Here we report on the existence and functionality of the immune checkpoint signal regulatory protein α (SIRPα) in NK cells and describe how it can be modulated for cell therapy. NK cell SIRPα is up-regulated upon IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counters other stimulatory signals, including IL-2, CD16, or NKG2D. Elevated expression of CD47 protected K562 tumor cells and mouse and human MHC class I–deficient target cells against SIRPα + primary NK cells, but not against SIRPα − NKL or NK92 cells. SIRPα deficiency or antibody blockade increased the killing capacity of NK cells. Overexpression of rhesus monkey CD47 in human MHC-deficient cells prevented cytotoxicity by rhesus NK cells in a xenogeneic setting. The SIRPα–CD47 axis was found to be highly species specific. Together, the results demonstrate that disruption of the SIRPα–CD47 immune checkpoint may augment NK cell antitumor responses and that elevated expression of CD47 may prevent NK cell–mediated killing of allogeneic and xenogeneic tissues.

The advance of science is dependent upon collaboration, which does not have a visa attached to it. Indeed, over 40% of all American-based Nobel Prize winners are immigrants, and data from the National Science Foundation show that 49% of postdocs and 29% of science and engineering faculty in the US are foreign-born. However, restrictive new immigration policies in the US have left many scientists deeply concerned about their future and many American-based laboratories worried about attracting the best talent. At JEM , we’re celebrating immigration by sharing the experiences of immigrant and nonimmigrant scientists on our editorial board. Alexander Rudensky and Jean-Laurent Casanova give their firsthand perspective on immigrating to the US, while Jedd Wolchok, Carl Nathan, David Holtzman, Susan Kaech, Lewis Lanier, and David Tuveson reflect on how immigration has affected their laboratories.

In this issue of JEM , Nagasawa et al. ( https://doi.org/10.1084/jem.20190490 ) have undertaken a detailed study of the cell surface phenotype, transcriptional profile, and cytokine secretion of ILC progenitor populations in human peripheral blood and tissues and describe markers that highlight the heterogeneity of these chameleons.

Endoplasmic reticulum (ER) stress is commonly observed in intestinal epithelial cells (IECs) and can, if excessive, cause spontaneous intestinal inflammation as shown by mice with IEC-specific deletion of X-box–binding protein 1 ( Xbp1 ), an unfolded protein response–related transcription factor. In this study, Xbp1 deletion in the epithelium ( Xbp1 ΔIEC ) is shown to cause increased expression of natural killer group 2 member D (NKG2D) ligand (NKG2DL) mouse UL16-binding protein (ULBP)–like transcript 1 and its human orthologue cytomegalovirus ULBP via ER stress–related transcription factor C/EBP homology protein. Increased NKG2DL expression on mouse IECs is associated with increased numbers of intraepithelial NKG2D-expressing group 1 innate lymphoid cells (ILCs; NK cells or ILC1). Blockade of NKG2D suppresses cytolysis against ER-stressed epithelial cells in vitro and spontaneous enteritis in vivo. Pharmacological depletion of NK1.1 + cells also significantly improved enteritis, whereas enteritis was not ameliorated in Recombinase activating gene 1 −/− ;Xbp1 ΔIEC mice. These experiments reveal innate immune sensing of ER stress in IECs as an important mechanism of intestinal inflammation.

Natural killer (NK) cells provide important host defense and can generate long-lived memory NK cells. Here, by using novel transgenic mice carrying inducible Cre expressed under the control of Ncr1 gene, we demonstrated that two distinct long-lived NK cell subsets differentiate in a mouse model of cytomegalovirus (MCMV) infection. NK cells expressing the MCMV-specific Ly49H receptor differentiated into memory NK cells by an activating signaling through Ly49H and Ly49H − NK cells differentiated into cytokine-activated NK cells by exposure to inflammatory cytokines during infection. Interleukin-12 is indispensable for optimal generation of both antigen-specific memory NK cells and cytokine-activated NK cells. MCMV-specific memory NK cells show enhanced effector function and augmented antitumor activity in vivo as compared with cytokine-activated NK cells, whereas cytokine-activated NK cells exhibited a more robust response to IL-15 and persisted better in an MCMV-free environment. These findings reveal that NK cells are capable of differentiation into distinct long-lived subsets with different functional properties.

Type I interferon (IFN) is crucial in host antiviral defense. Previous studies have described the pleiotropic role of type I IFNs on innate and adaptive immune cells during viral infection. Here, we demonstrate that natural killer (NK) cells from mice lacking the type I IFN-α receptor ( Ifnar −/− ) or STAT1 (which signals downstream of IFNAR) are defective in expansion and memory cell formation after mouse cytomegalovirus (MCMV) infection. Despite comparable proliferation, Ifnar −/− NK cells showed diminished protection against MCMV infection and exhibited more apoptosis compared with wild-type NK cells. Furthermore, we show that Ifnar −/− NK cells express increased levels of NK group 2 member D (NKG2D) ligands during viral infection and are susceptible to NK cell–mediated fratricide in a perforin- and NKG2D-dependent manner. Adoptive transfer of Ifnar −/− NK cells into NK cell–deficient mice reverses the defect in survival and expansion. Our study reveals a novel type I IFN–dependent mechanism by which NK cells evade mechanisms of cell death after viral infection.

Natural killer (NK) cells play a key role in the host response to cytomegalovirus (CMV) and can mediate an enhanced response to secondary challenge with CMV. We assessed the ability of mouse CMV (MCMV)–induced memory Ly49H + NK cells to respond to challenges with influenza, an acute viral infection localized to the lung, and Listeria monocytogenes , a systemic bacterial infection. MCMV-memory NK cells did not display enhanced activation or proliferation after infection with influenza or Listeria, as compared with naive Ly49H + or Ly49H − NK cells. Memory NK cells also showed impaired activation compared with naive cells when challenged with a mutant MCMV lacking m157, highlighting their antigen-specific response. Ex vivo, MCMV-memory NK cells displayed reduced phosphorylation of STAT4 and STAT1 in response to stimulation by IL-12 and type I interferon (IFN), respectively, and IFN-γ production was reduced in response to IL-12 + IL-18 compared with naive NK cells. However, costimulation of MCMV-memory NK cells with IL-12 and m157 antigen rescues their impaired response compared with cytokines alone. These findings reveal that MCMV-primed memory NK cells are diminished in their response to cytokine-driven bystander responses to heterologous infections as they become specialized and antigen-specific for the control of MCMV upon rechallenge.

Natural killer (NK) cells provide important host defense against microbial pathogens and can generate a population of long-lived memory NK cells after infection or immunization. Here, we addressed whether NK cells can expand and differentiate after alloantigen stimulation, which may be important in hematopoietic stem cell and solid tissue transplantation. A subset of NK cell in C57BL/6 mice expresses the activating Ly49D receptor that is specific for H-2D d . These Ly49D + NK cells can preferentially expand and differentiate when challenged with allogeneic H-2D d cells in the context of an inflammatory environment. H-2D d is also recognized by the inhibitory Ly49A receptor, which, when coexpressed on Ly49D + NK cells, suppresses the expansion of Ly49D + NK cells. Specificity of the secondary response of alloantigen-primed NK cells was defined by the expression of activating Ly49 receptors and regulated by the inhibitory receptors for MHC class I. Thus, the summation of signals through a repertoire of Ly49 receptors controls the adaptive immune features of NK cells responding to allogeneic cells.

Apoptosis is critical for the elimination of activated lymphocytes after viral infection. Proapoptotic factor Bim ( Bcl2l11 ) controls T lymphocyte contraction and the formation of memory T cells after infection. Natural killer (NK) cells also undergo antigen-driven expansion to become long-lived memory cells after mouse cytomegalovirus (MCMV) infection; therefore, we examined the role of Bim in regulating the MCMV-driven memory NK cell pool. Despite responding similarly early after infection, Bcl2l11 −/− Ly49H + NK cells show impaired contraction and significantly outnumber wild-type (WT) cells after the expansion phase. The inability to reduce the effector pool leads to a larger Bcl2l11 −/− NK memory subset, which displays a less mature phenotype (CD11b lo , CD27 + ) and lower levels of NK cell memory-associated markers KLRG1 and Ly6C. Bcl2l11 −/− memory NK cells demonstrate a reduced response to m157-mediated stimulation and do not protect as effectively as WT memory NK cells in an MCMV challenge model. Thus, Bim-mediated apoptosis drives selective contraction of effector NK cells to generate a pool of mature, MCMV-specific memory cells.

Viruses suppress host responses to increase infection, and understanding these mechanisms has provided insights into cellular signaling and led to novel therapies. Many viruses (e.g., Influenza virus, Rhinovirus [RV], Cytomegalovirus, Epstein-Barr virus, and Hepatitis C virus) activate epithelial epidermal growth factor receptor (EGFR), a tyrosine kinase receptor, but the role of EGFR in viral pathogenesis is not clear. Interferon (IFN) signaling is a critical innate antiviral host response and recent experiments have implicated IFN-λ, a type III IFN, as the most significant IFN for mucosal antiviral immune responses. Despite the importance of IFN-λ in epithelial antiviral responses, the role and mechanisms of epithelial IFN-λ signaling have not been fully elucidated. We report that respiratory virus-induced EGFR activation suppresses endogenous airway epithelial antiviral signaling. We found that Influenza virus– and RV-induced EGFR activation suppressed IFN regulatory factor (IRF) 1–induced IFN-λ production and increased viral infection. In addition, inhibition of EGFR during viral infection augmented IRF1 and IFN-λ, which resulted in decreased viral titers in vitro and in vivo. These findings describe a novel mechanism that viruses use to suppress endogenous antiviral defenses, and provide potential targets for future therapies.

Although natural killer (NK) cells are classified as innate immune cells, recent studies demonstrate that NK cells can become long-lived memory cells and contribute to secondary immune responses. The precise signals that promote generation of long-lived memory NK cells are unknown. Using cytokine receptor-deficient mice, we show that interleukin-12 (IL-12) is indispensible for mouse cytomegalovirus (MCMV)-specific NK cell expansion and generation of memory NK cells. In contrast to wild-type NK cells that proliferated robustly and resided in lymphoid and nonlymphoid tissues for months after MCMV infection, IL-12 receptor–deficient NK cells failed to expand and were unable to mediate protection after MCMV challenge. We further demonstrate that a STAT4-dependent IFN-γ–independent mechanism contributes toward the generation of memory NK cells during MCMV infection. Understanding the full contribution of inflammatory cytokine signaling to the NK cell response against viral infection will be of interest for the development of vaccines and therapeutics.

Although carcinoembryonic antigen (CEA)–related cell adhesion molecule 1 (CEACAM1) has been viewed as a tumor suppressor, increasing clinical evidence shows that high levels of CEACAM1 expression on tumors correlates with poor prognosis and high risk of metastasis. Here, we examined the consequences of CEACAM1 expression on tumor cells. We show that tumor cell–associated CEACAM1 causes intracellular retention of various NKG2D ligands in mouse and human tumor cells. CEACAM1-silenced tumor cells expressed more cell surface NKG2D ligands and exhibited greater sensitivity to natural killer cell–mediated cytolysis in vitro and rejection in vivo. Our studies reveal a novel mechanism through which CEACAM1-bearing tumor cells may escape immune-surveillance.

Natural killer (NK) and invariant NK T (iNKT) cells are critical in host defense against pathogens and for the initiation of adaptive immune responses. miRNAs play important roles in NK and iNKT cell development, maturation, and function, but the roles of specific miRNAs are unclear. We show that modulation of miR-150 expression levels has a differential effect on NK and iNKT cell development. Mice with a targeted deletion of miR-150 have an impaired, cell lineage–intrinsic defect in their ability to generate mature NK cells. Conversely, a gain-of-function miR-150 transgene promotes the development of NK cells, which display a more mature phenotype and are more responsive to activation. In contrast, overexpression of miR-150 results in a substantial reduction of iNKT cells in the thymus and in the peripheral lymphoid organs. The transcription factor c-Myb has been shown to be a direct target of miR-150. Our finding of increased NK cell and decreased iNKT cell frequencies in Myb heterozygous bone marrow chimeras suggests that miR-150 differentially controls the development of NK and iNKT cell lineages by targeting c-Myb.

DAP12, an immunoreceptor tyrosine-based activation motif–bearing adapter protein, is involved in innate immunity mediated by natural killer cells and myeloid cells. We show that DAP12-deficient mouse B cells and B cells from a patient with Nasu-Hakola disease, a recessive genetic disorder resulting from loss of DAP12, showed enhanced proliferation after stimulation with anti-IgM or CpG. Myeloid-associated immunoglobulin-like receptor (MAIR) II (Cd300d) is a DAP12-associated immune receptor. Like DAP12-deficient B cells, MAIR-II–deficient B cells were hyperresponsive. Expression of a chimeric receptor composed of the MAIR-II extracellular domain directly coupled to DAP12 into the DAP12-deficient or MAIR-II–deficient B cells suppressed B cell receptor (BCR)–mediated proliferation. The chimeric MAIR-II–DAP12 receptor recruited the SH2 domain–containing protein tyrosine phosphatase 1 (SHP-1) after BCR stimulation. DAP12-deficient mice showed elevated serum antibodies against self-antigens and enhanced humoral immune responses against T cell–dependent and T cell–independent antigens. Thus, DAP12-coupled MAIR-II negatively regulates B cell–mediated adaptive immune responses.

Cells of the immune system undergo homeostatic proliferation during times of lymphopenia induced by certain viral infections or caused by chemotherapy and radiation treatment. Natural killer (NK) cells are no exception and can rapidly expand in number when placed into an environment devoid of these cells. We explored the lifespan and function of mouse NK cells that have undergone homeostatic proliferation in various settings of immunodeficiency. Adoptive transfer of mature NK cells into lymphopenic mice resulted in the generation of a long-lived population of NK cells. These homeostasis-driven NK cells reside in both lymphoid and nonlymphoid organs for >6 mo and, similar to memory T cells, self-renew and slowly turn over at steady state. Furthermore, homeostatically expanded NK cells retained their functionality many months after initial transfer and responded robustly to viral infection. These findings highlight the ability of mature NK cells to self-renew and possibly persist in the host for months or years and might be of clinical importance during NK cell adoptive immunotherapy for the treatment of certain cancers.

Cytomegaloviruses (CMVs) are renowned for interfering with the immune system of their hosts. To sidestep antigen presentation and destruction by CD8 + T cells, these viruses reduce expression of major histocompatibility complex class I (MHC I) molecules. However, this process sensitizes the virus-infected cells to natural killer (NK) cell–mediated killing via the “missing self” axis. Mouse cytomegalovirus (MCMV) uses m152 and m06 encoded proteins to inhibit surface expression of MHC I molecules. In addition, it encodes another protein, m04, which forms complexes with MHC I and escorts them to the cell surface. This mechanism is believed to prevent NK cell activation and killing by restoring the “self” signature and allowing the engagement of inhibitory Ly49 receptors on NK cells. Here we show that MCMV lacking m04 was attenuated in an NK cell– and MHC I–dependent manner. NK cell–mediated control of the infection was dependent on the presence of NK cell subsets expressing different inhibitory Ly49 receptors. In addition to providing evidence for immunoevasion strategies used by CMVs to avoid NK cell control via the missing-self pathway, our study is the first to demonstrate that missing self–dependent NK cell activation is biologically relevant in the protection against viral infection in vivo.

During a screen for ethylnitrosourea-induced mutations in mice affecting blood natural killer (NK) cells, we identified a strain, designated Duane, in which NK cells were reduced in blood and spleen but increased in lymph nodes (LNs) and bone marrow (BM). The accumulation of NK cells in LNs reflected a decreased ability to exit into lymph. This strain carries a point mutation within Tbx21 (T-bet), which generates a defective protein. Duane NK cells have a 30-fold deficiency in sphingosine-1-phosphate receptor 5 (S1P 5 ) transcript levels, and S1P 5 -deficient mice exhibit an egress defect similar to Duane. Chromatin immunoprecipitation confirms binding of T-bet to the S1pr5 locus. S1P-deficient mice exhibit a more severe NK cell egress block, and the FTY720-sensitive S1P 1 also plays a role in NK cell egress from LNs. S1P 5 is not inhibited by CD69, a property that may facilitate trafficking of activated NK cells to effector sites. Finally, the accumulation of NK cells within BM of S1P-deficient mice was associated with reduced numbers in BM sinusoids, suggesting a role for S1P in BM egress. In summary, these findings identify S1P 5 as a T-bet–induced gene that is required for NK cell egress from LNs and BM.

Plasmacytoid dendritic cells (pDCs) produce copious type I interferon (IFN) upon sensing nucleic acids through Toll-like receptor (TLR) 7 and TLR9. Uncontrolled pDC activation and IFN production are implicated in lymphopenia and autoimmune diseases; therefore, a mechanism controlling pDC IFN production is essential. Human pDCs specifically express an orphan receptor, immunoglobulin-like transcript 7 (ILT7). Here, we discovered an ILT7 ligand expressed by human cell lines and identified it as bone marrow stromal cell antigen 2 (BST2; CD317). BST2 directly binds to purified ILT7 protein, initiates signaling via the ILT7–FcεRIγ complex, and strongly inhibits production of IFN and proinflammatory cytokines by pDCs. Readily induced by IFN and other proinflammatory cytokines, BST2 may modulate the human pDC’s IFN responses through ILT7 in a negative feedback fashion.

The activating natural killer (NK) cell receptor Ly49H recognizes the mouse cytomegalovirus (MCMV) m157 glycoprotein expressed on the surface of infected cells and is required for protection against MCMV. Although Ly49H has previously been shown to signal via DAP12, we now show that Ly49H must also associate with and signal via DAP10 for optimal function. In the absence of DAP12, DAP10 enables Ly49H-mediated killing of m157-bearing target cells, proliferation in response to MCMV infection, and partial protection against MCMV. DAP10-deficient Ly49H + NK cells, expressing only Ly49H–DAP12 receptor complexes, are partially impaired in their ability to proliferate during MCMV infection, display diminished ERK1/2 activation, produce less IFN-γ upon Ly49H engagement, and demonstrate reduced control of MCMV infection. Deletion of both DAP10 and DAP12 completely abrogates Ly49H surface expression and control of MCMV infection. Thus, optimal NK cell–mediated immunity to MCMV depends on Ly49H signaling through both DAP10 and DAP12.

Natural killer (NK) cells are crucial in resistance to certain viral infections, but the mechanisms used to recognize infected cells remain largely unknown. Here, we show that the activating Ly49P receptor recognizes cells infected with mouse cytomegalovirus (MCMV) by a process that requires the presence of H2-D k and the MCMV m04 protein. Using H2 chimeras between H2-D b and -D k , we demonstrate that the H2-D k peptide-binding platform is required for Ly49P recognition. We identified m04 as a viral component necessary for recognition using a panel of MCMV-deletion mutant viruses and complementation of m04 -deletion mutant (Δ m04 ) virus infection. MA/My mice, which express Ly49P and H2-D k , are resistant to MCMV; however, infection with Δm04 MCMV abrogates resistance. Depletion of NK cells in MA/My mice abrogates their resistance to wild-type MCMV infection, but does not significantly affect viral titers in mice infected with Δ m04 virus, implicating NK cells in host protection through m04-dependent recognition. These findings reveal a novel mechanism of major histocompatability complex class I–restricted recognition of virally infected cells by an activating NK cell receptor.