Oliver Nussbaumer

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. Clin Cancer Res; 18(13); 3524–31. ©2012 AACR.

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

Summary 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 TCRαβ+ 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.

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-1β 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.

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.

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-1β, 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.

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, CD56brightCD11c+ 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 104 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.

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.

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-2positive 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.

Abstract IA20: Targeting molecules mediating the dialogue between epithelial cells and local T cells

Some extraordinary clinical successes of immunotherapy have had game-changing effects on the way we view cancer biology and the interaction of the immune system with our tissues. The conventional context for considering cancer immunology is one in which dendritic cells and/or monocytes present cancer antigens, particularly neo-antigens generated by somatic mutation, to systemic T cells within cancer-draining lymph nodes, as a result of which an expansive adaptive, cancer-specific T lymphocyte-driven immune response develops. Furthermore, cancer-specific T cells may become tissue-associated, maintaining a so-called Tissue-Resident Memory (TRM) pool. However, although this perspective readily explains many basic biological and clinical data, it leaves several issues unanswered. For example, does the immune system recognize tumor cells that do not carry high mutational loads, many of which cancers have shown disappointing responses to immunotherapy? Related to this, what is the nature of cancer immunogenicity that is required to first activate the immune system, thereby promoting the presentation of cancer antigens by DC and/ or monocyte? We have addressed these issues by considering a separate T cell compartment that is constitutively tissue-resident, rather than being primed in the lymphoid circulation. Prominent within this T cell compartment are intraepithelial gamma delta T cells that have been particularly well characterized in mice, wherein they have been shown to limit sensitivity to carcinogens, and to promote tissue integrity 1,2 . Moreover, a recent bio-informatic analysis showed a tumor-associated gamma delta T cell signature to be the strongest gene expression correlate with overall survival in over 18,000 patients across 39 tumor types 3 . Furthermore, another very strong correlate was the expression of NKG2D, a cell surface receptor expressed on tissue-resident gamma delta T cells and whose ligands are upregulated on tumor cells by dysregulation of the EGF-receptor pathway 4 . Given these observations, we have asked several questions: [1] what is the nature of tissue-resident gamma delta T cell compartments in human skin, gut, and breast tissue? [2] do such cells become activated solely by the upregulation of NKG2D ligands, thereby offering an explanation for tumor immunogenicity? [3] what are the effector functions and potentials of such cells? [4] what are the molecular checkpoints by which the cells are regulated? [5] can the cells be explanted, grown, and cultured in a manner that preserves their function(s) and that can provide a source of such cells to be used for adoptive immunotherapy? [6] might such cells be applied “off-the-shelf” rather than requiring autologous transfusions? Emerging answers to these questions will be provided. Thus, by examining over 100 donors, we have found that human skin, gut, and breast reproducibly harbor large and complex T cell subsets that include substantive gamma delta T cell compartments clearly distinct from most blood-borne gd T cells. These tissue-resident gamma delta T cells show strong, innate-like responses to NKG2D-ligands and to cytokines that were not shown by CD8 + TRM cells harvested in parallel from the same sites. Additionally, however, tissue-resident gamma delta T cells are uniquely and powerfully regulated at steady-state by novel, CD80/B7/PDL1-like molecules known as butyrophilin-like (BTNL) proteins that are expressed by epithelial cells in a tissue-specific fashion, and that de facto constitute local checkpoints. These emerging insights create a new context in which to consider the interaction of immune cells with developing cancers, and possibly to explain the patient benefit of a strong gamma delta T cell signature. At the same time, we have developed novel methods for maintaining and expanding tissue-derived gamma delta T cells that offer a practical off-the-shelf approach to tissue-targeted cellular immunotherapy, and that may be particularly suited to adenocarcinomas of low mutation load or with high rates of tumor evolution and antigenic drift. Citation Format: Oliver Nussbaumer, Yin Wu, Fernanda Kyle, Rick Woolf, Robin Dart, Deborah Enting, Pierre Vantourout, Adrian Hayday. Targeting molecules mediating the dialogue between epithelial cells and local T cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Targeting the Vulnerabilities of Cancer; May 16-19, 2016; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(1_Suppl):Abstract nr IA20.

Potential for innate-like responsiveness of resident T cells in human skin: a new perspective on tissue immune-surveillance

Abstract Background The skin contains many resident immune cells that are key for tissue homoeostasis. In mouse skin, a distinct population of T cells have the innate-like capacity to respond rapidly to markers of tissue stress, rather than requiring clonal, antigen-specific activation. This lymphoid stress surveillance response (LSSR) is key to host immunity, contributing to cancer immunosurveillance and cutaneous atopy. We sought to identify whether the potential for LSSR exists in human skin. Methods We adapted a novel skin explant protocol to study the skin-resident lymphocyte compartments of over 100 healthy donors. Normal adult skin was obtained as discarded material after cutaneous surgery. Skin-resident lymphocytes were characterised by flow cytometry, and their functional potentials assessed in vitro with different models of activation. Ethics approval was granted by a local research ethics committee (REC 06/Q0704/18) Findings We established that human skin contains a large reproducible population of γδ T cells, which were distinct from γδ T cells in the blood. These cells displayed features of tissue-resident memory T cell receptor (TCR) αβ+ cells, but strikingly had the capacity to become activated by ligands for the NKG2D receptor, independent of the TCR, which is analogous to innate-like T cells in mouse skin. Such responsiveness was not seen in other NKG2D+ T cells. Upon NKG2D-mediated activation, skin-resident γδ T cells showed robust T helper-1-like and cytotoxic effector responses, and in some donors interleukin 13 production. These cells also displayed profound cytolytic activity against transformed epithelial cells in vitro, in part mediated via NKG2D. Interpretation To our knowledge, these data provide the first clear identification of human tissue-resident innate-like T cells, which strongly argues for the existence of LSSR in human tissues and places skin-resident γδ T cells in the early afferent phase of the immune response. This finding offers a new perspective on tissue immune surveillance, possibly representing a common feature of barrier tissues, and has implications for future studies in human skin immunobiology. Funding Medical Research Council, Wellcome Trust, Cancer Research UK.