- T CELL RECEPTOR

The structure of the T cell receptor was not elucidated until the 1980s. It was much more difficult to isolate the TcR than Ig because the T cell does not secrete its receptor and because the receptor is specific for both antigen and MHC.

Monoclonal antibodies and nucleic acid probes were both vital to the purification / isolation of the TcR.

The TcR was isolated using monoclonal antibodies against T cell clones. In other words, T cells derived from a single T cell bearing a single type of TcR. Just as in the isolation of Ig, a homogeneous preparation of TcRs was required.

STRUCTURE OF T CELL RECEPTOR

The T cell receptor is a heterodimer composed either of alpha and beta or gamma and delta polypeptide chains. Amino acid sequencing analysis shows a surprising similarity to the domain structure of the Igs. Each chain has a variable region domain and a constant region domain (designated Va and Ca, Vb and Cb etc.).

Three complementarity determining regions which appear to be equivalent to the CDRs in Ig heavy and light chains have been identified in both the alpha beta and gamma delta chains.

The variable region domains of alpha & beta or gamma & delta come together to form the antigen binding cleft.
The TcR has been visualized by X-ray crystallography and is quite similar in the way in which the antigen binding site is formed by the CDRs of Ig variable region domains.

The TcR heterodimer has a mw of ~ 85,000 - 90,000 D.
The 2 polypeptide subunits are ~40,000 D each and the 2 subunits have different pI points so can be separated by
isoelectric focusing.

The alpha/beta TcR is present on more than 95% of peripheral T cells and the vast majority of TcR+ thymocytes.

The gama /delta TcR is abundant in various epithelia (epidermis of mice/not humans, intestinal epithelium, and
epithelium of uterus and tongue).

Current evidence indicates that the gama /delta TcR may recognize bacterial peptides - perhaps presented on
non-classical MHC proteins--- OR----may recognize HSPs (heat shock proteins) produced in areas of infection.

The gamma / delta T cells may be our first line of defense, limiting the extent of infection until an MHC-restricted
alpha / beta T cell response can be mounted.

IDENTIFYING AND CLONING THE TCR GENES

Hedrick and Davis
Wanted to isolate mRNA for the TcR
The T cell does not secrete the TcR so there is not much message around.
Used the Approach of Subtractive Hybridization

Assumptions:
1) TcR genes expressed in T cells but not B cells (mRNA for the TcR will not be found in B cells)
2) mRNA for the TcR would be found on polyribosomes (attached to RER)
3) T cell Receptor genes will be rearranged in mature T cells but not in other cell types

The investigators isolated the polyribosomal fraction from a Th cell clone and used reverse transcriptase to make
³²P labeled cDNA probes. Only 3% of the mRNA was in the polyribosomal fraction - this eliminated 97% of the
mRNA.

B cells and T cells are derived from a common progenitor cell and have many genes in common. ~98% of the
genes expressed are the same!

The investigators hybridized B cell mRNA with their Th cell clone[³²P] cDNA. They removed the hybridized material
and the unhybridized [³²P]cDNA was greatly enriched for uniquely expressed T cell genes, including the mRNA
for the TcR. They then cloned the unhybridized [³²P] cDNA. In this attempt they were able to generate 10 different clones.
They then used the cloned cDNAs themselves as probes to look for gene sequences that rearranged, and that were expressed only in T cells. For this analysis they used genomic DNA isolated from various cell types. The genomic DNA was cut with restriction enzymes and then the restriction fragments were analyzed by Southern blotting. Only one of their clones hybridized with sequences that rearranged in T cells. This clone turned out to be the gene for the b chain of the TcR.
The gene which encodes the beta chain was discovered first. Later the genes for alpha, gamma, and delta were successfully
cloned and sequenced.

ORGANIZATION AND REARRANGEMENT OF GERM-LINE TCR GENES

The germline DNA contains four multigene families each encoding one of the TcR chains. Functional genes are
produced by gene rearrangements involving:

V and J ( alpha and gamma chains)
V, D and J (beta and delta chains)

Mouse TcR germline organization (alpha & delta)

5'---L--Va1---L--Va2-----L--Van----//---L--Vd1-----L--Vdn--//---Dd1--Dd2--Jd1--Jd2--Cd--L--Vd5----Ja1--Ja2--Ja3------Jan-----Ca----3'

The location of the delta chain genes is significant because a productive rearrangement of the alpha chain gene
segments deletes delta sequences so that the alpha /beta TcR cannot be coexpressed with the gamma /delta TcR in a
given T cell.

Mouse TcR (beta)

5'--L-V1---L-V2------L-Vn-----//-D1----J1-J2-J3-J4-J5-J6----C1-----D2----J1-J2-----Jn-- C2----L--V---3'

VARIABLE REGION GENE REARRANGEMENT

Similar mechanisms for rearrangement as for Immunoglobulin.

Obeys the heptamer- nonamer 12/23 rule (one turn - two turn rule)
RAG 1 and RAG 2 are expressed in the pre T cell
Shows allelic exclusion [for the most part] - 1 functional type of TcR per cell

Allelic exclusion is stringent for the beta chain of the TcR but appears to be not as stringent for the alpha chain. It
is rare, but occasionally you will find a single T cell expressing the same beta chain with two different alpha
chains. However, it is believed that only one of the TcRs is self MHC restricted and therefore only one of the
TcRs is functional.

MECHANISMS FOR GENERATING TCR DIVERSITY

Recombinatorial Diversity
Generated by multiple V gene segments and J gene segments for the alpha and gamma chain and by multiple V, D,
and J gene segments for the beta and delta chain.

Diversity in D region Usage

Due to the arrangement of the heptamer 12 nonamer and heptamer 23 nonamer sequences in the beta and delta
genes, differential D region usage has been observed. (see figure in text)

In beta chain gene rearrangement both direct V-J rearrangement and V-D-J rearrangement occurs. In delta chain
gene rearrangement, V-J, V-D-J, and V-D-D-J rearrangement is observed.

Combinatorial Diversity
Due to the random combination of alpha and beta chains or gamma and delta chains.

Junctional Diversity

N- nucleotide addition occurs at the V-J, D-J, and V-DJ junctions due to the action of terminal deoxynucleotidyl transferase (tDt).
This mechanism occurs with all 4 of the TcR gene families.
P-nucleotide addition also occurs following rearragement with all 4 of the TcR gene families.

Little to no Somatic Mutation occurs within V region gene segments- so TcRs do not show affinity maturation.

T CELL RECEPTOR COMPLEX

Other polypeptides are closely associated with the TcR

CD3 complex

A group of proteins collectively known as CD3 forms a complex with the receptor. The TcR and the CD3 must be co-expressed. In other words, a cell that is CD3+ is also TcR +. Appearance of CD3 is a very good marker for cells expressing the T cell receptor.

The CD3 is actually a complex of 5 polypeptides. Gamma, delta, and epsilon occur as 2 dimers [gamma-epsilon and delta-epsilon] complexed with either a homodimer of two zeta chains or a heterodimer of zeta and eta chains. Zeta and eta are encoded by the same gene and only differ in their carboxyl terminus. The two forms are generated by differential RNA splicing of the primary transcript.
About 90% or greater of the CD3 complexes examined to date incorporate the homodimer.

Short length of the cytoplasmic tail of the alpha & beta chains suggests that they are unsuitable for signal transduction.
CD3 functions in this regard.

The TcR exists on the membrane as a molecular complex with CD3. Mutation in either the CD3 or the TcR results
in loss of the entire complex from the membrane.

The transmembrane domain of all CD3 polypeptides contains a - charged aspartic acid. Correspondingly, there are
either 1 or 2 + charged amino acids in the transmembrane domain of each TcR chain. The alpha chain contains a
lysine and an arginine while the beta chain contains a single lysine.

Once the TcR recognizes an antigen-MHC complex, the associated CD3 complex is thought to transmit a signal to
the cell interior that contributes to cellular activation. Monoclonal antibody specific for CD3 bypasses the
antigen-specific T cell receptor. Following activation, several of the CD3 polypeptides are phosphorylated at
tyrosine or serine residues. Phosphorylation is thought to lead to the activation of second messengers involved in
T cell activation. The phosphorylation occurs at sequences within the cytoplasmic domains of the CD3 glycoproteins which are termed ITAMS (immuno-receptor tyrosine-based activation motifs). These ITAM sequences (also found in the cytoplasmic domains of the Iga / Igb heterodimers of the B cell receptor complex) interact with tyrosine kinases (the enzymes which catalyze phosphorylation at tyrosine or serine).

CD4 and CD8 Accessory Molecules

T helper cells express CD4 but not CD8
T cytotoxic cells express CD8 but not CD4

CD4 and CD8 are associated in the membrane with the TcR. Both CD4 and CD8 function as adhesion molecules;
CD4 binds to Class II MHC molecules and CD8 binds to Class I MHC molecules. Binding of the TcR to the
peptide/MHC complex is greatly augmented if CD4 or CD8 are assisting. Their cytoplasmic domains may also
allow for signal transduction to occur. The signal-transduction property of CD4 and CD8 is mediated through their
cytoplasmic domains. Both CD4 and CD8 are noncovalently associated with the protein kinase Lck.

Other TcR accessory molecules
A variety of other membrane molecules play an important accessory role in antigen recognition and T cell
activation.

In addition to CD4 and CD8, T cells possess several other accessory molecules including LFA-1 and CD28.

LFA-1 is an adhesion molecule which strengthens the interaction of the T cell with an APC or target cell.

LFA-1 is an integrin which binds to ICAM-1 (inter-cell adhesion molecule on the APC or target cell.

CD28 on Th cell binds to B7 on an APC or target cell. This interaction functions as a co-stimulatory signal leading to activation of the T lymphocyte. Without this co-stimulatory signal, the T lymphocyte experiences anergy (non-responsive, basically the opposite of activation). The display of B7 by an APC or target cell is an important "danger" signal for the immune system. Cells presenting self peptides should not be co-expressing B7.


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