Highly Active Microbial Phosphoantigen Induces Rapid yet Sustained MEK/Erk- and PI-3K/Akt-Mediated Signal Transduction in Anti-Tumor Human γδ T-Cells

Background: The unique responsiveness of V gamma 9V delta 2 T-cells, the major gamma delta subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current gamma delta T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human gamma delta T-cells remain unclear. In particular, previous reports have described a very slow kinetics of activation of T-cell receptor (TCR)-associated signal transduction pathways by isopentenyl pyrophosphate and bromohydrin pyrophosphate, seemingly incompatible with direct binding of these antigens to the V gamma 9V delta 2 TCR. Here we have studied the most potent natural phosphoantigen yet identified, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), produced by Eubacteria and Protozoa, and examined its gamma delta T-cell activation and anti-tumor properties. Methodology/Principal Findings: We have performed a comparative study between HMB-PP and the anti-CD3 epsilon monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular gamma delta T-cell activation events. We show that HMB-PP activates MEK/Erk and PI-3K/Akt pathways as rapidly as OKT3, and induces an almost identical transcriptional profile in V gamma 9(+) T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including gamma delta T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the V gamma 9(+) TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains gamma delta T-cell activation upon re-stimulation. This ultimately translates in potent human gamma delta T-cell anti-tumor function both in vitro and in vivo upon transplantation of human leukemia cells into lymphopenic mice, Conclusions/Significance: The development of efficient cancer immunotherapy strategies critically depends on our capacity to maximize anti-tumor effector T-cell responses. By characterizing the intracellular mechanisms of HMB-PP-mediated activation of the highly cytotoxic V gamma 9(+) T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials.