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

Molecular insight into targeting the NK cell immune response to cancer

Natural Killer (NK) cells have long been considered an important part of the anti-tumor immune response due to their potent cytolytic and cytokine-secreting abilities. To date, a clear demonstration of the role NK cells play in human cancer is lacking, and there are still very few examples of therapies that efficiently exploit or enhance the spontaneous ability of NK cells to destroy the autologous cancer cells. Given the paradigm shift toward cancer immunotherapy over the past decade, there is a renewed push to understand how NK cell homeostasis and function are regulated in order to therapeutically harness these cells to treat cancer. This review will highlight recent advancements in our understanding of how growth factors impact on NK cell development, differentiation, survival and function with an emphasis on how these pathways may influence NK cell activity in the tumor microenvironment and control of cancer metastasis

The antitumor effect of heparin is not mediated by direct NK cell activation

Natural killer (NK) cells are innate lymphocytes responsible for the elimination of infected or transformed cells. The activation or inhibition of NK cells is determined by the balance of target cell ligand recognition by stimulatory and inhibitory receptors on their surface. Previous reports have suggested that the glycosaminoglycan heparin is a ligand for the natural cytotoxicity receptors NKp30, NKp44 (human), and NKp46 (both human and mouse). However, the effects of heparin on NK cell homeostasis and function remain unclear. Here, we show that heparin does not enhance NK cell proliferation or killing through NK cell activation. Alternatively, in mice models, heparin promoted NK cell survival in vitro and controlled B16-F10 melanoma metastasis development in vivo. In human NK cells, heparin promisingly increased interferon (IFN)-γ production in synergy with IL-12, although the mechanism remains elusive. Our data showed that heparin is not able to increase NK cell cytotoxicity

Targeting cytokine signaling checkpoint CIS activates NK cells to protect from tumor initiation and metastasis

The cytokine-induced SH2-containing protein CIS belongs to the suppressor of cytokine signaling (SOCS) protein family. Here, we show the critical role of CIS in suppressing natural killer (NK) cell control of tumor initiation and metastasis. Cish-deficient mice were highly resistant to methylcholanthrene-induced sarcoma formation and protected from lung metastasis of B16F10 melanoma and RM-1 prostate carcinoma cells. In contrast, the growth of primary subcutaneous tumors, including those expressing the foreign antigen OVA, was unchanged in Cish-deficient mice. The combination of Cish deficiency and relevant targeted and immuno-therapies such as combined BRAF and MEK inhibitors, immune checkpoint blockade antibodies, IL-2 and type I interferon revealed further improved control of metastasis. The data clearly indicate that targeting CIS promotes NK cell antitumor functions and CIS holds great promise as a novel target in NK cell immunotherapy

GM-CSF Quantity Has a Selective Effect on Granulocytic vs. Monocytic Myeloid Development and Function

GM-CSF promotes myeloid differentiation of cultured bone marrow cells into cells of the granulocytic and monocytic lineage; the latter can further differentiate into monocytes/macrophages and dendritic cells. How GM-CSF selects for these different myeloid fates is unresolved. GM-CSF levels can change either iatrogenically (e.g., augmenting leukopoiesis after radiotherapy) or naturally (e.g., during infection or inflammation) resulting in different immunological outcomes. Therefore, we asked whether the dose of GM-CSF may regulate the development of three types of myeloid cells. Here, we showed that GM-CSF acted as a molecular rheostat where the quantity determined which cell type was favored; moreover, the cellular process by which this was achieved was different for each cell type. Thus, low quantities of GM-CSF promoted the granulocytic lineage, mainly through survival. High quantities promoted the monocytic lineage, mainly through proliferation, whereas moderate quantities promoted moDCs, mainly through differentiation. Finally, we demonstrated that monocytes/macrophages generated with different doses of GM-CSF differed in function. We contend that this selective effect of GM-CSF dose on myeloid differentiation and function should be taken into consideration during pathophysiological states that may alter GM-CSF levels and during GM-CSF agonistic or antagonistic therapy

Generation of novel Id2 and E2-2, E2A and HEB antibodies reveals novel Id2 binding partners and species-specific expression of E-proteins in NK cells

NK cells are cytotoxic lymphocytes with a key role in limiting tumour metastases. In mice, the NK cell lineage continually expresses high levels of the Inhibitor of DNA-binding 2 (Id2) protein and loss of Id2 is incongruous with their survival due to aberrant E-protein target gene activity. Using novel Id2 and E-protein antibodies that detect both mouse and human proteins, we have extensively characterised Id2 and E-protein expression in murine and human NK cells. We detected clear expression of E2 A and HEB, and to a lesser extent E2-2 in murine NK cells. In contrast HEB appears to be the major E-protein expressed in human NK cells, with minor E2-2 expression and surprisingly, no E2 A detected in primary NK cells nor human NK cell lines. These novel antibodies are also functional in immunofluorescence and immunoprecipitation. Mass spectrometry analysis of Id2 immuno-precipitated from murine NK cells revealed a number of novel associated proteins including several members of the SWI/SNF-related matrix-associated actin-dependent regulator chromatin (SMARC) and Mediator complex (MED) families. Taken together, these data highlight the utility of novel Id2 and E-protein antibodies and caution against mouse models for understanding Id2/E-protein biology in NK cells given their clearly disparate expression patternbetween species