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 Vγ9Vδ2 T-cells, the major γδ subset of human peripheral blood, to non-peptidic prenyl pyrophosphate antigens constitutes the basis of current γδ T-cell-based cancer immunotherapy strategies. However, the molecular mechanisms responsible for phosphoantigen-mediated activation of human γδ 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 Vγ9Vδ2 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 γδ T-cell activation and anti-tumor properties. Methodology/Principal Findings: We have performed a comparative study between HMB-PP and the anti-CD3ε monoclonal antibody OKT3, used as a reference inducer of bona fide TCR signaling, and followed multiple cellular and molecular γδ 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 Vγ9 T-cells. Moreover, MEK/Erk and PI-3K/Akt activities are indispensable for the cellular effects of HMB-PP, including γδ T-cell activation, proliferation and anti-tumor cytotoxicity, which are also abolished upon antibody blockade of the Vγ9 TCR Surprisingly, HMB-PP treatment does not induce down-modulation of surface TCR levels, and thereby sustains γδ T-cell activation upon re-stimulation. This ultimately translates in potent human γδ 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 Vγ9 T-cell subset, our data strongly support the usage of this microbial antigen in novel cancer clinical trials. © 2009 Correia et al.