Role of the Human Leukocyte Antigen System in Hematopoietic Stem Cell Transplantation

Introduction

The human leukocyte antigen (HLA) system is the major histocompatibility complex (MHC) gene in humans. The HLA gene complex comprises about 3.6 Mb deoxyribonucleic acid (DNA) located on the short arm of chromosome 6 (6p21.3). It received its name because genes embedded in the complex play a major role in tissue graft rejection. The HLA system is highly polymorphic, with genes responsible for identifying self versus non-self. The most well-known members of the complex control histocompatibility are the classic MHC class I and class II genes. Class I genes consist of the HLA-A, -B, and -C loci, and class II genes consist of the HLA-DR, -DQ, and -DP loci. Class I MHC molecules are expressed on the surface of essentially all nucleated human cells and interact with CD8+ T-lymphocytes, and class II MHC molecules are expressed on cells of the immune system and interact with CD4+ T-lymphocytes. Class I HLA molecules are composed of an α polypeptide chain and the invariant light chain beta-2 microglobulin (β ₂ m). Class II HLA molecules are composed of two noncovalently associated polypeptide chains (α-chain and β-chain). The HLA system is an important part of the immune system. HLA genes encode cell surface molecules specialized to present antigenic peptides to the T-cell receptor (TCR) on T cells, CD8+ cytotoxic T-cells by class I molecules and CD4+ T-cells by class II molecules.

HLA alleles are expressed in a codominant manner; that is, alleles inherited from both parents are expressed equally. Each person carries two alleles at an HLA locus, which can be the same allele (homozygous) or two different alleles (heterozygous). The entire MHC is inherited as an HLA haplotype in Mendelian fashion from each parent. A haplotype consists of a set of HLA alleles that are found on the same chromosome strand and inherited together. A haplotype can be obtained through family analysis.

The naming of HLA genes and alleles has evolved over the years from serologic specificsities identified by serologic typing to HLA alleles identified by molecular DNA typing. A new nomenclature system of using colon-delimited HLA allele names was introduced in 2010. Each HLA allele name has a unique number corresponding to up to four sets of digits separated by a colon. There are four fields after the gene and locus, representing the allele group in the first field, specific HLA protein in the second field, a synonymous DNA substitution within the coding region in the third field, and differences in a noncoding region in the fourth field. Alleles that are not expressed—“null” alleles—have been given the suffix N. Alleles that have been shown to be alternatively expressed may have the suffix L, S, C, A, or Q ( www.ebi.ac.uk/ipd/imgt/hla/nomenclature ).

Some HLA alleles, alleles that belong to the same group (e.g., HLA-A*02), share the same reaction patterns by a given typing method. An allele code system was developed and introduced by the National Bone Marrow Be the Match Program for reporting HLA typing. An allele code represents the possibility of the presence of any of these alleles in the code (e.g., HLA-DQB1*02:CGYWP = DQB1*02:02/02:156/02:163 N). A decode tool is available at https://bioinformatics.bethematchclinical.org/MacUI . In addition, there are two ways of reporting ambiguous HLA allele typing. One uses an uppercase P, which follows the allele designation of the lowest-numbered allele in the group (e.g., HLA-A*02:06 P = A*02:06/02:718/02:768 using IMGT 3.37) to represent HLA alleles having nucleotide sequences that encode the same protein sequence for the peptide-binding domains (exons 2 and 3 for HLA class I alleles and exon 2 only for HLA class II alleles). The second uses an uppercase G, which follows the allele designation of the lowest-numbered allele in the group HLA alleles (e.g., HLA-A*02:04:01 G = A*02:04/02:664/02:710 N using IMGT 3.33) to represent a set of alleles that share identical nucleotide sequences for the exons encoding the peptide-binding domains. Both P and G groups can also be coded (e.g., HLA-A*02:06 P = A*02:BKSXJ, HLA-A*02:04:01 G = A*02:BFBYC).

Histocompatibility Testing in Support of Hematopoietic Stem Cell Transplantation

HLA genes are highly polymorphic. HLA class I and II alleles have been increasingly described over the years, and there are now many known genes (alleles) in both the HLA class I and the HLA class II loci. As of January 2021, there were more than 20,500 alleles at HLA class I loci (HLA-A, B, C) and 7200 at HLA class II loci (DRB1, DRB3/4/5, DQB1, DPB1).

HLA typing was started in the 1960s using the lymphocyte microcytotoxicity test, a serologic method, which allows characterization of HLA antigens. In the 1980s, with the development of the polymerase chain reaction (PCR) technique, which allows a small amount of DNA to be rapidly amplified and replicated to millions or billions of copies for further study, molecular DNA typing of HLA alleles became a reality.

The methods most commonly used for HLA allele typing in clinical histocompatibility laboratories are based on the recognition of locus-specific polymorphism in genomic DNA by sequence-specific primers (SSP), hybridization of sequence-specific oligonucleotide (SSO) probes, sequence-based typing (SBT), or next-generation sequencing (NGS) with DNA that has been selectively amplified by PCR. Different DNA-based molecular techniques are used depending on the clinical application.

Sequence-Specific Primers

The use of SSP is commonly referred to as PCR-SSP . A set of SSP pairs is designed according to target sequences for a selected HLA locus to allow identification of a specific allele or a group of alleles. DNA is amplified by PCR using these primers, and the amplified product is either visually identified in agarose gel electrophoresis or measured using a quantitative PCR technique. The presence of the amplified DNA fragment indicates that an allele-specific sequence is present in the genomic DNA, and hence an allele or a group of alleles is present. SSP can be used for a specific allele or a group of alleles.

SSO Probes

A set of short SSO probes, usually 12 to 20 base pairs long, is designed according to target sequences of an allele or group of alleles at an HLA locus. After PCR amplification using a locus-specific primer pair, the amplified DNA is hybridized with these probes. The hybridization results, which are obtained by visual identification of a dot on the membrane or by quantitative measurement, are analyzed. HLA type can be determined from the reaction pattern of the positive probes. The SSO probe method is mostly used as a low- to intermediate-resolution HLA typing strategy.

Reverse SSO DNA Typing by Bead-Based Multiplexing

This SSO typing method uses the Luminex technology. SSO probes are chemically conjugated on polystyrene microspheres (beads). Each microsphere is color-coded by two fluorescent dyes. The DNA is PCR-amplified using a mixture of primers that are designed to target regions of a gene at HLA class I and class II loci. The PCR products are then biotinylated, hybridized to the probes conjugated to fluorescently coded microspheres. A flow analyzer, the Luminex, identifies the fluorescent intensity of phycoerythrin on each microsphere. Assignment of HLA typing is based on the reaction patterns. Currently, this is a widely used method, with commercial products available (One Lambda, Inc., Thermo Fisher Scientific; Immucor GTI Diagnostics, Inc.), in many histocompatibility laboratories for solid organ transplantations and HCT. This reverse SSO method is commonly used as a low- to intermediate-resolution typing approach.

Sequence-Based Typing

The most commonly used SBT method for HLA typing is Sanger sequencing, which was developed by two-time Nobel Laureate Frederick Sanger and his colleagues in 1977. This method of DNA sequencing is based on the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during DNA replication, the chain-termination method. This method involves PCR amplification of DNA and sequencing of PCR product using locus- or group-specific primer pairs that are designed to amplify target sequences, that is, exons of HLA genes, the key regions for peptide-binding and TCR interaction. A single HLA allele or a group of alleles is obtained. The commonly targeted areas are exons 2 and 3 for HLA class I-A, -B, and -C loci, and exon 2 for class II-DR, -DQ, -DP loci. For each HLA locus, both alleles are amplified and sequenced either together using locus-specific primers or separately using group-specific primers. SBT is mostly used as a high-resolution HLA typing method.

Next-Generation Sequencing

In recent years, NGS methods on various platforms have been developed and used for HLA allele typing. Several commercial NGS products for HLA typing have become available. NGS HLA typing has been implemented for routine clinical service in support of transplantation programs, especially in HCT. Most NGS technologies for HLA typing involve long-range PCR amplification of the whole genome or the clinically relevant class I and II HLA genes, library preparation, fragmentation, size selection, adaptor ligation, sequencing, and data analysis. NGS technologies provide a highly targeted and multiplexed analysis, and most NGS products run on Illumina MiSeq. NGS technologies allow simultaneous characterization of all HLA loci in one single test through barcoding. Using NGS HLA typing, the long-range PCR product covers the full gene from 5’-UTR to 3’-UTR for most class I and II HLA genes (HLA-A, -B, -C, -DQ, -DPA1), or the key regions for some genes (DRB1, DRB3/4/5, DPB1). Allelic typing by NGS can be obtained for most HLA class I and class II genes.

Selecting HLA Typing Methods in the Histocompatibility Laboratory to Support HCT

HLA matching at the allele level of HLA class I and II loci is the key for a successful outcome in HCT. For many years, SBT was the method of choice for high-resolution typing. However, when sequencing only selected areas (exons 2 and 3 for class I loci, exon 2 for class II loci), and given the heterozygous nature of SBT analysis, the combinations of many pairs of alleles may give an ambiguous typing result. In such cases, additional typing by another method, for example, SSP, is needed to resolve the ambiguities. The current standard for HCT with an unrelated donor requires characterization of all common and well-documented HLA alleles, including certain null alleles and the Common, Intermediate and Well-Documented HLA Alleles in World Populations (CIWD version 3.0.0) is available at https://onlinelibrary.wiley.com/doi/abs/10.1111/tan.13811 ). With NGS methods, allelic typing can be obtained for nearly all HLA loci. Ambiguous typing results can be expected in some cases owing to lack of sequence data (e.g., exon 1 in class II loci) or inability to phase sequence data because of breaking between regions.

A combination of low- to intermediate-resolution typing for donor screening and confirmatory tests for the recipient, or high-resolution typing by SBT or allelic typing by NGS for the recipient and the selected donor, is common practice in many histocompatibility laboratories to support the HCT program.

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