T Cell Receptors and MHC Molecules
THE TWO TYPES OF TCR MAY HAVE DISTINCT FUNCTIONS
The two types of TCR tend to populate different tissue sites and are thought to perform distinct functions.
Theαβ heterodimer is the antigen recognition unit of the αβ TCR
The αβ TCR is the predominant receptor found in the thymus and peripheral lymphoid organs of mice and humans. It is a disulfide-linked heterodimer of α (40–50 kDa) and β (35–47 kDa) subunits and its structural features have been determined by X-ray crystallography (Fig. 5.2).
Each polypeptide chain of the αβ TCR contains two extracellular immunoglobulin-like domains of approxi- mately 110 amino acids, anchored into the plasma membrane by a transmembrane domain that has a short cytoplasmic tail.
Q. How can receptors that lack intracytoplasmic domains signal to the cell? Give some examples.
A. They signal by associating with other membrane molecules that do have intracytoplasmic domains. For example, immuno- globulin associates with Igα and Igβ (see Fig. 3.1), and FcγRI associates with its γ chain dimer (see Fig. 3.20).
The extracellular portions of the α and β chains fold into a structure that resembles the antigen-binding portion (Fab) of an antibody (see Fig. 3.12). Indeed, as in antibodies, the amino acid sequence variability of the TCR resides in the N terminal domains of the α and β (and also the γ and δ) chains.
The regions of greatest variability correspond to immunoglobulin hypervariable regions and are also known as complementarity determining regions
Similarities and differences between T cell receptors and immunoglobulins
Fig. 5.1 TCRs are very similar to Fab fragments of B cell receptors. Both receptor types are composed of two different peptide chains and have variable regions for binding antigen, constant regions, and hinge regions. The principal differences are that TCRs remain membrane-bound and contain only a single antigen-binding site.
antibody T cell receptor
antigen-binding sites variable region constant region hinge antigen-binding site VL VH CL CH Vαorγ Cαorγ Vβorδ Cβorδ
(CDRs). They are clustered together to form an antigen-
binding site analogous to the corresponding site on antibodies (see Fig. 5.2). Note, however, that:
• the CDR3 loops from both the α and β chains lie at the center of the antigen-binding site.
These CDR3 loops make contact with antigen, which in the case of the TCR, is a peptide (see below).
The disulfide bond that links the α and β chains is in a peptide sequence located between the constant domain
of the extracellular portion of the receptor and the transmembrane domain (shown as the C terminal residue in the α and β chain in Fig. 5.2).
One remarkable feature of the transmembrane portion of the receptor is the presence of positively charged residues in both the α and β chains. Unpaired charges would be unfavorable in a transmembrane segment. Indeed, these positive charges are neutralized by assembly of the complete TCR complex, which contains additional polypeptides bearing complementary negative charges (see below).
The CD3 complex associates with the antigen-bindingαβ or γδ heterodimers to form the complete TCR
The αβ or γδ heterodimers must associate with a series of polypeptide chains collectively termed the CD3 complex for the antigen-binding domains of the TCR to form a complete, functional receptor that is stably expressed at the cell surface and is capable of transmitting a signal upon binding to antigen.
The four members of the CD3 complex (γ, δ, ε, and ζ) are sometimes termed the invariant chains of the TCR because they do not show variability in their amino acid sequences. (The γ and δ chains of the CD3 complex should not be confused with the quite distinct antigen-binding variable chains of the TCR that bear the same names.)
THE TWO TYPES OF TCR MAY HAVE DISTINCT FUNCTIONS
The T cell antigen receptor
Fig. 5.2 Three-dimensional structure of an αβ TCR – only extracellular domains are shown. The α chain is colored blue (residues 1–213), and the β chain is colored green (residues 3–247). The β strands are represented as arrows and labeled according to the standard convention used for the
immunoglobulin fold. The disulfide bonds (yellow balls for sulfur atoms) are shown within each domain and for the C terminal interchain disulfide. The complementarity determining regions (CDRs) lying at the top of the diagram are numerically labeled (1–4) for each chain. These form the binding site for antigen/MHC molecule. (Adapted from Garcia KC, Degano M, Stanfield RL, et al. Science 1996;274:209–219. Copyright AAAS) b c" d 4 2 1 3 3 2 1 4 c" d e b a f c c' c' c f V V C C a e c d e d c f g a b c" e a b f g
Rapid Reference Box 5
CDRs (complementarity determining regions) – the sections of an antibody or T cell receptor V region responsible for antigen or antigen–MHC molecule binding.
H-2 – the mouse MHC.
histocompatibility – the ability to accept grafts between individuals.
HLA – the human MHC.
ITAMs (immunoreceptor tyrosine activation motifs) and ITIMs (immunoreceptor tyrosine inhibitory motifs) – target sequences for phosphorylation by kinases involved in cell activation or inhibition.
MHC (major histocompatibility complex) – a genetic region found in all mammals whose products are primarily responsible for the rapid rejection of grafts between individuals; it functions in the signaling between lymphocytes and cells expressing antigen.
MHC class I and II molecules – molecules coded within the MHC – class I molecules have one MHC-encoded peptide complexed with β2-microglobulin; class II molecules have
two MHC-encoded peptides, which are non-covalently associated.
MHC restriction – a characteristic of many immune reactions in which cells cooperate most effectively with other cells that share an MHC haplotype.
RAG-1 and RAG-2 – recombination activating genes, required for recombination of V, D, and J gene segments during the generation of functional antigen receptor genes.
V, D, and J genes – variable (V), joining (J), and diversity (D) genes.
The CD3 chains are assembled as heterodimers of γε and δε subunits with a homodimer of ζ chains, giving an overall TCR stoichiometry of (αβ)2, γ, δ, ε2, ζ2. Current
data suggest that the TCR complex exists as a dimer (Fig. 5.3).
The CD3 γ, δ, and ε chains are the products of three closely linked genes, and similarities in their amino acid sequences suggest that they are evolutionarily related.
Indeed, all three are members of the immunoglobulin superfamily, each containing an external domain followed by a transmembrane region and a substantial, highly conserved, cytopasmic tail of 40 or more amino acids.
As with the transmembrane domains of the variable chains of the TCR, the membrane-spanning regions of these CD3 chains contain charged amino acids.
It is thought that the negatively charged residues in the transmembrane region of the CD3 chains interact with (and neutralize) the positively charged amino acids in the αβ polypeptides, leading to the formation of a stable TCR complex (Fig. 5.3).
The CD3 ζ gene is on a different chromosome from the CD3 γδε gene complex, and the ζ protein is struc- turally unrelated to the other CD3 components. The ζ chains possess:
• a small extracellular domain (nine amino acids); • a transmembrane domain carrying a negative charge;
and
• a large cytoplasmic tail.
An alternatively spliced form of CD3ζ, called CD3η, possesses an even larger cytoplasmic tail (42 amino acids longer at the C terminus).
The cytoplasmic portions of and chains contain ITAMs
These ζ and η chains may associate in all three possible combinations (ζζ, ζη, or ηη) and play a critical role in signal transduction through the TCR. The cytoplasmic portions of these subunits contain particular amino acid sequences called immunoreceptor tyrosine-based
activation motifs (ITAMs), and each chain contains
three of these motifs.
Q. Which other group of cell surface molecules contains ITAMs?
A. The Fcγ receptors, either as an intrinsic intracellular domain of the receptor, or because they associate with signaling mole- cules that have ITAMs (see Fig 3.20).
The conserved tyrosine residues in the ITAM motifs are targets for phosphorylation by specific protein kinases. When the TCR is bound to its cognate antigen–MHC complex, the ITAM motifs become phosphorylated within minutes in one of the first steps in T cell activation (see Fig. 7.21).
ITAMs:
• are essential for T cell activation, and mutational substi- tution of the tyrosines in the motif prevents activation; • play critical roles in B cell activation, and are present in the B cell receptor chains, Igα and Igβ (see Fig. 3.1 and Chapter 8).
CD3ζ also functions in another signaling pathway, asso- ciating with the low-affinity FcγRIIIa receptor (CD16), which is involved in the activation of macrophages and natural killer (NK) cells (see Fig. 3.20).
Other subunits of the CD3 complex (γ, δ, ε), though lacking in ITAMs, may also become phosphorylated following TCR engagement. Phosphorylation of the CD3γ chain downregulates TCR expression on the cell surface via a mechanism involving increased receptor internalization.
The␥␦ TCR structurally resembles the ␣ TCR but may function differently
The overall structure of the γδ TCR is similar to that of its αβ counterpart. Each chain is organized into:
• extracellular V and C domains;
The T cell receptor complex
Fig. 5.3 The TCR α and β (or γ and δ) chains each comprise an external V and C domain, a transmembrane segment containing positively charged amino acids, and a short cytoplasmic tail. The two chains are disulfide linked on the membrane side of their C domains. The CD3 γ, δ, and ε chains comprise an external immunoglobulin-like C domain, a transmembrane segment containing a negatively charged amino acid, and a longer cytoplasmic tail. A dimer of ζζ, ηη, orζη is also associated with the complex. Several lines of evidence support the notion that the TCR–CD3 complex exists at the cell surface as a dimer. The transmembrane charges are important for the assembly of the complex. A plausible arrangement that neutralizes opposite charges is shown.
transmembrane segment V V C C TCR TCR
• a transmembrane segment containing positively charged amino acids; and
• a short cytoplasmic tail.
One indication that the two types of T cell (i.e. T cells with αβ TCRs and T cells with γδ TCRs) might perform dif- ferent functions comes from their anatomic distribution: • in humans and mice, αβ TCRs are present on more
than 95% of peripheral blood T cells and on the majority of thymocytes;
• T cells bearing γδ TCRs are relatively rare in spleen, lymph nodes, and peripheral blood but predominate at epithelial surfaces – they are common in skin and in the epithelial linings of the reproductive tract and are especially numerous in the intestine, where they are found as intraepithelial lymphocytes.
It is further believed that there are distinct subsets of γδ T cells that can perform different functions.
Antigen recognition by ␥␦ T cells is unlike that of their␣ counterparts
The fact that γδ T cells are rare in anatomic locations known to support the classical mechanisms of antigen presentation and lymphocyte clonal expansion suggests the possibility that γδ cells might not need to rely upon nor- mal antigen presentation mechanisms for their activation.
Several lines of evidence support the hypothesis that γδ T cells can recognize antigen in an MHC-independent fashion, for example:
• γδ T cells can be found in normal numbers in MHC class I and class II-deficient mice;
• their cognate antigens are not necessarily peptides, and do not require classical processing – indeed, some murine γδ T cells have been found to recognize proteins directly, including MHC molecules and viral proteins, in a manner that requires neither antigen processing nor presentation by MHC.
γδ T cells therefore appear to be able to follow a different paradigm for T cell recognition of antigen than that employed by αβ T cells.
γδ T cells recognize at least two classes of ligand: • molecules that signal the presence of cellular stress; and • small organic molecules that serve as signifiers of
infection.
For example, human intraepithelial γδ T cells have been found to respond to MHC class I-related antigens (MICA and MICB) expressed on the surface of stressed cells.
In addition, some human γδ T cells recognize small, non-peptidic, organic compounds secreted by myco- bacteria, such as monoethylphosphate and isopentenyl pyrophosphate. These ligands are secreted by a number of bacteria and may also be produced by some eukaryotic pathogens.
The γδ T cell arm of the adaptive immune system therefore appears to share some key characteristics of innate immune responses.
γδ T CELLS HAVE A VARIETY OF BIOLOGICAL ROLES – γδ T cells:
• are essential for primary immune responses to certain viral and bacterial pathogens in mouse models, but in many cases their contribution to the primary response
can be substituted for by αβ T cells, and they rarely contribute to memory responses;
• interact with a variety of lymphocytes, and have been implicated in stimulating immunoglobulin class switch recombination by B cells in response to T-dependent antigens;
• provide regulatory signals to αβ T cells and have been implicated in shaping immune responses (e.g. γδ cells appear to be involved in downregulating inflammation and in this role may be responding to epithelial cells stressed by inflammatory processes rather than to specific antigens borne by pathogens).
The unique ability of γδ T cells to, on the one hand, sense tissue damage and, on the other, to recognize antigens without the normal constraints of antigen processing/ MHC restriction (see below), may allow them to fill several key biological roles such as immunoregulation. In particular, γδ T cells may downregulate potentially damaging inflammatory responses, providing immuno- protection:
• when MHC function is compromised by, for example, viral infections that downregulate MHC;
• in early life when αβ T cell function is immature and when the antigen processing and antigen sampling systems have not yet matured.
TCRs ARE ENCODED BY SEVERAL SETS OF GENES
The general arrangement of the genes encoding the α, β, γ, and δ chains of the TCR is remarkably similar to that of the immunoglobulin heavy chain genes (see Chapter 3, p. 81), suggesting a common origin from a primordial rearranging antigen receptor locus.
Fig. 5.4 illustrates the murine TCR genes, which are similar to those of humans. All four TCR gene families have been strongly conserved across more than 400 million years of evolution of the jawed vertebrates, which suggests a strong selective pressure for the preservation of both αβ and γδ T cell functions.
The nomenclature of the TCR genes is simple – the
TCRA locus encodes the α gene, TCRB the β gene, and so
on. Interestingly, the TCRD locus is nested within the
TCRA cluster.
The α and γ loci have sets of V and J gene segments (analogous to immunoglobulin light chain loci), whereas the β and δ loci have V, D, and J gene segments (analogous to immunoglobulin heavy chains).
TCR variable region gene diversity is generated by V(D)J recombination
As with antibody genes, a highly diverse repertoire of TCR variable region genes is generated during T cell differentiation by a process of somatic gene rearrange- ment termed V(D)J recombination (see Chapter 3). Variable (V), joining (J), and sometimes diversity (D) gene segments are joined together to form a completed variable region gene.
Junctional diversity (imprecise joining of V, D, and J with loss and/or addition of nucleotides) contributes an enormous amount of variability to the TCR repertoire in
addition to the variation that results from combinatorial assortment of the various gene segments. There is minor variation in detail for each locus (see Fig. 5.4).
TCRA recombination entails joining of V to J gene segments
As in the kappa light chain (IgK locus), joining of a Vα segment to a Jα segment produces a complete variable region gene. The large number of Jα segments con- tributes to the ultimate diversity of TCRα specificities.
The TCRB locus includes two sets of D, J, and C genes
Most of the Vβ genes are grouped together, but one (Vβ14) is present at the extreme 3′ end of the locus. The tandem duplication of Dβ, Jβ, and Cβ must have occurred early in mammalian evolution because it is present in both mice and humans. Extensive diversity is generated by recombination because not only are VDJ arrangements possible, but also VJ and VDDJ combinations.
The D segments are used in all three reading frames, further contributing to β chain diversity.
The arrangement of the TCRG locus differs in mice and in humans
The murine γ locus bears a striking similarity to the antibody light chain locus, with four Cγ genes (including a pseudogene), each associated with one J gene and from one to four Vγ genes. There are no D genes.
In humans there are eight Vγ genes, followed by three Jγ and the first Cγ, then two additional Jγ genes before Cγ2.
The TCRD locus possesses only five V␦, two D␦, and six J␦ genes
The TCRD locus was discovered during studies on the TCRA locus and possesses only five Vδ, two Dδ, and six Jδ genes. Despite this relative paucity of genetic material, more than 1000 different δ chains can be generated.
Q. How may the great number of δ chains be generated when there is only a limited number of V, D, and J gene segments in the TCRD locus?
A. As described in Chapter 3, the two D region segments can be used in all three possible reading frames, and imprecise joining of the VD and DJ junctions produces further diversity.
The mechanism of V(D)J recombination is the same in both T cells and B cells
The TCR genes are flanked by recombination signal sequences, just like their immunoglobulin cousins (see Fig. 3.29), and the same recombination machinery (the RAG proteins) operates in both B and T cells. Indeed, experiments have shown that TCR Dβ and Jγ genes can rearrange appropriately even if transfected into B cells.
Analysis of the amino acid sequences of many different TCRs shows that the greatest diversity lies within the third CDR (CDR3), which is also the case for B cell receptors. Addition of N regions (non-templated nucleotides added to the junctions by terminal deoxynucleotidyl transferase, TdT) is much more pronounced in TCRs, however. It is important to note, too, that neither somatic hyper- mutation nor class switching occur in T cells.
Q. Why is it that, unlike B cells, T cells have not evolved a class switching mechanism?
A. Class switching is irrelevant because there is no secreted form of the TCR and hence no interaction analogous to that of immunoglobulin and FcR.
RECOMBINATION YIELDS GREAT DIVERSITY – Hunkapiller and Hood have calculated that it is possible to construct about:
• 4.4× 1013different forms of TCR Vβ; and
• 8.5× 1012forms of TCR Vα.
They estimate that if only 1% of the sequences coded for viable proteins this would still give 2.9× 1022receptors.
Even if 99% of these viable receptors were rejected due
Murine T cell receptor genes
Fig. 5.4 Theδ chain loci are embedded within the α loci and tandem duplication has occurred in the β chain loci. The last of each set of Jβ genes and the Vγ3 gene are pseudogenes.
V1 TCRA, TCRD locus TCRB locus TCRG locus V2 Vn C V(~100) V1 V2 D V4 V5 J1 C1 V1 V(~20) J J J J J J J C1 D J J J J J J J C2 V14 D1 J1(7) J2(7) Vn J J J J J J J(~100) D2 V1 V2 V(1~5) V5 D D D(2) J J J(6) C V7 V6 V3 J3 C3 C2 J2 V2 J4 C4
to autoreactivity or other defects, recombination would still yield 2.9× 1020 possible murine TCRs. This would
seem to be more than enough potential diversity, given that the thymus produces fewer than 109thymocytes over
the lifetime of a mouse.
TCR V genes used in the responses against