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The Major Histocompatibility Complex (MHC)


The Major Histocompatibility Complex (MHC)
Molecules within this complex were originally defined by their ability to provoke vigorous rejection of grafts exchanged between different members of a species (Milestone 4.2). We have already referred to the necessity for antigens to be associated with class I or class II MHC molecules in order that they may be recognized by T‐lymphocytes (Figure 4.8). How antigenic peptides are processed and selected for presentation within MHC molecules and how the TCR sees this complex are discussed in detail in Chapter 5, but let us run through the major points briefly here so that reader will appreciate why these molecules are of huge importance within the immune system.
MHC molecules assemble within the cell, where they associate with short peptide fragments derived either from proteins being made by the cell (MHC class I molecules bind to peptides derived from proteins being synthesized within the cell) or proteins that have been internalized by the cell through phagocytosis or pinocytosis (MHC class II molecules bind to peptides derived from proteins made external to the cell). There are some exceptions to these general rules, which we deal with in Chapter 5. We have already made the analogy that this process represents a type of “quality control” checking system where a fraction of proteins present in the cell at any given moment are presented to T‐cells for inspection to ensure that none of these is derived from nonself. Of course, if a cell happens to harbor a nonself peptide, we want the immune system to know about this as quickly as possible, so that the appropri­ ate course of action can be taken. Thus, MHC class I molecules display peptides that are either self, or that are being made by an intracellular virus or bacterium. MHC class II molecules display peptides that are either extracellular self proteins or proteins being made by extracellular microorganisms. The whole point is to enable a T‐cell to inspect what is going on, antigenically speaking, within the cell.
As we shall see, MHC class I molecules serve an important role presenting peptides for inspection by CD8 T‐cells that are mainly preoccupied with finding virally infected or “abnormal” cells to kill. Should a TCR‐bearing CD8 T‐cell recognize a class I MHC–peptide combination that is a good “fit” for its TCR, it will attack and kill that cell. MHC class II molecules, on the other hand, are not expressed on the general cell population but are restricted to cells of the immune system, such as DCs, that have an antigen‐presenting function as we already outlined in Chapter 1. Upon recognition of an appropriate MHC class II–peptide combination by a CD4 T‐cell, this will result in activation of the latter and maturation to an effector T‐cell that can give help to B‐cells to make antibody for example. Although this is an oversimplification, as we will learn in later chapters, please keep in mind the general idea that MHC class I and II molecules present peptides to CD8‐ and CD4‐ restricted T‐cells, respectively, for the purposes of allowing these cells to determine whether they should become “activated” and differentiate to effector cells. Let us now look at these molecules in greater detail.
 
Figure M4.2.1 Main genetic regions of the major histocompatibility complex (MHC).

 


Class I and Class II Molecules Are Membrane Bound Heterodimers
MHC class I
Class I molecules consist of a heavy polypeptide chain of 44 kDa noncovalently linked to a smaller 12 kDa polypep­ tide called β2‐microglobulin. The largest part of the heavy chain is organized into three globular domains (α1, α2, and α3) that protrude from the cell surface, a hydrophobic section anchors the molecule in the membrane, and a short hydrophilic sequence carries the C‐terminus into the cytoplasm (Figure 4.19).
Class I and class II MHC molecules
Figure 4.19 Class I and class II MHC molecules. (a) Diagram showing domains and transmembrane segments; the α‐helices and β‐sheets are viewed end on. (b) Schematic bird’s eye representation of the top surface of human class I molecule (HLA‐A2) based on the X‐ray crystallographic structure. The strands making the β‐pleated sheet are shown as thick gray arrows in the amino to carboxy direction; α‐helices are represented as dark red helical ribbons. The inside‐facing surfaces of the two helices and the upper surface of the β‐sheet form a cleft. The two black spheres represent an intrachain disulfide bond. (c) Side view of the same molecule clearly showing the anatomy of the cleft and the typical Ig‐type folding of the α ‐ and β ‐microglobulin (β m) domains (four antiparallel β‐strands on one face and three on the other). (Source: Bjorkman P.J. et al. (1987) Nature 329, 506. Reproduced with permission of Nature Publishing Group.)

The solution of the crystal structure of a human class I molecule provided an exciting leap forwards in our understanding of MHC function. Both β2‐microglobulin and the α3 region resemble classic Ig domains in their folding pattern (see Figure 4.19c). However, the α1 and α2 domains, which are most distal to the membrane, form two extended α‐helices above a floor created by strands held together in a β‐pleated sheet, the whole forming an undeniable groove (Figure 4.19b,c). The appearance of these domains is so striking, we doubt whether the reader needs the help of gastronomic analogies such as “two sausages on a barbecue” to prevent any class I structural amnesia. Another curious feature emerged. The groove was occupied by a linear molecule, now known to be a peptide, which had co‐crystallized with the class I protein (Figure 4.20).
 
 Surface view of mouse class I and class II MHC molecules in complex with peptide
Figure 4.20 Surface view of mouse class I and class II MHC molecules in complex with peptide. Surface solvent‐accessible areas of the mouse class I molecule (H‐2Kb) in complex with a virus‐derived peptide and the mouse class II molecule I‐Ag7 in complex with an endogenous peptide. The views shown here are similar to that schematically depicted in Figure 4.19b and look down upon the surface of the MHC molecules. Note that the peptide‐binding cleft of class I molecules is more restricted than that of class II molecules, with the result that class I‐binding peptides are typically shorter than those that bind to class II molecules. (Source: Dr. Robyn Stanfield and Dr. Ian Wilson, Department of Molecular Biology, The Scripps Research Institute, La Jolla, California, USA. Reproduced with permission.)
MHC class II
Class II MHC molecules are also transmembrane glycoproteins, in this case consisting of α and β polypeptide chains of molecular weight 34 kDa and 29 kDa, respectively.
There is considerable sequence homology with class I, and structural studies have shown that the α2 and β2 domains, the ones nearest to the cell membrane, assume the characteristic Ig fold, while the α1 and β1 domains mimic the class I α1 and α2 in forming a groove bounded by two α‐helices and a β‐pleated sheet floor (Figure 4.19a and Figure 4.20).
The organization of the genes encoding the α chain of the human class II molecule HLA‐DR and the main regulatory sequences that control their transcription are shown in Figure 4.21.
 
Figure 4.21 Genes encoding human HLA‐DR α chain (darker blue) and their controlling elements (regulatory sequences in light blue and TATA box promoter in yellow). α1/α2 encode the two extracellular domains; TM and CYT encode the transmembrane and cytoplasmic segments, respectively. 3′‐UT represents the 3′‐untranslated sequence. Octamer motifs are also found in virtually all heavy and light chain immunoglobulin V gene promoters and in the promoters of other B‐cell‐specific genes such as B29 and CD20.
MHC class I and class II molecules are polygenic
Several different flavors of MHC class I and class II proteins are expressed by most cells. There are three different class I α‐chain genes, referred to as HLA‐A, HLA‐B, and HLA‐C in humans and H‐2 K, H‐2D, and H‐2 L in the mouse, which can result in the expression of at least three different class I proteins in every cell. This number is doubled if an individual is heterozygous for the class I alleles expressed at each locus; indeed, this is often the case because of the polymorphic nature of class I genes, as we shall discuss later in this chapter.
There are also three different types of MHC class II α‐ and β‐chain genes expressed in humans, HLA‐DQ, HLA‐ DP, and HLA‐DR, and two pairs in mice, H2‐A (I‐A) and H2‐E (I‐E). Thus, humans can express a minimum of three different class II molecules, with this number increasing significantly when polymorphisms are considered; this is because different α‐ and β‐chain combinations can be gener­ ated when an individual is heterozygous for a particular class II gene.
The different types of class I and class II molecules all exhibit the same basic structure as depicted in Figure 4.19a and all participate in presenting peptides to T‐cells but, because of significant differences in their peptide‐binding grooves, each presents a different range of peptides to the immune system. This has the highly desirable effect of reducing the probability that peptides derived from pathogen proteins will fail to be presented.
Class I and class II MHC molecules probably evolved from a single ancestral gene that underwent serial gene duplications, followed by diversification owing to selective pressure, to generate the different class I and class II genes that we see today (Figure 4.22). Genes that failed to confer any selective advantage or that suffered deleterious mutations were either deleted from the genome or are still present as pseudogenes (genes that fail to express a functional protein); indeed many pseudogenes are present within the MHC region. This type of gene evolution pattern has been termed the birth and death model or the accordion model because of the way in which this gene region expanded and contracted during evolution.

Several immune response‐related genes contribute to the remaining class III region of the MHC
A variety of other genes that congregate within the MHC chromosome region are grouped under the heading of class III. Broadly, one could say that many are directly or indirectly related to immune defense functions. A notable cluster involves four genes coding for complement components, two of which are for the C4 isotypes C4A and C4B and the other two for C2 and factor B. The cytokines tumor necrosis factor (TNF, sometimes referred to as TNFα) and lymphotoxin (LTα and LTβ) are encoded under the class III umbrella, as are three members of the human 70 kDa heat‐shock proteins. As ever, things do not quite fit into the nice little boxes we would like to put them in. Even if it were crystal clear where one region of the MHC ends and another begins (and it isn’t), some genes located in the middle of the “classical” (see Figure 4.24) class I or II regions should more correctly be classified as part of the class III cohort. For example, the LMP and TAP genes concerned with the intracellular processing and transport of T‐cell epitope peptides are found in the class II region but do not have the classical class II structure, nor are they expressed on the cell surface.

The genes of the MHC display remarkable polymorphism
Unlike the immunoglobulin system where, as we have seen, variability is achieved in each individual by a multigenic system, the MHC has evolved in terms of variability between individuals with a highly polymorphic (literally “many shaped”) system based on multiple alleles (i.e., alternative genes at each locus). This has likely arisen through pathogendriven selection to form new alleles that may offer increased “fitness” for the individual; in this context, fitness could mean increased protection from an infectious organism. The class I and class II genes are the most polymorphic genes in the human genome; for some of these genes over 600 allelic variants have been identified (Figure 4.26). This implies that there has been intense selective pressure on the MHC gene region and that genes within this region are mutating at rates much faster than those at other gene loci.
As is amply illustrated in Figure 4.26, class I HLA‐A, ‐B, and ‐C molecules are highly polymorphic and so are the class II β chains (HLA‐DRβ most, ‐DPβ next, and ‐DQβ third) and, albeit to a lesser extent than the β chains, the α chains of ‐DP and ‐DQ. HLA‐DRα and β2‐microglobulin are invariant in structure. The amino acid changes responsible for this poly­ morphism are restricted to the α1 and α2 domains of class I and to the α1 and β1 domains of class II. It is of enormous significance that they occur essentially in the β‐sheet floor and on the inner surfaces of the α‐helices that line the central cavity (Figure 4.19a) and also on the upper surfaces of the helices; these are the very surfaces that make contact with the peptides that these MHC molecules offer up for inspection by TCRs (Figure 4.20). The nonrandom location at which MHC alleles diverge from one another is as a result of positive selection over the course of animal evolution due to host–pathogen interactions. As a consequence of the polymorphic nature of MHC molecules, the spectrum of peptides bound by these molecules is highly variable. In Chapter 5 we will explore in greater detail how peptide interacts with the β‐pleated sheet floor of MHC molecules, as these interactions dramatically influence the type of peptides that can be presented by particular molecules. The ongoing drive towards creating new MHC molecules, with slightly altered peptide‐binding grooves, is akin to a genetic  arms  race  where  the  immune  system is  constantly trying to keep one step ahead of its foe. This genetic one‐upmanship has been termed pathogen‐driven balancing selection because heterozygotes typically have a selective advantage over homozygotes at a given locus.
The MHC region represents an outstanding hotspot with mutation rates two orders of magnitude higher than non‐ MHC loci. These multiple allelic forms can be generated by a variety of mechanisms: point mutations, recombination, homologous but unequal crossing over, and gene conversion.
The degree of sequence homology and an increased occur­ rence of the dinucleotide motif 5′‐cytosine–guanine‐3′ (to produce what are referred to as CpG islands) seem to be important for gene conversion, and it has been suggested that this might involve a DNA‐nicking activity that targets CpG‐rich DNA sequences. MHC genes that lack these sequences, for example H‐2Ead and HLA‐DRA, do not appear to undergo gene conversion, whereas those that possess CpG islands act as either donors (e.g., H‐2Ebb, H‐2Q2k, H‐2Q10b), acceptors
 
Figure 4.22 Birth and death model of MHC evolution. Different major histocompatibility complex (MHC) genes most likely arose though duplication events that resulted in diversification of the duplicated genes as a result of selective pressure. Genes that confer no selective advantage can suffer deleterious mutations resulting in pseudogenes or may be deleted from the genome altogether. Different environments impose distinct selective pressures, due to different pathogens for example, resulting in a high degree of polymorphism within this gene family. MHC polymorphism is seen primarily within the peptide‐binding regions of MHC class I and class II molecules.
Gene map of the MHC
The complete sequence of a human MHC was published at the very end of the last millennium after a gargantuan collaborative effort involving groups in England, France, Japan, and the United States. The entire sequence, which represents a composite of several MHC haplotypes, comprises 224 gene loci. Of the 128 of these genes that are predicted to be expressed, it is estimated that about 40% of them have functions related to the immune system. It is not clear why so many immune response‐related genes are clustered within this relatively small region, although this phenomenon has also been observed with housekeeping genes that share related functions. Because the location of a gene within chromatin can profoundly influence its transcriptional activity, perhaps it has something to do with ensuring that the genes within this region are expressed at similar levels. Genes found within con­ densed regions of chromatin are often expressed at relatively low levels and in some cases may not be expressed at all. The region between class II and class I in the human contains 60 or so class III genes. An overall view of the main clusters of class I, II, and III genes in the MHC of the mouse and human may be gained from Figure M4.2.1 in Milestone 4.2. More detailed maps of each region are provided in Figure 4.23, Figure 4.24, and Figure 4.25. A number of pseudogenes have been omitted from these gene maps in the interest of simplicity.
Figure 4.23  MHC class I gene map. The “classical” polymorphic class I genes, HLA‐A, ‐B, ‐C in humans and H‐2K, ‐D, ‐L in mice, are highlighted with orange shading and encode peptide chains that, together with β2‐microglobulin, form the complete class I molecules originally identified in earlier studies as antigens by the antibodies they evoked on grafting into another member of the same species. Note that only some strains of mice possess an H‐2L gene. The genes expressed most abundantly are HLA‐A and ‐B in the human and H‐2K and ‐D in the mouse. The other class I genes (“class Ib”) are termed “nonclassical” or “class I chain‐related.” They are oligorather than polymorphic or sometimes invariant, and many are silent or pseudogenes. In the mouse there are approximately 15 Q (also referred to as Qa) genes, 25 T (also referred to as TL or Tla) genes and 10 M genes. MICA and MICB are ligands for NK cell receptors. Tapasin is involved in peptide transport. The gene encoding this molecule is at the centromeric end of the MHC region and therefore is shown in  this gene map with respect to the mouse, but in Figure 4.24, the class II gene map is shown with respect to the human. Look at Figure M4.2.1 to see why.

Figure 4.24  MHC class II gene map. With “classical” HLA‐DP,‐DQ,‐DR in the human and H‐2A (I‐A) and H‐2E (I‐E) in mice more heavily shaded. Both α and β chains of the class II heterodimer are transcribed from closely located genes. There are usually two expressed DRB genes, DRB1 and one of either DRB3, DRB4, or DRB5. A similar situation of a single α chain pairing with different β chains is found in the mouse I‐E molecule. The LMP2 and LMP7 genes encode part of the proteasome complex that cleaves cytosolic proteins into small peptides that are transported by the TAP gene products into the endoplasmic reticulum. HLA‐DMA and‐DMB (mouse H‐2DMa,‐DMb1 and‐DMb2) encode the DM αβ heterodimer that removes class II‐associated invariant chain peptide (CLIP) from classical class II molecules to permit the binding of high affinity peptides. The mouse H‐2DM molecules are often referred to as H‐2M1 and H‐2M2, although this is a horribly confusing designation because the term H‐2M is also used for a completely different set of genes that lie distal to the H‐2T region and encode members of the class Ib family (see Figure 4.23). The HLA‐DOA (alternatively called HLA‐ DNA) and ‐DOB genes (H‐20a and ‐Ob in the mouse) also encode an αβ heterodimer that may play a role in peptide selection or exchange with classical class II molecules. (Source: Horton R. et al. (2004) Nature Reviews Genetics 5, 889–899. Reproduced with permission of Nature Publishing Group.)

The cell surface class I molecule, based on a transmembrane chain with three extracellular domains associated with β2‐microglobulin, has clearly proved to be a highly useful structure judging by the number of variants on this theme that have arisen during evolution. It is helpful to subdivide them, first into the classical class I molecules (sometimes referred to as class Ia), HLA‐A, ‐B, and ‐C in the human and H‐2 K, ‐D, and ‐L in the mouse. These were defined serologically by the antibodies arising in grafted individuals using methods developed from Gorer’s pioneering studies (Milestone 4.2).
Other molecules, sometimes referred to as class Ib, have related structures and are either encoded within the MHC locus itself (“nonclassical” MHC molecules, for example the human HLA‐E, ‐F, and ‐G, HFE, MICA and MICB, the murine H‐2 T, ‐Q, and ‐M), or elsewhere in the genome (“class I chain‐related,” including the CD1 family and FcRn). Nonclassical MHC genes are far less polymorphic than the classical MHC, are often invariant, and many are pseudogenes. Many of these nonclassical MHC class I molecules form structures that are very similar to class I molecules and have also been found to either present nonpeptide antigens or canonical (i.e., invariant) peptides that serve roles in monitoring overall cell stress levels. We will discuss these non­ classical MHC molecules in more detail towards the end of this chapter. 
 MHC class III gene map
Figure 4.25 MHC class III gene map. This region is something of a “rag bag.” Aside from immunologically “respectable” products such as C2, C4, factor B (encoded by the BF gene), tumor necrosis factor (TNF), lymphotoxin‐α and lymphotoxin‐β (encoded by LTA and LTB, respectively) and three 70 kDa heat‐shock proteins (the HSPA1A, HSPA1B, and HSPA1L genes in humans, HSP70–1, HSP70–3, and Hsc70t genes in mice), genes not shown in this figure but nonetheless present in this locus include those encoding valyl tRNA synthetase (G7a), NOTCH4, which has a number of regulatory activities, and tenascin, an extracellular matrix protein. Of course many genes may have drifted to this location during the long passage of evolutionary time without necessarily having to act in concert with their neighbors to subserve some integrated defensive function. The 21‐hydroxylases (21OHA and B, encoded by CYP21A and CYP21B, respectively) are concerned with the hydroxylation of steroids such as cortisone. Slp (sex‐limited protein) encodes a murine allele of C4, expressed under the influence of testosterone.

The genes of the MHC display remarkable polymorphism
Unlike the immunoglobulin system where, as we have seen, variability is achieved in each individual by a multigenic system, the MHC has evolved in terms of variability between individuals with a highly polymorphic (literally “many shaped”) system based on multiple alleles (i.e., alternative genes at each locus). This has likely arisen through pathogen‐driven selection to form new alleles that may offer increased “fitness” for the individual; in this context, fitness could mean increased protection from an infectious organism. The class I and class II genes are the most polymorphic genes in the human genome; for some of these genes over 600 allelic variants have been identified (Figure 4.26). This implies that there has been intense selective pressure on the MHC gene region and that genes within this region are mutating at rates much faster than those at other gene loci.
As is amply illustrated in Figure 4.26, class I HLA‐A, ‐B, and ‐C molecules are highly polymorphic and so are the class II β chains (HLA‐DRβ most, ‐DPβ next, and ‐DQβ third) and, albeit to a lesser extent than the β chains, the α chains of ‐DP and ‐DQ. HLA‐DRα and β2‐microglobulin are invariant in structure. The amino acid changes responsible for this poly­ morphism are restricted to the α1 and α2 domains of class I and to the α1 and β1 domains of class II. It is of enormous significance that they occur essentially in the β‐sheet floor and on the inner surfaces of the α‐helices that line the central cavity (Figure 4.19a) and also on the upper surfaces of the helices; these are the very surfaces that make contact with the peptides that these MHC molecules offer up for inspection by TCRs (Figure 4.20). The nonrandom location at which MHC alleles diverge from one another is as a result of positive selection over the course of animal evolution due to host–pathogen interactions. As a consequence of the polymorphic nature of MHC molecules, the spectrum of peptides bound by these molecules is highly variable. In Chapter 5 we will explore in greater detail how peptide interacts with the β‐pleated sheet floor of MHC molecules, as these interactions dramatically influence the type of peptides that can be presented by particular molecules. The ongoing drive towards creating new MHC molecules, with slightly altered peptide‐binding grooves, is akin to a genetic  arms  race  where  the  immune  system is  constantly trying to keep one step ahead of its foe. This genetic one‐upmanship has been termed pathogen‐driven balancing selection because heterozygotes typically have a selective advantage over homozygotes at a given locus.
The MHC region represents an outstanding hotspot with mutation rates two orders of magnitude higher than non‐ MHC loci. These multiple allelic forms can be generated by a variety of mechanisms: point mutations, recombination, homologous but unequal crossing over, and gene conversion.
The degree of sequence homology and an increased occurrence of the dinucleotide motif 5′‐cytosine–guanine‐3′ (to produce what are referred to as CpG islands) seem to be impor­ tant for gene conversion, and it has been suggested that this might involve a DNA‐nicking activity that targets CpG‐rich DNA sequences. MHC genes that lack these sequences, for example H‐2Ead and HLA‐DRA, do not appear to undergo gene conversion, whereas those that possess CpG islands act as either donors (e.g., H‐2Ebb, H‐2Q2k, H‐2Q10b), acceptors (e.g., H‐2Ab) or both (e.g., H‐2Kk, HLA‐DQB1). The large number of pseudogenes within the MHC may represent a stockpile of genetic information for the generation of polymorphic diversity in the “working” class I and class II molecules.

Polymorphism within human HLA (human leukocyte antigen) class I and class II genes
Figure 4.26 Polymorphism within human HLA (human leukocyte antigen) class I and class II genes. Number of distinct human HLA class I (A, B, C) and class II (DRA, DRB, DQA, DQB, DPA, DPB) alleles at each locus as of January 2005. (Adapted from Marsh S.G. et al. (2005) Tissue Antigens 65, 301. Reproduced with permission of Wiley.)

Nomenclature
As much of the experimental work relating to the MHC is based on experiments in our little laboratory friend, the mouse, it may be helpful to explain the nomenclature used to describe the allelic genes and their products. If someone says to you in an obscure language “we are having free elections,” you fail to understand, not because the idea is complicated but because you do not comprehend the language. It is much the same with the shorthand used to describe the H‐2 system, which looks unnecessarily frightening to the uninitiated. In order to identify and compare allelic genes within the H‐2 complex in different strains, it is usual to start with certain pure homozygous inbred strains, obtained by successive brother–sister matings, to provide the prototypes. The collection of genes in the H‐2 complex is called the haplotype and the haplotype of each prototypic inbred strain will be allotted a given superscript. For example, the DBA strain haplotype is designated H‐2d and the genes constituting the complex are therefore H‐2Kd, H‐2Aad, H‐2Abd, H‐2Dd, and so on; their products will be H‐2Kd, H‐2Ad, and H‐2Dd, and so forth (Figure 4.27). When new strains are derived from these by genetic recombination during breeding, they are assigned new haplotypes, but the individual genes are designated by the haplotype of the prototype strain from which they were derived. Thus the A/J strain produced by genetic cross‐over during interbreeding between (H‐2k × H‐2d) F1 mice (Figure 4.28) is arbitrarily assigned the haplotype H‐2a, but Table 4.4 shows that individual genes in the complex are identified by the haplotype symbol of the original parents.
 
Figure 4.27 How the definition of H‐2 haplotype works. Pure strain mice homozygous for the whole H‐2 region through prolonged brother–sister mating for at least 20 generations are each arbitrarily assigned a haplotype designated by a superscript. Thus the particular set of alleles that happens to occur in the strain named C57BL is assigned the haplotype H‐2b and the particular nucleotide sequence of each allele in its MHC is labeled as geneb (e.g., H‐2Kb). It is obviously more convenient to describe a given allele by the haplotype than to trot out its whole nucleotide sequence, and it is easier to follow the reactions of cells of known H‐2 make‐up by using the haplotype terminology (see, for example, the interpretation of the experiment in Figure 4.28).
 Inheritance and co‐dominant expression of MHC genes
Figure 4.28 Inheritance and co‐dominant expression of MHC genes. Each homozygous (pure) parental strain animal has two identical chromosomes bearing the H‐2 haplotype, one paternal and the other maternal. Thus in the present example we designate a strain that is H‐2k as k/k. The first familial generation (F1) obtained by crossing the pure parental strains CBA (H‐2k) and DBA/2 (H‐2d) has the H‐2 genotype k/d. As 100% of F1 lymphocytes are killed in the presence of complement by antibodies to H‐2k or to H‐2d (raised by injecting H‐2k lymphocytes into an H‐2d animal and vice versa), the MHC molecules encoded by both parental genes must be expressed on every lymphocyte. The same holds true for other tissues in the body.
 Inheritance of the MHC

Pure strain mice derived by prolonged brother sister mating are homozygous for each pair of homologous chromosomes. Thus, in the present context, the haplotype of the MHC derived from the mother will be identical to that from the father; animals of the C57BL strain, for example, will each bear two chromosomes with the H‐2b haplotype (see Table 4.4).

Let us see how the MHC behaves when we cross two pure strains of haplotypes H‐2k and H‐2d, respectively. We find that the lymphocytes of the offspring (the F1 generation) all display both H‐2k and H‐2d molecules on their surface (i.e., there is co‐dominant expression) (Figure 4.28). If we go further and breed F1s together, the progeny have the genotypes k, k/d, and d in the proportions to be expected if the haplotype segregates as a single mendelian trait. This happens because the H‐2 complex spans 0.5 centimorgans, equivalent to a recombina­ tion frequency between the K and D ends of 0.5%, and the haplotype tends to be inherited en bloc. Only the relatively infrequent recombinations caused by meiotic cross‐over events, as described for the A/J strain above, reveal the complexity of the system.

The tissue distribution of MHC molecules
Essentially, all nucleated cells carry classical class I molecules. These are abundantly expressed on both lymphoid and myeloid cells, less so on liver, lung, and kidney and only sparsely on brain and skeletal muscle. In the human, the surface of the placental extravillous cytotrophoblast lacks HLA‐A and ‐B, although there is now some evidence that it may express HLA‐C. What is well established is that the extravillous cytotrophoblast and other placental tissues bear HLA‐G, a molecule that generally lacks allodeterminants and that does not appear on most other body cells, except for medullary and sub­ capsular epithelium in the thymus, and on blood monocytes following activation with interferon‐γ. The role of HLA‐G in the placenta is not fully resolved, but it appears to function as a replacement for classical class I molecules serving to inhibit immune responses against paternal MHC alleles carried by the fetus. Class II molecules, on the other hand, are highly restricted in their expression, being present only on B‐cells, dendritic cells, macrophages, and thymic epithelium. However, when activated by agents such as interferon‐γ, capillary endothelia and many epithelial cells in tissues other than the thymus express surface class II and increased levels of class I.

The nonclassical MHC and class I chain‐related molecules
These molecules include the CD1 family that utilize β2‐ microglobulin and have an overall structure similar to the classical class I molecules (Figure 4.29). They are, however, encoded by a set of genes on a different chromosome to the MHC, namely on chromosome 1 in humans and chromosome 3 in the mouse. Like its true MHC counterparts, CD1 is involved in the presentation of antigens to T‐cells, but the anti­ gen‐binding groove is to some extent covered over, contains mainly hydrophobic amino acids, and is accessible only through a narrow entrance. Instead of binding peptide antigens, the CD1 molecules generally present lipids or glycolipids. At least four different CD1 molecules are found expressed on human cells; CD1a, b, and c are present on cortical thymocytes, dendritic cells and a subset of B‐cells, whereas CD1d is expressed on intestinal epithelium, hepatocytes, and all lymphoid and myeloid cells. Mice appear to only express two different CD1 molecules that are both similar to the human CD1d in structure and tissue distribution and are referred to as CD1d1 and CD1d2 (or CD1.1 and CD1.2).
Genes in the MHC itself that encode nonclassical MHC molecules include the H‐2 T, H‐2Q, and H‐2 M loci in mice, each of which encodes a number of different molecules. The T22 and T10 molecules, for example, are induced by cellular activation and are recognized directly by γδ TCR without a requirement for antigen, possibly suggesting that they are involved in triggering immunoregulatory γδ T‐cells. Other nonclassical class I molecules do bind peptides, such as H‐2 M3 that presents N‐formylated peptides produced either in mito­ chondria or by bacteria.
In the human, HLA‐E binds a nine‐amino‐acid peptide derived from the signal sequence of HLA‐A, ‐B, ‐C, and ‐G molecules, and is recognized by the CD94/NKG2 receptors on NK cells and cytotoxic T‐cells, as well as by the αβ TCR on some cytotoxic T‐cells. HLA‐E is upregulated when other HLA alleles provide the appropriate leader peptides, thereby allowing NK cells to monitor the expression of polymorphic class I molecules using a single receptor. The murine homolog, Qa‐1, has a similar function.
The stress‐inducible MICA and MICB (MHC class I chain‐related molecules) have the same domain structure as classical class I and display a relatively high level of polymorphism. They are present on epithelial cells, mainly in the gastrointestinal tract and in the thymic cortex, and are recog­ nized by the NKG2D‐activating molecule. One possible role for this interaction is in the promotion of NK cell and T‐cell antitumor responses.
The function of HLA‐F is unclear, although its expression in placental trophoblasts has led some to suggest that it may play a role in protecting the developing fetus from attack by the maternal immune system. A more definitive role for HLA‐G in this context has been found. This HLA molecule is also preferentially expressed on placental trophoblast cells where it plays a role in shielding the fetus from the unwanted attentions of the maternal NK cells and cytotoxic T‐cells. It has long been a puzzle why mothers tolerate their genetically non‐identical fetuses, as one would normally expect a strong immune response to foreign (i.e., paternal) HLA molecules. Although this is partially solved through downregulation of the expression of MHC class I A, B, and C molecules on placenta, this would normally attract the attentions of NK cells on the prowl for cells with such missing‐self characteristics, as we discussed earlier when dealing with NK receptors. HLA‐G expression on the placen­ tal–maternal trophoblast interface appears to be a solution to this. The interaction between the immunoglobulin‐like tran­ script‐2 (ILT2) molecule on NK cells, which is an inhibitory NK receptor, with HLA‐G expressed on placental trophoblasts confers protection against NK cell‐mediated cytolysis.
HFE, previously referred to as HLA‐H, possesses an extremely narrow groove that is unable to bind peptides, and it may serve no role in immune defense. However, it binds to the transferrin receptor and appears to be involved in iron uptake. A point mutation (C282Y) in HFE is found in 70–90% of patients with hereditary hemochromatosis.
Comparison of the crystal structures of CD1 and MHC class I
Figure 4.29 Comparison of the crystal structures of CD1 and MHC class I. (a) Backbone ribbon diagram of mouse CD1d1 (red, α‐helices; blue, β‐strands). (b) Ribbon diagram of the mouse MHC class I molecule H‐2Kb (cyan, α‐helices; green, β‐strands). (c) Superposition using alignment of β2‐microglobulin highlights some of the differences between CD1d1 and H‐2Kb. Note in particular the shifting of the α‐helices. This produces a deeper and more voluminous groove in CD1d1, which is narrower at its entrance compared with H‐2Kb. (Source: Porcelli ) Immunology Today 19, 362. Reproduced with permission of Elsevier.)
Nonclassical MHC molecules may be the precursors to classical MHC molecules
Analysis of vertebrate genomes suggests that invariant nonclas­ sical MHC molecules are probably the primordial forerunner to modern polymorphic MHC class I and class II molecules and rather than playing a role in antigen presentation, these molecules were most likely used as primitive “danger signals” involved in conveying stress signals to innate immune cells. Thus, expression of these molecules on the cell surface signified a stressed or potentially transformed cell that should be eliminated in the interests of overall organismal fitness. During the course of evolution, such molecules then most likely evolved the ability to bind self peptides, which were initially relatively invariant, followed by the ability to bind highly variable peptides, as we now see with classical MHC class I and class II gene products. The appearance of polymorphic MHC molecules, as a consequence of gene duplication events followed by divergence, would have enabled much greater diversity in the range of peptides bound by these molecules. Thus, invariant MHC‐like molecules (such as HLA‐E, ‐F, ‐G, and MICA, MICB) tend not to have antigen‐presenting functions, but perform homeostatic or regulatory roles, permitting cells of the innate immune system to monitor cell health in a relatively antigen‐nonspecific way.
A good example, which was discussed in the context of NK receptors but is worth going over again, is the HLA‐E molecule that binds a nine‐amino‐acid peptide derived from the signal sequence of HLA‐A, ‐B, and ‐C molecules. Should HLA‐E– peptide complexes be absent from cells, this suggests that an infectious agent may be present or that cells are stressed in some way. This results in activation of NK cells via the activat­ ing CD94/NKG2 receptors, with consequent NK‐mediated killing of such cells. In the absence of class I leader peptides, HLA‐E can be stabilized on the surface of stressed cells by heat‐shock treatment because the HSP‐60 signal peptide can also bind in place of HLA class I peptides. However, such HLA‐E/ HSP‐60 leader peptide complexes fail to be recognized by the CD94/NKG2 receptor, once again precipitating attack by the NK cell. Thus, cell stress can override the presentation of class I‐derived peptides through competition for HSP‐60‐derived peptides that would not normally be present at levels high enough to compete effectively in unstressed cells. If this isn’t a clever molecular security system, we don’t know what is.

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