CIS is a potent checkpoint in NK cell-mediated tumor immunity

The detection of aberrant cells by natural killer (NK) cells is controlled by the integration of signals from activating and inhibitory ligands and from cytokines such as IL-15. We identified cytokine-inducible SH2-containing protein (CIS, encoded by Cish) as a critical negative regulator of IL-15 signaling in NK cells. Cish was rapidly induced in response to IL-15, and deletion of Cish rendered NK cells hypersensitive to IL-15, as evidenced by enhanced proliferation, survival, IFN-γ production and cytotoxicity toward tumors. This was associated with increased JAK-STAT signaling in NK cells in which Cish was deleted. Correspondingly, CIS interacted with the tyrosine kinase JAK1, inhibiting its enzymatic activity and targeting JAK for proteasomal degradation. Cish -/- mice were resistant to melanoma, prostate and breast cancer metastasis in vivo, and this was intrinsic to NK cell activity. Our data uncover a potent intracellular checkpoint in NK cell-mediated tumor immunity and suggest possibilities for new cancer immunotherapies directed at blocking CIS function

Innate immunodeficiency following genetic ablation of Mcl1 in natural killer cells

The cytokine IL-15 is required for natural killer (NK) cell homeostasis; however, the intrinsic mechanism governing this requirement remains unexplored. Here we identify the absolute requirement for myeloid cell leukaemia sequence-1 (Mcl1) in the sustained survival of NK cells in vivo. Mcl1 is highly expressed in NK cells and regulated by IL-15 in a dose-dependent manner via STAT5 phosphorylation and subsequent binding to the 3'-UTR of Mcl1. Specific deletion of Mcl1 in NK cells results in the absolute loss of NK cells from all tissues owing to a failure to antagonize pro-apoptotic proteins in the outer mitochondrial membrane. This NK lymphopenia results in mice succumbing to multiorgan melanoma metastases, being permissive to allogeneic transplantation and being resistant to toxic shock following polymicrobial sepsis challenge. These results clearly demonstrate a non-redundant pathway linking IL-15 to Mcl1 in the maintenance of NK cells and innate immune responses in vivo

The SPRY domain–containing SOCS box protein SPSB2 targets iNOS for proteasomal degradation

Macrophages lacking SPSB2 have increased NO production and enhanced pathogen-killing capabilities due to decreased ubiquitin-mediated destruction of iNOS

DNAM-1 Expression Marks an Alternative Program of NK Cell Maturation

Natural killer (NK) cells comprise a heterogeneous population of cells important for pathogen defense and cancer surveillance. However, the functional significance of this diversity is not fully understood. Here, we demonstrate through transcriptional profiling andfunctional studies that the activating receptor DNAM-1 (CD226) identifies two distinct NK cell functional subsets: DNAM-1 and DNAM-1 NK cells. DNAM-1 NK cells produce high levels of inflammatory cytokines, have enhanced interleukin 15 signaling, and proliferate vigorously. By contrast, DNAM-1 NK cells that differentiate from DNAM-1 NK cells have greater expression of NK-cell-receptor-related genes and are higher producers of MIP1 chemokines. Collectively, our data reveal the existence of a functional program of NK cell maturation marked by DNAM-1 expression

Elevated cytokine and chemokine production in lungs of <i>Socs4^R108X/R108X</i> is associated with an increased influx of T cells.

<p>(A) Cytokine and chemokine levels were analysed by ELISA and Bioplex in lung homogenates at day 3 post-infection with X31 virus. Mean data ± S.E.M. are shown for biological replicates (n = 4 for Balb/c, n = 5 for <i>Socs4^R108X/R108X</i>), * indicates p<0.05, **<0.005. (B) Phenotypic analysis of lung hematopoietic subsets in <i>Socs4^R108X/R108X</i> and Balb/c mice at day 3 post-infection. Flow cytometry analysis was performed on homogenized lungs and extracted BAL. Data plotted include combined cell numbers from lungs and BAL, * indicates p<0.05, **<0.005. (C) Expression of <i>Socs4</i> mRNA in immune cells recovered from BAL and in the lungs of Balb/c mice infected with X31 virus. Mean data ± S.E.M. are shown for n = 3 biological replicates, u/inf = uninfected.</p

Reduced downregulation of CD62L expression on <i>Socs4^R108X/R108X</i> CD8 cells in following X31 influenza infection.

<p>(A) Flow cytometric analysis showing the gating strategy and percentages of CD62L^hi and CD62L^l°CD8 T cells on day 5 post-infection with X31 influenza virus. Representative dot plots are shown from control Balb/c and <i>Socs4^R108X/R108X</i> mice. (B) Total CD8 T cell numbers in the lymph node (MLN), lungs (BAL) and spleen (SPL) of <i>Socs4^R108X/R108X</i> and Balb/C mice on day d5, d6 and d7 following infection with X31 influenza virus. (C) Total number and percentages of CD62L^hi and CD62L^l°CD8 cells in the MLN of <i>Socs4^R108X/R108X</i> and Balb/C mice following X31 influenza virus infection.</p

Altered tissue distribution of virus-specific CD8 T cells in <i>Socs4^R108X/R108X</i> mice.

<p>(A) Total numbers (BAL+MLN+SPL) of virus-specific (K^dNP₁₄₇ positive) CD8 T cells following X31 influenza virus infection. Mean values (n = 5) are plotted, error bars represent SEM. (B) Significant differences were observed in the distribution of K^dNP₁₄₇ positive CD8 T cells in lungs (BAL) and spleens of <i>Socs4^R108X/R108X</i> and Balb/c control mice following infection. *p<0.05, **<0.005, ***<0.001 (C) Percentage of GzmB positive K^dNP₁₄₇ positive CD8 T cells in various tissues on d10 post-infection (left hand panel); percentage of CD107a positive (middle panel) or GzmB positive (right hand panel) IFN-γ positive CD8 T cells on d10 post-infection following <i>ex vivo</i> stimulation with K^dNP₁₄₇ peptide. Mean values (n = 5) are plotted, error bars represent SD.</p

Defective TCR responses in <i>Socs4^R108X/R108X</i> CD8 T cells.

<p>CD4 or CD8 T cells were purified from <i>Socs4^R108X/R108X</i> or wild-type spleens by negative selection, plated onto anti-CD3 coated plates in the presence of IL-2 and analysed at the indicated timepoints. (A) Q-PCR analysis showing increased expression of <i>Socs4</i> mRNA in purified T cells from Balb/c mice following TCR engagement (mean±SEM, n = 3). (B) Cells were analysed by flow cytometry for the expression of the T cell activation marker, CD62L (mean±S.D.), n = cells derived from 3 mice. (C–D) Cells were labelled with Cell Trace Violet (CTV) for analysis of proliferative responses. Cell numbers per division are shown in the left hand panels; total cell numbers in the centre panels, (C) CD4 T cells, (D) CD8 T cells. Results are shown as the mean±S.E.M. using pooled data from 2 independent experiments (n = 5 mice per group per experiment). Representative histograms are plotted for each subset (day 4; right hand panels).</p

The Helix-Loop-Helix Protein ID2 Governs NK Cell Fate by Tuning Their Sensitivity to Interleukin-15

International audienceThe inhibitor of DNA binding 2 (Id2) is essential for natural killer (NK) cell development with its canonical role being to antagonize E-protein function and alternate lineage fate. Here we have identified a key role for Id2 in regulating interleukin-15 (IL-15) receptor signaling and homeostasis of NK cells by repressing multiple E-protein target genes including Socs3. Id2 deletion in mature NK cells was incompatible with their homeostasis due to impaired IL-15 receptor signaling and metabolic function and this could be rescued by strong IL-15 receptor stimulation or genetic ablation of Socs3. During NK cell maturation, we observed an inverse correlation between E-protein target genes and Id2. These results shift the current paradigm on the role of ID2, indicating that it is required not only to antagonize E-proteins during NK cell commitment, but constantly required to titrate E-protein activity to regulate NK cell fitness and responsiveness to IL-15

RIPLET, and not TRIM25, is required for endogenous RIG-I-dependent antiviral responses.

The innate immune system is our first line of defense against viral pathogens. Host cell pattern recognition receptors sense viral components and initiate immune signaling cascades that result in the production of an array of cytokines to combat infection. Retinoic acid-inducible gene-I (RIG-I) is a pattern recognition receptor that recognizes viral RNA and, when activated, results in the production of type I and III interferons (IFNs) and the upregulation of IFN-stimulated genes. Ubiquitination of RIG-I by the E3 ligases tripartite motif-containing 25 (TRIM25) and Riplet is thought to be requisite for RIG-I activation; however, recent studies have questioned the relative importance of these two enzymes for RIG-I signaling. In this study, we show that deletion of Trim25 does not affect the IFN response to either influenza A virus (IAV), influenza B virus, Sendai virus or several RIG-I agonists. This is in contrast to deletion of either Rig-i or Riplet, which completely abrogated RIG-I-dependent IFN responses. This was consistent in both mouse and human cell lines, as well as in normal human bronchial cells. With most of the current TRIM25 literature based on exogenous expression, these findings provide critical evidence that Riplet, and not TRIM25, is required endogenously for the ubiquitination of RIG-I. Despite this, loss of TRIM25 results in greater susceptibility to IAV infection in vivo, suggesting that it may have an alternative role in host antiviral defense. This study refines our understanding of RIG-I signaling in viral infections and will inform future studies in the field