Human γδ T cells can be activated by phospho-antigens and aminobisphosphonates such as zoledronate. Because they can kill tumor cells in a major histocompatibility complex (MHC)-unrestricted manner, adoptive transfer of activated γδ T cells may represent a novel cancer immunotherapy. We tested whether γδ T cells from advanced cancer patients can be expanded by zoledronate. Peripheral blood mononuclear cells from healthy donors and patients with advanced non-small cell lung cancer, bone metastatic breast or prostate cancer, or lung metastatic colorectal cancer, were stimulated with zoledronate (5 µM) and interleukin (IL)-2 (1000 IU/mL) for 14 days. The phenotype and function of the expanded γδ T-cell populations from healthy donors and cancer patients were compared. γδ T cells from cancer patients and healthy donors responded to zoledronate equally well in terms of both phenotype and function. γδ T cells grew rapidly in vitro and expression of effector molecules, such as interferon (IFN)-γ, tumor necrosis factor (TNF)-α, perforin, granzyme B, FasL and TRAIL, increased over time. Cytotoxicity peaked on days 12–14, and proliferation continued up to 14 days, during which time>1 × 10⁹ γδ T cells could be obtained from a starting sample of 45–70 mL peripheral blood. Using the agent zoledronate, already widely used in the clinic, we have established that efficient large-scale ex vivo expansion of γδ T cells from cancer patients is possible. These cells exert potent cytotoxicity and may be used for autologous cellular immunotherapy of cancer.

Microbes and their byproducts have been reported to regulate host health and immune functions. Here we demonstrated that microbial exopolysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus OLL1073R-1 (EPS-R1) induced CCR6+ CD8+ T cells of mice and humans. In mice, ingestion of EPS-R1 augmented antitumor effects of anti–CTLA-4 or anti–PD-1 monoclonal antibody against CCL20-expressing tumors, in which infiltrating CCR6+ CD8+ T cells were increased and produced IFNγ accompanied by a substantial immune response gene expression signature maintaining T-cell functions. Of note, the antitumor adjuvant effect of EPS-R1 was also observed in germ-free mice. Furthermore, the induction of CCR6 expression was mediated through the phosphorylated structure in EPS-R1 and a lysophosphatidic acid receptor on CD8+ T cells. Overall, we find that dietary EPS-R1 consumption induces CCR6+ CD8+ T cells in Peyer's patches, favoring a tumor microenvironment that augments the therapeutic effect of immune-checkpoint blockade depending on CCL20 production by tumors. Significance: Gut microbiota- and probiotic-derived metabolites are attractive agents to augment the efficacy of immunotherapies. Here we demonstrated that dietary consumption of Lactobacillus-derived exopolysaccharide induced CCR6+ CD8+ T cells in Peyer's patches and improved the tumor microenvironment to augment the therapeutic effects of immune-checkpoint blockade against CCL20-producing tumors. See related commentary by Di Luccia and Colonna, p. 1189. This article is highlighted in the In This Issue feature, p. 1171

Activation of the WNT pathway allows highly immunogenic cancer cells to escape antitumor immunity and induce cancer progression.

Malignant ascites caused by peritoneal dissemination of gastric cancer is chemotherapy‐resistant and associated with poor prognosis. We conducted a pilot study to evaluate the safety of weekly intraperitoneal injections of in vitro expanded Vγ9Vδ2 T cells together with zoledronate for the treatment of such malignant ascites. Patient peripheral blood mononuclear cells were stimulated with zoledronate (5 μmol/L) and interleukin‐2 (1000 IU/mL). After 14 days culture, Vγ9Vδ2 T‐cells were harvested and administered intraperitoneally in four weekly infusions. The day before T‐cell injection, patients received zoledronate (1 mg) to sensitize their tumor cells to Vγ9Vδ2 T‐cell recognition. Seven patients were enrolled in this study. The number of Vγ9Vδ2 T‐cells in each injection ranged from 0.6 to 69.8 × 10⁸ (median 59.0 × 10⁸). There were no severe adverse events related to the therapy. Intraperitoneal injection of Vγ9Vδ2 T cells allows them access to the tumor cells in the peritoneal cavity. The number of tumor cells in the ascites was significantly reduced even after the first round of therapy and remained substantially lower over the course of treatment. IFN‐γ was detected in the ascites on treatment. Computed tomography revealed a significant reduction in volume of ascites in two of seven patients. Thus, injection of these antitumor Vγ9Vδ2 T‐cells can result in local control of malignant ascites in patients for whom no standard therapy apart from paracentesis is available. Adoptively transferred Vγ9Vδ2 T‐cells do indeed recognize tumor cells and exert antitumor effector activity in vivo, when they access to the tumor cells.

To understand global effector mechanisms of CTL therapy, we performed microarray gene expression analysis in a murine model using pmel-1 T-cell receptor (TCR) transgenic T cells as effectors and B16 melanoma cells as targets. In addition to upregulation of genes related to antigen presentation and the MHC class I pathway, and cytotoxic effector molecules, cell-cycle–promoting genes were downregulated in the tumor on days 3 and 5 after CTL transfer. To investigate the impact of CTL therapy on the cell cycle of tumor cells in situ, we generated B16 cells expressing a fluorescent ubiquitination-based cell-cycle indicator (B16-fucci) and performed CTL therapy in mice bearing B16-fucci tumors. Three days after CTL transfer, we observed diffuse infiltration of CTLs into the tumor with a large number of tumor cells arrested at the G1 phase of the cell cycle, and the presence of spotty apoptotic or necrotic areas. Thus, tumor growth suppression was largely dependent on G1 cell-cycle arrest rather than killing by CTLs. Neutralizing antibody to IFNγ prevented both tumor growth inhibition and G1 arrest. The mechanism of G1 arrest involved the downregulation of S-phase kinase-associated protein 2 (Skp2) and the accumulation of its target cyclin-dependent kinase inhibitor p27 in the B16-fucci tumor cells. Because tumor-infiltrating CTLs are far fewer in number than the tumor cells, we propose that CTLs predominantly regulate tumor growth via IFNγ-mediated profound cytostatic effects rather than via cytotoxicity. This dominance of G1 arrest over other mechanisms may be widespread but not universal because IFNγ sensitivity varied among tumors. Cancer Immunol Res; 3(1); 26–36. ©2014 AACR.See related commentary by Riddell, p. 23