The Core Gene Family Underlying HLA Matching: Structure, Function and Significance

In the dynamic regulation system of the human immune system, the Human Leukocyte Antigen (HLA) gene family is the core regulatory unit, which accurately mediates the cascade reaction of immune recognition and response by encoding the major histocompatibility complex (MHC) molecule. The evolution of HLA matching technology is closely related to the structural analysis and functional interpretation of this gene family. From the basic research on classical HLA genes to the frontier exploration of non-classical HLA genes, its theoretical breakthroughs and technological innovations continue to drive the development of transplantation medicine and the in-depth study of immunobiology.

This paper systematically expounds the core gene family of HLA matching, including its genetic organization in chromosome 6p21.3, the structure and function of class I and class II genes, the key role in transplantation matching.

Genetic Organization of HLA Loci

HLA locus is the genetic center of the immune system, located in the short arm 6p21.3 region of human chromosome 6, and constitutes a MHC of about 3.6 Mb. Its gene cluster is distributed in a modular way, containing class I, class II, and class III genes. It is characterized by high polymorphism and close linkage and regulates immune response through fine genetic organization, which is the core target of transplantation matching and immune research.

Distribution of Gene Clusters in Chromosome 6p21.3 Region

The HLA gene family is located in the short arm 6p21.3 region of chromosome 6, which constitutes a gene-rich region with a length of about 3.6 Mb, that is, the MHC. The gene cluster presents a strict modular organizational structure from centromere to telomere, and its spatial arrangement mode is not only the molecular imprint of evolutionary events such as gene replication and recombination but also plays a key role in the regulation of immune response with specificity and efficiency.

• In the HLA gene cluster structure system, the class I gene region is located on the telomere side, and the three core gene sites of HLA-B, HLA-C, and HLA-A are linearly distributed along the chromosome, with a distance of about 1.5 Mb. As a key component of the immune monitoring system, MHC I molecules encoded by these genes assume the core function of presenting antigenic peptides to endogenous antigen-specific CD8+ T lymphocytes.
• The class III gene region next to the centromere of the class I gene region contains immune regulatory elements such as complement system-related genes (such as C4 and C2), cytokine coding genes (such as TNF-α), and heat shock protein genes. Although the genes in this region are not directly involved in the antigen presentation process, they play an important regulatory role in the inflammatory response cascade regulatory network.
• The class II gene region closest to the centromere consists of three subregions, namely HLA-DP, HLA-DQ, and HLA-DR, with a total span of about 1 Mb. The MHC II molecule encoded by it is responsible for the presentation of exogenous antigenic peptides to CD4+ T lymphocytes and plays a bridge role in the synergistic regulation mechanism of humoral immunity and cellular immunity.

HLA region of chromosome 6 (Xie et al., 2010)

This kind of gene cluster organization has important functional significance: on the one hand, closely linked genes tend to be inherited in the form of Haplotype, which ensures the cooperative expression of immune response-related genes; On the other hand, there are many recombination hotspots in the gene cluster, such as the recombination rate between HLA-A and HLA-B is about 1%, which provides a mechanism basis for population genetic diversity. Studies have shown that the recombination events of HLA gene clusters are positively selected in human evolution, which may be related to the rapid evolutionary pressure of pathogens. This genetic plasticity enables the human immune system to continuously adapt to the changing pathogenic environment.

Immune Monitoring Functional Framework of Class I Gene

HLA-A, HLA-B, and HLA-C are the core members of the Class I gene, and the Class I molecules encoded by them are highly conservative and specific in structure and function. Class I molecules are heterodimeric proteins, which are composed of heavy chain (α chain) and light chain (β2-microglobulin). The α chain contains three extracellular domains (α1, α2, and α3), in which α1 and α2 domains are folded together into a peptide-binding groove with a depth of about 2.5 nm, which can accommodate endogenous antigenic peptides of 8-10 amino acids. The α3 domain and β2-microglobulin are responsible for maintaining molecular stability and mediating the interaction with CD8+ T cells.

These three class I genes have overlapping functions and division of labor:

• HLA-B locus has the highest polymorphism (9,783 alleles were included in the IMGT/HLA database by 2024), and the flexibility of its peptide binding groove enables it to present a wider range of antigenic peptides.
• HLA-A sites tend to present longer antigenic peptides and play a leading role in virus immunity.
• HLA-C loci interact with the inhibitory receptors of natural killer cells (NK cells) to regulate the balance between innate immunity and adaptive immunity.

This functional differentiation enables class I molecules to monitor the intracellular environment in an all-around way and identify abnormal cells (such as virus infection or canceration) in time.

Type of classical HLA-I alterations in cancer cells (Hazini et al., 2021)

Construction of Antigen Presentation Network of Class II Gene

Class II molecules encoded by HLA-DR, HLA-DQ, and HLA-DP subregions in the Class II gene region constitute the core network of exogenous antigen presentation. Class II molecules are also heterodimers, but both chains (α chain and β chain) are encoded by Class II genes, and each contains two extracellular domains (α1, α2, and β1, β2). The peptide binding groove formed by α1 and β1 domains is more open, which can accommodate exogenous antigenic peptides with 13-17 amino acids. This structural feature makes it particularly suitable for presenting peptide fragments degraded by pathogens.

The HLA-DR subregion is the region with the most abundant polymorphism in class II genes. There are 5,342 alleles at the HLA-DRB1 locus, and there are associated genes such as HLA-DRB3, DRB4, and DRB5, which form a complex gene family. The strong association between the DQB103:02 allele of the HLA-DQ subregion and type 1 diabetes mellitus makes it a model of pharmacogenomics research. Although the polymorphism of the HLA-DP subregion is relatively low, the role of the DPB104:01 allele in renal transplant rejection has attracted widespread attention. Cell-specific expression of class II molecules (mainly limited to antigen-presenting cells) ensures the accuracy of exogenous antigen presentation and avoids the over-activation of immune response.

HLA class II clusters (supertypes) identified using an agglomerative hierarchical clustering algorithm (Greenbaum et al., 2011)

The innovation of HLA typing technology has greatly promoted the study of gene clusters. High-throughput sequencing revealed the genetic association pattern of HLA gene clusters by analyzing linkage disequilibrium. Allele annotation in IMGT/HLA database is highly dependent on whole genome sequencing (WGS) data. By integrating high-throughput sequencing results, accurate allele typing and discovery of new alleles are realized.

Key HLA Genes in Transplantation Matching

In the field of transplantation immunity, the HLA gene is the core antigen target that triggers rejection, and its polymorphism directly affects the success or failure of transplantation. Genes such as HLA-A/-B/-C and HLA-DRB1 become the core of the matching strategy because they encode key antigen-presenting molecules, while secondary histocompatibility antigen genes assist in regulating the intensity of immune response, thus forming a gene target network for transplantation matching.

Dominant Factors of Rejection of Class I Molecules

In transplantation immunology, HLA-A, HLA-B, and HLA-C are the cornerstones of the matching strategy, and their coded class I molecules are the main antigen targets to mediate graft rejection. Relevant data show that the high-resolution mismatch (4-digit allele mismatch) of these three loci in hematopoietic stem cell transplantation (HSCT) can increase the risk of acute graft-versus-host disease (aGVHD) by 1.5-2 times, while the mismatch of class I molecules in solid organ transplantation is closely related to the occurrence of chronic rejection. This difference in immunogenicity stems from the widespread expression of class I molecules- almost all nuclear cells express class I molecules on their surfaces, making every cell in the graft a possible target of immune attack.

The core mechanism of class I molecules causing rejection lies in the difference in peptide presentation between donors and recipients. When the HLA-A/-B/-C allele of the donor is different from that of the recipient, class I molecules on the donor cell surface will present unique antigenic peptides to the recipient T cells, which may come from normal cell proteins, virus antigens, or graft-specific tissue antigens. Once the recipient T cell receptor (TCR) recognizes this "non-self" HLA-peptide complex, it will start the killing process of cytotoxic T lymphocytes (CTL), resulting in graft damage.

The cytoplasmic domain of the HLA-I heavy chain mediates the interaction between HLA-I and integrin β4 (Zhang et al., 2010)

Core-related Genes of GVHD in Class II Molecules

HLA-DRB1 gene locus plays a key role in the matching strategy of allogeneic transplantation with its unique genetic characteristics among the major MHC class II gene clusters. There is a significant correlation between the high polymorphism of this gene locus and the occurrence and development of graft-versus-host disease (GVHD), and its association strength is statistically superior in the class II gene family (p<0.001).

From the perspective of molecular structure, the DRβ1 chain encoded by HLA-DRB1, as the key subunit of the MHC class II peptide binding region, directly regulates the antigen recognition threshold of CD4+ T cells through its polymorphic amino acid residues. This molecular interaction mechanism plays a signal cascade amplification effect in the process of allogeneic immune response, and then significantly affects the pathological process of transplant rejection.

The mechanism of GVHD induced by HLA-DRB1 is closely related to its expression on antigen-presenting cells (APC). APC in the graft (such as donor-derived dendritic cells) carries donor HLA-DRB1 molecules, which can present recipient tissue antigen to donor CD4+ T cells, initiate Th1/Th17 immune response, and then activate the release of CTL and inflammatory factors. Studies have shown that HLA-DRB1 mismatch can increase the risk of aGVHD by 30-50%, especially in unrelated donor transplantation.

Survival analysis and correlation analysis of HLA-DRB1 expression (Deng et al., 2022)

Potential Influence of Non-classical HLA Genes

In addition to the classic HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, and HLA-DP genes, the HLA gene family also contains a series of non-classic class I genes (such as HLA-E, HLA-F, HLA-G) and other immune-related genes. Although these genes are not directly involved in traditional matching, they play an important potential role in immune regulation and transplantation tolerance.

Immune Monitoring Function of HLA-E

HLA-E is the most deeply studied member of non-classical class I genes, and its coded molecular structure is similar to that of classical class I molecules, but its polymorphism is extremely low (only 12 alleles have been found so far). The main function of the HLA-E molecule is to combine with the inhibitory receptor CD94/NKG2A on the surface of natural killer cells (NK cells), transmit inhibitory signals, and prevent NK cells from attacking normal cells. This immune monitoring mechanism is of great significance in tumor immunity and transplant rejection.

In the field of transplantation, the expression level of HLA-E is closely related to the rejection mediated by NK cells. It was found that the higher the affinity between the donor HLA-E molecule and recipient NKG2A receptor, the stronger the inhibitory effect of NK cells, and the more acceptable the transplanted organ. An animal experimental model shows that the survival time of a transplanted heart overexpressing HLA-E is twice as long as that of the control group (28 days vs 14 days), which proves the potential of HLA-E in inducing transplantation tolerance. In addition, HLA-E is also involved in regulating the activation threshold of T cells, and its peptide binding groove mainly binds peptide segments from other class I molecular signal peptides, forming a double checkpoint for recognition.

HLA-E overexpression and impact on immune recognition in vitro (Seliger et al., 2016)

Mechanism of Maternal-fetal Immune Tolerance of HLA-G

HLA-G is another non-classical class I gene that has attracted much attention. Its product is mainly expressed in placental villous trophoblast cells, which play a key role in maternal and fetal immune tolerance. HLA-G molecules bind to inhibitory receptors (such as ILT2 and ILT4) on the surface of NK cells, T cells, and macrophages, and inhibit the rejection of the maternal immune system to the fetus. This special expression pattern and function make HLA-G an ideal target for the study of transplantation immune tolerance.

Discovery of New Matching Related HLA Genes

With the progress of genomics technology, more and more new matching-related genes have been found, which affect transplantation matching through interaction with classical HLA genes or independent immune regulation. Linkage analysis between microsatellite markers and HLA haplotypes is one of the important directions. Microsatellites are repetitive short-sequence DNA in the genome, which can be used as genetic markers of HLA haplotypes because of their high polymorphism and genetic stability.

Using 10 microsatellite markers linked to the HLA gene cluster, the researchers analyzed the HLA haplotypes of 100 families, and successfully improved the accuracy of haplotype reconstruction from 85% to 98% by traditional methods. This technique is of great value in haploidentical transplantation, which can help to determine the small genetic differences between donors and recipients and optimize the immunosuppression scheme.

In addition, the combined effect of the non-classical HLA gene and KIR (killer cell immunoglobulin-like receptor) gene has also become a research hotspot. KIR gene encodes the receptor on the surface of NK cells and regulates the activity of NK cells together with HLA molecules. The preliminary study shows that the matching degree between donor KIR and recipient HLA-C may affect the risk of GVHD after HSCT, which provides a new idea for the expansion of the matching strategy in the future.

Nucleotide diversity and phylogenetic analyses using the HLA-DPB1 allele sequences (Suzuki et al., 2018)

Conclusion

The research on the core gene family of HLA matching is still deepening, from the functional analysis of classical class I and class II genes to the potential mining of non-classical genes, every breakthrough promotes the progress of transplantation medicine. In the future, with the development of long reading and long sequencing, single-cell omics, and other technologies, HLA matching will move from gene sequence comparison to immune function matching, and individual rejection risk prediction will be realized by combining AI algorithms. These advances not only provide new ideas for solving the shortage of donors but also promote the paradigm innovation of transplantation treatment in the era of precision medicine, making the HLA gene family a key link between genetic diversity and immune regulation.