Increasing immunological knowledge and advances in techniques lay the ground for more efficient and broader application of immunotherapies. gamma delta (γδ) T-cells possess multiple favorable anti-tumor characteristics, making them promising candidates to be used in cellular and combination therapies of cancer. They recognize malignant cells, infiltrate tumors, and depict strong cytotoxic and pro-inflammatory activity. Here, we focus on human Vγ9Vδ2 T-cells, the most abundant γδ T-cell subpopulation in the blood, which are able to inhibit cancer progression in various models in vitro and in vivo. For therapeutic use they can be cultured and manipulated ex vivo and in the following adoptively transferred to patients, as well as directly stimulated to propagate in vivo. In clinical studies, Vγ9Vδ2 T-cells repeatedly demonstrated a low toxicity profile but hitherto only the modest therapeutic efficacy. This review provides a comprehensive summary of established and newer strategies for the enhancement of Vγ9Vδ2 T-cell anti-tumor functions. We discuss data of studies exploring methods for the sensitization of malignant cells, the improvement of recognition mechanisms and cytotoxic activity of Vγ9Vδ2 T-cells. Main aspects are the tumor cell metabolism, antibody-dependent cell-mediated cytotoxicity, antibody constructs, as well as activating and inhibitory receptors like NKG2D and immune checkpoint molecules. Several concepts show promising results in vitro, now awaiting translation to in vivo models and clinical studies. Given the array of research and encouraging findings in this area, this review aims at optimizing future investigations, specifically targeting the unanswered questions.

Gamma delta (γδ) T-cell based immunotherapy is a promising concept for the treatment of hematologic malignancies. Not only in vitro but also in early phase clinical trials, zoledronic acid (Zol) and interleukin-2 (IL-2) have been successfully used to activate human γδ T-cells and to induce clinical anti-tumor effects. Aiming to improve the effectiveness of future γδ T-cell based immunotherapies against leukemia, we analyzed the impact of programmed cell death protein 1 (PD-1) signaling, on the different phases of γδ T-cell activation, of proliferation, production of anti-tumor cytokines and cytotoxic function in vitro.PD-1 expression was found significantly upregulated between day 2 and day 4 following stimulation with Zol and IL-2. However, proliferation or expression of activation markers of γδ, αβ and NK-cells are not altered by additional PD-1 blockade. Pembrolizumab increases interferon-γ (IFN-γ) production in γδ T-cells upon direct stimulation with Zol and in response to Zol treated primary acute myeloid leukemia (AML) cells by approximately 57% and 30%, respectively. Zol sensitized primary AML cells also induce PD-1 expression in co-cultured γδ T-cells and such PD-1(+) cells contain more IFN-γ. In contrast, PD-1 blockade does not have a significant effect on direct cell dependent lysis of leukemia cells by γδ T-cells.This study demonstrates that PD-1 blockade impacts cell dependent cytotoxicity and cytokine production in response to leukemia cells differently. While Pembrolizumab did not increase cell lysis of stimulated and expanded γδ T-cells, it induces significant upregulation of the potent pro-inflammatory and anti-tumor cytokine IFN-γ, which might facilitate anti-leukemia effects. ARTICLE HISTORY

Alterations of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and mitochondrial respiration are possible triggers of pulmonary vascular remodeling in pulmonary hypertension (PH). We investigated the role of MMP in PH and hypothesized that deletion of the mitochondrial uncoupling protein 2 (UCP2) increases MMP, thus promoting pulmonary vascular remodeling and PH. MMP was measured by JC-1 in isolated pulmonary arterial smooth muscle cells (PASMCs) of patients with PH and animals with PH induced by exposure to monocrotaline (MCT) or chronic hypoxia. PH was quantified in vivo in UCP2-deficient (UCP2(-/-)) mice by hemodynamics, morphometry, and echocardiography. ROS were measured by electron spin resonance spectroscopy and proliferation by thymidine incorporation. Mitochondrial respiration was investigated by high-resolution respirometry. MMP was increased in PASMCs of patients and in animal models of PH. UCP2(-/-) mice exhibited pulmonary vascular remodeling and mild PH compared with wild-type (WT) mice. PASMCs of UCP2(-/-) mice showed increased proliferation, MMP, and ROS release. Increased proliferation of UCP2(-/-) PASMCs could be attenuated by ROS inhibitors and inhibited by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, which decreased MMP to the level of WT mice. Mitochondrial respiration was altered in PASMCs from MCT rats and PASMCs exposed to hypoxia but not in isolated pulmonary mitochondria of UCP2(-/-) mice or PASMCs after treatment with small interfering RNA for UCP2. Our data suggest that increased MMP causes vascular remodeling in UCP2(-/-) mice partially via increased ROS. In chronic hypoxia and MCT-induced PH, additional pathomechanisms such as decreased respiration may play a role.