Thierry Walzer

Identification, activation, and selective in vivo ablation of mouse NK cells via NKp46

Natural killer (NK) cells contribute to a variety of innate immune responses to viruses, tumors and allogeneic cells. However, our understanding of NK cell biology is severely limited by the lack of consensus phenotypic definition of these cells across species, by the lack of specific marker to visualize them in situ, and by the lack of a genetic model where NK cells may be selectively ablated. NKp46/CD335 is an Ig-like superfamily cell surface receptor involved in human NK cell activation. In addition to human, we show here that NKp46 is expressed by NK cells in all mouse strains analyzed, as well as in three common monkey species, prompting a unifying phenotypic definition of NK cells across species based on NKp46 cell surface expression. Mouse NKp46 triggers NK cell effector function and allows the detection of NK cells in situ. NKp46 expression parallels cell engagement into NK differentiation programs because it is detected on all NK cells from the immature CD122+NK1.1+DX5− stage and on a minute fraction of NK-like T cells, but not on CD1d-restricted NKT cells. Moreover, human NKp46 promoter drives NK cell selective expression both in vitro and in vivo. Using NKp46 promoter, we generated transgenic mice expressing EGFP and the diphtheria toxin (DT) receptor in NK cells. DT injection in these mice leads to a complete and selective NK cell ablation. This model paves a way for the in vivo characterization and preclinical assessment of NK cell biological function.

Fate mapping analysis of lymphoid cells expressing the NKp46 cell surface receptor.

NKp46 is a cell surface receptor expressed on natural killer (NK) cells, on a minute subset of T cells, and on a population of innate lymphoid cells that produce IL-22 and express the transcription factor retinoid-related orphan receptor (ROR)-γt, referred to as NK cell receptor (NKR)+ROR-γt+ cells. Here we describe Nkp46iCre knock-in mice in which the gene encoding the improved Cre (iCre) recombinase was inserted into the Nkp46 locus. This mouse was used to noninvasively trace cells expressing NKp46 in vivo. Fate mapping experiments demonstrated the stable expression of NKp46 on NK cells and allowed a reappraisal of the sequential steps of NK cell maturation. NKp46 genetic tracing also showed that gut NKR+ROR-γt+ and NK cells represent two distinct lineages. In addition, the genetic heterogeneity of liver NK cells was evidenced. Finally, Nkp46iCre mice also represent a unique mouse model of conditional mutagenesis specifically in NKp46+ cells, paving the way for further developments in the biology of NKp46+ NK, T, and NKR+ROR-γt+ cells.

The metabolic checkpoint kinase mTOR is essential for IL-15 signaling during the development and activation of NK cells

Interleukin 15 (IL-15) controls both the homeostasis and the peripheral activation of natural killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we found that the metabolic checkpoint kinase mTOR was activated and boosted bioenergetic metabolism after exposure of NK cells to high concentrations of IL-15, whereas low doses of IL-15 triggered only phosphorylation of the transcription factor STAT5. mTOR stimulated the growth and nutrient uptake of NK cells and positively fed back on the receptor for IL-15. This process was essential for sustaining NK cell proliferation during development and the acquisition of cytolytic potential during inflammation or viral infection. The mTORC1 inhibitor rapamycin inhibited NK cell cytotoxicity both in mice and humans; this probably contributes to the immunosuppressive activity of this drug in different clinical settings.

Cutting Edge: CD8+ T Cell Priming in the Absence of NK Cells Leads to Enhanced Memory Responses

It is uncertain whether NK cells modulate T cell memory differentiation. By using a genetic model that allows the selective depletion of NK cells, we show in this study that NK cells shape CD8+ T cell fate by killing recently activated CD8+ T cells in an NKG2D- and perforin-dependent manner. In the absence of NK cells, the differentiation of CD8+ T cells is strongly biased toward a central memory T cell phenotype. Although, on a per-cell basis, memory CD8+ T cells generated in the presence or the absence of NK cells have similar functional features and recall capabilities, NK cell deletion resulted in a significantly higher number of memory Ag-specific CD8+ T cells, leading to more effective control of tumors carrying model Ags. The enhanced memory responses induced by the transient deletion of NK cells may provide a rational basis for the design of new vaccination strategies.

Interactions between Human NK Cells and Macrophages in Response to Salmonella Infection

NK cells play a key role in host resistance to a range of pathogenic microorganisms, particularly during the initial stages of infection. NK cell interactions with cells infected with viruses and parasites have been studied extensively, but human bacterial infections have not been given the same attention. We studied crosstalk between human NK cells and macrophages infected with intracellular Salmonella. These macrophages activated NK cells, resulting in secretion of IFN-γ and degranulation. Reciprocally, NK cell activation led to a dramatic reduction in numbers of intramacrophagic live bacteria. We identified three elements in the interaction of NK cells with infected macrophages. First, communication between NK cells and infected macrophages was contact-dependent. The second requirement was IL-2- and/or IL-15-dependent priming of NK cells to produce IFN-γ. The third was activation of NK cells by IL-12 and IL-18, which were secreted by the Salmonella-infected macrophages. Adhesion molecules and IL-12Rβ2 were enriched in the contact zone between NK cells and macrophages, consistent with contact- and IL-12/IL-18-dependent NK activation. Our results suggest that, in humans, bacterial clearance is consistent with a model invoking a “ménage à trois” involving NK cells, IL-2/IL-15-secreting cells, and infected macrophages.

TGF-β inhibits the activation and functions of NK cells by repressing the mTOR pathway

Blocking TGF-β signaling in natural killer cells enhances their metabolism and ability to kill tumor cells. Relieving NK cell suppression The immunosuppressive cytokine transforming growth factor–β (TGF-β) has beneficial effects when it resolves inflammation and prevents autoimmunity, but not when it inhibits antitumor immune responses. Viel et al. found that TGF-β signaling in mouse and human natural killer (NK) cells, cytotoxic cells that target tumor cells, inhibited the activation of the kinase mTOR, a central regulator of cellular metabolism and cytotoxic function. NK cells deficient in a TGF-β receptor subunit showed enhanced antitumor activity in mouse models of metastasis, suggesting that enhancing metabolism in NK cells may provide a therapeutic strategy to kill cancer cells. Transforming growth factor–β (TGF-β) is a major immunosuppressive cytokine that maintains immune homeostasis and prevents autoimmunity through its antiproliferative and anti-inflammatory properties in various immune cell types. We provide genetic, pharmacologic, and biochemical evidence that a critical target of TGF-β signaling in mouse and human natural killer (NK) cells is the serine and threonine kinase mTOR (mammalian target of rapamycin). Treatment of mouse or human NK cells with TGF-β in vitro blocked interleukin-15 (IL-15)–induced activation of mTOR. TGF-β and the mTOR inhibitor rapamycin both reduced the metabolic activity and proliferation of NK cells and reduced the abundances of various NK cell receptors and the cytotoxic activity of NK cells. In vivo, constitutive TGF-β signaling or depletion of mTOR arrested NK cell development, whereas deletion of the TGF-β receptor subunit TGF-βRII enhanced mTOR activity and the cytotoxic activity of the NK cells in response to IL-15. Suppression of TGF-β signaling in NK cells did not affect either NK cell development or homeostasis; however, it enhanced the ability of NK cells to limit metastases in two different tumor models in mice. Together, these results suggest that the kinase mTOR is a crucial signaling integrator of pro- and anti-inflammatory cytokines in NK cells. Moreover, we propose that boosting the metabolic activity of antitumor lymphocytes could be an effective strategy to promote immune-mediated tumor suppression.

Regulation of mouse NK cell development and function by cytokines

Natural Killer (NK) cells are innate lymphocytes with an important role in the early defense against intracellular pathogens and against tumors. Like other immune cells, almost every aspects of their biology are regulated by cytokines. Interleukin (IL)-15 is pivotal for their development, homeostasis, and activation. Moreover, numerous other activating or inhibitory cytokines such as IL-2, IL-4, IL-7, IL-10, IL-12, IL-18, IL-21, Transforming growth factor-β (TGFβ) and type I interferons regulate their activation and their effector functions at different stages of the immune response. In this review we summarize the current understanding on the effect of these different cytokines on NK cell development, homeostasis, and functions during steady-state or upon infection by different pathogens. We try to delineate the cellular sources of these cytokines, the intracellular pathways they trigger and the transcription factors they regulate. We describe the known synergies or antagonisms between different cytokines and highlight outstanding questions in this field of investigation. Finally, we discuss how a better knowledge of cytokine action on NK cells could help improve strategies to manipulate NK cells in different clinical situations.

Intrasplenic trafficking of natural killer cells is redirected by chemokines upon inflammation.

The spleen is a major homing site for NK cells. How they traffic to and within this site in homeostatic or inflammatory conditions is, however, mostly unknown. Here we show that NK cells enter the spleen through the marginal sinus and home to the red pulp via a pertussis toxin‐insensitive mechanism. Upon inflammation induced by poly(I:C) injection or mouse cytomegalovirus infection, many NK cells left the red pulp while others transiently entered the white pulp, predominantly the T cell area. This migration was dependent on both CXCR3 and CCL5, suggesting a synergy between CXCR3 and CCR5, and followed the path lined by fibroblastic reticular cells. Thus, the entry of NK cells in the white pulp is limited by the expression of pro‐inflammatory chemokines. This phenomenon ensures the segregation of NK cells outside of the white pulp and might contribute to the control of immunopathology.

Terminal NK cell maturation is controlled by concerted actions of T-bet and Zeb2 and is essential for melanoma rejection.

The transcription factor Zeb2 cooperates with T-bet to control NK cell maturation, viability, and exit from the bone marrow and is essential for rejection of melanoma lung metastasis.

Efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in the treatment of vasculopathy associated with TMEM173-activating mutations in 3 children

Marie-Louise Frémond, MD, Mathieu Paul Rodero, PhD, Nadia Jeremiah, PhD, Alexandre Belot, MD, PhD, Eric Jeziorski, MD, Darragh Duffy, PhD, Didier Bessis, MD, Guilhem Cros, MD, Gillian I. Rice, PhD, Bruno Charbit, MSc, Anne Hulin, PharmD, PhD, Nihel Khoudour, MD, Consuelo Modesto Caballero, MD, Christine Bodemer, MD, PhD, Monique Fabre, MD, Laureline Berteloot, MD, Muriel Le Bourgeois, MD, Philippe Reix, MD, Thierry Walzer, PhD, Despina Moshous, MD, PhD, Stéphane Blanche, MD, PhD, Alain Fischer, MD, PhD, Brigitte Bader-Meunier, MD, Fréderic Rieux-Laucat, PhD, Yanick Joseph Crow, MD, PhD, Bénédicte Neven, MD, PhD