Cristina Cerboni

ATM-ATR-dependent up-regulation of DNAM-1 and NKG2D ligands on multiple myeloma cells by therapeutic agents results in enhanced NK-cell susceptibility and is associated with a senescent phenotype.

There is much evidence to support a role for natural killer (NK) cells in controlling the progression of multiple myeloma (MM), a malignancy characterized by an abnormal plasma cell proliferation in the bone marrow (BM). Induction of DNA damage response has been recently shown capable of enhancing NKG2D ligand (NKG2DL) expression, but nothing is known about DNAM-1 ligand (DNAM-1L) regulation. In this study, we show that myeloma cells treated with low doses of therapeutic agents commonly used in the management of patients with MM, such as doxorubicin, melphalan, and bortezomib, up-regulate DNAM-1 and NKG2D ligands. Accordingly, therapeutic drug treatment of MM cells increases NK-cell degranulation, the NKG2D and DNAM-1 receptors being the major triggering molecules. Similar data were also obtained using ex vivo primary plasma cells derived from MM patients. Drug-induced DNAM-1 and NKG2D ligand expression was abolished after treatment with the ATM (ataxia telangiectasia mutated) and ATR (ATM- and RAD3-related) pharmacologic inhibitors caffeine and KU-55933, and was preferentially associated with senescent cells arrested in the G2 phase of the cell cycle. Altogether, our findings have identified a common pathway that can trigger the up-regulation of different NK cell-activating ligands and suggest that NK cells represent an immunosurveillance mechanism toward cells undergoing stress-induced senescent programs.

Effects of Human Cytomegalovirus Infection on Ligands for the Activating NKG2D Receptor of NK Cells: Up-Regulation of UL16-Binding Protein (ULBP)1 and ULBP2 Is Counteracted by the Viral UL16 Protein

Human CMV (HCMV) interferes with NK cell functions at various levels. The HCMV glycoprotein UL16 binds some of the ligands recognized by the NK-activating receptor NKG2D, namely UL16-binding proteins (ULBP) 1 and 2 and MHC class I-related chain B, possibly representing another mechanism of viral immune escape. This study addressed the expression and function of these proteins in infected cells. HCMV induced the expression of all three ULBPs, which were predominantly localized in the endoplasmic reticulum of infected fibroblasts together with UL16. However, while at a lower viral dose ULBP1 and 2 surface expression was completely inhibited compared to ULBP3, at a higher viral dose cell surface expression of ULBP1 and ULBP2 was delayed. The induction of ULBPs correlated with an increased dependency on NKG2D for recognition; however, the overall NK sensitivity did not change (suggesting that additional viral mechanisms interfere with NKG2D-independent pathways for recognition). Infection with a UL16 deletion mutant virus resulted in a different pattern compared to the wild type: all three ULBP molecules were induced with similar kinetics at the cell surface, accompanied by a pronounced, entirely NKG2D-dependent increase in NK sensitivity. Together our findings show that upon infection with HCMV, the host cell responds by expression of ULBPs and increased susceptibility to the NKG2D-mediated component of NK cell recognition, but UL16 limits these effects by interfering with the surface expression of ULBP1 and ULBP2.

Pivotal Role of KARAP/DAP12 Adaptor Molecule in the Natural Killer Cell–mediated Resistance to Murine Cytomegalovirus Infection

Natural killer (NK) cells are major contributors to early defense against infections. Their effector functions are controlled by a balance between activating and inhibiting signals. To date, however, the involvement of NK cell activating receptors and signaling pathways in the defense against pathogens has not been extensively investigated. In mice, several NK cell activating receptors are coexpressed with and function through the immunoreceptor tyrosine-based activation motif (ITAM)-bearing molecule KARAP/DAP12. Here, we have analyzed the role of KARAP/DAP12 in the early antiviral response to murine cytomegalovirus (MCMV). In KARAP/DAP12 mutant mice bearing a nonfunctional ITAM, we found a considerable increase in viral titers in the spleen (30–40-fold) and in the liver (2–5-fold). These effects were attributed to NK cells. The formation of hepatic inflammatory foci appeared similar in wild-type and mutant mice, but the latter more frequently developed severe hepatitis with large areas of focal necrosis. Moreover, the percentage of hepatic NK cells producing interferon γ was reduced by 56 ± 22% in the absence of a functional KARAP/DAP12. This is the first study that shows a crucial role for a particular activating signaling pathway, in this case the one induced through KARAP/DAP12, in the NK cell–mediated resistance to an infection. Our results are discussed in relation to recent reports demonstrating that innate resistance to MCMV requires the presence of NK cells expressing the KARAP/DAP12-associated receptor Ly49H.

The Human Immunodeficiency Virus Type 1 Nef and Vpu Proteins Downregulate the Natural Killer Cell-Activating Ligand PVR

ABSTRACT The human immunodeficiency virus type 1 (HIV-1) evades the immune responses of natural killer (NK) cells through mechanisms that have been partially deciphered. Here we show that in HIV-1-infected T lymphocytes, the early viral Nef protein downmodulates PVR (CD155, Necl-5), a ligand for the activating receptor DNAM-1 (CD226) expressed by all NK cells, CD8+ T cells, and other cell types. This novel Nef activity is conserved by Nef proteins of laboratory HIV-1 strains (NL4-3, SF2) and of a patient-derived virus, but it is not maintained by HIV-2. Nef uses the same motifs to downregulate PVR and HLA-I molecules, likely by the same mechanisms. Indeed, as previously demonstrated for HLA-I, Nef reduces the total amounts of cell-associated PVR. Optimal downregulation of cell surface PVR by Nef also requires the presence of the late viral factor Vpu. In line with PVR reduction, the NK cell-mediated lysis of T cells infected by a wild-type but not Nef-deficient virus is virtually abrogated upon blocking of both DNAM-1 and another activating receptor, NKG2D, previously shown to mediate killing of HIV-infected cells. Together, these data demonstrate that the PVR downmodulation by Nef and Vpu is a strategy evolved by HIV-1 to prevent NK cell-mediated lysis of infected cells. The PVR downregulation reported here has the potential to affect the immune responses of other DNAM-1-positive cells besides NK cells and to alter multiple PVR-mediated cellular processes, such as adhesion and migration, and may thus greatly influence HIV-1 pathogenesis.

Human Cytomegalovirus Strain-Dependent Changes in NK Cell Recognition of Infected Fibroblasts

NK cells play a key role in the control of CMV infection in mice, but the mechanism by which NK cells can recognize and kill CMV-infected cells is unclear. In this study, the modulation of NK cell susceptibility of human CMV (hCMV)-infected cells was examined. We used a human lung and a human foreskin fibroblast cell line infected with clinical isolates (4636, 13B, or 109B) or with laboratory strains (AD169, Towne). The results indicate that all three hCMV clinical isolates confer a strong NK resistance, whereas only marginal or variable effects in the NK recognition were found when the laboratory strains were used. The same results were obtained regardless of the conditions of infection, effector cell activation status, cell culture conditions, and/or donor-target cell combinations. The NK cell inhibition did not correlate with HLA class I expression levels on the surface of the target cell and was independent of the leukocyte Ig-like receptor-1, as evaluated in Ab blocking experiments. No relevant changes were detected in the adhesion molecules ICAM-I and LFA-3 expressed on the cell surface of cells infected with hCMV clinical and laboratory strains. We conclude that hCMV possesses other mechanisms, related neither to target cell expression of HLA-I or adhesion molecules nor to NK cell expression of leukocyte Ig-like receptor-1, that confer resistance to NK cell recognition. Such mechanisms may be lost during in vitro passage of the virus. These results emphasize the differences between clinical hCMV isolates compared with laboratory strains.

Synergistic effect of IFN-γ and human cytomegalovirus protein UL40 in the HLA-E-dependent protection from NK cell-mediated cytotoxicity

Human CMV (HCMV) has evolved several strategies to evade the immune system of the infected host. Here, we investigated the role of the HCMV‐encoded protein UL40 in the modulation of NK cell lysis. UL40 carries in its leader sequence a nonameric peptide similar to that found in many HLA class I molecules leader sequences. This peptide up‐regulates the expression of HLA‐E, the ligand for the NK cell inhibitory receptor CD94 / NKG2A. The UL40‐encoded HLA‐E‐binding peptide was present in all HCMV clinical (4636, 13B, 109B, 3C) and laboratory (AD169) strains analyzed. However, transfection of UL40 in different cell lines (293T, 721.221, K562) did not consistently confer protection from NK lysis (as measured using NKL and the newly generated NK line Nishi), despite a moderate up‐regulation of HLA‐E. Interestingly, combined transfection and treatment with IFN‐γ increased the inhibitory effect, via an HLA‐E‐ and CD94 / NKG2A‐dependent mechanism. Although cells transfected with UL40 derived from either AD169 or 3C showed protection from NK cell lysis, infection of fibroblasts with the viruses resulted in a strong inhibition only with the clinical strain 3C. Our results suggest that UL40 and IFN‐γ‐dependent up‐regulation of HLA‐E is only one possible mechanism to avoid NK cell recognition of HCMV infected cells.

DAP12 Signaling Directly Augments Proproliferative Cytokine Stimulation of NK Cells during Viral Infections

NK cells vigorously proliferate during viral infections. During the course of murine CMV infection, this response becomes dominated by the preferential proliferation of NK cells that express the activation receptor Ly49H. The factors driving such selective NK cell proliferation have not been characterized. In this study, we demonstrate that preferential NK cell proliferation is dependent on DAP12-mediated signaling following the binding of Ly49H to its virally encoded ligand, m157. Ly49H signaling through DAP12 appears to directly augment NK cell sensitivity to low concentrations of proproliferative cytokines such as IL-15. The impact of Ly49H-mediated signaling on NK cell proliferation is masked in the presence of high concentrations of proproliferative cytokines that nonselectively drive all NK cells to proliferate.

DNAM-1 ligand expression on Ag-stimulated T lymphocytes is mediated by ROS-dependent activation of DNA-damage response: relevance for NK–T cell interaction

An important role for natural killer (NK) cells in the regulation of T-cell responses is emerging, although the receptor pairs regulating the NK-T-cell interaction have still not been identified. We found that superantigen-stimulated T cells express Nectin-2 (CD112) and poliovirus receptor (PVR; CD155), the ligands of the activating NK receptor DNAX accessory molecule-1 (DNAM-1; CD226). Interestingly, only PVR was present at the T cell surface, particularly on cells in the S and G(2)/M phases of the cell cycle. The up-regulation of PVR expression involves DNA-damage response (DDR)-dependent pathways, because we found that pharmacologic inhibition of ATM and ATR kinases reduced PVR expression and that PVR was almost exclusively induced on cells expressing the DDR marker γH2AX. Oxidative stress contributed to DDR activation, and our results showed impaired PVR levels in the presence of the reactive oxygen species (ROS) scavenger N-acetyl-cysteine (NAC), being monocytes the main ROS source needed for optimal PVR expression on activated T cells. Interestingly, in accordance with ligand expression, NK cells lysed allogeneic proliferating more efficiently than nonproliferating T lymphocytes, with a mechanism requiring the cooperation between DNAM-1 and NKG2D. These results could contribute to unraveling the role of NK cells in the down-regulation of T-cell responses in physiologic and pathologic processes such as autoimmunity or GVHD.

The DNA damage response: a common pathway in the regulation of NKG2D and DNAM-1 ligand expression in normal, infected, and cancer cells

NKG2D and DNAM-1 are two activating receptors, present on the surface of NK cells and other cells of the immune system. Their ligands – MICA, MICB, ULBP1-6 for NKG2D, PVR/CD155 and Nectin-2/CD112 for DNAM-1 – can be constitutively expressed at low levels in some normal cells, but they are more often defined as “stress-induced,” since different stimuli can positively regulate their expression. In this review, we describe the molecular mechanisms involved in the up-regulation of NKG2D and DNAM-1 ligands under different physiological and pathological “stress” conditions, including mitosis, viral infections, and cancer. We will focus on the DNA damage response, as recent advances in the field have uncovered its important role as a common signaling pathway in the regulation of both NKG2D and DNAM-1 ligand expression in response to very diverse conditions and stimuli.

Natural killer (NK) cells from killers to regulators: Distinct features between peripheral blood and decidual NK cells

Natural killer (NK) cells are a key component of innate immunity, particularly crucial during the early phase of immune responses against certain viruses, parasites, and microbial pathogens. The role of NK cell during pregnancy has been vividly discussed over the past years and it is now becoming increasingly clear that NK cells control pregnancy maintenance at several levels. In normal pregnancy, it appears that they provide benefit by properly secreting cytokines, chemokines and angiogenic factors rather than functioning as cytotoxic effector cells. However, as they are endowed with all the cytolytic weapons, they promptly become capable of attacking fetal and maternal tissues during infection and inflammation.