Non-productive human TCR beta chain genes represent V-D-J diversity before selection upon function: insight into biased usage of TCRBD and TCRBJ genes and diversity of CDR3 region length.

The aim of the study was to assess the influence of constraints of V-D-J rearrangement on the nonrandom junctional diversity of productive T-cell receptor beta-chain genes in peripheral T-cells. Mature peripheral T lymphocytes are expected to display a biased repertoire of T cell receptors (TCRs), enriched for those that can recognize peptides presented by the major histocompatibility complex (MHC) molecules. Therefore, functional TCR rearrangements of peripheral T-cells are unsuitable to reveal the bias of the TCR repertoire, introduced by V-D-J rearrangement. To overcome this problem, we have studied nonfunctional TCR genes representing a repertoire of rearranged TCR gene sequences without any known post-rearrangement selection. Detailed molecular analysis of a database generated from more than 500 functional (TCRBV20S1) and nonfunctional (TCRBV10S1P and TCRBV19S1P) T-cell receptor genes from peripheral blood T-cells permitted a comparative analysis of recombination frequencies of each germline-encoded V, D, and J-segments, as well as exonucleolytic nibbling and addition of nucleotides in functional and nonfunctional transcripts. Our data demonstrate that V-D-J recombination generates a more diverse CDR3 length distribution than found among productive TCRBV genes, suggesting that selection constrains the CDR3 to an optimal junctional region length. Furthermore, the well established biased patterns of D- and J-usage in the rearranged TCRBV genes in human peripheral blood lymphocytes were also present in nonfuncrional transcripts. Therefore, V-D-J diversity is biased mainly by constraints of the rearrangement process rather than intrathymic T-cell selection and peripheral expansion of particular T-cell clones.