Activation of biological functions in T lymphocytes is determined by the molecular dynamics occurring at the T cell͞opposing cell interface. In the present study, a central question of cytotoxic T lymphocyte (CTL) biology was studied at the single-cell level: can two distinct activation thresholds for cytotoxicity and cytokine production be explained by intercellular molecular dynamics between CTLs and targets? In this study, we combine morphological approaches with numerical analysis, which allows us to associate specific patterns of calcium mobilization with different biological responses. We show that CTLs selectively activated to cytotoxicity lack a mature immunological synapse while exhibiting a low threshold polarized secretion of lytic granules and spike-like patterns of calcium mobilization. This finding is contrasted by fully activated CTLs, which exhibit a mature immunological synapse and smooth and sustained calcium mobilization. Our results indicate that intercellular molecular dynamics and signaling characteristics allow the definition of two activation thresholds in individual CTLs: one for polarized granule secretion (lytic synapse formation) and the other for cytokine production (stimulatory synapse formation).

Vgamma9Vdelta2 T lymphocytes, a major gammadelta T lymphocyte subset in humans, display cytolytic activity against various tumor cells upon recognition of yet uncharacterized structures. Here, we show that an entity related to the mitochondrial F1-ATPase is expressed on tumor cell surface and promotes tumor recognition by Vgamma9Vdelta2 T cells. When immobilized, purified F1-ATPase induces selective activation of this lymphocyte subset. The Vgamma9Vdelta2 T cell receptors (TCR) and the F1-ATPase also bind a delipidated form of apolipoprotein A-I (apo A-I), as demonstrated by surface plasmon resonance. Moreover, the presence of apo A-I in the culture medium is required for optimal activation of Vgamma9Vdelta2 T cells by tumors expressing F1-ATPase. This study thus describes an unanticipated tumor recognition mechanism by Vgamma9Vdelta2 lymphocytes and a possible link between gammadelta T cell immunity and lipid metabolism.

Helper T cells discriminate among different antigen-presenting cells to provide their help in a selective fashion. The molecular mechanisms leading to this exquisite selectivity are still elusive. Here, we demonstrate that immunological synapses are dynamic and adaptable structures allowing T cells to communicate with multiple cells. We show that T cells can form simultaneous immunological synapses with cells presenting different levels of antigenic ligands but eventually polarize toward the strongest stimulus. Remarkably, living T cells form discrete foci of signal transduction of different intensities during the interaction with different antigen-presenting cells and rapidly relocate TCR and Golgi apparatus toward the cell providing the strongest stimulus. Our results illustrate that, although T cell activation requires sustained signaling, T cells are capable of rapid synapse remodeling and swift polarization responses. The combination of sustained signaling with preferential and rapid polarization provides a mechanism for the high sensitivity and selectivity of T cell responses.

Most human blood ␥␦ T cells react without major histocompatibility complex restriction to small phosphorylated nonpeptide antigens (phosphoantigens) that are abundantly produced by mycobacteria and several other microbial pathogens. Although isopentenyl pyrophosphate has been identified as a mycobacterial antigen for ␥␦ T cells, the structure of several other stimulating compounds with bioactivities around 1000-fold higher than isopentenyl pyrophosphate remains to be elucidated. This paper describes the structural identification of 3-formyl-1-butyl-pyrophosphate as the core of several non-prenyl mycobacterial phosphoantigens bioactive at the nM range. Recognition of this molecule by ␥␦ T cells is very selective and relies on its aldehyde and pyrophosphate groups. This novel pyrophosphorylated aldehyde most probably corresponds to a metabolic intermediate of the non-mevalonate pathway of prenyl phosphate biosynthesis in eubacteria and algae. The reactivity to 3-formyl-1-butyl-pyrophosphate supports the view that human ␥␦ T cells are physiologically devoted to antimicrobial surveillance.Although the vast majority of T lymphocytes recognize via their ␣␤ TCR 1 antigenic peptides associated to major histocompatibility complex molecules, the so-called unconventional T cells that often express ␥␦ TCR recognize their ligands in a different way. The prominent ␥␦ T cell subset in human blood expresses the V␥9/V␦2 TCR and responds to nonpeptide antigens produced by various microbial pathogens, such as mycobacteria. The mycobacterial stimuli for these T cells have been characterized independently by two groups as nonpeptide phosphoesters, collectively referred to as phosphoantigens. On the one hand, isoprenoid-PP such as isopentenyl-PP, dimethylallyl-PP, farnesyl-PP, and geranyl-PP have been characterized as V␥9/V␦2 T cell-stimulating ligands in bioactive fractions from mycobacteria (1-4). On the other hand, we have purified from several mycobacterial species a set of four phosphoantigens composed of two pyrophosphates of an unidentified monoester (X, in the so-called TUBag1 and TUBag2) and of the corresponding X-phosphodiesters of ␥-UTP (5) and ␥-TTP (6) (respectively, TUBag3 and TUBag4). These TUBag compounds have been shown to be active at the nM range (i.e. with bioactivities about 1000-fold higher than that of IPP), thus suggesting that these molecules could account for most of the ␥␦ T cell-stimulating activity recovered from mycobacteria. Poor yields and intrinsic lability of purified TUBag1-4 have considerably slowed the identification of X. However, several biochemical lines of evidence indicated that this mycobacterial X moiety was distinct from prenyl phosphates (5, 7). Accordingly, a molecular analysis of phosphoantigen recognition has evidenced a pattern of TCR ␥␦ cell reactivity that distinguishes alkyl-PP from mycobacterial phosphoantigens (8). To understand the fine specificity of ␥␦ T cell reactivity to mycobacteria, we have identified the hitherto referred-to X moiety as 3-formyl-1-butyl-PP. The activation...