Human Leukocyte Antigens

HLA (human leukocyte antigen) is the human major histocompatibility complex (MHC), a multigene family involved in the defense of humans (and all vertebrae) against pathogens. The HLA molecule’s role is to present peptides to T cells. Depending on the peptide, antigen presentation can lead to activation of T cells and initiation of an adaptive immune response. HLA molecules interact with NK cells too, inhibiting cytotoxicity when HLA molecules (mainly HLA-C) link the NK inhibitor receptor. They also have a primordial role in lymphocyte maturation in the thymus.

HLA Genes

The HLA complex is located within the 6p21.3 region of the short arm of chromosome 6 ( Fig. 32.1 ) and contains >240 genes of diverse functions. Many of the genes encode immune system proteins. The MHC gene family is divided into three subgroups or classes, named I, II, and III. The classical loci, routinely studied in human medicine, are HLA-A, HLA-B, and HLA-C for class I, and HLA-DRB1, HLA-DQB1, and HLA-DPB1 for class II. HLA genes are closely linked to one another and are inherited en bloc as a genetic unit. The series of HLA alleles on a single chromosome 6 is called haplotype . Combination of maternal and paternal haplotypes inherited creates the individual’s HLA genotype.

Figure 32.1, Genetic organization of the human leukocyte antigen (HLA) complex and structure of HLA class I and II molecules.

HLA Antigens

HLA antigens are glycoproteins and belong to the immunoglobulin superfamily, meaning that they form domains ( Fig. 32.1 ). The genes’ organization in a sequence of exons and introns reflects that particular protein structure.

HLA class I molecules are composed of a polymorphic α-chain combined with a monomorphic β-globulin chain and are expressed on almost all nucleated cells. They present endogenously processed peptides (derived from cytoplasmic protein degradation from self, as well viral proteins, for example) to CD8 ⁺ T cells.

HLA class II molecules are composed of two polymorphic chains (α and β) and are expressed constitutively only on professional antigen-presenting cells (such as macrophages, dendritic cells, and B cells, as well as activated T cells and thymic epithelial cells). Their expression can be induced on other cells, in case of stress, for example. They present peptides derived from endosomal and lysosomal protein degradation (from extracellular content, such as bacteria or parasites) to CD4 ⁺ T cells.

The combination of peptide and peptide-binding groove of the HLA molecule forms the epitope that is recognized by the T-cell receptor. Antigen recognition is restricted to the MHC , as neither peptide nor MHC alone can stimulate T-cell responses, which require formation of an MHC–peptide complex.

Polymorphism

The MHC region is the most polymorphic of the human genome. Polymorphism is mainly located in the peptide-binding groove, where it affects antigen presentation and confers selective advantage to the population. More than 17,000 HLA alleles have been described (12,893 for class I and 4802 for class II as of March 2018). Frequencies of individual HLA alleles vary greatly within a population and between populations.

Common alleles are those that appear with gene frequencies >0.001 in any reference population. Well-documented alleles are those having been described more than five times in unrelated individuals. The current common/well-documented HLA allele list is available on ImMunoGeneTics database ( https://www.ebi.ac.uk/ipd/imgt/hla/ ).

Alleles are not randomly combined in haplotypes, as certain HLA alleles are found associated with one another more frequently than would be predicted by chance alone. This linkage disequilibrium is particularly found for loci that are close together (HLA-B and HLA-C, HLA-DRB1 and HLA-DQB1), defining frequent and rare allele associations that affect donor searches.

HLA Typing

Historically, HLA antigens were defined by serological methods using a complement-dependent microcytotoxicity assay and panels of alloantisera-containing specific HLA antibodies.

DNA Typing Methods

Most HLA typing methods in use are based on amplification of specific HLA gene portions from genomic DNA using polymerase chain reaction (PCR). DNA-based typing methods vary in regard to the level of discrimination (resolution) they provide in defining the nucleotide sequence of an HLA gene.

Sequence-specific oligonucleotide probe (SSOP) hybridization is based on amplification of the most relevant portions of exons 2 and 3 (coding for the peptide-binding domain in class I molecules) or exon 2 (coding for the peptide-binding domain in class II molecules), followed by hybridization (on nylon membranes, plates, or flow cytometry beads) using sequences specific for a certain allele or group of alleles.
Sequence-specific primers (SSP) use multiple PCR reactions, each specific for an allele or group of alleles. Presence or absence of amplification is detected by electrophoresis. The number of reactions needed depends on locus polymorphism and degree of resolution.
Sequencing-based typing (SBT ) involves sequencing HLA genes and comparing the sequence with published libraries (IMGT/HLA database). Degree of resolution will depend on the length of the sequence obtained. It is the only method with ability to detect and characterize new alleles.
Next generation sequencing (NGS) methods have recently been applied to HLA typing, increasing throughput and allele identification capabilities, and allowing for easier suitable donor identification.

HLA Nomenclature

The WHO Nomenclature Committee for Factors of the HLA System is responsible for naming HLA genes and allele sequences ( http://www.hla.alleles.org ). Each HLA allele name has a unique number corresponding to up to four sets of digits (two or three), separated by colons ( Fig. 32.2 ). The first two digits describe the type (allele family), usually corresponding to a serological antigen. The Next two or three digits are assigned in the order those sequences were determined. Subsequent digits may be used in some typings ( Fig. 32.2 ).

Low Resolution

DNA-based typing result at the level of digits composing the first field in DNA-based nomenclature, or serologically defined equivalent (e.g., HLA-A2).

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