Vγ9Vδ2⁺ lymphocytes are among the first T-cells to develop in the human fetus and are the predominant peripheral blood γδ T-cell population in most adults. Capable of broad polyclonal responses to pyrophosphate antigens (pAg), they are implicated in immunity to a diverse range of infections. Previously Vγ9Vδ2⁺ development was thought to involve postnatal selection and amplification of public Vγ9 clonotypes in response to microbial stimuli. However, recent data indicate the Vγ9Vδ2⁺ T-cell receptor (TCR) repertoire, which is generated early in gestation, is dominated by public Vγ9 clonotypes from birth. These chains bear highly distinct features compared to Vγ9 chains from Vδ1⁺ T-cells, due either to temporal differences in recombination of each subset and/or potentially prenatal selection of pAg-reactive clonotypes. While these processes result in a semi-invariant repertoire featuring Vγ9 sequences preconfigured for pAg recognition, alterations in TCRδ repertoires between neonate and adult suggest either peripheral selection of clonotypes responsive to microbial antigens or altered postnatal thymic output of Vγ9Vδ2⁺ T-cells. Interestingly, some individuals demonstrate private Vγ9Vδ2⁺ expansions with distinct effector phenotypes, suggestive of selective expansion in response to microbial stimulation. The Vγ9Vδ2⁺ T-cell subset, therefore, exhibits many features common to mouse γδ T-cell subsets, including early development, a semi-invariant TCR repertoire, and a reliance on butyrophilin-like molecules in antigen recognition. However, importantly Vγ9Vδ2⁺ T-cells retain TCR sensitivity after acquiring an effector phenotype. We outline a model for Vγ9Vδ2⁺ T-cell development and selection involving innate prenatal repertoire focusing, followed by postnatal repertoire shifts driven by microbial infection and/or altered thymic output.

Activation of human Vγ9/Vδ2 T cells by “phosphoantigens” (pAg), the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP) and the endogenous isoprenoid intermediate isopentenyl pyrophosphate, requires expression of butyrophilin BTN3A molecules by presenting cells. However, the precise mechanism of activation of Vγ9/Vδ2 T cells by BTN3A molecules remains elusive. It is not clear what conformation of the three BTN3A isoforms transmits activation signals nor how externally delivered pAg accesses the cytosolic B30.2 domain of BTN3A1. To approach these problems, we studied two HLA haplo-identical HeLa cell lines, termed HeLa-L and HeLa-M, which showed marked differences in pAg-dependent stimulation of Vγ9/Vδ2 T cells. Levels of IFN-γ secretion by Vγ9/Vδ2 T cells were profoundly increased by pAg loading, or by binding of the pan-BTN3A specific agonist antibody CD277 20.1, in HeLa-M compared to HeLa-L cells. IL-2 production from a murine hybridoma T cell line expressing human Vγ9/Vδ2 T cell receptor (TCR) transgenes confirmed that the differential responsiveness to HeLa-L and HeLa-M was TCR dependent. By tissue typing, both HeLa lines were shown to be genetically identical and full-length transcripts of the three BTN3A isoforms were detected in equal abundance with no sequence variation. Expression of BTN3A and interacting molecules, such as periplakin or RhoB, did not account for the functional variation between HeLa-L and HeLa-M cells. Instead, the data implicate a checkpoint controlling BTN3A1 stability and protein trafficking, acting at an early time point in its maturation. In addition, plasma membrane profiling was used to identify proteins upregulated in HMB-PP-treated HeLa-M. ABCG2, a member of the ATP-binding cassette (ABC) transporter family was the most significant candidate, which crucially showed reduced expression in HeLa-L. Expression of a subset of ABC transporters, including ABCA1 and ABCG1, correlated with efficiency of T cell activation by cytokine secretion, although direct evidence of a functional role was not obtained by knockdown experiments. Our findings indicate a link between members of the ABC protein superfamily and the BTN3A-dependent activation of γδ T cells by endogenous and exogenous pAg.