Author response for "Loss of NKG2D in murine NK cells leads to increased perforin production upon long‐term stimulation with IL‐2"
Daniela PrinzKlara R. KleinJulia ListVanessa M. KnabIngeborg MenzlNicoletta LeidenfrostGerwin HellerBojan PolićEva Maria PutzAgnieszka Witalisz‐SieprackaVeronika SexlDagmar Gotthardt
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NKG2D
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Cytolysin
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NK cells are innate lymphocytes responsible for lysis of pathogen-infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46+ cells (NKG2DΔNK ). NKG2DΔNK NK cells develop normally, have an unaltered IFN-γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long-term stimulation with IL-2, NKG2D-deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL-2 activated NK cells.
NKG2D
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Conformational change
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Fibroblasts are known to eliminate intracellular bacteria, but the lethal hit of the bactericidal mechanism has not been defined. We show that primary embryonic and established fibroblasts can be induced by interferons or by intracellular bacterial infection to express a perforin-like mRNA previously described as macrophage-expressed gene 1 (Mpeg1). The presence and level of the perforin-like mRNA correlate with the ability of primary mouse embryonic fibroblasts (MEF) to eliminate intracellular bacteria. In addition, siRNA knockdown of the perforin-like molecule abolishes bactericidal activity and allows intracellular bacterial replication. Complementation of MEF in which the endogenous perforin-like molecule has been knocked down with a red fluorescent protein-tagged version restores bactericidal activity. The perforin-like molecule has broad bactericidal specificity for pathogenic and non-pathogenic bacteria, including Gram-positive and -negative, and acid fast bacteria. The perforin-like molecule renders previously lysozyme-resistant bacteria sensitive to lysis by lysozyme suggesting physical damage of the outer cell wall by the perforin-like protein. MEF damage cell walls of intracellular bacteria by insertion, polymerization, and pore formation of the perforin-like protein, analogous to pore formers of complement and perforin-1 of cytolytic lymphocytes. We propose the name perforin-2.
Intracellular parasite
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Fas ligand
Granzyme
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Although natural killer (NK) cells play an important antitumor role, melanoma cells may affect their effector functions. In this study, we analyzed the expression of various receptors and effector molecules in NK cells and their subsets in metastatic melanoma (MM) patients compared with healthy controls (HCs). In HC and MM patients, we analyzed NK cell activity using a chromium release assay and the expression of CD107a degranulation marker, activating NKG2D, NKp46, DNAM-1, and inhibitory CD158a and CD158b receptors, IL-12R beta 1, IL-12R beta 2, intracellular interferon (IFN)-γ, perforin, and STAT-1 in CD3-CD56+ NK cells, and cytotoxic CD3-CD56 and immunoregulatory CD3-CD56 subsets by flow cytometry. MM patients compared with HC not only had significantly decreased NK cell activity, lower expression of CD107a, and impaired IFN-γ production but also had decreased expression of activating NKG2D, NKp46, and DNAM-1 receptors, which was followed by lower expression of perforin, STAT-1, and both IL-12R subunits in NK cells. In MM patients only, there was a positive correlation between NKG2D expression and degranulation capacity, as well as IFN-γ production in NK cells. Analysis of the expression of various parameters of NK cell effector functions between MM patients with different localization of distant metastases showed that patients in the unfavorable M1c subclass had decreased expression of NKG2D and NKp46 on NK cells compared with patients in the M1a+b group. Downregulated NKG2D, NKp46, and DNAM-1 receptors associated with impaired NK cell effector function are important biomarkers of advanced disease with a poor prognosis in melanoma patients.
NKG2D
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Lymphokine-activated killer cell
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Background: Three substitutions at either Gly305 or Gly306 within the membrane attack/complex perforin domain (MACPF) of perforin have been previously identified in a number of patients with hemophagocytic lymphohistiocytosis (HLH). However, their pathogenic impact remains unclear since all the cases reported so far carried heterozygous genotypes and showed very heterogeneous clinical presentations. Here, we report a new substitution (p.Gly306Asp) and use in silico tools to elucidate the pathogenic mechanisms and severity associated with human Gly306 and Gly305 mutations. Methods: The immunological workup included perforin expression and perforin gene (PRF1) mutation analysis. Computer algorithms based on conservation, secondary, and tertiary protein structure analyses were applied to assess the role of the mutations in disease pathogenesis. Results: In our patient, we found a previously undescribed homozygous c. 917G>A (p.Gly306Asp) mutation in the PRF1 gene that was associated with null perforin expression in her natural killer lymphocytes. Sequence alignments revealed that Gly306 and Gly305 are highly conserved positions among vertebrate perforins, as well as in other related pore-forming proteins such as bacterial cytolysins. Further in silico analyses consistently predicted mutations in these 2 positions to be pathogenic due to diminished stability of the perforin molecule. Conclusion: Age of HLH onset, severity of the disease and undetectable perforin in our p.Gly306Asp homozygous patient along with the in silico results unmask this novel mutation as highly detrimental. Our results highlight the need of combining all clinical features, in vitro phenotypes and computer based approaches to classify human perforin mutations accurately. Statement of novelty: The study of these PRF1 mutations points to an important role of the 2 glycine amino acids (Gly305 and Gly306) in the molecular stability of perforin, which may also be likely in other pore-forming proteins. Our in silico results conclude that the pathogenicity of mutations in highly conserved Gly305 and Gly306 is likely to be associated with a serious destabilization of the native perforin conformation.
Hemophagocytic Lymphohistiocytosis
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The granule exocytosis pathway of T cell cytotoxicity is absent in mice whose perforin gene has been ablated by targeted mutagenesis. The ability of activated naive T cells to undergo apoptosis in vitro following reaggregation of the TCR complex with anti-TCR mAbs via a Fas-independent pathway was found to be defective in the absence of perforin. Protection from death was most marked in CD8+ T cells. In wild-type cells, perforin was expressed at the same time that apoptosis occurred, and blockade of perforin expression by either incubation with perforin antisense oligonucleotides or with anti-IL-2 Abs resulted in increased viability of activated T cells. The role of perforin was not via perforin-dependent fratricidal killing. The results suggest a model in which perforin acts internally to cause a form of activation-induced T cell death distinct from that caused by members of the TNFR superfamily.
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