Immunology_Janeway

Immunology_Janeway. Like MHC class I molecules, MHC class II molecules that lack bound peptide are unstable. Peptide binding to MHC class II molecules has also been analyzed by elution of bound peptides and by X-ray crystallography, and differs in several ways from peptide binding to MHC class I molecules. Natural peptides that bind to MHC class II molecules are at least 13 amino acids long and can be much longer. The clusters of conserved residues that bind the two ends of a peptide in MHC class I molecules are not found in MHC class II molecules, and the ends of the peptide are not bound. Instead, the peptide lies in an extended conformation along the peptide-binding cleft. It is held there both by peptide side chains that protrude into shallow and deep pockets lined by polymorphic residues, and by interactions between the peptide backbone and the side chains of conserved amino acids that line the peptide-binding cleft in all MHC class II molecules (Fig. 4.22). Structural data show that amino acid side


Immunology_Janeway

Immunology_Janeway. between the peptide backbone and the side chains of conserved amino acids that line the peptide-binding cleft in all MHC class II molecules (Fig. 4.22). Structural data show that amino acid side chains at residues 1, 4, 6, and 9 of an MHC class II-bound peptide can be held in these binding pockets.


Immunology_Janeway

Immunology_Janeway. The binding pockets of MHC class II molecules accommodate a greater variety of side chains than those of MHC class I molecules, making it more difficult to define anchor residues and to predict which peptides will be able to bind


Immunology_Janeway

Immunology_Janeway. Fig. 4.23 Peptides that bind MHC class II molecules are peptides can vary, and so by convention the first anchor residue variable in length and their anchor residues lie at various is denoted as residue 1. note that all of the peptides share a distances from the ends of the peptide. The sequences of a hydrophobic residue in position 1, a negatively charged residue set of peptides that bind to the mouse Mhc class ii Ak allele are [aspartic acid (D) or glutamic acid (e)] in position 4, and a tendency shown in the upper panel. All contain the same core sequence to have a basic residue [lysine (K), arginine (R), histidine (h), (shaded) but differ in length. in the lower panel, different peptides glutamine (Q), or asparagine (n)] in position 6 and a hydrophobic binding to the human Mhc class ii allele hLA-DR3 are shown. residue [for example, tyrosine (Y), leucine (L), phenylalanine (f)] in Anchor residues are shown as green circles. The lengths of these position 9.


Immunology_Janeway

Immunology_Janeway. a particular MHC class II variant (Fig. 4.23). Nevertheless, by comparing the sequences of known binding peptides it is usually possible to detect patterns of amino acids that permit binding to different MHC class II variants, and to model how the amino acids of this peptide sequence motif will interact with the amino acids of the peptide-binding cleft. Because the peptide is bound by its backbone and allowed to emerge from both ends of the binding groove, there is, in principle, no upper limit to the length of peptides that could bind to MHC class II molecules. An example of this is the protein known as invariant chain, part of which lies entirely across the peptide-binding groove of nascent MHC class II molecules during their synthesis in the endoplasmic reticulum. We will return in Chapter 6 to the role of the invariant chain in the loading of peptides onto MHC class II molecules. In most cases, long peptides bound to MHC class II molecules are trimmed by peptidases to a length of


Immunology_Janeway

Immunology_Janeway. 6 to the role of the invariant chain in the loading of peptides onto MHC class II molecules. In most cases, long peptides bound to MHC class II molecules are trimmed by peptidases to a length of around 13–17 amino acids.


Immunology_Janeway

Immunology_Janeway. 4-17 The crystal structures of several peptide:MHC:T-cell receptor complexes show a similar orientation of the T-cell receptor over the peptide:MHC complex. At the time that the first X-ray crystallographic structure of a T-cell receptor was published, a structure of the same T-cell receptor bound to a peptide:MHC class I ligand was also produced. The orientation revealed by these structures showed that the T-cell receptor is aligned diagonally over the peptide and the peptide-binding cleft (Fig. 4.24). The TCRα chain lies over the α2 domain of the MHC molecule at the amino-terminal end of the bound peptide, as seen from the side view shown in Fig. 4.24a. The TCRβ chain lies over the MHC molecule’s α1 domain, closer to the carboxy-terminal end of the peptide. Figure 4.24b shows a view of this structure as if looking down through a transparent T-cell receptor to indicate where it contacts the MHC molecule. The CDR3 loops of both TCRα and TCRβ chains come together and lie over


Immunology_Janeway

Immunology_Janeway. Fig. 4.24 The T-cell receptor binds to the peptide:MHC complex. Panel a: the T-cell receptor binds to the top of a peptide:Mhc class i molecule, touching both the α1 and α2 domain helices. The cDRs of the T-cell receptor are shown in color: the cDR1 and cDR2 loops of the β chain are light and dark blue, respectively; and the cDR1 and cDR2 loops of the α chain are light and dark purple, respectively. The α-chain cDR3 loop is yellow, and the β-chain cDR3 loop is green. The β-chain hV4 loop is in red. The thick yellow line P1–P8 is the bound peptide. Panel b: the outline of the T-cell receptor’s antigen-binding site (thick black line) is superimposed over the top surface of the peptide:Mhc complex (the peptide is shaded dull yellow). The T-cell receptor lies at a somewhat diagonal angle across the peptide:Mhc complex, with the α and β cDR3 loops of the T-cell receptor (3α, 3β, yellow and green, respectively) contacting the center of the peptide. The α-chain cDR1 and cDR2 loops (1α, 2α,


Immunology_Janeway

Immunology_Janeway. the peptide:Mhc complex, with the α and β cDR3 loops of the T-cell receptor (3α, 3β, yellow and green, respectively) contacting the center of the peptide. The α-chain cDR1 and cDR2 loops (1α, 2α, light and dark purple, respectively) contact the Mhc helices at the amino terminus of the bound peptide, whereas the β-chain cDR1 and cDR2 loops (1β, 2β, light and dark blue, respectively) make contact with the helices at the carboxy terminus of the bound peptide. courtesy of i.A. Wilson.


Immunology_Janeway

Immunology_Janeway. central amino acids of the peptide. The T-cell receptor is threaded through a valley between the two high points on the two surrounding α helices that form the walls of the peptide-binding cleft. This can be seen in Fig. 4.25, which shows a view from the end of the peptide-binding groove of a peptide:MHC class II:T-cell receptor complex. Comparison of various peptide: MHC:T-cell receptor complexes shows that the axis of the TCR as it binds the surface of the MHC molecule is rotated somewhat relative to the peptide-binding groove of the MHC molecule (see Fig. 4.24b). In this orientation, the Vα domain makes contact primarily with the amino-terminal half of the bound peptide, whereas the Vβ domain contacts primarily the carboxy-terminal half. Both chains also interact with the α helices of the MHC class I molecule (see Fig. 4.24). The T-cell receptor contacts are not symmetrically distributed over the MHC molecule: whereas the Vα CDR1 and CDR2 loops are in close contact with the helices


Immunology_Janeway

Immunology_Janeway. MHC class I molecule (see Fig. 4.24). The T-cell receptor contacts are not symmetrically distributed over the MHC molecule: whereas the Vα CDR1 and CDR2 loops are in close contact with the helices of the peptide:MHC complex around the amino terminus of the bound peptide, the β-chain CDR1 and CDR2 loops, which interact with the complex at the carboxy terminus of the bound peptide, have variable contributions to the binding.


Immunology_Janeway

Immunology_Janeway. Comparison of the three-dimensional structure of an unliganded T-cell receptor and the same T-cell receptor complexed to its peptide:MHC ligand shows that the binding results in some degree of conformational change, or ‘induced fit,’ particularly within the Vα CDR3 loop. Subtle variations at amino acids that contact the T-cell receptor can have strikingly different effects on the recognition of an otherwise identical peptide:MHC ligand by the same T cell. The flexibility in the CDR3 loop demonstrated by these two structures helps to explain how the T-cell receptor can adopt conformations that recognize related, but different, peptide ligands.


Immunology_Janeway

Immunology_Janeway. The specificity of T-cell recognition involves both the peptide and its presenting MHC molecule. Kinetic analysis of T-cell receptor binding to peptide:MHC ligands suggests that the interactions with MHC molecules might predominate at the start of the contact, but that subsequent interactions with the peptide as well as the MHC molecule dictate the final outcome—binding or dissociation. As with antibody–antigen interactions, only a few amino acids at the interface might provide the essential contacts that determine the specificity and strength of binding. Simply changing a leucine to isoleucine in the peptide, for example, is sufficient to alter a T-cell response from strong killing to no response at all. Mutations of single residues in the presenting MHC molecules can have the same effect. This dual specificity for T-cell recognition of antigen underlies the MHC restriction of T-cell responses, a phenomenon that was observed long before the peptide-binding properties of MHC molecules


Immunology_Janeway

Immunology_Janeway. This dual specificity for T-cell recognition of antigen underlies the MHC restriction of T-cell responses, a phenomenon that was observed long before the peptide-binding properties of MHC molecules were known. Another consequence of this dual specificity is a need for T-cell receptors to exhibit some inherent specificity for MHC molecules in order to be able to interact appropriately with the antigen-presenting surface of MHC


Immunology_Janeway

Immunology_Janeway. Fig. 4.25 The T-cell receptor interacts with MHC class I and MHC class II molecules in a similar fashion. Shown is the structure of a T-cell receptor, specific for a peptide derived from chicken cytochrome c, bound to an Mhc class ii molecule. This T-cell receptor’s binding is at a site and orientation similar to that of the T-cell receptor bound to the Mhc class i molecule shown in fig. 4.24. The α and β chains of the T-cell receptor are colored in light and dark blue, respectively. The cytochrome c peptide is light orange. The T-cell receptor sits in a shallow saddle formed between the α-helical regions of the Mhc class ii α (brown) and β (yellow) chains at roughly 90° to the long axis of the Mhc class ii molecule and the bound peptide. Structure derived from PDB 3QiB. courtesy of K.c. garcia.


Immunology_Janeway

Immunology_Janeway. molecules. We will return to these issues in Chapter 6, where we recount the discovery of MHC restriction in the context of T-cell recognition and MHC polymorphisms, and in Chapter 8, where we discuss the impact of these phenomena on T-cell development in the thymus. 4-18 The CD4 and CD8 cell-surface proteins of T cells directly contact MHC molecules and are required to make an effective response to antigen.


Immunology_Janeway

Immunology_Janeway. As we introduced in Section 1-21, T cells fall into two major classes distinguished by the expression of the cell-surface proteins CD4 and CD8. CD8 is expressed by cytotoxic T cells, while CD4 is expressed by T cells whose function is to activate other cells. CD4 and CD8 were known as markers for these functional sets for some time before it became clear that the distinction was based on the ability of T cells to recognize different classes of MHC molecules. We now know that CD8 recognizes MHC class I molecules and CD4 recognizes MHC class II. During antigen recognition, CD4 or CD8 (depending on the type of T cell) associates with the T-cell receptor on the T-cell surface and binds to invariant sites on the MHC portion of the composite peptide:MHC ligand, away from the peptide-binding site. This binding contributes to the overall effectiveness of the T-cell response, and so CD4 and CD8 are called co-receptors.


Immunology_Janeway

Immunology_Janeway. CD4 is a single-chain protein composed of four Ig-like domains (Fig. 4.26). The first two domains (D1 and D2) are packed tightly together to form a rigid


Immunology_Janeway

Immunology_Janeway. Fig. 4.26 The structures of the CD4 and CD8 co-receptor molecules. The cD4 molecule contains four ig-like domains, shown in schematic form in panel a and as a ribbon diagram from the crystal structure in panel b. The amino-terminal domain, D1, is similar in structure to an immunoglobulin V domain. The second domain, D2, although related to an immunoglobulin domain, is different from both V and c domains and has been termed a c2 domain. The first two domains of cD4 form a rigid rodlike structure that is linked to the two carboxy-terminal domains by a flexible link. The binding site for Mhc class ii molecules involves mainly the D1 domain. The cD8 molecule is a heterodimer of an α and a β chain covalently linked by a disulfide bond. An alternative form of cD8 exists as a homodimer of α chains. The heterodimer is depicted in panel a, whereas the ribbon diagram in panel b is of the homodimer. cD8α and cD8β chains have very similar structures, each having a single domain resembling an


Immunology_Janeway

Immunology_Janeway. chains. The heterodimer is depicted in panel a, whereas the ribbon diagram in panel b is of the homodimer. cD8α and cD8β chains have very similar structures, each having a single domain resembling an immunoglobulin V domain and a stretch of polypeptide chain, believed to be in a relatively extended conformation, that anchors the V-like domain to the cell membrane.


Immunology_Janeway

Immunology_Janeway. rod about 6 nm long, which is joined by a flexible hinge to a similar rod formed by the third and fourth domains (D3 and D4). The MHC-binding region on CD4 is located mainly on a lateral face of the D1 domain, and CD4 binds to a hydrophobic crevice formed at the junction of the α2 and β2 domains of the MHC class II molecule (Fig. 4.27a). This site is well away from the site where the T-cell receptor binds, as shown by the complete crystal structure of a T-cell receptor bound to peptide:MHC class II with bound CD4 (Fig. 4.28). This structure demonstrates that the CD4 molecule and the T-cell receptor can bind simultaneously to the same peptide:MHC class II complex. CD4 enhances sensitivity to antigen, as the T cell is about 100-fold more sensitive to the antigen when CD4 is present. The enhancement process results from the ability of the intracellular portion of CD4 to bind to a cytoplasmic tyrosine kinase called Lck. As we will discuss in detail Chapter 7, bringing Lck into proximity


Immunology_Janeway

Immunology_Janeway. process results from the ability of the intracellular portion of CD4 to bind to a cytoplasmic tyrosine kinase called Lck. As we will discuss in detail Chapter 7, bringing Lck into proximity with the T-cell receptor complex helps activate the signaling cascade induced by antigen recognition.


Immunology_Janeway

Immunology_Janeway. The structure of CD8 is quite different. It is a disulfide-linked dimer of two different chains, called αand β, each containing a single Ig-like domain linked to the membrane by a segment of extended polypeptide (see Fig. 4.26). This segment


Immunology_Janeway

Immunology_Janeway. The binding sites for cD4 and cD8 on the Mhc class ii and class i molecules, respectively, lie in the ig-like domains nearest to the membrane and distant from the peptide-binding cleft. The binding of cD4 to an Mhc class ii molecule is shown as a ribbon structure in panel a and schematically in panel c. The α chain of the Mhc class ii molecule is shown in pink, and the β chain in white, while cD4 is in gold. only the D1 and D2 domains of the cD4 molecule are shown in panel a. The binding site for cD4 lies at the base of the β2 domain of an Mhc class ii molecule, in the hydrophobic crevice between the β2 and α2 domains. The binding of cD8αβ to an Mhc class i molecule is shown in panel b and schematically in panel d. The class i heavy chain and β2-microglobulin are shown in white and pink, respectively, and the two chains of the cD8 dimer are shown in light (cD8β) and dark (cD8α) purple. The binding site for cD8 on the Mhc class i molecule lies in a similar position to that of cD4 in


Immunology_Janeway

Immunology_Janeway. respectively, and the two chains of the cD8 dimer are shown in light (cD8β) and dark (cD8α) purple. The binding site for cD8 on the Mhc class i molecule lies in a similar position to that of cD4 in the Mhc class ii molecule, but cD8 binding also involves the base of the α1 and α2 domains, and thus the binding of cD8 to Mhc class i is not completely equivalent to the binding of cD4 to Mhc class ii. Structures derived from PDB 3S4S (cD4/ Mhc class ii) and PDB 3DMM (cD8αβ/ Mhc class i). courtesy of K.c. garcia.


Immunology_Janeway

Immunology_Janeway. Fig. 4.27 The binding sites for CD4 and CD8 on MHC class II and class I molecules lie in the Ig-like domains. is extensively glycosylated, which is thought to maintain it in an extended conformation and protect it from cleavage by proteases. CD8α chains can form homodimers, although these are usually not found when CD8β is expressed. Naive T cells express CD8αβ, but the CD8αα homodimer can be expressed by highly activated effector and memory T cells. CD8αα is also expressed by a population of intraepithelial lymphocytes known as mucosal associated invariant T cells (MAIT cells); these cells recognize metabolites of folic acid that are produced by bacteria in association with the nonclassical MHC class I molecule MR1, which we will describe in Chapter 6.


Immunology_Janeway

Immunology_Janeway. CD8αβ binds weakly to an invariant site in the α3 domain of an MHC class I molecule (see Fig. 4.27b). The CD8β chain interacts with residues in the base of the α2 domain of the MHC class I molecule, while the αchain is in a lower position interacting with the α3 domain of the MHC class I molecule. The strength of binding of CD8 to the MHC class I molecule is influenced by the glycosylation state of the CD8 molecule; increasing the number of sialic acid residues on CD8 carbohydrate structures decreases the strength of the interaction. The pattern of sialylation of CD8 changes during the maturation of T cells and also on activation, and this likely has a role in modulating antigen recognition.


Immunology_Janeway

Immunology_Janeway. Like the interactions with MHC class II molecules, the T-cell receptor and CD8 can interact simultaneously with one MHC class I molecule (Fig. 4.29). Like CD4, CD8 binds Lck through the cytoplasmic tail of the α chain, and CD8αβ increases the sensitivity of T cells to antigen presented by MHC class I molecules about 100-fold. Although the molecular details are unclear, the CD8αα homodimer appears to function less efficiently than CD8αβ as a co-receptor, and may negatively regulate activation. In contrast to CD8, CD4 is not thought to dimerize. Fig. 4.28 CD4 and the T-cell receptor bind to distinct regions of the MHC class II molecule. A ribbon diagram is shown from a crystal structure of a complete α:β TcR:peptide-Mhc:cD4 ternary complex. The α and β chains of the T-cell receptor (TcR) are blue and red, respectively. The Mhc class ii molecule is green, with the bound peptide shown in gray. cD4 is shown in orange. Structure derived from PDB 3T0e. courtesy of K.c. garcia.


Immunology_Janeway

Immunology_Janeway. Fig. 4.29 CD8 binds to a site on MHC class I molecules that is distant from where the T-cell receptor binds. The relative binding positions of both the T-cell receptor and cD8 molecules can be seen in this hypothetical reconstruction of their interaction with Mhc class i (α chain in dark green and β2-microglobulin in light green). The α and β chains of the T-cell receptor are shown in brown and purple, respectively. The cD8αβ heterodimer is shown bound to the Mhc class i α3 domain. The cD8 α chain is in blue, and the cD8β chain is in red. courtesy of chris nelson and David fremont.


Immunology_Janeway

Immunology_Janeway. Mhc class i molecules are expressed on all nucleated cells, although they are most highly expressed in hematopoietic cells. Mhc class ii molecules are normally expressed only by a subset of hematopoietic cells and by thymic stromal cells, although they may be expressed by other cell types on exposure to the inflammatory cytokine ifn-γ. *in humans, activated T cells express Mhc class ii molecules, whereas in mice all T cells are Mhc class ii-negative. †in the brain, most cell types are Mhc class ii-negative, but microglia, which are related to macrophages, are Mhc class ii-positive. 4-19 The two classes of MHC molecules are expressed differentially on cells.


Immunology_Janeway

Immunology_Janeway. MHC class I and MHC class II molecules have distinct distributions among cells, and these reflect the different effector functions of the T cells that recognize them (Fig. 4.30). MHC class I molecules present peptides from pathogens, commonly viruses, to CD8 cytotoxic T cells, which are specialized to kill any cell that they specifically recognize. Because viruses can infect any nucleated cell, almost all such cells express MHC class I molecules, although the level of constitutive expression varies from one cell type to the next. For example, cells of the immune system express abundant MHC class I on their surface, whereas liver cells (hepatocytes) express relatively low levels (see Fig. 4.30). Nonnucleated cells, such as mammalian red blood cells, express little or no MHC class I, and thus the interior of red blood cells is a site in which an infection can go undetected by cytotoxic T cells. Because red blood cells cannot support viral replication, this is of no great consequence for


Immunology_Janeway

Immunology_Janeway. the interior of red blood cells is a site in which an infection can go undetected by cytotoxic T cells. Because red blood cells cannot support viral replication, this is of no great consequence for viral infection, but it might be the absence of MHC class I that allows the Plasmodium parasites that cause malaria to live in this privileged site.


Immunology_Janeway

Immunology_Janeway. In contrast, a major function of the CD4 T cells that recognize MHC class II molecules is to activate other effector cells of the immune system. Thus, MHC class II molecules are normally found on dendritic cells, B lymphocytes, and macrophages—antigen-presenting cells that participate in immune responses—but not on other tissue cells (see Fig. 4.30). The peptides presented by MHC class II molecules expressed by dendritic cells can function to activate naive CD4 T cells. When previously activated CD4 T cells recognize peptides bound to MHC class II molecules on B cells, the T cells secrete cytokines that can influence the isotype of antibody that those B cells will choose to produce. Upon recognizing peptides bound to MHC class II molecules on macrophages, CD4 T cells activate these cells, again in part through cytokines, to destroy the pathogens in their vesicles.


Immunology_Janeway

Immunology_Janeway. The expression of both MHC class I and MHC class II molecules is regulated by cytokines, in particular, interferons, released in the course of an immune response. Interferon-α (IFN-α) and IFN-β increase the expression of MHC class I molecules on all types of cells, whereas IFN-γ increases the expression of both MHC class I and MHC class II molecules, and can induce the expression of MHC class II molecules on certain cell types that do not normally express them. Interferons also enhance the antigen-presenting function of MHC class I molecules by inducing the expression of key components of the intracellular machinery that enables peptides to be loaded onto the MHC molecules. 4-20 A distinct subset of T cells bears an alternative receptor made up of γ and δ chains.


Immunology_Janeway

Immunology_Janeway. 4-20 A distinct subset of T cells bears an alternative receptor made up of γ and δ chains. During the search for the gene for the TCRα chain, another T-cell receptor-like gene was unexpectedly discovered. This gene was named TCRγ, and its discovery led to a search for further T-cell receptor genes. Another receptor chain was identified by using antibody against the predicted sequence of the γ chain and was called the δ chain. It was soon discovered that a minority population of T cells bore a distinct type of T-cell receptor made up of γ:δ heterodimers rather than α:β heterodimers. The development of these cells is described in Sections 8-11 and 8-12.


Immunology_Janeway

Immunology_Janeway. Like α:β T cells, γ:δ T cells can be found in the lymphoid tissues of all vertebrates, but they are also prominent as populations of intraepithelial lymphocytes, particularly in the skin and female reproductive tracts, where their receptors display very limited diversity. Unlike α:β T cells, γ:δ T cells do not generally recognize antigen as peptides presented by MHC molecules, and γ:δ T-cell receptors are not restricted by the ‘classical’ MHC class I and class II molecules that function in binding and presenting peptides to T cells. Instead, γ:δ T-cell receptors seem to recognize their target antigens directly and thus probably are able to recognize and respond rapidly to molecules expressed by many different cell types. Their ligands have been difficult to identify, but several have now been described and seem to indicate that γ:δ T cells play an intermediate, or transitional, role between wholly innate and fully adaptive immune responses.


Immunology_Janeway

Immunology_Janeway. Like NK-cell receptor ligands, such as the proteins MIC and RAET1 (see Section 3-27), many of the ligands seen by γ:δ T cells are induced by cellular stress or damage. γ:δ T cells may also bind antigens presented by ‘nonclassical’ MHC class Ib molecules, which we will discuss in Chapter 6. These proteins are related structurally to the MHC proteins we have already discussed, but have functions besides binding peptides for presentation to T cells. Additional ligands may include heat-shock proteins and nonpeptide ligands such as phosphorylated ligands or mycobacterial lipid antigens. γ:δ T cells can also respond to unorthodox nucleotides and phospholipids. Recognition of molecules expressed as a consequence of infection, rather than recognition of pathogen-specific antigens themselves, distinguishes intraepithelial γ:δT cells from other lymphocytes, and this would place them in the innate-like class. For these reasons, the term ‘transitional immunity’ has been proposed to clarify the


Immunology_Janeway

Immunology_Janeway. intraepithelial γ:δT cells from other lymphocytes, and this would place them in the innate-like class. For these reasons, the term ‘transitional immunity’ has been proposed to clarify the role of γ:δ T cells, since the function of these cells seems to be someplace between innate and adaptive responses.


Immunology_Janeway

Immunology_Janeway. The crystallographic structure of a γ:δ T-cell receptor reveals that, as expected, it is similar in shape to α:β T-cell receptors. Figure 4.31 shows a crystal structure of a γ:δ T-cell receptor complex bound to one of the nonclassical MHC class I molecules mentioned above, called T22. This structure shows that the overall orientation of the γ:δ T-cell receptor with the MHC molecule is strikingly different from that of an α:β T-cell receptor, in that it interacts primarily with one end of the T22 molecule. However, the CDR3 regions of the γ:δ T-cell receptor still play a critical role in recognition, similar to that of antibodies and of α:β T-cell receptors. Further, the CDR3 of the γ:δ T-cell receptor is longer than either of these other two antigen receptors, and this could have implications toward the type of ligand that the γ:δ T-cell receptor recognizes, as there is enormous combinatorial diversity from the CDR3 within the γ:δ T-cell receptor repertoire. We will return to discuss


Immunology_Janeway

Immunology_Janeway. toward the type of ligand that the γ:δ T-cell receptor recognizes, as there is enormous combinatorial diversity from the CDR3 within the γ:δ T-cell receptor repertoire. We will return to discuss further the ligands and the development of γ:δ T cells in Chapters 6 and 8.


Immunology_Janeway

Summary.

Immunology_Janeway. Summary.


Immunology_Janeway

Immunology_Janeway. The receptor for antigen on most T cells, the α:β T-cell receptor, is composed of two protein chains, TCRα and TCRβ, and resembles in many respects a single Fab fragment of immunoglobulin. α:β T-cell receptors are always membrane-bound and recognize a composite ligand of a peptide antigen bound to an MHC molecule. Each MHC molecule binds a wide variety of different peptides, but the different variants of MHC each preferentially recognize sets of peptides with particular sequences and physical features. The peptide antigen is generated intracellularly, and is bound stably in a peptide-binding cleft on the surface of the MHC molecule. There are two classes of MHC molecules, and these are bound in their nonpolymorphic domains by CD8 and CD4 molecules that distinguish two different functional classes of α:β T cells. CD8 binds MHC class I molecules and can bind simultaneously to the same peptide:MHC class I complex being recognized by a T-cell receptor, thus acting as a co-receptor and


Immunology_Janeway

Immunology_Janeway. classes of α:β T cells. CD8 binds MHC class I molecules and can bind simultaneously to the same peptide:MHC class I complex being recognized by a T-cell receptor, thus acting as a co-receptor and enhancing the T-cell response; CD4 binds MHC class II molecules and acts as a co-receptor for T-cell receptors that recognize peptide:MHC class II ligands. A T-cell receptor interacts directly both with the antigenic peptide


Immunology_Janeway

Immunology_Janeway. Fig. 4.31 Structures of γ:δ T-cell receptor bound to the nonclassical MHC class I molecule T22. The γ:δ T-cell receptor has a similar overall structure to the α:β T-cell receptor and the fab fragment of an immunoglobulin. The cδ domain is more like an immunoglobulin domain than is the corresponding cα domain of the α:β T-cell receptor. in this structure, the overall orientation of the γ:δ T-cell receptor with respect to the nonclassical Mhc molecule T22 is very different from the orientation of an α:β T-cell receptor with either Mhc class i or class ii molecules. Rather than lying directly over the peptide-binding groove, the γ:δ T-cell receptor is engaged with one end much more than the other; this is consistent with a lack of peptide contact and absence of Mhc-restricted recognition.


Immunology_Janeway

Immunology_Janeway. and with polymorphic features of the MHC molecule that displays it, and this dual specificity underlies the MHC restriction of T-cell responses. A second type of T-cell receptor, composed of a γand a δ chain, is structurally similar to the α:β T-cell receptor, but it binds to different ligands, including nonpeptide ligands, nonpolymorphic nonclassical MHC molecules, and certain lipids. The receptor is thought not to be MHC-restricted and is found on a minority population of lymphoid and intraepithelial T cells, the γ:δ T cells. Summary to Chapter 4.


Immunology_Janeway

Immunology_Janeway. Summary to Chapter 4. B cells and T cells use different, but structurally similar, molecules to recognize antigen. The antigen-recognition molecules of B cells are immunoglobulins, and are made both as a membrane-bound receptor for antigen, the B-cell receptor, and as secreted antibodies that bind antigens and elicit humoral effector functions. The antigen-recognition molecules of T cells, in contrast, are made only as cell-surface receptors and so elicit only cellular effector functions. Immunoglobulins and T-cell receptors are highly variable molecules, with the variability concentrated in that part of the molecule—the variable (V) region—that binds to antigen. Immunoglobulins bind a wide variety of chemically different antigens, whereas the major type of T-cell receptor, the α:β T-cell receptor, predominantly recognizes peptide fragments of foreign proteins bound to MHC molecules, which are ubiquitous on cell surfaces.


Immunology_Janeway

Immunology_Janeway. Binding of antigen by immunoglobulins has chiefly been studied with antibodies. The binding of antibody to its antigen is highly specific, and is determined by the shape and physicochemical properties of the antigen-binding site. Located at the other end of the antibody from the antigen-binding site is the constant, or Fc, region, which influences the types of effector function the antibody can elicit. There are five main functional classes of antibodies, each encoded by a different type of constant region. As we will see in Chapter 10, these interact with different components of the immune system to incite an inflammatory response and eliminate the antigen.


Immunology_Janeway

Immunology_Janeway. T-cell receptors differ in several respects from the B-cell immunoglobulins. One is the absence of a secreted form of T-cell receptor, reflecting the functional differences between T cells and B cells. B cells deal with pathogens and their protein products circulating within the body; secretion of a soluble antigen-recognition molecule enables the B cell to act in the clearance of antigen effectively throughout the extracellular spaces of the body. T cells, in contrast, are specialized for active surveillance of pathogens, and T-cell recognition does not involve a soluble, secreted receptor. Some, such as CD8 T cells, are able to detect intracellular infections and are able to kill infected cells that bear foreign antigenic peptides on their surface. Others, such as CD4 T cells, interact with cells of the immune system that have taken up foreign antigen and are displaying it on the cell surface.


Immunology_Janeway

Immunology_Janeway. T-cell receptors also recognize a composite ligand made up of the foreign peptide bound to a self MHC molecule, and not intact antigen. This means that T cells can interact only with a body cell displaying the antigen, not with the intact pathogen or protein. Each T-cell receptor is specific for a particular combination of peptide and a self MHC molecule. MHC molecules are encoded by a family of highly polymorphic genes. Expression of multiple variant MHC molecules, each with a different peptide-binding repertoire, helps to ensure that T cells from an individual will be able to recognize at least some peptides generated from nearly every pathogen. Questions. 4.1 True or False: An antibody proteolytically cleaved by papain yields a fragment with higher avidity to the cognate antigen than an antibody cleaved by pepsin. 4.2 Short Answer: how is cD4 and cD8 co-receptor binding to Mhc important for T-cell receptor signaling?


Immunology_Janeway

Immunology_Janeway. 4.2 Short Answer: how is cD4 and cD8 co-receptor binding to Mhc important for T-cell receptor signaling? 4.3 Short Answer: Why and how is it advantageous to have heterozygosity in the Mhc locus? 4.4 Matching: Match the term to the best description: A. Antigenic determinant i. The structure recognized by an antibody (that is, the epitope) B. conformational/ ii. Regions of the V region C. continuous/linear iii. An epitope composed epitope of a single segment of a polypeptide chain


Immunology_Janeway

Immunology_Janeway. B. conformational/ ii. Regions of the V region C. continuous/linear iii. An epitope composed epitope of a single segment of a polypeptide chain D. hypervariable region iv. An epitope composed of amino acids from different parts of a polypeptide chain brought together by protein folding 4.5 Fill-in-the-Blanks: Most vertebrates, including humans and mice, produce antibodies composed of ________ and ________ chains. These bear ____ regions that recognize the antigen and ____ regions that dictate the antibody class and isotype. camelids and cartilaginous fish, however, produce ________________ and _______________, respectively, which are forming the basis for single-chain antibody production for clinical applications. 4.6 Multiple Choice: Which of the following statements is not true? A. T-cell receptor α and β chains pair together, but the α chain can be switched out for a γ or a δ chain. B. electrostatic interactions (for example, a salt bridge) occur between charged amino acids.


Immunology_Janeway

Immunology_Janeway. B. electrostatic interactions (for example, a salt bridge) occur between charged amino acids. C. hydrophobic interactions occur between two hydrophobic surfaces and exclude water. D. Antibodies often have many aromatic amino acids such as tyrosine in their antigen-binding sites. E. Mhc restriction is the phenomenon where T cells will recognize a unique set of peptides bound to a particular Mhc molecule. 4.7 Multiple Choice: Which of the following is the most abundant immunoglobulin class in healthy adult humans and mice? A. igA B. igD C. ige D. igg E. igM 4.8 Multiple Choice: Which of the following describes the structure of an immunoglobulin fold? A. Two antiparallel β sheets with an α-helical linker and a disulfide bond link B. Two β strands linked by a disulfide bond C. four α helices linked by two disulfide bonds D. Seven antiparallel α helices in series


Immunology_Janeway

Immunology_Janeway. B. Two β strands linked by a disulfide bond C. four α helices linked by two disulfide bonds D. Seven antiparallel α helices in series E. one β sandwich of two β sheets folded together and linked by a disulfide bond 4.9 Multiple Choice: Antibodies have flexibility at various points in the molecule, particularly the hinge region between the fc and fab portion and, to some extent, the junction between the V and c regions. Which of the following properties of an antibody are not affected by its flexibility? A. Binding to small antigens (haptens) B. Avidity to antigen C. Affinity to antigen D. interaction with antibody-binding proteins E. Binding to distantly spaced antigens 4.10 Multiple Choice: Which region of the antigen receptor of B cells and T cells is most critical in antigen recognition and specificity? A. fR1 B. cDR1 C. fR2 D. cDR2 E. fR3 F. cDR3 G. fR4 General references.


Immunology_Janeway

Immunology_Janeway. A. fR1 B. cDR1 C. fR2 D. cDR2 E. fR3 F. cDR3 G. fR4 General references. Garcia, K.C., Degano, M., Speir, J.A., and Wilson, I.A.: Emerging principles for T cell receptor recognition of antigen in cellular immunity. Rev. Immunogenet. 1999, 1:75–90. Garcia, K.C., Teyton, L., and Wilson, I.A.: Structural basis of T cell recognition. Annu. Rev. Immunol. 1999, 17:369–397. Moller, G. (ed): Origin of major histocompatibility complex diversity. Immunol. Rev. 1995, 143:5–292. Poljak, R.J.: Structure of antibodies and their complexes with antigens. Mol. Immunol. 1991, 28:1341–1345. Rudolph, M.G., Stanfield, R.L., and Wilson, I.A: How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 2006, 24:419–466. Sundberg, E.J., and Mariuzza, R.A.: Luxury accommodations: the expanding role of structural plasticity in protein-protein interactions. Structure 2000, 8:R137–R142. Section references. 4-1 IgG antibodies consist of four polypeptide chains.


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Immunology_Janeway. Section references. 4-1 IgG antibodies consist of four polypeptide chains. Edelman, G.M.: Antibody structure and molecular immunology. Scand. J. Immunol. 1991, 34:4–22. Faber, C., Shan, L., Fan, Z., Guddat, L.W., Furebring, C., Ohlin, M., Borrebaeck, C.A.K., and Edmundson, A.B.: Three-dimensional structure of a human Fab with high affinity for tetanus toxoid. Immunotechnology 1998, 3:253–270. Harris, L.J., Larson, S.B., Hasel, K.W., Day, J., Greenwood,A., and McPherson,A.: The three-dimensional structure of an intact monoclonal antibody for canine lymphoma. Nature 1992, 360:369–372. Barclay, A.N., Brown, M.H., Law, S.K., McKnight, A.J., Tomlinson, M.G., and van der Merwe, P.A. (eds): The Leukocyte Antigen Factsbook, 2nd ed. London: Academic Press, 1997. Brummendorf, T., and Lemmon, V.: Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion. Curr. Opin. Cell Biol. 2001, 13:611–618.


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Immunology_Janeway. Brummendorf, T., and Lemmon, V.: Immunoglobulin superfamily receptors: cis-interactions, intracellular adapters and alternative splicing regulate adhesion. Curr. Opin. Cell Biol. 2001, 13:611–618. Marchalonis, J.J., Jensen, I., and Schluter, S.F.: Structural, antigenic and evolutionary analyses of immunoglobulins and T cell receptors. J. Mol. Recog. 2002, 15:260–271. Ramsland, P.A., and Farrugia, W.: Crystal structures of human antibodies: a detailed and unfinished tapestry of immunoglobulin gene products. J. Mol. Recog. 2002, 15:248–259. 4-4 The antibody molecule can readily be cleaved into functionally distinct fragments. Porter, R.R.: Structural studies of immunoglobulins. Scand. J. Immunol. 1991, 34:382–389. Yamaguchi, Y., Kim, H., Kato, K., Masuda, K., Shimada, I., and Arata, Y.: Proteolytic fragmentation with high specificity of mouse IgG—mapping of proteolytic cleavage sites in the hinge region. J. Immunol. Methods. 1995, 181:259–267.


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Immunology_Janeway. 4-5 The hinge region of the immunoglobulin molecule allows flexibility in binding to multiple antigens. Gerstein, M., Lesk, A.M., and Chothia, C.: Structural mechanisms for domain movements in proteins. Biochemistry 1994, 33:6739–6749. Jimenez, R., Salazar, G., Baldridge, K.K., and Romesberg, F.E.: Flexibility and molecular recognition in the immune system. Proc. Natl Acad. Sci. USA 2003, 100:92–97. Saphire, E.O., Stanfield, R.L., Crispin, M.D., Parren, P.W., Rudd, P.M., Dwek, R.A., Burton, D.R., and Wilson, I.A.: Contrasting IgG structures reveal extreme asymmetry and flexibility. J. Mol. Biol. 2002, 319:9–18. 4-6 Localized regions of hypervariable sequence form the antigen-binding site. Chitarra, V., Alzari, P.M., Bentley, G.A., Bhat, T.N., Eiselé, J.-L., Houdusse, A., Lescar, J., Souchon, H., and Poljak, R.J.: Three-dimensional structure of a heteroclitic antigen-antibody cross-reaction complex. Proc. Natl Acad. Sci. USA 1993, 90:7711–7715.


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Immunology_Janeway. Decanniere, K., Muyldermans, S., and Wyns, L.: Canonical antigen-binding loop structures in immunoglobulins: more structures, more canonical classes? J. Mol. Biol. 2000, 300:83–91. Gilliland, L.K., Norris, N.A., Marquardt, H., Tsu, T.T., Hayden, M.S., Neubauer, M.G., Yelton, D.E., Mittler, R.S., and Ledbetter, J.A.: Rapid and reliable cloning of antibody variable regions and generation of recombinant single-chain antibody fragments. Tissue Antigens 1996, 47:1–20. Johnson, G., and Wu, T.T.: Kabat Database and its applications: 30 years after the first variability plot. Nucleic Acids Res. 2000, 28:214–218. Wu, T.T., and Kabat, E.A.: An analysis of the sequences of the variable regions of Bence Jones proteins and myeloma light chains and their implications for antibody complementarity. J. Exp. Med. 1970, 132:211–250.


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Immunology_Janeway. Xu, J., Deng, Q., Chen, J., Houk, K.N., Bartek, J., Hilvert, D., and Wilson, I.A.: Evolution of shape complementarity and catalytic efficiency from a primordial antibody template. Science 1999, 286:2345–2348. Ban, N., Day, J., Wang, X., Ferrone, S., and McPherson, A.: Crystal structure of an anti-anti-idiotype shows it to be self-complementary. J. Mol. Biol. 1996, 255:617–627. Davies, D.R., and Cohen, G.H.: Interactions of protein antigens with antibodies. Proc. Natl Acad. Sci. USA 1996, 93:7–12. Decanniere, K., Desmyter, A., Lauwereys, M., Ghahroudi, M.A., Muyldermans, S., and Wyns, L.: A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops. Structure Fold. Des. 1999, 7:361–370. Padlan, E.A.: Anatomy of the antibody molecule. Mol. Immunol. 1994, 31:169–217.


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Immunology_Janeway. Padlan, E.A.: Anatomy of the antibody molecule. Mol. Immunol. 1994, 31:169–217. Saphire, E.O., Parren, P.W., Pantophlet, R., Zwick, M.B., Morris, G.M., Rudd, P.M., Dwek, R.A., Stanfield, R.L., Burton, D.R., and Wilson, I.A.: Crystal structure of a neutralizing human IgG against HIV-1: a template for vaccine design. Science 2001, 293:1155–1159. Stanfield, R.L., and Wilson, I.A.: Protein–peptide interactions. Curr. Opin. Struct. Biol. 1995, 5:103–113. Tanner, J.J., Komissarov, A.A., and Deutscher, S.L.: Crystal structure of an antigen-binding fragment bound to single-stranded DNA. J. Mol. Biol. 2001, 314:807–822. Wilson, I.A., and Stanfield, R.L.: Antibody–antigen interactions: new structures and new conformational changes. Curr. Opin. Struct. Biol. 1994, 4:857–867. 4-9 Antibody interaction with intact antigens is influenced by steric constraints.


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Immunology_Janeway. 4-9 Antibody interaction with intact antigens is influenced by steric constraints. Braden, B.C., Goldman, E.R., Mariuzza, R.A., and Poljak, R.J.: Anatomy of an antibody molecule: structure, kinetics, thermodynamics and mutational studies of the antilysozyme antibody D1.3. Immunol. Rev. 1998, 163:45–57. Braden, B.C., and Poljak, R.J.: Structural features of the reactions between antibodies and protein antigens. FASEB J. 1995, 9:9–16. Diamond, M.S.,Pierson,T.C., and Fremont, D.H.: The structural immunology of antibody protection against West Nile virus. Immunol Rev. 2008, 225:212–225. Lok, S.M., Kostyuchenko, V., Nybakken, G.E., Holdaway, H.A., Battisti, A.J., Sukupolvi-Petty, S., Sedlak, D., Fremont, D.H., Chipman, P.R., Roehrig, J.T., et al.: Binding of a neutralizing antibody to dengue virus alters the arrangement of surface glycoproteins. Nat. Struct. Mol. Biol. 2008, 15:312–317.


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Immunology_Janeway. Ros, R., Schwesinger, F., Anselmetti, D., Kubon, M., Schäfer, R., Plückthun, A., and Tiefenauer, L.: Antigen binding forces of individually addressed single-chain Fv antibody molecules. Proc. Natl Acad. Sci. USA 1998, 95:7402–7405. 4-10 Some species generate antibodies with alternative structures. Hamers-Casterman, C., Atarhouch, T., Muyldermans, S., Robinson, G., Hamers, C., Songa, E.B., Bendahman, N., and Hamers, R.: Naturally occurring antibodies devoid of light chains. Nature 1993, 363:446–448. Muyldermans, S.: Nanobodies: natural single-domain antibodies. Annu. Rev. Biochem. 2013, 82:775–797. Nguyen, V.K., Desmyter, A., and Muyldermans, S.: Functional heavy-chain antibodies in Camelidae. Adv. Immunol. 2001, 79:261–296. 4-11 The TCRα:β heterodimer is very similar to a Fab fragment of immunoglobulin. Al-Lazikani, B., Lesk, A.M., and Chothia, C.: Canonical structures for the hypervariable regions of T cell αβreceptors. J. Mol. Biol. 2000, 295:979–995.


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Immunology_Janeway. Al-Lazikani, B., Lesk, A.M., and Chothia, C.: Canonical structures for the hypervariable regions of T cell αβreceptors. J. Mol. Biol. 2000, 295:979–995. Kjer-Nielsen, L., Clements, C.S., Brooks, A.G., Purcell, A.W., McCluskey, J., and Rossjohn, J.: The 1.5 Å crystal structure of a highly selected antiviral T cell receptor provides evidence for a structural basis of immunodominance. Structure (Camb.) 2002, 10:1521–1532. Machius, M., Cianga, P., Deisenhofer, J., and Ward, E.S.: Crystal structure of a T cell receptor Vα11 (AV11S5) domain: new canonical forms for the first and second complementarity determining regions. J. Mol. Biol. 2001, 310:689–698. 4-12 A T-cell receptor recognizes antigen in the form of a complex of a foreign peptide bound to an MHC molecule. Garcia, K.C., and Adams, E.J.: How the T cell receptor sees antigen—a structural view. Cell 2005, 122:333–336.


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Immunology_Janeway. Garcia, K.C., and Adams, E.J.: How the T cell receptor sees antigen—a structural view. Cell 2005, 122:333–336. Hennecke, J., and Wiley, D.C.: Structure of a complex of the human αβ T cell receptor (TCR) HA1.7, influenza hemagglutinin peptide, and major histocompatibility complex class II molecule, HLA-DR4 (DRA*0101 and DRB1*0401): insight into TCR cross-restriction and alloreactivity. J. Exp. Med. 2002, 195:571–581. Luz, J.G., Huang, M., Garcia, K.C., Rudolph, M.G., Apostolopoulos, V., Teyton, L., and Wilson, I.A.: Structural comparison of allogeneic and syngeneic T cell receptor–peptide–major histocompatibility complex complexes: a buried alloreactive mutation subtly alters peptide presentation substantially increasing Vβinteractions. J. Exp. Med. 2002, 195:1175–1186.


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Immunology_Janeway. Reinherz, E.L., Tan, K., Tang, L., Kern, P., Liu, J., Xiong, Y., Hussey, R.E., Smolyar, A., Hare, B., Zhang, R., et al.: The crystal structure of a T cell receptor in complex with peptide and MHC class II. Science 1999, 286:1913–1921. Rudolph, M.G., Stanfield, R.L., and Wilson, I.A.: How TCRs bind MHCs, peptides, and coreceptors. Annu. Rev. Immunol. 2006, 24:419–466. 4-13 There are two classes of MHC molecules with distinct subunit compositions but similar three-dimensional structures. & 4-14 Peptides are stably bound to MHC molecules, and also serve to stabilize the MHC molecule on the cell surface. Bouvier, M.: Accessory proteins and the assembly of human class I MHC molecules: a molecular and structural perspective. Mol. Immunol. 2003, 39:697–706. Dessen, A., Lawrence, C.M., Cupo, S., Zaller, D.M., and Wiley, D.C.: X-ray crystal structure of HLA-DR4 (DRA*0101, DRB1*0401) complexed with a peptide from human collagen II. Immunity 1997, 7:473–481.


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Immunology_Janeway. Zhu, Y., Rudensky, A.Y., Corper, A.L., Teyton, L., and Wilson, I.A.: Crystal structure of MHC class II I-Ab in complex with a human CLIP peptide: prediction of an I-Ab peptide-binding motif. J. Mol. Biol. 2003, 326:1157–1174. 4-15 MHC class I molecules bind short peptides of 8–10 amino acids by both ends. Bouvier, M., and Wiley, D.C.: Importance of peptide amino and carboxyl termini to the stability of MHC class I molecules. Science 1994, 265:398–402. Govindarajan, K.R., Kangueane, P., Tan, T.W., and Ranganathan, S.: MPID: MHC-Peptide Interaction Database for sequence–structure–function information on peptides binding to MHC molecules. Bioinformatics 2003, 19:309–310. Saveanu, L., Fruci, D., and van Endert, P.: Beyond the proteasome: trimming, degradation and generation of MHC class I ligands by auxiliary proteases. Mol. Immunol. 2002, 39:203–215.


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Immunology_Janeway. Saveanu, L., Fruci, D., and van Endert, P.: Beyond the proteasome: trimming, degradation and generation of MHC class I ligands by auxiliary proteases. Mol. Immunol. 2002, 39:203–215. Weiss, G.A., Collins, E.J., Garboczi, D.N., Wiley, D.C., and Schreiber, S.L.: A tricyclic ring system replaces the variable regions of peptides presented by three alleles of human MHC class I molecules. Chem. Biol. 1995, 2:401–407. 4-16 The length of the peptides bound by MHC class II molecules is not constrained. Conant, S.B., and Swanborg, R.H.: MHC class II peptide flanking residues of exogenous antigens influence recognition by autoreactive T cells. Autoimmun. Rev. 2003, 2:8–12. Guan, P., Doytchinova, I.A., Zygouri, C., and Flower, D.R.: MHCPred: a server for quantitative prediction of peptide–MHC binding. Nucleic Acids Res. 2003, 31:3621–3624.


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Immunology_Janeway. Rudensky, A.Y., Preston-Hurlburt, P., Hong, S.C., Barlow, A., and Janeway, C.A., Jr.: Sequence analysis of peptides bound to MHC class II molecules. Nature 1991, 353:622–627. Sercarz, E.E., and Maverakis, E.: MHC-guided processing: binding of large antigen fragments. Nat. Rev. Immunol. 2003, 3:621–629. Sinnathamby, G., and Eisenlohr, L.C.: Presentation by recycling MHC class II molecules of an influenza hemagglutinin-derived epitope that is revealed in the early endosome by acidification. J. Immunol. 2003, 170:3504–3513. 4-17 The crystal structures of several peptide:MHC:T-cell receptor complexes show a similar orientation of the T-cell receptor over the peptide:MHC complex. Buslepp, J., Wang, H., Biddison, W.E., Appella, E., and Collins, E.J.: A correlation between TCR Vαdocking on MHC and CD8 dependence: implications for T cell selection. Immunity 2003, 19:595–606.


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Immunology_Janeway. Wu, H., Kwong, P.D., and Hendrickson, W.A.: Dimeric association and segmental variability in the structure of human CD4. Nature 1997, 387:527–530. Yin, Y., Wang, X.X., and Mariuzza, R.A.: Crystal structure of a complete ternary complex of T-cell receptor, peptide-MHC, and CD4. Proc. Natl Acad. Sci. USA 2012, 109:5405–5410. Zamoyska, R.: CD4 and CD8: modulators of T cell receptor recognition of antigen and of immune responses? Curr. Opin. Immunol. 1998, 10:82–86. 4-19 The two classes of MHC molecules are expressed differentially on cells. Steimle, V., Siegrist, C.A., Mottet, A., Lisowska-Grospierre, B., and Mach, B.: Regulation of MHC class II expression by interferon-γ mediated by the trans-activator gene CIITA. Science 1994, 265:106–109. 4-20 A distinct subset of T cells bears an alternative receptor made up of γand δchains. Adams, E.J., Chien, Y.H., and Garcia, K.C.: Structure of a γδT cell receptor in complex with the nonclassical MHC T22. Science 2005, 308:227–231.


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Immunology_Janeway. Adams, E.J., Chien, Y.H., and Garcia, K.C.: Structure of a γδT cell receptor in complex with the nonclassical MHC T22. Science 2005, 308:227–231. Allison, T.J., and Garboczi, D.N.: Structure of γδ T cell receptors and their recognition of non-peptide antigens. Mol. Immunol. 2002, 38:1051–1061. Allison, T.J., Winter, C.C., Fournie, J.J., Bonneville, M., and Garboczi, D.N.: Structure of a human γδ T-cell antigen receptor. Nature 2001, 411:820–824. Das, H., Wang, L., Kamath, A., and Bukowski, J.F.: Vγ2Vδ2 T-cell receptor-mediated recognition of aminobisphosphonates. Blood 2001, 98:1616–1618. Luoma, A.M., Castro, C.D., Mayassi, T., Bembinster, L.A., Bai, L., Picard, D., Anderson, B., Scharf, L., Kung, J.E., Sibener, L.V., et al.: Crystal structure of Vδ1 T cell receptor in complex with CD1d-sulfatide shows MHC-like recognition of a self-lipid by human γδT cells. Immunity 2013, 39:1032–1042.


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Immunology_Janeway. Vantourout, P., and Hayday, A.: Six-of-the-best: unique contributions of γδT cells to immunology. Nat. Rev. Immunol. 2013, 13:88–100. Wilson, I.A., and Stanfield, R.L.: Unraveling the mysteries of γδ T cell recognition. Nat. Immunol. 2001, 2:579–581. Wingren, C., Crowley, M.P., Degano, M., Chien, Y., and Wilson, I.A.: Crystal structure of a γδT cell receptor ligand T22: a truncated MHC-like fold. Science 2000, 287:310–314. Wu, J., Groh, V., and Spies, T.: T cell antigen receptor engagement and specificity in the recognition of stress-inducible MHC class I-related chains by human epithelial γδT cells. J. Immunol. 2002, 169:1236–1240. The Generation of Lymphocyte Antigen Receptors


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Immunology_Janeway. A lymphocyte expresses many exact copies of a single antigen receptor that has a unique antigen-binding site (see Section 1-12). The clonal expression of antigen receptors means that each lymphocyte is unique among the billions of lymphocytes that each person possesses. Chapter 4 described the structural features of immunoglobulins and T-cell receptors, the antigen receptors on B cells and T cells, respectively. We saw that the vast repertoire of antigen receptors results from variations in the amino acid sequence at the antigen-binding site, which is composed of the two variable regions from the two chains of the receptor. In immunoglobulins, these are the heavy-chain variable region (VH) and the light-chain variable region (VL), and in T-cell receptors, the Vα and Vβ regions. The immunoglobulin domains of these regions contain three loops that comprise three hypervariable regions, or complementarity-determining regions (CDRs) (see Section 4-6) that determine the receptor’s antigen


Immunology_Janeway

Immunology_Janeway. domains of these regions contain three loops that comprise three hypervariable regions, or complementarity-determining regions (CDRs) (see Section 4-6) that determine the receptor’s antigen binding site and allow for seemingly limitless diversity in specificity.


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Immunology_Janeway. In the 1960s and 1970s, immunologists recognized that the limited size of the genome (at roughly 3 billion nucleotides) meant that the genome could not directly encode a sufficient number of genes to account for the observed diversity of antigen receptors. For example, encoding each distinct antibody by its own gene could easily fill the genome with nothing but antibody genes. As we will see, variable regions of the receptor chains are not directly encoded as a complete immunoglobulin domain by a single DNA segment. Instead, the variable regions are initially specified by so-called gene segments that encode only a part of the immunoglobulin domain. During the development of each lymphocyte, these gene segments are rearranged by a process of somatic DNA recombination to form a complete and unique variable-region coding sequence. This process is known generally as gene rearrangement. A fully assembled variable region sequence is produced by combining two or three types of gene segments,


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Immunology_Janeway. unique variable-region coding sequence. This process is known generally as gene rearrangement. A fully assembled variable region sequence is produced by combining two or three types of gene segments, each of which is present in multiple copies in the germline genome. The final diversity of the receptor repertoire is the result of assembling complete antigen receptors from the many different gene segments of each type during the development of each individual lymphocyte. This process gives each new lymphocyte only one of many possible combinations of antigen receptors, providing the repertoire of diverse antigen specificities of naive B cells and T cells.


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Immunology_Janeway. The first and second parts of this chapter describe the gene rearrangements that generate the primary repertoire of immunoglobulins and T-cell receptors. The mechanism of gene rearrangement is common to both B cells and T cells, and its evolution was probably critical to the evolution of the vertebrate adaptive immune system. The third part of the chapter explains how the transition from production of transmembrane immunoglobulins by activated B cells results in the production of secreted antibodies by plasma cells. Immunoglobulins can be synthesized as either transmembrane receptors or secreted antibodies, unlike T-cell receptors, which exist only as trans-membrane receptors. Antibodies can also be produced with different types of constant regions, or isotypes (see Section 4-1). Here, we describe how the Primary immunoglobulin gene rearrangement. T-cell receptor gene rearrangement. Structural variation in immunoglobulin constant regions.


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Immunology_Janeway. Primary immunoglobulin gene rearrangement. T-cell receptor gene rearrangement. Structural variation in immunoglobulin constant regions. Evolution of the adaptive immune response. Fig. 5.1 Three hypervariable regions are encoded within a single V-region exon. Panel a: the variable region is based on the immunoglobulin (Ig) fold that is supported by framework regions (yellow) composed of nine β sheets and contains three hypervariable (HV) regions (red) that determine its antigen specificity. Panel b: the three HV regions exist as loops of amino acids between the β sheets of B and C, between Cʹ and Cʹʹ, and between F and G. Panel c: a complete variable region in a lymphocyte is encoded within a single exon of the full antigen-receptor gene. The three HV regions are interspersed between four framework regions (FRs) made up of the β sheets of the Ig domain.


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Immunology_Janeway. expression of the isotypes IgM and IgD is regulated, but we postpone describing how isotype switching occurs until Chapter 10, since that process and the affinity maturation of antibodies occurs normally in the context of an immune response. The last part of this chapter briefly examines alternative evolutionary forms of gene rearrangements that give rise to different forms of adaptive immunity in other species. Primary immunoglobulin gene rearrangement.


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Immunology_Janeway. Primary immunoglobulin gene rearrangement. Virtually any substance can be the target of an antibody response, and the response to even a single epitope comprises many different antibody molecules, each with a subtly different specificity for the epitope and a unique affinity, or binding strength. The total number of antibody specificities available to an individual is known as the antibody repertoire or immunoglobulin repertoire, and in humans is at least 1011 and probably several orders of magnitude greater. The number of antibody specificities present at any one time is, however, limited by the total number of B cells in an individual, as well as by each individual’s previous encounters with antigens.


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Immunology_Janeway. Before it was possible to examine the immunoglobulin genes directly, there were two main hypotheses for the origin of this diversity. The germline theory held that there is a separate gene for each different immunoglobulin chain and that the antibody repertoire is largely inherited. In contrast, somatic diversification theories proposed that the observed repertoire is generated from a limited number of inherited V-region sequences that undergo alteration within B cells during the individual’s lifetime. Cloning of the immunoglobulin genes revealed that elements of both theories were correct and that the DNA sequence encoding each variable region is generated by rearrangements of a relatively small group of inherited gene segments. Diversity is further enhanced by the process of somatic hypermutation in mature activated B cells. Thus, the somatic diversification theory was essentially correct, although the germline theory concept of the existence of multiple germline genes also proved


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Immunology_Janeway. in mature activated B cells. Thus, the somatic diversification theory was essentially correct, although the germline theory concept of the existence of multiple germline genes also proved true.


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Immunology_Janeway. 5-1 Immunoglobulin genes are rearranged in the progenitors of antibody-producing cells.


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Immunology_Janeway. Figure 5.1 shows the relationships between a light-chain variable region’s antigen-binding site, its domain structure, and the gene that encodes it. The variable regions of immunoglobulin heavy and light chains are based on the immunoglobulin fold, which is composed of nine β sheets. The anti-body-binding site is formed by three loops of amino acids known as hyper-variable regions HV1, HV2, and HV3, or also CDR1, CDR2, and CDR3 (see Fig. 5.1a). These loops are located between the pairs of β sheets B and C, Cʹ and Cʹʹ, and F and G (see Fig. 5.1b). In a mature B cell, the variable regions for heavy and light chains are encoded by a single exon, but are separated from one another within this coding sequence (see Fig. 5.1c). This exon is the gene’s second exon (exon 2). The first exon of the variable regions encodes the antibody’s leader sequence, which directs the antibody into the endoplasmic reticulum for surface expression or secretion.


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Immunology_Janeway. Unlike most genes, the complete DNA sequence of the variable-region exon is not present in the germline of the individual, but is originally encoded by two separate DNA segments, as illustrated in Fig. 5.2. These two DNA segments are spliced together to form the complete exon 2 as the B cell develops in the bone marrow. The first 95–101 amino acids of the variable region, encoding βsheets A–F and the first two complete hypervariable regions, originate from a


Immunology_Janeway

Immunology_Janeway. Fig. 5.2 The CDR3 region originates from two or more individual gene segments that are joined during lymphocyte development. Panel a: a complete light-chain variable region encoding the CDR1, CDR2, and CDR3 loops resides in a single exon. Panel b: the complete variable region is derived from distinct germline DNA sequences. A V gene segment encodes the CDR1 and CDR2 loops, and the CDR3 loop is formed by sequences from the end of the V gene segment and the beginning of the J gene segment, and by nucleotides added or lost when these gene segments are joined during lymphocyte development. The exon for the CDR3 loop of the heavy chain is formed by the joining of sequences from V, D, and J gene segments (not shown).


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Immunology_Janeway. variable or V gene segment (see Fig. 5.2). This segment also contributes part of the third hypervariable region. Other parts of the third hypervariable region, and the remainder of the variable region including β sheet G (up to 13 amino acids), originate from a joining or J gene segment. By convention, we will refer to the exon encoding the complete variable region formed by the splicing together of these gene segments as the V-region gene.


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Immunology_Janeway. In nonlymphoid cells, the V-region gene segments remain in their original germline configuration, and are a considerable distance away from the sequence encoding the C region. In mature B lymphocytes, however, the assembled V-region sequence lies much closer to the C region, as a consequence of a splicing event of the gene’s DNA. Rearrangement within the immunoglobulin genes was originally discovered almost 40 years ago, when the techniques of restriction enzyme analysis first made it possible to study the organization of the immunoglobulin genes in both B cells and nonlymphoid cells. Such experiments showed that segments of genomic DNA within the immunoglobulin genes are rearranged in cells of the B-lymphocyte lineage, but not in other cells. This process of rearrangement is known as ‘somatic’ DNA recombination to distinguish it from the meiotic recombination that takes place during the production of gametes.


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Immunology_Janeway. 5-2 Complete genes that encode a variable region are generated by the somatic recombination of separate gene segments. The rearrangements that produce the complete immunoglobulin light-chain and heavy-chain genes are shown in Fig. 5.3. For the light chain, the joining of a VL and a JL gene segment creates an exon that encodes the whole light-chain VL region. In the unrearranged DNA, the VL gene segments are located relatively far away from the exons encoding the constant region of the light chain (CL region). The JL gene segments are located close to the CL region, however, and the joining of a VL gene segment to a JL gene segment also brings the VL gene segment close to a CL-region sequence. The JL gene segment of the rearranged VL region is separated from a CL-region sequence only by a short intron. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription.


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Immunology_Janeway. For the heavy-chain, there is one additional complication. The heavy-chain V region (VH) is encoded in three gene segments, rather than two. In addition to the V and J gene segments (denoted VH and JH to distinguish them from the light-chain VL and JL), the heavy chain uses a third gene segment called the diversity or DH gene segment, which lies between the VH and JH gene segments. The recombination process that generates a complete heavy-chain V region is shown in Fig. 5.3 (right panel), and occurs in two separate stages. First, a DH gene segment is joined to a JH gene segment; then a VH gene segment rearranges to DJH to make a complete VH-region exon. As with the light-chain genes, RNA splicing joins the assembled V-region sequence to the neighboring C-region gene.


Immunology_Janeway

Immunology_Janeway. Fig. 5.3 V-region genes are constructed from gene segments. Heavy-chain V regions are constructed from three gene segments Light-chain V-region genes are constructed from two segments (right panel). First, the diversity (D) and J gene segments join, and (center panel). A variable (V) and a joining (J) gene segment in the then the V gene segment joins to the combined DJ sequence, genomic DNA are joined to form a complete light-chain V-region forming a complete VH exon. A heavy-chain C-region gene is exon. Immunoglobulin chains are extracellular proteins, and the encoded by several exons. The C-region exons, together with the V gene segment is preceded by an exon encoding a leader peptide leader sequence, are spliced to the V-domain sequence during (L), which directs the protein into the cell’s secretory pathways processing of the heavy-chain RNA transcript. The leader sequence and is then cleaved. The light-chain C region is encoded in a is removed after translation, and the disulfide


Immunology_Janeway

Immunology_Janeway. secretory pathways processing of the heavy-chain RNA transcript. The leader sequence and is then cleaved. The light-chain C region is encoded in a is removed after translation, and the disulfide bonds that link the separate exon and is joined to the V-region exon by splicing of polypeptide chains are formed. The hinge region is shown in purple. the light-chain RNA to remove the L-to-V and the J-to-C introns.


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Immunology_Janeway. 5-3 Multiple contiguous V gene segments are present at each immunoglobulin locus.


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Immunology_Janeway. For simplicity we have discussed the formation of a complete V-region sequence as though there were only a single copy of each gene segment. In fact, there are multiple copies of the V, D, and J gene segments in germline DNA. It is the random selection of just one gene segment of each type that produces the great diversity of V regions among immunoglobulins. The numbers of functional gene segments of each type in the human genome, as determined by gene cloning and sequencing, are shown in Fig. 5.4. Not all the gene segments discovered are functional, as some have accumulated mutations that prevent them from encoding a functional protein. Such genes are termed ‘pseudogenes.’ Because there are many V, D, and J gene segments in germline DNA, no single gene segment is essential, resulting in a relatively large number of pseudogenes. Since some of these can undergo rearrangement just like a functional gene segment, a significant proportion of rearrangements incorporate a pseudogene and


Immunology_Janeway

Immunology_Janeway. in a relatively large number of pseudogenes. Since some of these can undergo rearrangement just like a functional gene segment, a significant proportion of rearrangements incorporate a pseudogene and will thus be nonfunctional.


Immunology_Janeway

Immunology_Janeway. We saw in Section 4-1 that there are three sets of immunoglobulin chains— the heavy chain, and two equivalent types of light chains, the κ and λ chains. The immunoglobulin gene segments that encode these chains are organized into three clusters or genetic loci—the κ, λ, and heavy-chain loci—each of which can assemble a complete V-region sequence. Each locus is on a different chromosome and is organized slightly differently, as shown for the human loci in Fig. 5.5. At the λ light-chain locus, located on human chromosome 22, a cluster of Vλ gene segments is followed by four (or in some individuals five) sets of Jλ gene segments each linked to a single Cλ gene. In the κ light-chain locus, on chromosome 2, the cluster of Vκ gene segments is followed by a cluster of Jκ gene segments, and then by a single Cκ gene.


Immunology_Janeway

Immunology_Janeway. The organization of the heavy-chain locus, on chromosome 14, contains separate clusters of VH, DH, and JH gene segments and of CH genes. The heavy-chain locus differs in one important way: instead of a single C region, it contains a series of C regions arrayed one after the other, each of which corresponds to a different immunoglobulin isotype (see Fig. 5.19). While the Cλ locus contains several distinct C regions, these encode similar proteins, which function similarly, whereas the different heavy-chain isotypes are structurally quite distinct and have different functions.


Immunology_Janeway

Immunology_Janeway. B cells initially express the heavy-chain isotypes μ and δ (see Section 4-1), which is accomplished by alternative mRNA splicing and which leads to the expression of immunoglobulins IgM and IgD, as we shall see in Section 5-14. The expression of other isotypes, such as γ (giving IgG), occurs through DNA rearrangements referred to as class switching, and takes place at a later stage, after a B cell is activated by antigen in an immune response. We describe class switching in Chapter 10. The human V gene segments can be grouped into families in which each member shares at least 80% DNA sequence identity with all others in the


Immunology_Janeway

Immunology_Janeway. The human V gene segments can be grouped into families in which each member shares at least 80% DNA sequence identity with all others in the Fig. 5.4 The number of functional gene segments for the V regions of human heavy and light chains. The numbers shown are derived from exhaustive cloning and sequencing of DNA from one individual and exclude all pseudogenes (mutated and nonfunctional versions of a gene sequence). As a result of genetic polymorphism, the numbers will not be the same for all people.


Immunology_Janeway

Immunology_Janeway. Fig. 5.5 The germline organization of the immunoglobulin heavyand light-chain loci in the human genome. Depending on the individual, the genetic locus for the λ light chain (chromosome 22) has between 29 and 33 functional Vλ gene segments and four or five pairs of functional Jλ gene segments and Cλ genes. The κ locus (chromosome 2) is organized in a similar way, with about 38 functional Vκ gene segments accompanied by a cluster of five Jκ gene segments but with a single Cκ gene. In approximately 50% of individuals, the entire cluster of Vκ gene segments has undergone an increase by duplication (not shown, for simplicity). The heavy-chain locus (chromosome 14) has about 40 functional VH gene segments and a cluster of around 23 DH segments lying between these VH gene segments and 6 JH gene segments. The heavy-chain locus also contains a large cluster of CH genes (see Fig. 5.19). For simplicity, all V gene segments have been shown in the same chromosomal orientation; only the first


Immunology_Janeway

Immunology_Janeway. gene segments. The heavy-chain locus also contains a large cluster of CH genes (see Fig. 5.19). For simplicity, all V gene segments have been shown in the same chromosomal orientation; only the first CH gene (for Cμ) is shown, without illustrating its separate exons; and all pseudogenes have been omitted. This diagram is not to scale: the total length of the heavy-chain locus is more than 2 megabases (2 million bases), whereas some of the D gene segments are only 6 bases long.


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Immunology_Janeway. Fig. 5.6 Recombination signal sequences are conserved heptamer and nonamer sequences that flank the gene segments encoding the V, D, and J regions of immunoglobulins.


Immunology_Janeway

Immunology_Janeway. Recombination signal sequences (RSSs) are composed of heptamer (CACAGTG) and nonamer (ACAAAAACC) sequences that are separated by either 12 bp or approximately 23 bp of nucleotides. The heptamer–12-bp spacer–nonamer motif is depicted here as an orange arrowhead; the motif that includes the 23-bp spacer is depicted as a purple arrowhead. Joining of gene segments almost always involves a 12-bp and a 23-bp RSS—the 12/23 rule. The arrangement of RSSs in the V (red), D (green), and J (yellow) gene segments of heavy (H) and light (λ and κ) chains of immunoglobulin is shown here. The RAG-1 recombinase (see Section 5-5) cuts the DNA precisely between the last nucleotide of the V gene segment and the first C of the heptamer; or between the last G of the heptamer and the first nucleotide of the D or J gene segment. Note that according to the 12/23 rule, the arrangement of RSSs in the immunoglobulin heavy-chain gene segments precludes direct V-to-J joining.


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Immunology_Janeway. family. Both the heavy-chain and the κ-chain V gene segments can be subdivided into seven families, and there are eight families of Vλ gene segments. The families can be grouped into clans, made up of families that are more similar to each other than to families in other clans. Human VH gene segments fall into three clans. All the VH gene segments identified from amphibians, reptiles, and mammals also fall into the same three clans, suggesting that these clans existed in a common ancestor of these modern animal groups. Thus, the V gene segments that we see today have arisen by a series of gene duplications and diversification through evolutionary time. 5-4 Rearrangement of V, D, and J gene segments is guided by flanking DNA sequences.


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Immunology_Janeway. For a complete immunoglobulin or T-cell receptor chain to be expressed, DNA rearrangements must take place at the correct locations relative to the V, D, or J gene segment coding regions. In addition, these DNA rearrangements must be regulated such that a V gene segment is joined to a D or a J and not joined to another V gene segment. DNA rearrangements are guided by conserved noncoding DNA sequences, called recombination signal sequences (RSSs), that are found adjacent to the points at which recombination takes place. The structure and arrangements of the RSSs are shown in Fig. 5.6 for the λ and κ light-chain loci and the heavy-chain loci. An RSS consists of a conserved block of seven nucleotides—the heptamer 5ʹCACAGTG3ʹ, which is always contiguous with the coding sequence; followed by a nonconserved region known as the spacer, which is either 12 or 23 base pairs (bp) long; followed by a second conserved block of nine nucleotides, the nonamer 5ʹACAAAAACC3ʹ.


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Immunology_Janeway. The sequences given here are the consensus sequences, but they can vary substantially from one gene segment to another, even in the same individual, as there is some flexibility in the recognition of these sequences by the enzymes that carry out the recombination. The spacers vary in sequence, but their conserved lengths correspond to one turn (12 bp) or two turns (23 bp) of the DNA double helix. This is thought to bring the heptamer and nonamer sequences to the same side of the DNA helix to allow interactions with proteins that catalyze recombination, but this concept still lacks structural proof. The heptamer– spacer–nonamer sequence motif—the RSS—is always found directly adjacent to the coding sequence of V, D, or J gene segments. Recombination normally occurs between gene segments located on the same chromosome. A gene segment flanked by an RSS with a 12-bp spacer typically can be joined only to one flanked by a 23-bp spacer RSS. This is known as the 12/23 rule.


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Immunology_Janeway. It is important to recognize that the pattern of 12and 23-bp spacers used by the various gene segments is different between the λ, κ, and heavy-chain loci (see Fig. 5.6). Thus, for the heavy chain, a DH gene segment can be joined to a JH gene segment and a VH gene segment to a DH gene segment, but VH gene segments cannot be joined to JH gene segments directly, as both VH and JH gene segments are flanked by 23-bp spacers. However, they can be joined with a DH gene segment between them, as DH segments have 12-bp spacers on both sides (see Fig. 5.6).


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Immunology_Janeway. In the antigen-binding region of an immunoglobulin, CDR1 and CDR2 are encoded directly in the V gene segment (see Fig. 5.2). CDR3 is encoded by the additional DNA sequence that is created by the joining of the V and J gene segments for the light chain, and the V, D, and J gene segments for the heavy chain. Further diversity in the antibody repertoire can be supplied by CDR3 regions that result from the joining of one D gene segment to another D gene segment, before being joined by a J gene segment. Such D–D joining is infrequent and seems to violate the 12/23 rule, suggesting that such violations of the 12/23 rule can occur at low frequency. In humans, D–D joining is found in approximately 5% of antibodies and is the major mechanism accounting for the unusually long CDR3 loops found in some heavy chains.


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Immunology_Janeway. The mechanism of DNA rearrangement is similar for the heavyand light-chain loci, although only one joining event is needed to generate a light-chain gene but two are required for a heavy-chain gene. When two gene segments are in the same transcriptional orientation in the germline DNA, their rearrangement involves the looping out and deletion of the DNA between them (Fig. 5.7, left panels). By contrast, when the gene segments have opposite transcriptional orientations, the rearrangement retains the intervening DNA in the chromosome but with an inverted orientation (see Fig. 5.7, right panels). This mode of recombination is less common, but it accounts for about half of all Vκ to Jκ joins in humans because the orientation of half the Vκ gene segments is opposite to that of the Jκ gene segments. 5-5 The reaction that recombines V, D, and J gene segments modifying enzymes.


Immunology_Janeway

Immunology_Janeway. The overall enzymatic mechanisms involved in V-region rearrangement, or V(D)J recombination, are illustrated in Fig. 5.8. Two RSSs are brought together by interactions between proteins that specifically recognize the length of the spacers and thus enforce the 12/23 rule for recombination. The DNA molecule is then precisely cleaved by endonuclease activity at two locations and is then rejoined in a different configuration. The ends of the heptamer sequences are joined in a head-to-head fashion to form a signal joint. In the majority of cases, no nucleotides are lost or added between the two heptamer sequences, creating a double-heptamer sequence 5ʹCACAGTGCACAGTG3ʹ within the DNA molecule. When the joining segments are in the same orientation, the signal joint is contained in a circular piece of extrachromosomal DNA (see Fig. 5.7, left panels), which is lost from the genome when the cell divides. The V and J gene segments, which remain on the chromosome, join to form what is called the


Immunology_Janeway

Immunology_Janeway. of extrachromosomal DNA (see Fig. 5.7, left panels), which is lost from the genome when the cell divides. The V and J gene segments, which remain on the chromosome, join to form what is called the coding joint. When the joining segments are in the opposite relative orientation to each other within the chromosome (see Fig. 5.7, right panels), the signal joint is also retained within the chromosome, and the region of DNA between the V gene segment and the RSS of the J gene segment is inverted to form the coding joint. This situation leads to rearrangement by inversion. As we shall see later, the coding joint junction is imprecise, meaning that nucleotides can be added or lost between joined segments during the rearrangement process. This imprecise nature of coding joint formation adds to the variability in the V-region sequence, called junctional diversity.


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MOVIE 5.1

Immunology_Janeway. MOVIE 5.1


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Immunology_Janeway. Fig. 5.7 V-region gene segments are joined by recombination. Top panel: in every V-region recombination event, the recombination signal sequences (RSSs) flanking the gene segments are brought together to allow recombination to take place. The 12-bp-spaced RSSs are shown in orange, the 23-bp-spaced RSSs in purple. For simplicity, the recombination of a light-chain gene is illustrated; for a heavy-chain gene, two separate recombination events are required to generate a functional V region. Left panels: in most cases, the two segments undergoing rearrangement (the V and J gene segments in this example) are arranged in the same transcriptional orientation in the chromosome, and juxtaposition of the RSSs results in the looping out of the intervening DNA. Recombination occurs at the ends of the heptamer sequences in the RSSs, creating the so-called signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates


Immunology_Janeway

Immunology_Janeway. heptamer sequences in the RSSs, creating the so-called signal joint and releasing the intervening DNA in the form of a closed circle. Subsequently, the joining of the V and J gene segments creates the coding joint in the chromosomal DNA. Right panels: in other cases, the V and J gene segments are initially oriented in opposite transcriptional directions. In this case, alignment of the RSSs requires the coiled configuration shown, rather than a simple loop, so that joining the ends of the two heptamer sequences now results in the inversion and integration of the intervening DNA into a new position on the chromosome. Again, the joining of the V and J segments creates a functional V-region exon.


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Immunology_Janeway. direction of transcription L2 V2 Vn Ln L2 V2 L1 V1 JL1 V1 direction of transcription JLn Vn deleted inverted signal joint coding joint After recombination this loop is excised from the chromosome, taking the two RSS regions with it After recombination the coiled region is retained in the chromosome in an inverted orientation L1 V1 L2 L2 V2 V2 23 23 23 12 L2 V2 Vn Vn Ln Ln J J L1 V1 J Ln Vn L1 V1 V gene segments may be in either forward or reverse transcriptional orientation relative to downstream gene segments When a forward-oriented V gene segment recombines with a downstream gene segment, alignment of the two RSS regions loops out the intervening DNA When a reverse-oriented V gene segment recombines with a downstream gene segment, alignment of the RSS regions forms the intervening DNA into a coiled conÿguration


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Immunology_Janeway. The complex of enzymes that act in concert to carry out somatic V(D)J recombination is termed the V(D)J recombinase. The lymphoid-specific components of the recombinase are called RAG-1 and RAG-2, and they are encoded by two recombination-activating genes, RAG1 and RAG2. This pair of genes is essential for V(D)J recombination, and they are expressed in developing lymphocytes only while the lymphocytes are engaged in assembling their antigen receptors, as described in more detail in Chapter 8. Indeed, the RAG genes expressed together can confer on nonlymphoid cells such as fibroblasts the capacity to rearrange exogenous segments of DNA containing the appropriate RSSs; this is how RAG-1 and RAG-2 were initially discovered.


Immunology_Janeway

Immunology_Janeway. DNA ligase IV:XRCC4 ligates DNA ends Ku70:Ku80 binds DNA ends Precise signal joint DNA ligase:XRCC4 Signal joints Ku80 Ku70 5´-phosphorylated blunt ends TdT processes DNA ends Ku70:Ku80 binds DNA ends DNA ligase IV:XRCC4 ligates DNA ends terminal deoxynucleotidyl transferase (TdT) DNA ligase:XRCC4 1223 JV RAG-1:2 binds RSS Synapsis of two RSSs Germline confguration Cleavage of RSSs Coding joints DNA-PK:Artemis opens hairpin RAG-1/2 Ku70 Ku80 Artemis DNA-PK covalently closed DNA hairpin ends Imprecise coding joint Fig. 5.8 Enzymatic steps in RAG-dependent V(D)J rearrangement.


Immunology_Janeway

Immunology_Janeway. Recombination of gene segments containing recombination signal sequences (RSSs, triangles) begins with the binding of a complex of RAG-1 (purple), RAG-2 (blue), and high-mobility-group (HMG) proteins (not shown) to one of the RSSs flanking the coding sequences to be joined (second row). The RAG complex then recruits the other RSS. In the cleavage step, the endonuclease activity of RAG makes single-stranded cuts in the DNA backbone precisely between each coding segment and its RSS. At each cutting point this creates a 3ʹ-OH group, which then reacts with a phosphodiester bond on the opposite DNA strand to generate a hairpin, leaving a blunt double-stranded break at the end of the RSS. These two types of DNA ends are resolved in different ways. At the coding ends (left panels), essential repair proteins such as Ku70:Ku80 (green) bind to the hairpin. Ku70:80 forms a ringlike structure as a heterodimer, but the monomers do not encircle the DNA. The DNA-PK:Artemis complex (purple) then


Immunology_Janeway

Immunology_Janeway. repair proteins such as Ku70:Ku80 (green) bind to the hairpin. Ku70:80 forms a ringlike structure as a heterodimer, but the monomers do not encircle the DNA. The DNA-PK:Artemis complex (purple) then joins the complex, and its endonuclease activity opens the DNA hairpin at a random site, yielding either two flush-ended DNA strands or a single-strand extension. The cut end is then modified by terminal deoxynucleotidyl transferase (TdT, pink) and exonuclease, which randomly add and remove nucleotides, respectively (this step is shown in more detail in Fig. 5.11). The two coding ends are finally ligated by DNA ligase IV in association with XRCC4 (turquoise). At the signal ends (right panels), Ku70:Ku80 binds to the RSS but the ends are not further modified. Instead, a complex of DNA ligase IV:XRCC4 joins the two ends precisely to form the signal joint.


Immunology_Janeway

Immunology_Janeway. The other proteins in the recombinase complex are members of the ubiquitously expressed nonhomologous end joining (NHEJ) pathway of DNA repair known as double-strand break repair (DSBR). In all cells, this process is responsible for rejoining the two ends at the site of a double-strand break in DNA. The DSBR joining process is imprecise, meaning that nucleotides are frequently gained or lost at the site of joining. This has evolutionary relevance as in most cells it would not be advantageous to gain or lose nucleotides when repairing DSBs. However, in lymphocytes, the imprecise nature of DSBR is critical for junctional diversity and adaptive immunity. Thus, this may be the driving pressure for NHEJ to mediate imprecise joining. One ubiquitous protein contributing to DSBR is Ku, which is a heterodimer (Ku70:Ku80); this forms a ring around the DNA and associates tightly with a protein kinase catalytic subunit, DNA-PKcs, to form the DNA-dependent protein kinase (DNA-PK). Another protein


Immunology_Janeway

Immunology_Janeway. heterodimer (Ku70:Ku80); this forms a ring around the DNA and associates tightly with a protein kinase catalytic subunit, DNA-PKcs, to form the DNA-dependent protein kinase (DNA-PK). Another protein that associates with DNA-PKcs is Artemis, which has nuclease activity. The DNA ends are finally joined together by the enzyme DNA ligase IV, which forms a complex with the DNA repair protein XRCC4. DNA polymerases μ and λ participate in DNA-end fill-in synthesis. In addition, polymerase μ can add nucleotides in a template-independent manner. In summary, lymphocytes have adapted several enzymes used in common DNA repair pathways to help complete the process of somatic V(D)J recombination that is initiated by the RAG-1 and RAG-2 V(D)J recombinases.


Immunology_Janeway

Immunology_Janeway. The first reaction is an endonucleolytic cleavage that requires the coordinated activity of both RAG proteins. Initially, a complex of RAG-1 and RAG-2 proteins, together with high-mobility group chromatin protein HMGB1 or HMGB2, recognizes and aligns the two RSSs that are the target of the cleavage reaction. RAG-1 operates as a dimer, with RAG-2 acting as a cofactor (Fig. 5.9). RAG-1 specifically recognizes and binds the heptamer and the nonamer of the RSS and contains the Zn2+-dependent endonuclease activity of the RAG protein complex. As a dimer, RAG-1 seems to align the two RSSs that will undergo rearrangement. Recent models suggest that the 12/23 rule may be established because an essential asymmetric orientation of the RAG-1:RAG-2 complex induces a preference for binding to RSS elements of different types (Fig. 5.10). The bound RAG complex makes a single-strand DNA break at the nucleotide just 5ʹ of the heptamer of the RSS, thus creating a free 3ʹ-OH group at the end of the