No abstract

Adoptive cell transfer utilizing tumour-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against haematological malignancies, but significant clinical success has not yet been achieved in solid tumours due in part to the strong immunosuppressive tumour microenvironment. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is in part mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Highly selective optical control of Ca2+ signalling in adoptively transferred CTLs enhances T cell activation and IFN-γ production in vitro, leading to a significant reduction in tumour growth in mice. Altogether, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses at the tumour sites.

Phospholipase C (PLC) enzymes hydrolyze the plasma membrane (PM) lipid phosphatidylinositol 4,5-bisphosphate (PI4,5P) to generate the second messengers inositol trisphosphate (IP) and diacylglycerol (DAG) in response to receptor activation in almost all mammalian cells. We previously found that stimulation of G protein-coupled receptors (GPCRs) in cardiac cells leads to the PLC-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) at the Golgi, a process required for the activation of nuclear protein kinase D (PKD) during cardiac hypertrophy. We hypothesized that GPCR-stimulated PLC activation leading to direct PI4P hydrolysis may be a general mechanism for DAG production. We measured GPCR activation-dependent changes in PM and Golgi PI4P pools in various cells using GFP-based detection of PI4P. Stimulation with various agonists caused a time-dependent reduction in PI4P-associated, but not PI4,5P-associated, fluorescence at the Golgi and PM. Targeted depletion of PI4,5P from the PM before GPCR stimulation had no effect on the depletion of PM or Golgi PI4P, total inositol phosphate (IP) production, or PKD activation. In contrast, acute depletion of PI4P specifically at the PM completely blocked the GPCR-dependent production of IPs and activation of PKD but did not change the abundance of PI4,5P Acute depletion of Golgi PI4P had no effect on these processes. These data suggest that most of the PM PI4,5P pool is not involved in GPCR-stimulated phosphoinositide hydrolysis and that PI4P at the PM is responsible for the bulk of receptor-stimulated phosphoinositide hydrolysis and DAG production.

No abstract

Adoptive cell transfer utilizing tumor-targeting cytotoxic T lymphocytes (CTLs) is one of the most effective immunotherapies against hematological malignancies, but significant clinical success has not yet been achieved in solid tumors due in part to the strong immunosuppressive tumor microenvironment. Systemic or intratumoral delivery of an immune boosting molecule to overcome local suppression has been proposed, but the full potential is limited by non-specific stimulation of tumor growth, metastasis, and angiogenesis. Here, we show that suppression of CTL killing by CD4+CD25+Foxp3+ regulatory T cell (Treg) is mainly mediated by TGFβ-induced inhibition of inositol trisphosphate (IP3) production, leading to a decrease in T cell receptor (TCR)-dependent intracellular Ca2+ response. Both in vitro and in vivo assays revealed that highly selective optical control of Ca2+ signaling in adoptively transferred CTLs was sufficient to overcome immunosuppression at the tumor site by enhancing T cell activation, IFN-γ production and antitumor cytotoxicity, leading to a significant reduction in tumor growth in mice. Together, our findings indicate that the targeted optogenetic stimulation of intracellular Ca2+ signal allows for the remote control of cytotoxic effector functions of adoptively transferred T cells with outstanding spatial resolution by boosting T cell immune responses only at the targeted tumor sites.