Generation of TREC Molecules

As mentioned above, during the thymocyte maturation process, highly diverse TCR molecules are generated to ensure that all T cells leaving the thymus harbor a specific TCR endowed with a unique antigenic specificity. Such diversity is made possible through multiple chromosomal rearrangements both at the TCRB and TCRA loci. At the TCRB locus, two subsequent genetic rearrangements occur. The fusion of a DP (diversity) segment with a JP (junction) segment, followed by the junction of a VP (variable) segment to the already rearranged DPJP segments leads to the generation of the third hypervariable domain (CDR3) of the TCRB chain (see Fig. 1). Similarly, at the TCRA locus, the junction of a Va segment to a Ja segment defines the TCRA CDR3 region (see Fig. 2). During each of these rearrangement processes, the excised DNA sequence is circularized as stable episomal DNA, termed TCR rearrangement excision circles (TRECs). According to the number of Va, Ja, VP, DP, and JP segments present on the chromosomes, many different TRECs can be produced by the different rearrangements (several hundreds for VJaTRECs, dozens for VDPTRECs, and at least 13 for DJPTRECs). Moreover, before the rearrangement at the TCRA locus, a vast majority of precursor T cells will delete the TCRD locus, that is flanked by Va and Ja segments. This TCRD locus excision is mainly realized through specific rearrangement of the SRec and TJa

Tcr Rearrangement

Fig. 1. Rearrangements at the human T-cell receptor (TCR)B locus. Rearrangement of the TCR germline DNA is a stochastic procedure by which noncontiguous variable (V), diversity (D) and joining (J) segments are fused to form complete and, more importantly, variable TCR sequences. The 685 kb human TCRB chain sequence is composed of 2 Dp -JP clusters that are both accompanied by nearly identical constant CP sequences (DP -JP1.1-1.6- CP1 and Dp2- Jp2.1-2.7- CP2). Sixty-five VP segments (grouped into 32 families according to sequence homology) are found upstream of these clusters and enhance the multiplicity of possible combinations. The rearrangement of the TCRBD to TCRBJ segments occurs first and generates a by-product: a specific DJPTRECs that can be quantified by PCR. This is followed by the recombination of the V to DJ segments, which also generates a specific VDPTREC. These VDPTRECs can also be quantified, but they are not used in the evaluation of the sj/PTREC ratio as with 65 different VP segments, the possibilities are much too numerous to cover all rearrangements.

Fig. 1. Rearrangements at the human T-cell receptor (TCR)B locus. Rearrangement of the TCR germline DNA is a stochastic procedure by which noncontiguous variable (V), diversity (D) and joining (J) segments are fused to form complete and, more importantly, variable TCR sequences. The 685 kb human TCRB chain sequence is composed of 2 Dp -JP clusters that are both accompanied by nearly identical constant CP sequences (DP -JP1.1-1.6- CP1 and Dp2- Jp2.1-2.7- CP2). Sixty-five VP segments (grouped into 32 families according to sequence homology) are found upstream of these clusters and enhance the multiplicity of possible combinations. The rearrangement of the TCRBD to TCRBJ segments occurs first and generates a by-product: a specific DJPTRECs that can be quantified by PCR. This is followed by the recombination of the V to DJ segments, which also generates a specific VDPTREC. These VDPTRECs can also be quantified, but they are not used in the evaluation of the sj/PTREC ratio as with 65 different VP segments, the possibilities are much too numerous to cover all rearrangements.

deleting elements and leads to the generation of a specific TREC molecule that is present in a majority of cells (about 70%) exiting the thymus: the latter has justified the use of the sjTREC (signal joint TCR rearrangement excision circle) to quantify thymic output (4,5).

TRECs are stable episomal DNA molecules that persist in a cell until mitosis. As TRECs are not duplicated during cell division, only one of the daughter cells will inherit a TREC molecule. Accordingly, the TREC frequency in a given T cell population is inversely proportional to its proliferative history subsequent to TREC generation. Concerning the sjTREC molecule that is produced late during thymopoiesis, most of this proliferation occurs in the periphery and corresponds to homeostatic proliferation of mature naive T cells. However, the DJPTRECs are also diluted during their intrathymic maturation as TCRB+

VÔ4 VÔ6 Vôl Vô5 SRec VÔ2 D61-3 CÔ J61-4 V63 WJa Jas

VÔ4 VÔ6 Vôl Vô5 SRec VÔ2 D61-3 CÔ J61-4 V63 WJa Jas

Fig. 2. Rearrangements at the human T-cell receptor (TCR)A locus. The TCRA chain sequence, devoid of D segments, is composed of approx 70 Va and 60 Ja segments that, interestingly, border the TCRD locus. To successfully rearrange a TCRA chain and draw the V and J segments nearer, the small DP thymocytes must first excise the TCRD loci, committing the T cell to aP T cell lineage. In most thymocytes (70%), the removal of the TCRD loci is governed by the rearrangement of two deleting elements of the a locus, SRec and ¥Ja. This generates the sjTREC, that can be quantified by PCR. Va and Ja segments are then rearranged and a structurally in-frame product of the TCRa chain pairs with the TCRP chain and form the complete TCR.

Fig. 2. Rearrangements at the human T-cell receptor (TCR)A locus. The TCRA chain sequence, devoid of D segments, is composed of approx 70 Va and 60 Ja segments that, interestingly, border the TCRD locus. To successfully rearrange a TCRA chain and draw the V and J segments nearer, the small DP thymocytes must first excise the TCRD loci, committing the T cell to aP T cell lineage. In most thymocytes (70%), the removal of the TCRD loci is governed by the rearrangement of two deleting elements of the a locus, SRec and ¥Ja. This generates the sjTREC, that can be quantified by PCR. Va and Ja segments are then rearranged and a structurally in-frame product of the TCRa chain pairs with the TCRP chain and form the complete TCR.

thymocytes undergo massive clonal expansion at late DN, intermediate single positive (ISP) and early DP differentiation stages (termed P-selection), before the excision of the TCRD locus and the generation of the sjTREC molecule.

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