**3. The human leukocyte antigen system**

#### **3.1. Organization of the human MHC genetic loci**

As previously described, the human MHC, also known as HLA, located on the short arm of chromosome 6 (6p21), is a highly polymorphic gene dense genetic system. The HLA gene products are globular glycoproteins, each composed of two noncovalently linked chains. These proteins are ligand molecules, cell surface receptors and other factors involved in inflammatory response; recognition, processing, and presentation of foreign antigens to T cells, as part of the adaptive immune response; and also in innate immunity.

In addition to protein encoding genes, the MHC genetic loci contains pseudogenes and also transposon, retro-transposon and regulatory elements [11].

The HLA system comprises of almost 220 genes, with 21 of them being genes of major interest. These are located within genomic location 6p21.3 and their protein products mediate human response to infectious disease and influence the outcome of cell and organ transplants [12].

The human MHC genetic loci are divided in three distinct regions.

The class I region consists of genes that encode for HLA class I molecules, namely the HLA-A, HLA-B, and HLA-C (and also the nonclassical HLA-E, HLA-F, HLA-G, and the class I-like molecules MIC-A and MIC-B). These are expressed on the surface of almost all nucleated cells and are responsible for presenting intracellular derived peptides to CD8+ T cells.

The class II region includes genes that encode for HLA class II molecules, namely HLA-DR (DRA, DRB1, and depending on the haplotype the DRB3, DRB4, or DRB5), HLA-DQ (DQA1, DQB1), and HLA-DP (DPA1, DPB1) molecules. These are expressed in professional antigen presenting cells (APCs), such as macrophages, dendritic cells and B lymphocytes, in order to present extracellular derived peptides to CD4+ T cells.

Located between these two, is the class III region that contains non-HLA genes with immune function, such as complement components (C2, C4, factor B), cytokines, tumor necrosis factor (TNF), and lymphotoxins and heat shock proteins [13].

#### **3.2. HLA nomenclature**

KIRs can be subdivided into two main categories based on the strength of their affinity to the

KIRs seem to play important role in transplantation outcome. Transplant recipients missing KIR-ligands, especially in the absence of allo-reactive T cells (e.g., in T-cell-depleted HLAhaplo HSCT) were proven to present decreased rate of disease recurrence and improved

To conclude, the presence of activating KIR genes in the donor favorably affects recurrence

As previously described, the human MHC, also known as HLA, located on the short arm of chromosome 6 (6p21), is a highly polymorphic gene dense genetic system. The HLA gene products are globular glycoproteins, each composed of two noncovalently linked chains. These proteins are ligand molecules, cell surface receptors and other factors involved in inflammatory response; recognition, processing, and presentation of foreign antigens to T cells, as part of the

In addition to protein encoding genes, the MHC genetic loci contains pseudogenes and also

The HLA system comprises of almost 220 genes, with 21 of them being genes of major interest. These are located within genomic location 6p21.3 and their protein products mediate human response to infectious disease and influence the outcome of cell and organ transplants [12].

The class I region consists of genes that encode for HLA class I molecules, namely the HLA-A, HLA-B, and HLA-C (and also the nonclassical HLA-E, HLA-F, HLA-G, and the class I-like molecules MIC-A and MIC-B). These are expressed on the surface of almost all nucleated cells

The class II region includes genes that encode for HLA class II molecules, namely HLA-DR (DRA, DRB1, and depending on the haplotype the DRB3, DRB4, or DRB5), HLA-DQ (DQA1, DQB1), and HLA-DP (DPA1, DPB1) molecules. These are expressed in professional antigen presenting cells (APCs), such as macrophages, dendritic cells and B lymphocytes, in order to

Located between these two, is the class III region that contains non-HLA genes with immune function, such as complement components (C2, C4, factor B), cytokines, tumor necrosis factor

and are responsible for presenting intracellular derived peptides to CD8+ T cells.

ligands. It has been observed that Group A binds more effectively than group B.

rates in myeloid, but not lymphoid neoplasms [10].

48 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

**3. The human leukocyte antigen system**

**3.1. Organization of the human MHC genetic loci**

adaptive immune response; and also in innate immunity.

transposon, retro-transposon and regulatory elements [11].

present extracellular derived peptides to CD4+ T cells.

(TNF), and lymphotoxins and heat shock proteins [13].

The human MHC genetic loci are divided in three distinct regions.

survival.

The complexity of HLA requires the development of a more sophisticated nomenclature for locating the specific genomic region addressed each time:

	- **i.** Field/digit 3. Alleles that differ only by synonymous nucleotide substitutions within the coding sequence (CDS) are distinguished by the use of the fifth and sixth digits (six-digit typing).
	- **ii.** Field/digit 4. Alleles that differ only by sequence polymorphisms in noncoding regions (e.g., introns) are distinguished by the use of the seventh and eighth digits (eight-digit typing). This is level of resolution distinguishes the specific HLA genome sequence.

**Figure 1.** Paradigm of a complete MHC molecule nomenclature where exact information regarding the specific antigen expressed on the cell surface are enclosed between prefix and suffix information, which concern the type of protein produced, as well as its expression levels, respectively.

#### **3.3. The diversity of HLA**

The HLA genes located on a single chromosome, meaning the entire set of A, B, C, DR, DQ, and DP genes, also called a haplotype, are inherited in a typical Mendelian fashion altogether. So, each parent passes on a specific HLA haplotype to their descendants. This way in 50% of the cases two siblings are HLA haplo-identical (share one haplotype), whereas siblings with the same HLA genotype (both haplotypes are the same) or totally different HLA haplotypes equally share the remaining percentage (25% each).

This first level of genetic variation may be further enhanced with random genetic crossovers (chromosomal recombination) in the HLA region during meiotic division of gametic cells, though this is usually uncommon.

Additionally, amino acid variation which is mainly found in the extracellular antigen-binding grooves, as well as their surrounding regions, on the HLA protein molecules, alters the antigen binding specificity of the cells. This possibly contributes to enhanced diverse response after exposure to a variety of environmental infectious and noninfectious agents in the different areas of the world.

This amino acid variation stems from nucleotide sequence alterations such as single nucleotide polymorphisms (SNPs), copy number variations (CNVs), insertion/deletion events (InDels), and inversions, especially within the HLA class I and II gene regions [5, 11, 17].

#### **3.4. HLAs in HSCT: the purpose of HLA typing**

It has become clear by now that HLA molecules play an important role in HSCs, the success of which highly relies on the degree to which donor and recipient are HLA matched. HLA genotypically identical sibling is the gold standard. Whenever this is not the case, a perfect or well-matched unrelated donor is preferred over mismatched unrelated donors, haplo-identical donors and UCB.

Thus HLA matching is especially crucial when it comes to HSCT between unrelated persons. This is because allo-recognition of HLA allelic differences by T cells is related with acute and chronic GVHD, impaired engraftment, and higher mortality [12, 18].

To address this issue, molecular typing technologies have evolved substantially in order to more accurately determine the HLA genotype of both patients and donors, before HSCT. Older techniques provided limited information compared to more advanced high throughput sequencing methods, which dramatically increased the list of known HLA alleles. More than 14,000 HLA alleles have been identified, the vast majority of whom is being variants of the HLA class I genes. These encode for more than 10,000 different HLA proteins [8, 17].

#### **3.5. HLA typing resolution**

Levels of HLA typing resolution have been established by expert consortiums. These include:

**•** Low-resolution typing, or two-digit typing, is equivalent to serological typing, provides limited information that correspond to identification of broad families of alleles and is also called antigen level typing.

**3.3. The diversity of HLA**

though this is usually uncommon.

areas of the world.

donors and UCB.

**3.5. HLA typing resolution**

equally share the remaining percentage (25% each).

50 Umbilical Cord Blood Banking for Clinical Application and Regenerative Medicine

**3.4. HLAs in HSCT: the purpose of HLA typing**

The HLA genes located on a single chromosome, meaning the entire set of A, B, C, DR, DQ, and DP genes, also called a haplotype, are inherited in a typical Mendelian fashion altogether. So, each parent passes on a specific HLA haplotype to their descendants. This way in 50% of the cases two siblings are HLA haplo-identical (share one haplotype), whereas siblings with the same HLA genotype (both haplotypes are the same) or totally different HLA haplotypes

This first level of genetic variation may be further enhanced with random genetic crossovers (chromosomal recombination) in the HLA region during meiotic division of gametic cells,

Additionally, amino acid variation which is mainly found in the extracellular antigen-binding grooves, as well as their surrounding regions, on the HLA protein molecules, alters the antigen binding specificity of the cells. This possibly contributes to enhanced diverse response after exposure to a variety of environmental infectious and noninfectious agents in the different

This amino acid variation stems from nucleotide sequence alterations such as single nucleotide polymorphisms (SNPs), copy number variations (CNVs), insertion/deletion events (InDels),

It has become clear by now that HLA molecules play an important role in HSCs, the success of which highly relies on the degree to which donor and recipient are HLA matched. HLA genotypically identical sibling is the gold standard. Whenever this is not the case, a perfect or well-matched unrelated donor is preferred over mismatched unrelated donors, haplo-identical

Thus HLA matching is especially crucial when it comes to HSCT between unrelated persons. This is because allo-recognition of HLA allelic differences by T cells is related with acute and

To address this issue, molecular typing technologies have evolved substantially in order to more accurately determine the HLA genotype of both patients and donors, before HSCT. Older techniques provided limited information compared to more advanced high throughput sequencing methods, which dramatically increased the list of known HLA alleles. More than 14,000 HLA alleles have been identified, the vast majority of whom is being variants of the

Levels of HLA typing resolution have been established by expert consortiums. These include:

HLA class I genes. These encode for more than 10,000 different HLA proteins [8, 17].

and inversions, especially within the HLA class I and II gene regions [5, 11, 17].

chronic GVHD, impaired engraftment, and higher mortality [12, 18].


Today, when adult donor HSCT is considered, the gold standard is high-resolution typing at the HLA-A, HLA-B, HLA-C, HLA-DRB1, and HLA-DQB1 genetic loci (10/10 match). Single discrepancies for these regions are associated with increased risk of post-transplant complications, although HLA-DQB1 and in some cases HLA-C mismatches seem to be better tolerated, compared to mismatches in the other regions. Also, not all mismatches are of the same risk, some of them appear to have little or no increased risk, the so called permissive mismatches, which will be discussed later on.

HLA-DPB1 and KIR are also taken into account whenever possible. HLA-DPB1 is not tightly linked to the other genomic regions, so it is more difficult to find a perfect donor (12/12) when this genetic locus is also taken into account. The positive aspect of this misfortune is the fact that there are some permissive HLA-DPB1 mismatches that do not impact overall survival rates in case of perfectly matched donor unavailability (11/12).

Nowadays, HLA-DQA1 and HLA-DPA1 are not taken into account during HLA typing because of the strong linkage disequilibrium (LD) they present with the corresponding HLA-DQB1 and HLA-DPB1 loci. LD refers to certain alleles inherited together with increased frequency than that expected only by chance.

A treatment algorithm has been developed to address the complicated issue of selecting the fittest available unrelated donor for HSCT:


#### **3.6. Seeking for permissive (relatively well-tolerated) HLA mismatches**

It is well understood that even single nucleotide substitutions might impact the course of transplantation significantly either on the aspect of GVHD, engraftment success and transplant related mortality, delayed immune reconstitution, but not disease relapse.

Of course the extent of their impact is driven not only by the kind of genetic alteration (SNP, CNV, InDel, inversion), the effect it exerts on the final protein product (synonymous versus nonsynonymous polymorphisms) and the gene it appears at (HLA class I or II), but also the exact nucleotide position it is located, since this might affect more or less important amino acid sequences, regarding the protein's function.

With the advent of novel sequencing techniques allele level typing provides extensive nucleotide sequence data, which in correlation with previously available clinical data, is going to provide significant information in the context of retrospective studies [8, 12, 19].
