Genetics, Human Major Histocompatibility Complex (MHC)

Development

The foundations of the HLA complex were established in 1958 by 3 scientists: Jean Dausset, Jon van Rood, and Rose Payne. In 3 separate publications, the investigators described alloantigens on leukocytes detected in serum samples from multiparous women and patients who had received multiple transfusions.[5][6][7] Jean Dausset received recognition for discovering the first HLA antigen and was awarded the Nobel Prize in 1980. The discovery of HLA antigens represented the first major milestone in the field of histocompatibility. However, the importance of histocompatibility testing in transplant was not established until 1969. Patel and Terasaki demonstrated a strong association between positive lymphocyte crossmatch results and hyperacute rejection among kidney transplant donor-recipient pairs.[8] These compelling findings led to the adoption of mandatory prospective crossmatching before kidney transplant to identify antibodies reactive against donor lymphocytes that could cause graft loss.

Biochemical

Structure

Class I and class II molecules differ structurally. HLA class I molecules are composed of a polymorphic α chain encoded by class I genes and a β2-microglobulin chain encoded by a gene located on chromosome 15. The α chain, also known as the heavy chain, contains 3 domains: α1, α2, and α3. The α1 and α3 domains contain most of the polymorphic regions that confer HLA antigen specificity.[3][9] The class II region contains the HLA-DR, HLA-DQ, and HLA-DP loci. Each class II molecule is encoded by 2 genes, an A gene and a B gene, which encode the α and β chains, respectively.[9][10]

Function

The major histocompatibility complex (MHC) genes that act as transplant antigens are the classic HLA genes. The classic HLA class I genes (HLA-A, HLA-B, and HLA-C) are expressed on most somatic cells in the body. The classic HLA class II genes (HLA-DR, HLA-DQ, and HLA-DP) are expressed on antigen-presenting cells, including B cells, activated T cells, dendritic cells, macrophages, and thymic epithelial cells. In the presence of interferon, other cell types may also express HLA class II molecules.

The primary function of HLA molecules is to distinguish self from nonself. Both class I and class II molecules present short peptides derived from pathogens to T cells, thereby initiating the adaptive immune response. HLA class I molecules display peptides derived from the degradation of cytosolic proteins on the cell surface, where they are recognized by CD8-positive T cells. HLA class II molecules present peptides derived from the degradation of endocytosed proteins on the cell surface, where they are recognized by CD4-positive T cells.[3]

Testing

HLA Typing

HLA typing is defined as polymorphisms of class I and class II loci.[11]  Serology was the first HLA typing methodology. The microcytotoxicity method detects HLA antigens through complement-mediated cytotoxic cell death. In this in vitro assay, T or B lymphocytes are isolated from an individual and incubated with serum containing antibodies of known HLA specificity, historically obtained from multiparous women.[12][13] Advances in molecular methods have eliminated the need to perform HLA typing with panels of sera containing antibodies against numerous HLA antigens. Although HLA polymorphisms are distributed throughout the genes, most are concentrated within exons 2 and 3. Modern molecular methods identify nucleotide sequence polymorphisms specific to individual HLA alleles. Artificially synthesized, commercially available DNA probes or primers are then used to detect these polymorphisms.[13]

Three categories of DNA-based molecular HLA typing methods are available. All 3 methods determine HLA types based on polymorphisms identified by polymerase chain reaction (PCR) technology, which uses a heat-stable DNA polymerase to rapidly amplify target sequences within HLA genes.

1. Sequence-specific primer typing: This technique uses sequence-specific primer pairs that target and amplify a particular DNA sequence. The PCR primers are designed to bind only to a specific HLA allele or group of alleles. Amplified alleles are identified by the presence or absence of DNA amplification with a particular primer pair, as visualized using agarose gel electrophoresis.[2][13] Because the primers are directed at specific targets, amplification indicates the presence of the designated allele or alleles. Commercially available primer sets can define an individual’s complete HLA type (HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP). The most common application is deceased donor typing because this method provides rapid results. Sequence-specific primer typing provides low-resolution HLA typing, represented in DNA-based nomenclature as HLA-A01.[2][14] A disadvantage is that the method is not well suited for testing large numbers of samples.[2]

2. Sequence-specific oligonucleotide probe typing: This technique is frequently used in clinical histocompatibility laboratories. The method identifies HLA polymorphisms by hybridizing oligonucleotide probes attached to a solid-phase matrix, such as microbeads, with broadly amplified exon- and locus-specific PCR products.[13] Sequence-specific oligonucleotide probe typing provides low- to high-resolution DNA-based results, including antigen-level typing. This methodology is suitable for testing large numbers of samples in batches.

3. Sequence-based typing: This technique can be performed on several platforms, including Sanger DNA sequencing and next-generation sequencing. The most common application of sequence-based typing is allelic-level HLA typing for hematopoietic stem cell transplant recipients and donors. An allele refers to a unique nucleotide sequence for a gene. In DNA-based HLA nomenclature, allelic resolution uses all digits in the current allele name, such as HLA-A01:01:01:01. This methodology also permits the identification and confirmation of new allelic sequences.[2][13][14]

As HLA typing methods advanced and molecular methods replaced serologic testing, HLA nomenclature changed to reflect the greater complexity and resolution of modern typing results.[11]

HLA Antibody Testing

Identification of HLA antibodies is important before and after solid organ transplant. Currently, single-antigen bead assays are the most common approach to identifying HLA antibodies. These solid-phase assays use microbeads coated with recombinant single HLA antigens. The analysis is performed on a specialized flow cytometry platform.[15] Interpretation of the results is highly complex and requires specially trained laboratory professionals.

Crossmatching

Flow cytometric crossmatch is an essential test for assessing compatibility between a donor and a recipient. This assay is frequently performed before solid organ transplant.[2]

Clinical Significance

HLA compatibility represents a major barrier to hematopoietic progenitor cell (HPC) transplant. Therefore, HLA matching and compatibility between the donor and recipient are essential for successful HPC transplant. HLA antigens also contribute to graft-versus-host disease (GVHD), a potentially serious complication of allogeneic stem cell transplant. GVHD occurs when donor T cells recognize host antigens presented by antigen-presenting cells (APCs) and attack host tissues, leading to the sequential activation of donor T cells and monocytes and macrophages.[16]

In solid organ transplant, antibodies against HLA antigens play an important role, whereas the importance of HLA matching varies by organ type. Exposure to foreign HLA antigens through pregnancy, blood transfusion, or transplant can stimulate the production of antibodies against epitopes on those HLA molecules, a process known as HLA alloimmunization. The presence of donor-specific HLA antibodies can cause antibody-mediated rejection and graft loss.[17]

HLA alloimmunization can also cause platelet refractoriness. This condition can make it difficult to identify compatible platelet units, especially for transfusion-dependent patients undergoing HPC transplant. In platelet refractoriness, HLA class I antibodies, particularly those directed against HLA-A and HLA-B antigens, play an important role.[18] HLA antibodies also play a role in febrile nonhemolytic transfusion reactions and transfusion-related acute lung injury (TRALI).[19]