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'{6, '@r. '{otr This book and other books may be purchased at a discount from the publisher when ordered in bulk quantities. Contact: IEEE Press Marketing Attn. Flexible Alternating Current. Transmission Systems (FACTS). FACTS. AC transmission systems incorporating the power electronic-based to enhance. Read the edition of Brain Facts as a PDF file, or listen to previous US and Canada can request one free hard copy of the updated Brain Facts book here.

In this serological context the MHC glycoproteins behaved as alloantigens and were called major histocompatibility antigens. Two very different kinds of cell-surface glycoprotein serve as the inhibitory class I receptors of human NK cells. Today 18, 43 2 Trowsdale, J. Forgot password? Student Companion Site. An important effect of the evolution of multiple polymorphic class I and II HLA genes is that it provides individual human beings with a diversity of class I and II antigen-presenting functions.

Although HLA class II molecules are restricted in their expression to a few kinds of cell in the human body, they are expressed at high levels by the one kind of transformed cell that is easy to make from any healthy person.

Such cells are immortalized B-cell lines made by co-culturing peripheral blood lymphocytes with Epstein-Barr virus EBV in the presence of an immunosuppressive drug such as cyclosporin A to prevent T-cell-mediated killing of the newly transformed B cells. Once the transformed B cells grow out they can be maintained in medium without the immunosuppressive drug and are hardy growers in comparison to many cultured cells. The many favourable properties of B-cell lines have arguably been some of the most important factors in furthering research and knowledge of HLA class II genes and proteins.

Even more valuable for studies of HLA class II have been cell lines obtained from people who have inherited precisely the same HLA type from their mother and father. Such people are usually the children of consanguineous marriages between first cousins. Included in this panel were many cell lines that were consanguineous homozygotes. For these cell lines the complexity of HLA variation in all methods of analysis is reduced by half. More importantly, they permitted unambiguous definition of HLA haplotypes while alleles could be defined by PCR amplification and direct sequencing of the products.

At the 10th Workshop, numerous studies on the panel of homozygous B-cell lines were presented and a few of the cells proved to be heterozygous in the face of such intensive study.

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The principles of the class II nomenclature are the same as that outlined for class I in the previous chapter. As well as the beginning of a nomenclature based upon the precise chemical structures of HLA alleles, the nomenclature report from the 10th International HLA Workshop in can, in retrospect, be seen as the beginning of the end of serological HLA typing. To both accommodate and encourage this development. Marsh and assign names to new alleles on an ongoing basis.

Table 5. Workshops and Nomenclature A total of class I alleles and class II alleles are currently defined. Since all this information has also been made available on the World Wide Web and can be accessed at either www. The development and maintenance of the class I and II sequence databases is now being consolidated by Steven Marsh at the Anthony Nolan Research Institute, London, which will also act as a clearing house for the assignment of HLA nomenclature.

Table 8 lists the names that have been abandoned. Table 6. Workshops and Nomenclature Because of their polymorphism and functions, the naming of alleles for two types of gene in the HLA complex that do not have HLA names has recently become the responsibility of the HLA nomenclature committee. So far, no functional differences between TAP alleles have been defined.

Names of the TAP alleles are given in Table 9. Table 9. Table 10 gives the 15 alleles that are currently defined.

Table This approach was subsequently and successfully emulated by investigators studying macromolecules of human leukocyte surfaces using mouse monoclonal antibodies. Using serological methods based on those used to define HLA antigens, monoclonal antibody reactivities were grouped according to their tissue distribution and other properties into 'clusters of differentiation' defining particular antigenic molecules or their subunits.

Each cluster of differentiation, or CD for short, is assigned a number in a single series, well-known examples being the CD4 and CDS markers of T-cell subsets.

However, certain non-polymorphic class I-like molecules were first discovered as leukocyte antigens and are included in the CD series, CDl for example. Springer-Verlag, New York, pp. Today 11, By definition, 'typing' involves the recognition and distinction of entities that have already been described.

So the necessary first step in developing DNA-typing systems for class II genes was determination of nucleotide sequences for the alleles to be typed. From this database polymorphic motifs that distinguish alleles can be identified, as can their combination, to define individual alleles. The amplified DNA is then covalently attached to a nylon membrane by ultraviolet irradiation and incubated with an oligonucleotide designed to detect a particular polymorphic motif and modified so that it can be detected when bound to the membrane.

After incubation and washing to remove unbound oligonucleotide the membrane is assessed for oligonucleotide binding. Only if the amplified DNA contains the sequence complementary to that of the oligonucleotide will it bind the probe.

This method uses sequence-specific oligonucleotide probes and is called SSOP. In practice, it is common to dot PCR products from many different individuals in a matrix upon a single membrane which can then be successively hybridized with different probes.

Alternatively, the reactions can be carried out with PCR products attached to the wells of microtitre plates. The typing of individual samples is then obtained from the combinations of probes to which they hybridize. This type of assay, sometimes called a dot blot, is particularly suited to typing large numbers of samples, for example, when typing prospective unrelated donors for bone marrow transplantation. Here, the set of oligonucleotides is attached to a membrane in a matrix of dots, or to the wells of microtitre plates and the PCR products obtained from the person to be typed are used as the probe.

This system is suited for situations in which only one or a small number of persons need to be typed. In forensic applications this is usually the case, for example, when blood or semen samples obtained from the scene of a crime are compared to those of a suspect. In a second type of assay, pairs of synthetic oligonucleotides based upon polymorphic sequences are tested for their capacity to prime in PCRs. To determine the extent and specificity of the PCR, the products are analyzed on agarose gels.

To distinguish between a set of alleles requires a series of different PCRs, the number being at least half that of the alleles to be typed. In typing a person these PCRs are performed simultaneously and the DNA products of amplification are compared for their abundance and electrophoretic mobility on gels.

Like the reverse dot blot assay, PCR-SSP is better suited for typing small numbers of samples and is less practical for high-volume typing. Phototyping is an example of a high-resolution DNA-typing method. By contrast, low-resolution DNA typing is at a level comparable to that of serology.

In DNA typing performed at intermediate resolution, certain groups of alleles and some individual alleles are distinguished. In matching donors and recipients for transplantation it is common for initial assessments to be made at low resolution and for higher resolution typing to be subsequently applied to distinguish amongst donors that match at low resolution.

Increased discrimination of the reactions in PCR-SSP can be achieved by using the so-called 'nested' PCR strategy, in which each reaction is performed in two steps with different oligonucleotide primers. The polymorphisms targeted in the first step flank those targeted in the second step. With this strategy a typing reaction is dependent upon the linkage of four sets of substitution within an allele. A third approach to DNA typing is based upon the differences in electrophoretic mobility of DNA molecules caused by non-complementary bases in the two strands of the double helix''.

Thus when PCR products of identical length from the same allele are reannealed with each other, the mixed product gives the same banding pattern on electrophoresis as either of the alleles alone. By contrast, when PCR products from different alleles are reannealed, the banding pattern is more complicated than the sum of the two components.

Extra bands arise from the formation of heteroduplex molecules in which one strand is derived from one allele and the complementary strand is derived from the second allele.

This approach is well-suited to the assessment of identity or difference between two samples. In developing it as a routine method for typing, a useful strategy has been to simplify the banding patterns by using reference strands or duplexes to which test samples are annealed. If the reference strand is used in excess or labelled for selective detection, then only molecules containing the reference are seen.

Because the mobility of DNA duplexes can be sensitive to single nucleotide mismatches, typing systems which discriminate all alleles are potentially possible with this approach. A PCR is performed that amplifies with comparable yield all the alleles of a locus.

As most people are heterozygous, this PCR amplifies both alleles. The two alleles are then sequenced as a mixture and analyzed using a computer program that identifies and analyzes positions of heterozygosity. From comparison of the patterns obtained with those expected for all combinations of alleles, the program can determine the possible types. For the vast majority of people, sequence-based typing SBT permits assignment of an unambiguous type.

Since then, DNA typing has essentially superseded serology both as the method of choice for class II typing and as the routine clinical method.

A variety of methods are used, the choice being dictated by the number of samples being typed and the desired level of resolution. In general, the HLA laboratories involved in bone marrow transplantation are those that are typing the largest number of samples and are also using the methods with highest resolution.

A major consequence of the application of DNA typing is that it has led to the discovery and characterization of many new alleles. Most new HLA alleles arise by genetic events that recombine substitutions in the existing alleles.

Because all the methods of DNA typing discriminate between combinations of polymorphic substitutions, they have the capacity to detect novel combinations. When a novel allele is inferred from DNA typing it must be confirmed and defined by cloning and sequencing before it can be named. As new alleles are added to the sequence database they stimulate modification of DNA-typing systems to accommodate the new allele.

In this manner there is a two-way feedback process which continually acts to refine and expand the databases of allele sequences and the methods for DNA typing. In it was still widely believed that serological typing performed adequately for HLA class I, although it was appreciated that many subtypic differences could not be distinguished serologically.

This view gradually changed as continued molecular analysis of HLA-A, -B and -C showed that serological typing was rarely able to discriminate at the level of individual alleles". In addition, certain HLA-A and -B alleles that had evaded almost 40 years of serological typing were very distinctive in nucleotide sequence.

Most worrying was increasing evidence of poor performance of serological HLA-C typing. The methods being explored are all based upon those that have been successfully used for class II typing. The major difference is that two exons need to be analyzed for each class I gene, whereas only one was necessary for each class II gene.

If exons 2 and 3 of class I genes are analyzed separately then the experimental methods can be simply transferred from class II.

However, the interpretation of data becomes more challenging because the linkage between the two exons is lost. Conversely, if the linkage between the exons is to be preserved and the interpretation of data eased, then modified methods with longer PCR reactions must be developed to perform the typing reactions.

USA 88, 3 Trachtenberg, E. T cells develop in the thymus gland to express one of two types of antigen receptor: T cells expressing aP receptors recognize peptide antigens bound either to an HLA class I or II molecule and they provide adaptive immunity. By contrast, Y6 T cells recognize a different range of antigens and are implicated in innate immunity and functions associated with the homeostasis of mucosal tissues, principally the gut. Throughout this book T cells will refer to aP T cells,- y6 T cells will be referred to as such.

The genes for T-cell receptor chains consist of families of gene segments which need to rearrange in order to become functional. This situation is analogous to that of the genes encoding immunoglobulin heavy and light chains. T-cell development in the thymus involves first the rearrangement of the P-chain gene. If one of the two copies of the p-chain gene makes a functional rearrangement then rearrangement of the other copy is prevented.

Subsequently, rearrangement of the a-chain genes is initiated. As a consequence of these sequential rearrangements T cells can express only one P chain and either one or two a chains. Negative selection eliminates cells bearing receptors that interact too strongly with any autologous self class I or II allotype, while positive selection drives the maturation of cells bearing receptors that interact with intermediate affinity with an autologous class I or II allotype.

Cells with receptors that interact poorly with all the autologous class I and II allotypes fail to mature and die. P indicates the peptide antigen bound by the HLA molecule. An individual T cell is restricted by a single class I or II allotype, the one on which it was positively selected in the thymus. Determining the patterns of restriction are the cell-surface glycoproteins CDS and CD4, which distinguish the two kinds of T cell and specifically bind to class I and class II respectively Figure 2.

CDS T cells have a cytotoxic function that enables them to kill cells infected with viruses or other intracellular pathogens. CD4 T cells have a wider range of effector functions, all of which involve the targeted delivery of cytokines to other cells of the immune system. Because their common role is to induce activation of other cell types, CD4 T cells are often called helper T cells. In experimental systems, two distinct types of CD4 T cell have been distinguished: The sites on HLA molecules bound by the co-receptors are distinct from those bound by T-cell receptors.

Efficient activation of T cells requires simultaneous interactions of an HLA molecule with peptide antigen P , T-cell receptor and co-receptor. These may represent extremes of a range in which there are CD4 T cells of various intermediate types.

CD4 T cells are activated in response to extracellular pathogens. Fundamental differences in both tissue distribution and the intracellular mechanism for binding peptide antigens distinguish class I and II molecules. These differences allow the immune system to initiate a T-cell response, either CD4 or CD8, that is effective against the type of pathogen from which antigenic peptides are being derived. In general, the foreign antigens presented by class I molecules are derived from intracellular infection of the type caused by viruses.

These antigens initiate cytolytic CDS T-cell responses which kill off the infected cells and prevent viral replication and the spread of infection. Because all nucleated cells are potential targets for viral infection, class I molecules are constitutively expressed by almost all types of cell.

In general, the foreign antigens presented by class II molecules are derived from pathogens present in the extracellular spaces. These include organisms that live and replicate in the extracellular spaces, for example many bacteria, and others, such as virions, which are in transit between cells. These antigens stimulate CD4 T-cell responses that serve to activate macrophages and B cells. This mode of antigen presentation does not need to be carried out by every type of cell and thus HLA class II molecules are selectively expressed on cells specialized for the purpose and collectively called professional antigen-presenting cells APC.

Macrophages, B cells and dendritic cells are the major professional APC. These cells have specialized mechanisms for engulfing, killing and degrading extracellular pathogens.

For example, macrophages phagocytose bacteria and degrade them in endosomes and lysosomes. Peptides derived from these processes tend to be bound by class II molecules because they bind peptides within endosomal vesicles. This mode of presentation activates CD4 T cells that can facilitate macrophage function in two distinct ways: The constituent polypeptides of both classes of HLA molecule are made on ribosomes of the rough endoplasmic reticulum and then translocated into the lumen of the endoplasmic reticulum ER.

Most of the peptides bound by class I are generated in the cytosol by proteasomes, proteolytic particles made up of many small subunits. By themselves, HLA class I heavy chains are unstable, as are complexes of heavy chains with Pi-m that have not bound peptide. These intermediate forms in the assembly of HLA class I molecules are stabilized in the ER by interaction with calnexin and calreticulin, two chaperonins resident in the ER. The tight binding of peptides to HLA class I molecules means that the pathogen-derived peptides presented by HLA class I molecules on infected cells infrequently dissociate and bind to the HLA class I molecules of healthy cells.

As a consequence, healthy cells do not passively acquire antigen and become vulnerable to cytolytic attack from pathogen-specific CDS T cells.

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The in vivo pathway for peptide generation and binding to class I molecules is depicted in Figure 3. Instead the a and P chains assemble with a third polypeptide, called the invariant chain also termed li. The interaction with the invariant chain has the effect of stabilizing the structure of the HLA class II molecule while preventing the binding of peptides within the ER. The invariant chain forms a trimer in which each constituent interacts with one a and one p chain.

Schematic of a cell showing the pathway by which intracellular antigens are processed and presented by HLA class I molecules. Proteins in the cytosol are degraded by proteasomes into small peptides which are transported by the TAP protein into the lumen of the endoplasmic reticulum ER. HLA class I heavy chains and P2-microglobulin are synthesized on ribosomes and translocated into the lumen of the ER where they assemble with each other and bind peptide.

As an example of how this pathway is used, in virus-infected cells breakdown of malfolded viral proteins in the cytosol leads to the presentation of viral peptides by HLA class I molecules at the cell surface. CDS T cells are stimulated to kill virus-infected cells, thereby stopping the replication of virus within those cells and thus the spread of infection. Within these vesicles the invariant chain is degraded and can be replaced by a peptide derived from degradation in the endosomes or lysosomes of endocytosed material.

The in vivo pathway for peptide generation and binding to class II molecules is depicted in Figure 4. Schematic of a cell showing the pathway by which extracellular antigens are processed and presented by HLA class II molecules.

Extracellular proteins are taken into the cell by endocytosis or phagocytosis and are then degraded to peptides within endosomes and lysosomes. HLA class II a and P chains and the invariant chain li are synthesized on ribosomes and translocated into the lumen of the ER where they assemble into heterotrimers that cannot bind peptides because the invariant chain occupies the peptide-binding site.

This pathway is used to respond to infection by the species of bacteria that live and replicate in the connective tissues. Macrophages phagocytose bacteria and present bacterial peptides to CD4 T cells. Some CD4 T cells are activated to secrete cytokines that directly act on macrophages to improve the rate at which they kill bacteria, other CD4 T cells stimulate B cells to produce bacteria-specific antibodies which by coating bacteria make them more susceptible to phagocytosis by macrophages.

Such interaction leads to cellular activation, proliferation and differentiation of effector CDS T cells that kill cells presenting the specific peptide antigens. The cell-surface phenotype of NK cells is distinguished from those of B and T cells by their lack of clonally expressed antigen receptors encoded by rearranging genes. Thus NK cells are most accurately defined in a negative way: No cell-surface molecule uniquely distinguishes NK cells from other lymphocytes; all known components of the NK cell surface are present on other cell types, most often on T cells.

NK cells circulate in the peripheral blood. They are bigger than circulating B and T cells, due to a more voluminous cytoplasm containing cytotoxic granules. The granules and their contents are similar to those of cytotoxic CDS T cells.

Natural killers are cells of innate immunity that enter sites of inflammation and function soon after the onset of infection.

In addition to their cytotoxic function, NK cells secrete certain cytokines. For example, at early times in infection, before the T-cell response develops, NK cells are the principal source of interferon y. A few patients have been described who lack NK cells,- they suffer from protracted, life-threatening viral infections that they cannot terminate despite the presence of adaptive T-cell immunity.

Such correlation indicates that NK cells are an essential early defence against viral infection, one that complements the activities of cytolytic T cells. The NK cells in freshly drawn peripheral blood can kill certain types of target cell, for example the human erythroleukaemia cell line K Susceptibility of cells to NK cell-mediated killing inversely correlates with the expression of class I molecules.

When certain of these receptors engage a class I ligand they deliver inhibitory signals to the NK cell which prevent both cytotoxicity and cytokine secretion Figure 1. One purpose of this mechanism is to prevent NK cells from killing healthy cells. In terms of defence, this mechanism directs NK cell attack at cells that have a pathological loss of HLA class I expression.

Natural killer cell function is regulated by inhibitory receptors with specificity for polymorphic determinants of HLA class I molecules. Healthy cells are not killed by autologous natural killer NK cells because their HLA class I molecules interact with inhibitory receptors expressed on the surface of NK cells. Two very different kinds of cell-surface glycoprotein serve as the inhibitory class I receptors of human NK cells.

They are type 2 membrane proteins and thus have their amino-termini in the cell's cytosol and their carboxy-termini outside the cell.

The class I ligand bound by the CD The binding site of HLA-E is highly specific for this kind of peptide. Despite the structural similarities between CD HLA-E is oligomorphic, only four heavy-chain allotypes having been described and they differ by substitutions at just three positions see p.

The type of intracellular signal that is generated by ligand recognition is determined by the NKG2 polypeptide. NKG2A receptor Figure 2. Fiowever, whereas the latter receptor delivers inhibitory signals to NK cells, the CD NKG2C receptor delivers an activating signal.

The cause of this difference lies in the cytoplasmic tails of the NKG2 polypeptides. In the region of chromosome 12 containing the CD94 and NKG2 genes there are related gene families encoding further lectin-like molecules of the NK cell surface.

The orthologous region on chromosome 6 of the mouse also contains similar families of genes, including the Ly gene family which encodes lectin-like class I receptors of mouse NK cells. Whereas Ly genes provide all the known class I receptors for the mouse, this family of genes is represented by a single pseudogene in the human NKC.

The second kind of class I receptor on human NK cells are type 1 membrane proteins amino-terminus outside the cell and carboxy-terminus inside , whose extracellular mass consists of two or three immunoglobulin Ig C2-like domains Figure 3. KIR vary in the number of extracellular immunoglobulin-like domains and the length of the cytoplasmic tail. The names for the four groups of KIR are shown. The numbers 2 or 3 give the number of extracellular Ig-like domains; L or S refers to the possession of either a long or short cytoplasmic tail.

On the basis of simple diallelic polymorphisms at position 77 and 80 of the heavy chain, HLA-C allotypes can be sorted into two groups.

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This is because in the region including residues 77 and 80 this allotype has a sequence which is like that found in HLA-C allotypes and unlike the sequences found in other HLA-B allotypes. Amino acid sequence motifs determining the Bw4 and Bw6 epitopes. However, this specificity remains less well defined. Some residues within this region are located on the outside surface of the class I molecule where they can interact directly with KIRs.

Although a bound peptide must be present, unlike the HLA-E: As in the case of the lectin-like receptors, this family includes inhibitory receptors with long cytoplasmic tails containing ITIM motifs and activating receptors with short cytoplasmic tails.

Closely linked to the KIR gene family are other families of structurally similar molecules. Most closely linked are the leukocyte immunoglobulin-receptor LIR gene family also called ILT for immunoglobulinlike transcripts , for which at least one member is both a class I receptor and expressed on NK cells.

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The number of receptors expressed by NK cells varies, as does the combination of receptors. Certain KIRs appear to be ubiquitously expressed, but most are not. As a consequence there is considerable heterogeneity within a person's NK cell population due to the combinations of receptors that individual cells express. This variation in receptor combination defines a person's NK-cell receptor repertoire. One rule governing the NK-cell receptor repertoire is that each NK cell expresses at least one inhibitory receptor with specificity for an autologous class I allotype.

NKG2 gene possessed by a person is expressed by some subset of NK cells. These haplotypes differ in the number of genes and in the type of genes, the number of genes encoding non-inhibitory KIRs being particularly variable. In addition, a number of the KIR loci exhibit genetic polymorphism.

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Regulation of NK-cell function involves interaction between polymorphic ligands encoded by class I genes and polymorphic receptors encoded by KIR genes. That the two sets of genes are unlinked means that the combination of ligands and receptors is itself highly variable within human populations, producing considerable diversity in NK-cell receptor repertoires.

Both the CD They can be found on both aP and 76 T cells. The ap cells that express these receptors are largely CDS T cells having a memory phenotype.

The amino-terminus of the heavy chain is on the outside of the cell while the carboxyterminus is on the inside,- thus it is a type I membrane glycoprotein.

Whereas the heavy chain has a single site of N-linked glycosylation at asparagine 86, p2-m is not glycosylated. All HLA class I molecules at the cell surface contain a tightly bound peptide, usually consisting of amino acid residues.

In healthy cells the peptides are derived from normal cellular components whose routine turnover and degradation takes place in the cytoplasm. The peptides, called self peptides, are transported by TAP into the endoplasmic reticulum where they bind to assembling complexes of class I heavy chains and p2-ni- Although a class I molecule only binds one peptide, each class I allotype can bind peptides of different amino acid sequence.

Consequently thousands of different self peptides are presented at the cell surface by the molecules of a single class I allotype. If a cell is infected with a pathogen, some of the peptides bound by class I are derived from proteolytic degradation of the foreign proteins. The structure of class I molecules reflects a compromise between the need for tight peptide binding and the requirement to bind a broad range of peptides to provide effective surveillance for the presence of pathogenderived peptides.

Three-dimensional structures for the four extracellular domains aj, a2, aa and Pj-m of HLA-A, -B and -C allotypes have been determined by X-ray crystallography. The carboxy-terminal segments of these domains adopt an a-helical conformation that forms the two flanking walls of the groove. The membraneproximal a3 domain pairs with Pj-m to form a pedestal-like structure that supports the peptide-binding site.

Peptides occupy the binding site in an extended conformation with the two termini pinned down into the ends of the groove. This mode of binding limits the length of peptides that can be bound by HLA class I molecules to residues. The disulfide bond connecting residues is indicated as two linked filled circles. Residue numbers and the N-linked glycosylation site CHO at position 86 are labelled. Three-Dimensional Structures oi HLA Class I Molecules chain the backbone of peptide bonds at the ends of the peptide and conserved tyrosine residues clustered at the extremities of the peptide-binding groove.

Highaffinity binding ensures that the same peptide remains bound for the Ufe of the class I molecule. This prevents the class I molecules of healthy cells from exchanging self peptides for foreign peptides acquired from the extracellular environment, a mechanism that might inadvertently target a cell for destruction even though it is not infected by a pathogen.

Although a single HLA class I allotype can bind numerous different peptide sequences, there is some specificity to the interaction. This is manifest by a preference for certain amino acid residues at particular positions within the peptides sequences. These preferences are described by peptide-binding motifs. The peptide-binding groove is divisible into six pockets denoted A, B, C, D, E and F Table 1 , although for any given peptide not all of the pockets are necessarily occupied.

Most HLA-A and -B allotypes exert selectivity at position 2 and the carboxy-terminal position of the peptide. The side-chains of these two residues are accommodated within pockets B and F of the binding site respectively. Residues in the middle of bound peptides are not buried within the site and consequently there is little or no restriction of the amino acids found at these positions.

This flexibility facilitates binding of a broad spectrum of peptides. Leader sequences are exceedingly hydrophobic and the peptides which bind to HLA-E consist almost entirely of hydrophobic amino acids. A subset of peptide: NKG2 receptors expressed by NK cells. When peptide is bound to an HLA class I molecule the exposed middle portion of the peptide and the upper faces of the two a helices form a planar surface that interacts with T-cell receptors.

The variable domains of the T-cell receptor a and p chains are seen to contact both the a helices and the exposed residues of the bound peptide. The hypervariable loops of the T-cell receptor a chain interact with the section of the groove containing the amino-terminal half of the peptide, while the hypervariable loops complementarity-determining regions, CDRs of the T-cell receptor 3 chain interact with the section of the groove containing the carboxyterminal half of the peptide.

Centrally placed on the groove are the third hypervariable loops CDRs 3 of the a and 3 chains, which form a pocket that binds the side-chain of one of the central amino acid residues of the bound peptide residue 5 in a nonamer peptide. From the structures so far studied, the interactions of the T-cell receptor a chains with the class I molecule appear more conserved than do those of the 3 chain.

The CDS molecule can consist either of a heterodimer of a and p chains or a homodimer of two a chains. Each CDS chain consists of an extracellular immunoglobulinlike domain which is connected by a long stalk to a transmembrane anchor and a cytoplasmic tail.

The immunoglobulin-like domains are the site of interaction with class I molecules. Cell 1, 3 Ding, Y. Orientated with their amino-terminal ends on the outside of the cell, both chains comprise two extracellular domains, each of amino acids, connected to a short cytoplasmic tail by a hydrophobic sequence that makes a single pass through the cell membrane.

Likewise, in the 3 chain the membrane-distal domain is known as Pi and the membrane-proximal domain as p2. Both membrane-proximal domains possess structural characteristics of CI-type immunoglobulin domains.

The a chain has two N-linked glycosylation sites, one in each domain and an intradomain disulfide bond in the aj domain. The P chain has a single N-linked glycosylation site, which is in the pi domain, and two intradomain disulfide bonds, one in each extracellular domain. Each of the four extracellular domains of the class II molecule is similar to one of the four extracellular domains of the class I molecule. Three-dimensional structure of the HLA class II molecule, a In this diagram the polypeptide backbones of the extracellular domains of the a and P chains are depicted as ribbons.

The disulfide bond connecting residues pi5 to P79 is indicated as two linked solid-filled circles. Residue numbers and the N-linked glycosylation site CHO at positions a78 and pi9 are labelled. Determination of the three-dimensional structures of several HLA-DR allotypes, each complexed with a single peptide, has confirmed that the membranedistal domains of HLA class II molecules form a peptide-binding groove Figure 2.

The amino-terminal portions of tti and pi fold into P-pleated strands that form the floor of the groove, while the carboxy-terminal regions of these domains adopt an ahelical conformation and form its two walls.

In HLA class I molecules the peptide termini are buried within the groove and much of the binding affinity is derived from hydrogen bonds between the peptide termini and pockets at the ends of the groove.

In the absence of such interactions in HLA class II molecules, conserved hydrogen bonds between the peptide and residues in the groove are formed at regular intervals throughout that central portion of the peptide's main chain which occupies the binding site.

Because peptides bound by HLA class I are held by their ends, their length is limited to amino acids. In contrast, because peptides bind to HLA class II by being gripped in the middle they can be much longer and more variable in their length.

Typically the peptides bound to HLA class II molecules are amino acids in length but longer peptides are not uncommon Figure 3. Each bead represents an amino acid. The hydrogen bonds formed between conserved residues in the HLA class II groove and the peptide backbone provide a component of the peptide-binding affinity that is independent of class II allotype and common to all of them.

Positions of polymorphism within the peptide-binding site provide a second component of the binding affinity, one that causes different HLA class II allotypes to bind peptides of different amino acid sequence. This pocket has essentially two kinds of specificity dependent upon the amino acid residue at position 86 of the P chain. Every DRp allotype has either glycine or valine at position S6. By contrast, when residue 86 is valine, this residue's more bulky side-chain reduces the size of the pocket which then prefers smaller aliphatic residues.

All binding of peptides to HLA-DR molecules appears dependent upon this pocket interacting with a side-chain of an amino acid residue in the peptide. The side-chain which interacts with this pocket is located towards the peptide's amino-terminus, and is designated as relative position 1 in the peptide residue numbering. In general, constraints on the amino acids accommodated by pockets within the peptide-binding site are less restrictive for HLA class II than for class I.

The lower selectivity of the HLA class II binding site combined with the potential for binding longer peptides enables certain 'promiscuous' peptides to bind to several HLA class II allotypes. Many more promiscuous peptides are found amongst those that bind to class II than those that bind to class I. The so-called CLIP peptide class Il-associated invariant-chain peptide , comprising amino acids of the invariant chain, is an example of a promiscuous peptide.

Invariant chain associates with class II molecules during their biosynthesis and serves to promote assembly, induce transport out of the endoplasmic reticulum and inhibit the binding of peptides before class II molecules enter the endosomal compartments. The part of the invariant chain that corresponds to CLIP prevents peptide binding. Consistent with this property, CLIP or extensions thereof have been isolated from numerous allotypes. With knowledge of the crystallographic structures, this type of analysis was refined to show that nucleotide substitutions are selectively concentrated at sites that change amino acids in direct contact with either bound peptide or the T-cell receptor.

Although not proven, it is generally believed that the pressures exerted by epidemics of infectious disease select for new HLA alleles that have distinctive peptide-binding properties. Differences in amino acid sequence between allotypes and isotypes do not change the basic structure of HLA class I or class II molecules.

Conserved amino acids in the peptide-binding site allow a network of hydrogen bonds to be formed with the peptide main chain which provides most of the binding affinity.

This enables a broad spectrum of peptides to be displayed by HLA molecules for effective surveillance of pathogen-derived peptides. However, the diversity is not infinite. The peptide-binding site comprises a variety of pockets, clefts and ridges and the peptides bound are therefore restricted to those whose amino acid sequences complement the architecture of the binding site.

The precise size, shape and charge of the peptide-binding site varies dependent upon the side-chains of residues at positions of variability that modify the surface properties of the groove. In this way polymorphism changes the types of peptides that are selectively bound by HLA allotypes, and influences interactions with T-cell receptors.

Class I and class II residue positions that form the surface of the peptide-binding site are shown in Tables 1 and 2, respectively. Such endogenously bound peptides are usually obtained from preparations of HLA class I or II that have been isolated from cultured B-cell lines. Monoclonal antibodies with specificity for one or a few related allotypes can be used to purify a desired allotype from a cell expressing a normal set of alleles.

When a suitable antibody is not available, transfected cells are generated in which only the HLA class I or class II allele under study is expressed and these molecules can then be purified with a monoclonal antibody specific for all allotypes.

Purified HLA molecules are denatured to release the bound peptides. After removal of the HLA proteins themselves by size fractionation, the mixture of small peptides can be analysed in various ways Figure 1. The peptide mixture can be further fractionated by reversed-phase highperformance liquid chromatography HPLC and individual peak fractions, as detected spectrophotometrically, are then sequenced by Edman degradation.

However, this type of analysis is limited to a few abundant peptides that are wellresolved on HPLC. Amino acid substitutions in other class I allotypes may result in a different classification for some positions.

Amino acid substitutions in other DR allotypes may result in a different classification for some positions. An additional problem is the frequent failure to identify the carboxy-terminal sequence because the amount of a peptide is insufficient for complete sequence determination. Minimally, a mass spectrometer determines the molecular weight of each peptide in a sample, which often helps to interpret data obtained from Edman analysis.

In addition, this simple application estimates the total number of different peptides present in a mixture. More importantly, when two mass spectrometers are placed in tandem they can be used to determine the amino acid sequence of peptides isolated from HLA molecules.

The peptide mixture is first fractionated by reversed-phase HPLC in a microcapillary tube which directly feeds into the first mass spectrometer. This machine is used to purify an individual peptide for analysis and to direct it to a HLA Polymorphism, Peptide-biiidini; Motiis and T-Cell Epitopes chamber where collisions with molecules of an inert gas cause its fragmentation. The mass of each fragment and its abundance is then measured in the second mass spectrometer.

From these data the structure of the peptide is inferred. The validity of the sequence assignment is tested by synthesis of the peptide and comparison of its pattern of fragmentation with that of the natural peptide. In contrast to Edman degradation, the combination of microcapillary reversed-phase HPLC coupled to an electrospray ionization tandem mass spectrometer is a powerful tool for sequencing low-abundance peptides from complex mixtures.

Estimates based upon mass spectrometry indicate that a typical HLA class I allotype binds between and different peptides within a cell. Most peptides represent 0.

Searching gene and protein sequence databases with the sequences of endogenous peptides can identify the cellular proteins from which the peptides derive. Some of the cellular proteins are already known, others have been identified as 'expressed-sequence tags' ESTs in the random sequencing of cDNAs performed as part of the analysis of the human genome, and for some peptide sequences there is no match.

It is not uncommon to find peptides derived from other HLA proteins. Although the B-cell lines commonly used for the isolation of HLA allotypes were transformed with Epstein-Barr virus EBV and carry the viral genome, peptides of viral origin have rarely been encountered in the analysis of endogenously bound peptides.

This reflects the quiescence of the virus in these cells, which enables it to persist in B cells in vivo without being eliminated by T cells or other immune mechanisms. The normal locations of the proteins furnishing endogenously bound peptides are generally consistent with the different pathways for processing and presentation of antigens by class I and II molecules see Chapter 7, Figures 3 and 4.

Peptides bound by class I derive from cytoplasmic and nuclear proteins that were synthesized within the cell, such as ribosomal proteins, heat shock proteins and histones. These proteins undergo partial proteolysis in the cytoplasm as part of the routine turnover of cellular constituents. Peptides thus generated are transported into the endoplasmic reticulum where they bind nascent class I molecules. Class II molecules bind peptides from extracellular or integral membrane proteins which are not necessarily synthesized by the cell but gain access to endosomal compartments.

Examples include albumin, apolipoprotein, transferrin receptor, and class I and class II molecules themselves. When material has been purified from cultured cells it is not unusual to find peptides derived from bovine serum proteins, components of the media in which cells are grown. Fragments of the invariant chain called CLIP class Il-associated invariant-chain peptide encompassing residues are also frequently isolated from class II molecules.

There the invariant chain is cleaved, leaving CLIP in the binding site. Shared features of the peptides bound by an HLA allotype can also be deduced by Edman degradation-based sequencing of the peptides as a mixture - so-called pool sequencing. The results are determined from comparison of the relative yield of all amino acids at each cycle of amino acid sequencing. At positions of selectivity, one or a few amino acids dominate, whereas at other positions many amino acids at more even abundance are detected.

Positions of selectivity are said to be occupied by anchor residues. Dominant anchor residues are classified as those where one or a few closely related amino acids exclusively occupy a position.

Calculators

Amino acids that are enriched at a position are described as strong anchor residues. Preferences are rarely absolute and so allotypes can have a hierarchy of preferred amino acid residues at particular positions in the peptide. For the pool of peptides bound by an HLA class I or II allotype, a description of the anchor residues and their positions within the peptide sequence is called the peptide-binding motif of the allotype.

The dominant anchors of proline P at position 2 and leucine L at the carboxy-terminus are shown in bold. These data are taken from the entry on page The sequences of additional endogenously bound peptides can be found in that entry.

The uniform length makes them tractable to pool-sequence analysis and because of its relative simplicity and ease many class I peptide binding motifs have been characterized. The peptide-binding motifs of most HLA-A and -B allotypes have two dominant anchors, one at the carboxyterminus and a second that is usually at position 2.

The type of side-chain bound by each pocket depends upon the class I allotype, more specifically on the residues at positions of polymorphism that line the pockets. The size, shape and character of pockets correlate with the peptide side-chains they bind.

The anchor residue at peptide position 2 is commonly accommodated by the B pocket whose specificity is conferred by the residues at polymorphic positions 9, 45, 63 and 67 of the class I heavy chain.

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The carboxy-terminal peptide anchor interacts with the F pocket, whose specificity is determined by polymorphisms at positions 11, 80, 81, 95, 97, and of the class I heavy chain. The residue at position appears particularly influential. Peptides bound by class II allotypes are typically longer and more heterogeneous in size amino acids than the peptides bound by class I molecules. These differences arise because the extremities of the class n peptide-binding site are open and while peptides are gripped in the middle, their ends are free to extend out of the groove in a variable fashion.

As a consequence, class II molecules typically bind sets of overlapping peptides which derive from a protein and share a common core but have different overall lengths because they start and end at different places Figure 3. Anchor residues and peptide-binding motifs for class II allotypes cannot simply be determined by pool sequencing because of the variable distance between the amino-terminus of different peptides and the anchor residues.

Consequently, anchor residues are not seen as dominant amino acids at a single cycle of Edman degradation. Instead, the identification of potential anchors requires a search for clusters of enrichment for certain types of amino acids over several sequencing cycles. Anchor positions are shown as black boxes. Alignment of individual sequences can be informative for characterizing class II pep tide-binding motifs.

However, the determination of complete sequences for class Il-bound peptides is also problematic. The increased length of the class IIbound peptides greatly complicates sequencing by mass spectrometry and reduces the likelihood of obtaining a complete, unambiguous sequence. Likewise, the increased length of the peptides reduces the likelihood that complete sequences will be obtained by Edman degradation or that the critical region which interacts with the class II binding groove will be present in partial sequences.

Moreover, some of the most prominent peptides derive from the invariant chain which interacts with the binding groove of all class II allotypes and they do not provide information on allotypic differences in peptide binding. Although comparison of the sequences of individual binding peptides can reveal allotype preferences at particular peptide positions, these are less clear-cut than the peptide-binding motifs of class I molecules.

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HLA class II peptide-binding motifs generally include more anchor residues than class I motifs with less rigid specificity at each position.

Selectivities can be described in terms of amino acids that are excluded because of inhibitory contact residues with pockets as well as the more conventional description of favoured amino acids. This aspect of the complexity is a function of the biology of the class II molecule rather than a limitation of the methods. Strict residue preferences are not observed because the specificity of the class II peptidebinding site is intrinsically more degenerate than that of the class I molecule.

Most of the endogenously bound peptides sequenced are from normal cellular proteins so called self peptides. Even in infected cells, pathogen-derived peptides often only constitute a very small fraction of the peptides presented by HLA molecules for surveillance. As few as copies of a peptide per cell can stimulate a T-cell response. Instead, investigations usually start with a T-cell response to a pathogen, tumour or autoantigen and seek to define the epitope to which individual clones of T cell respond using synthetic peptides.

Lymphocytes obtained from peripheral blood or tissue biopsies are cultured under conditions to give clonal populations of CDS or CD4 T cells whose functions provide the assay for defining the antigen-presenting molecule and the peptide it presents. Responses of CDS T cells are assessed by screening for cytolytic activity or cytokine production whilst functional assays for CD4 T cells involve measurement of cellular proliferation or cytokine production.

Epitopes recognized by CDS T cells can be identified by examination of the amino acid sequences of a pathogen's proteins for the presence of peptides possessing a particular class I binding motifs. Recognition by T cells is tested using synthetic versions of the predicted class I ligands, which are exogenously provided to cells expressing the appropriate HLA class I allotype.

However, the approach has limitations. Some peptides bound by class I molecules do not conform to the relevant motif, and recognition of a synthetic peptide does not necessarily mean that the peptide will be available for binding within cells because of the influence of the patterns of proteolysis and transport on peptide supply.

The ambiguous nature of class II peptide-binding motifs means that their application to the identification of CD4 T-cell epitopes is often not informative. Many T-cell epitopes have been defined by systematic strategies which use synthetic peptides that cover the region of interest in a known protein sequence. Although laborious, this kind of approach provides reliable methods for identification of the minimum peptide epitope required for T-cell recognition and also of the HLA allotype which presents the antigen.

Studies of the CDS T-cell response to infectious agents such as viruses use autologous B cells infected with pathogen or transfected with segments of the pathogen genome as antigen-presenting target cells.

The presenting allotype can be deduced by testing for T-cell recognition a panel of infected target cells of known HLA type, each of which shares one class I allotype with the autologous cells.

A simpler alternative is to use a panel of target cells derived from a class Ideficient cell and expressing the product of a single transfected class I allele. Definition of the epitope recognized by a clone of CTL is done in a series of steps which gradually limit the possibilities.

The first objective is to identify which protein is the source of the antigenic peptide. This typically involves a comparison of target cells transfected with different pathogen genes. Having identified the protein, a series of overlapping synthetic peptides that cover the protein's sequence are tested for their capacity to sensitize uninfected cells for recognition by the CTL. A variety of exercises and games as well as dictionary activities and collocations recycle key vocabulary found throughout the readings.

ExamView Pro test-generating software allows instructors to create custom tests and quizzes. Learners develop useful and Product Information. Reading and Vocabulary Development Book 1 Back to series. Language s: American English Level s: Beginning Authors: New to this Edition. Companion Site. Student Companion Site. Authors Patricia Ackert. Linda Lee. Animals 2.