Structure was finally elucidated in the 1980s. Difficulties 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 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. 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.
SEE DIAGRAM IN TEXT
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 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.
Hedrick and Davis
Needed 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 on polyribosomes (attached to ER)
3) T cell Receptor genes will be rearranged in mature T cells
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 [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.
Cloned the unhybridized [32P] cDNA.
They then looked for gene sequence that rearranges, and that is expressed only in T cells.
The gene which encodes the beta chain was discovered first. Later the genes for alpha, gamma, and delta were cloned.
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)
V, D and J (beta and delta)
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'
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'
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.
VARIABLE REGION GENE REARRANGEMENT
Similar mechanisms for rearrangement as for Immunoglobulin.
Obeys the heptamer- nonamer 12/23 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
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