γδ T cells constitute a small (~5–10%) but unique subpopulation of T cells. Because of their features, including antigen recognition through a somatically rearranged T cell receptor (TCR) and several NK cell receptors1, cytokine production and immunoregulation2, they act as a bridge between innate and adaptive immunity, rapidly responding to infected or transformed cells in a major histocompatibility complex (MHC)-independent manner1.
Contrary to their αβ counterparts, there is little evidence supporting the hypothesis of human γδ T cells being positively and/or negatively selected in the thymus. Indeed, post-natal human γδ T cells seem to only complete their maturation program in the periphery, especially upon infection challenge that triggers clonal expansion3,4,5,6,7. Thus, the goal of post-natal thymic development seems to be the generation of a highly diverse, naïve and immature human γδ T cell repertoire. As such, the main event for developing γδ T cells is the generation of a functional TCR through the rearrangement of the variable (V), diversity (D) and joining (J) segments and subsequent pairing of the rearranged γ- and δ-chains. These events alone could lead to the establishment of a large number of diverse antigen receptors, but the addition and/or subtraction of non-templated (N) and palindromic (P) nucleotides at the gene segment junctions contribute substantially to increasing diversity, providing nearly limitless potential to the TCRγδ repertoire8.
There are several subsets of human γδ T cells, identified by the combination of rearranged TCRγ- and δ-chains. 14 TRGV genes, of which only 6 are functional (Vγ2, Vγ3, Vγ4, Vγ5, Vγ8 and Vγ9), can rearrange with 5 TRGJ (JP1, JP, J1, JP2 and J2) genes, whereas 7 TRDV genes (Vδ1, Vδ2, Vδ3 and Vα14/Vδ4, Vα23/Vδ6, Vα29/Vδ5, Vα36/Vδ7, Vα38-2/Vδ8) can rearrange with three TRDD (D1, D2 and D3) and with four TRDJ genes (J1, J4, J2 and J3)9. Although the thymus can generate all the possible combinations of TRG and TRD genes, the major γδ T cell population in peripheral blood (PB) expresses a TCR composed by Vγ9 recombined with JP and paired with Vδ210. This specifically rearranged Vγ9Vδ2 T cell subset is mostly produced during foetal life but still constitutes the major γδ T cell subset in adults. Antigen-driven stimulation in the periphery underlies a strong and specific expansion of this subset after birth and during the lifespan of each individual11. Indeed, Vγ9Vδ2 T cells can rapidly recognize, in a TCR-dependent manner, cellular dysregulation resulting from infection or malignant transformation1,10.
Vδ1+ γδ T cells are the second most abundant subset in the human PB but the predominant γδ T cell subset in the post-natal thymus and in peripheral tissues (such as the intestine or the liver), where Vδ1 is mainly paired to Vγ8 or Vγ9 chains. These γδ T cells play an important role during viral infections, especially CMV3,4, and tumour progression12. Indeed, a subpopulation of Vδ1+ T cells recognizes nonpolymorphic MHC-like (class Ib) proteins presenting lipids, such as CD1 proteins, in a similar way to other unconventional T cells like NKT or MAIT cells1,13,14,15.
Thanks to recent technical advances in the comprehensive analysis of TCR repertoires using next-generation sequencing (NGS) approaches, it has become more accessible to understand the dynamics of T cell development and homeostasis5,16, and T cell expansion in response to infections4,17 or tumours18. In this context, the purpose of this study is to characterize the Vδ1+ TCR repertoire in the thymus of young children, providing a detailed description of transcribed (mRNA) V(D)J recombination products in highly (FACS-)purified thymic Vδ1+ T cells. This allowed us to reveal unsuspected aspects of the rearranged and expressed TRG and TRD repertoires of this cell population with the striking presence of a big fraction (~20%) of Vδ2 sequences in the thymic TRD repertoire of all 8 donors. While a small fraction of Vδ2 sequences, like that found in the PB Vδ1+ T cell pool, could be due to contamination with Vδ2+ cells, this cannot explain the large fraction of Vδ2 sequences in the highly FACS-purified thymic CD3+ Vδ1+ Vδ2− samples.
Moreover, PB and cord blood Vδ1+ T cells were described to display private TRD repertoires, in contraposition to the respective TRG repertoire that showed a fraction of common (shared among individuals) sequences4,6. Here, we show that is also the case for purified Vδ1+ thymocytes, albeit these shared TRG clonotypes consist of TRGJ1 gene segments. This raises the interesting question whether the public TRG clonotypes of Vδ1+ T cells are selected (upon ligand encounter) in the thymus; or simply the output of a favoured TCR rearrangement. This, in fact, is an open question also on the Vγ9-JPVδ2 rearrangement that is prevalent in foetal life10.
In sum, this study constitutes a resource providing new data and qualifying previous conclusions on the TCR repertoire of human thymic γδ T cells16. Critically, the unexpected presence of a large fraction of Vδ2 sequences in Vδ1+ thymocytes strongly advocates for the use of highly purified cell populations, ideally complemented by single-cell validation experiments, to avoid misinterpretations of NGS data in future studies.