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If we inherit both maternal and paternal MHC haplotypes, the functional (non-pseudo)genes are expressed and all of their products (MHC molecules) are exhibited on the cytoplasmic membrane of any nucleated cell.
In case of MHC type I molecules our all nucleated cells bear one (or more? how many?) types of polypeptide(s) (corresponding to and) encoded by the inherited allels of each of A, B, C, E, F, G MHC-I genes.
In other words each nucleated cell expresses all MHC type I molecules encoded by our inherited MHC haplotype.
Is it true?
Whole-genome molecular haplotyping of single cells
Conventional experimental methods of studying the human genome are limited by the inability to independently study the combination of alleles, or haplotype, on each of the homologous copies of the chromosomes. We developed a microfluidic device capable of separating and amplifying homologous copies of each chromosome from a single human metaphase cell. Single-nucleotide polymorphism (SNP) array analysis of amplified DNA enabled us to achieve completely deterministic, whole-genome, personal haplotypes of four individuals, including a HapMap trio with European ancestry (CEU) and an unrelated European individual. The phases of alleles were determined at ∼ 99.8% accuracy for up to ∼ 96% of all assayed SNPs. We demonstrate several practical applications, including direct observation of recombination events in a family trio, deterministic phasing of deletions in individuals and direct measurement of the human leukocyte antigen haplotypes of an individual. Our approach has potential applications in personal genomics, single-cell genomics and statistical genetics.
REVIEW articleIva Trenevska, Demin Li* and Alison H. Banham*
- Nuffield Division of Clinical Laboratory Sciences, Radcliffe Department of Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
Monoclonal antibodies are among the most clinically effective drugs used to treat cancer. However, their target repertoire is limited as there are relatively few tumor-specific or tumor-associated cell surface or soluble antigens. Intracellular molecules represent nearly half of the human proteome and provide an untapped reservoir of potential therapeutic targets. Antibodies have been developed to target externalized antigens, have also been engineered to enter into cells or may be expressed intracellularly with the aim of binding intracellular antigens. Furthermore, intracellular proteins can be degraded by the proteasome into short, commonly 8 amino acid long, peptides that are presented on the cell surface in the context of major histocompatibility complex class I (MHC-I) molecules. These tumor-associated peptide–MHC-I complexes can then be targeted by antibodies known as T-cell receptor mimic (TCRm) or T-cell receptor (TCR)-like antibodies, which recognize epitopes comprising both the peptide and the MHC-I molecule, similar to the recognition of such complexes by the TCR on T cells. Advances in the production of TCRm antibodies have enabled the generation of multiple TCRm antibodies, which have been tested in vitro and in vivo, expanding our understanding of their mechanisms of action and the importance of target epitope selection and expression. This review will summarize multiple approaches to targeting intracellular antigens with therapeutic antibodies, in particular describing the production and characterization of TCRm antibodies, the factors influencing their target identification, their advantages and disadvantages in the context of TCR therapies, and the potential to advance TCRm-based therapies into the clinic.
Definition of MHC and HLA
The major histocompatibility complex (MHC) is a large gene complex present in all jawed vertebrates with an integral role in the immune system. The antigen-presenting molecules encoded by the MHC class I and class II genes are cell-surface glycoproteins that bind intracellular and extracellular peptides, respectively 1 . The human MHC is located on chromosome 6 and contains more than 200 genes 2 . The human MHC-encoded glycoproteins are known as human leukocyte antigen (HLA) and are specialized in presentation of short peptides to T cells and play a key role in the body's immune defence 3 (Fig. 1).
MHC name is derived from its role in graft rejection and tissue compatibility between donor–recipient pair. MHC compatibility between individuals is responsible for successful graft transplant. MHC is characterized by extensive polymorphism which in one hand is obstacle for finding matched pairs and in the other hand enables the immune system to recognize any invading pathogen.
Histocompatibility: HLA and other systems
What has become known as the major histocompatibility complex (MHC) was initially identified in the early 1900s, but it was not until the late 1930s that studies began to focus on graft acceptance (histocompatibility) and antigen response phenotypes (H-2) in different strains of mice. 1, 2 In the 1950s, Dausset detected the first histocompatibility antigens in humans, the MHC class I antigens, with antibodies from multiply transfused patients. 3, 4 These antibodies revealed in the human population differing patterns of binding to white blood cells (leukocytes) and each pattern of binding came to define a human leukocyte antigen (HLA) specificity. 5, 6 These HLA specificities were later determined to be encoded by three distinct polymorphic loci, HLA-A, HLA-B and HLA-C. The human MHC class II antigens were initially described via their ability to stimulate the proliferation of T-cells from one individual when mixed with lymphocytes from a second individual. 7 Each pattern of T-cell reactivity (allorecognition) to a panel of homozygous typing cells (HTC) was assigned an HLA-D phenotype. 8 It is now known that the HLA-D phenotypes are due to T-cell allorecognition of the products of the MHC class II loci, primarily HLA-DR. HLA-DQ and HLA-DP make minor contributions to these phenotypes. The genes specifying both class I and class II antigens are tightly clustered in a single chromosomal region, the MHC.
The human MHC is a genetic region located on the short arm of chromosome 6 (6p21.3) extending approximately 4 megabases (Mb) (Fig. 39.1). The MHC encodes over 250 genes and pseudogenes of which at least 150 are expressed as proteins. 9, 10 This genetic complex is divided into three regions: (centromeric) class II, class III and class I (telomeric). Although the proteins encoded within the MHC participate in a variety of functions, approximately 40% are devoted to immune system functions.
(The figure was generated from data contained in references. 9, 13 )
1.2 Mb) contains at least 34 expressed genes and 16 pseudogenes and spans from SynGAP (Ras-GTPase-activating protein, centromeric) to TSBP (testis-specific basic protein, telomeric). 11 This region includes the genes that encode for the classical class II molecules (HLA-DR, -DQ and -DP). In addition, gene products involved in MHC class I antigen processing (LMP2 and LMP7, the large multifunctional proteosome genes), peptide transport (TAP1 and TAP2, the transporter associated with antigen processing genes) and complex assembly (tapasin) and gene products involved in MHC class II complex assembly (HLA-DM and HLA-DO) are encoded in this region.
1 Mb) extends from NOTCH4 (transmembrane receptor involved in cell differentiation and development, centromeric) to MCCD1 (mitochondrial coiled-coil domain protein 1, telomeric). 12, 13 On average, this region contains 1 gene every 10 kilobases (kb) and is the most gene dense region in the human genome with approximately 72% of it being transcribed. The class III region encodes at least 62 expressed genes including complement components (e.g., C2 and C4B), heat shock proteins (e.g., Hsp70-1 and Hsp70-2) and cytokines of the tumor necrosis factor family (e.g., TNF and LTA). Some individuals have duplications in the area encoding complement component C4B thus, this area can vary in length.
1.85 Mb) from MICB (centromeric) to HLA-F (telomeric) encodes at least 118 genes 57 expressed genes and 61 pseudogenes. 14 In some instances, an additional
0.95 Mb to TRIM27 (tripartite motif 27, a transcription factor, telomeric) is included and called the extended class I region. 9 This region includes the classical class I genes (HLA-A, -B and -C) and the nonclassical class I genes (HLA-E, -F, and -G). This region also encodes the MHC class I chain-related (MIC) genes (MICA and MICB). The focus of this chapter is on the MHC encoded gene products involved in histocompatibility, primarily the classical human leukocyte antigens. Other MHC and nonMHC encoded genes that participate in histocompatibility are also covered.
The heavy chains of the classical MHC class I (class Ia) molecules are encoded in the class I region of the MHC and associate with beta 2 microglobulin (encoded on chromosome 15) to form the mature class I molecule. The gene order within the MHC is shown in Fig. 39.1. Each of the classical MHC class I heavy chains is encoded by a single gene that is divided into 8 exons. 15 Exon 1 encodes the 5′ untranslated region (UTR) and the hydrophobic signal sequence. The signal sequence directs insertion of the protein into the membrane at the cell surface and is cleaved from the mature protein. The extracellular portion of the class I heavy chain is encoded by exons 2–4. Exon 5 encodes the transmembrane region and exons 6 and 7 encode the intracellular cytoplasmic tail. The 3′ UTR and the polyadenylation (poly(A)) site are encoded by exon 8. The mRNA, which is translated into protein, includes all eight exons after removal by splicing of the intervening sequences (introns).
The class II region of the MHC contains three subregions (centromeric) HLA-DP, -DQ and -DR (telomeric). Each encodes at least one cell surface class II molecule (Fig. 39.1). The class II molecules are noncovalently associated heterodimers that consist of an α chain and a β chain. 15, 16 Each chain is encoded by a separate gene, an A gene for the α chain and a B gene for the β chain. The expressed HLA-DP heterodimer is encoded by the DPA1 and DPB1 genes. The HLA-DP subregion contains two DP pseudogenes, DPA2 and DPB2. The HLA-DQ subregion contains two A (DQA1 and DQA2) and three B (DQB1, DQB2 and DQB3) genes. The expressed HLA-DQ heterodimer is encoded by the DQA1 and DQB1 genes, while the remaining DQ genes are pseudogenes. Each individual has two copies of chromosome 6 and, thus, two copies of each of the expressed HLA-DP and HLA-DQ genes. These genes are polymorphic and, consequently, an individual can have two different expressed A genes and two different expressed B genes for HLA-DP and for HLA-DQ. While not all combinations form, 17 the products of some of these genes can associate in several αβ combinations, regardless of chromosomal origin. Therefore, an individual could express up to four different HLA-DP and up to four different HLA-DQ molecules.
The HLA-DR subregion is more complex. 18, 19 A HLA-DR molecule composed of a conserved α chain encoded by the DRA gene and a polymorphic β chain encoded by the DRB1 gene is almost always present. This is the major class II molecule expressed on the cell surface. An additional eight DRB genes and pseudogenes have been identified in the HLA-DR subregion. The number of DRB genes present and the number of expressed DRB gene products is characteristic of each chromosome (haplotype) a person inherits (Fig. 39.2). For example, the DR1 haplotype carries two DRB genes, the expressed DRB1 gene and a DRB6 pseudogene. The DR8 haplotype carries only one DRB gene, the expressed DRB1 gene. Other DR subregion haplotypes can encode a second expressed HLA-DR molecule composed of the DRA gene product associated with a DRB3 gene product (DR52 molecule), a DRB4 gene product (DR53 molecule) or a DRB5 gene product (DR51 molecule) and can contain one to three DRB pseudogenes. The designations DR51, DR52 and DR53 are antibody (serologically) defined.
The A genes, which encode the α chains of class II molecules, contain five exons. 15, 16 The 5′ UTR and hydrophobic signal sequence are encoded by exon 1, like the class I genes. Exons 2 and 3 encode the extracellular domains. Exon 4 encodes the connecting peptide, the transmembrane region, the intracellular cytoplasmic tail and a portion of the 3′ UTR. The remainder of the 3′ UTR and the poly(A) signal are encoded by exon 5. Each class II β chain is encoded by a B gene divided into six exons. Exons 1–3 are similar to that of the A genes. Exon 4 encodes the connecting peptide and transmembrane region, while exon 5 encodes the cytoplasmic tail. The 3′ UTR and poly(A) signal are encoded by exon 6. All exons and introns are transcribed into RNA for the class II A and B genes. Again, introns are removed by splicing to form the mRNA that is translated into protein.
The nucleotide sequences of many of the HLA genes differ among individuals. These sequence variants are termed alleles genes with many alleles are termed polymorphic. Alleles of a locus may differ by a single nucleotide to many nucleotides potentially resulting in changes in the amino acid sequence of the protein specified by that gene. The classical HLA class I and class II loci are the most polymorphic loci in humans. The HLA-B and HLA-DRB1 loci have over 1100 and 650 known alleles, respectively (Tables 39.1, 39.2). 20, 21 In contrast to non-HLA genes, the nucleotide differences found among HLA alleles are usually nonsynonymous (alter the amino acid sequence) and are focused in the exon(s) encoding the most functionally important region of the HLA molecule, the antigen binding site. 18 It is thought that this diversity has been maintained to provide the human population with the capacity to recognize a diverse repertoire of pathogenic peptides. 22 – 24 Unfortunately, the allelic differences in HLA molecules expressed on the cells of different individuals can be recognized as foreign when tissue is grafted from one individual to another.
|DRA*0101–*010202 c||DQA1*010101–0107 h||DPA1*010301–*0110 j|
a Listing of class II alleles assigned by July 2009. 20, 21 The number of HLA class II alleles continues to increase as more individuals are studied. Alleles are defined by DNA sequencing. An expanded and updated table can be found at hla.alleles.org. A description of the nomenclature in this table can be found in Table 39.9.
The alleles in the MHC complex can be reshuffled by crossing over between homologous chromosomes during the generation of sperm or eggs. The frequency of recombination across the MHC from HLA-A to HLA-DPB1 can range from 0.7 to 4.3%. 26 Studies in humans suggest that there are several sites at which recombination preferentially occurs within the MHC, particularly between HLA-B and HLA-DRB1 and between HLA-DQB1 and HLA-DPB1. 26, 27 Studies comparing MHC-identical sib pairs versus haplotype mismatched sib pairs and unrelated individuals show that recombination rates can vary up to sixfold between individuals with different MHC haplotypes and suggest a genetic influence within the MHC on recombination rates. 26 On average, the frequency of recombination between HLA-A and HLA-B is 0.7%, between HLA-B and HLA-DRB1 is 1.0%, and between DQB1 and DPB1 is 0.8%. Recombinations between DQA1 and DRB1 loci and between B and C loci are very rare.
The HLA alleles and haplotypes found in individuals depend on their racial and ethnic backgrounds. 28, 29 For example, the allele DRB1*0302 is found in African Americans, but is only rarely observed in individuals of northern European or Asian descent. Likewise, the frequency of a combination of alleles on a single copy of chromosome 6 can differ among population groups. Table 39.3 lists the ten most common haplotypes identified in the African American population compared to the ranking of these haplotypes in several other US populations. 29 For example, the most common haplotype in African Americans is A*3001, Cw*1701, B*4201, DRB1*0302 found at a frequency of 1.5%. This haplotype was ranked 37th in Hispanic Americans but was not observed in this sampling of European Americans and Asian Americans.
When large databases of HLA typed individuals are analyzed, only a small percent of potential HLA phenotypes are found. Using serologic assignments from an unrelated donor registry, of the predicted 19 536 660 HLA-A,-B,-DR phenotypes, only 1.6% were observed. 30 This suggests that not all HLA allele combinations will be found. Indeed, some HLA haplotypes appear more frequently than expected. Linkage disequilibrium measures the degree of non-random association between alleles of separate loci. Apparently high disequilibrium across the DR-DQ subregion coupled with a lack of recombination have resulted in specific associations between DQA1 and DQB1 alleles and between DRB1 and DQ alleles although a single allele such as DQB1 may be associated with one of several partner alleles. For example, DQB1*0602 is found on the same chromosome as the DQA1 alleles DQA1*0102, or *0103 or *0104, but has not been observed with DQA1*0201, *0301, *0401, or *0501. 31, 32 These associations may differ among individuals of different racial/ethnic backgrounds. For example, DRB1*0901 is associated with DQB1*0201 in African Americans but with DQB1*0303 in individuals of northern European descent. Within the DR subregion, specific allele combinations at the several DRB loci are associated with families of DR haplotypes (Fig. 39.2). For example, the DRB3 locus is found in haplotypes carrying specific DRB1 alleles including DRB1*0301, *1101, *1201, *1301, and *1401 alleles (Table 39.4). In the class I region, associations between HLA-B and -C alleles have also been noted. 29
|DR molecules encoded||Expressed DR loci included in haplotype||DRB1 alleles associated with haplotype|
|DR, DR51||DRA, DRB1, DRB5||DRB1*15, *16|
|DR, DR52||DRA, DRB1, DRB3||DRB1*03, *11, *12, *13, *14|
|DR, DR53||DRA, DRB1, DRB4||DRB1*04, *07, *09|
|DR||DRA, DRB1||DRB1*01, *08, *10|
Extension of linkage disequilibrium across longer regions of the MHC has resulted in associations between specific class I and class II alleles. The associations of multiple alleles result in extended haplotypes. 33, 34 The most well known extended haplotype is A*0101, Cw*0701, B*0801, DRB1*0301 which is common in northern Europe, appearing at a frequency of approximately 5–15%. 28 It has been hypothesized that these associations may have been maintained in the population by selection, that is, associations between DR and DQ as well as associations within an extended haplotype might represent optimal combinations of immune response molecules. It is also likely that features of the genome structure limiting recombination or changes in the structure of the population, such as through admixture of different ethnic groups, have caused the linkage disequilibrium. Because alleles at various HLA loci are non-randomly associated, these associations enhance the frequency with which individuals share alleles across multiple HLA loci facilitating the selection of HLA identical individuals as tissue donors.
Classical MHC class I proteins are expressed by most nucleated cells, but the level of expression on the cell surface varies for different cell types. Cis -acting sequence blocks (enhancer A, interferon-stimulated response element (ISRE), W/S box, X box (previously known as site α) and Y box (previously known as enhancer B)) in the regulatory (promoter) region upstream of each class I gene control gene expression (Fig. 39.5A). 35 Each promoter sequence block binds numerous proteins (transcription factors) that regulate the level of transcription of the gene and ultimately the amount of protein at the cell surface. For example, enhancer A binds stimulating protein 1 (Sp1) and various members of the NF-κB/rel family of transcription factors. Collectively, the complex is termed NF-κB. Normal class I gene expression requires the coordinated action of each of these regulatory elements however, disruption of any one sequence block reduces, but does not appear to ablate, MHC class I expression.
The amounts of HLA-A, -B and -C molecules expressed at the surface of a cell are not equal. 36 HLA-A and -B are abundant with HLA-A expressed at somewhat higher levels than HLA-B, in many instances. HLA-C is expressed at very low levels in comparison accounting for about 10% of cell surface class I molecules. This is due to sequence variations in the regulatory blocks of each class I locus (Fig. 39.5B) which alter the type and affinity of transcription factor binding. For example, only NF-κB/rel family members bind to enhancer A in the HLA-A promoter, while enhancer A in the HLA-B promoter binds SP1 in addition to NF-κB/rel family members. The inclusion of SP1 binding may lead to less efficient expression of the HLA-B gene. There are also allele specific differences in the nucleotide sequence of these regulatory elements such that, for example, different HLA-B alleles are expressed at different levels. Additionally, some HLA-B allele promoter regions encode an E box regulatory element that binds the transcription factors upstream stimulatory factor 1 (USF-1) and USF-2. The presence of the E box correlates with reduced basal expression levels of these HLA-B alleles. 35
MHC class I gene expression can be up-regulated by various cytokines. 35, 36 Interferon-γ (IFN-γ) performs a fundamental role in enhancing MHC class I expression by inducing increased gene transcription via transactivation of the ISRE. Again, locus and allele specific sequence differences in the ISRE result in different levels of transcriptional enhancement for each of the class I genes. Additionally, IFN-γ up-regulates expression of the class II transactivator (CIITA) further enhancing MHC class I gene expression. Other cytokines, such as tumor necrosis factor (TNF), can enhance the stimulatory effect of INF-γ on MHC class I gene expression via up-regulation of NF-κB that acts through enhancer A.
MHC class II protein expression is more limited than that of MHC class I. Cell surface HLA-DR, -DQ and -DP molecules are found primarily on professional antigen presenting cells (APC) and on other immune system cells such as T-lymphocytes. 36, 37 Professional APC are bone marrow derived cells dedicated to the task of peptide presentation by MHC molecules and include B-lymphocytes, macrophages, dendritic cells, thymic epithelial cells and Kupffer cells. IFN-γ can induce class II expression in other cell types. Like the class I genes, the promoter regions of the class II genes contain the cis -acting sequence blocks W/S box, X box and Y box (termed the SXY module) that regulate gene expression (Fig. 39.5C). This is a conserved regulatory module present in the promoters of a wide range of genes involved in antigen presentation which functions as a single regulatory block. The X and Y boxes bind several ubiquitous transcription factors, such as regulatory factor X (RFX) and nuclear factor Y (NF-Y), respectively, in a complex termed the enhanceosome. 38, 39
Occupancy of the class II gene regulatory elements by the enhanceosome is absolutely required, but not adequate for expression and IFN-γ induction of the A and B genes of HLA-DR, -DQ and -DP. CIITA also is required for class II gene expression. 35, 38 Cell type specific and IFN-γ induction of class II expression is the direct result of CIITA expression patterns. Constitutive expression of CIITA is confined to professional APC and other immune system cells and can be induced by IFN-γ in other cell types, paralleling expression of MHC class II. CIITA is recruited to the enhanceosome of the class II promoter by the S box. This is not the case for class I gene expression for which the S box apparently plays no role. All of the other transcription factors that regulate class II gene transcription are ubiquitously expressed and constitutively occupy the regulatory sequence blocks in the MHC class II gene promoters. Of interest, patients with bare lymphocyte syndrome (MHC class II deficiency) can have a defect in any one of a number of the transcription factors that bind to the SXY module or in CIITA. These patients still express MHC class I albeit at reduced levels.
HLA-DR, -DQ and -DP are not expressed at the same levels on cell surfaces, similar to expression of the different MHC class I molecules. HLA-DR is the most abundant MHC class II molecule expressed by cells. DRB1 is expressed at a higher level compared to DRB3 and DRB4. 40, 41 HLA-DQ is expressed at reduced levels and HLA-DP is the least abundant cell surface class II molecule. Like the regulatory elements in the promoters of class I genes, there are both locus and allele specific sequence differences in the regulatory elements of the class II genes. 36 These sequence differences account for the dissimilar levels of class II molecule expression in two ways: 1) alter the binding affinity of the transcription factors and 2) allow binding of proteins that repress gene transcription of specific class II loci. For example, the X box in the HLA-DPA1 gene promoter region specifically binds the X box repressor protein which diminishes transcription of the HLA-DPA1 gene and reduces the overall level of HLA-DP on the cell surface.
Some pathogenic micoorganisms and many malignant cells down-regulate HLA gene expression to avoid recognition by the immune system. 42 – 44 For example, human cytomegalovirus (CMV) interferes with IFN-γ induction of MHC class I and class II gene expression. 45 To avoid detection by T-cells, many carcinomas and lymphomas lack cell surface HLA-A and HLA-B molecules due to defects in the expression or the binding of specific transcription factors to the promoter regulatory blocks of these genes. In many instances, expression of HLA-C in these malignant cells is unaffected, allowing the cell to avoid recognition by natural killer (NK) cells . In fact, alterations in MHC class I expression is very common (reported to be 70% or greater) in a variety of tumor types such as cervical, breast and colorectal cancers. 42
44 kilodaltons (kD)) that belong to the immunoglobulin (Ig) superfamily of proteins. 15, 46 The extracellular portion of the class I heavy chain is composed of the amino-terminal 275 amino acids. The following 40 amino acids make up the hydrophobic transmembrane region and the carboxy-terminal 25 amino acids comprise the intracellular cytoplasmic tail. As an aside, soluble isoforms of the classical HLA molecules are produced and may have immunoregulatory roles. 47
The extracellular portion of the class I heavy chain is divided into three domains, termed α1, α2 and α3 (Fig. 39.6A). Each domain is encoded by a separate exon (exons 2–4, respectively) and is approximately 90 amino acids long. The 3D structures of the extracellular portion of several class I molecules were resolved by X-ray crystallography. 48, 49 The α1 and α2 domains fold together to form a groove (termed the antigen binding groove) distal to the cell membrane that consists of a floor of eight antiparallel beta strands topped by two alpha helices fashioned into the walls. The membrane proximal α3 domain folds into a structure which is similar to that of the constant region domains of immunoglobulins (antibodies). This domain is composed of two antiparallel beta sheets, one with four strands and one with three strands. The sheets are linked by a disulfide bond. β 2 m (
12 kD) is non-covalently associated with the α3 domain of the class I heavy chain and is required for cell surface expression. Its 3D structure is identical to that of the α3 domain of the heavy chain.
The peptide, the third component of the trimolecular complex, is generally 8–10 amino acids in length and non-covalently bound to the class I heavy chain. 48, 49 It lies in the groove formed by the α1 and α2 domains (Fig. 39.6C). The peptide is anchored at its amino- and carboxy-terminal ends by non-covalent bonds to amino acid residues in the class I heavy chain. There are pockets along the groove which accommodate amino acid side chains at various positions along the peptide. The pockets are unique for each class I molecule because polymorphic residues from the α1 and α2 domains participate in their formation. Each pocket has specific physical and chemical characteristics that are determined by the conserved and polymorphic class I residues that form the pocket. These characteristics, in turn, dictate which amino acid side chains are accommodated at the corresponding peptide position. This defines the peptide binding motif of each class I molecule and defines the overall character of the set of peptides bound (Table 39.5). 50, 51 For example, the protein encoded by A*1101 will accommodate a variety of ‘small’ amino acids at peptide postions 2, 3 and 6 and prefers basic amino acids at peptide position 9. However, each pocket does not make an equal contribution to peptide binding. Certain pockets, specific to each class I molecule, play a more predominant role and the corresponding peptide position is termed an anchor position. The preferred amino acids at an anchor position are termed anchor residues. Using the protein encoded by A*1101 again as an example, although peptide positions 2, 3 and 6 contribute to peptide binding, it is peptide position 9 that is the anchor position. Lysine and arginine are the anchor residues at this position with lysine preferred over arginine. It is of note that not all peptides that bind to an HLA molecule fully adhere to the defined peptide binding motif and that amino acids other than anchor residues can be found at anchor positions in these peptides. In the end, each class I molecule does bind a unique, large set of peptides and the peptide set shares particular sequence characteristics which are dictated by the amino acid residues that make up the groove of the HLA class I heavy chain.
There are benefits to determining the peptide binding motif for HLA molecules. For example, these motifs can be used to identify antigenic peptides from pathogen proteins as candidates for use in peptide based vaccines. As another example, expression of specific HLA allelic products is associated with an increased risk of developing many autoimmune diseases. 52, 53 In most cases, this is thought to be the result of the differential binding capacity of HLA molecules for particular peptides. Thus, knowing the binding motif for a HLA molecule aids in the identification of the culprit peptide and allows for the design of synthetic peptides that mimic disease associated peptides for use in blocking autoimmune responses. Because the number of HLA allelic products is so large, algorithms have been developed for prediction of peptide binding to HLA molecules. 54 – 57
The class II molecules are expressed on cell surfaces as a trimolecular complex, structurally analogous to the class I molecules, and consist of the class II α chain, the class II β chain and a peptide. Both the class II α (34 kD) and β (28 kD) chains are transmembrane glycoproteins and are Ig superfamily members, comparable to the class I heavy chain. 15, 16 The extracellular portion of the α and β chains are divided into two domains, the membrane distal α1 and β1 domains and the membrane proximal α2 and β2 domains. Similar to the class I heavy chains, each domain is encoded by a separate exon (exons 2 and 3, respectively) and is about 90 amino acids in length. Three additional regions complete each chain of the class II molecule a connecting peptide of 12 amino acids which is highly hydrophilic and links the membrane proximal domain to the transmembrane region, a 23 amino acid hydrophobic transmembrane region and an intracellular cytoplasmic tail that consists of the carboxy terminal 8–15 amino acids.
The 3D structures of the extracellular portion of several class II molecules have been determined by X-ray crystallography. 48, 49, 58 These structures are strikingly similar to that of class I molecules. The α1 and β1 domains fold together to form a peptide binding groove like the groove formed by the class I heavy chain α1 and α2 domains (Fig. 39.6B). The α2 and β2 domains of the class II chains fold to form Ig constant region domain like structures similar to that of the class I α3 domain and β 2 m.
The peptides that bind to class II molecules are anchored to the class II antigen binding groove by non-covalent bonds to the peptide backbone and by binding of peptide amino acid side chains into pockets along the groove (Fig. 39.6D), similar to the class I molecules. 49, 58, 59 Because of the polymorphic nature of the MHC class II proteins, each class II molecule, like each class I molecule, also binds a large set of peptides which share a peptide binding motif specific to that class II molecule (Table 39.5). The peptides that bind to class II molecules are heterogenous in length and generally 13–25 amino acids long. The low and open ends of the class II groove allow peptides of varying lengths to bind in an extended conformation with the ends of the peptide overhanging the ends of the groove. This is in contrast to the class I molecules which bind peptides of 8–10 amino acids. The ends of the class I groove are high and closed thus, MHC class I molecules optimally accommodate shorter peptides whose ends are tucked into the groove (compare Fig. 39.6C and 39.6D).
Mature cell surface class I molecules are formed in the endoplasmic reticulum (ER) with the aid of several resident ER proteins including tapasin, calnexin and calreticulin. 60, 61 Initially, the class I heavy chain and β 2 m fold and associate facilitated by calnexin and calreticulin (Fig. 39.7). This complex then transiently associates with the transporter associated with antigen processing (TAP) where a peptide is loaded into the groove of the class I heavy chain. Finally, the trimolecular complex is dispatched to the cell surface.
Peptides, derived from both self (normal cellular) proteins (potential autoantigens) or foreign proteins (antigens), are generated in the cytosol by the proteosome. 60, 61 The proteosome is a macromolecular structure that proteolytically cleaves proteins into peptides (a process termed antigen processing) and consists of members of the large multifunctional proteosome (LMP) family and other protein subunits. Two LMP family members (LMP2 and LMP7) are encoded in the class II region of the MHC. The proteosome is tightly associated with the TAP molecule which shuttles the peptides into the lumen of the ER. 60, 61 TAP is formed by the association of the products of the TAP1 and TAP2 genes also encoded in the MHC class II region. Another MHC encoded gene product, tapasin, stabilizes the TAP heterodimers, links the class I heavy chain to TAP for peptide loading and facilitates loading of peptides onto the class I molecule. An endoplasmic reticulum aminopeptidase plays a dual function in class I antigen presentation. It trims longer peptides that are more readily shuttled by TAP to the optimal length (8–9 amino acids) enhancing binding to class I molecules, but also destroys many peptides limiting the pool available for binding to class I. 62, 63
The class II α and β chains fold and associate in the ER with the assistance of resident ER chaperones such as calnexin (Fig. 39.7), 64 similar to class I molecules. Unlike class I, full maturation of the class II molecule does not take place in the ER. Instead, class II heterodimers are directed primarily to specialized endosomal compartments (MHC class II compartments, MIIC), or, in some instance, first traffic to the cell surface and then to the MIIC. 65 Once in the MIIC, peptides are loaded into the antigen binding groove and mature class II molecules are dispatched to the cell surface.
MHC class II molecules bind peptides derived from endocytosed microorganisms and from self and foreign proteins degraded by proteases in the endocytic pathway. 66 This is in contrast, yet complementary, to the peptides bound by MHC class I molecules. In general, class II molecules bind peptides from cell surface and extracellular sources, while class I molecules bind peptides from intracellular sources. Thus, proteins from the whole environment of a cell can be surveyed by the immune system. Invariant chain (Ii), a nonMHC encoded glycoprotein, plays a key role in facilitating this division of function.
Ii performs several functions in assuring proper antigen presentation by class II molecules. Ii chain serves as a chaperone in the folding and assembly of class II αβ heterodimers and protects the class II peptide binding groove from binding peptide in the ER via a 25 residue internal peptide segment termed CLIP (class II associated invariant chain peptide). 65, 66 Ii also provides the intracellular targeting signals that direct the complex to the MIIC. Under the acidic conditions of the MIIC, Ii is proteolytically cleaved and dissociates from the class II molecule, while CLIP remains bound to the class II antigen binding groove. CLIP is exchanged for antigenic peptides in a reaction catalyzed by HLA-DM, a resident MIIC protein that tightly associates with the class II heterodimer. 67 – 69 HLA-DM is a critical component in shaping the repertoire of peptides bound to class II molecules as it retains the class II molecules in the MIIC until a stable, high affinity complex between the class II molecule and a peptide is formed. In certain APC types, B-cells and thymic epithelial cells, another resident MIIC protein, HLA-DO, negatively regulates the actions of HLA-DM. 67, 68
Analogous to the MHC class II molecules, both HLA-DM and HLA-DO are class II related Ig superfamily members expressed as heterodimers that consist of an α chain and a β chain. 67 – 69 The HLA-DM and HLA-DO α and β chains are encoded by A and B genes, respectively, in the MHC class II region and regulation of these genes is similar to that of the MHC class II genes. 69 The 3D structure of HLA-DM resembles that of the MHC class II molecules except that its peptide binding groove is almost entirely obscured. 48
Components of the peptide processing and binding pathways of MHC class I and class II molecules are a favorite target of disruption by many pathogens and malignant cells to avoid detection by the immune system. 42 – 44 For example, two proteins (US3 and US6) encoded by human CMV block cell surface expression of MHC class I molecules and thus, detection of the infected cell by cytotoxic T-cells. US3 binds to MHC class I molecules and retains them in the ER and US6 inhibits peptide transport into the ER by TAP. Lack of cell surface MHC class I, however, renders the CMV infected cell susceptible to lysis by NK cells. To circumvent NK cell recognition, CMV encodes a class I like decoy termed UL18 which is recognized by NK cells and inhibits their function. 45 Other pathogens and malignant cells employ a variety of unique strategies to block MHC expression.
Is the whole MHC haplotype expressed and so their proteins are exhibited on each nucleated cell membrane? - Biology
ABSTRACT. The major histocompatibility complex (MHC) in sheep, Ovar-Mhc, is poorly characterised, when compared to other domestic animals. However, its basic structure is similar to that of other mammals, comprising class I, II and III regions. Currently, there is evidence for the existence of four class I loci. The class II region is better characterised, with evidence of one DRA, four DRB (one coding and three non-coding), one DQA1, two DQA2, and one each of the DQB1, DQB2, DNA, DOB, DYA, DYB, DMA, and DMB genes in the region. The class III region is the least characterised, with the known presence of complement cascade (C4, C2 and Bf), TNF a and CYP21 genes. Products of the class I and II genes, MHC molecules, play a pivotal role in antigen presentation required for eliciting immune responses against invading pathogens. Several studies have focused on polymorphisms of Ovar-Mhc genes and their association with disease resistance. However, more research emphasis is needed on characterising the remaining Ovar-Mhc genes and developing simplified and cost-effective methods to score gene polymorphisms. Haplotype screening, employing multiple markers rather than single genes, would be more meaningful in MHC-disease association studies, as it is well known that most of the MHC loci are tightly linked, exhibiting very little recombination. This review summarises the current knowledge of the structure of Ovar-Mhc and polymorphisms of genes located in the complex.
Key words: Ovar-Mhc, MHC, OLA, Sheep, Structure, Gene polymorphisms, Review
The major histocompatibility complex (MHC) is an organised cluster of tightly linked genes with immunological and non-immunological functions, and is present in all vertebrates, except the jawless fish (Tizard, 2004). The MHC was discovered during tissue transplantation studies in mice (Gorer, 1937) and was first known for its role in histocompatibility. Subsequently, its role in immune regulation (Benacerraf and McDevitt, 1972) and several other functions (Bonner, 1986 Zavazava and Eggert, 1997 Penn and Potts, 1999) were discovered. The primary function of the MHC is to code for specialised antigen-presenting receptor glycoproteins, known as histocompatibility molecules or MHC molecules. These molecules bind processed peptide antigens and present them to T lymphocytes, thereby triggering immune responses.
The ovine MHC was first identified about 27 years ago by serological studies on sheep lymphocyte antigens (Millot, 1978). Since then, it has been generally referred to as ovine leukocyte antigen (OLA) or sheep lymphocyte antigen. In accordance with a nomenclature system for the MHC of vertebrates (Klein et al., 1990), it has now been designated as &lsquoOvar-Mhc&rsquo (&lsquoOvar&rsquo representing Ovis aries). However, this system of nomenclature has not been universally adopted amongst animal immunogeneticists (Rothschild et al., 2000). Ovar has been localised by in situ hybridisation to chromosome 20 between bands q15 and q23 (Mahdy et al., 1989 Hediger et al., 1991).
Owing to the immunological importance of MHC genes and their possible role in disease resistance, research on the ovine MHC received an impetus in the late 1980s and was comprehensively reviewed in 1996 (Schwaiger et al., 1996). Since that review, there have been a number of studies investigating the polymorphisms of genes within the Ovar-Mhc and their association with resistance to infectious diseases. This paper reviews the literature pertaining to the structure of the ovine MHC and polymorphisms of genes located in the region. A review of literature pertaining to the association of genes within the Ovar-Mhc with disease resistance has recently been completed (Dukkipati et al., 2006).
STRUCTURE OF THE OVINE MHC
Human and mouse MHCs have been investigated in much more detail than those of other mammals (Deverson et al., 1991), and among the domesticated species, the sheep MHC is poorly characterised (Kostia et al., 1998). The MHC of humans, designated as the human leukocyte antigens (HLA), covers a region of about 3.6 megabasepairs. Its complete sequence and gene map locus have been reported (MHC Sequencing Consortium, 1999). It serves as a valuable reference for intra-species and inter-species comparative studies (Kulski et al., 2002). With over 224 gene loci (128 predicted to be expressed), it is the most gene-dense region of the human genome. The average gene density, including pseudogenes, over the entire region is one gene per 16 kilobasepairs. It is believed that about 40% of the expressed HLA genes are involved in immune system function.
The HLA complex is divided into three regions, the telomeric class I, the centromeric class II and the central class III (Klein, 1976). Analysis of the immediate-flanking regions has revealed that the classical class I and class II regions extend much further than originally thought and are referred to as extended class I and class II regions (Stephens et al., 1999). A set of more than 7 genes involved in inflammation, including the three members of the tumor necrosis factor (TNF) superfamily that is located at the telomeric end of the class II region, is sometimes specified as the class IV region (Gruen and Weissman, 1997).
The general structure of the MHC is conserved among mammalian species, including three main regions with different functional roles (Amills et al., 1998). However, when MHCs of different mammals are compared, some regions appear to be well conserved and others vary widely (Kelley et al., 2005). In general, the class II and class III regions are orthologous, i.e., they are clearly derived from a single ancestor without being subjected to major rearrangements (except in ruminants) and their gene order is conserved. In ruminants, the class II region is unique in that it is split into two distinct sub-regions, &lsquoa&rsquo and &lsquob&rsquo, separated by a distance of at least 15 cM (Andersson et al., 1988 van Eijk et al., 1995). The class I genes, in contrast, are paralogous, i.e., they are derived by duplication and have been reorganised several times (Kelley et al., 2005). The schematic structure of the ovine MHC is illustrated in Figure 1. Details of the genes harboured in the three regions and their known polymorphisms are summarised below.
The class I loci include both classical and non-classical genes. The classical class I genes are members of the immunoglobulin gene family that are involved in the presentation of peptides, predominantly derived from intracellular proteins and parasites, to CD8+ cytotoxic T cells. They have also been found to interact with natural killer (NK) cells to prevent NK-mediated cell lysis (Reyburn et al., 1997). The non-classical class I genes are evolutionarily related and appear to have distinct functions related to immune response and NK cell recognition in specific settings (Lee et al., 1998). There are three classical (HLA-A, B and C) and three non-classical (HLA-E, F and G) class I genes in the HLA complex (Rhodes and Trowsdale, 1998).
In sheep, the class I region is poorly characterised and there is a significant controversy over the number of classical class I loci. Initial studies in this regard relied mainly on the use of alloantisera in micro-lymphocytotoxicity assays. Evidence for the presence of two closely linked class I loci, designated as OLA-A and B, was provided in 1978 (Millot, 1978). Several other studies confirmed the existence of two class I loci (Stear and Spooner, 1981 Cullen et al., 1982 Garrido et al., 1995 Stear et al., 1996 Jugo and Vicario, 2001 Jugo et al., 2002). Three different studies, one based on the micro-lymphocytotoxicity assay (Millot, 1984), one based on immunoprecipitation followed by 2-dimensional gel analysis (Puri et al., 1987a) and another based on restriction fragment length polymorphism (RFLP) (Grossberger et al., 1990), have indicated the existence of a third class I locus. In a recent study aimed at haplotype characterisation of transcribed ovine MHC class I genes, at least four distinct polymorphic loci were identified (Miltiadou et al., 2005).
Several molecular genetic investigations have been undertaken to study polymorphisms of class I genes. An RFLP study conducted employing a human class I probe revealed polymorphic bands co-segregating and correlating with serologically defined lymphocyte antigens (Chardon et al., 1985). This was the first evidence that the serologically detected class I sheep leukocyte antigens are coded by MHC genes. In a different study, a sheep thymus cDNA library was screened with a human cDNA probe derived from HLA-B27 (Grossberger et al., 1990). Thirteen clones were identified and partially sequenced. Based on the sequences, the clones could be categorised into 5 distinct groups, requiring the expression of at least 3 loci. These sequences were found to be more similar to bovine than to murine class I genes.
A purine-pyrimidine repeat of the form (CA)20 was identified in an ovine class I (Ovar-MHC I)-positive clone from a sheep genomic library (Groth and Wetherall, 1994). Polymerase chain reaction (PCR) amplification of this microsatellite region revealed the presence of 11 alleles at the locus, segregating in a Mendelian fashion. This microsatellite (SMHCC) was found to be highly polymorphic in different breeds of sheep (Buitkamp et al., 1996 Paterson, 1998 Paterson et al., 1998 Charon et al., 2001 Gruszczynska et al., 2002a). These studies revealed allele numbers ranging from 5 to 13, with high heterozygosity coefficients, indicating the usefulness of this locus as a genetic marker. This locus was found by recombination frequency to be 5.8 cM from the DRB1 locus (Buitkamp et al., 1996).
Recently, molecular genetic analyses in two heterozygous Scottish Blackface rams revealed 12 novel MHC class I transcripts (Miltiadou et al., 2005). Based on the class I sequence-specific genotypes of their progeny, these transcripts could be assigned to four individual haplotypes. Phylogenetic analyses of the more conserved exons (4 to 8) grouped the transcripts into four clusters, while a combination of phylogenetic analyses, haplotype data and transcription levels suggested the transcripts to be products of at least four loci, three of which appeared together in a number of combinations in individual haplotypes.
Classical MHC molecules have four characteristics by which their function is defined: a high degree of polymorphism, high-level expression in particular cells, and the ability to bind small peptide molecules and present them to T cells (Kaufman et al., 1994). The class I MHC molecules, called class Ia molecules or class I classical molecules, are glycoproteins expressed on the surface of all nucleated somatic cells. They are found in highest concentration on lymphocytes and macrophages. The structure of the class I molecule was originally derived by X-ray crystallography (Bjorkman et al., 1987a,b). It is a heterodimer (Figure 2) consisting of an a - or heavy chain, non-covalently linked to a light b 2-microglobulin chain. The chain is composed of three extracellular domains ( a 1, a 2 and a 3), a transmembrane domain and a cytoplasmic domain. The a 1 and a 2 domains form the peptide-binding region (PBR), lying above the a 3 domain. The groove is formed by two a helices bordering a b -pleated sheet, and residues from both a 1 and a 2 domains contribute to the groove (Bjorkman et al., 1987a). The microglobulin chain has a single extracellular domain and probably serves to stabilise the structure. The known polymorphisms of the molecule, i.e., variations in the amino acid sequence, are concentrated in three or four discrete hypervariable regions within the PBR. The rest of the molecule is highly conserved and shows little sequence variation. The a -chains are encoded by polymorphic class I loci within the MHC, while b 2-microglobulin is encoded by a non-polymorphic locus outside the MHC (Hughes and Yeager, 1998).
The class I molecules present antigenic peptides (8 or 9 amino acids long) to T cell receptors (TCRs) of CD8+ cytotoxic T lymphocytes (CTLs), the principal immune function of which is considered to be the killing of virus-infected cells and tumour cells (Rammensee et al., 1995). In all cells, there is constant turnover of cellular proteins that are broken down into small peptides by a multimeric proteolytic complex in the cytoplasm, known as a proteasome (Rivett, 1993). In mammals, there are two proteasome components encoded within the MHC class II region, called the low-molecular mass polypeptide 2 (LMP2) and LMP7. The expression of class I molecules and LMPs is enhanced by the cytokine interferon gamma. The peptides derived in the proteasome are transported across the membrane of endoplasmic reticulum (ER) by a dimeric transporter associated protein (TAP), encoded within the class II MHC region. In the ER, a complex involving the class I molecule, the peptide and b 2-microglobulin is formed, and then transported to the cell surface.
The CTLs exercise a continual surveillance in the body by means of their TCRs. In the absence of any infection, the peptides bound by class I molecule are self-peptides. During infection by a virus or other intracellular parasite, some of the proteins broken down by the proteasome are of parasitic origin (non-self or foreign peptides). When CTLs encounter the complex of self-class I MHC and foreign peptide, a cytotoxic reaction is initiated that kills the infected cells. CTLs can only recognise foreign peptides in the context of self-class I MHC, a phenomenon referred to as class I MHC restriction of CTL (Zinkernagel and Doherty, 1974).
The molecular structure and tissue distribution of sheep classical class I molecules were studied using a panel of three monoclonal antibodies (Gogolin-Ewens et al., 1985). The class I heterodimer comprised a heavy chain of 44 kDa and a smaller b 2-microglobin of 12 kDa. In similarity to the class I MHC molecules of other species, these molecules were found to be distributed on all sheep lymphocytes and many non-lymphoid tissues, with differential expression on mature and immature lymphocytes. They were found to be expressed equally on normal lymphocytes and antigen-activated lymphoblasts (Hopkins and Dutia, 1990).
The biosynthesis of sheep class I molecules was analysed by sodium dodecyl (lauryl) sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of immunoprecipitates of splenocytes pulse-chase labelled with (35)S-methionine and (35)S-cysteine (Puri et al., 1987a). Two biosynthetic intermediates (39-40 and 41-42 kDa), finally resulting in a heavy chain (44 kDa) were noticed. Throughout the period of pulse-chase labelling, b -microglobulin could not be detected along with the heavy chain, indicating that sheep b -microglobulin either possesses very few methionine and cysteine residues or has a very low synthesis/turnover rate. A similar finding with b -microglobulin, that was corroborated in cattle (Joosten et al., 1988) but found to be contrary to that in humans, was reported in a study pertaining to immunoprecipitation and isoelectric focusing of sheep class I antigens (Jugo et al., 2002).
Another interesting aspect of sheep b -microglobulin is that it displays heterogeneity in 2-dimensional non-equilibrium pH gradient electrophoresis (NEPHGE)/SDS-PAGE analysis of class I molecules from (125)I-surface-labelled cells (Puri et al., 1987a). b -microglobulin can be resolved into two forms of varying charge and intensity, being consistent with either two primary gene products or allelic variation.
Class II genes are members of the immunoglobulin superfamily of genes which are functionally specialised for presentation of antigenic peptides mainly derived from extracellular proteins and parasites to the TCR on CD4+ helper T cells. In the HLA complex, these include five sets of the classical genes DP, DM, DO, DQ, and DR and non-classical genes such as LMP, TAP and TAPBP. Within each set of classical genes, genes for the a -chain are designated A, while genes for the b -chain are called B. The a - and b -chain genes in each set are located close together and resemble a two-gene duplication unit, with the exception of DOA and DOB genes, which are well separated from each other. Not all sets contain genes for both chains, although some contain many pseudogenes (Tizard, 2004).
Among the three regions of the ovine MHC, genes of the class II region are the best characterised. They are classified into different families, as in other mammalian species, using nomenclature adapted from humans and include DQ, DR, DP, etc. (Hein, 1997). Early studies of the class II region by genomic Southern analysis employing HLA gene probes resulted in a complex pattern of cross-hybridising bands, which suggested that sheep contained homologues of DQ and DR genes but probably not DP (Chardon et al., 1985 Puri et al., 1987d Scott et al., 1987). In a subsequent study on two unrelated sheep, 7 distinct class II a and 24 distinct class II b or b -related sequences were identified (Deverson et al., 1991). Consistent with earlier predictions, DQ and DR homologues were detected but not DP. The ovar-DQ and ovar-DR loci, which constitute the class IIa sub-region, have been studied in detail. A number of other ovar-MHC II genes of the class IIb type have also been identified. These include DY (Wright et al., 1994), DM (Schwaiger et al., 1996) and DN/DO (Wright et al., 1995, 1996).
The DR genes are highly polymorphic and the classical class II molecules encoded by these genes are expressed in higher concentrations than the DQ molecules on the cell membranes of macrophages and B cells (Outteridge et al., 1996). Several studies have been undertaken to characterise DRA and DRB genes of sheep.
An early Southern hybridisation study (Scott et al., 1987) employing human HLA-D probes provided evidence for the existence of a single DRA gene in sheep that was later isolated and found to be expressed (Deverson et al., 1991 Ballingall et al., 1992). Although there was an indication for the presence of a second DRA gene in sheep (Deverson et al., 1991) that might have been the result of gene duplication (Ballingall et al., 1992), it has not been confirmed in any subsequent studies. Initial sequencing of exons 1 to 4 of the expressed DRA gene indicated that it was homologous to the human DRA gene. Complete sequencing of the gene (Fabb et al., 1993) has revealed that it could code for a polypeptide of 253 amino acids of which 24 constitute the signal peptide and the remaining 229 form the mature polypeptide. The DRA clones in the two studies differed at only two amino acid positions, one within exon2 (H 50 /A 50 ) and the other in exon3 (T 109 /I 109 ). This low level of Ovar-DRA sequence polymorphism was similarly reflected in RFLP studies (Fabb et al., 1993 Escayg et al., 1993, 1996). Three allelic fragments of 6.1, 4.9 and 2.4/2.8 kb with respective frequencies of 0.05, 0.875 and 0.075 were found to be associated with the enzyme BgIII in Merino and Romney sheep.
The most polymorphic among the MHC genes is the DRB locus (Andersson and Rask, 1988). Ovar-DRB genes have been reported to exist in multiple copies, some functional and others non-functional. Early serological and biochemical work on sheep MHC class II molecules detected seven b -polypeptides in association with DRA chains that provided evidence for the existence of more than one locus encoding them. Two distinct DRB-like genes were identified using RFLP studies on bacteriophage clones of a sheep genomic library (Scott et al., 1987), while a different study provided evidence for the expression of two distinct Ovar-DRB genes (Dutia et al., 1994). RFLP studies employing probes specific for Ovar-DRB exon 2 revealed 10 DRB alleles that required the presence of at least three DRB genes (Grain et al., 1993). Further evidence for the presence of two copies of the expressed DRB1 gene was provided in a study on single strand conformational polymorphism (SSCP) and sequence polymorphism of MHC-DRB exon 2 in Latxa and Karrantzar sheep (Jugo and Vicario, 2000). Apart from red deer (Swarbrick et al., 1995), sheep are the only ruminants in which the existence of two expressed DRB genes has been described, although a second DRB gene (DRB2) in cattle has been found to be expressed at very low levels (Groenen et al., 1990).
Four Ovar-DRB loci have been described by Scott et al. (1991b). The functional DRB1 gene is located at one of them and pseudogenes, DRB2, DRB3 and DRB4, are found at the remaining three loci. The pseudogenes lack defined exons 1 and 2, and also show numerous mutations in their sequences as well as stop codons in exons 3 and 4. There are indications that additional DRB pseudogenes exist (Schwaiger et al., 1996).
The whole Ovar-DRB region numbers several thousand base pairs and its basic structure is considered similar to other mammalian species (Schwaiger et al., 1996). However, almost all the studies on this region have concentrated on the polymorphisms found in exon 2 and adjoining intron 2 of the expressed gene DRB1. This is because DRB1 exon 2 encodes the b 1 domain, which constitutes part of the PBR of the DR molecules. The highly variable residues concentrated in this region are in close contact with the peptides presented in the PBR or the TCR (Brown et al., 1993), and therefore, they are likely to be related to functionality such as disease resistance/susceptibility.
Another characteristic feature of Ovar-DRB1 is that a simple tandem repeat (STR) of the form [(GT)n(GA)m] exists in intron 2, 30 bp downstream from the 3&rsquo splice site of exon 2 (Schwaiger and Epplen, 1995). This STR with the same basic structure is present at virtually the identical positions in all the expressed DRB alleles of cattle, sheep, goat, red deer, and humans, indicating that it remained unchanged at a specific location across various species for nearly 100 million years of mammalian evolution. Two sheep DRB pseudogenes, DRB3 and DRB4, also harbour this STR either in the same or degenerated forms, while another pseudogene (DRB2) lacks it (Schwaiger and Epplen, 1995). In DRB3, the STR structure is highly disintegrated, and in DRB4 only three copies of each dinucleotide [(GT)3(GA)3] are detectable.
A different microsatellite of the form (AC)n is present in intron 5, adjacent to the 5&rsquoend of exon 6 of Ovar-DRB2 (Scott et al., 1991b Blattman and Beh, 1992). Typing of this microsatellite together with that found in intron 2 of DRB1 in sheep belonging to the international mapping flock, AgResearch, New Zealand indicated a distance of 2.6 cM between the two loci (Schwaiger et al., 1996). This distance is almost the same as that between Ovar-DRB2 and Ovar-MHC I. Also, haplotype analysis of unrelated animals has identified several haplotypes of the DRB region combining different DRB1 and DRB2 alleles, which underscores the genomic instability of the DRB sub-region. Similar distances between these loci have also been reported in a subsequent study (Paterson et al., 1998).
Typing of Ovar-DRB1 genes employing different methods in various sheep breeds has revealed extensive polymorphism at these loci (Table 1). Initial studies employed RFLP techniques utilizing DRB1 exon 2-specific probes (Blattman et al., 1993 Grain et al., 1993). However, this method has been considered unsuitable to study variation at the DRB1 owing to extensive cross-hybridisation between the DRB1 probe and the DQB locus (Escayg et al., 1996). Sequencing of the PCR-amplified DRB1 exon 2, either alone or together with the adjacent STR in intron 2, has revealed extensive polymorphism within the locus (Schwaiger et al., 1993b, 1994 Paterson, 1998 Konnai et al., 2003a Sayers et al., 2005). SSCP and sequence analysis of DRB1 exon 2 is another method for DRB typing (Kostia et al., 1998 Tkacikova et al., 2005). However, in one of the studies employing this method (Jugo and Vicario, 2000), alleles from more than one DRB locus could be detected.
Another method for typing DRB1 alleles of farm animals, using PCR-RFLP analysis, has been suggested (Amills et al., 1996 Rasool et al., 2000 Konnai et al., 2003b Dongxiao and Yuan, 2004 Gruszczynska et al., 2005). Using a pair of bovine specific primers, DRB1 exon 2 was amplified from cattle, buffalo, sheep, and guinea pig DNA samples. The amplified fragment was the same size in all the animals from the different species. Polymorphisms in exon 2 were detected by RFLP of the amplified product. Two recent studies looked at polymorphisms in exon 2 of the Ovar-DRB3 gene employing PCR-RFLP (Sun et al., 2003 Liu et al., 2004). An oligonucleotide method has also been described as a means for typing DRB genes (Schwaiger et al., 1993a). PCR fragments including exon 2 plus adjacent intron 2 are first separated on a polyacrylamide gel based on length variations of the microsatellite repeat and then hybridised with probes for both the intron repeat and exonic sequence. This polymorphism-specific oligonucleotide typing has been utilised for Ovar-DRB1 typing in various studies (Schwaiger et al., 1995 Stear et al., 1996 Buitkamp and Epplen, 1996 McCririe et al., 1997).
PCR amplification of exon 2 together with microsatellite in intron 2 and determination of the exact length of the amplified product using an automatic capillary sequencer is another method for typing Ovar-DRB1 alleles (Gruszczynska, 1999 Gruszczynska et al., 2000 Charon et al., 2002). Length polymorphism of the microsatellite in intron 2 of the expressed DRB gene in various artiodactyl species has been found to be strongly associated with sequence polymorphisms in exon 2 and thus could be utilised for DRB typing (Ellegren et al., 1993). This method was employed in several studies to detect Ovar-DRB1 alleles (Outteridge et al., 1996 Paterson, 1998 Paterson et al., 1998 Saberivand et al., 1998 Griesinger et al., 1999).
Length polymorphisms of the microsatellite in intron 5 of the pseudogene Ovar-DRB2 has also been studied in different breeds of sheep (Table 2). High heterozygosity (>78%) at this locus, reported in these studies, suggests the potential application of this locus as a genetic marker, especially for disease resistance. It has been shown in cattle that the resolution of microsatellite-based DRB3 typing was much better when the length polymorphism of another microsatellite located in DRB1 pseudogene was included (van Haeringen et al., 1999). However, no such typing studies in sheep involving the microsatellites located at DRB1 and DRB2 have been reported.
The existence of DQ genes in sheep was first demonstrated by genomic Southern blot analysis employing probes homologous to the HLA DQ region (Chardon et al., 1985 Scott et al., 1987). In the latter study, the presence of three DQA-like and four DQB-like genes was indicated. RFLP and sequence data derived from genomic clones (Scott et al., 1991a) and cDNA clones (Fabb et al., 1993) indicated the existence of two DQA genes per haplotype in sheep. This is consistent with a detailed genomic map of the ovine DQ sub-region (Wright and Ballingall, 1994), which revealed two DQ loci each containing one DQA and one DQB gene arranged in tail to tail orientation (Figure 3). The two loci are 22 kb apart and are linked on a linear tract of 130 kbp of DNA. The Ovar-DQ sub-region is more compact than the HLA-DQ sub-region, since a distance of 70 kb separates the two HLA-DQ loci (Campbell and Trowsdale, 1993). The Ovar-DQA1 and DQB1 genes at the first locus are separated by 11 kb, while the DQA2 and DQB2 genes at the second locus are 25 kb apart. The HLA-DQ1 genes and Bota (Bos Taurus)-DQ1 genes are also separated by a similar distance, while the HLA-DQ2 genes lie much closer together than the Ovar-DQ2 genes. The equivalents of Bota-DQA3 (Andersson, 1988) and HLA-DQB3 pseudogene (Ando et al., 1989) could not be detected in sheep.
In a study on the linkage analysis between the Ovar-DQA1, DQA2, DQB1, DQB2, and DRA loci, no recombinants were observed between DQA1 and DQA2 loci or between DQA and DQB genes (Escayg et al., 1996). Also, there was no evidence of recombination between the DRA locus and any of the DQ loci. This finding, despite the lack of any available information on the distance between the DQ and DR subregion, would suggest that these loci are physically close.
Ample evidence exists for both in vitro (Wright and Ballingall, 1994) and in vivo (Scott et al., 1991a Fabb et al., 1993 Wright and Ballingall, 1994) transcription of Ovar-DQA genes. However, cell surface expression of DQ products has been detected only for the DQ1 locus (Wright and Ballingall, 1994). It is probable that despite expression of genes at the DQ2 locus, the lack of suitable monoclonal antibody (Wright and Ballingall, 1994) or the possibility of the DQ2 a - and b -chains mis-pairing (Snibson et al., 1998) may be the reason(s) for failure in detecting their products. This view is further supported by the fact that about 10 to 18% of sheep from different breeds (Scott et al., 1991a Fabb et al., 1993 Escayg et al., 1996) lack the DQA1 gene in their haplotypes, indicating that any functional DQ molecule in these sheep would be the product of expressed genes at DQ2 locus (Snibson et al., 1998).
The nucleotide sequence of all exons and introns, excluding exon 1 of Ovar-DQA1 and DQA2 genes, has been determined and was found to be similar to respective analogues in humans (Scott et al., 1991a). The second exons in these two genes were less similar in terms of nucleotide (78%) and coding amino acid (71%) identities between them. Subsequently, full-length cDNA clones coding for these two Ovar-DQA genes, together with that for the Ovar-DRA gene, have been isolated and sequenced (Fabb et al., 1993). All of these encode polypeptides of 255 amino acids, with 23 of them accounting for signal peptide and the other 232 encoding the mature polypeptide. DQA1 and DQA2 could be discriminated mainly based on the nucleotide sequence of exon 2. The exon 2 nucleotide dissimilarity between DQA1 and DQA2 genes (19.5%) is far more than that between the alleles within either DQA1 (8.0%) or DQA2 (10.0%). Nucleotide variation was found to be minimal in exon 4 of both genes. Similar sequence polymorphisms in exon 2 were also observed in a different study (Snibson et al., 1998).
Several alleles of Ovar-DQA1 and DQA2 have been identified based on sequence variation of the PCR amplified exon 2. Twenty-three different DQA2 sequence alleles (1-Scott et al., 1991a 1-Fabb et al., 1993 1-Wright and Ballingall, 1994 7-Snibson et al., 1998 13-Hickford et al., 2004) and sixteen DQA1 sequence alleles (1-Scott et al., 1991a 1-Fabb et al., 1993 3-Wright and Ballingall, 1994 2-Snibson et al., 1998 3-Zhou and Hickford, 2001 6-Zhou and Hickford, 2004) have been identified. PCR-SSCP is an ideal method for typing DQA sequence alleles (Snibson et al., 1998). A single set of PCR primers could amplify all known DQA2 alleles, while a separate set of primers amplified only the DQA1 gene. Two new DQA1 and nine DQA2 alleles were identified in the study using this method.
Employing PCR-SSCP, an extensive investigation on the DQA2 gene was carried out in 2000 sheep belonging to Merino, Corriedale, Borderdale, Romney, Awassi, and Finnish Landrace breeds (Hickford et al., 2004). As many as 23 exon 2 sequences could be identified, of which 5 were found to be more similar to bovine DQA3 or DQA4 sequences than to other sheep DQA2 and were designated as DQA2-like sequences. However, there was no evidence for the presence of the bovine DQA5-like sequences in sheep. Three or four unique DQA2 sequences could be recovered from individual sheep, suggesting the presence of two DQA2 loci.
A different study, but employing the same technique, on DQA1 in 300 sheep belonging to Merino, Corriedale, Borderdale, Romney, Awassi, and Finnish Landrace breeds revealed extensive polymorphism in the exon 2 sequence, with as many as 14 alleles (Zhou and Hickford, 2004). Comparison of the sheep DQA1 exon 2 sequences with those available from cattle revealed several clusters of ovine DQA1 sequences, with some of the sheep alleles being more similar to cattle alleles than to the other sheep alleles. It was suggested that this trans-species polymorphism might be the result of balancing selection at the DQA1 locus.
Polymorphisms of DQA genes have also been reflected in RFLP studies employing exon 2-specific probes (Scott et al., 1991a Fabb et al., 1993 Escayg et al., 1996 Hickford et al., 2000). DQA2 was found to be more polymorphic than DQA1. Up to 8 and 16 alleles have been reported for DQA1 and DQA2, respectively. Another interesting feature of these studies is that in 11 to 36% of the sheep screened, no DQA1 allele could be detected and the allele in such animals was considered as null. Thus, sheep do have a variable number of DQA genes in their haplotypes. In some of the sheep that possessed null DQA1 allele, two DQA2-like sequences could be detected (Hickford et al., 2000), retaining the pattern of two DQA loci per haplotype. Duplication of DQA2 gene was suggested in these animals. Also, the similarity between the two DQA2 sequences would suggest that DQA1/DQA2 haplotype is ancestral to DQA1 null/DQA2 (duplicated) haplotype. Similarly, since the DQA1/DQA2 haplotype is more diverse than the DQA1 null/DQA2, it seems likely that DQA1 null/DQA2 haplotype preceded DQA1/DQA2. The presence of two DQA2-like sequences in animals with DQA1 null alleles has also been reported in cattle (Ballingall et al., 1997). However, the two DQA2 sequences were diverse and had been categorised as DQA2 and DQA3. In sheep, it was shown that some ovine DQA2 sequences exhibited much closer similarity to the cattle DQA3 gene than to other DQA2 sequences (Snibson et al., 1998). This suggests that the duplicated ovine DQA2 gene in animals with DQA1 null allele may be analogous to the cattle DQA3 gene (Hickford et al., 2000). However, there is no evidence with regard to the expression of this gene. The presence of two additional DQA loci in cattle, Bota-DQA4 (Ballingall et al., 1997) and DQA5 (Gelhaus et al., 1999) has been reported, but their homologues in sheep are yet to be identified.
The nucleotide sequence of Ovar-DQB gene, excluding exon 1 and parts of the introns, has been reported (Scott et al., 1991b). Comparison with human sequences revealed similarity with both HLA-DQB1 and DQB2, suggesting the presence of a common ancestor. Subsequently, exon 2 nucleotide sequences of two separate Ovar-DQB genes (DQB1 and DQB2), derived from cosmid clones, have been determined (Wright and Ballingall, 1994). The two genes could not be assigned to separate loci based on the nucleotide sequences, owing to >90% similarity. However, their proximity to an Ovar-DQA1 or DQA2 gene could be used to discriminate between these genes.
Several new DQB sequences have been determined in subsequent studies. Difficulty still exists in assigning these sequences to separate loci because of the high similarity between the two DQB genes. Ten distinct sequences were identified from an SSCP sequence analysis of PCR-amplified DQB exon 2 in 13 Merino sheep, demonstrating considerable variation in the ovine DQB region (van Oorschot et al., 1994). Twenty-nine percent of the total 267 nucleotide sites in exon 2 of these alleles, translating to 46% of amino acid sites, are polymorphic. The presence of at least two separate OLA-DQB genes was demonstrated in that study. Phylogenetic analyses of the exon 2 nucleotide and amino acid sequences from sheep, cattle and humans showed that the ovine and bovine sequences are more closely related to each other than either are to the human sequences. The SSCP technique was shown to be capable of discriminating between all the Ovar-DQB sequences identified in the study.
Sixteen distinct PCR-amplified Ovar-DQB exon 2 sequences have been characterised from only 18 sheep in another study (Schwaiger et al., 1996). While three of these sequences could be assigned to DQB1 and two to DQB2, the rest could not be assigned to either locus. Reference-strand-mediated conformation analysis or double-strand conformational analysis, employing two reference alleles, has been shown to be a new method for high resolution typing of the Ovar-DQB genes (Feichtlbauer-Huber et al., 2000). The use of two different reference alleles would enable high resolution of many and probably all alleles and reduce the probability of missing new alleles. Using this method, 16 new sequences (from that of van Oorschot et al., 1994) were obtained from 10 unrelated Scottish black-faced sheep, increasing the number of known alleles to 28. However, the alleles could not be assigned to separate loci.
Ovar-DNA and DOB genes
The presence of the DNA (formerly DZA) gene in sheep had been inferred from Southern analysis of genomic DNA (Scott et al., 1987). Cosmid clones from the sheep MHC class II region were found to contain this gene (Deverson et al., 1991). Subsequently, the nucleotide sequence of the DNA gene, together with its predicted amino acid translations, were reported (Wright et al., 1995). It had all the salient features of a class IIA gene, including two exons coding for the two extracellular domains, and one coding for a proline rich connecting peptide, a hydrophobic transmembrane region and a cytoplasmic tail. Also, it has two conserved N-linked glycosylation sites NGT and NAT, and two conserved cysteine residues, forming a disulphide bond in the a 2 domain. The ovine and human genes share 83% nucleotide identity (translating to 78% amino acid identity) at exons 2 and 3. Though transcription of the Ovar-DNA gene was detected by Northern hybridisation with an Ovar-DNA probe, there was no evidence of expression of the gene. Like that of the Ovar-DRA, the Ovar-DNA gene appears to be monomorphic (Schwaiger et al., 1996).
The B gene partner for HLA-DNA gene is the non-polymorphic HLA-DOB gene (Tonnelle et al., 1985), while the murine homologue of the Ovar-DNA gene expresses in combination with the H2-OB gene (Karlsson et al., 1991). There was an early indication in sheep for the existence of Ovar-DOB gene (Scott et al., 1987). The gene has been cloned and subsequently sequenced (Wright et al., 1996). Exons 1 and 2 have been found to exhibit amino acid identities of 62 and 80%, respectively, in comparison with the HLA-DOB gene. Neither transcription of the gene nor its expression in combination with Ovar-DNA gene could be detected in the study.
DYA and DYB (DIB) genes which are absent in HLA have been detected in cattle (Andersson et al., 1988). These were shown to segregate with the DOB gene in one region separated by a recombination distance of 17 cM from the region that contains DQ, DR and C4 loci. The Bota-DYA gene has been cloned and sequenced (van der Poel et al., 1990), while there has been no report of cloning of its B gene partner. A unique single copy class IIB gene, Bota-DIB has been cloned and sequenced from a phage library (Stone and Muggli-Cockett, 1990). The homologues of Bota-DYA and DIB genes in sheep, designated as Ovar-DYA and DYB, have been identified in sheep by screening a cosmid library with Ovar- and HLA-DQ probes at low stringency (Wright et al., 1994). The presence of DY genes, together with the absence of DP genes and variability in the number of DQ genes between haplotypes, has been considered as a distinguishing feature of the ruminant class II region.
The Ovar-DYA gene have shown high sequence similarity to the bovine and caprine DYA genes and much less so to the Ovar-DRA, DNA and DQA genes (Wright et al., 1994). Similarly, the Ovar-DYB gene exhibited a higher degree of sequence similarity to the Bota-DIB and was different from the Ovar-DQB and DRB genes. It was named DYB rather than DIB because of its close proximity to DYA gene. The DYA and DYB genes lie tail to tail with a distance of 11 kb between them. While transcription of the gene could be detected, there was no evidence for its expression. The authors suggested that evolution of the DY locus may be the result of duplication of a pair of DQ genes, with subsequent rapid divergence.
A polymorphic microsatellite (DYMS1) of the form (CA)n was found to be located in the region 5&rsquo of the DYA gene, 19 cM from the DRB1 locus (Buitkamp et al., 1996). Nineteen alleles were identified at the locus in this study. The polymorphism at this microsatellite locus was later confirmed in a different study in German Rhonschaf sheep that revealed 6 alleles (JanBen et al., 2002).
Studies on the second exon of DY genes employing SSCP have revealed 3 alleles for Ovar-DYA and 4 in DYB, with respective heterozygosities of 0.67 and 0.61 (Maddox, 1999). A recent study assessed the degree of conservation between ovine and bovine DYA gene sequences (Ballingall and McKeever, 2005). Nucleotide similarities of 97% in the immediate promoter, 94% in the coding and 91% in the intronic regions were observed between the species. The Ovar-DYA full length transcript revealed an open frame encoding a 288 amino acid protein compared with a 253 amino acid protein associated with the bovine DYA transcript.
The existence of DMA and DMB genes in sheep has been indicated based on PCR amplification of fragments from exons 2 and 3 of the Ovar-DMB gene and exon 2 of the Ovar-DMA gene, employing primers derived from murine and human gene sequences (Schwaiger et al., 1996). Only two exon 2 alleles could be detected in the case of the DMB gene by SSCP (Maddox, 1999).
Class II molecules have a much more restricted expression pattern than do class I molecules, in that they are expressed primarily on cells deemed to have antigen uptake, processing and presentation functions (macrophages, dendritic cells and B cells). Their expression varies among species and is enhanced in rapidly dividing cells and in cells treated with interferon (Tizard, 2004). Class II molecules are also heterodimers (Figure 2), but in contrast to class I molecules, are composed of an a - and a b -peptide chain. Each chain has two extracellular domains, a connecting peptide, a transmembrane domain and a cytoplasmic domain. A third protein chain, called g or invariant chain (Li or CD74), is associated with intracellular class II molecules (Tizard, 2004).
The class II PBR consists of two a helices bordering a b -pleated sheet (Hughes and Yeager, 1998), as with the class I molecule. The difference is that in class II, one of the a helices and about half of the b -pleated sheet are contributed by the a -chain, whereas the other a helix and other half of the b -pleated sheet come from the b -chain. Polymorphisms in the class II molecules result from variation in the amino acid sequences of the a helices at the sides of the groove. The a - and b -chains are encoded by genes in the class II region. In mammals, the class II subregions (designated as DR, DP and DQ in humans), each contain a functional a -chain gene and one or more functional b -chain genes.
The class II molecules present peptides derived from exogenous proteins to the TCR of CD4+ helper T cells (Germain and Margulies, 1993). In response to a foreign peptide, the helper T cells release cytokines that trigger the production of antibodies and cell-mediated immune responses. The class II molecules also possess the property of MHC restriction, in which the antigens bound to MHC molecules also need to be recognised by a TCR on a helper cell, in order to trigger an immune response. The peptides presented by class II molecules can vary substantially in length, between 11 and 17 residues (Rammensee et al., 1995).
The complex between the class II molecule and its peptide ligand is created by a mechanism quite different from that of class I. Before transport to the cell surface, the class II dimer forms a complex with the invariant chain (Li) in the ER, which acts as a chaperone to stabilise the heterodimer and prevents premature peptide loading. This complex then travels to an acidic endosome-like compartment (Peters et al., 1991), where the Li is degraded by a series of proteolytic cleavage events, leaving a residual peptide (class II-associated invariant chain peptide) occupying the PBR of the MHC molecule. The release of class II-associated invariant chain peptide and its replacement with antigenic peptides is catalysed by HLA-DM, which is independently targeted to endosomal compartments. The resultant MHC class II-peptide complex is then transported to the cell surface, where it awaits interaction with antigen-specific T cells. The expression of MHC class II, Li and HLA-DM genes is coordinately regulated at the level of transcription by a conserved set of factors and defined cis-acting elements (Boss and Jensen, 2003).
Immunoprecipitation and SDS-PAGE analysis of ovine class II molecules have revealed a non-covalently associated glycoprotein complex with a 30-32 kDa a -chain and a 24-26 kDa b -chain (Puri et al., 1985). A similar finding on the structure of class II molecules was reported by Hopkins et al. (1986). An interesting feature in both these studies, in contrast to that in humans, was that the sheep class II a - and b -chains could only be resolved under non-reducing conditions. Under reducing conditions, the b -polypeptide appeared to undergo a shift to a molecular mass of 30 kDa and thus co-migrated with the a -chain. Under non-reducing conditions, three bands, one corresponding to the a -chain, one to the b -chain and one to a probable invariant chain could be identified.
Another significant difference in the structure of the class II heterodimer of sheep, compared to mouse and human, is that it is unstable in the presence of 1% SDS at 20°C (Puri et al., 1987c). Under these conditions, 75% of the molecules were found to be dissociated into a - and b -chains and at a temperature of 100°C, almost all the molecules were found to be dissociated. The mouse and human class II molecules, on the other hand, are stable in SDS up to 38°C (Shackelford et al., 1982). Also, the rate of biosynthesis of sheep class II molecules appeared to be similar to or slightly faster than that in humans (Puri et al., 1987a).
Studies have been undertaken to categorise the sheep MHC class II molecules (Puri et al., 1987a,b,d Puri and Brandon, 1987). Sequential immunodepletion by a panel of monoclonal antibodies, followed by two-dimensional NEPHGE/SDS-PAGE analysis, revealed four structurally and serologically distinct subsets of class II molecules, similar to those found in humans. Also, these molecules exhibited structurally detectable allelic polymorphism. Three of the subsets displayed allelic polymorphism in b -polypeptides, while the fourth set showed allelic variation in both of their a - and b -polypeptides (Puri et al., 1987a). Approximately 10-12 different class II molecules were found to be expressed by a single sheep (Puri and Brandon, 1987). Subgroup-specific monoclonal antibodies against sheep MHC class II molecules, nine specific for the b -chain and four for the a -chain, have been developed (Dutia et al., 1990).
Relative to other parts of the MHC, this region has the highest gene density, with the least number of pseudogenes (Kulski et al., 2002). However, some of the genes located in this region are not involved with the immune system. Class III genes with an obvious role in immunobiology include members of the complement cascade (C4A, C4B, C2, and Bf) and genes such as TNF a , LTA and LTB. C4, C2 and Bf are genes for complement proteins (Campbell et al., 1986). TNF a , LTA and LTB encode cachectin, lymphotoxin A and B molecules, respectively (Webb and Chaplin, 1990). Other genes of interest located in the region include HSP70, CYP21, G15, cytochrome p450, LST1, and 1C7. Of these, HSP70 is important as it encodes heat shock protein 70, which presents intracellular contents of cancer cells to the immune system and thus has a role in tumor rejection (Srivastava et al., 1998). The gene coding for HSP70 is duplicated and it has been shown recently that the loss of one of the duplicated genes in Holstein cattle is responsible for hereditary myopathy of diaphragmatic muscles (Sugimoto et al., 2003).
The class III region is poorly characterised in sheep. The existence of this region is based on circumstantial evidence derived from comparisons with related species, namely goats and cattle, and synteny between several loci (Schwaiger et al., 1996). The authors described a preliminary map of the Ovar-class III region. Cosmid clones containing C4 genes were isolated from a sheep genomic library by hybridisation with a bovine C4 cDNA probe. Additional cosmid clones containing the genes for 21-hydroxylase (CYP21), complement factor 2 (C2) and factor B (Bf) could also be obtained by a cosmid walking procedure employing respective human DNA probes. Relative positions of these loci were mapped within an approximate 150-kb DNA segment. Evidence could be obtained for duplication of C4 and CYP21 loci. Also, the order of CYP and C4 loci in sheep (CYP21B𠉬YP21) is quite different from that in humans, mouse and cattle (CYP21B𠉬YP21A). Furthermore, the two Ovar-C4 loci lie in tail-to-tail orientation. This evidence suggests the occurrence of a chromosomal inversion in this region of the sheep chromosome.
Complement cascade genes
The presence of the C4 gene in sheep was first indicated in RFLP studies employing human C4 cDNA probe (Chardon et al., 1985). Neither any polymorphism nor linkage to MHC could be demonstrated. Subsequently, linkage between the C4 gene and OLA-SY1b antigen was established (Groth et al., 1987a). The presence of two polymorphic C4 loci has been indicated in a study on C3 and C4 concentrations in Merino and Suffolk sheep (Groth et al., 1987b). A rapid procedure for the isolation of complement factor, C4, from ovine plasma has been described, and two isotypes of C4 molecules, C4A and C4B, have been detected (Groth et al., 1988). The isotypes differed in the molecular mass of the a -chain (108 and 95 kDa, respectively). An RFLP of the C4 gene, employing Taq1 enzyme and the HLA-C4 probe, revealed linkage disequilibrium between C4 and DQB genes in unrelated sheep. Similar linkage of the C4 and DRB genes has also been reported (Wetherall et al., 1991). A C4*A2 phenotypic allele was found to be associated with a 19-kb DRB RFLP fragment in 18 of the 27 sheep studied.
In another study based on cloning and sequencing of DNA fragments obtained by PCR amplification of thioester and isotype determining sites of the sheep C4 genes, up to five distinct C4 gene loci were detected (Ren et al., 1993). The number of C4 genes per haplotype is thus similar to that in both humans and mice (Schwaiger et al., 1996). However, the sheep and cattle genes are believed to have evolved independent of those in primates and mice (Ren et al., 1993). Close to another complement factor gene, Bf, a polymorphic microsatellite locus, BfMS, has been detected (Groth and Wetherall, 1995). Eight alleles, differing in base-pair length, were detected at the locus in an Australian fine-wool Merino flock (Bot et al., 2004).
TNF a gene
TNF a is a cytokine with a wide range of effects on both lymphoid and non-lymphoid cell types. The existence of a single copy of the TNF a gene in sheep has been demonstrated (Nash et al., 1991). Ovine TNF a cDNAs were cloned and sequenced by three independent groups (Young et al., 1990 Green and Sargan, 1991 Nash et al., 1991). The sequences obtained in the first two studies were exactly the same, encoding for a 76-amino acid leader sequence and a 157-amino acid mature protein. The amino acid sequence was up to 88% homologous to the human TNF a protein. The cDNA sequence obtained in the third study was similar to that obtained in the first two studies, except that it lacked one amino acid in the leader sequence.
A recent study investigated allelic variation at the Ovar-TNF a locus (Alvarez-Busto et al., 2004). SSCP and sequence analysis of a 273-bp fragment, comprising part of the fourth exon and the 3&rsquo untranslated region of the gene, revealed three different alleles. These alleles differed in one deletion and one single nucleotide polymorphism. However, no difference was found in their frequencies in Latxa and Rasa breeds. An earlier attempt to detect polymorphism at this locus, employing RFLP with the use of human cDNA probes, was unsuccessful (Engwerda et al., 1996).
Other class III genes
There has been little research in the characterisation of genes other than the complement cascade and TNF genes of the Ovar-class III region. A dinucleotide microsatellite of the form (CA)n has been found to occur in at least one of the two cattle CYP21 genes (Moore et al., 1991). However, no such microsatellite could be detected in any of the Ovar-CYP21 genes either by PCR using an oligonucleotide primer (Moore et al., 1991) or by Southern hybridisation (Schwaiger et al., 1996).
INHERITANCE AND POLYMORPHISM OF MHC GENES
A characteristic feature of the MHC antigens is their co-dominant expression, i.e., both the alleles at a given locus are expressed in a heterozygote individual. Also, the MHC is inherited en bloc as a haplotype with the exception of rare recombination (1-3% frequency). Hence, in the case of MHC genes, an association based on haplotypes is usually stronger and more meaningful than an allelic association (Dorak, 2005). Despite the enormous number of alleles at each expressed locus, the number of haplotypes observed in a population is much smaller than the theoretical expectations. This is because of certain alleles tending to occur together on the same haplotype rather than randomly segregating, a phenomenon referred to as linkage disequilibrium (Begovich et al., 1992).
Among the expressed loci in the human genome, the MHC shows the greatest degree of polymorphism (Dorak, 2005). The level of polymorphism is at such a degree that it is theoretically possible for each human to possess a different set of MHC alleles. Certain of the class I and class II loci that are involved in antigen presentation show extraordinarily high levels of polymorphism with several hundreds of allelic variants of the genes within the population (Klein, 1986). The genes at these loci are usually present as multiple copies, many of them being pseudogenes. The pseudogenes lack either one or more exons in them and even in the exons that exist, numerous mutations occur, rendering them non-functional. The presence of multiple copies is of evolutionary significance. Since it involves a birth and death process, new genes are created and some of them are maintained in the genome for a long time, while others are deleted or become non-functional through deleterious mutations (Klein et al., 1998). Class I loci undergo a faster rate of birth and death evolution than class II loci, and hence, it is difficult to establish the orthologous relationships of different class I genes among different orders of mammals (Hughes and Nei, 1989). On the other hand, the high longevity of class II genes enables such orthologous class II loci to be shared by different orders of mammals (Takahashi et al., 2000).
The mechanisms responsible for polymorphism in the MHC genes have been intensely debated and reviewed (Hughes and Yeager, 1998 Meyer and Thomson, 2001 Bernatchez and Landry, 2003). Parasite-mediated balancing selection and reproductive mechanisms constitute the two main types of mechanisms that operate to maintain the unusually high level of MHC polymorphism. Three different non-exclusive forms of balancing selection, symmetrical overdominance, negative frequency-dependent selection and fluctuation in selection pressure, are known to exist (Charbonnel and Pemberton, 2005). According to the hypothesis of heterozygote advantage or symmetrical overdominance (Doherty and Zinkernagel, 1975), an individual that is heterozygous, rather than homozygous, at the MHC loci has better immune surveillance against infectious organisms. The domains a 1/ a 2 and a 1/ b 1 of class I and class II molecules, respectively, that form the peptide-binding groove in each case, constitute the driving force for heterozygote selection, in the presence of challenge from infectious agents. Several studies (Thursz et al., 1997 Carrington et al., 1999 Penn et al., 2002 Stear et al., 2005) have confirmed this selective advantage of MHC heterozygosity against infectious agents.
Under negative frequency-dependent selection or rare allele advantage (Clarke and Kirby, 1996), MHC genotypes with a rare allele are supposed to have a strong selective advantage as few pathogens have been exposed and adapted to it. Conversely, the relative fitness of the common genotypes would be decreased. A study on the association between class II DRB alleles and resistance to gastro-intestinal parasitism in Soay sheep (Paterson et al., 1998) has provided evidence for rare allele advantage. The third form of balancing selection results from fluctuation in the selection pressure. Spatial and(or) temporal variation in the presence or density of pathogens could result in constant changes in the intensity of pathogen-mediated selection, thus maintaining polymorphism at the level of metapopulation (Hedrick, 2002). A recent study pertaining to a long-term genetic survey of Soay sheep supported this hypothesis (Charbonnel and Pemberton, 2005).
One early hypothesis explaining the high level of polymorphism within the MHC was the neutral theory of molecular evolution (Kimura, 1968). This theory suggested that the molecular mechanisms that result in polymorphism include point mutations, reciprocal recombination and gene conversion. However, the point mutation rate in MHC is by no means higher than elsewhere in the genome (Parham et al., 1995). However, accumulation of point mutations over millions of years as a result of the sharing of allelic lineages by related species, a fact referred to as trans-species polymorphism, brings about this extensive allelic polymorphism (Klein et al., 1993).
Other mechanisms that may bring about and maintain MHC gene diversity include MHC-based non-assortative mating preferences (Penn and Potts, 1999) and maternal-foetal incompatibility (Ober et al., 1998). However, these mechanisms together with the neutral theory have been discarded as the main cause of MHC polymorphism, as these processes should affect gene regions at random, rather than being concentrated in the PBRs (Jeffery and Bangham, 2000).
Several studies, over the past two and a half decades, have focused on the Ovar-Mhc. However, when compared to other domestic species, the ovine MHC is still poorly characterised. Several genes, across the three regions of the Ovar-Mhc, are yet to be characterised. Recent advances in large-scale cloning and large-scale sequencing have helped generate long genomic sequences, even complete MHC sequences, in several species (Kumanovics et al., 2003). The genomic sequences, in contrast to the cDNA sequences, provide the complete and ordered set of the MHC genes, including pseudogenes. The complete sequence of the HLA complex was available in late 1990s (MHC Sequencing Consortium, 1999) and it was evident that the class I and II regions extend well beyond the original boundaries (Stephens et al., 1999). Among the domesticated species, such large MHC genomic sequences have been reported for the B locus of the chicken (Kaufman et al., 1999), the class I region of the quail (Shiina et al., 1999), the class II region of the cat (Beck et al., 2001), and the class I region of the pig (Renard et al., 2001 Chardon et al., 2001). However, there are no reports of such sequences with regard to the Ovar-Mhc. Availability of the complete sequence of the Ovar-Mhc would enable the design of multiple markers that are more dense, equidistant and expansive throughout the region. This would facilitate the characterisation of individuals in terms of haplotypes rather than individual genes. MHC haplotypes are more meaningful, considering the existence of linkage disequilibrium among the MHC genes.
Most of the studies, aimed at characterizing the Ovar-Mhc, have focused on the class II region in general and on DR and DQ genes, in particular. The length polymorphisms of three microsatellites (one each at the DRB1, DRB2 and DYA genes), and the exon 2 sequence variations at DRB1, DQA and DQB genes have been extensively studied in different breeds of sheep. In contrast, the class I region is poorly characterised. Controversy still exists with regard to the number of classical class I loci and there is no information on the non-classical class I genes. Studies pertaining to this region have focused mainly on the length polymorphism of a microsatellite located at one of the loci. Associations of the Ovar-Mhc genes with disease resistance have been reported in various studies (reviewed by Dukkipati et al., 2006). Several associations (especially of MHC antigens) with resistance/susceptibility to gastrointestinal nematodes have been revealed. However, those could not be utilised in screening sheep flocks for increased genetic resistance, owing to the complexity and labour intensiveness of MHC antigen serotyping methods. Hence, the development of accurate, simplified and cost-effective typing methods for various MHC loci enables more meaningful association studies to be carried out, followed by marker-assisted selection on a commercial basis.
Financial support to the first author in the form of a Doctoral Scholarship from Massey University, New Zealand is gratefully acknowledged. The authors are also thankful to Annual Reviews, for permission to reprint the Figure 2 depicted in the review.
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The authors thank Cristina Ambrós and Eduard Palou, Pilar Armengol and Roger Colobran, and Mariona Pascal for providing some SSO-PCR typed DNA samples, for their general support in the laboratory and for critical reading, respectively. This study was supported by a grant from the “Fundación para la Investigación y la Prevención del Sida en España”, (project number 36487/05), by the Fondo de Investigaciones Sanitarias del Instituto de Salud Carlos III (PI07/0329), by the PROFIT (FIT 010000–2006–38) of the BST and by the “Departament d'Educació i Universitats de la Generalitat de Catalunya” to Manel Juan and NIH contract HHSN 266200400028C to William W Kwok.
Conflict of interest: The authors declare no financial or commercial conflict of interest.
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The major histocompatibility complex (MHC) is a region unique to the genomes of jawed vertebrates and contains genes that are critical to the generation of immune responses. It is the most gene dense and polymorphic region in the genome (reviewed in ). The MHC is named for its role in recognition of ‘self’ and ‘non-self’, and was first identified in connection with tumour transplant rejection . Genes in the MHC are also associated with resistance to infectious diseases, autoimmunity, reproductive success, inflammatory response and innate immunity (reviewed in [3, 4]).
The genes of the MHC are sub-divided into class I, II and III. The MHC class I genes are particularly noteworthy for having undergone gene duplication and divergence, resulting in an extended gene family whose members perform a broad range of functions. The classical role of class I molecules is to present endogenously-derived peptides to CD8 + T cells to stimulate cytotoxic responses against virus-infected or tumour cells. The class I molecules performing this role are sometimes referred to as classical MHC class I. Examples of classical class I genes include HLA-A, -B and -C in humans and H2-K, H2-D and H2-L in mouse. Classical MHC class I genes are generally broadly expressed in nucleated cells and highly polymorphic. Class I molecules performing other functions, collectively known as non-classical MHC class I, generally have low polymorphism, may have tissue-specific expression and in some cases have evolved functions other than antigen-presentation, including immuno-regulatory and non-immune roles. Examples of non-classical class I genes include HLA-E, -F and -G in human, B1 and Qa1 in mouse, as well as MIC. The function of non-classical molecules is not limited to the immune system. The HFE gene, for example, serves as part of the transferrin complex involved in iron storage (reviewed in ). Others, such as the neonatal Fc receptor, FcRN, that transports maternal IgG to fetal or neonatal mammals, has a role in the immune system that is distinctly different from conventional class I (reviewed in ). Typically, classical and some non-classical genes are located in the MHC, although many of the non-classical are located elsewhere in the genome .
In humans, the MHC is located on chromosome 6p . Additionally, there are three regions of the genome that are paralogues of the MHC, indicative of the two rounds of whole genome duplication thought to have occurred in early vertebrate evolution . These paralogous regions are located on chromosomes 1q, 9q, and 19p. They contain additional non-classical class I genes, including the CD1 gene family, MR1 and FCGRT. Other non-classical class I genes are found on chromosome 20 (PROCR), chromosome 7 (AZGP1) and chromosome 6q (ULBP and RAET families), suggesting that duplication and translocation have acted to further distribute MHC class I genes throughout the genome.
In other species, similar processes have acted to spread class I genes from the MHC. Two tightly linked, classical class I-like genes (UB and UC) in the opossum, Monodelphis domestica, for example, were translocated outside the MHC although they remain syntenic to the MHC on chromosome 2 [9, 10]. In a more extreme example, in the tammar wallaby, Macropus eugenii, the classical class I-like genes have been completely translocated out of the MHC and are distributed across multiple chromosomes .
Both classical and non-classical class I molecules have a conserved and distinctive protein domain structure. MHC class I genes typically have 5–9 exons encoding proteins with well-defined domain organization (Fig. 1a and 1b). The first exon encodes a signal peptide. Exons 2 and 3 encode the α1 and α2 domains, which together make up the antigen-presenting domain (APD). An immunoglobulin domain (Ig or α3) is encoded by exon 4. Additional exons may encode one or more transmembrane domains and the final exon contains a conserved cytoplasmic domain at the C-terminal of some MHC class I genes. The α1, α2 and Ig domains are the hallmark of MHC class I genes. However, different isoforms of some MHC class I genes exist. These may splice out some of these domains to produce other membrane bound versions of the protein or secreted forms. Additionally, the UL16-binding protein (ULBP) and retinoic acid early transcript (RAET) families, known in eutherians, are MHC class I-related genes that lack immunoglobulin domains and may utilize a GPI-anchor, rather than a transmembrane domain [12–15].
Sensitive pan-genome search for MHC class I genes. a The canonical domain structure of MHC class I proteins and (b) genes. c The location in the opossum genome and score of matches to profile hidden Markov models representing the antigen-presenting domain (split into α1 and α2 regions), C-type immunoglobulin domain and C-terminal domain. d Example of a high-scoring run of α1, α2, Ig and C-terminal domains in the opossum genome. e Finite state automata of the alignment algorithm to search for runs of α1, α2, Ig and C-terminal domains, taking domain score and distance between domains into account. The nodes (circles) show match states. Symbols on edges show scores/penalties: +m is the match score, which is based on the HMM match score -γ is a distance-dependent affine gap penalty, which models introns and allows the alignment to skip over matches that interrupt a run of domains -ψ is a constant penalty for dropping the C-terminal domain
To better understand the evolution of MHC class I genes, particularly in mammals, we undertook to catalogue the class I genes. Here, we describe a sensitive comparative genomics analysis of MHC class I genes spanning vertebrate life. This was achieved using a novel approach based upon combining profile hidden Markov models (HMMs), which represent the separate domains characteristic of MHC class I genes. Our results reveal a new sub-family of MHC class I genes in marsupials and monotremes, which are not found in non-mammals and have been lost from the eutherian lineage. We show that these genes are transcribed in immune tissues in the gray short-tailed opossum, tammar wallaby, brushtail possum and Tasmanian devil. Structural homology mapping is used to begin to investigate the function of these genes.
“Dark side” in the title can be considered a reference to the evil force in the famous Star Wars saga. Due to many of its intrinsic characteristics, including a well-established desmoplastic reaction and an immunosuppressive microenvironment, pancreatic cancer actually represents the dark side of immunotherapy. Compared to other solid tumors, indeed, many immunotherapy approaches drastically fail when applied to pancreatic cancer patients. However, thanks to intensive worldwide research focused on this deadly disease, in the last decade our knowledge has greatly increased, which has paralleled an increase in 5-year survival rate to 10% at least in the USA. The scientific community has developed sophisticated tools to study the biology of pancreatic cancer such as Avatars, genetically engineered mice, and organoids, in which novel therapeutic drugs can be tested. Genetically engineered mouse models represent the best choice for studying antitumor immune responses and novel immunotherapy approaches, as they mimic the complex relationship between the immune system and tumor cells. Of equal importance is the possibility to develop novel clinical studies and to enroll patients in clinical trials.
Therefore, our title better refers to the famous Pink Floyd album, “The Dark Side of the Moon”, which experimented with multiple sounds to create this masterpiece. The huge increase in funds focused on PDAC, together with advanced efforts by the scientific community, can provide hope for patients affected by this tumor.
The repertoire of MHC class I genes in the common marmoset: evidence for functional plasticity
In humans, the classical antigen presentation function of major histocompatibility complex (MHC) class I molecules is controlled by the human leukocyte antigen HLA -A, HLA-B and HLA-C loci. A similar observation has been made for great apes and Old World monkey species. In contrast, a New World monkey species such as the cotton-top tamarin (Saguinus oedipus) appears to employ the G locus for its classical antigen presentation function. At present, little is known about the classical MHC class I repertoire of the common marmoset (Callithrix jacchus), another New World monkey that is widely used in biomedical research. In the present population study, no evidence has been found for abundant transcription of classical I class genes. However, in each common marmoset, four to seven different G-like alleles were detected, suggesting that the ancestral locus has been subject to expansion. Segregation studies provided evidence for at least two G-like genes present per haplotype, which are transcribed by a variety of cell types. The alleles of these Caja-G genes cluster in separate lineages, suggesting that the loci diversified considerably after duplication. Phylogenetic analyses of the introns confirm that the Caja-G loci cluster in the vicinity of HLA-G, indicating that both genes shared an ancestor. In contrast to HLA-G, Caja-G shows considerable polymorphism at the peptide-binding sites. This observation, together with the lack of detectable transcripts of A and B-like genes, indicates that Caja-G genes have taken over the function of classical class I genes. These data highlight the extreme plasticity of the MHC class I gene system.
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