Interdependence of sequential cytotoxic T lymphocyte and natural killer cell cytotoxicity against melanoma cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells recognize and eliminate cancer cells. However, immune evasion, downregulation of immune function by the tumour microenvironment and resistance of cancer cells are major problems. Although CTL and NK cells are both important to eliminate cancer, most studies address them individually. We quantified sequential primary human CTL and NK cell cytotoxicity against the melanoma cell line SK-Mel-5. At high effector-to-target ratios, NK cells or melan-A (MART-1)-specific CTL eliminated all SK-Mel-5 cells within 24 h, indicating that SK-Mel-5 cells are not resistant initially. However, at lower effector-to-target ratios, which resemble numbers of the immune contexture in human cancer, a substantial number of SK-Mel-5 cells survived. Pre-exposure to CTL induced resistance in surviving SK-Mel-5 cells to subsequent CTL or NK cell cytotoxicity, and pre-exposure to NK cells induced resistance in surviving SK-Mel-5 cells to NK cells. Higher human leucocyte antigen class I expression or interleukin-6 levels were correlated with resistance to NK cells, whereas reduction in MART-1 antigen expression was correlated with reduced CTL cytotoxicity. The CTL cytotoxicity was rescued beyond control levels by exogenous MART-1 antigen. In contrast to the other three combinations, CTL cytotoxicity against SK-Mel-5 cells was enhanced following NK cell pre-exposure. Our assay allows quantification of sequential CTL and NK cell cytotoxicity and might guide strategies for efficient CTL-NK cell anti-melanoma therapies. KEY POINTS: Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells eliminate cancer cells. Both CTL and NK cells attack the same targets, but most studies address them individually. In a sequential cytotoxicity model, the interdependence of antigen-specific CTL and NK cell cytotoxicity against melanoma is quantified. High numbers of antigen-specific CTL and NK cells eliminate all melanoma cells. However, lower numbers induce resistance if secondary CTL or NK cell exposure follows initial CTL exposure or if secondary NK cell exposure follows initial NK cell exposure. On the contrary, if secondary CTL exposure follows initial NK cell exposure, cytotoxicity is enhanced. Alterations in human leucocyte antigen class I expression and interleukin-6 levels are correlated with resistance to NK cells, whereas a reduction in antigen expression is correlated with reduced CTL cytotoxicity; CTL cytotoxicity is rescued beyond control levels by exogenous antigen. This assay and the results on interdependencies will help us to understand and optimize immune therapies against cancer.

MCU controls melanoma progression through a redox-controlled phenotype switch.

Melanoma is the deadliest of skin cancers and has a high tendency to metastasize to distant organs. Calcium and metabolic signals contribute to melanoma invasiveness; however, the underlying molecular details are elusive. The MCU complex is a major route for calcium into the mitochondrial matrix but whether MCU affects melanoma pathobiology was not understood. Here, we show that MCUA expression correlates with melanoma patient survival and is decreased in BRAF kinase inhibitor-resistant melanomas. Knockdown (KD) of MCUA suppresses melanoma cell growth and stimulates migration and invasion. In melanoma xenografts, MCUA_KD reduces tumor volumes but promotes lung metastases. Proteomic analyses and protein microarrays identify pathways that link MCUA and melanoma cell phenotype and suggest a major role for redox regulation. Antioxidants enhance melanoma cell migration, while prooxidants diminish the MCUA_KD -induced invasive phenotype. Furthermore, MCUA_KD increases melanoma cell resistance to immunotherapies and ferroptosis. Collectively, we demonstrate that MCUA controls melanoma aggressive behavior and therapeutic sensitivity. Manipulations of mitochondrial calcium and redox homeostasis, in combination with current therapies, should be considered in treating advanced melanoma.

Lrig1- and Wnt-dependent niches dictate segregation of resident immune cells and melanocytes in murine tail epidermis.

The barrier-forming, self-renewing mammalian epidermis comprises keratinocytes, pigment-producing melanocytes and resident immune cells as first-line host defense. In murine tail skin, interfollicular epidermis patterns into pigmented 'scale' and hypopigmented 'interscale' epidermis. Why and how mature melanocytes accumulate in scale epidermis is unresolved. Here, we delineate a cellular hierarchy among epidermal cell types that determines skin patterning. Already during postnatal development, melanocytes co-segregate with newly forming scale compartments. Intriguingly, this process coincides with partitioning of both Langerhans cells and dendritic epidermal T cells to interscale epidermis, suggesting functional segregation of pigmentation and immune surveillance. Analysis of non-pigmented mice and of mice lacking melanocytes or resident immune cells revealed that immunocyte patterning is melanocyte and melanin independent and, vice versa, immune cells do not control melanocyte localization. Instead, genetically enforced progressive scale fusion upon Lrig1 deletion showed that melanocytes and immune cells dynamically follow epithelial scale:interscale patterns. Importantly, disrupting Wnt-Lef1 function in keratinocytes caused melanocyte mislocalization to interscale epidermis, implicating canonical Wnt signaling in organizing the pigmentation pattern. Together, this work uncovers cellular and molecular principles underlying the compartmentalization of tissue functions in skin.

Protein Signatures of NK Cell-Mediated Melanoma Killing Predict Response to Immunotherapies.

Despite impressive advances in melanoma-directed immunotherapies, resistance is common and many patients still succumb to metastatic disease. In this context, harnessing natural killer (NK) cells, which have thus far been sidelined in the development of melanoma immunotherapy, could provide therapeutic benefits for cancer treatment. To identify molecular determinants of NK cell-mediated melanoma killing (NKmK), we quantified NK-cell cytotoxicity against a panel of genetically diverse melanoma cell lines and observed highly heterogeneous susceptibility. Melanoma protein microarrays revealed a correlation between NKmK and the abundance and activity of a subset of proteins, including several metabolic factors. Oxidative phoshorylation, measured by oxygen consumption rate, negatively correlated with melanoma cell sensitivity toward NKmK, and proteins involved in mitochondrial metabolism and epithelial-mesenchymal transition were confirmed to regulate NKmK. Two- and three-dimensional killing assays and melanoma xenografts established that the PI3K/AKT/mTOR signaling axis controls NKmK via regulation of NK cell-relevant surface proteins. A "protein-killing-signature" based on the protein analysis predicted NKmK of additional melanoma cell lines and the response of patients with melanoma to anti-PD-1 checkpoint therapy. Collectively, these findings identify novel NK cell-related prognostic biomarkers and may contribute to improved and personalized melanoma-directed immunotherapies. SIGNIFICANCE: NK-cell cytotoxicity assays and protein microarrays reveal novel biomarkers of NK cell-mediated melanoma killing and enable development of signatures to predict melanoma patient responsiveness to immunotherapies.

Oxidative Stress-Induced STIM2 Cysteine Modifications Suppress Store-Operated Calcium Entry.

Store-operated calcium entry (SOCE) through STIM-gated ORAI channels governs vital cellular functions. In this context, SOCE controls cellular redox signaling and is itself regulated by redox modifications. However, the molecular mechanisms underlying this calcium-redox interplay and the functional outcomes are not fully understood. Here, we examine the role of STIM2 in SOCE redox regulation. Redox proteomics identify cysteine 313 as the main redox sensor of STIM2 in vitro and in vivo. Oxidative stress suppresses SOCE and calcium currents in cells overexpressing STIM2 and ORAI1, an effect that is abolished by mutation of cysteine 313. FLIM and FRET microscopy, together with MD simulations, indicate that oxidative modifications of cysteine 313 alter STIM2 activation dynamics and thereby hinder STIM2-mediated gating of ORAI1. In summary, this study establishes STIM2-controlled redox regulation of SOCE as a mechanism that affects several calcium-regulated physiological processes, as well as stress-induced pathologies.

Redox signals at the ER-mitochondria interface control melanoma progression.

Reactive oxygen species (ROS) are emerging as important regulators of cancer growth and metastatic spread. However, how cells integrate redox signals to affect cancer progression is not fully understood. Mitochondria are cellular redox hubs, which are highly regulated by interactions with neighboring organelles. Here, we investigated how ROS at the endoplasmic reticulum (ER)-mitochondria interface are generated and translated to affect melanoma outcome. We show that TMX1 and TMX3 oxidoreductases, which promote ER-mitochondria communication, are upregulated in melanoma cells and patient samples. TMX knockdown altered mitochondrial organization, enhanced bioenergetics, and elevated mitochondrial- and NOX4-derived ROS. The TMX-knockdown-induced oxidative stress suppressed melanoma proliferation, migration, and xenograft tumor growth by inhibiting NFAT1. Furthermore, we identified NFAT1-positive and NFAT1-negative melanoma subgroups, wherein NFAT1 expression correlates with melanoma stage and metastatic potential. Integrative bioinformatics revealed that genes coding for mitochondrial- and redox-related proteins are under NFAT1 control and indicated that TMX1, TMX3, and NFAT1 are associated with poor disease outcome. Our study unravels a novel redox-controlled ER-mitochondria-NFAT1 signaling loop that regulates melanoma pathobiology and provides biomarkers indicative of aggressive disease.

A calcium optimum for cytotoxic T lymphocyte and natural killer cell cytotoxicity.

KEY POINTS: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to eliminate cancer cells. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity and found that in particular CTLs have a very low optimum of [Ca2+ ]i (between 122 and 334 nm) and [Ca2+ ]o (between 23 and 625 µm) for efficient cancer cell elimination, well below blood plasma Ca2+ levels. As predicted from these results, partial down-regulation of the Ca2+ channel Orai1 in CTLs paradoxically increases perforin-dependent cancer cell killing. Lytic granule release at the immune synapse between CTLs and cancer cells has a Ca2+ optimum compatible with this low Ca2+ optimum for efficient cancer cell killing, whereas the Ca2+ optimum for CTL migration is slightly higher and proliferation increases monotonously with increasing [Ca2+ ]o . We propose that a partial inhibition of Ca2+ signals by specific Orai1 blockers at submaximal concentrations could contribute to tumour elimination. ABSTRACT: Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are required to protect the human body against cancer. Ca2+ is a key metabolic factor for lymphocyte function and cancer homeostasis. We analysed the Ca2+ dependence of CTL and NK cell cytotoxicity against cancer cells and found that CTLs have a bell-shaped Ca2+ dependence with an optimum for cancer cell elimination at rather low [Ca2+ ]o (23-625 µm) and [Ca2+ ]i (122-334 nm). This finding predicts that a partial inhibition of Orai1 should increase (rather than decrease) cytotoxicity of CTLs at [Ca2+ ]o higher than 625 µm. We tested this hypothesis in CTLs and indeed found that partial down-regulation of Orai1 by siRNA increases the efficiency of cancer cell killing. We found two mechanisms that may account for the Ca2+ optimum of cancer cell killing: (1) migration velocity and persistence have a moderate optimum between 500 and 1000 µm [Ca2+ ]o in CTLs, and (2) lytic granule release at the immune synapse between CTLs and cancer cells is increased at 146 µm compared to 3 or 800 µm, compatible with the Ca2+ optimum for cancer cell killing. It has been demonstrated in many cancer cell types that Orai1-dependent Ca2+ signals enhance proliferation. We propose that a decrease of [Ca2+ ]o or partial inhibition of Orai1 activity by selective blockers in the tumour microenvironment could efficiently reduce cancer growth by simultaneously increasing CTL and NK cell cytotoxicity and decreasing cancer cell proliferation.

Measuring Mitochondrial ROS in Mammalian Cells with a Genetically Encoded Protein Sensor.

Reactive oxygen species (ROS) are not only known for their toxic effects on cells, but they also play an important role as second messengers. As such, they control a variety of cellular functions such as proliferation, metabolism, differentiation and apoptosis. Thus, ROS are involved in the regulation of multiple physiological and pathophysiological processes. It is now apparent that there are transient and local changes in ROS in the cell; in so-called 'microdomains' or in specific cellular compartments, which affect signaling events. These ROS hotspots need to be studied in more depth to understand their function and regulation. Therefore, it is necessary to identify and quantify redox signals in single cells with high spatial and temporal resolution. Genetically encoded fluorescence-based protein sensors provide such necessary tools to examine redox-signaling processes. A big advantage of these sensors is the possibility to target them specifically. Mitochondria are essential for energy metabolism and are one of the major sources of ROS in mammalian cells. Therefore, the evaluation of redox potential and ROS production in these organelles is of great interest. Herein, we provide a protocol for the real-time visualization of mitochondrial hydrogen peroxide (H2O2) using the H2O2-specific ratiometric sensor mitoHyPer in adherent mammalian cells.

Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription.

The channel Orai1 requires Ca2+ store depletion in the endoplasmic reticulum and an interaction with the Ca2+ sensor STIM1 to mediate Ca2+ signaling. Alterations in Orai1-mediated Ca2+ influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca2+ influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca2+ permeation associated with gene transcription and provide a gating mechanism for Orai1.