IMMUNOLOGY - Roitt I. - Mir 2000

Chapter 7. T-Cell Antigen-Recognition Receptors and MHC Molecules

■ The T-Cell antigen-recognition receptor is a heterodimeric (αβ or γδ) glycoprotein that enables T Cells to recognize a diverse array of Antigens. On the T-cell surface, it is associated with a polypeptide complex collectively known as CD3.

■ The Major Histocompatibility Complex (MHC) encodes two sets of highly polymorphic cell-surface Proteins termed Class I and class II MHC molecules. The αβ-TCR recognizes processed antigen in the form of peptide fragments bound to class I or class II MHC molecules. Both the MHC molecule and the antigen fragment make contact with the TCR.

■ The folding of the MHC polypeptide chains creates a cleft that binds peptide fragments of the processed antigen for presentation to T cells. Class I molecules typically bind Peptides of 8-9 amino acid residues, whereas class II molecules accommodate somewhat longer ones.

■ The pockets of the antigen-binding cleft can accommodate peptides of various structures depending on the MHC haplotype. The high polymorphism of MHC molecules, coupled with the ability of each APC to express several different MHC molecules, ensures the presentation of a vast array of diverse antigenic peptides to T cells.

T-CELL RECEPTORS

Specific antigen recognition by T lymphocytes is the central event in triggering and regulating an effective Immune Response. Unraveling The Nature of the T-cell receptors that perform this function is key to resolving many fundamental problems in immunology.

T-cell receptors (TCRs) were identified and isolated in purified form (using appropriate antireceptor Antibodies) as surface structures specific to each individual T-cell clone. Initially, the TCR was designated αβ because it was found to be a heterodimer composed of one α chain and one β chain linked by a disulfide bond. In a parallel line of research, complementary DNA (cDNA) libraries yielded genes putative for encoding TCR chains—each such pair of genes being expressed by cells of only one specific clone. The Amino Acid Sequence deduced from The nucleotide sequence of these genes matched sequencing data from α- and β-chain fragments of TCRs isolated using Monoclonal Antibodies. Thus, two alternative methodological approaches identified the exact same Structure. Subsequently, another variant of the TCR, designated γδ, was also discovered and isolated.

Both the αβ and γδ forms of the T-cell receptor are associated with the CD3 complex

On The Cell surface, both the αβ and γδ forms of the TCR lie in close proximity to the polypeptide complex collectively known as CD3. This juxtaposition is essential for the surface expression of the entire receptor complex. The CD3 Polypeptides share identical Amino acid sequences across all T cells and, consequently, upon associating with the TCR, cannot introduce additional diversity into the receptor complexes. Most importantly, they are believed to mediate the signals generated when the TCR heterodimer recognizes an antigen. The CD3 complex consists of four invariant polypeptides designated γ, δ, ε, and ζ. Alternative Splicing can yield the expression of the η polypeptide (also identified) instead of the ζ chain. The architecture of the T-cell receptor complex is schematically illustrated in Fig. 7.1.

Fig. 7.1. Each α and β chain (or γ and δ chain) within the TCR features one extracellular V and C domain, a transmembrane segment containing positively charged amino acid residues, and a short cytoplasmic tail. The chains are interconnected by a disulfide bond formed by their C domains near The cell membrane. Each of the CD3 polypeptide components—γ, δ, and ε—possesses an extracellular immunoglobulin-like C domain, a transmembrane segment containing a negatively charged amino acid residue, and a long cytoplasmic tail. The CD3 complex is also associated with a ζζ, ηη, or ζη dimer. Cumulative evidence Supports the existence of the complete TCR/CD3 receptor complex as a dimer on the T-cell surface. The charges of the transmembrane segments are thought to be crucial for the assembly and expression of the complex. The diagram depicts a chain arrangement that neutralizes oppositely charged residues.

The CD3 γ, δ, and ε polypeptides are encoded by three tightly linked genes, are very similar in amino acid sequence, and belong to the immunoglobulin superfamily. Each possesses an extracellular domain followed by a transmembrane segment and a cytoplasmic tail of 40 or more amino acid residues with a highly conserved sequence. A notable feature of the transmembrane segments is that each contains a single negatively charged (polar) amino acid, making them partially polar rather than entirely hydrophobic.

Unlike the CD3 γδε Gene cluster, the CD3 ζ gene is located on a different chromosome, and there are substantial structural differences between the ζ and γδε polypeptides. The ζ and η polypeptides, which are alternative splicing products, contain a small extracellular domain consisting of only nine amino acid residues, a transmembrane segment with a single negatively charged residue, and a long cytoplasmic tail whose C-terminal region is 42 amino acid residues longer in the η polypeptide than in the ζ polypeptide. The CD3 ζ and η polypeptides exist as three disulfide-linked dimers: ζ-ζ, η-η, and ζ-η.

The stoichiometry (composition and ratio of structural components) of the T-cell receptor complex and the putative mechanism of interaction between the CD3 polypeptides and the αβ or γδ heterodimer are discussed below.

The antigen-recognition center of the TCR is formed by the chains of the αβ or γδ heterodimer

The αβ-TCR complex includes a heterodimer of two disulfide-linked polypeptide chains: α (40–50 kDa) and β (35–47 kDa). A model of this heterodimer is shown in Fig. 7.1. The extracellular portion of each chain folds into two immunoglobulin-like domains containing approximately 110 amino acid residues each. The chains are anchored in the cell membrane by their transmembrane segments, which feature a short cytoplasmic tail. In humans, additional N-linked carbohydrate moieties impart a higher molecular mass to α chains than to β chains. The function of these carbohydrate components remains unclear.

Like the variable domains of IMMUNOGLOBULINS, the N-terminal domains of α and β chains exhibit marked sequence diversity. Each such domain is encoded by genes generated through the recombination of V, D, and J segments for β chains, and V and J segments for α chains (see Chapter 8). The amino acid sequences of the TCR V domains reveal regions of heightened Variability that correspond to the hypervariable regions of Ig chains, also known as complementarity-determining regions (CDRs).

The disulfide bond linking the α and β chains is formed in the region between the constant domain and the transmembrane segment of each monomer. A characteristic feature of both chains is the presence of positively charged amino acid residues within the transmembrane segment (see Fig. 7.1), which play a critical role in the assembly and Intracellular Transport of the T-cell receptor complex.

X-ray crystallography has elucidated the three-dimensional structures of two known TCR variants. The αβ form of the TCR, which recognizes antigen complexed with a class I MHC molecule, is depicted in Fig. 7.2. The overall folding pattern of the α and β chains that constitute the TCR closely resembles that of the Fab fragment of an antibody molecule. The Significance of this TCR architecture for antigen recognition is discussed in Chapter 9.

Fig. 7.2. Three-dimensional STRUCTURE OF THE α form of the TCR—a ribbon-diagram two-chain model. The α chain (amino acid residues 1–213) is colored blue, and the β chain (amino acid residues 3–247) is colored green. Arrows indicate antiparallel β-sheet strands denoted by lowercase letters of the Latin alphabet, following the standard convention for describing immunoglobulin spatial folding. Four intradomain and one C-terminal interdomain Disulfide Bonds are shown in purple, with sulfur atoms represented as yellow spheres.

Hypervariable regions are numbered 1 through 4 on each chain. (After Garcia, Degano, Stanfield et al. 1996. An open T cell receptor structure at 2.5 Å and its orientation in the TCR-MHC complex. Science 1996; 274: 209-19.)

Structure of the T-cell receptor complex

The stoichiometry and interaction dynamics of the subunits forming the T-cell receptor (TCR) complex remain an area of extensive research. In vitro mutagenesis studies have demonstrated that charged amino acid residues within the transmembrane domains of polypeptide chains play a critical role in the assembly and surface expression of the complete receptor complex. It is hypothesized that ionic or Hydrogen Bonds are formed within The Lipid Bilayer during assembly, specifically between the basic amino acid residues of the TCR $\alpha$- and $\beta$-chains and their complementary acidic residues within the CD3 polypeptides.

The immunoglobulin-like extracellular domains of the TCR polypeptide chains ($\alpha\beta$ or $\gamma\delta$) and CD3 ($\gamma$, $\delta$, and $\varepsilon$) also appear to associate with one another. Notably, the V-domains of the TCR $\alpha$- and $\beta$-chains associate in a manner remarkably similar to the V_H and V_L domains of an immunoglobulin molecule, bringing their six hypervariable regions into close spatial proximity to form the antigen-binding site (Fig. 7.2). Unlike immunoglobulins, the TCR antigen recognition center binds not only the antigen itself but also portions of the MHC molecule presenting it.

On the surface of the T cell, the TCR/CD3 complex likely represents a higher-order structure compared to its constituent components. Stoichiometric studies have shown that in its mature state, the complex contains two copies of the CD3$\varepsilon$ polypeptide, with these polypeptides present in a 1:1 ratio relative to the $\alpha\beta$ heterodimer. Based on this, as well as molecular weight determinations of the solubilized TCR/CD3 complex, the most probable composition is: ($\alpha\beta$)₂, $\gamma$, $\delta$, $\varepsilon$₂, $\zeta$₂. This exact model is illustrated in Fig. 7.1, reflecting the precise distribution of opposite charges on the transmembrane segments of The polypeptide chains.

The $\gamma\delta$ and $\alpha\beta$ forms of the TCR are structurally very similar

In its overall architecture, the $\gamma\delta$ form of the TCR closely resembles its $\alpha\beta$ counterpart. Each chain consists of extracellular V and C domains, a transmembrane segment containing positively charged amino acid residues, and a short cytoplasmic tail.

In humans, The structure of the $\gamma\delta$ TCR is more variable than in mice, and the $\gamma$ and $\delta$ chains themselves can be linked by a disulfide bond to form a dimer or exist as unlinked monomers. The presence of the disulfide bond is determined by the C$\gamma$2 exon rather than C$\delta$1, as only the C$\gamma$2 sequence contains a Cysteine residue. (For details on TCR gene formation, see Chapter 8.) As a result of C$\gamma$2 exon duplication or triplication, the Molecular Weight of TCR $\gamma$-chains can vary. The Biological Significance of these structural differences remains unclear. To date, there have been no reports of a disulfide-linked $\gamma\delta$ TCR form in mice.

T cells bearing either αβ or γδ receptors exhibit distinct tissue distribution patterns

The anatomical localization of T cells varies depending on their TCR isotype. The αβ form is expressed by the majority of TCR-bearing thymocytes and over 95% of peripheral T cells. In contrast, the γδ form is carried by T cells found exclusively in specific body Tissues: they constitute a small fraction of T cells in the Thymus and Secondary Lymphoid Organs, yet make up a significant proportion of T cells in epithelial barriers, such as the epidermis (in mice, but not humans), as well as the epithelia lining the mucosa of the intestine, Uterus, and Tongue.

Within each Epithelial Tissue, γδ TCR-bearing T cells belong to distinct subpopulations characterized by unique receptor structures, specifically the V regions of their γ and δ chains. The differential expression of V genes across γδ T cell subpopulations likely arises during ontogeny. For instance, in mice, γδ T cells that colonize the Skin (dendritic epidermal T cells) express exclusively Vγ3 and Vδ1 regions (see Chapter 8), whereas intraepithelial lymphocytes of the gastrointestinal tract display, with rare exceptions, only Vγ5 regions (most commonly combined with Vδ4, Vδ5, Vδ6, or Vδ7 regions). It is believed that these subpopulations may emerge at specific stages of T cell maturation in the thymus.

Antigen recognition by γδ T cells

The Nature of the antigenic structures recognized and bound by γδ T cells remains a subject of debate. These cells have been shown to recognize a remarkably diverse array of antigens, including N-formylated bacterial peptides, autoantigens such as heat Shock proteins, or non-classical (non-traditional) MHC class I antigens, such as products of the mouse TL locus or human CD1 molecules. Certain human and murine γδ T cells also recognize classical MHC class I and II antigens, though conclusive evidence that this is a universal property of the entire population is lacking.

Overall, γδ T cells may play a vital role in anti-infective Immunity by recognizing bacterial peptides (likely presented via non-classical MHC molecules) or stress-induced proteins (such as heat shock proteins) synthesized by host cells at the site of bacterial infection. Essentially, these cells provide a first line of defense, containing the spread of infection until an immune response mediated by antigen recognition through αβ T cells in complex with MHC molecules can fully develop.