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Monday, May 3, 2021

The T‐Cell Surface Receptor For Antigen (TCR)

The T‐Cell Surface Receptor For Antigen (TCR)
As alluded to earlier, T‐cells interact with antigen in a manner that is quite distinct from the way in which B‐cells do; the receptors that most T‐cells are equipped with cannot directly engage soluble antigens but instead “see” fragments of antigen that are immobilized within a narrow groove on the surface of MHC molecules (Figure 4.1b). 

As we shall discuss in detail in Chapter 5, MHC molecules bind to short 8–20 amino acid long peptide fragments that represent “quality control” samples of the proteins a cell is expressing at any given time, or what it has internalized through phagocytosis, depending on the type of MHC molecule. In this way, T‐cells can effectively inspect what is going on, antigenically speaking, within a cell at any given moment by surveying the range of peptides being presented within MHC molecules. Another major difference between B‐ and T‐cell receptors is that T‐cells cannot secrete their receptor molecules in the way that B‐cells can switch production of Ig from a membrane‐bound form to a secreted form. These differences aside, T‐cell receptors are structurally quite similar to antibody as they are also built from modules that are based upon the immunoglobulin fold.
Before we explore the structural aspects of T‐cell receptors, please keep in mind that the practical function of these recep­ tors is to enable a T‐cell to probe the surfaces of cells looking for nonself peptides. If a T‐cell finds a peptide–MHC combination that is a good match for its TCR it will become acti­ vated, undergo clonal expansion, and differentiate to a mature effector T‐cell capable of joining the fight against the infectious agent generating these nonself peptides. In practice, such an eventuality is a very low probability event because, as we shall see, TCRs are generated in such a way as to produce an enor­ mous variety of these receptors, each with their own exquisite specificity for a particular peptide–MHC combination. Moreover, because the majority of peptides presented on MHC molecules at any one time will be derived from self (unless the antigen‐presenting cell is infected with a microorganism), this further reduces the probability of a T‐cell encountering a per­ fect nonself peptide–MHC combination to trigger a response.

Milestone 4.1 The T‐cell receptor
As T‐lymphocytes respond by activation and proliferation when they contact antigen presented by cells such as macrophages, it seemed reasonable to postulate that they do so by receptors on their surface. In any case, it would be difficult to fit T‐cells into the clonal selection club if they lacked such receptors. Guided by Occam’s razor (the law of parsimony, which contends that it is the aim of science to present the facts of nature in the simplest and most economical conceptual formulations), most investigators plumped for the hypothesis that nature would not indulge in the extravagance of evolving two utterly separate molecular recognition species for B‐ and T‐cells, and many fruitless years were spent looking for the “Holy Grail” of the T‐cell receptor with anti‐immunoglobulin serums or monoclonal antibodies. Success only came when a monoclonal antibody directed to the idiotype of a T‐cell was used to block the response to antigen. This was identified by its ability to block one individual T‐cell clone out of a large number, and it was correctly assumed that the structure permitting this selectivity would be the combining site for antigen on the T‐cell receptor. Immunoprecipitation with this antibody brought down a disulfide‐linked heterodimer composed of 40–44 kDa subunits (Figure M4.1.1).
Figure M4.1.1 Antibody (Ab) to T‐cell receptor (anti‐idiotype) blocks antigen (Ag) recognition. (Adapted from Haskins K. et al. (1983) Journal of Experimental Medicine 157, 1149; simplified a little.)

Isolation of T‐cell receptor genes
Figure M4.1.2 Isolation of T‐cell receptor genes. DNA fragments of differing sizes, produced by a restriction enzyme, are separated by electrophoresis and probed with the T‐cell gene. The T‐cells show rearrangement of one of the two germline genes found in liver or B‐cells. (Source: Hendrick S.M. et al. (1984) Nature 308, 149. Reproduced with permission of Nature Publishing Group.)

The other approach went directly for the genes, arguing as follows. The T‐cell receptor should be an integral membrane protein not present in B‐cells. Hence, T‐cell polysomal mRNA from the endoplasmic reticulum, which should provide an abundant source of the appropriate transcript, was used to prepare cDNA from which genes common to B‐ and T‐cells were subtracted by hybridization to B‐cell mRNA. The resulting T‐specific clones were used to probe for a T‐cell gene that is rearranged in all functionally mature T‐cells but is in its germline configuration in all other cell types (Figure M4.1.2). In such a way were the genes encoding the β‐subunit of the T‐cell receptor uncovered.

The receptor for antigen is a transmembrane heterodimer
Identification of the TCR proved more difficult than initially anticipated (Milestone 4.1), but eventually the receptor was found to be a membrane‐bound molecule composed of two disulfide‐linked chains, α and β. Each chain folds into two Ig‐ like domains, one having a relatively invariant structure and the other exhibiting a high degree of variability, so that the αβ TCR has a structure really quite closely resembling an Ig Fab fragment. This analogy stretches even further – each of the two variable regions has three hypervariable regions (or complementarity determining regions, CDRs) that X‐ray diffraction data have defined as incorporating the amino acids that make contact with the peptide–MHC ligand. Plasticity of the CDR loops is an important factor, enabling TCRs to mold around structurally diverse peptide MHC combinations.
Although the manner in which the TCR makes contact with peptide–MHC is still not fully understood, it appears that in some TCRs CDRs 1 and 2 of the TCR bear much of the responsibility for making contact with the MHC molecule itself, while CDR3 makes contact with the peptide; however in other TCRs  the  reverse  is  true. Whatever  CDRs bear  the responsibility for contacting MHC versus peptide, it is clear that these are the recognition components of the receptor and so it follows that it is here that much of the variability is seen between TCRs, as we shall discuss later.
Both α and β chains are required for antigen specificity as shown by transfection of the T‐receptor genes from a cytotoxic T‐cell clone specific for fluorescein to another clone of a different specificity; when it expressed the new α and β genes, the transfected clone acquired the ability to lyse the fluoresceinated target cells. Another type of experiment utilized T‐cell hybridomas formed by fusing single antigen‐specific T‐cells with T‐cell tumors to achieve “immortality.” One hybridoma recog­ nizing chicken ovalbumin, presented by a macrophage, gave rise spontaneously to two variants, one of which lost the chromosome encoding the α chain, and the other, the β chain. Neither variant recognized antigen but, when they were physically fused together, each supplied the complem ntary receptor chain, and reactivity with antigen was restored.

CD4 and CD8 molecules act as co‐receptors for TCRs
In addition to the TCR, the majority of peripheral T‐cells also express one or other of the membrane proteins CD4 or CD8 that act as co‐receptors for MHC molecules (Figure 4.7). CD4 is a single‐chain polypeptide containing four Ig‐like domains packed tightly together to form an extended rod that projects from the T‐cell surface. The cytoplasmic tail of the CD4 molecule is important for TCR signaling as this region is constitutively bound by a protein tyrosine kinase, Lck, that initiates the signal transduction cascade that follows upon encounter of a T‐cell with antigen (Figure 4.8). CD8 plays a similar role to CD4, as it also binds Lck and recruits this kinase to the TCR complex, but is structurally quite distinct; CD8 is a disulfidelinked heterodimer of α and β chains, each of which contains a single Ig‐like domain connected to an extended and heavily glycosylated polypeptide projecting from the T‐cell surface (Figure 4.7).
CD4 and CD8 act as co‐receptors for MHC molecules and define functional subsets of T‐cells
Figure 4.7 CD4 and CD8 act as co‐receptors for MHC molecules and define functional subsets of T‐cells. (a) Schematic representation of CD4 and CD8 molecules. CD4 is composed of four Ig‐like domains (D1 to D4, as indicated) and projects from the T‐cell surface to interact with MHC class II molecules. CD8 is a disulfide‐linked heterodimer composed of Ig‐like α and β subunits connected to a heavily glycosylated rod‐like region that extends from the plasma membrane. CD8 interacts with MHC class I molecules. The cytoplasmic tails of CD4 and CD8 are associated with the tyrosine kinase Lck. (b) Ribbon diagram representations of the extracellular portions of CD4 and CD8. The Ig‐like domains (D1 to D4) of CD4 are colored blue, green, yellow, and red, respectively. A CD8 homodimer of two α subunits is shown. (Source: Dr. Dan Leahy.Reproduced with permission.)

The T‐cell receptor (TCR) complex, assisted by CD4 or CD8 receptors, recognizes peptide antigen in the context of MHC molecule
Figure 4.8 The T‐cell receptor (TCR) complex, assisted by CD4 or CD8 receptors, recognizes peptide antigen in the context of MHC molecules. TCR activation signals are propagated via the CD3 co‐receptor complex, which is made up of CD3 γ, ε, δ, and ζ chains. Co‐clustering of CD4 or CD8, which are constitutively associated with the Lck kinase, with the TCR complex facilitates Lck‐initiated signal propagation through phosphorylation of immunoreceptor tyrosine‐based activation motifs (ITAMs) within the CD3 ζ chain.

CD4 and CD8 molecules play important roles in antigen recognition by T‐cells as these molecules dictate whether a T‐ cell can recognize antigen presented by MHC molecules that obtain their peptide antigens primarily from intracellular (MHC class I), or extracellular (MHC class II), sources. This has major functional implications for the T‐cell, as those lymphocytes that become activated upon encounter with antigen presented within MHC class I molecules (CD8+ T‐cells) invariably become cytotoxic T‐cells, and those that are activated by peptides presented by MHC class II molecules (CD4+ T‐cells) become helper T‐cells (see Figure 7.1).

There are two classes of T‐cell receptors
Not long after the breakthrough in identifying the αβ TCR, reports came of the existence of a second type of receptor composed of γ and δ chains. As it appears earlier in thymic ontogeny, the γδ receptor is sometimes referred to as TCR1 and the αβ receptor as TCR2.
The γδ cells make up only 1–5% of the T‐cells that circulate in blood and peripheral organs of most adult animals; however these cells are much more common in epithelial‐rich tissues such as the skin, intestine, reproductive tract, and the lungs where they can comprise almost 50% of the T‐cell population. It cannot be denied that γδ T‐cells are somewhat of an oddity among T‐cells; unlike αβ T‐cells, γδ cells do not appear to require antigen to be presented within the context of MHC molecules and are thought to be able to recognize soluble antigen akin to B‐cells. Perhaps because of this lack of dependence on MHC for antigen presentation, the majority of γδ T‐cells do not express either of the MHC co‐receptors, CD4 or CD8 (Table 4.1).
The mechanism of antigen recognition by γδ T‐cells is still somewhat mysterious but these cells are known to be able to interact with MHC‐related molecules, such as the mouse T10 and T22 proteins, in a manner that does not require antigen. Because the latter MHC‐like molecules are upregulated upon may have an important immunoregulatory function; by becoming activated by molecules that appear on activated T‐ cells, γδ T‐cells may help to regulate immune responses in a positive  or  negative  manner.  γδ T‐cells can  also  recognize pathogen‐derived lipids, organic phosphoesters, nucleotide conjugates, and other nonpeptide ligands.
Certain γδ T‐cells (the Vγ1 Vδ1 subset, which are enriched in epithelial tissues) also share some of the same recognition features of NK cells of the innate immune system, as they can both recognize the MHC class I‐like proteins MICA and MICB, which do not function as antigen‐presenting molecules. Rather, MICA and MICB are typically present at low levels on epithelial tissues but are upregulated in response to cellular stress, including heat shock and DNA damage. Infection with cytomegalovirus or Mycobacterium tuberculosis is also capable of inducing the surface appearance of these primitive MHC‐like molecules and other stress‐inducible γδ T‐cell ligands are almost certain to exist. As we shall see later in this chapter, MICA and MICB are also used by NK cells as activation ligands, although in this case a very different receptor is responsible.
Genes encoding αβ and γδ T‐cell receptors (TCRs)
Figure 4.9 Genes encoding αβ and γδ T‐cell receptors (TCRs). Genes encoding the δ chains lie between the and clusters and some V segments in this region can be used in either δ or α chains (i.e., as either or ). TCR genes rearrange in a manner analogous to that seen with immunoglobulin genes, including N‐region diversity at the V(D)J junctions. One of the genes is found downstream (3′) of the gene and rearranges by an inversional mechanism.
The encoding of TCRs is similar to that of immunoglobulins
The gene segments encoding the TCR β chains follow a broadly similar arrangement of V, D, J, and constant segments to that described for the immunoglobulins (Figure 4.9). In a parallel fashion, as an immunocompetent T‐cell is formed, rearrangement of V, D, and J genes occurs to form a continuous VDJ sequence. The firmest evidence that B‐ and T‐cells use similar recombination mechanisms comes from mice with severe combined immunodeficiency (SCID) that have a single autosomal recessive defect preventing successful recombination of V, D, and J segments. Homozygous mutants fail to develop immuno­ competent B‐ and T‐cells and identical sequence defects in VDJ joint formation are seen in both pre‐B‐ and pre‐T‐cell lines.
Looking first at the β chain cluster, one of the two genes rearranges next to one of the genes. Note that, because of the way the genes are organized, the first gene, Dβ1, can utilize any of the 13 genes, but Dβ2 can only choose from the seven Jβ2 genes (Figure 4.9). Next, one of the 50 or so genes is rear­ ranged to the preformed DβJβ segment. Variability in junction formation and the random insertion of nucleotides to create N‐region diversity either side of the D segment mirror the same phenomenon seen with Ig gene rearrangements. Sequence analysis emphasizes the analogy with the antibody molecule; each V segment contains two hypervariable regions, while the DJ junctional sequence provides the very hypervariable CDR3 structure, making a total of six potential CDRs for antigen bind­ ing in each TCR (Figure 4.10). As in the synthesis of antibody, the intron between VDJ and C is spliced out of the mRNA before translation with the restriction that rearrangements involving genes in the Dβ2Jβ2 cluster can only link to Cβ2.
All the other chains of the TCRs are encoded by genes formed through similar translocations. The α chain gene pool lacks D segments but possesses a prodigious number of J segments. The number of and genes is small in comparison with and Vβ. Like the α chain pool, the β chain cluster has no D segments. The awkward location of the δ locus embedded within the α gene cluster results in T‐cells that have undergone Vα–Jα combination having no δ genes on the rearranged chromosome; in other words, the δ genes are completely excised.
The T‐cell receptor (TCR)/CD3 complex.
Figure 4.10 The T‐cell receptor (TCR)/CD3 complex. The TCR resembles the immunoglobulin Fab antigen‐binding fragment in structure. The variable and constant segments of the TCR α and β chains (VαCα/VβCβ), and of the corresponding γ and δ chains of the γδ TCR, belong structurally to the immunoglobulin‐type domain family. (a) In the model the α chain CDRs are colored magenta (CDR1), purple (CDR2), and yellow (CDR3), whilst the β chain CDRs are cyan (CDR1), navy blue (CDR2) and green (CDR3). The fourth hypervariable region of the β chain (CDR4), which constitutes part of the binding site for some superantigens, is colored orange. (Reproduced from Garcia K. et al. (1998) Science 279, 1166; with permission.) The TCR α and β CDR3 loops encoded by (D)J genes are both short; the TCR γ CDR3 is also short with a narrow length distribution, but the δ loop is long with a broad length distribution, resembling the Ig light and heavy chain CDR3s, respectively. (b) The TCRs may be expressed in pairs linked to the CD3 complex. Negative charges on trans­ membrane segments of the invariant chains of the CD3 complex contact the opposite charges on the TCR Cα and Cβ chains conceivably as depicted. (c) The cytoplasmic domains of the CD3 peptide chains contain immunoreceptor tyrosine‐based activation motifs (ITAMs; see BCR, Figure 4.4) that contact src protein tyrosine kinases. Try not to confuse the TCR γδ and the CD3 γδ chains.
The CD3 complex is an integral part of the T‐cell receptor
The T‐cell antigen recognition complex and its B‐cell counterpart can be likened to army scouts whose job is to let the main battalion know when the enemy has been sighted. When the TCR “sights the enemy” (i.e., ligates antigen), it relays a signal through an associated complex of transmembrane polypeptides (CD3) to the interior of the T‐lymphocyte, instructing it to awaken from its slumbering G0 state and do something useful – like becoming an effector cell. In all immunocompetent T‐cells, the TCR is noncovalently but still intimately linked with CD3 in a complex that, as current wisdom has it, may contain two heterodimeric TCR αβ or γδ recognition units closely apposed to one molecule of the invariant CD3 polypep­ tide chains γ and δ, two molecules of CD3ε, plus the disulfidelinked ζ–ζ dimer. The total complex therefore has the structure TCR2–CD3γδε2–ζ2 (Figure 4.8 and Figure 4.10b).
Similar to the BCR‐associated Ig–α/β heterodimer, the CD3 chains also contain one or more ITAMs and these motifs, once again, are instrumental in the propagation of activation signals into the lymphocyte. Upon encounter of the TCR with peptide–MHC, the ITAMs within the CD3 complex become phosphorylated at tyrosine residues; these then act as a platform for the recruitment of a veritable multitude of phosphotyrosine‐binding proteins that further disseminate the signal throughout the T‐cell. It is here that the role of the CD4 and CD8 co‐receptors becomes apparent; phosphorylation of the ITAMs within the CD3 ζ (zeta) chain is accomplished by the Lck tyrosine kinase that, you may recall, is associated with the cytoplasmic tails of CD4 and CD8 (Figure 4.7 and Figure 4.8). In mice, either or both of the ζ chains can be replaced by a splice variant from the ζ gene termed η. The ζ chain also associates with the FcγRIIIA receptor in natural killer (NK) cells where it functions as part of the signal transduction mechanism in that context also. We shall discuss TCR‐initiated signal transduction in much greater detail in Chapter 7.

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