Natural killer cells induce distinct modes of cancer cell death: Discrimination, quantification, and modulation of apoptosis, necrosis, and mixed forms.

Immune therapy of cancer is among the most promising recent advances in medicine. Whether the immune system can keep cancer in check depends on, among other factors, the efficiency of immune cells to recognize and eliminate cancer cells. We describe a time-resolved single-cell assay that reports the quality, quantity, and kinetics of target cell death induced by single primary human natural killer (NK) cells. The assay reveals that single NK cells induce cancer cell death by apoptosis and necrosis but also by mixed forms. Inhibition of either one of the two major cytotoxic pathways, perforin/granzyme release or FasL/FasR interaction, unmasked the parallel activity of the other one. Ca2+ influx through Orai channels is important for tuning killer cell function. We found that the apoptosis/necrosis ratio of cancer cell death by NK cells is controlled by the magnitude of Ca2+ entry and furthermore by the relative concentrations of perforin and granzyme B. The possibility to change the apoptosis/necrosis ratio employed by NK cells offers an intriguing possibility to modulate the immunogenicity of the tumor microenvironment.

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.

NFATc1 controls the cytotoxicity of CD8+ T cells.

Cytotoxic T lymphocytes are effector CD8+ T cells that eradicate infected and malignant cells. Here we show that the transcription factor NFATc1 controls the cytotoxicity of mouse cytotoxic T lymphocytes. Activation of Nfatc1 -/- cytotoxic T lymphocytes showed a defective cytoskeleton organization and recruitment of cytosolic organelles to immunological synapses. These cells have reduced cytotoxicity against tumor cells, and mice with NFATc1-deficient T cells are defective in controlling Listeria infection. Transcriptome analysis shows diminished RNA levels of numerous genes in Nfatc1 -/- CD8+ T cells, including Tbx21, Gzmb and genes encoding cytokines and chemokines, and genes controlling glycolysis. Nfatc1 -/- , but not Nfatc2 -/- CD8+ T cells have an impaired metabolic switch to glycolysis, which can be restored by IL-2. Genome-wide ChIP-seq shows that NFATc1 binds many genes that control cytotoxic T lymphocyte activity. Together these data indicate that NFATc1 is an important regulator of cytotoxic T lymphocyte effector functions.NFAT nuclear translocation has been shown to be required for CD8+ T cell cytokine production in response to viral infection. Here the authors show NFATc1 controls the cytotoxicity and metabolic switching of activated CD8+ T cells required for optimal response to bacteria and tumor cells.

The extracellular adherence protein (Eap) of Staphylococcus aureus acts as a proliferation and migration repressing factor that alters the cell morphology of keratinocytes.

Staphyloccocus aureus is a major human pathogen and a common cause for superficial and deep seated wound infections. The pathogen is equipped with a large arsenal of virulence factors, which facilitate attachment to various eukaryotic cell structures and modulate the host immune response. One of these factors is the extracellular adherence protein Eap, a member of the "secretable expanded repertoire adhesive molecules" (SERAM) protein family that possesses adhesive and immune modulatory properties. The secreted protein was previously shown to impair wound healing by interfering with host defense and neovascularization. However, its impact on keratinocyte proliferation and migration, two major steps in the re-epithelialization process of wounds, is not known. Here, we report that Eap affects the proliferation and migration capacities of keratinocytes by altering their morphology and adhesive properties. In particular, treatment of non-confluent HaCaT cell cultures with Eap resulted in cell morphology changes as well as a significant reduction in cell proliferation and migration. Eap-treated HaCaT cells changed their appearance from an oblong via a trapezoid to an astral-like shape, accompanied by decreases in cell volume and cell stiffness, and exhibited significantly increased cell adhesion. Eap had a similar influence on endothelial and cancer cells, indicative for a general effect of Eap on eukaryotic cell morphology and functions. Specifically, Eap was found to interfere with growth factor-stimulated activation of the mitogen-activated protein kinase (MAPK) pathway that is known to be responsible for cell shape modulation, induction of proliferation and migration of epithelial cells. Western blot analyses revealed that Eap blocked the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2) in keratinocyte growth factor (KGF)-stimulated HaCaT cells. Together, these data add another antagonistic mechanism of Eap in wound healing, whereby the bacterial protein interferes with keratinocyte migration and proliferation.

How ORAI and TRP channels interfere with each other: interaction models and examples from the immune system and the skin.

Four types of Ca(2+) selective ion channels are known, ten voltage gated Ca(2+) (CaV) channels, four CatSper channels, three store operated CRAC channels (ORAI channels) and at least two members of the TRPV subfamily (TRPV5, TRPV6). Some of the other TRP channels also show some Ca(2+) selectivity like certain splice variants of TRPM3. In addition to Ca(2+) selective channels, various cation channels play an important role for Ca(2+) entry and furthermore, they may also regulate Ca(2+) entry through other channels by modulating the membrane potential or other means as outlined in this review. Of the different types of cation channels, TRP channels form one of the most prominent families of non-selective cation channels with functional relevance in electrically non-excitable and electrically excitable cell types. Among these, the seven channels of the TRPC subfamily are rather non-selective with very modest Ca(2+) selectivity, whereas in the other subfamilies, cation selectivity ranges from monovalent selectivity (i.e. TRPM4, TRPM5) to divalent selectivity (i.e. TRPM6, TRPM7) or Ca(2+) selectivity (i.e. TRPV5, TRPV6). Rather than discussing the heavily reviewed individual functions of ORAI or TRP channels, we summarize data and present models how TRP and ORAI may functionally interact to guide cellular functions. We focus on T lymphocytes representing a more ORAI-dominated tissue and skin as model system in which both ORAI and TRP channel have been reported to control relevant functions. We present several interaction models how ORAI and TRP may interfere with each other's function.