Activation Receptor-Dependent IFN-γ Production by NK Cells Is Controlled by Transcription, Translation, and the Proteasome.

NK cells can recognize target cells such as virus-infected and tumor cells through integration of activation and inhibitory receptors. Recognition by NK cells can lead to direct lysis of the target cell and production of the signature cytokine IFN-γ. However, it is unclear whether stimulation through activation receptors alone is sufficient for IFN-γ production. In this study, we show that NK activation receptor engagement requires additional signals for optimal IFN-γ production, which could be provided by IFN-ß or IL-12. Stimulation of murine NK cells with soluble Abs directed against NK1.1, Ly49H, Ly49D, or NKp46 required additional stimulation with cytokines, indicating that a range of activation receptors with distinct adaptor molecules require additional stimulation for IFN-γ production. The requirement for multiple signals extends to stimulation with primary m157-transgenic target cells, which triggers the activation receptor Ly49H, suggesting that NK cells do require multiple signals for IFN-γ production in the context of target cell recognition. Using quantitative PCR and RNA flow cytometry, we found that cytokines, not activating ligands, act on NK cells to express Ifng transcripts. Ly49H engagement is required for IFN-γ translational initiation. Results using inhibitors suggest that the proteasome-ubiquitin-IKK-TPL2-MNK1 axis was required during activation receptor engagement. Thus, this study indicates that activation receptor-dependent IFN-γ production is regulated on the transcriptional and translational levels.

Dual Requirement of Cytokine and Activation Receptor Triggering for Cytotoxic Control of Murine Cytomegalovirus by NK Cells.

Natural killer (NK) cells play a critical role in controlling murine cytomegalovirus (MCMV) and can mediate both cytokine production and direct cytotoxicity. The NK cell activation receptor, Ly49H, is responsible for genetic resistance to MCMV in C57BL/6 mice. Recognition of the viral m157 protein by Ly49H is sufficient for effective control of MCMV infection. Additionally, during the host response to infection, distinct immune and non-immune cells elaborate a variety of pleiotropic cytokines which have the potential to impact viral pathogenesis, NK cells, and other immune functions, both directly and indirectly. While the effects of various immune deficiencies have been examined for general antiviral phenotypes, their direct effects on Ly49H-dependent MCMV control are poorly understood. To specifically interrogate Ly49H-dependent functions, herein we employed an in vivo viral competition approach to show Ly49H-dependent MCMV control is specifically mediated through cytotoxicity but not IFNγ production. Whereas m157 induced Ly49H-dependent degranulation, efficient cytotoxicity also required either IL-12 or type I interferon (IFN-I) which acted directly on NK cells to produce granzyme B. These studies demonstrate that both of these distinct NK cell-intrinsic mechanisms are integrated for optimal viral control by NK cells.

Interferon-γ mediates chemokine-dependent recruitment of natural killer cells during viral infection.

Natural killer (NK) cells provide in vivo control of orthopoxvirus infections in association with their expansion in the draining lymph node (LN), where they are normally very rare. The mechanism of this expansion is unclear. Herein, we determined that NK-cell depletion results in enhanced infection following footpad inoculation of cowpox virus, a natural pathogen of rodents. Following cowpox virus infection in normal mice, NK cells were greatly expanded in the draining LN, were not replicating, and displayed markers similar to splenic NK cells, suggesting specific recruitment of splenic NK cells rather than in situ proliferation. Moreover, NK-cell expansion was abrogated by prior injection of clodronate-loaded liposomes, indicating a role for subcapsular sinus macrophages. Furthermore, recruitment of transferred splenic NK cells to the draining LN was pertussis toxin-sensitive, suggesting involvement of chemokine receptors. Comprehensive analysis of chemokine mRNA expression in the draining LN following infection suggested the selective involvement of CCR2, CCR5, and/or CXCR3. Mice deficient for CCR2 or CCR5 had normal NK-cell recruitment, whereas CXCR3-deficient mice displayed a major defect, which was NK cell-intrinsic. Interestingly, both induction of transcripts for CXCR3 ligands (Cxcl9 and Cxcl10) and NK-cell recruitment required IFN-γ. These data indicate that NK-cell recruitment is mediated by subcapsular sinus macrophages, IFN-γ, and CXCR3 during orthopoxvirus infection.

Isolation of murine natural killer cells.

This unit describes the isolation of natural killer (NK) cells from mouse spleen. The basic protocol describes a method for preparing a highly purified NK cell population from mouse spleen by depletion of contaminating cells with selected monoclonal antibodies (MAbs) and magnetic separation. There are several advantages to this negative selection process. One of these is that the NK cells are not coated with antibody and, therefore, are not at risk of functional perturbation by antibody cross-linking. Additionally, negative selection provides a way to isolate diverse subpopulations of NK cells without selectively purifying a specific subpopulation. Following enrichment, NK cell purity can be assessed by cell surface phenotype using flow cytometry.

Tissue-resident natural killer (NK) cells are cell lineages distinct from thymic and conventional splenic NK cells.

Natural killer (NK) cells belong to the innate immune system; they can control virus infections and developing tumors by cytotoxicity and producing inflammatory cytokines. Most studies of mouse NK cells, however, have focused on conventional NK (cNK) cells in the spleen. Recently, we described two populations of liver NK cells, tissue-resident NK (trNK) cells and those resembling splenic cNK cells. However, their lineage relationship was unclear; trNK cells could be developing cNK cells, related to thymic NK cells, or a lineage distinct from both cNK and thymic NK cells. Herein we used detailed transcriptomic, flow cytometric, and functional analysis and transcription factor-deficient mice to determine that liver trNK cells form a distinct lineage from cNK and thymic NK cells. Taken together with analysis of trNK cells in other tissues, there are at least four distinct lineages of NK cells: cNK, thymic, liver (and skin) trNK, and uterine trNK cells. DOI: http://dx.doi.org/10.7554/eLife.01659.001.