Human NK cells responses are enhanced by CD56 engagement.

Natural Killer (NK) cells are important innate lymphocytes for effective immune responses against intracellular pathogens and tumors. CD56 is a well-known marker for human NK cells, but there is very limited information about a functional role of this surface receptor. Here, we show that engagement of CD56 can induce NK cell activation resulting in degranulation, IFN-γ secretion and morphological changes, making CD56 a potential co-activating receptor in NK cells. Interestingly, this effect was only observed in cytokine pre-activated and not in freshly isolated human NK cells, demonstrating that NK cell reactivity upon CD56 engagement was dependent on cytokine stimulation. Inhibition of Syk, PI3K, Erk, and src-family-kinases impaired CD56-mediated NK cell stimulation. Finally, we used CRISPR/Cas9 to delete CD56 from primary human NK cells. While this abolished the stimulatory effect of CD56 on pre-activated NK cells, the cytotoxic activity of NK cells against several tumor target cells was not affected by the absence of CD56. This demonstrates that the stimulating effect of CD56 on pre-activated NK cells does not have a major impact on their cytotoxic activity, but it may contribute to the function of CD56 as a fungal recognition receptor and in the NK cell developmental synapse.

Dopamine induces in vitro migration of synovial fibroblast from patients with rheumatoid arthritis.

Preventing synovial fibroblast (SF) migration into the adjacent cartilage is a desirable therapeutic target in rheumatoid arthritis (RA). As previous studies demonstrated that RASF and SF from osteoarthritis (OA) patients express dopamine receptors (DR), aim of the present study was to investigate the impact of dopamine on mobility of fibroblasts from patients with chronic arthritides. Synovial tissue and fibroblasts were obtained from RA and OA patients. Immunohistochemistry was performed for all DR-subtypes in the invasion zone. Migration- and motility-assays were performed under DR-stimulation. Cytokines were evaluated using ELISA. Expression of DRs was evaluated by flow cytometry, and DR activation was measured by xCELLigence real-time analysis. All DRs were expressed in RA invasion zone. Migration and motility of RASF and OASF were increased after DR stimulation in patients ≤ 75 years old. Synovial fibroblasts from older RA patients (> 75 years old) expressed lower levels of D1-, D2- and D4-DR than patients ≤ 75 years old. DR activation was not altered in older patients. Our results suggest a possible involvement of dopamine on migration of fibroblasts from arthritis patients. Therefore, the synovial dopaminergic pathway might represent a potential therapeutic target to interfere with progressive joint damage in RA patients.

Enhanced activation of human NK cells by drug-exposed hepatocytes.

Drug-induced liver injury (DILI) represents one of the major causes why drugs have to be withdrawn from the market. In this study, we describe a new interaction between drug-exposed hepatocytes and natural killer (NK) cells. In a previous genome-wide expression analysis of primary human hepatocytes that had been exposed to clinically relevant concentrations of 148 drugs, we found that several activating ligands for NK cell receptors were regulated by various drugs (e.g., valproic acid, ketoconazole, promethazine, isoniazid). Especially expression of the activating NKG2D ligands (MICA, MICB and ULBPs) and the NKp30 ligand B7-H6 were upregulated in primary human hepatocytes upon exposure to many different drugs. Using the human hepatocyte cell lines Huh7 and HepG2, we confirmed that protein levels of activating NK cell ligands were elevated after drug exposure. Hepatocyte cell lines or primary human hepatocytes co-cultivated with NK cells caused enhanced NK cell activation after pretreatment with drugs at in vivo relevant concentrations compared to solvent controls. Enhanced NK cell activation was evident by increased cytotoxicity against hepatocytes and interferon (IFN)-γ production. NK cell activation could be blocked by specific antibodies against activating NK cell receptors. These data support the hypothesis that NK cells can modulate drug-induced liver injury by direct interaction with hepatocytes resulting in cytotoxicity and IFN-γ production.

NK cells switch from granzyme B to death receptor-mediated cytotoxicity during serial killing.

NK cells eliminate virus-infected and tumor cells by releasing cytotoxic granules containing granzyme B (GrzB) or by engaging death receptors that initiate caspase cascades. The orchestrated interplay between both cell death pathways remains poorly defined. Here we simultaneously measure the activities of GrzB and caspase-8 in tumor cells upon contact with human NK cells. We observed that NK cells switch from inducing a fast GrzB-mediated cell death in their first killing events to a slow death receptor-mediated killing during subsequent tumor cell encounters. Target cell contact reduced intracellular GrzB and perforin and increased surface-CD95L in NK cells over time, showing how the switch in cytotoxicity pathways is controlled. Without perforin, NK cells were unable to perform GrzB-mediated serial killing and only killed once via death receptors. In contrast, the absence of CD95 on tumor targets did not impair GrzB-mediated serial killing. This demonstrates that GrzB and death receptor-mediated cytotoxicity are differentially regulated during NK cell serial killing.

SLAM family receptors in natural killer cells - Mediators of adhesion, activation and inhibition via cis and trans interactions.

SLAM family receptors are important for the fine-tuning of immune reactions. Their expression is restricted to cells of hematopoietic origin and most SLAM family receptors are their own ligand. Here we review how these receptors are involved in regulating the functions of Natural Killer (NK) cells. We discuss that promoting cellular adhesion may be a main function of SLAM family receptors in NK cells. The homophilic interactions of SLAM family receptors can not only occur in trans between different cells, but also in cis on the surface of the same cell. This cis interaction additionally modulates the function of the receptors and subsequently affects the activities of NK cells. Finally, SLAM-family receptors can also mediate inhibitory signals under certain conditions. These inhibitory signals can contribute to the functional maturation of NK cells during NK cell education. Therefore, SLAM family receptors are critically involved in many aspects of NK cell functionality.

Impedance-based analysis of Natural Killer cell stimulation.

The use of impedance-based label free cell analysis is increasingly popular and has many different applications. Here, we report that a real-time cell analyzer (RTCA) can be used to study the stimulation of Natural Killer (NK) cells. Engagement of NK cells via plate-bound antibodies directed against different activating surface receptors could be measured in real time using the label-free detection of impedance. The change in impedance was dependent on early signal transduction events in the NK cells as it was blocked by inhibitors of Src-family kinases and by inhibiting actin polymerization. While CD16 was the only receptor that could induce a strong change in impedance in primary NK cells, several activating receptors induced changes in impedance in expanded NK cells. Using PBMCs we could detect T cell receptor-mediated T cell activation and CD16-mediated NK cell activation in the same sample. Performing a dose-response analysis for the Src-family kinases inhibitor PP1 we show that T cells are more sensitive to inhibition compared to NK cells. Our data demonstrate that the RTCA can be used to detect physiological activation events in NK cells in a label-free and real-time fashion.

Differential Requirements for Src-Family Kinases in SYK or ZAP70-Mediated SLP-76 Phosphorylation in Lymphocytes.

In a synthetic biology approach using Schneider (S2) cells, we show that SLP-76 is directly phosphorylated at tyrosines Y113 and Y128 by SYK in the presence of ITAM-containing adapters such as CD3ζ, DAP12, or FcεRγ. This phosphorylation was dependent on at least one functional ITAM and a functional SH2 domain within SYK. Inhibition of Src-kinases by inhibitors PP1 and PP2 did not reduce SLP-76 phosphorylation in S2 cells, suggesting an ITAM and SYK dependent, but Src-kinase independent signaling pathway. This direct ITAM/SYK/SLP-76 signaling pathway therefore differs from previously described ITAM signaling. However, the SYK-family kinase ZAP70 required the additional co-expression of the Src-family kinases Fyn or Lck to efficiently phosphorylate SLP-76 in S2 cells. This difference in Src-family kinase dependency of SYK versus ZAP70-mediated ITAM-based signaling was further demonstrated in human lymphocytes. ITAM signaling in ZAP70-expressing T cells was dependent on the activity of Src-family kinases. In contrast, Src-family kinases were partially dispensable for ITAM signaling in SYK-expressing B cells or in natural killer cells, which express SYK and ZAP70. This demonstrates that SYK can signal using a Src-kinase independent ITAM-based signaling pathway, which may be involved in calibrating the threshold for lymphocyte activation.

Natural Killer Cells and Liver Fibrosis.

In the 40 years since the discovery of natural killer (NK) cells, it has been well established that these innate lymphocytes are important for early and effective immune responses against transformed cells and infections with different pathogens. In addition to these classical functions of NK cells, we now know that they are part of a larger family of innate lymphoid cells and that they can even mediate memory-like responses. Additionally, tissue-resident NK cells with distinct phenotypical and functional characteristics have been identified. Here, we focus on the phenotype of different NK cell subpopulations that can be found in the liver and summarize the current knowledge about the functional role of these cells with a special emphasis on liver fibrosis. NK cell cytotoxicity can contribute to liver damage in different forms of liver disease. However, NK cells can limit liver fibrosis by killing hepatic stellate cell-derived myofibroblasts, which play a key role in this pathogenic process. Therefore, liver NK cells need to be tightly regulated in order to balance these beneficial and pathological effects.

Natural killer cell regulation - beyond the receptors.

Natural killer (NK) cells are lymphocytes that are important for early and effective immune responses against infections and cancer. In the last 40 years, many receptors, their corresponding ligands and signaling pathways that regulate NK cell functions have been identified. However, we now know that additional processes, such as NK cell education, differentiation and also the formation of NK cell memory, have a great impact on the reactivity of these cells. Here, we summarize the current knowledge about these modulatory processes.