Incorporation of multiple diversity genes in the TCRδ chain is highly regulated and evolutionarily conserved.

In the canonical model of VDJ recombination, a single diversity (D) gene is incorporated into the complementarity-determining region 3 (CDR3) to produce a T cell receptor δ (TCRδ; TRD) or TCRβ (TRB) chain. But by constructing a dataset of more than 2.7 million TRD CDR3 sequences from 6 species (human, mouse, cattle, sheep, naked mole-rat, and rabbit), we found that incorporation of more than one D gene in the same CDR3 is strikingly high and evolutionarily conserved in the TRD CDR3, while remaining minimal in the TRB CDR3. These high frequencies are favored by TRD locus architecture and configuration of 12/23-bp recombination signal sequences (RSSs). Multiple D gene incorporation is further shaped by V (variable) gene usage and age, and is especially enriched in adaptive-like γδ T cells. This regulation is established in the thymus at the level of hematopoietic stem and precursor cells, driven by the RNA-binding protein LIN28B. Thus, genomic design, recombination constraints, and developmental timing regulate the frequencies of multiple D gene incorporation, closely associated with γδ T cell biology.