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T Cell Receptor (TCR)Comments

T Cell Receptor (TCR)

The T cell receptor (TCR) is a T cell surface receptor that recognizes antigens presented by MHC molecules.

t cell receptor diagram

It is a heterodimer composed of either α and β chains or γ and δ chains, and which interacts with another T cell surface component CD3. αβ T cells are usually highly specific (adaptive immunity), while γδ T cells usually recognize broader antigen classes (innate immunity). TCR chains each contain two 60-75 amino acid domains: the amino-terminal variable (V) domain and the constant (C) domain. The V domain contains three hypervariable regions, similar to antibody complementarity determining regions.

The transmembrane region is 21 or 22 amino acids and positively charged, anchoring the TCR into the cell membrane and promoting TCR-CD3 interaction. Lastly, the cytoplasmic tail (carboxy-terminal end) is 5 to 12 amino acids. αβ and γδ TCR heterodimers resemble an Fab fragment attached to the cell membrane; due to this structural homology, they are included under the immunoglobulin superfamily.

αβ TCRs react only with peptide antigens. However, γδ TCRs can react to glycolipids and phospholipids (nonpeptides) presented by CD1. Furthermore, some γδ cells react with unprocessed peptide antigens not even presented by an MHC. Also, the most common γδ TCRs within a given species bind the most common pathogens that species encounters. These γδ TCRs directly binds microbes, and the γδ cells might directly kill pathogens.

Both innate and adaptive immunity rely heavily upon binding of the TCR to a peptide-MHC complex.

γδ cells are characterized as part of innate immunity due to their rapid responses, low specificity and ability to bind free antigens. αβ cells, conversely, are clearly part of adaptive immunity. In addition to these functional differences, γδ cells and αβ cells are also structurally different.

Allison, Garboczi and their coworkers found that a γδ receptor specific for non-presented microbial phospholipids has a deep binding cleft. Also, separate research found that γδ TCRs have a 111° bend between their V and C regions while αβ TCRs have a 147° bend.

T Cell Receptor Accessory Molecules

In B cells, membrane-bound antibodies associate with the Ig-α/Ig-β heterodimer to form the B cell antigen receptor. In T cells, the T cell receptor associates with its fellow membrane-bound component CD3 to form the TCR-CD3 membrane complex.

Please note that CD3 is another component of the cell surface on T cells -- TCR and CD3 are together on the T cell surface and interact together to transduce signals into the T cell. CD3 is essential for membrane expression of the TCR and for signal transduction. All CD3 chains contain a negatively charged amino acid (aspartic or glutamic acid) which interacts with one or two positively charged transmembrane TCR residues. Please note that B and T cell accessory molecules only participate in signal transduction after antigen interaction and do not actually influence antigen interaction.

Alone, the T cell receptor has low affinity for the peptide-MHC complex. For this reason, additional accessory molecules known as coreceptors or cell-adhesion molecules strengthen the bond between a T cell and a target or antigen-presenting cell.

CD4 and CD8 are coreceptors which also distinguish cytotoxic T cells from helper T cells; CD4 binds Class II MHC molecules and CD8 binds Class I MHC molecules. Once bound, CD4 and CD8 also activate signal transduction. Additional coreceptors include CD2, LFA-1, CD28 and CD45R, all of which bind independently to other ligands on target or antigen-presenting cell. Once these coreceptors bind, the TCR scans for its appropriate peptide-MHC complex. Once TCR binds, membrane expression of cell-adhesion molecules is activated to tighten the intracellular bond. Cell-adhesion molecules play a role in tissue graft acceptance or rejection. If the grafted tissue bears MHC molecules which are similar to the host, then T cells will still respond to the MHC molecules.

T Cell Receptor Genomics

Genes encoding α, β, γ and δ chains are expressed only in T cells.

Functional TCR genes are formed by rearrangements of V and J segments in α and γ genes, and of V, D and J segments (like IgH) in β and δ genes. The α, β, γ and δ genes also include C segments which do not rearrange. The C region contains a long exon encoding much of the C domain, followed by shorter exons encoding the connecting, transmembrane and cytoplasmic regions. In humans, α and δ gene segments are located on chromosome 14, while β and γ gene segments are located on chromosome 7. In mice, the α, β and γ gene segments are located on chromosomes 14, 6 and 13.

In both mice and humans, δ gene segments are located between V and J α segments. The location of the δ gene segments is important: functional rearrangement of the α gene segments deletes Cδ, so that a T cell cannot co-express αβ TCRs with γδ TCRs. The table below describes human and murine TCR genes, and counts both pseudogenes (nonfunctional mutants) and functional genes.

# of gene segments
Gene Xsm V D J C Overview
Human α Chain 14 54 61 1 Jα region is enormous, with over 75 segments over 50kB.
Mouse α Chain 14 80 80 1
Human β Chain 7 67 2 14 2 30-50 V segments and two near-identical repeats of D, J and C segments.
Mouse β Chain 6 20 2 2 2 Contains two almost identical repeats of D, J and C segments.
Human γ Chain 7 14 5 2 Contains two almost identical repeats of J and C segments.
Mouse γ Chain 13 7 3 3 Contains three different functional J-C repeats.
Human δ Chain 14 3 3 3 1 δ gene segments are located between α gene V and J segments.
Mouse δ Chain 14 10 2 2 1

Like the antibody L chain, the α chain is encoded by V, J and C segments. Like the antibody H chain, the β chain is encoded by V, D, J and C gene segments. Rearrangement of the TCR gene segments results in α VJ joining and β VDJ joining. After the rearranged TCR genes are transcribed, the α and β chains are expressed as a disulfide-linked heterodimer on the T cell membrane. Immunoglobulins can be bound or secreted, but TCRs are only membrane-bound. The TCR constant region, as shown in the figure above, includes constant (C), connecting, transmembrane and cytoplasmic sequences. Also, as opposed to the multiple different immunoglobulin C gene segments encoding different isotypes, α DNA has only one C segment and β DNA has duplicated J and C segments.

Pre-T cells expresses the recombination activating genes RAG-1 and RAG-2, as do pre-B cells. The RAG-1/2 recombinase recognizes conserved recombination signal sequences (RSSs) flanking each V, D and J gene segments in non-recombined TCR DNA. RAG-1/2 catalyzes V-J and V-D-J joining via the same deletions and inversions which occur in immunoglobulin genes. First, Rag-1/2 knicks one DNA strand between the coding and signal sequences and excises the resulting DNA loops. SCID mice (which lack B and T cells) are defective for a gene which repairs double-stranded DNA breaks; as a result, in SCID mice, the Ab and TCR D and J gene segments are not rejoined. RAG-1/2 only recombines TCR genes in T cells and Ig genes in B cells. This specificity is due to different recombinase regulatory systems, and uniquely configured chromatin in each lineage that allows recombinase to access only appropriate sites.

δ genes are located between the V and J segments of the &alpha gene. Thus, when the α gene recombines, the δ DNA is excised. In a phenomenon known as allelic exclusion, this mechanism prevents αβ TCRs and γδ TCRs from co-expressing. Another instance of allelic exclusion involves the two duplicate β gene J and C clusters. Having two duplicates means that at least one functional rearrangement is likely to take place. Once a functional rearrangement occurs, then rearrangement of the other cluster is inhibited. However, more than one α allele can undergo functional recombination; while this can lead to different α chains being expressed on the same cell surface, only one allele is MHC restricted and therefore functional. The mechanisms of TCR diversity are described below.

Mechanism Overview
Alternative Joining In addition to combinatorial joining, δ genes can alternatively join. Although impossible with antibodies, there are functional (VDDDJ)δ and other processed δ genes.
Combinatorial Joining This simply refers to the various ways that α V and J segments and β V, D and J segments can combine.
Junctional Flexibility The junctions between gene segments are prone to nucleotide addition during rearrangement.
Nucleotide Addition In addition to junctional flexibility, palindromic sequences known as p-region nucleotide additions can be added between gene segments. Also, nucleotides can be added at the ends of TCR genes via n-region nucleotide addition.

Isolating the T Cell Receptor

Step Overview
MHC Restriction Cytotoxic T lymphocytes (CD8+ T cells) only kill infected cells with a self MHC haplotype. T cells do not kill infected cells with non-self MHC haplotypes, nor bind free antigens. This phenomenon — MHC Restriction — was identified in a classic 1974 experiment by Zinkernagel and Doherty (which led to the 1996 Nobel Prize). Using cytotoxic T lymphocytes (CTLs) specific for virally infected cells, immunologists found that the CTLs lysed target cells in vitro but did not bind free virus antigens. Zinkernagel and Doherty built upon this, realizing that CTLs are also specific only for virally infected cells presenting a self MHC molecule.
Heterodimers Another study was performed to isolate the T cell receptor. Researchers created monoclonal antibodies to various T cell clones, then isolated antibodies which bound a specific clone population. Assuming that the T cell clones differed only in their T cell receptors, it was concluded that these antibodies bound the T cell receptors. This approach illuminated that T cell receptors are heterodimers, and the two chains were labeled α and β.
αβ and γδ αβ heterodimers were isolated from the membranes of various T cell clones. Antibodies were generated to these heterodimers; some antibodies bound to only one clone population, while other antibodies bound all the clone populations. This suggested that T cell receptors contained variable and constant regions, which is logical due to their antigen specificity. Later, a second T cell receptor heterodimer was identified, and its two chains were labeled δ and γ. Most T cells expressed αβ heterodimers, but depending on the organ there can be just as many or more δγ T cells.
TCR cDNA Immunologists Hedrick and Davis next isolated the genes encoding the T cell receptor. After isolating mRNA from T cells, the researchers eluted only mRNA associated with membrane-bound polyribosomes (as opposed to free cytoplasmic ribosomes). This step removed ∼97% of total cellular mRNA. Next, reverse transcriptase synthesized labeled cDNA probes from these mRNA samples. The following step was DNA subtractive hybridization: they hybridized labeled B cell mRNA to the cDNA probes; unhybridized labeled cDNA was thus unique to T cells. After eliminating ∼97% of cellular mRNA, this step removed ∼98% of cDNA probes.
TCR Genes Approximately ten cDNA probes remained, and it was assumed that within these probes were the genes encoding the T cell receptor. The probes were used to identify genomic DNA in T cells and other cells; a certain region of DNA was found to rearrange in T cells but not in any other cells. This region was putatively the T cell receptor gene: it encoded a membrane-bound protein, was expressed only in T cells and rearranged only in T cells. It was later found that the cDNA clone encoded the β chain — subsequent research identified α, γ and finally δ chain genes.