Clinical Information

The rearrangement of the T-cell receptor (TCR) through somatic recombination of V (variable), D (diversity), J (joining), and C (constant) regions is a defining event in the development and maturation of a T cell. TCR gene rearrangement takes place in the thymus. During the process of rearrangement, DNA byproducts are generated called T-cell receptor excision circles (TREC) and these are used as markers of T cells that have recently emigrated from the thymus (TRECS / T-Cell Receptor Excision Circles Analysis, Blood). T cells, as part of the adaptive immune system, recognize foreign antigens when they are displayed on the surface of the body's own cells. T cells recognize these foreign antigens as peptides presented in the context of major histocompatibility complex (MHC) molecules through their TCRs. Each TCR exists as 2 different polypeptide chains (heterodimers) called the TCR alpha chain and TCR beta chain, and these are linked by disulfide bonds. The majority of T cells (approximately 90%) in the body express TCRs with alpha and beta chains. A minority of T cells express other T-cell receptors made of different polypeptide chains, gamma and delta. Each T cell has approximately 30,000 identical antigen receptors on its cell surface. A TCR has only one antigen-binding site, in contrast to the B-cell receptor, which has two. TCRs are never secreted and always remain on the cell surface. The alpha and beta chains are encoded by different gene loci (alpha and beta TCR gene locus). The beta chain locus rearranges before the alpha chain and a functional beta chain has to be produced in order for the T cell to form a pre-T-cell receptor. The expression of the rearranged beta chain with an alpha chain precursor suppresses additional gene rearrangement at the TCR beta locus. The TCR alpha chain locus rearrangement can proceed even with production of a functional alpha chain until there is positive selection of the particular T cell. However, it is important to note that each T cell has a single functional specificity for its TCR.

A key concept in understanding the immune response is that there is enormous diversity in the immune system to enable protection against a huge array of pathogens. Since the germline genome is limited in size, diversity is achieved not only by the process of V(D)J recombination but also by junctional (junctions between V-D and D-J segments) deletion of nucleotides and addition of pseudo-random, non-templated nucleotides. In particular, the CDR3 (complementarity determining region 3), which is the most critical determinant of antigenic specificity in T cells (and B cells) is short (between 66-90 nucleotides, approximately 20-30 amino acids) and amenable to assessment of length by fragment length analysis, which provides a size resolution of up to one base pair between different CDR3 regions. It is thought that the CDR3-TCR beta chain repertoire in healthy adults contains somewhere between 3 and 4 million unique sequences.(1) Other reports suggest that the unique TCR repertoire after thymic selection is between 10 to 100 million in humans.(2) There is, however, a bias in TCR selection with overrepresentation of certain TCRs that are widely used in individuals who share the same major histocompatibility types and these are called "public TCRs." Public TCRs generally have fewer random nucleotide additions in their sequence. The TCR V beta repertoire varies significantly between individuals and populations because of 7 frequently occurring inactivating alterations (ie, polymorphisms) in functional gene segments and a large deletion/insertion-related alteration encompassing 2 V beta gene segments. With this latter situation, the TCR Vb 6-2/6-3 and TCR Vb 4-3 genes are frequently deleted from all ethnic groups.(3) It has been reported that the total number of functional TCR V beta gene segments expressed by an individual varies from 42 to 47.(4)

Deep sequencing technologies are evolving to analyze this large diversity in the adaptive immune receptors.(5,6) However, deep sequencing of the T-cell and B-cell receptor genes is not yet widely available and is expensive. Flow cytometry-based analysis to assess TCR V beta diversity is available. However, the antibodies are limited and therefore the assay cannot assess the entire TCR V beta repertoire. On the other hand, TCR beta chain repertoire analysis by fragment length analysis (spectratyping) using fluorescent primers to measure CDR3 length variability, while unable to provide the extreme high resolution of deep sequencing, can provide a global "snapshot" of TCR repertoire diversity, which is useful for most clinical applications where this level of assessment is required.(7-14) It is important to note that this method uses polymerase chain reaction to amplify the rearranged variable regions to provide adequate template for sequencing (fragment length analysis), and this can introduce bias due to the more efficient amplification of certain templates compared to others. Despite this limitation, since this assay is not quantitative, it is still able to provide an assessment of diversity by measuring the CDR3 length in various TCR V beta genes, which are organized into 24 families.