Mariella Della Chiesa

Transforming growth factor β1 inhibits expression of NKp30 and NKG2D receptors: Consequences for the NK-mediated killing of dendritic cells

The surface density of the triggering receptors responsible for the natural killer (NK)-mediated cytotoxicity is crucial for the ability of NK cells to kill susceptible target cells. In this study, we show that transforming growth factor β1 (TGFβ1) down-regulates the surface expression of NKp30 and in part of NKG2D but not that of other triggering receptors such as NKp46. The TGFβ1-mediated inhibition of NKp30 surface expression reflects gene regulation at the transcriptional level. NKp30 has been shown to represent the major receptor involved in the NK-mediated killing of dendritic cells. Accordingly, the TGFβ1-dependent down-regulation of NKp30 expression profoundly inhibited the NK-mediated killing of dendritic cells. On the contrary, killing of different NK-susceptible tumor cell lines was variably affected, reflecting the differential usage of NKp30 and/or NKG2D in the lysis of such tumors. Our present data suggest a possible mechanism by which TGFβ1-producing dendritic cells may acquire resistance to the NK-mediated attack.

The natural killer cell-mediated killing of autologous dendritic cells is confined to a cell subset expressing CD94/NKG2A, but lacking inhibitory killer Ig-like receptors

The cognate NK–DC interaction in inflamed tissues results in NK cell activation and acquisition of cytotoxicity against immature DC (iDC). This may represent a mechanism of DC selection required for the control of downstream adaptive immune responses. Here we show that killing of monocyte‐derived iDC is confined to the NK cell subset that expresses CD94/NKG2A, but not killer Ig‐like receptors (KIR). Consistent with these data, the expression of HLA‐E (i.e. the cellular ligand of CD94/NKG2A) was down‐regulated in iDC. On the other hand, HLA‐B and HLA‐C down‐regulation in iDC was not sufficient to induce cytotoxicity in NK cells expressing KIR3DL1 or KIR2DL. Remarkably, CD94/NKG2A+KIR– NK cells were heterogeneous in their ability to kill iDC and an inverse correlation existed between their CD94/NKG2A surface density and the magnitude of their cytolytic activity. It is conceivable that the reduced CD94/NKG2A surface density enables these cells to efficiently sense the decrease of HLA‐E surface expression in iDC. Finally, most NK cells that lysed iDC did not kill mature DC that express higher amounts of HLA class I molecules (including HLA‐E)as compared with iDC. However, a small NK cell subset was capable of killing not only iDC but also mature DC.

Preclinical characterization of 1-7F9, a novel human anti―KIR receptor therapeutic antibody that augments natural killer―mediated killing of tumor cells

Inhibitory-cell killer immunoglobulin-like receptors (KIR) negatively regulate natural killer (NK) cell-mediated killing of HLA class I-expressing tumors. Lack of KIR-HLA class I interactions has been associated with potent NK-mediated antitumor efficacy and increased survival in acute myeloid leukemia (AML) patients upon haploidentical stem cell transplantation from KIR-mismatched donors. To exploit this pathway pharmacologically, we generated a fully human monoclonal antibody, 1-7F9, which cross-reacts with KIR2DL1, -2, and -3 receptors, and prevents their inhibitory signaling. The 1-7F9 monoclonal antibody augmented NK cell-mediated lysis of HLA-C-expressing tumor cells, including autologous AML blasts, but did not induce killing of normal peripheral blood mononuclear cells, suggesting a therapeutic window for preferential enhancement of NK-cell cytotoxicity against malignant target cells. Administration of 1-7F9 to KIR2DL3-transgenic mice resulted in dose-dependent rejection of HLA-Cw3-positive target cells. In an immunodeficient mouse model in which inoculation of human NK cells alone was unable to protect against lethal, autologous AML, preadministration of 1-7F9 resulted in long-term survival. These data show that 1-7F9 confers specific, stable blockade of KIR, boosting NK-mediated killing of HLA-matched AML blasts in vitro and in vivo, providing a preclinical basis for initiating phase 1 clinical trials with this candidate therapeutic antibody.

The small subset of CD56brightCD16- natural killer cells is selectively responsible for both cell proliferation and interferon-γ production upon interaction with dendritic cells

The encounter of NK cells with dendritic cells (DC) undergoing maturation may result in the induction of NK cell proliferation. Whether such proliferation involves most NK cells or just a subset has yet to be determined. In the present study we analyzed the nature of such proliferating NK cells by combining carboxyfluorescein succinimidyl ester staining and double‐fluorescence cytofluorimetric analysis. Freshly isolated peripheral blood NK cells cultured with LPS and immature DC underwent proliferation; however, proliferating cells were confined to a minor NK cell subset. This subset is characterized by the CD56brightCD16–NKG2A+KIR– surface phenotype (KIR, killer Ig‐like receptor). This was further confirmed by the fact that, after cell sorting, only the CD56bright NK cells were able to proliferate in response to the DC stimulus, whereas the CD56dull were not. We also provide evidence that the CD56bright subset is the main source of IFN‐γ‐producing NK cells, upon interaction with DC. The CD56brightCD16– NK cells express a panel of surface molecules including CD62L, CCR7 and CXCR3 that may allow their homing either to secondary lymphoid compartments or to inflamed tissues. This implies that, in vivo, the interactions between DC undergoing maturation and CD56bright NK cells may occur in different tissues and have different functional implications.

Phenotypic and functional heterogeneity of human NK cells developing after umbilical cord blood transplantation: a role for human cytomegalovirus?

Natural killer (NK) cells play a crucial role in early immunity after hematopoietic stem cell transplantation because they are the first lymphocyte subset recovering after the allograft. In this study, we analyzed the development of NK cells after intrabone umbilical cord blood (CB) transplantation in 18 adult patients with hematologic malignancies. Our data indicate that, also in this transplantation setting, NK cells are the first lymphoid population detectable in peripheral blood. However, different patterns of NK-cell development could be identified. Indeed, in a group of patients, a relevant fraction of NK cells expressed a mature phenotype characterized by the KIR(+)NKG2A(-) signature 3-6 months after transplantation. In other patients, most NK cells maintained an immature phenotype even after 12 months. A possible role for cytomegalovirus in the promotion of NK-cell development was suggested by the observation that a more rapid NK-cell maturation together with expansion of NKG2C(+) NK cells was confined to patients experiencing cytomegalovirus reactivation. In a fraction of these patients, an aberrant and hyporesponsive CD56(-)CD16(+)p75/AIRM1(-) NK-cell subset (mostly KIR(+)NKG2A(-)) reminiscent of that described in patients with viremic HIV was detected. Our data support the concept that cytomegalovirus infection may drive NK-cell development after umbilical CB transplantation.

Activation of human NK cells by plasmacytoid dendritic cells and its modulation by CD4+ T helper cells and CD4+ CD25hi T regulatory cells

Plasmacytoid dendritic cells (pDC) represent a specialized cell population that produce type I interferon (IFN) in response to virus. Although type I IFN is a natural killer (NK) cell modulator, a direct role for pDC in coordinating NK cell functions has not yet been elucidated in detail, especially in humans. Here we report that human pDC, following engagement of Toll‐like receptor (TLR) 9, not only activate autologous NK cells, as indicated by the induction of CD69 expression, but also enhance their effector functions, especially cytotoxicity. Moreover, they can induce selective proliferation of CD56bright CD16– NK cells. This activity can be strongly augmented by the addition of autologous CD4+ CD25– T helper cells in an IL‐2‐dependent fashion. Strikingly, CD4+ CD25hi T regulatory (Treg) cells completely abrogate this IL‐2‐dependent proliferation of NK cells, while they are not able to influence NK cell activation or proliferation solely induced by pDC. Our data show that TLR9‐engaged pDC represent a critical stimulus for human NK cells and that CD4+ Th cells and CD4+ CD25hi Treg cells play an important role in modulating human NK cell responses.

IL-12 or IL-4 Prime Human NK Cells to Mediate Functionally Divergent Interactions with Dendritic Cells or Tumors

In the course of inflammatory responses in peripheral tissues, NK cells may be exposed to cytokines such as IL-12 and IL-4 released by other cell types that may influence their functional activities. In the present study we comparatively analyzed purified human peripheral blood NK cells that had been exposed to either IL-12 or IL-4 during short (overnight) incubation. We show that although IL-12-cultured NK cells produced abundant IFN-γ, TNF-α, and GM-CSF in response to stimuli acting on the NKp46-activating receptor, IL-4-cultured NK cells did not release detectable levels of these cytokines. In contrast, IL-4-cultured NK cells produced significant levels of TNF-α and GM-CSF only when stimulated with PMA and ionomycin. In no instance could the production of IL-5 and IL-13 be detected. Importantly, IL-12-cultured, but not IL-4-cultured, NK cells displayed strong cytolytic activity against various tumor cells or immature dendritic cells (DCs). Moreover, only NK cells that had been cultured in IL-12 were able to induce substantial DC maturation. Our data suggest that NK cells exposed to IL-12 for a time interval compatible with in vivo responses may favor the selection of appropriate mature DCs for subsequent Th1 cell priming in secondary lymphoid organs. On the contrary, NK cells exposed to IL-4 do not exert DC selection, may impair efficient Th1 priming, and favor either tolerogenic or Th2-type responses.

Human Cytomegalovirus Infection Promotes Rapid Maturation of NK Cells Expressing Activating Killer Ig–like Receptor in Patients Transplanted with NKG2C−/− Umbilical Cord Blood

NK cells are the first lymphoid population recovering after allogeneic hematopoietic stem cell transplantation and play a crucial role in early immunity after the graft. Recently, it has been shown that human CMV (HCMV) infection/reactivation can deeply influence NK cell reconstitution after umbilical cord blood transplantation by accelerating the differentiation of mature NKG2A−killer Ig-like receptor (KIR)+ NK cells characterized by the expression of the NKG2C-activating receptor. In view of the hypothesis that NKG2C could be directly involved in NK cell maturation driven by HCMV infection, we analyzed the maturation and function of NK cells developing in three patients with hematological malignancies given umbilical cord blood transplantation from donors carrying a homozygous deletion of the NKG2C gene. We show that HCMV infection can drive rapid NK maturation, characterized by the expansion of CD56dimNKG2A−KIR+ cells, even in the absence of NKG2C expression. Interestingly, this expanded mature NK cell subset expressed surface-activating KIR that could trigger NK cell cytotoxicity, degranulation, and IFN-γ release. Given the absence of NKG2C, it is conceivable that activating KIRs may play a role in the HCMV-driven NK cell maturation and that NK cells expressing activating KIRs might contribute, at least in part, to the control of infections after transplantation.

Human NK cell receptors/markers: A tool to analyze NK cell development, subsets and function

Natural killer (NK) cells are important components of the innate immunity and play a key role in host defense by virtue of their ability to release cytokines and to mediate cytolytic activity against tumor cells and virus‐infected cells. NK cells were first described more than 30 years ago on the basis of their peculiar functional capabilities. Subsequently, thanks to the production of a variety of monoclonal antibodies, it became possible to identify surface receptors and markers expressed by NK cells as well as to characterize their functional properties. Here, we provide a brief historical overview about the discovery of human NK cell receptors and we delineate the main phenotypic features of differentiating and mature NK cells in healthy donors as well as their alterations in certain pathologic conditions.

Human NK Cells Induce Neutrophil Apoptosis via an NKp46- and Fas-Dependent Mechanism

Polymorphonuclear neutrophils (PMN) are potent inflammatory effector cells essential to host defense, but at the same time they may cause significant tissue damage. Thus, timely induction of neutrophil apoptosis is crucial to avoid tissue damage and induce resolution of inflammation. NK cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and neutrophils. Coculture experiments revealed that human NK cells could trigger caspase-dependent neutrophil apoptosis in vitro. This event was dependent on cell–cell contact, and experiments using blocking Abs indicated that the effect was mediated by the activating NK cell receptor NKp46 and the Fas pathway. CD56-depleted lymphocytes had minimal effects on neutrophil survival, suggesting that the ability to induce neutrophil apoptosis is specific to NK cells. Our findings provide evidence that NK cells may accelerate neutrophil apoptosis, and that this interaction may be involved in the resolution of acute inflammation.