Introduction␥␦ T cells are key players in the immune surveillance of cellular distress, thanks to their ability to recognize conserved determinants up-regulated after inflammation, infection, or cell transformation. 1,2 Although ␥␦ T-cell receptors (TCRs) contribute to detection of danger-associated determinants, ligands for these receptors have been identified in a few cases only. 3 Thus, the antigenic specificity of ␥␦ T cells and their fine activation modalities in response to cell stress remain largely unknown.One of the best studied ␥␦ T-cell subsets in humans expresses V␥9V␦2 TCR and predominates in blood, composing several percent of the whole peripheral lymphoid pool in most adults. V␥9V␦2 T cells are activated by nonpeptidic phosphorylated isoprenoid pathway metabolites, 4-6 hereafter referred to as phosphoagonists (PAg). Natural V␥9V␦2-stimulating PAg include isopentenyl pyrophosphate (IPP), 7 a metabolite of the mevalonate pathway found in mammalian cells and the desoxyxylulose phosphate pathway shared by many microorganisms, and hydroxy-methyl-butyl-pyrophosphate, 8 an intermediate of the latter pathway. PAg detection by ␥␦ T cells underlies their broad reactivity toward infected and transformed cells. Indeed, tumor cell recognition by V␥9V␦2 T cells is linked to enhanced production of the weak agonist IPP, resulting from increased cell metabolism and cholesterol biosynthesis. Accordingly, pharmacologic inhibitors of the mevalonate pathway that up-regulate (eg, aminobisphosphonates, NBP) or down-regulate (eg, statins) IPP production, respectively, increase or decrease antitumor V␥9V␦2 T-cell responses. 9,10 Moreover, because of the high V␥9V␦2 T cell-stimulating activity of the microbial agonist hydroxy-methyl-butyl-pyrophosphate, V␥9V␦2 T-cell responses are elicited by infected cells producing even traces of this PAg. 8 Although PAg-induced activation is restricted to V␥9V␦2 T cells and can be conferred by V␥9V␦2 TCR gene transfer, 11,12 attempts to detect cognate interactions between PAg and V␥9V␦2 TCR have failed so far. 13 So how V␥9V␦2 T cells sense PAg remains an enigma. PAg rapidly induce Ca 2ϩ signaling and activation of V␥9V␦2 T-cell clones, but this requires cell-to-cell contact, suggesting the implication of additional target cell surface receptors in this phenomenon. 11,14 PAg elicit V␥9V␦2 T-cell responses against basically all human cells, irrespective of their tissue origin, but do not induce recognition of any murine target cells. Therefore, the putative target cell receptors involved in PAg-mediated T-cell activation are expected to be broadly expressed by human, but not murine, cells.Activation of antigen-stimulated T cells is tuned by interactions involving T cell-derived CD28-related receptors and target cellderived B7-related counter-receptors, 15 which family includes members, such as Skint and butyrophilin (BTN) receptors. The mandatory role played by Skint-1 in the intrathymic positive There is an Inside Blood commentary on this article in this issue.The online version of this...

Summary In humans, Vγ9Vδ2 T cells detect tumor cells and microbial infections including Mycobacterium tuberculosis through recognition of small pyrophosphate containing organic molecules known as phosphoantigens (pAgs). Key to pAg-mediated activation of Vγ9Vδ2 T cells is the butyrophilin 3A1 (BTN3A1) protein that contains an intracellular B30.2 domain critical to pAg reactivity. Here, we have demonstrated through structural, biophysical and functional approaches that the intracellular B30.2 domain of BTN3A1 directly binds pAg through a positively-charged surface pocket. Charge-reversal of pocket residues abrogates binding and Vγ9Vδ2 T cell activation. We have also identified a gain-of-function mutation within this pocket that when introduced into B30.2 domain of the non-stimulatory BTN3A3 isoform, transfers pAg binding ability and Vγ9Vδ2 T cell activation. These studies demonstrate that internal sensing of changes in pAg metabolite concentrations by BTN3A1 molecules is a critical step in Vγ9Vδ2 T cell detection of infection and tumorigenesis.