Creating Tcell receptor diversity

Defining the TCR binding region: T-cell receptor fingerprinting measures the length of the complementarity determining region 3 (CDR3) (Figure 1). In most cases, rearrangement of the TCR loci results from deletional joining. The V genes are localized upstream from the J (or D and C) genes.

Two genes, the recombination activating gene 1 and 2 (RAG1 and RAG2) were identified in 1989 and 1990. RAG1 and RAG2 are transcribed in lymphocytes that show recombinase activity (i.e. B or T-cells). These genes are - with few exceptions- only active in thymic tissue pertaining to T-lymphocytes (2, 3) Junctional diversity is responsible for creation of the TCR diversity associated with antigen recognition (i.e. MHC/peptide complexes). The N-region diversity results from the deletion of nucleotides at the extremities of the coding V, D and J genes by activation of an exonuclease and the random addition of nucleotides by the terminal deoxynucleotidyl transferase (TdT). The enzyme preferentially adds G and C nucleotides at the junctions and is specifically transcribed during lymphocytic maturation. This region is determined complementarity determining region 3 (CDR3). In brief, the specificity of a T-cell resides in the CDR3 region, the length of this regions defines the diversity of the TCR repertoire. The CDR3 region in humans measures approximately 11 amino acid residues (i.e. 33 base pairs) and shows a Gauss-distribution: the most abundant transcript measures approximately 6 amino acid residues (see Figure 1).

Figure 1. Determination of the TCR repertoire. A primer panel is used to amplify all possible TCR VB families using primers in the constant and in the respective variable region of the TCR. Example of the TCR CDR 3 analysis of the TCR VB16 family in CD4+ sorted T-cells. The major middle peak is 263 bp, there are 5 peaks on the left and right side respectively: a typical Gauss distribution of 11 aa coding for the CDR3 area. Note that each peak identifies 3 base pairs, coding for one amino acid residue. A single peak may suggest monoclonality. This has to be confirmed by DNA sequence analysis, since even a single peak (one amino acid residue) indicates that the length of the CDR3 region is 1 amino acid residue, however, different amino acid residues may be possible.

Figure 1. Determination of the TCR repertoire. A primer panel is used to amplify all possible TCR VB families using primers in the constant and in the respective variable region of the TCR. Example of the TCR CDR 3 analysis of the TCR VB16 family in CD4+ sorted T-cells. The major middle peak is 263 bp, there are 5 peaks on the left and right side respectively: a typical Gauss distribution of 11 aa coding for the CDR3 area. Note that each peak identifies 3 base pairs, coding for one amino acid residue. A single peak may suggest monoclonality. This has to be confirmed by DNA sequence analysis, since even a single peak (one amino acid residue) indicates that the length of the CDR3 region is 1 amino acid residue, however, different amino acid residues may be possible.

The CDR3 composition in a defined TCR VB "pool" may either be poly-, oligo- or monoclonal (see Figure 2). It is estimated that the theoretically possible TCR repertoire is not used in humans (4), but T-cells in human peripheral blood can carry up to 106 different TCR VB chains (5). Of note, most of the studies available today address the question of TCR diversity in the TCR VB chain, based on the presumption that each individual T-cell expresses only a single TCR VA chain paired with a single TCR VB chain. This is true for most T-lymphocytes in the peripheral circulation, although double TCR VB chains paired with a single TCR VA chain (and vice versa) have been reported (6, 7).

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