PD-1 defines a distinct, functional, tissue-adapted state in Vδ1+ T cells with implications for cancer immunotherapy.

Checkpoint inhibition (CPI), particularly that targeting the inhibitory coreceptor programmed cell death protein 1 (PD-1), has transformed oncology. Although CPI can derepress cancer (neo)antigen-specific αß T cells that ordinarily show PD-1-dependent exhaustion, it can also be efficacious against cancers evading αß T cell recognition. In such settings, γδ T cells have been implicated, but the functional relevance of PD-1 expression by these cells is unclear. Here we demonstrate that intratumoral TRDV1 transcripts (encoding the TCRδ chain of Vδ1+ γδ T cells) predict anti-PD-1 CPI response in patients with melanoma, particularly those harboring below average neoantigens. Moreover, using a protocol yielding substantial numbers of tissue-derived Vδ1+ cells, we show that PD-1+Vδ1+ cells display a transcriptomic program similar to, but distinct from, the canonical exhaustion program of colocated PD-1+CD8+ αß T cells. In particular, PD-1+Vδ1+ cells retained effector responses to TCR signaling that were inhibitable by PD-1 engagement and derepressed by CPI.

Functional Phenotypes of Human Vγ9Vδ2 T Cells in Lymphoid Stress Surveillance.

Butyrophilin and butyrophilin-like proteins select γδ T cells and direct the migration of γδ T cell subsets to distinct anatomical sites. γδ T cells expressing Vδ2 paired with Vγ9 (Vγ9Vδ2 T cells) are the predominant γδ T cell type in human peripheral blood. Vγ9Vδ2 T cells, which cannot be studied easily in vivo because they do not exist in rodents, are often referred to as innate-like T cells. The genetically recombined γδ T cell receptor (TCR) that responds to isoprenoid-derived pyrophosphates (phosphoantigens) produced by infected and malignant cells in a butyrophilin-dependent manner qualifies them as therapeutically relevant components of the adaptive immune system. On the other hand, cell-surface proteins such as the C-type lectin CD161 mark a functional phenotype of Vγ9Vδ2 T cells that mediates TCR-independent innate-like responses. Moreover, CD56 (neural cell adhesion molecule, NCAM) and the G protein-coupled receptor GPR56 define Vγ9Vδ2 T cells with increased cytolytic potential and, like CD161, may also be expressed by dendritic cells, principally facilitating the generation of an innate-like immunological synapse. In this review, we summarise current knowledge of Vγ9Vδ2 T cell functional phenotypes that are critical to lymphoid stress surveillance.

The emerging role of γδ T cells in cancer immunotherapy.

The recent successes of chimeric antigen receptor T cells in the treatment of hematological malignancies have clearly led to an explosion in the field of adoptive cell therapy for cancer. Current efforts are focused on the translation of this exciting technology to the treatment of solid tumors and the development of allogeneic 'off-the-shelf' therapies. γδ T cells are currently gaining considerable attention in this field as their unique biology and established role in cancer immunosurveillance place them in a unique position to potentially overcome these challenges in adoptive cell therapy. Here, we review the relevant aspects of the function of γδ T cells in cancer immunity, and summarize clinical observations and clinical trial results that highlight their emerging role as a platform for the development of safe and effective cancer immunotherapies.

The γδTCR combines innate immunity with adaptive immunity by utilizing spatially distinct regions for agonist selection and antigen responsiveness.

T lymphocytes expressing γδ T cell antigen receptors (TCRs) comprise evolutionarily conserved cells with paradoxical features. On the one hand, clonally expanded γδ T cells with unique specificities typify adaptive immunity. Conversely, large compartments of γδTCR+ intraepithelial lymphocytes (γδ IELs) exhibit limited TCR diversity and effect rapid, innate-like tissue surveillance. The development of several γδ IEL compartments depends on epithelial expression of genes encoding butyrophilin-like (Btnl (mouse) or BTNL (human)) members of the B7 superfamily of T cell co-stimulators. Here we found that responsiveness to Btnl or BTNL proteins was mediated by germline-encoded motifs within the cognate TCR variable γ-chains (Vγ chains) of mouse and human γδ IELs. This was in contrast to diverse antigen recognition by clonally restricted complementarity-determining regions CDR1-CDR3 of the same γδTCRs. Hence, the γδTCR intrinsically combines innate immunity and adaptive immunity by using spatially distinct regions to discriminate non-clonal agonist-selecting elements from clone-specific ligands. The broader implications for antigen-receptor biology are considered.

Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific γδ T Cell Compartments.

Many body surfaces harbor organ-specific γδ T cell compartments that contribute to tissue integrity. Thus, murine dendritic epidermal T cells (DETCs) uniquely expressing T cell receptor (TCR)-Vγ5 chains protect from cutaneous carcinogens. The DETC repertoire is shaped by Skint1, a butyrophilin-like (Btnl) gene expressed specifically by thymic epithelial cells and suprabasal keratinocytes. However, the generality of this mechanism has remained opaque, since neither Skint1 nor DETCs are evolutionarily conserved. Here, Btnl1 expressed by murine enterocytes is shown to shape the local TCR-Vγ7(+) γδ compartment. Uninfluenced by microbial or food antigens, this activity evokes the developmental selection of TCRαß(+) repertoires. Indeed, Btnl1 and Btnl6 jointly induce TCR-dependent responses specifically in intestinal Vγ7(+) cells. Likewise, human gut epithelial cells express BTNL3 and BTNL8 that jointly induce selective TCR-dependent responses of human colonic Vγ4(+) cells. Hence, a conserved mechanism emerges whereby epithelia use organ-specific BTNL/Btnl genes to shape local T cell compartments.

Ecto-ATPase CD39 Inactivates Isoprenoid-Derived Vγ9Vδ2 T Cell Phosphoantigens.

In humans, Vγ9Vδ2 T cells respond to self and pathogen-associated, diphosphate-containing isoprenoids, also known as phosphoantigens (pAgs). However, activation and homeostasis of Vγ9Vδ2 T cells remain incompletely understood. Here, we show that pAgs induced expression of the ecto-ATPase CD39, which, however, not only hydrolyzed ATP but also abrogated the γδ T cell receptor (TCR) agonistic activity of self and microbial pAgs (C5 to C15). Only mevalonate-derived geranylgeranyl diphosphate (GGPP, C20) resisted CD39-mediated hydrolysis and acted as a regulator of CD39 expression and activity. GGPP enhanced macrophage differentiation in response to the tissue stress cytokine interleukin-15. In addition, GGPP-imprinted macrophage-like cells displayed increased capacity to produce IL-1ß as well as the chemokine CCL2 and preferentially activated CD161-expressing CD4(+) T cells in an innate-like manner. Our studies reveal a previously unrecognized immunoregulatory function of CD39 and highlight a particular role of GGPP among pAgs.

Stress-related and homeostatic cytokines regulate Vγ9Vδ2 T-cell surveillance of mevalonate metabolism.

The potentially oncogenic mevalonate pathway provides building blocks for protein prenylation and induces cell proliferation and as such is an important therapeutic target. Among mevalonate metabolites, only isopentenyl pyrophosphate (IPP) has been considered to be an immunologically relevant antigen for primate-specific, innate-like Vγ9Vδ2 T cells with antitumor potential. We show here that Vγ9Vδ2 T cells pretreated with the stress-related, inflammasome-dependent cytokine interleukin 18 (IL-18) were potently activated not only by IPP but also by all downstream isoprenoid pyrophosphates that exhibit combined features of antigens and cell-extrinsic metabolic cues. Vγ9Vδ2 T cells induced this way effectively proliferated even under severe lymphopenic conditions and the antioxidant N-acetylcysteine significantly improved reconstitution of γδ T cells predominantly with a central memory phenotype. The homeostatic cytokine IL-15 induced the differentiation of effector cells in an antigen-independent fashion, which rapidly produced abundant interferon γ (IFNγ) upon antigen re-encounter. IL-15 induced effector γδ T cells displayed increased levels of the cytotoxic lymphocyte-associated proteins CD56, CD96, CD161 and perforin. In response to stimulation with isoprenoid pyrophosphates, these effector cells upregulated surface expression of CD107a and exhibited strong cytotoxicity against tumor cells in vitro. Our data clarify understanding of innate immunosurveillance mechanisms and will facilitate the controlled generation of robust Vγ9Vδ2 T cell subsets for effective cancer immunotherapy.

Essential requirements of zoledronate-induced cytokine and γδ T cell proliferative responses.

The potent nitrogen-containing bisphosphonate zoledronate inhibits farnesyl pyrophosphate synthase, a key enzyme of the mevalonate pathway that is often hyperactive in malignant cells. Zoledronate activates human Vγ9Vδ2 T cells, which are immune sentinels of cell stress and tumors, through upstream accumulation of the cognate Ag isopentenyl pyrophosphate. IL-18 was shown to enhance zoledronate-induced γδ T cell activation. Although monocytes have been considered important accessory cells that provide the Ag isopentenyl pyrophosphate, CD56(bright)CD11c(+) NK cells were postulated to mediate the costimulatory effects of IL-18. We report in this article that downstream depletion of geranylgeranyl pyrophosphate (GGPP), which is required for protein prenylation, caused cell stress in monocytes, followed by caspase-1-mediated maturation and release of IL-18, which, in turn, induced γδ T cell CCL2. Likewise, zoledronate caused a substantial delay in γδ T cell expansion, which could be skipped by GGPP supplementation. Moreover, repletion of GGPP, which prevented acute zoledronate toxicity, and supplementation with IL-18, which strongly upregulated IL-2Rα (CD25) and favored the central memory phenotype, were sufficient to enable zoledronate-induced expansion of highly purified γδ T cells, even when starting cell numbers were as low as 10(4) γδ T cells. Our study reveals essential components of γδ T cell activation and indicates that exogenous IL-18, which can directly costimulate γδ T cells, eliminates the need for any accessory cells. Our findings will facilitate the generation of robust γδ T cells from small blood or tissue samples for cancer immunotherapy and immune-monitoring purposes.

Regulation of mevalonate metabolism in cancer and immune cells.

The mevalonate pathway is a highly conserved metabolic cascade and provides isoprenoid building blocks for the biosynthesis of vital cellular products such as cholesterol or prenyl pyrophosphates that serve as substrates for the posttranslational prenylation of numerous proteins. The pathway, which is frequently hyperactive in cancer cells, is considered an important target in cancer therapy, since prenylated members of the Ras superfamily are crucially involved in the control of proliferation, survival, invasion and metastasis of tumour cells. Upstream accumulation and downstream depletion of mevalonate pathway intermediates as induced for instance by aminobisphosphonates translate into different effects in cancer and immune cells. Thus, mevalonate pathway regulation can affect tumour biology either directly or exhibit indirect antitumour effects through stimulating cancer immune surveillance. The present review summarizes major effects of pharmacologic mevalonate pathway regulation in cancer and immune cells that may collaboratively contribute to the efficacy of cancer therapy.

Novel aspects of mevalonate pathway inhibitors as antitumor agents.

The mevalonate pathway for cholesterol biosynthesis and protein prenylation has been implicated in various aspects of tumor development and progression. Certain classes of drugs, such as statins and bisphosphonates, inhibit mevalonate metabolism and therefore have also been tested as antitumor agents. This concept is strongly supported by the recent finding that mutant p53, which is present in more than half of all human cancers, can significantly upregulate mevalonate metabolism and protein prenylation in carcinoma cells. The first evidence that mevalonate pathway inhibitors may have the potential to reverse the malignant phenotype has already been obtained. Moreover, recently discovered immunomodulatory properties of statins and bisphosphonates may also contribute to their known anticancer effects. Drug-induced inhibition of protein prenylation may induce sequential cellular stress responses, including the unfolded protein response and autophagy, that eventually translate into inflammasome-dependent and caspase-1-mediated activation of innate immunity. This review focuses on these novel capabilities of mevalonate pathway inhibitors to beneficially affect tumor biology and contribute to tumor immune surveillance.

DC-like cell-dependent activation of human natural killer cells by the bisphosphonate zoledronic acid is regulated by γδ T lymphocytes.

Bisphosphonates are mainly used for the inhibition of osteoclast-mediated bone resorption but also have been shown to induce γδ T-cell activation. Using IL-2-primed cultures of CD56(+) peripheral blood mononuclear cells, we show here that zoledronic acid (zoledronate) could induce IFN-γ production not only in γδ T lymphocytes but, surprisingly, also in natural killer (NK) cells in a manner that depended on antigen-presenting cells, which share properties of inflammatory monocytes and dendritic cells (DCs; here referred to as DC-like cells). In the presence of γδ T lymphocytes, DC-like cells were rapidly eliminated, and NK cell IFN-γ production was silenced. Conversely, in the absence of γδ T lymphocytes, DC-like cells were spared, allowing NK cell IFN-γ production to proceed. γδ T cell-independent NK cell activation in response to zoledronate was because of downstream depletion of endogenous prenyl pyrophosphates and subsequent caspase-1 activation in DC-like cells, which then provide mature IL-18 and IL-1ß for the activation of IL-2-primed NK cells. Pharmacologic inhibition of caspase-1 almost abolished IFN-γ production in NK cells and γδ T lymphocytes, indicating that caspase-1-mediated cytokine maturation is the crucial mechanism underlying innate lymphocyte activation in response to zoledronate.

Real-Time Live Confocal Fluorescence Microscopy as a New Tool for Assessing Platelet Vitality.

BACKGROUND: Assessment of platelet vitality is important for patients presenting with inherited or acquired disorders of platelet function and for quality assessment of platelet concentrates. METHODS: Herein we combined live stains with intra-vital confocal fluorescence microscopy in order to obtain an imaging method that allows fast and accurate assessment of platelet vitality. Three fluorescent dyes, FITC-coupled wheat germ agglutinin (WGA), tetramethylrhodamine methyl ester perchlorate (TMRM) and acetoxymethylester (Rhod-2), were used to assess platelet morphology, mitochondrial activity and intra-platelet calcium levels. Microscopy was performed with a microlens-enhanced Nipkow spinning disk-based system allowing live confocal imaging. RESULTS: Comparison of ten samples of donor platelets collected before apheresis and platelets collected on days 5 and 7 of storage showed an increase in the percentage of Rhod-2-positive platelets from 3.6 to 47 and finally to 71%. Mitochondrial potential was demonstrated in 95.4% of donor platelets and in 92.5% of platelets stored for 7 days. CONCLUSION: Such fast and accurate visualization of known key parameters of platelet function could be of relevance for studies addressing the quality of platelets after storage and additional manipulation, such as pathogen inactivation, as well as for the analysis of inherited platelet function disorders.

IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells.

Statins are inhibitors of cholesterol biosynthesis and protein prenylation that also have been studied in cancer therapy and chemoprevention. With regard to natural killer (NK) cells, only inhibitory effects of statins such as suppression of granule exocytosis have been reported so far. In this study, we show that statins can cooperate with IL-2 to potently induce the activation of CD56(dim) NK cells in a synergistic, time- and dose-dependent fashion. Supplementation experiments revealed that the statin effect was specific to inhibition of their target hydroxymethylglutaryl coenzyme A reductase and that downstream depletion of geranylgeranyl pyrophosphate was responsible for cooperating with IL-2 in NK cell activation. Mechanistic studies revealed that CD56(+)HLA-DR(+)CD14(+) dendritic cell (DC)-like accessory cells mediated the ability of statin to activate NK cells. In contrast, BDCA-1(+) (CD1c(+)) myeloid DCs, which partially expressed CD56, were somewhat less potent. Conventional blood monocytes, which lack CD56, exhibited the lowest accessory cell capacity. NK cell IFN-γ production was IL-12 independent but required endogenous IL-18, IL-1ß, and caspase-1 activity. Statins directly induced apoptosis in human cancer cell lines and cooperated with NK cell-derived IFN-γ to generate potent cytotoxic antitumor effects in vitro even in the presence of statin-mediated inhibitory effects on granule exocytosis. Our work reveals novel and unexpected immunomodulatory properties of statins, which might be harnessed for the treatment of cancer.