About 1–5% of human blood T cells are Vγ9Vδ2 T cells. Their hallmark is the expression of T cell antigen receptors (TCR) whose γ-chains contain a rearrangement of Vγ9 with JP (TRGV9JP or Vγ2Jγ1.2) and are paired with Vδ2 (TRDV2)-containing δ-chains. These TCRs respond to phosphoantigens (PAg) such as (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is found in many pathogens, and isopentenyl pyrophosphate (IPP), which accumulates in certain tumors or cells treated with aminobisphosphonates such as zoledronate. Until recently, these cells were believed to be restricted to primates, while no such cells are found in rodents. The identification of three genes pivotal for PAg recognition encoding for Vγ9, Vδ2, and butyrophilin (BTN) 3 in various non-primate species identified candidate species possessing PAg-reactive Vγ9Vδ2 T cells. Here, we review the current knowledge of the molecular basis of PAg recognition. This not only includes human Vγ9Vδ2 T cells and the recent discovery of BTN2A1 as Vγ9-binding protein mandatory for the PAg response but also insights gained from the identification of functional PAg-reactive Vγ9Vδ2 T cells and BTN3 in the alpaca and phylogenetic comparisons. Finally, we discuss models of the molecular basis of PAg recognition and implications for the development of transgenic mouse models for PAg-reactive Vγ9Vδ2 T cells.

Butyrophilin (BTN)–3A and BTN2A1 molecules control the activation of human Vγ9Vδ2 T cells during T cell receptor (TCR)-mediated sensing of phosphoantigens (PAg) derived from microbes and tumors. However, the molecular rules governing PAg sensing remain largely unknown. Here, we establish three mechanistic principles of PAg-mediated γδ T cell activation. First, in humans, following PAg binding to the intracellular BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the extracellular V-domain of BTN3A2/BTN3A3. Moreover, the localization of both protein domains on different chains of the BTN3A homo-or heteromers is essential for efficient PAg-mediated activation. Second, the formation of BTN3A homo-or heteromers, which differ in intracellular trafficking and conformation, is controlled by molecular interactions between the juxtamembrane regions of the BTN3A chains. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and the division of labor in BTN proteins improves our understanding of PAg sensing and elucidates a mode of action that may apply to other BTN family members.

Most circulating human gamma delta T cells are Vγ9Vδ2 T cells. Their hallmark is the expression of T cell antigen receptors (TCR) whose γ-chains show a Vγ9-JP (Vγ2-Jγ1.2) rearrangement and are paired with Vδ2-containing δ-chains, a dominant TCR configuration, which until recently seemed to occur in primates only. Vγ9Vδ2 T cells respond to phosphoantigens (PAg) such as (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), which is produced by many pathogens and isopentenyl pyrophosphate (IPP), which accumulates in certain tumors or cells treated with aminobisphosphonates such as zoledronate. A prerequisite for PAg-induced activation is the contact of Vγ9Vδ2 T cells with cells expressing butyrophilin-3 A1 (BTN3A1). We will first critically review models of how BTN3 might act in PAg-mediated Vγ9Vδ2 T cell activation and then address putative co-evolution of Vγ9, Vδ2, and BTN3 genes. In those rodent and lagomorphs used as animal models, all three genes are lost but a data-base analysis showed that they emerged together with placental mammals. A strong concomitant conservation of functional Vγ9, Vδ2, and BTN3 genes in other species suggests co-evolution of these three genes. A detailed analysis was performed for the new world camelid alpaca (Vicugna pacos). It provides an excellent candidate for a non-primate species with presumably functional Vγ9Vδ2 T cells since TCR rearrangements share features characteristic for PAg-reactive primate Vγ9Vδ2 TCR and proposed PAg-binding sites of BTN3A1 have been conserved. Finally, we analyze the possible functional relationship between the butyrophilin-family member Skint1 and the γδ TCR-V genes used by murine dendritic epithelial T cells (DETC). Among placental mammals, we identify five rodents, the cow, a bat, and the cape golden mole as the only species concomitantly possessing potentially functional homologs of murine Vγ3, Vδ4 genes, and Skint1 gene and suggest to search for DETC like cells in these species.

Vγ9Vδ2 T cells comprise 1–10% of human peripheral blood T cells. As multifunctional T cells with a strong antimicrobial and antitumor potential, they are of strong interest for immunotherapeutic development. Their hallmark is the eponymous Vγ9Vδ2 T‐cell antigen receptor (TCR), which mediates activation by so‐called “phosphoantigens” (PAg). PAg are small pyrophosphorylated intermediates of isoprenoid synthesis of microbial or host origin, with the latter elevated in some tumors and after administration of aminobisphosphonates. This review summarizes the progress in understanding PAg‐recognition, with emphasis on the interaction between butyrophilins (BTN) and PAg and insights gained by phylogenetic studies on BTNs and Vγ9Vδ2 T cells, especially the comparison of human and alpaca. It proposes a composite ligand model in which BTN3A1‐A2/A3‐heteromers and BTN2A1 homodimers form a Vγ9Vδ2 TCR activating complex. An initiating step is the binding of PAg to the intracellular BTN3A1‐B30.2 domain and formation of a complex with the B30.2 domains of BTN2A1. On the extracellular surface this results in BTN2A1‐IgV binding to Vγ9‐TCR framework determinants and BTN3A‐IgV to additional complementarity determining regions of both TCR chains. Unresolved questions of this model are discussed, as well as questions on the structural basis and the physiological consequences of PAg‐recognition.

Butyrophilin (BTN)-3A and BTN2A1 molecules control TCR-mediated activation of human Vγ9Vδ2 T-cells triggered by phosphoantigens (PAg) from microbes and tumors, but the molecular rules governing antigen sensing are unknown. Here we establish three mechanistic principles of PAg-action. Firstly, in humans, following PAg binding to the BTN3A1-B30.2 domain, Vγ9Vδ2 TCR triggering involves the V-domain of BTN3A2/BTN3A3. Moreover, PAg/B30.2 interaction, and the critical γδ-T-cell-activating V-domain, localize to different molecules. Secondly, this distinct topology as well as intracellular trafficking and conformation of BTN3A heteromers or ancestral-like BTN3A homomers are controlled by molecular interactions of the BTN3 juxtamembrane region. Finally, the ability of PAg not simply to bind BTN3A-B30.2, but to promote its subsequent interaction with the BTN2A1-B30.2 domain, is essential for T-cell activation. Defining these determinants of cooperation and division of labor in BTN proteins deepens understanding of PAg sensing and elucidates a mode of action potentially applicable to other BTN/BTNL family members.