracellular peptide to CD8⁺ T cells. Class II molecules process and present extracellular peptide to CD4⁺ T cells. This process is described in more detail in Chapter 3 on transplantation immunology.

Potential recipients and donors are tested for HLA class I (HLA-A, HLA-B and HLA-Cw) and HLA class II (HLA-DR, HLA-DQ and in some instances HLA-DP) to facilitate organ allocation (HLA matching), crossmatching and antibody screening. There are two means of determining the HLA type of an individual: by serological methods or DNA polymerase chain reaction (PCR)-based techniques.

HLA nomenclature

Nomenclature for both accepted and novel HLA alleles is regulated by the World Health Organisation (WHO) Nomenclature Committee for factors of the HLA system.¹ HLA sequences are officially recorded on the IMGT/HLA Sequence Database (www.ebi.ac.uk/imgt), which is updated quarterly and is part of the International Immunogenetics Project (IMGT). HLA genes are highly polymorphic. In January 2008, this database contained sequences for 2047 class I alleles and 944 class II alleles.

Over time, major revisions of HLA nomenclature are necessary due to the ever-increasing numbers of alleles identified. A guide to the most recent nomenclature for HLA antigens and alleles is summarised in Table 4.1, where resolution of HLA alleles to the four-digit level is shown. In practice matching beyond this level, even in stem cell transplantation, is impractical. Resolution to eight digits can be carried out and in some instances an alphabetical suffix is used to describe the biological expression of an encoded molecule.

Table 4.1 HLA nomenclature: basic overview of the levels of HLA typing performed in the histocompatibility laboratory

WHO nomenclature	Interpretation
HLA-B	Identification of HLA locus
HLA-B44	HLA defined by serology-based technique
HLA-B*44	Asterisk denotes HLA alleles defined by analysis of DNA
HLA-B*44 two-digit resolution	Denotes the allele family
	Corresponds where possible to the serological group
	Often termed ‘low resolution’
	Level used for matching in solid organ transplants
HLA-B*4402 four-digit resolution	Allele sequence variation results in amino acid substitutions, coding variation or non-synonymous changes
HLA-B*4402 four-digit resolution	Level of matching used in haemopoietic stem cell transplantation

HLA typing

Serologically based techniques: complement-dependent cytotoxicity (CDC)

In a CDC test, antisera with specificity for HLA antigens are incubated with peripheral blood lymphocytes in the presence of complement. The antiserum used is usually monoclonal. If the target lymphocytes carry HLA antigen(s) to which the antiserum has specificity the test leads to killing of the target cells. In order to visualise the cell death a cocktail of fluorescent dyes is added to distinguish between live cells (negative test) and dead cells (positive test). CDC serological HLA typing identifies HLA protein polymorphisms as expressed at the cell surface.

Molecular techniques

Molecular techniques for HLA typing of DNA sequence polymorphisms have largely replaced serology since they offer flexibility of resolution, much improved reproducibility and greater accuracy. The invention of the polymerase chain reaction (PCR)² has revolutionised HLA typing techniques by facilitating identification of HLA polymorphisms at the single nucleotide polymorphism (SNP) level.

Principle of the polymerase chain reaction

Use of the PCR allows amplification of selected regions of interest within a length of target DNA. The technique involves heat separation of double-stranded DNA, primer annealing and extension, resulting in exponential amplification of the template DNA. The essential agent is Taq polymerase, which is a thermostable enzyme that facilitates nucleotide extension from primer pairs, constructing a DNA copy of the template DNA strand. Primers are chemically synthesised oligonucleotides, usually 17–30 nucleotides in length. The primers are designed to flank the region of interest by binding to complementary sequences on the target DNA. Adenine (A) binds to thymine (T) and guanine (G) to cytosine (C) via hydrogen bonds. The PCR mixture contains the target DNA, the primers, the four deoxyribonucleotide triphosphate building blocks (A, T, G and C), Taq polymerase enzyme and reaction buffer.

This process is performed in a thermocycler, which creates rapid (millisecond) changes in temperature in a controlled environment.

• Double-stranded DNA is denatured by heating to 90–95 °C.

• Cooling to 40–60 °C allows the primers to anneal to their complementary sequences on the target DNA.

• Taq polymerase synthesises new strands from the primed sequences using target DNA as template.

• Denaturation, annealing and extension steps are repeated through many cycles and at each cycle new strands can act as templates for the next round of synthesis. This allows exponential increases in product.

PCR sequence-specific primers (PCR-SSP)

PCR-SSP is currently the HLA typing system of choice in most histocompatibility and immunogenetics laboratories for typing deceased organ donors. A result can be generated in 3 hours. There are various commercially available PCR-SSP kits in use, such as those manufactured by Invitrogen (www.invitrogen.com), Olerup (www.alphahelix.co.uk) and BAG Health Care GmbH (www.bag-germany.com). These kits are constantly updated by the manufacturer to incorporate new WHO-recognised alleles. Some laboratories construct in-house SSP trays using their own design primers; however, as more alleles are defined it has become almost impossible for individual laboratories to keep their own primer design up to date.

The underlying concept of PCR-SSP typing of HLA alleles is based on the fact that Taq polymerase lacks 3′ to 5′ exonuclease proofreading activity. Therefore only primers that are matched to the 3′ end of the template will facilitate DNA extension.

To perform HLA typing on one individual, multiple PCRs are performed simultaneously using different combinations of sequence-specific primers. The combinations utilised should allow amplification of all known HLA alleles.

The assignment of alleles is based on the presence or absence of amplified product. To ensure that the absence of a specific product is due to the individual lacking the corresponding sequence and is not simply due to a technical error, a control is included in each of the SSP reactions. The control is an invariant region of a gene that is constant between all individuals, such as human growth hormone. The PCR-SSP product is visualised by size differences using agarose gel electrophoresis, as shown in Fig. 4.1. Electrophoresis through agarose relies on the movement of negatively charged DNA (due to the phosphate backbone) towards the anode. Fragments of DNA differentially migrate and thus can be identified according to their size. DNA is visualised on the gel by staining with ethidium bromide, which intercollates between the strands of DNA and fluoresces under ultraviolet light. In Fig. 4.1, control bands are visible in each of the SSP reactions and the specific product is visible as a second band in the well. By knowing which wells contain which SSP, the HLA type of an individual can be allocated.

Figure 4.1 A PCR sequence-specific primer (PCR-SSP) reaction. A control band (DNA) can be seen in each well and specific product bands are visible in wells 3, 8 and 11. By knowing the specific primers in each well, the HLA type of an individual can be determined.

SBT involves typing of HLA genes at the nucleotide or allele level. The technique utilises the elegant way in which DNA is copied by a DNA polymerase enzyme. Bases are incorporated into an extending DNA strand in a PCR through the formation of a phosphodiester bridge between the 3′ hydroxyl group at the growing end of a primer and the 5′ phosphate group of the nucleotide that is being incorporated.⁵ In SBT, DNA polymerases incorporate analogues of A, G, T and C that do not have a 3′ hydroxyl group. This results in termination of the strand.⁶ The analogues of A, G, T and C are labelled with a fluorescent dye. Detection of the dye then reveals which base is incorporated at each position. In this way the entire sequence can be determined. There are various SBT techniques and software options available from manufacturers such as Abbott Diagnostics (www.abbottmolecular.com) and Protrans (www.protrans.com) for HLA typing.

Clinical application of HLA typing

HLA typing and evaluation of HLA mismatching are essential prerequisites to kidney and stem cell transplantation.

The level of HLA typing employed for solid organ transplantation is generally low to medium resolution (two digits). HLA matching is not applied in pancreas, heart, liver or lung transplantation unless the recipient is sensitised to donor HLA antigens. In the case of haemopoietic stem cell transplants (HSCTs), medium- to high-resolution (four digits) HLA typing is performed. The level of matching is increased for HSCTs due to the risk of graft vs. host disease, when immunocompetent T cells transferred with the graft attack the recipient’s own cells and tissues.

Causes of HLA-specific alloimmunisation

HLA-specific antibodies can be produced in response to blood transfusions, pregnancy or transplantation.⁷^–⁹

The method of antigen presentation and the dose of the antigen influence the nature of the immune response, which will differ for each of these stimuli.

The antigen source in a blood transfusion is the lymphocyte content of the transfused blood.¹⁰ A number of studies have indicated that a low number of transfusions are a minimal risk for sensitisation whilst multiple transfusions are required to cause alloimmunisation.^11,¹² These data suggest that one or a few – usually less than 10 – blood transfusions induce minimal clonal expansion with limited proliferation of T and B cells. This is usually a transient IgM antibody, or IgM antibody followed by IgG antibody production, which is promptly downregulated. Multiple transfusions induce significant clonal expansion where antibody production is more likely and may result in a sustained level of IgG antibody production.¹³