Diversity in T-lymphocyte antigen receptors is generated by somatic rearrangement of T-cell receptor (TCR) genes and is concentrated within the third complementarity-determining region (CDR3) of each chain of the TCR heterodimer. We sequenced the CDR3 regions from millions of rearranged TCR β chain genes in naïve and memory CD8+ T-cells of seven adults. The CDR3 sequence repertoire realized in each individual is strongly biased toward specific Vβ-Jβ pair utilization, dominated by sequences containing few inserted nucleotides, and drawn from an effective sequence space 250-fold smaller than predicted. Surprisingly, the overlap in the naïve CD8+ TCRβ CDR3 sequence repertoires of any two of the individuals is ~1000-fold larger than predicted and essentially independent of the degree of HLA matching.

Advances in high-throughput sequencing have enabled technologies that probe the adaptive immune system with unprecedented depth. We have developed a multiplex PCR method to sequence tens of millions of T cell receptors (TCRs) from a single sample in a few days. A method is presented to test the precision, accuracy, and sensitivity of this assay. T cell clones, each with one fixed productive TCR rearrangement, are doped into complex blood cell samples. TCRs from a total of eleven samples are sequenced, with the doped T cell clones ranging from 10% of the total sample to 0.001% (one cell in 100,000). The assay is able to detect even the rarest clones. The precision of the assay is demonstrated across five orders of magnitude. The accuracy for each clone is within an overall factor of three across the 100,000 fold dynamic range. Additionally, the assay is shown to be highly repeatable.

The two main lineages of T lymphocytes develop from multi potent precursors in the human thymus. The most common type in blood are αβ T cells, which bind to antigenic peptides displayed on the surface of cells by human leukocyte antigen (HLA) molecules. Far less well understood are γδ T cells, which do not bind HLA: peptide complexes and are more prevalent in the gut mucosa. For both lineages, their ability to recognize a diverse array of antigens is mediated by a rearranged Y-like receptor on their surface, the T cell receptor (TCR), composed and of an α and β chain for αβ T cells or a γ and δ chain for γδ T cells. The canonical model for commitment from the precursor to one these two lineages assumes that γ, δ, and β chains rearrange prior to commitment to αβ or γδ T cells. A crucial step towards better understanding the role of γδ T cells is to work out the developmental process. To test the standard model and to understand the γδ TCR repertoire, we use high-throughput sequencing to catalog millions of TCRγ and TCRβ chains from peripheral blood αβ and γδ T cells, from three unrelated individuals. Almost all sampled αβ and γδ T cells have rearranged TCRγ sequences. While sampled αβ T cells have a diverse repertoire of rearranged TCRβ chains, less than 10% of γδ T cells in peripheral blood have a rearranged TCRβ chain. Our data indicate that TCRγ rearranges in all T lymphocytes, consistent with TCRγ rearranging prior to T cell lineage commitment, while rearrangement of the TCRβ locus is restricted, and occurs after T cell precursors commit to the αβ T cell lineage. This result explains the conundrum in T cell leukemia and lymphoma that TCRγ is almost always rearranged and TCRβ is only rearranged in a subset of cancers. As high-throughput sequencing of TCRs is translated into the clinic for monitoring minimal residual for leukemia/lymphoma, our data suggests the sequencing target needs to be TCR γ.